The regulation of intracelMar calcium ion concentration and mitochondrial fvnction by cyclosporh A:

a putative mechanisrn for the pathogenesis of gingival overgrowth

Livia Silvestri

A thesis submitted in conformity with the requirements for the degree of Master of Science Graduate Department of Dentistry

University of Toronto

O Copyright by Livia Silvestri 2000

National Library I * m of Canada Bibliothèque nationale du Canada

Acquisitions and Acquisitions et Bibliographie Services services bibliographiques

395 Wellington Street 395, ~e Wellington Ottawa ON K I A ON4 Ottawa ON KIAON4 Canada Canada

The author has granted a non- L'auteur a accordé une licence non exclusive licence allowing the exclusive permettant à la National Library of Canada to Bibliothèque nationale du Canada de reproduce, loan, distribute or selI reproduire, prêter, distribuer ou copies of this thesis in microform, vendre des copies de cette thèse sous paper or elec~onic fomats. la forme de microfiche/filrn, de

reproduction sur papier ou sur format électronique.

The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts fiom it Ni la thèse ni des extraits substantiels may be printed or othexwise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation.

Abstract

The regulation of intracellular calcium ion concentration and mitochondrial function by cyclosporin A: a putative mechanisrn for the pathogenesis of gingival overgrowth, by Livia Silvestri. Degree: Master of Science, Department of Periodontology, Faculty of Dentistry, University of Toronto, 2000

Gingival overgrowth is associated with loss of homeostasis of collagen

metabolism çubsequent to the administration of several drugs, including the

immunosuppressant cyclosporin A. Currently, the mechanisms by w hich CsA induces

gingival overgrowth have not been resolved. My hypothesis is that the induction of

gingival overgrowth by cyclosporin A (CsA) involves the deregulation of collagen

turnover due to a disturbance of calcium utilization in the mitochondria of gingival

fibroblasts. CsA (1 0nM) inhibited collagen bead phagocytosis (Ffold; p c 0.05) in

cultured human gingival fibroblasts (HGF) and Rat2 cells. Thapsigargin (Tg; 1 FM;

pc0.01) and carbonyl cyanide mchlorophenyi hydrazone (CCCP; 10 FM; pe 0.02) also

inhibited phagocytosis of collagen beads, indicating the importance of mitochondrial

function in phagocytosis. Collagen-coated beads (CCB) induced equivalent increases

of intracellular calcium [ca2'li in HGF and Rat2 cells. Tg inhibited the collagen-bead

induced [ca2']i response 3.7-fold. Pre-treatment with CsA inhibited collagen-induced

[ca2'li and [ca2']sERCA res ponses. Collagen bead-induced p hagocytosis induced a tirne-

dependent decrease in [~a~'],i~, in both Rat2 cells and Rat2 cells pre-treated with CsA.

CsA prevented the loss of mitochondrial membrane potential of cells in suspension

indicating that CsA was indeed acting upon the mitochondrial permeability transition

pore (MPTP). Phagocytosis was decreased in mitochondria-depleted Rat2 cells

compared to Rat2 cells by Bfold ( ~ ~ 0 . 0 5 ) and by at least 2-fold when they were pre-

treated with CsA (pe 0.05). Collectively, these data indicate that CsA acts at an

intracellular locus where it: (i) inhibits calcium flux through the MPTP; (ii) inhibits calcium

exchange between the SERCA and mitochondria; and (iii) inhibits collagen

phagocytosis through a mitochondrial-dependent, calcium-regulated pathway. Future

work could focus on how the interaction of mitochondria and SERCA calcium stores

contributes to the anti-phagocytic activity of CsA.

Ackno wledgements

This is truly a project that could not have been accomplished without the

intellectual leadership, technical insight and friendship of my supen/isor, Dr. Christopher

McCulloch. The exceptional generosity that he demonstrated with his tirne and

knowledge enriched my experience as a graduate student. I am also grateful for the

technical assistance of the laboratory staff, including Pam Arora for fluorescence

microscopy, Wilson Lee for flow cytometry, Cheung Lo for the culture of mitochondria-

depleted cells and Elisabeth Lukse for immunoblotting.

I would like to thank rny family for al1 their support, encouragement, concern and

love over the years. The cornfort and security that stems from a caring farnily has

allowed me to achieve rny goals. For his unfailing motivation and understanding, I thank

rny fiancé, Frank Nicolais.

iii

Table of Contents

Abstract Acknowledgements List of Tables List of Figures List of Ab breviations

1. Literature Review

A. Periodontal Diseases

1 . l nflammatory periodontal diseases

2. Drug-induced lesions a) Drugs which cause gingival overgrowth b) Cyclosporin A

3. Common mechanisms for fibrosis a) The developrnent of fibrosis

B. Mechanisms of phagocytosis in fibroblasts

1. How is phagocytosis mediated?

2. Physiological regulation a) Growth factors and cytokines b) Integrins

C. Calcium regulation of phagocytosis

1. Calcium ion regulation

2. Calcium response to collagen phagocytosis a) calcium response within the mitochondria

3. Mitochondrial calcium metabolism

4. Calcium responses to CsA

II m..

Ill

v i vii

D. Mode1 systems and rationale

II.

III.

IV*

v.

VI.

VI I

Staternent of the Problem

Materials and Methods

Results

Discussion

Conclusions

References

List of Tables

Table 1: Drugs that induce gingival overgrowth

Table II: Propeities of the most common drugs which induce gingival overgrowth

Table III: Adverse effects of CsA therapy

List of Figures

Figure 1 :

Figure 2:

Figure 3:

Figure 4:

Figure 5:

Figure 6A:

Figure 6B:

Figure 6C:

Figure 7A:

Figure 78:

Figure 7C:

Figure 7D:

Figure 8:

Figure 9A:

Figure 9B:

Examples of possible activation mechanisms of T and B lymphocytes.

Maintenance of collagen homeostasis.

Regulation of collagen degradation.

Pathways for calcium transport in mitochondria.

Model system.

Flow cytometry - Analysis of the collagen bead binding step of phagocytosis after incubation with cyclosporin A in human gingival fibroblasts (HGFs).

CsA (10 nM) causes a marked decrease in the phagocytic capacity of HGF.

CsA causes a dose-dependent inhibition of phagocytosis.

Collagen-coated beads (CCBs) cause an equivalent rise of [ca2'li in human gingival fibroblasts (HGFs) and Rat2 cells.

Treatment with increasing concentrations of CsA induces dose-dependent increases of [ca2+li in HGF.

The addition of collagen-coated beads (CCBs) to fibroblasts in a calcium-containing medium induces a substantial calcium flux (-1 30 nM ca2') which is reduced 3.4-fold by CsA (1 0 nM).

Pre-incubation of Rat2 fibroblasts with CsA induces decreased [ca2'lsERCA response to collagen-coated beads.

Effect of agents that inhibit calcium signalling on phagocytosis in Rat2 fibroblasts.

Co-localization of MitoTracker Green and rhod 2 dyes in Rat2 fibroblasts.

The addition of collagen-coated beads to Rat2 and Rat2 fibroblasts treated with 10 nM CsA induces a time-dependent decrease in [~a~+]+l,ito.

vii

Figure 10: CsA prevents the loss of mitochondrial membrane potentiai of Rat2 fibroblasts in suspension.

Figure 1 1 A: Treatment of Rat2 fibroblasts with ethidium bromide for over 30 passages of continuous culture induces inhibition of mitochondrial DNA synthesis and reduced synthesis of the mitochondria-associated protein COX 1.

Figure 1 1 €3: Phagocytic capacity of Rat2 and Rat2EtBr fibroblasts.

Figure 12A: Incubation with CCCP inhibits [ca2+li response to collagen-coated beads (CCBs) in both Rat2 and Rat2EtBr fibroblasts.

Figure 128: Pre-incubation with 10 nM cyclosporin A (CsA) induces a large decrease in [ ~ a ~ + ] ~ ~ ~ ~ ~ in Rat2 cells but not in Rat2EtBr cells responding to CCBs.

Figure 13A: Stimulation with collagen-coated beads (CCBs) in Rat2EtSr fibroblasts stained with rhod 2/AM induces a decrease in [ ~ a ~ + ] ~ i ~ ~ of the few remaining mitochondria. 64 and Rat2EtBr.

Figure 138: CCBs induce decreased [~a*+ ]mi~~ in Rat2 and RatPEtBr. 64

Figure 13C: Addition of CCBs to Rat2 or Rat2EtBr fibroblasts pre-treated with 10 nM CsA induces decreased [ ~ a ~ ' ] m i ~ ~ . 64

Figure 14: Schematic diagram of fibroblasts with and without CsA treatment.

viii

BAPTA

CCB

CCCP

COX -1

DMSO

HGF

MPTP

PBS

SERCA

mitochondrial membrane potential

dibromo-i,2-bis-(eaminophenoxy)ethane-N,N,N', N I tetraacetic acid

intraceilular calcium concentration

rnitochondrial calcium concentration

SERCA calcium concentration

collagen-coated beads

carbonyl cyanide mchlorophenyl hydrazone

cyciosporin A

cytochrorne oxidase I

dimethylsulphoxide

human gingival fibroblasts

using 5,5',6,6'-tetrachloro-l , 1 '3,3'- tetraethylbenzirnidazolecarbocyanine iodide

membrane permeability transition pore

phosphate buffered solution

Rat2 cells treated with ethidium bromide

smooth endoplasmic reticulum ca2+ ATPase pump

I. Literature Re vie w

The specific problem addressed in this thesis is the mechanism of action of

cyclosporin A (CsA) on mitochondrial calcium signalling and collagen phagocytosis in

the context of CsA-induced gingival overgrowth. As any one of these specific topics

entalis a large literature which cannot be surveyed in detail due tu çpace restrictions, I

will focus this review on those studies which are specific to the central problem. I will

begin with an introduction on the main forms of drug-induced gingival overgrowth and a

discussion of the specific characteristics of CsA as an immunosuppressant and a

rnodulator of gingival overgrowth. A brief overview of the mechanisms for fibrosis will

lead into a discussion of collagen phagocytosis and its regulation. As the ionic

regulation of phagocytosis is a lengthy topic to review, this discussion will be restricted

to regulation of phagocytosis by calcium. This last topic will be expanded to include

calcium responses to collagen phagocytosis within different organelles, rnitochondrial

calcium metabolism, and calcium response to CsA. I will conclude the review with a

discussion of collagen phagocytic model systems and their rationale.

A. Periodontal Diseases

1. lnflammatory periodontal diseases

Periodontal diseases encompass a group of conditions which affect the

su pporting structures of the teeth including alveolar bone, root cementum, periodontal

ligament, and gingiva. The clinical signs associated with periodontal diseases may

include gingival inflammation, gingival fibrosis, epithelial ulceration and resorption of

alveolar bone. A recent classification of these diseases indicates that 'gingival

diseases' are separate from 'periodontitis'. This view is based primarily on the absence

of bone resorption with gingival diseases. As the gingiva can exhibit many altered

features with disease, gingival diseases are segmented into smaller groups based on

their etiology. Gingival diseases modified by medications include the drug-influenced

gingival enlargements (Armitage, 1 999).

Although the most common cause of gingival enlargement is an inflammatory

response to bacterial plaque (Loe, 1965), there are systemic factors which predispose

to enlargement. Specifically, the administration of certain drugs produces gingival

overgrowth (Carranza, 1 996). Kimball (1 939) first described drug-induced gingival

overgrowth due to phenytoin and until 1981 this continued to be the only drug

associated with enlargement. Subsequently, other drugs, specifically anticonvulsants,

calcium channel blockers and the immunosuppressant cyclosporin A, have been shown

to produce gingival overgrowth as a side effect. Notably, gingival overgrowth was

referred to as 'gingival hyperplasia' before histological examination revealed normal

numbers of fibroblasts surrounded by an abundance of collagen (Bonnaure-Mallet et ai.,

1995)

2. Drug-induced lesions

a) Drugs which cause gingival overgrowth

The categories of drugs which are associated with gingival overgrowth are

anticonvulsants, calcium channel blockers, and immunosuppressants (which only

includes CsA). Table I provides a list of common drugs within these categories. I will

provide a brief overview of one drug from each category to introduce a possible

common link with calcium metabolism in their mode of action.

Table 1. Drugs that induce gingival overgrowth

Drug Category 1 Agent 1 Trade name . --

Anticonvulsant Hydantoins

Succinimides

Val~roic acid

Calcium channet blockers Dihydropyridine derivatives

Benzothiazine derivatives

Phenylal kylamine de rivat ives

Ethotoin Mep henytoin Phenytoin EthosuxirnIde Methsuximide P hensuxirnide Valproic acid Cyclosporine A

Amlodipine

Felodipine Nicardi pine Nifedipine

Nimodipine Nisooldipine Nitrendipine Diltiazem

Verapamil HCI

?aganone@ ~ e s a n t o i n ~ ~ilantin" ~erontin@ celontinm Not reoorted@

~otrel@ ~orvasc@ plendi? cardene@ ~dalat@ procardia@ Nimotop@ suiar@ Not reported ~ardizern" Dilacor XR@ ~iazac@ calan@ lsoptine verelana Covera HS@

Phenytoin is an anticonvulsant which blocks or interferes with calcium influx

across cell membranes. This in turn stabilizes neuronal discharge and limits the

progression of neuronal excitation, thus depressing the motor cortex of the central

nervous system (Wilson and Kornman, 1996). Nifedipine is a calcium channel blocker

used prknarily to treat acute and chronic coronary insufficiency, including angina

pectoris and refractory hypertension (Hancock and Swan, 1992). CsA is a potent

immunosuppressive drug used primarily to prevent rejection after organ transplantation

and for treatment of autoimmune diseases (Calne et al., 1979). While the exact

immunoçuppressive mechanism of action of CsA is not completely understood, it

appears selectively and reversibly to inhibit T helper cells by interfering with the

response of these cells to an elevation of intracellular calcium ([câ2+li ) (Granelli-Piperno

et a/., 1984).

The characteristic clinicat picture of drug-induced gingival overgrowth usually

includes corn bined enlargement and inflammation. This overgrowth consists of a

primary expansion of connective tissue and epithelium which is related to the effects of

the drug and a secondary inflarnmatory component. Due to the enlargement, the

gingival sulcus is deepened which impedes effective hygienic measures. Consequently

the entire lesion takes on an inflammatory appearance because of increased

colon izat ion by su b-g ingival plaque. Consequently, the pockets (or pseudopocks?r)

become deeper, the inflammatory response is more severe, and the area becomes

more difficult to debride (Carranza, 1996). CsA-specific gingival overgrowth is also

characterized by initial enlargement of the interproximal papillae which is frequently

restricted to the anterior facial areas and may include partial coverage of the crowns.

The tissue is usually pink, dense, and resilient, with a stippled or granular surface.

There is little bleeding on probing (Raeste et al., 1978). Table II summarizes the

epidemiological and clinical properties of phenytoin, nifedipine, and CsA.

Clinical management of gingival fibrosis is difficult as the treatment often employs

surgical removal of excess tissue. This approach only addresses the signs and does

not control the source of the problem. Recurrence is common as long as drug

administration persists and treatment often consists of repeated surgical excision. If the

mechanism of fibrosis associated with gingival overgrowth were to be unraveled,

treatment outcornes could likely be improved.

b) Cyclosporin A

CsA is an immunosuppressant used after bone marrow and organ

transplantation that causes gingival overgrowth (Kahan, 1984). There is controversy

with respect to the role of plaque in CsA-induced gingival overgrowth. McGaw et al.

(1 987) reported that plaque-induced gingival inflammation exacerbated the expression

of CsA-induced gingival overgrowth. However, this notion was contested by Seymour

et al. (1987) who concluded that the magnitude of CsA-induced gingival enlargement is

related more to the plasma concentration of CsA than to the patient's periodontal status.

CsA was first studied as an immunosuppressant by Borel et al. (1976). Subsequent to

successful clinical trials in the 1970s, CsA became the drug of choice to prevent

rejection after organ transplantation. The trade name of CsA was originally

andi immune? A new oral formulation ( ~ e o r a n has been developed which is better

absorbed from the digestive systern and can be monitored by blood drug

Table Il. Properties of the most cornmon drugs which induce gingival overgrowth

CsA

Phenyfoln

Niledipine

Drug

17% il CsA blood levels c 400 ndmL, 59% il CsA b l d levels 400 nglrnL (Helti el al., 1094) 70% over 2.5 years (Daley el

al., 1986) 81% (Friskopp and

Kllnlmalm, 1986)

varies from 3% Io 84.5% (Angetopoulos and Goaz, 1072; Glkkman and Lewilus, 1941; Panuska et al, 1961)

Incidence

15% Io 83% (krak el al.. 1 087; Barclay et al., 1892; Fattore et al., 1991 ; Slavin and Taylor, 1987)

Greater risk for development in adolescents and young lemales ( Daley et al., 1986; Hetli el al., 1994; Seymour and Heasman, 1988)

Gender or Age Predilectlon

more wmmon among younger patients and Instilulionalized people (Babcock, 1965; DahHoI and Modder, 1986)

spontaneous disappearance within lew monlhs after disconlinualion 01 phenyloin

Re verslbility

mild improvemenl within 1 week ol dixonlinuation ol nitedipine, reinslilulion ol dru9 lollowing wilhdrawal resuits in recurrence within 4 weeks (Lederman el al., 1984)

rnarked reduction ol grngival enlargement requm much longer (Lainson, 1986)

no recurrence alter extraction 01 teeth (Raieilschak-Pluss el al., 1983) not presenl in edentulous

patients (Friskopp and Klinimalm, 1986)

Presen t in edentulous sites

no1 presenl in edantulous spaces and the overgrowth disappears when leeth are extracied enlargement in

edentulous mouths is rare (Oreyer el al.. 1978)

does no1 aflect edenlulous areas ( Lucas et al., 1985; Wilson and Kornman. 1996) enlargemenl seen about

dental implanls (Silverstein el al., 1995)

Dose which causes overgro wlh

CsA blood levels z 400 n! (Helti el al., 1994; Seymou~ Heasman, 1988)

CsA >5M) mgiday (Daley i sas) magnitude ot CsA-induce

gingival enlargement is reli more loo Ihe plasma concentralion lhan Io the p, periodonlal stalus (Seymoi al., 1987) some reports show a dire

relationship belween tho d ot gingival enlargement am ol Ihe phenytoin (Kapur et a

1973) others show thal the occu

and severily ol gingival enlargemenl are not neces rdated Io the dosage, concentration of phenytoin serum or saliva, of duration drug ttierapy

Rate of recurrence

blood levels >800

llmL r and

el al..

d tted

,itienI's II et

1: t egree 1 dose ,dl.,

rrenco

sarily

in i 04

combined drug therapy (concomitant adminislration il niledipirie) is a significanl risk faclor for recurrence (Rossmann et al., 1994)

recurrence aiîer surgical removal of gingival enlargement can be altenuated with dillgenl plaque control (Hall, 1969) , but no1 entirely eliminated (Slaple el al., 1978)

levels more accurately. Lindholm and Kahan (1 993) showed that a 25% increase in the

bioavailability of CsA resulted in a significant increase in the one-year graft survival rate

and the number of patients who were rejection-free. As such, NeoralQ, which is

absorbed more predictably and is not degraded by bile acids, is the preferred form of

CsA (Sells, 1994).

CsA is a highly hydrophobic cyclic endecapeptide which binds to many types of

cells with low affinity (16 = 1-5 x 10 '~ M). Since lymphocytes do not possess specific

receptors for CsA, and as its binding affinity is similar to that of phospholipid vesicles,

LeGrue et al. (1 983) suggested that the hydrophobic CsA molecule intercalates into the

phospholipid bilayer of the cell membrane and is split up into intracytoplasmic lipid

droplets to facilitate crossing of the plasma membrane.

Histopathological analysis of CsA-induced gingival overgrowth reveals excessive

collagen covered by a parakeratinized, multilayered epithelium with elongated rete pegs

(Carranza, 1996). The connective tissue may appear highly vascularized with foci of

chronic inflammatory cells (Rateitschak-PIüss, 1 983), especially plasma cells (Deliliers

et al., 1986) that are presumably a response to sub-gingival plaque.

The mode of action of CsA as an immunosuppressant has been investigated and

relies on its capacity to inhibit proliferation of activated B and T lymphocytes. However

the mechanisrn of CsA-induced gingival overgrowth has not been clarified and there is

speculation on the role of CsA as an inhibitor of the membrane permeability transition

pore in mitochondria (see below).

Both arms of the lymphocytic immune response are suppressed by CsA. T-

lymphocyte-dependent immunity (cell-mediated) and B lymphocyte-dependent

immunoglobulin synthesis (humoral immunity) are inhibited by CsA (Dongworth and

Klaus, 1982), i.e., upon stimulation, these cells respond less robustly in mounting an

immune response. Lymphocytes must be activated to respond appropriately to stimuli

and two important cytokines responsible for their activation are IL-2 and 11-4, for T and

B lymphocytes, respectively. After IL-2 or IL-4 bind to their cell surface receptors,

phosphatidylinositol bisphosphate is cleaved into diacylglycerol and inositol-1,4,5-

triphosphate (Isakov et al., 1987). Diacylglycerol can contribute to the downstream

activation of protein kinase C which then induces lymphocyte activation. lnositol-1,4,5-

triphosphate induces lymphocyte activation by elevation of cytoplasmic calcium through

release of calcium from interna1 cellular stores (King, 1988) and the maintenance of

calcium stores by leaking extracellular calcium through the cell membrane (Zilberman et

ai., 1987). In view of these findings, many investigators have tried to establish a locus

of action for CsA as an irnmunosuppressant based on its role as a modulator of [ca2'li .

CsA has been reported to not inhibit the generation of calcium signals from

intracellular (Kay et al., 1983) or extracellular sources (Gelfand et al., 1987). Based on

in vitro studies, its irnmunosuppressive behavior may be dependent on the availability of

calcium in the culture medium andlor on its ability to enter the cell (Kay, 1987).

Further, lymphocyte proliferation does not generally occur without generation of a

calcium signal. Therefore it was suggested that CsA inhibits proliferation of activated T

and B lymphocytes by interfering with calcium permeability of the cell membrane (Figure

1). Since IL-2 is an important activator of T lymphocytes, and as this process is

dependent on a rise of [ca2+li, the finding of highly selective inhibition of IL-2 synthesis

by CsA in activated T lymphocytes was significant (Granelli-Piperno et ab, 1 984).

Figure 1. Examples of possible activation rnechanisms of T and B lymphocytes

Phosphatidyi in itol bisphosphate

f i Diacylglycerol lnositol-l,4,5-trisphosphate

Lymphocyte activation

Emrnel et al. (1989) characterized the CsA-induced 11-2 suppression further and

demonstrated that transcription of 11-2 was arrested due to the inhibition of

dephosphorylation of cytoplasmic nuclear factors, (e.g. NF-AT). These factors are

necessary for transcription of 11-2. CsA binds cyclophilin, a low molecular weight

protein. Cyclophilin and CsA inhibit activation of calcineurin, a calcium- and calmodulin-

dependent phosphatase which inhibits dephosphorylation of nuclear factors

(Hanschumaker et al., 1984). If this process is the route by which CsA causes

immunosuppression, then addition of exogenous IL-2 should reverse the

immunosuppression as shown by Bunjes et al. (1981). However others have shown

only a partial recovery of T lymphocyte activation after addition of 11-2 (Kay and Benzie,

1986). Therefore other signalling intermediates may be sensitive to CsA.

Aside from its main use as an immunosuppressant, CsA unfortunately exhibits

many adverse effects in vivo (Table III). While one of the more innocuous side effects is

gingival overgrowth, these lesions provide a biological system which can be analyzed in

vitro and which suggests alternative modes of action. Apart from its action on

lymphocytes, other possible cellular functions of CsA in cells have been investigated

and an important locus of cell function seems to be centered on mitochondria.

Accordingly, in cells with increased calcium and phosphate ion concentrations,

mitochondria may undergo a permeability transition in the conductance of mitochondrial

membrane channels which results in the loss of small molecules such as glutathione

and calcium. This loss can lead to mitochondrial dysfunction. CsA inhibits these

processes (Savage et al., 1991). Crompton et al. (1 988) showed that CsA binds to

cyclophilin D, a mitochondrial protein that mediates opening of the permeability

Table 111. Adverse effects of CsA therapy

Side Effect 1 Comments

Nep hrotoxicity

Hypertension Hepatotoxicity

Infection Malignant neoplasms

Dermato logical

Neurological toxicity

Gingival overgrowth

-dose-dependent -exact mechanism is unclear -reversible with dose reduction -may be secondary to nephrotoxicity -usually dose-dependent -usually reversible -related to toxicity of graft rejection -due to immunosuppression -CsA is not carcinogenic -due to imriiunosuppressive EBV proliferation - CsA enhances production of TGF-P which promotes cells to become cancerous (Nabel, 1999) -doseodependent hypeitrichosis on the face and upper trunk in 80°h of patients -tremor which resolves with dose reduction -convulsions associated with concornmitant use of steroids in children -dose-dependent

Adapted from Thiru, S., 1989, pgs 324-359.

transition pore in the mitochondrial membrane (MPTP). This interaction inhibits opening

of the MPTP thus preventing transit of small molecules and in consequence preventing

the loss of the mitochondrial membrane potential as well. If CsA regulates the traffic of

small molecules within the mitochondria, and if mitochondrial ion homeostasis is

important in the regulation of collagen metabolism, CsA may play a role in the

dereçlilation of collagen secretion or degradation seen with gingival overgrowth.

An irnbalance between collagen synthesis and collagen degradation may lead to

drug-induced gingival overgrowth (Jones, 1986). Many investigators have studied the

effects of overgrowth-inducing drugs on collagen synthesis and the results have been

inconclusive (Newell and Irwin, 1997; Bartold, 1989; McGaw and Porter, 1988; Tipton et

al., 1991 ; McGaw and Ten-Cate, 1983). Drug-induced effects on collagen degradation

have also been investigated. Tipton et al. (1 991) demonstrated a dose-dependent

decrease in collagenase activity after CsA administration, but conflicting studies from

lngman et al. (1 994) reported that matrix metalloproteinase-1 (MMP-1) and MMP-3

cannot be detected in inflamed marginal gingival tissue of patients with periodontal

disease. Thomason et al. (1998) demonstrated that MMPs are not detected within

fibroblasts from overgrown gingiva. As collagen degradation is mediated by both a

collagenase-dependent extracellular pathway and a collagenase-independent

intracellular pathway (i.e. phagocytosis), CsA-induced deregulation of collagen

phagocytosis may be important in the etiology of gingival overgrowth. Accordingly , the

mechanisms of fibrosis and phagocytosis will be reviewed below and the possible

deregulation of these processes by CsA will be introduced.

3. Cornmon mechanisms for fibrosis

a) The developrnent of fibrosis

Fibrosis is defined as the excessive deposition of matrix components that results

in distortion or disruption of normal tissue architecture and compromised tissue function.

The development of fibrosis follows a similar pathway to that of normal wound healing

but resolution (or scarring) is impaired (Mutsaers et a/., 1997). Fibrosis can be induced

by several growth factors, including insulin-like growth factor-l (Goldstein et al., 1989)

and transforming growth factor Pl (Border et al., 1992). After the administration of CsA,

the expression of both insulin-like growth factor-l and transforming growth factor Pl is

increased, thus prornoting fibrosis (Johnson et al., 1999). As mentioned above, gingival

fibrosis may be the result of an increased rate of collagen synthesis and/or decreased

rate of collagen degradation. One aspect of degradation which rnay be significant in the

context of CsA-induced gingival overgrowth is intracellular degradation by phagocytosis.

P hagocytosis is particularly important in tissue remodelling under p hysiological

conditions such as the final stages of wound healing or during scar resolution. In both

examples, the degradation of wound collagen and other matrix proteins by phagocytic

cells is mediated by phagocytosis (Miganatti et al., 1996). In pathological conditions,

such as fibrosis, the rate of collagen phagocytosis may be decreased, thereby resulting

in net collagen overgrowth.

When phenytoin- or CsA- induced fibrosis were exarnined at the ultrastructural

level with stereologic methods, both Hall and Squier (1982) and McGaw and Porter

(1 988) observed a decreased amount of phagocytosed collagen within the gingival

cells. McCulloch and Knowles (1993) provided further N, vitro support for these

observations when phagocytosis of collagen-coated beads was inhibited in fibroblasts

treated with phenytoin or nifedipine.

B. Mechanisms of phagocytosis in fibroblasts

1. How is phagocytosis mediated?

Gingival overgrowth is a term which describes the excessive accumulation of

collagen in affected tissues (Figure 2). As such it is useful to examine the mechanisms

of collagen turnover in physiological conditions.

The fibroblast is the cell responsible for the synthesis and secretion of collagen in

gingival overgrowth, however, the fibroblast can also act as a resorptive cell when it

degrades collagen by phagocytosis. lndeed phagocytosis by gingival fibroblasts is the

main mechanism of physiological collagen degradation in vivo (Melcher and Chan,

1981) and due to the ubiquitous occurrence of collagen phagocytosis by fibroblasts,

they may be considered as professional phagocytes (Rabinovitch, 1995).

Collagen degradation may be mediated by matrix metalloproteinases (MMPs;

e.g. collagenase, gelatinase, and stromelysin), cysteine proteinases (e.g. cathepsin B

and L), or serine proteinases (e.g. plasmin and plasminogen activator). As collagen

degradation is a common process in many physiological and pathological events, it is

not surprising that the remodeling of collagen exhibits wide variations of rate. For

example, MMPs have been implicated in rapid remodeling or digestion of large amounts

of collagen during the loss of tissue architecture in acute infiammatory processes

(Birkedal-Hansen et al., 1993), tumor cell invasion and metastasis (Aznavoorian et al.,

1993), and uterine involution (Brandes and Anton, 1969). This

Figure 2. Maintenance of collagen homeostasis

MMPs p hag ocytosis

Collagen Metabolisrn in the Balance

pathway is also called the collagenase-dependent extracellular pathway (Everts and

Beertsen, 1988; Sodek and Overall, 1988).

MMPs are not usually associated with equilibrium conditions in which collagen

synthesis is balanced by collagen degradation. More cornmonly, physiological turnover

of soft connective tissues involves phagocytosis of collagen fibrils and their final

intracellular lysosome-associated degradation by cysteine proteinases. This pathway

can be regulated by growth factors and cytokines and is called the collagenase-

independent intracellular pathway (Everts and Beertsen, 1988; Sodek and Overall,

1988).

Although gingival collagen does undergo rapid turnover, this does not lead to the

net loss of tissue because the collagen turnover is in a steady-state (Figure 2). Several

studies have demonstrated that collagen degradation in physiological conditions

employs the intracellular route. Svoboda et al. (1981) showed that cross-banded

collagen fibrils were present in fibroblast lysosomes, especially in gingiva and

periodontal ligament. They also demonstrated a positive correlation between the

arnount of intracellular collagen and the rate of collagen turnover. Further, calculation of

the digestion time of internalized collagen (30 minutes); (Everts et al, 1989) is consistent

with an expected half-life of collagen corresponding to the turnover time as assessed

biochemically (Everts and Beertsen, 1 988; Everts et ai., 1 989). Finally, Liu et al. (1 995)

used mutant mice with collagenase-resistant type I collagen to show that the

collagenase-dependent extracellular pathway played an insignificant role except during

periods of extensive connective tissue turnover. As mice aged, signs such as fibrosis of

the derm is and im paired postpartum involution of the uterus were evident. Therefore,

when steady state collagen turnover predominates, as is the case during growth and

development, the int racellular collagenase-inde pendent pathway prevailed, but when

rapid collagen degradation is necessary, the extracellular collagenase-dependent

pathway prevai!ed (Figure 3).

The collagenase-independent intracellular pathway of collagen phagocytosis

involves an initial recognition of the fibril by membrane-bound receptorslintegrins

(McKeown et al., 1990). This binding step initiates actin filament remodeling (Isberg

and Tran Van Nhieu, 1995) and an increase in the expression and release of gelatinase

A (Larjava et al., 1993). This step is followed in turn by segregation and partial

digestion of the fibril andlor its surrounding non-collagenous proteins by matrix

netalloproteinases and finally lysosomal fusion with the phagosome containing partially

digested collagen fibrils and eventual degradation by cysteine proteinases within the

lysosomes.

2. Physiological Regulation

a) Growth Factors and Cytokines

Van der Zee et ai. (1995) examined the effect of several growth factors and

cytokines on phagocytosis in vitro. They concluded that only two substances had an

effect on phagocytosis: (i) interleukin-lu inhibited phagocytosis and (ii) transfoning

growth factor+ enhanced phagocytosis. When combined, they were antagonistic.

Chou et al. (1 996) investigated the role of tumor necrosis factor-a and showed in hibition

of collagen adherence and phagocytosis.

Figure 3. Reg ula tion of collagen degradation

MMPs p haggcytosis

einterleukin -1 +concanavalin A +turnor necrosis factor- a +transforming growth factor+ +concanavalin A * tumor necrosis factor- a +b transforming growth factor-8 a

Hormones may also influence phagocytosis: (i) cortisol decreases collagen

phagocytosis in pig synovial tissue (Fell et al., 1986) and (ii) increased collagen

phagocytosis was associated with post-partum uterine involution (Brandes and Anton,

1969), a process which is associated with increased progesterone.

b) lntegrins

Integrins are ceIl surface heterodimeric adhesion receptors which bind to

extracellular matrix proteins. The integrins on fibroblasts which are involved in

recognition, adhesion, and signalling in response to collagen are the alPl, ct2P1, and

-Bi (Kirchhofer et al., 1990). However, in gingival fibroblasts the a2B1 is considered to

be particularly important for the process of collagen phagocytosis (Lee et al., 1996).

Using flow cytometry, they initially showed that a2 expression was ubiquitous among

phagocytic cells and collagen phagocytosis was inhibited by blocking access to the

a& The blockade of other integrins did not alter phagocytosis. To investigate the rote

of a2B1, after binding of collagen, fibroblasts were incubated with a large number of

collagen-coated beads to occupy as many a2P1 binding sites as possible. The levels of

a2 and phagocytic efficiency were decreased. Evidently, collagen must bind to the a2

subunit before being internalized. Evidence was also presented to suggest that the a:!

subunit was internalized in the process of collagen phagocytosis as bead internalization

led to a decrease in surface levels of an, which were restored within 3 hours. Further,

Chou et al. (1 996) showed that turnor necrosis factor-a inactivated a&, thereby

inhibiting collagen binding and phagocytosis. Despite these data, the presence of a2p1

alone is not sufficient to mediate phagocytosis. This is not surprising as other collagen

receptors rnay regulate different activities. Lee et al. (1996) suggested that the a1

subunit rnay regulate the initial contact of a2 with collagen since the level of collagen-

coated beads was shown to be twice as high in cells with high al staining than with high

a2 staining. The efficiency of phagocytosis was also dependent on the number of

available collagen-coated beads and a2 . With low bead:cell ratios, the presence of

apP1 did not necessarily promote phagocytosis, suggesting an effect due to collagen-

bead availability. Further, phagocytosis seemed to be inhibited unless the a2P1 integrin

was present in at least 60% of cells.

C. Calcium Regulation of Phagocytosis

1. Calcium ion regulation

Calcium levels are very tightly controlled within the cell since intracellular calcium

concentrations are approximately 100 nM and yet extracellular calcium concentration is

- 1-2 mM. To maintain calcium homeostasis the cell possesses many pathways of

regulated entry and exit for calcium but there are fundamentally two sources of calcium.

Calcium can be released from interna1 stores within the cell or can be allowed to enter

the cell from the extracellular medium when channels in the plasma membrane are

more permeable. Although the studies on calcium signalling reviewed below refer

mainly to "profcssional" phagocytes, namely macrophages and neutrophils, Rabinovitch

(1995) notes that due to the ubiquitous occurrence of collagen phagocytosis by

fibroblasts, they rnay also be considered as professional phagocytes. In the absence of

a substantial data set on calcium signalling in fibroblasts, I review data on macrophages

and neutrophils to provide some background on calcium signalling in phagocytosis.

Murine macrophages were used by Young et al. (1 984) to illustrate the need for

tight control of [ca2']i in the execution of phagocytosis. The clamping of both

intracellular and extracellular calcium to low levels led to the inhibition of phagocytosis.

When only extracellular calcium was chelated, there was only a partial decrease of

phagocytosis. This underlines the need for calcium fluxes from intracellular stores as

well. An excessive increase in [ca2']i by ionomycin treatrnent also led to inhibition of

phagocytosis. After binding of ligand to receptors, [ca2+]i was increased and the

amplitude of this increase was directly proportional to the extent of receptor

aggregation. Kobayashi et al. (1 995) obtained similar results in human neutrophils. To

induce maximum phagocytosis, both calcium entry from the extracellular medium and

release from intracellular stores were necessary.

From both of these reports it is evident that the same controls that are used to

maintain calcium homeostasis in the absence of a phagocytic target must be maintained

to execute phagocytosis. Although individual steps of phagocytosis may seem to

promote separate rises or falls of calcium in different cell compartments, many types of

cells contain signalling networks that involve a great deal of intracellular communication

(Berridge et al., 1999)

Kwiatkowska and Sobota (1 999) have described the signalling events for

phagocytosis specific to Fcy receptors. Once the receptor binds ligand, a phagocytic

signal is generated and the cytoskeleton is rernodelled to form a phagosome. Receptor

binding induces the activation of tyrosine kinase and protein kinase C. Phosphorylation

of cytoplasmic residues on Fcy serves to promote further binding of more ligand and the

phagocytic signal is amplified. In macrophages, protein kinase C accumulates around

phagosomes (Allen et al., 1996) and isoenzymes of the kinase are activated by calcium

(Mochly-Rosen and Gordon, 1998). Consequently, calcium can amplify the phagocytic

signal further, but these observations are in the context of Fcy signalling and not

integrins.

After formation of the phagosome (which contains the internalized particle, the

integrin and parts of the plasma membrane), the phagosome fuses with a lysosome

w hich will provide the acidic environment necessary for degradation by proteases.

Fusion is regulated by calcium. For example in human neutrophils, Jaconi et al. (1990)

showed a requirement for an increase in [ca2'li to promote fusion of phagosomes and

lysosomes. Initially, Bengtsson et al. (1 993) suggested that [ca2']i does not control the

initial actin polymerization seen during receptor-ligand adhesion and subsequent

phagocytosis, but [ca2'li does control the depolymerization of the actin network so as to

promote fusion of the phagosome and lysosome. Definitive evidence on this issue has

not been obtained because experimental-specific and cell-type variations of fusion

control preclude direct comparisons.

2. Calcium response to collagen phagocytosis

In the previous section, 1 have examined the regulatory effect of calcium on

phagocytosis. In this section the effect of calcium on phagocytosis in different cellular

compartments will be reviewed. Within the cytosol, the formation of receptor-ligand

complexes and pseudopods generates rises in [ca2+li within seconds. In ongoing

phagocytic processes, [ca2'li remains elevated especially around phagosomes. As

expected there is no rise in [ca2+> when both intracellular and extracellular calcium was

chelated (Bengtsson et a l , 1993). Thus [ca2+li rises do not originate solely from

intracellular stores. It is important to consider also the uptake of extracellular calcium

within endosomes. In fact, Gerasimenko et al. (1998) showed that endosomes

delivered a substantial amount of calcium into the cell within a few minutes.

Direct evidence to illustrate the behaviour of calcium within the endoplasmic

reticulum during phagocytosis is not available but several groups have shown a physical

movement of the receptors involved in calcium regulation during phagocytosis (Favre et

al., 1996). Calcium release from intracellular stores is mediated through inositol-1,4,5-

triphosphate (InsP3) and ryanodine receptors. Calcium accumulation in stores is

mediated by sarcoplasmic/endoplasmic reticulum ca2+-~~pases , some of which are

inhibited by thapsigargin. During phagocytosis in HL-60 cells, the SERCA2 receptor,

calreticulin (a calcium-buffering protein)(Stendahl et ai., 1994) and the InsPs receptor

were translocated to the periphagosomal space (Favre et al., 1996). This suggests that

calcium within the ER may play a regulatory role in phagocytosis since the receptors

which regulate calcium levels have translocated in order to remain proximal to the

phagosome and can thereby act upon it.

Putney (1986) first developed the notion that the process of emptying and

replenishing calcium stores must be linked. He proposed that empty calcium stores

must somehow trigger the opening of store-activated channels (SOCs) in the plasma

membrane and allow calcium into the cell. He thus coined the term 'capacitative

calcium entry'. The question of how the signal of empty calcium stores is transmitted to

the SOCs is still unanswered although lrvine et al. (1990) and Berridge et al. (1995)

suggested a mechanism called 'conformational coupling'. When lnsP3 binds to the

lnsPs receptor on the endoplasrnic reticulum, calcium is released from the internal

stores. The lnsPs receptor then sustains a conformational change. It is this change

that is detected by the SOCs which are located physically close to InsP3 receptors.

lndeed van Rossurn et al. (2000) have shown that the interaction of lnsPs receptors and

SOCs control calcium entry through the plasma membrane is possibly due to their close

approximation.

a) Calcium response within the mitochondria

There is a paucity of literature on the mitochondrial calcium response due to phagocytic

stimuli. However, there is indirect evidence describing the interaction and

interdependence between calcium in the mitochondria and the endoplasmic reticulum.

Subsequent to depletion of calcium in internal stores, ATP is needed to drive the refilling

of stores by the SERCA (Gamberucci et al., 1994). This energy is derived from the

energy-producing mitochondria (Gamberucci et ai., 1 994). An additional mitochondria-

endoplasmic reticulum interaction exists in the context of the lnsPs and ryanodine

receptors. These receptors indirectly influence [ ~ a ~ + ] ~ due to their capacity to release

calcium from internal stores when bound. The extent to which they cause calcium

release depends on the calcium buffering capacity of mitochondria (Bezprozvanny et

al., 1991). A lower [ca2+li will lead to binding of the lnsPs and ryanodine receptors and

thus cause release of calcium into the cytosol whereas a higher [ca2+]i leaves the

receptors unbound (Landolfi et al., 1998). These results demonstrate that mitochondrial

function is important for appropriate calcium responses within the cell and is needed to

amplify this response.

3. Mitochondriai calcium metabolism

Mitochondria have piqued the interest of researchers as these are the only

organelles (apart from the nucleus) that possess their own genetic material and are also

the essential "powerhouses" of the cell. They are excitable organelles capable of

generating calcium signals and actively participate in cellular calcium signalling (Ichas et

a l , 1997; Babcock et ai., 1997). There are several pathways for calcium influx and

efflux as shown in figure 4. The predominant pathways for calcium uptake consist of

the calcium uniporter and the rapid mode of uptake. Calcium transport by the calcium

uniporter is dependent on the rnitochondrial membrane potential that can be generated

by redox-linked H' pumps. Consequently the more that H' is pumped out of the

mitochondria, the greater is the negative charge inside the mitochondria. To preserve

the potential across the mitochondrial membrane, more ca2' can be pumped into the

mitochondria and removed from the cytosol. The calcium uniporter is thus driven by an

electroc hemical gradient comprised of a negative mitochondrial inner membrane

potential and a low concentration of calcium in the matrix. The rapid mode uptake also

transports calcium into the mitochondria but seems to show more specificity when

dealing with brief physiologic stimuli (Bernardi, 1999).

There are three pathways for calcium efflux, the Na+-independent pathway for

ca2' efflux (NICE), the Na+-dependent pathway for ca2' efflux (NCE), and the

membrane permeability transition pore (MPTP). The NICE is possibly a H'/ ca2+

antiporter which requires a transmembrane potential as part of its driving force. It

conducts very slow movernent of ions and mediates steady-state calcium cycling in

Adapted from Bernardi, 1999. Figure 4.. Pathways for calcium transport in mitochondria. H+ pump, redox-linked H+ pumps; JN, Ca2+ uni porter; Rab rapid uptake mode; NICE, Na* -independent pathway for Ca2+ efflux; NCE, Na+ "dependent pathway for Ca2+ effiux; NHE, H+/Na* exchanger; MPTP, membrane permeability transition pore, CsA, cyclosporin A; cyp, cyclophilin; [Ca2+], ; mitochondrial Ca2+ concentration; [Ca2+], ; cytosolic Ca2+ concentration t4

O\

mitochondria. The NCE

cycling through interplay

is a ~ a ' / ~ a ~ + exc hanger that mediates physiolog ical calcium

with the NHE (H'I Na' exchanger). The MPTP is a large

channel with a pore diameter of -3 nM (Haworth and Hunter, 1979). When the pore is

open, this is described as a permeability transition and solutes with molecular mass I

1500 Da are allowed to pass into and out of the mitochondria. The pore opening

depends on transmembrane potential difference, matrix pH, and mitochondrial calcium

concentration ([ca2'lm). Prolonged opening of the pore leads to dissipation of the

mitochondrial membrane potential, leakage of cytochrome c and ultimately cell death.

This can be simulated in vitro with the addition of carbonyl cyanide mchlorophenyl

hydrazone (CCCP), a protonophore which causes a short circuit of the electrochernical

potential for protons. As a result, the ion concentration gradient is dissipated and the

electrical potential can no longer be maintained. The pore favours opening with

increases of [ca2'lm and closing with CsA. As mentioned above, CsA induces

cyclophilin D to detach from the MPTP (Nicolli et al., 1996) and thus facilitates closure

of the MPTP. In view of their involvement in the maintenance of calcium levels and their

interaction with the endoplasmic reticulurn, mitochondria evidently play an important role

in calcium homeostasis and intracellular communication. lndeed using aequorin to

target and report mitochondrial calcium ([ca2'lmit0) specifically, mitochondria can sense

microdomains of high [ca2+lc which result when IP3-sensitive channels release calcium

from the ER (Riuutto et al., 1993).

As described above, CsA inhibits the MPTP and by doing so inhibits the passage

of cations via the MPTP. As a result, calcium either cannot exit the mitochondria using

this pathway or the rate of efflux is slowed down. Since dissipation of the mitochondrial

membrane potential can lead to cell death, CsA can prevent this event and this has

been shown in vitro in gingival fibroblasts (Kulkarni et al., 1998). Sullivan et al. (1999)

showed that the addition of CsA to cells subjected to traumatic brain injury is

neuroprotective by preventing mitochondrial dysf unction. When they looked at individual

synaptosomes, there was a lower in CsA-treated cells than controls. They

proposed that because CsA inhibits the MPTP, less calcium is allowed to leave the

mitochondria and this prevents the cycling of calcium across the mitochondrial

membrane using other channels. As the effect of CsA on [ ~ a ~ ' ] ~ ~ ~ ~ does not involve

inhibition of calcineurin (which is the case with immunosuppression; Nicolli et al., 1996),

the immunosuppressive effects of CsA and its ability to inhibit the MPTP are likely

separate. To date, no other mitochondrial functions other than inhibition of the MPTP

appear to be affected by CsA (Bernardi et al., 1994; Zoratti and Szabo, 1995).

4. Calcium responses to CsA

The response of [ca2']i to CsA has received little attention although rat cardiac

cells show an upregulation of angiotensin II receptors and an increased [ca2+], that is

independent of the irnmunosuppressive action of CsA, (Le., not dependent on

calcineurin; Avdonin et a/., 1999). They proposed that these data could explain CsA-

induced hypertension and nephrotoxicity due to the vasoconstriction induced by an

augmentation of angiotensin II receptors. In addition, the sarne group examined the

effect of CsA treatment on human vascular smooth muscle cells but did not detect any

change in [ca2']i .

Whereas the effect of CsA on the rnitochondria was determined to be separate

from its action as an immunosuppressant in that it binds calcineurin, this is not the case

in the sarcoplasmic reticulum. In rat hearts, CsA mediates the upregulation of

calcineurin. By binding more calcineurin, there should be reduced phosphorylation.

Park et al. (1999) used this finding to explain a decrease in the maximal ryanodine

binding and an increaçe in the dissociation constant (&) of ryanodine binding. Since

calcium-release channels in the sarcoplasrnic reticulum need to be phosphorylated in

order to function, decreased phosphorylation due to higher levels of calcineurin leads to

less ryanodine binding and consequently less calcium release from the interna1 stores.

Model systems and rationale

An in vivo model to quantify collagen phagocytosis using electron microscopic

stereology was used by Svoboda et al., (1 981). After obtaining a magnified version of a

stained micrograph of the area of interest, two grids were overlayed on this micrograph.

With the coarse grid, the point intersections was used to estimate the number of areas

falling on cytoplasm and extracellular collagen and the fine grid was used to count the

num ber of areas falling on individual collagen profiles. This analytical system provides

a good approach to assess collagen phagocytosis in vivo, but it is both labour-intensive

and carries many assumptions on uniformity of intracellular transport, intracellular

degradation rates and cellular homogeneity. In contrast, the collagen-coated bead

model is an in vitro system used in conjunction with flow cytometry. A cell which has

engulfed a collagen-coated fluorescent bead will report a larger fluorescence intensity

per cell. The advantages of using this model are; (i) the ability to analyze a large

number of samples in a very short time; (ii) the lack of bias as to whether phagocytosis

has occurred and; (iii) the ability to report how many beads are internalized per cell.

(McKeown et al., 1 990).

As mentioned above, in vivo models are very labour-intensive and cannot

provide kinetic analysis of the data. As well, investigators do not agree on whether

incomplete phagocytosis (i.e. just cytosegregation) should be considered a positive

finding. Svoboda et al., (1 981) argued that cytosegregation should be included as this

was a necessary step for phagocytosis to occur. An important limitation of the bead

model is that it is an in vitro reductionist system and, consequently some component(s)

of the more complex in vivo setting may be omitted.

McCulloch and Knowles (1993) used the collagen-coated bead model to show

that cells from fibrotic lesions have decreased phagocytic efficiency. They also

demonstrated a reduction in phagocytosis when control cells were treated with fibrosis-

inducing drugs, including phenytoin. These findings provide the basis for my studies.

11. Statement of the problem

Literature on the cellular effects of CsA suggests a defined mechanism for its main

action as an immunosuppressant. However the modes of action which mediate rnany of

the adverse effects resulting frorn CsA administration have not been described.

Gingival overgrowth is an adverse effect that can be studied in vitro using cultured

gingival fibroblasts. Notably, fibroblasts are central to the processes of collagen

synthesis and degradation and study of collagen metabolism and its deregulation by

CsA is a logical approach to understanding the pathobiology of this disorder.

CsA acts on the mitochondrial permeability transition pore and consequently

affects calcium transport. This is relevant as two other groups of drugs which cause

gingival overgrowth also affect calcium metabolism (i.e. ant iconvulsants and calcium

channel blockers). As a result, I have considered a role for calcium in the mechanisrn of

CsA-induced gingival overgrowth. My hypothesis is that the induction of gingival

overgrowth by cyclosporin A involves deregulation of collagen turnover due to a

disturbance of calcium utilization in the mitochondria of gingival fibroblasts. The

principal rationale for this study is that the mechanism by which CsA induces gingival

overgrowth is not known and consequently, rational approaches to treatment are not

possible. If CsA regulates [ca2+] and/or mitochondrial functions, this may suggest a

possible locus of action at which gingival overgrowth is initiated.

To test my hypothesis, I investigated the effect of CsA on the phagocytic capacity

of human gingival and Rat2 fibroblasts. Next, 1 studied the effect of CsA on calcium

regulation in different cellular compartments including the cytosol, mitochondria, and

SERCA stores. I used mitochondria- depleted cells to explore the functional

relationship between mitochondrial calcium regulation in other cell compartments and

phagocytosis. My specific objectives were to:

1. Determine if CsA can affect the phagocytic capacity of human gingival fibroblasts

and Rat2 fibroblasts and if mitochondria are important for phagocytosis.

2. Establish an in vitro system to measure CsA regulation of [ca2'li in gingival

fibroblasts and Rat2 fibroblasts responding to collagen-coated beads.

3. Determine if perturbation of calcium signalling in the cytosol and in mitochondria

regulates collagen bead internalization.

4. Evaluate [~â~+],il, and [ca2'lsERCA and their responses to collagen bead stimulation

and CsA deregulation and determine if integrin-induced calcium fluxes require

mitochondrial function and [ ~ â ~ ' ] , i ~ ~ fluxes.

111. Materials and Methods

Materials

Carbonyl cyanide mchlorophenyl hydrazone and cyclosporin A were obtained from

Sigma (St. Louis, MO). Anti-human cytochrome oxidase subunit I mouse monoclonal

antibodies (clone # 6403), BAPTNAM, fura UAM, JC-1, mag-fura 2/AM, MitoTracker

Green, ~ l u r o n i c ~ F-127 and rhod 2/AM were obtained from Molecular Probes (Eugene,

OR). lonomycin and thapsigargin were obtained from Calbiochem (LaJolla, CA).

Fluorescent and non-fluorescent 2 pm diameter polystyrene beads were obtained from

Polysciences (Warrington, PA) or from Molecular Probes (Eugene, OR). Bovine

collagen (>95% type 1) was obtained from Celtrix (Palo Alto, CA).

Methods

Cell Cultures Human gingival fibroblaçts (HG F; 8- 1 5'"assage) were derived from primary explant

cultures as described (Pender and McCulloch, 1991). Cells from passages 8-15 were

grown as monolayer cultures in T-75 flasks (Costar, Mississauga, ON) containing alpha

minimal essential medium, 1 5% heat-inactivated fetal bovine serum (v/v; Flow

Laboratories, Maclean, VA), and a M O dilution of an antibiotic solution (0.17% wtlvol;

penicillin V, 0.1 Oh (wüvol) gentamicin sulfate, 0.01 pg/mL amphotericin). The cells were

maintained at 37°C in a humidified incubator containing 5% CO2 and were passaged

with 0.01 % trypsin (Gibco BRL, Burlington, Ontario, Canada). Twenty-four hours before

each experiment, cells were harvested with 0.01% trypsin and -50,000 cells were

plated onto 0.1 -mm-thick, 31 -mm-diameter, round glass coverslips (VWR, Toronto,ON)

in 35-mm-diameter Petri dishes (Falcon, Becton Dickinson, Mississauga, Ontario,

Canada) for calcium measurements. Rat2 cells (ATCC CR1 1764; Amencan Type

Culture Collection, Rockville, MD) were incubated in Dulbecco's modified Eagle's

medium (DMEM; high glucose) containing 10% fetal bovine serum (FBS) and a 1 :10

dilution of the same antibiotic solution described above. The cells were propagated,

maintained and prepared for calcium measurements as described above. According to

the ATCC, Rat2 cells are phenotypically normal by al1 criteria tested and are diploid.

They have been used in these experiments because of their ease of passaging and

their phenotypic similarity to human periodontal cells based on collagen synthesis,

collagen phagocytosis, osteopontin synthesis and morphology (Hui et ai., 1997).

Mitochondrial Depletion

Rat2 cells were grown in Dulbecco's modified Eagle's medium (DMEM; high glucose)

containing 10% fetal bovine serum (FBS) and a 1 :10 dilution of the same an antibiotic

solution described above. Cells were grown in the presence of 100 ng/mL of ethidium

bromide for 20 passages as described (Biswas et al., 1999). The dose was increased

gradually to 1 O00 ng/mL between passages 21 -30. The cells were maintained at 1000

ng/mL of ethidium bromide for at least 4 weeks before using for experiments.

[~8][ measuremen t

For measurernent of whole cell [ca2+li, cells on glass cover slips were incubated at 37°C

with 3 pM fura 2/AM in bicarbonate-free medium for 25 minutes. The attached cells

were washed twice with bicarbonate-free calcium buffer which contained 145 mM NaCI,

5 mM KCI, 5 mM MgCl2, 10 mM D-glucose, 10 mM HEPES, and 1 mM CaC12, with pH

adjusted to 7.4 and an osmolarity of 291 mosM. CaCI2 was omitted from the buffer

solution where indicated. Inspection of cells by fluorescence microscopy demonstrated

no cornpartrnentalization of dye nor were estimates of [ca2'li changed by loading of

cells with fura 2AM in Pluronic detergent. Consequently, estimates of [ca2+li using

these loading methods very likely represent cytoplasmic [ca2'li and not simply calcium

ion concentration in membrane-bound compartments such as endosomes.

Measurements of [ca2'li were made on single cells using a Nikon Diaphot II inverted

microscope optically interfaced to an epifluorescence spectrofluorimeter and analysis

system (Photon Technology International, London, ON) operating on a 386SX personal

computer. Fura Bloaded cells were excited at alternating (approximately 100 Hz)

wavelengths of 346 and 380 nm from dual monochromators with slit widths set at 2 nm.

Emitted fluorescence was collected by a 40X quartz 1.30-NA oil-immersion Nikon Fluor

objective and passed throug h a 520/30-nm barrier filter (Omega Optical, Brattleboro,

VT). Signais from the photomultiplier tube (mode1 D104; Photon Technology) were

recorded at 2 5 points/s. A variable-aperture intrabeam mask was used to restrict

measurements to single cells.

Estimates of [ca2'li independent of the precise intracellular concentration of fur&

were calculated from dual-excitation ernitted fluorescence according to the equation of

Grynkiewicz et al. (1 985) (Le., [ca2+li = & x Sf2/Sb2 x (R-R,e)/(Rm, - R) (nM), where R

is fluorescence ratio, R,, is maximum R, Rmin is minimal R, and SfZSb2 is the ratio of

the fluorescence intensity of the free and bound dye at 380-nrn excitation). The

dissociation constant (Kd ; 305 nM) and SfZSb2 ratio (4.09) were calculated from 11

excitation wavelength scans of 1 pM fura 2 free acid (Molecular Probes) in buffers with

[ca2'] ranging from 0.0 to 39.8 mM (ca2' calibration kit; Molecular Probes). R,, of

3461380 was measured after saturation of intracellular fura 2 with ca2+ by adding 3 pM

ionomycin to allow equilibration with extracellular ca2+. R,in was measured in

ionomycin-treated cells after complete dissociation of fura 2 from [ca2'li by adding 2

mM EGTA to the calcium buffer bathing the measured cell and was estimated at 0.568.

Dye loading with mag-fura U-4M

Dye loading and data collection is similar to that described for fura 2/AM except that

intensity values are not converted to [ c ~ ~ + ] ~ ~ ~ ~ A values. Instead, the ratio of

fluorescence intensity collected at 3461380 nm was used to calculate changes in

[ca2*lsERCA as described previously (Hofer et a l , 1 998).

Dye loading with rhod-UA M

RhodQ is a single wavelength excitation and single wavelength emission dye. As ratio

imaging cannot be used to overcome problems of dye leakage and photobleaching over

time with this dye, the fluorescence intensity of rhod-2 was measured in small ( ~ 4 ~ r n ~ )

sampling grids in the lamellopodia of well-spread cells. Nucleoli (which stain brightly

with rhod 2) were not included in the measurement. Background fluorescence and

adjustments for time-dependent photo-bleaching were made for each measurement

calculating the bleach-induced rate constant (Le. time-dependent loss of fluorescence)

in untreated cell samples that were not treated with collagençoated beads.

Dye loading and coimalization with rhod-2, MitoTracker Green and JC-1

Rat2 fibroblasts were CO-loaded with 100 nM MitoTracker Green and 4.5 pM Rhod-2/AM

in 0.005% pluronic gel for 30 minutes in a 37°C incubator containing 5% CO2. The

cells were washed twice and left in calcium buffer before irnaging. The magnitude of

fluorescence of stained samples was observed on an inverted microscope (Nikon)

equipped with a CCD camera (Pentamax) and analyzed using Winview (Princeton)

software program or photographs were taken.

Collagen coating of beads

Experiments with collagen-coated beads were conducted with cells grown in T-25 tissue

culture flasks. All bead incubations with cells were conducted at 37OC with 5% CO2 ,

95% humidity. Aliquots (80 pl) of either yellow-green fluorescent (excitation max = 490

nm, emission rnax = 515 nm) or non-fluorescent 2 prn diameter polystyrene beads

were incubated with 1 mL of a 2.9 mg/mL acidic bovine collagen solution to produce

collagen-coated beads. To neutralize the collagen solution to pH=7.4 and thereby

induce fibril assembly on beads, an aliquot of 1 N NaOH was added to 1 rnL of collagen

with beads and mixed by vortexing. The beads were incubated at 37OC for 10 minutes,

followed by centrifugation at 8000 x g for 2 minutes. The supernatant was removed and

the pellet of coated beads was resuspended in 1 mL phosphate-buffered saline (PBS).

After sonication of the coated beads to produce single bead suspensions, the number of

beads per mL was estimated by counting beads in an aliquot of the sample with a

hernocytometer. Cells were incubated with collagen-coated fluorescencebeads at a

ratio of 2 beads per cell for phagocytosis experiments and assessed by flow cytometry.

For calcium experirnents, non-fluorescent collagen-coated beads were added to cells at

a ratio of -4 beads per cell.

Flow cytometric analysis of phagocytosis

Binding of collagen-coated beads to cells was assessed by flow cytometry. Cell

samples were analyzed with a FACStar Plus flow cytorneter (Becton Dickinson FACS

Systems, Mountain View, CA) at a sheath pressure of 11 psi with an Innova 70 argon

laser (Coherent Laser Products, Palo Alto, CA) at a light regulation mode setting of 250

mW and a wavelength of 488 nm. Emitted fluorescence was directed through a short

pass 560 beam splitter (al1 filters and beam splitters from Omega Optical Inc.,

Brattleboro, VT) and a 530DF30 filter for green fluorescence in channel 1.

Photomultiplier tube voltage settings were determined for each experiment on the basis

of thresholds established from negative and positive control samples for each sample

that was analyzed. To reduce the inclusion of cells with loosely bound beads during the

process of phagocytosis measurernents, cells were washed with calcium and

magnesium - free PBS and trypsinized.

Flow cytometric analysis of mitochondrial membrane potential fi).

Changes in the Ym were analyzed using 5,5',6,6'-tetrachloro-1 ,113,3'-

tetraethylbenzimidazolecarbocyanine iodide (JC-1). This cyanine dye accumulates in

the mitochondrial matrix under the influence of the Ym and forms J aggregates which

have characteristic absorption and emission spectra (Smiley et al., 1991). Untreated

cells and cells treated with CsA were incubated in 3 mL of PBS supplemented with 10%

serum containing 0.5 pM JC-1 for 1 hour. As a positive control for reduction of Y,, a

group of cells were treated with the uncoupling agent carbonyl cyanide m-

chlorophenylhydrazone (CCCP) after labeling with JC-1 (Zamzami et al , 1995). Cell

suspensions were prepared for flow cytometry (Kulkarni and McCulloch, 1995) and the

488-nm line of an argon ion laser was used for excitation. Orange and green emitted

fluorescence were collected through 585142 (FL2)- and 530130-nm (FLI) bandpass

filters. The FLI-FL2 compensation was 4% and the FL2-FL1 compensation was 44%.

Flow cytometry analysis was performed on a FACSTAR Plus flow cytometer and

analyzed using LYSYS II software (Becton-Dickinson lmmunocytochemistry Systems,

Moutainview, CA). After gating out srnall-sized (i.e. noncellular debris), 10,000 events

were collected for each analysis. Bivariate plots of FL2 versus FLI were used to

analyze Y, . Fonvard scatter was used to estimate relative cell volume. Based on

foward and side scatter, and JC-1 fluorescence levels of untreated Rat2 fibroblasts,

cells were gated into high and low FL1 and FL2 populations. The percentages and

mean fluorescence intensities of these populations were cornpared to establish the

effect of increasing concentrations of CsA.

lmmunoblotting

For assessments of the mitochondrial-specific protein cytochrome oxidase 1, the

immunoblotting methods of Marusich et ai. (1997) were used. Whole ce11 extracts were

prepared by rinsing trypsinized Rat2 cells with calcium and magnesium free (CMF) PBS

containing protease inhibitors (0.5 pg/mL leupeptin, 0.5 pglmL pepstatin, and 1 mM

PMSF). Cell pellets were solubilized for 30 min at 5 x 106 cells/mL in CMF-PBS

containing the above protease inhibitors plus 1.5% lauryl maltoside at 4OC, centrifuged

for 20 minutes at 16,600 x g, and the supernatants were saved. Equivalent amounts of

protein, determined by Bradford assay, were separated on 10% polyacrylamide gels

and transferred electrophoretically ont0 0.2 pn nitrocellulose membranes in CAPS

transfer buffer (1 0% methanol in 1 0 mM 3-[cyclo hexylaminoj-1 -propanesulfonic acid, pH

11) on ice. The blots were blocked with 5% non-fat dried milk powder in CMF-PBS,

incubated overnight at 4OC with anti-human cytochrome oxidase-1 monoclonal antibody

diluted in 5% milk CMF-PBS to 1:1000, washed with 0.05% Tween in CMF-PBS,

incubated for 1 hour in horse radish peroxidase conjugated goat anti-mouse IgG at 0.2

pg/mL in 5% milk CMF-PBS and washed with 0.05% Tween in CMF-PBS. Blots were

processed for cherniluminescence, exposed to X-ray film, developed and analyzed.

Data Analysis

Means and standard errors of the means were computed for phagocytic, calcium, and

mitochondrial membrane potential experiments. Comparisons between two groups

were perfoned with the unpaired Student's t-test. Corn parisons between more than

two groups were done using ANOVA.

Mode1 System - Fibroblast

Figure 5. Model system. Upon interaction with mitochondria, CsA will alter the calcium permeability of this organelle which in turn is likely to impact on calcium fluxes in the SERCA. The central question is: what is the effect of this perturbation on collagen phagocytosis? CC8 - collagen-coated beads; CsA - cyclosporin A; SERCA - smooth endoplasrnic reticulum calcium ATPase pump.

1 V. Results

Effect of CsA on collagen bead phagocytosis

In HGF incubated with collagen-coated beads, there was abundant binding of

beads that was inhibited -2-fold with as low as 10 nM CsA (1 hour pre-incubation

with CsA; 3 hour bead incubation; Fig. 6A). The phagocytic capacity of HGFs

treated with CsA was compared to that of Rat2 fibroblasts treated with CsA to

rationalize the use of Rat2 fibroblasts for ail subsequent experiments. Rat2 cells

were incubated with increasing doses of CsA prior to three-hour incubations with

collagen-coated beads, a protocol that enables quantitative evaluation of the

bead-binding step of phagocytosis. After the addition of 10 nM CsA, there was a

significant and comparable reduction in mean O h phagocytosis in both HGFs

(pc0.05) and Rat2 fibroblasts (pc0.05; Fig. 6B). CsA doses which are

substantially lower than those used therapeutically in humans (-200yM)

produced a significant decrease in the phagocytic capacity of Rat2 fibroblasts (pc

0.05 for 10-10,000 nM CsA; Fig. 6C). Dissipation of the mitochondrial membrane

potential using CCCP ( ~ ~ 0 . 0 2 ) or inhibition of ATP formation using sodium azide

also led to significant decreases in phagocytosis similar to those seen with CsA

(pc 0.01), suggesting an involvement of mitochondrial function in the bead-

binding step of phagocytosis.

[ca2+li and [ca2TsERCn responses to collagen beads

As integrin ligation with extracellular matrix ligands stimulates calcium rises

(Schwartz, 1993) that may be important in the bead binding step of phagocytosis,

the response of [ca2'li to collagen bead-induced phagocytosis in HGFs was

10" t o ~ loS II 'O ' RlHdpht

HGF + CCB HGF + 10 nM CsA+ CCB

untreated 10 nM CsA

untreated 10 "M 100 nM 1 pM 10 pM CCCP Sodium CsA CsA CsA CsA Azide

Figure 6. A. Flow cytometry - analysis of the collagen bead binding step of phagocytosis after incubation with cyclosporin A in human gingival f i broblasts (HGFs). B. CsA (1 0 nM) causes a marked decrease in the phagocytic capacity of HGFs. Data are means I SEM (n=3). C. CsA causes a dose-dependent inhibition of phagocytosis. To study the possible involvement of mitochondria, CCCP (10 FM) and sodium azide (1%) caused a marked decrease in the phagocytic capacity of Rat2 fibroblasts. Data are means I SEM (n=3). For figs. 6 B and C, *, p< 0.05; 7, pc0.02; $, p<0.01 when compared to vehicle-treated cells. ANOVA testing for untreated tells and increasing doses of CsA revealed that the groups are significantly different at p=0.001.

examined. The ratiometric calcium-sensitive dye fura 2 was used to measure [ca2'li as

it is relatively insensitive to photobleaching and dye leakage is minimal over time. In

Fig. 7A the insert depicts a representative tracing obtained when collagen-coated beads

were added to HGF cells and [ca2']i was measured over time. As cells were incubated

with beads at a ratio of -4: 1 (beads:cell), multiple asynchronous bead-cell interactions

occur over 1500 seconds and consequently the decrease in calcium to basal levels

occurs later (not shown). The mean [ca2+li rise above baseline was similar in HGFs

(124 i 31 nM) and Rat2 fibroblasts (1 30 2 9 nM; p>0.02). The rise to maximum levels

of [ca2']i also proceeded over a similar time course. The similarity of Rat2 fibroblasts

and HGF responses to collagen beads rationalized the use of Rat2 fibroblasts for al1

subsequent experiments which measured levels of calcium within the cell.

When CsA was added to HGFs or Rat2 cells, [ca2+]i rose steadily over time and

exhibited a dose-dependent increase of [ca2']i. The addition of CsA, even in small

doses, induced significant increases of [ca2'li compared to the DMSO vehicle (p<0.05

for 10 nM CsA; Fig. 78). When Rat2 fibroblasts were pre-treated with 10 nM CsA for 1

hour, there was a significantly reduced rise of [ca2'li in response to collagen bead-

induced phagocytosis (3.4-fold; pç0.01; Fig. 7C).

The levels of [ca2+li were investigated when intracellular stores of calcium were

depleted usinç 1 yM thapsigargin or when intracellular calcium was chelated using

BAPTAIAM. After addition of thapsigargin to either Rat2 fibroblasts or Rat2 fibroblasts

pre-treated with CsA, there was a robust rise of [ca2+li in both groups. The post-

Mean change in [Car*], tïme to maxlmum [Ca hl,

r h

Time (seconds) 1

HGF + CCB Rat2 + CCB

r l Y 1 i . I . !.., w'- n o . c --, 5 - L - 8-9. - - ..\CC*"-

0 0 . N + /*-+ ,">;F- B O . E 6 0 - %l. . . .- -.

I l .;un . - 111.1

* 4 0 .

2 0 . Thne (seconds) & a

0 . - d i I DMSO, 1nM 10nM 100nM 1pM 1OpM

untreated [CSAI O

0 ; RaPflbroblasts C

O Rat2 flbroblests + 0 . Y

10 nM CsA - 8 ,

0 . + _

O .

O * Time (seconds)

* - - + CCB, P O S ~ - ~ g

untreated + CCB

Mean change In 346i380 ratio

1 Time to maximum 3461380 ratio

-r i eoo I

- 1000 '-

; 800

400

Y I n

Rat2 + CCB Rat2 + CsA +%CS -

Figure 7. A. Collagen-coated beads (CCBs) cause an equivalent rise of [Ca2+Ii in human gingival fibroblasts (HGFs) and Rat2 cells. Note that the tirne required to reach maximum values are also similar. In contrast, cells stimulated with poly-L-lysine coated beads did not show any significant rise above baseline. Data are means I SEM (n-5). Insert shows an increase of [Ca2+], induced by collagen-coated beads in HGFs. B. Treatment with increasing concentrations of CsA induces dose-dependent increases of [Ca2+], in HG F. Data are means I SEM (n=5). *, pe 0.05; $, pe0.01 when compared to DMSO. Inset demonstrates representative tracing when CsA is added to human gingival fibroblasts loaded with fura 2/AM (2 FM) and [Ca2+ji is recorded. C. The addition of CCBs to fibroblasts in a calcium-containing medium induces a substantial calcium flux (-130 nM Ca2+) which is reduced 3.4-fold by CsA (10 nM). As expected, thapsigargin (Tg; 1 PM) induces a large calcium increase which is not affected by pre-treatment with CsA (10 nM). After depletion of intracellular calcium stores by thapsigargin, the collagen-induced calcium response is reduced -3.7-fold (post-Tg + CCB). In contrast, there is no significant reduction of collagen-induced calcium increase in CsA-t reated cells that are then treated wit h thapsigargin. Evidently CsA is not acting primarily at a thapsigargin-sensitive locus in the response to collagen beads. Data are means I SEM (n=5). *, pe 0.05; *, pc0.01 when compared to Rat2 + CCB; **, pc0.05 when compared to Rat2 fibroblasts treated with IO8 M CsA. Inset shows pronounced increase in [Ca2+], produced by thapsigargin and a very small rise due to the addition of collagen-coated beads after thapsigargin. D. Pre-incubation of Rat2 fibroblasts with CsA induces decreased [Ca2+],,,, response to collagen-coated beads. Rat2 cells were loaded with [Ca2+Is,,, indicator rnag-fura 2 and measured by ratio fluorimetry. Data are means r SEM (n=5). t, pe0.02 when compared to Rat2 + CCB.

thapsigargin response to collagen bead-induced phagocytosis was small in both groups

but showed a larger reduction of [ca2'li in Rat2 fibroblasts (3.7-fold; ~ ~ 0 . 0 5 ) than in

Rat2 fibroblasts pre-treated with CsA (2-fold). Calcium chelation with BAPTAIAM (5

PM) almost obliterated the [ca2+li rise induced by beads (Fig. 7C).

When 2 pM mag-fura 2/AM was used to Joad cells the response of [ca2'lsERcn to

collagen bead-induced phagocytosis was measured with ratio fluorimetry, pre-treatment

with CsA produced a > 5-fold reduction in the rise of [ ca2 ' ] s~~c~ (p < 0.02) over a similar

time span as vehicle-treated controls (-1 100 seconds; Fig. 7D).

Dependence of phagocytic response on calcium signalling

The addition of thapsigargin to release calcium from interna1 stores and to inhibit store

uptake dramatically reduced the phagocytic capacity of Rat2 fibroblasts (1 0.2-fold;

pc0.01) a reduction that was comparable to the 8-fold decrease seen when Rat2

fibroblasts were pre-treated with CsA (pc 0.02; Fig. 8). Pre-treatment with CsA (1 0 nM)

produced no additional inhibition of phagocytosis. A similar sized reduction of

phagocytosis was obtained after pre-treatment with BAPTAIAM. These data indicated

that intracellular calcium is important in regulating the bead-binding step of

phagocytosis.

Mitochondrial calcium ([ca27,)

Rat2 fibroblasts were CO-loaded with MitoTracker Green [which specifically stains

mitochondria, Babcock et al. (1997)l and rhod 2/AM [which reportedly stains

untreated lm Tg 10 nM CsA 5 PM + 1 yM Tg BAPTAIAM

Figure 8. Effect of agents that inhibit calcium signalling on phagocytosis in Rat2 fibroblasts. Cells treated with thapsigargin (Tg; 1pM) or BAPTNAM (5 PM) show 10- fold reduction of phagocytosis. Pre-incubation with CsA produces no additional inhibition of phagocytosis compared to thapsigargin alone. Data are means I SEM (n=3). ;t, p<0.02; $, p4.01 when compared to untreated cells.

mitochondrial calcium, Babcock et al. (1997)l to determine if these dyes CO-

localize and thereby assess if rhod 2 can be used to monitor changes in

[ca2'],ito. There was marked CO-localization of rhod 2 and MitoTracker Green

(Fig. 9Ai). After staining with rhod-2/AM for 30 minutes, Rat2 fibroblasts or Rat2

fibroblasts pre-treated with CsA for 1 hour exhibited an equivalent time-

dependent decrease of [ca2'],ito in response to collagen beads at > 10 minutes

(Fig. SAii, iii). Linear regression showed that mitochondrial calcium was lost at a

similar rate in both controls and CsA-treated cells. However, in control cells (Le.

not pre-treated with CsA). there was marked and non-linear reduction of

mitochondrial calcium loss in the 5-10 minute range (Fig. 9B).

CsA prevents loss of mitochondrial membrane potential (Y,)

As CsA is thought to affect the permeability of the MPTP (Zamzami et al., 1996),

I evaluated the efficacy of CsA to block the loss of Y, that occurs following an

apoptotic sirnulus (Kulkarni et al., 1998). Suspension-induced apoptosis

(anoikis) was used as the stimulus (Kulkarni and McCulloch, 1994) and JC-1

aggregate formation was used to estimate the mitochondrial membrane potential.

The addition of increasing concentrations of CsA to Rat2 fibroblasts in

suspension was accompanied by an elevated mitochondrial membrane potential

(pc0.01 for 1 and 10 yM CsA; pc 0.05 for 100 nM CsA; Fig. 10). Thus CsA

appears to preserve Y, as described (Zamzami et al., 1996) which indicates that

an important locus of action for CsA is the MPTP of fibroblasts.

Mean Fluorescence Intensity (x 105)

Figure 9. A. (i) Co-localization of MitoTracker Green and rhod 2 dyes in Rat2 fibroblasts. Cells were CO-loaded with both dyes and then irnaged by fluorescence microscopy using filters appropriate for either dye. (ii) Stimulation using collagen- coated beads (CCB) in Rat2 fibroblasts stained with rhod 2 induces a decrease in [Ca2+],,, over tirne. Arrows indicate decreased rhod 2 staining in discrete regions of the cell. Note that in the left panel, discrete mitochondria stained with rhod 2. In the right panel, after addition of collagen beads (2 fluorescent beads can be seen on left margin of cell) there is a generalized reduction of rhod 2 fluorescence. (iii) Stimulation using CCBs induces a decrease of [Ca2+],,, in Rat2 fibroblasts treated with CsA. B. The addition of collagen-coated beads to Rat2 and Rat2 fibroblasts treated with 10 nM CsA induces a time-dependent decrease in [Ca2+],,,. Data are means I SEM (n=5). Linear regression equations and Pearson correlation values for Rat2 and Rat2 fibroblasts treated with CsA, respectively, are y=-0.060~ + 2.22, r=-0.97 and y=-0.18~ + 4.84, r=- 0.98.

i contr I 10 nM CsA

10pM CCCP CsA

Figure 10. CsA prevents the loss of mitochondrial membrane potential of Rat2 fibroblasts in suspension. Controls are untreated cells. Analysis was conducted by flow cytometry of JC-1-loaded Rat2 cells in suspension to promote dissipation of Y, and thereby induce anoikis (suspension-induced apoptosis). Data are means I SEM (n=3). $, p<0.01 when compared to untreated cells.

Mitochondrial-depleted cells

The data in Figs. 6, 9 and 10 indicated that mitochondria may be an important organelle

for regulation of SERCA ca2+ and also of collagen bead phagocytosis. Consequently,

mitochondria-depleted Rat2 cells were produced using well-established methods

(Biswas et al., 1999) to determine if reduction of the numbers of mitochondria would

affect collagen bead-induced calcium signalling and phagocytosis. I first verified if the

standard protocol for mitochondrial depletion (Biswas et al., 1999) would in fact reduce

the numbers of mitochondria.

Rat2 fibroblasts or Rat2 fibroblasts treated with 1000 ng/mL ethidium bromide

were stained with JC-1 (0.5 PM) for visualizing mitochondria with a fluorescence

microscope. The EtBr-treated cells showed much smaller numbers of mitochondria

(Fig. 11Ai). Staining with rhod 2 AM (4.5 PM) to assess mitochondrial calcium stores

also showed a large decrease of fluorescence in the EtBr-treated cells (Fig. 11Aii).

Notably, rhod 2 staining of nucleoli was unchanged, indicating that the EtBr treatment

specifically depleted mitochondrial calcium stores, presumably due to the greatly

reduced numbers of mitochondria. Western blotting demonstrated that treated cells

also contained less of the mitochondria-specific protein, cytochrome oxidase I (Fig.

1 1 Aiii).

As shown in figure 11 B, mitochondria-depleted cells exhibited 50% less collagen

bead binding than normal cells in the absence of CsA (pc0.05). The addition of CsA

caused a dose-dependent decrease in the mean % phagocytosis of collagen-coated

beads in Rat2 fibroblasts treated with ethidium bromide (pe 0.05 for 100 and 1000 nM

CsA) similar to that seen in untreated cells. The mitochondria-depleted cells

Figure 11. A. Treatment of Rat2 fibroblasts with ethidiurn bromide for over 30 passages of continuous culture induces inhibition of mitochondrial DNA synthesis and reduced synthesis of the mitochondria-associated protein COX 1. (i) Staining of Rat2 fibroblasts and Rat2 fibroblasts treated with 1000 ng/mL ethidium bromide (RaQEtBr) with the mitochondrial specific dye, JC-1. (ii) Staining of Rat2 fibroblasts and Rat2EtBr fibroblasts with rhod 2/AM shows no change in rhod 2 staining of nucleoli but greatly reduced fluorescence in the cytoplasrn. (iii) Western blot for COX I protein in Rat2 and Rat2EtBr fibroblasts shows -3-fold reduction of this mitochondrial-specific protein. B. Phagocytic capacity of Rat2 and RatPEtBr fibroblasts. All cells were incubated with coiiagen-coated beads at a ratio of 2:1 (beads:cell) for 1 hour after indicated treatment. Treatments: 1- untreated; 2- 10 nM CsA; 3- 100 nM CsA; 4- 1 FM CsA; 5- 10 pM CsA;6- 10 nM CsA + 1 pM Tg (15 mins); 7- 1 pM Tg (15 rnins.); 8- 5 pM BAPTNAM (30 mins); 9- 500 FM CCCP (30 mins); 10- 1% sodium azide (30 mins); al1 CsA treatments were conducted for 1 hour. These data shows that compared to untreated controls, CsA induces a more marked inhibition of phagocytosis in normal Rat2 fibroblasts than in ethidium bromide-treated fibroblasts. The thapsigargin. BAPTA and CCCP effects are also proportionately less in the ethidiurn bromide-treated cells. Data are means I SEM (n=3). * ~~0.05 when compared to Rat2EtBr fibroblasts, ", pc0.05 when compared to Rat2 fibroblasts.

displayed reductions of phagocytosis after treatment with thapsigargin,

BAPTAIAM, CCCP or sodium azide (Fig. 11 B) but the magnitude of the reduction

was - one-half that of normal Rat2 cells.

Calcium signalling in mitochondria-depleted cells

Rat2 or mitochondria-depleted Rat2 fibroblasts were loaded with fura 2JAM (2

FM) and treated with CCCP (10 PM) for 10 minutes to depolarize mitochondria

and block oxidative phosphorylstion. As estimated by ratio fluorimetry, CCCP

reduced [ca2'li responses to collagen beads in Rat2 fibroblasts by -90%

(pc0.01). In contrast, collagen bead-induced [ca2']i responses in mitochondria-

depleted cells were < 10 nM and were reduced only an additional 5 nM by CCCP

(Fig . 1 2A).

As shown in Fig. 7D and Fig. 128, treatment of Rat2 fibroblasts with CsA

markedly reduces the maximal [ c a 2 + ] s ~ ~ ~ ~ response to collagen beads. In

contrast, when mitochondria-depleted Rat2 fibroblasts were treated with CsA,

there was no significant reduction of maximal [C~*']SER~A in response to beads

(Fig. 12B; pç0.2). Further, there was no significant difference between Rat2

fibroblasts and mitochondria-depleted fibroblasts with or without CsA treatment

(~>0.2).

The data shown above indicate that mitochondria regulate calcium

signalling in response to collagen beads but when the numbers of mitochondria

are reduced, SERCA stores continue to exhibit a response to beads that is

unaffected by CsA. I assessed if calcium stores in the remaining mitochondria of

Fïbroblasts + CCCP + CCB

Rat2 fibroblasts Rat2EtBr fibroblasts

Mean change in 3461380 iatlo O .I

ci 0.07 * Tirne to maximum 316/380

L

i ooo 7

Rat2EtBr Rat2EtBr + CCB + CsA + CCB

Rat2 + CCB

Rat2 + CsA + CCB

Figure 12. A. Incubation with CCCP inhibits [Ca2+Ii response to collagen-coated beads (CCBs) in both Rat2 and Rat2EtBr fibroblasts. CCBs induce only a minimal [Ca2+], response in Rat2 fibroblasts cultured with 1000 ng/mL ethidium bromide (RaPEtBr). Data are means I SEM (n=5). $, pc0.01 when compared to Rat2 fibroblasts + CCB. 8. Pre-incubation with 10 nM cyclosporin A (CsA) induces a large decrease in [Ca2+],,,, in Rat2 cells but not in Rat2EtBr cells responding to CCBs. [Ca2+],,,, was measured using ratio fluorimetiy of mag-fura 2-loaded cells. Data are means I SEM (n=5). t, pc0.02 when compared to Rat2 fibroblasts + CCB.

mitochondria-depleted cells would still discharge ca2' if stimulated with collagen

beads. Mitochondria-depleted Rat2 fibroblasts were loaded with rhod 2/AM (4.5

PM) and fluorescence intensity was recorded over 30 minutes after initiation of

collagen-bead induced phagocytosis as described (Fig. 96). In cells treated with

vehicle or CsA, the fluorescence intensity of rhod 2 decreased following

stimulation with collagen-coated beads (Fig. 13A). As expected, the baseline

fluorescence intensity due to rhod 2 reporting of [ ~ a " ' ] ~ i ~ ~ was significantiy lower

in mitochondria-depleted cells compared to control Rat2 cells. Linear regression

of fluorescence intensity data showed that [ca2'lmito in Rat2 fibroblasts dissipated

at the same rate as [~a*' ]mi~~ in RatPEtBr fibroblasts between 10-30 minutes. In

contrast to mitochondria-depleted cells which showed a linear reduction of

fluorescence between 0-30 minutes, control cells exhibited a pronounced 2-fold

reduction 5-10 minutes after bead incubation which thereafter showed a similar

linear reduction of fluorescence as the ethidium bromide-treated cells (Fig. 138).

After treatment with 10 nM CsA for 1 hour, the control Rat2 fibroblasts lost

mitochondrial calcium at a linear but faster rate than Rat2EtBr fibroblasts (slope =

-0.18 vs. slope = -0.055). The marked loss of mitochondrial calcium in the 5-10

minute range after stimulation with collagen beads was notably absent after CsA

pre-treatment.

Sasdine, non-stirnulated + CC8, t=lO minutes

10 15 20 Tirne (minutes)

Time (minutes)

Figure 13. A. (i) Stimulation with collagen-coated beads (CCB) in RatZEtBr fibroblasts stained with rhod 2/AM induces a decrease in [Ca2+],,, of the few remaining mitochondria. In this micrograp h I have included fluorescence beads (bright circles at cell periphery) to illustrate bead size and binding. Arrows point to rhod 2 stained mitochondria and indicate decreased rhod 2 staining. (ii) Stimulation with CCBs induces a decrease of [Ca2+],, in Rat2EtBr treated with CsA. B. CCBs induce decreased [Ca2+],, in Rat2 and RatPEtBr. In normal Rat2 cells, CCBs induce a s 2- fold reduction of rhod 2 fluorescence between 5-10 minutes which decreases lineariy thereafter (10-30 minutes). Mitochondria-depleted cells exhibit a linear reduction of rhod 2 fluorescence which has the sarne slope as the normal cells from 10-30 minutes. Linear regression equations and Pearson correlation values for Rat2 and Rat2EtBr fibroblasts, respectively, are y=-0.060~ + 2.22, r=-0.97 and y=-0.090~ + 2.88, r=-0.90. C. Addition of CCBs to Rat2 or Rat2EtBr fibroblasts pre-treated with 10 nM CsA induces decreased [Ca2+],,io. Note the continuous linear reduction of rhod 2 fluorescence in the normal Rat2 cells and the absence of the sharp drop from 5-10 minutes. Linear regression equations and Pearson correlation values for Rat2 and Rat2 fibroblasts both treated with CsA, respectively, are y=-0.1 8x + 4.84, r=-0.98 and y=-0.055~ + 2.1 6, r=-0.78.

Control fibroblast

'l' ytos i s

Fibroblast treated with CsA bead

Figure 14. Schematic diagram of fibroblasts with and without CsA treatment. Summary changes of [Ca2ri, [Ca2+],,,,,, [Ca2+],,, and rate of phagocytosis are represented by arrows. Subsequent to collagen bead-induced phagocytosis, different calcium responses are observed after treatment with CsA. In control cells, [Ca2+],,, decreases initially at a faster rate than CsA-treated cells. CsA sharply reduces whole cell calcium and SERCA calcium responses to collagen bead stimulation. 1 propose that binding of CsA to mitochondria inhibits mitochondrial release of calcium and therefore uptake by the SERCA store and a consequent reduced calcium response in the cytosol to collagen beads. The downstream effect is decreased phagocytic efficiency in CsA- treated fibroblasts which contributes to loss of collagen homeostasis and results in gingival overgrowth.

V. Discussion

Current data indicate that administration of CsA results in gingival overgrowth in as low

as 17% and as high as 81% of cases available for study (Hefti et al., 1994; Friskopp and

Klintmalm, 1986). The mechanisrn by which CsA induces gingival overgrowth is

unknown but some of the modes of action of CsA as an immunosuppressant are

understood (Kay, 1989). CsA inhibits the MPTP (Crompton et al., 1988) in the

mitochondrial membrane, and more recently, has been shown to act upon the internal

calcium stores and inhibit calcium release (Park et al., 1999). Further, several

functional interactions between mitochondria and internal calcium stores have been

characterized (Landolfi et al., 1998). With this background in mind, I propose that upon

entry into the cell, CsA interacts with mitochondria to affect the normal functional

relationship of mitochondrial calcium stores and other internal stores of calcium (Fig. 4).

As calcium signalling is important for integrin functions related to binding of extracellular

matrix molecules (Schwartz, 1993), this deregulation is likely to affect collagen

phagocytosis. Below I will discuss in more detail the functional relationships between

calcium deregulation and phagocytic processes.

Effect of CsA on phagocytosis

HGFs have been used previously to study collagen phagocytosis in vitro (McCulloch

and Knowles, 1993). These cells are the principal synthetic and degradative cell in

normal gingiva (Narayan and Page, 1993; Everts, 1996) and consequently are closely

lin ked to the de reg ulation of collagen rnetabolism seen in drug-induced gingival

overgrowth. I used Rat2 fibroblasts primarily for my studies since I showed that these

cells exhibit very similar calcium and phagocytic responses as HGFs. As Rat2 cells is a

stable and easily propagated cell line, problems of cell death and the phenotypic

variability associated with primary isolates were rninimized. Data from previous

collagen phagocytosis studies also dernonstrate the utility of Rat2 cells for phagocytic

studies and the similarity of the two cell types (Hui et al., 1997). For example I showed

that addition of 10 nM CsA caused a significant and similar decrease in phagocytosis

(~50%) for both cell types. Further, in Rat2 fibroblasts and HGFs, I showed that

addition of CsA, in concentrations less than those administered in vivo (Kay, l989),

caused a significant decrease of phagocytosis compared to untreated cells. These data

describe the final end-point effect of CsA upon fibroblasts studied here, (i.e. due to

some mechanism, CsA inhibits the collagen phagocytic capacity of gingival fibroblasts).

CCCP, a proton ionophore which dissipates the mitochondrial membrane potential

(Zamzami et al., 1995) and eventually promotes ce11 death (Kulkarni et al., 1998), also

reduced phagocytosis as was seen with CsA treatment. The mitochondrial dysfunction

caused by CCCP suggests the importance of mitochondria in phagocytic function.

lndeed the involvement of ATP production by mitochondria was shown by treating cells

with sodium azide which also caused a similar decrease of collagen bead phagocytosis.

Phagocytosis and [ c ~ J signals

When [ca2'li was monitored during collagen bead-induced phagocytosis, HGFs and

Rat2 fibroblasts exhibited a gradua1 similar rise in [ca2'li over 30 minutes but with no

return to baseline over this monitoring period, presumably because of the prolonged

period of bead-cell interactions and the progressive cellular contacts with more than one

bead. After intracellular calcium stores were depleted by thapsigarg in, collagen bead-

induced phagocytosis was still able to invoke a small but measurable calcium rise, an

effect that was seen in both Rat2 fibroblasts and Rat2 fibroblasts pre-treated with CsA.

If calcium was released into the cytosol through a thapsigargin-insensitive route,

collagen phagocytic stimuli may effect calcium release from other calcium stores or this

increase may be due to calcium influx from the extracellular medium (Schwartz, 1993).

Notably, the post-thapsigargin response in CsA-treated cells was not as large as

untreated control cells, suggesting that CsA interacts with thapsigargin-insensitive sites

which may cause an increase of [ca2']i due to phagocytosis. I propose that since

treated cells have more of these binding sites engaged by CsA, they cannot respond

fully to phagocytic stimuli. The chelation of [ca2']i with BAPTA also caused a significant

decrease in the [ca2*]i response compared to untreated cells and the magnitude of this

response was similar to that following thapsigargin. These data are consistent with

previous findings showing that [ca2'li responses to phagocytic stimuli are not due solely

to release from SERCA stores (Bengtsson et al., 1993). lndeed as SERCA calcium

stores were actually increased after collagen bead stimulation, it is possible that a large

part of the rise of [ca2'li due to phagocytosis originates from non-SERCA stores. Small

contributions may also come from both the extracellular medium and from other, as yet

unidentified, stores within the cell.

CsA induced a relatively rapid, dose-dependent rise of [ca2']i, likely reflecting the

transport of CsA into cells and its interaction with binding sites or receptors that regulate

intracellular calcium stores (LeGrue et al., 1983). Addition of thapsigargin to cells pre-

treated with CsA induced the same robust increase in [ca2+]i as seen in untreated cells.

However, collagen bead-induced phagocytosis aRer interna! store depletion by

thapsigargin produced a reduced response of [ca2*]i in Rat2 fibroblasts (p<0.05) and a

similar rise in Rat2 fibroblasts pre-treated with CsA. This finding indicates that pre-

treatment with CsA caused calcium release from thapsigargin-insensitive stores. As

collagen beads stimulated uptake of calcium into the SERCA, and as this uptake was

inhibited by CsA, it seems likely that mitochondrial calcium is released by collagen bead

stimuli and that the released calcium is taken up by the SERCA.

[ C ~ ~ ' ] S E R C A response to collagen phagocytic stimuli

As discussed above, pre-incu bation of Rat2 fibroblasts with CsA caused a significant

decrease in the [ C ~ ~ ' ] S E R C A response to collagen beads. Previous data by Schwartz

(1993) indicate that a large part of the increase in [ca2'li may be due to calcium release

from the SERCA stores. However, when mag-fura was used to observe the behaviour

of calcium within SERCA stores, I found an increase after collagen bead-induced

phagocytosis over 30 minutes. Therefore, both [ca2+li and [ca2'lsERCA were increased

after collagen bead-induced phagocytosis. My expectation was that release of SERCA

stores of calcium would provide the increased [ca2+li seen experimentally. However,

when cellç were pre- treated with CsA, [ca2+lsERCA and [ca2+li were reduced compared

to untreated cells. Thus CsA may act initially to release calcium from intracellular stores

thereby reducing the calcium ion pool for uptake by the SERCA. In this context,

mitochondria in untreated cells rnay sense high local concentrations of calcium and act

to buffer [ca2+], as has been shown earlier (Riuuto et al., 1993). Accordingly, in

mitochondria-depleted cells, CsA treatment did not alter [ c ~ ~ + ] ~ ~ R ~ A presumably

because productive interactions between mitochondrial and SERCA-related stores were

restricted (Biswas et al., 1 999).

[~fl],,,,~~ responses to collagen phagocytic stimuli

Collagen bead-induced phagocytosis provoked a time-dependent decrease of [ca2+lmi,

in untreated Rat2 cells or Rat2 cells pre-treated with CsA but there was a much more

rapid loss at early times (5-10 minutes) in untreated cells. Pre-incubation with CsA or

mitochondrial depletion attenuated the dissipation of mitochondrial calcium in the 5-1 0

minute range. This result may be a reflection of the initial obstruction to calcium efflux

due to inhibition of the MPTP by CsA, an effect that is probably overcome as calcium

ions exit via other channels or pumps (Bernardi, 1999). My data on CsA inhibition of

suspension-induced reduction of Y, indicate that CsA was indeed acting upon the

MPTP and consequently the supposition that CsA is altering the conductance of the

MPTP seems reasonable.

Phagocytosis in rnitochondria-depleted fibroblasts

Compared to untreated Rat2 cells, Rat2 fibroblasts treated with ethidium bromide

exhibited similar reductions of phagocytic efficiency as seen in cells treated with CsA.

This suggests that mitochondria play a role in regulating collagen phagocytosis. When

the rate of dissipation of [ca2+]mito due to collagen bead-induced phagocytosis was

compared between these two ceII types, there was no significant difference. This result

appears reasonable since the ethidium bromide-treated cells have a lower nurnber of

mitochondria but the function of the remaining mitochondria is not altered, otherwise the

cells would not proliferate in culture. A similar rate of rnitochondrial calcium dissipation

was seen in Rat2 fibroblasts treated with CsA, emphasizing that although reduced in

number, these mitochondria function normally.

The major conclusion of this thesis is that mitochondrial calcium signalling is an

important determinant of collagen bead phagocytosis and that consequently, CsA-

mediated gingival overgrowth may be mediated by the same or a similar pathway. Fig.

14 illustrates a model integrating data obtained for changes of [ca2'li, [ ~ a ~ ' ] ~ ~ ~ ~ ~ ,

[ca2'],im and phagocytic efficiency. Consistent with my overall conclusion, subsequent

to collagen bead-induced phagocytosis, very different calcium responses were

obsewed in Rat2 fibroblasts or Rat2 fibroblasts treated with CsA. [~a*'],~~, decreased

at a faster rate initially in the untreated cells. Both [C~~+]SERCA and [ca2']i increased to a

greater extent in controls than CsA-treated Rat2 fibroblasts. I propose that the effect of

CsA on mitochondria is transmitted to the SERCA stores, which then "relay" the

information and dampens the phagocytosis-induced calcium signal in the cytosol. The

effect of this perturbation on the whole cell is a decrease of phagocytic efficiency which,

in the CsA-treated gingival fibroblast, results in gingival overgrowth. While the actual

sequence of events is not shown by these data, the importance of signaling interactions

between discrete calcium stores is evident. When the sequence and regulation of these

phagocytic signaling events is deciphered, and the exact effect of CsA on the

mitochondria as a fibrosis-inducing agent is outlined, modifications to CsA therapy could

be approached more rationally. For example, if CsA could be modified so as to reduce

fibrosis while retaining its immunosuppressant properties, this would be a significant

clinical advance.

VI. Conclusions

1. CsA inhibits collagen bead phagocytosis dose-dependently in human gingival

and Rat2 fibroblasts. Phagocytosis is decreased in mitochondria-depleted

RatZEtBr fibroblasts, suggesting that mitochondria play a role in the regulation of

collagen phagocytosis.

2. Collagen-coated beads induce equivalent calcium rises in both human gingival

and Rat2 fibroblasts. Part of the calcium increase is due to thapsigargin-

sensitive and thapsigargin-insensitive stores. Mitochondria depleted Rat2EtBr

fibroblasts show reduced rise of [Cali in response to collagen-coated beads. CsA

prevents the loss of mitochondrial membrane potential of Rat2 and Rat2EtBr

fibroblasts in suspension indicating that CsA blocks the mitochondrial membrane

perrneability transition pore.

3. Depletion of internal calcium stores or dissipation of mitochondrial membrane

potential greatly reduce phagocytosis in both Rat2 and mitochondria-depleted

Rat2-EtBr fibroblasts.

4. Pre-incubation of Rat2 fibroblasts with CsA causes a decrease in [C~~* ]SERCA

which is probably related to CsA-induced release of calcium from internal stores.

In Rat2-EtBr cells, CsA treatment does not alter [C~~ ' ]SERCA because productive

interactions between mitochondrial and SERCA-related stores are restricted.

Collagen bead-induced phagocytosis induces a time-dependent decrease in

[Calmito in both Rat2 and Rat2 fibroblasts treated with CsA. Calcium release from

mitochondria is provoked by collagen phagocytosis in both cell types.

Vil. References

Allen, L.-A.H. and Aderem, A. (1 996) Molecular definition of distinct cytoskeletal structures involved in complemnt- and Fc Receptor-mediated phagocytosis in macrophages. J Exp Med 184:627-637.

Angelopoulos, A.P. and Goaz, P.W. (1972) Incidence of diphenyylhydantoin gingival hyperplasia. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 34: 898-906.

Armitage, G .C. (1 999) Developrnent of a classification system for periodontal diseases and conditions. Ann Periodontol4: 1 -6.

Avdonin, P.V., Cottet-Maire, F., Afanasjeva, GY., Loktionova, S.A. Lhote, P. and Ruegg, U.T. (1999) Cyclosporin A up-regulates angiotensin II receptors and calcium responses in human vascular smooth muscle cells. Kidney Int 55(6):2407-2414.

Aznavoorian, S., Murphy, A.N., Stetler-Stevenson, W.G. and Liotta, L.A. (1993) Molecular aspects of tumor cell invasion and metastasis. Cancer i l : 1368-1 381 .

Babcock, J. R. (1 965) l ncidence of gingival hyperplasia associated with dilantin therapy in a hospital population. J Am Dent Assoc 71 : 1447-1 450.

Babcock, D.F., Herrington, J., Goodwin, P.C., Park Y.B. and Hille B. (1997) Mitochondrial participation in the intracellular ca2+ network. Journal of Ce11 Biology 136:833-844.

Barak, S., Engelberg, I.S. and Hiss ,J. (1987) Gingival hyperplasia caused by nifedipine - histological findings. J Periodontol: 58: 639-642.

Bartold ,P. (1 989) Regulation of human gingival fibroblast growth and synthetic activity by cyclosporine-A in vitro. J Periodontol Res 24:314-321.

Bengtsson, T., Jaconi, M.E.E., Gustafson, M., Magnusson, K.-E., Theler, J.-M., Lew, D.P. and Stendhal, 0. (1 993) Actin dynamics in human neutrophils during adhesion and phagocytosis is controlled by changes in intracellular free calcium. Eur J Cell Biol62:49-58.

Bernardi, P. (1 999) Mitochond rial transport of cations: channels, exchangers, and perrneability transition. Physiol Rev 79: 1 127-1 155.

Bernardi, P., Broekemeier, K.M. and Pfeiffer, DR. (1 994) Recent progress on regulation of the mitochondrial peneability transition pore: a cyclosporin- sensitive pore in the inner mitochondrial membrane. J Bioenerg Biomembr 26:509-517.

Berridge, M.J. (1 995) Capacitative calcium entry. Biochem J 31 2:1-11.

Berridge, M., Lipp, P. and Bootman, M. (1999) Calcium signalling. Curr Biol 9(5): R I 57-1 59.

Bezprozvanny, I., Watras, J. and Ehrlich, B. E. (1 991 ) Bell-shaped calcium response curves of Ins(1 ,4,5)P3-and calcium-gated channels frorn endoplasrnic reticulum of cerebellum. Nature 351 :75l -754.

Birkedal-Hansen, H. (1 993) Role of cytokines and inflammatory mediators in tissue destruction. J Periodontol Res 28:500-510.

Biswas, G., Adebanjo, O.A., Freedman, B.D., Anandatheerthavarada, H.K., Vijayasarathy, C., Zaidi, M., Kotlikoff, M. and Avzdhani, G. (1999) Retrograde ca2+ signaling in C2C12 skeletal myocytes in response to mitochondrial genetic and metabolic stress: a novel mode of inter-organelle cross-talk. EM80 J 18(3):522-533.

Bonnaure-Mallet, M., Tricot-Doleux, S. and Godeau, G. (1 995) Changes in extracellular matrix macromolecules in human gingiva after treatment with drugs inducing gingival overgrowth. Arch Oral Bi01 40:393-400.

Border, W.A., Noble, N.A., Yamamoto, T., Harper, H.R., Yamaguchi, Y., Pierschbacher, M.D. and Ruoslahti, E. (1 992) Natural inhibitor of transforming growth factor-P protects against scarring in experimental kidney disease. Nature 3603361 -364.

Borel, J.F., Feurer, C., Gubler, H.U. and Stahelin, H. (1976) Biological efects of cyclosporin A: a new antilyrnphocytic agent. Agents Actions 6:468-475.

Brandes, D. and Anton, E. (1969) Lysosomes in uterine involution: intracytoplasmic degradation of myofilaments and collagen. J Gerontol24:55-69.

Bunjes, D., Hardt, C., Rollinghoff, M. and Wagner, H. (1981) Cyclosporin A mediates immunosuppression of primary cytotoxic T cell responses by impairing the release of interleukin-1 and interleu kin-2. Eur J lmmunol 1 1 :657-661.

Calne, R.Y., Rolles, K., White, D.J., Thiru, S., Evans, D.B., McMaster, P., Dunn, D.C., Craddock, G.N., Henderson, R.G., Aziz, S. and Lewis P. (1979). Cyclosporin A initially as the only immunosuppressant in 34 recipients of cadaveric organs: 32 kidneys, 2 pancreas and 2 livers. Lancet 2: 1033-1 036.

Carranza, F.A. Jr. Gingival enlargement. ln: Clinical Periodontoloav (eds. F.A. Carranza Jr and M.G. Newman, 8'h ed) Philadelphia, PA: W.B. Saunders Co., 1 996: 235-238.

Chou, D.H., Lee, W., and McCulloch, C.A.G. (1 996) TNF-alpha inhibits collagen synthesis and at high concentrations tirnulates collagenase synthesis in fibroblasts. J lmrnunol 1 56(11):4354-4362.

Crompton. M.. Ellinger. H. and Costi, A. (1988) Inhibition by cyclosporin A of a cas- dependent pore in heart mitochondria activated by inorganic phosphate and oxidative stress. Biochem J 255:357-360.

Daley, T.D., Wysocki, G.P. and May C. (1 986) Clinical and pharmacological correlations in cyclosporine-induced gingival hyperplasia. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 62: 41 7-421.

Dahllof, G. and Modéer, T. (1986) The effect of a plaque control program on the development of phenytoin-induced gingival overgrowth. A 2-year longitudinal study. J Clin Periodontol13: 845-849.

Deliliers, G.L., Santoro, F., Polli, N., Bruno, E., Fumagalli, L. and Risciotti, E. (1 986) Light and electron microscopie study of cyclosporin-A induced gingival hyperplasia. J Periodontok 57: 771 -775.

Dongworth, D.W. and Klaus, G.G.B. (1982) Effects of cyclosporin on the immune system of the mouse. 1. Evidence for a direct selective effect of cyclosporin A on B cells responding to anti-immunoglobulin antibodies. Eur. J. Immunol. 12:1018- 1022.

Dreyer, W.P., Dent, H.D. and Thomas, C.J. (1 978) Diphenylhydantoin hyperplasia of the masticatory mucosa in an edentulous epileptic patient. Oral Surg Oral Med Oral Pathol Oral Radiol Endod: 45: 701 -706.

Ernrnel, E. A., C. L. Verweij, D. B. Durand, K. M. Higgins, E. Lacy, and G. R. Crabtree. (1 989) Cyclosporin A specifically inhibits function of nuclear proteins involved in T cefl activation. Science 246: 1 61 7-1 620.

Everts, V. and Beertsen, W. (1988) The cellular basis of tooth eruption: the role of collagen phagocytosis. In Bioloaical Mechanisms of Tooth Eruotion and Root Resorption (ed. Z. Davidovitch) Birmingham, AL:EBSCO Media1237-242

Everts, V., Hembry, R.M., Reynolds, J.J. and Beertsen, W. (1 989) Metalloproteinases are not involved in the phagocytosis of collagen fibrils by fibroblasts. Matrix 9:266-276.

Everts, V. van der Zee, E., Creemers, L. and Beertsen, W. (1 996) Phagocytosis and intracellular digestion of collagen, its turnover and remodelling. Histochem J 28(4):229-245.

Fattore, L., Stableirn, M., Brenfeldt, G., Semla, T., Moran, B. and Doherty- Greenberg, J .M. (1 991 ) Gingival hyperplasia: a side effect of nifedipine and diltiazem. Spec Care Dent 11 : 107-1 09.

Favre, C.J., Jerstrom, P., Foti, M., Stendhal, O., Huggler, E., Lew, D.P. and Krause. K.-H. (1 996) Organization of ca2+ stores in rnyeloid cells: association of SERCA2b and the type-1 inositol-l,,4,5-trisphosphate receptor. Biochem J 31 6: 137-1 42.

Fell, H.B., Reynolds, J.J., Lawrence, C.E., Bagga, M.R. and Glauert, A.M. (1 986) The promotion and inhibition of collagen breakdown in organ cultures of pig synovium: The requirement for serum components and the involvement of cyclic adenosine-3',5'-monophosphate (CAMP). Collagen Re1 Res 6:51-57.

Friskopp, J. and Klintmalm, G. (1 986) Gingival enlargement: a cornparison between cyclosporine and azathioprine treated renal allograft recipients. Swed Dent J: 10: 85-92.

Gamerberucci, A., Innocenti, R., Fulcri, G., Banhegyi, R., Giunti, T., Pouan, T. and Benedetti A. (1 994) Modulation of ca2' influx dependent on store depletion by intracellular adenine-guanine nucleotide levels. J Biol Chem 269:23597- 23602.

Gelfand, E.W., Cheung, R.K., and Mills, G.B. (1987) The cyclosporins inhibit lymphocyte activation at more than one site. J lmmunol138:1115-1120.

Gerasimenko, J.V., Tepikin, A.V., Petersen, O.H. and Gerasimenko, O.V. (1998) Calcium uptake via endocytosis with rapid release from acidifying endosomes. Curr Bi0l8(24): 1335-1 338.

Glickman, 1. and Lewitus, M. (1941) Hyperplasia of the gingiva associated with Dilantin (sodium diphenyl hydantoinate) therapy. J Am Dent Assoc 28: 199-203.

Goldstein, R.H., Poliks, C.F., Pilch, P.F., Smith, B.D. and Fine, A. (1 989) Stimulation of collagen formation by insulin and insulin-like growth factor 1 in cultures of human lung fibroblasts. Endocrinology l24:964:97O.

Granelli-Piperno, A., Inaba, K. and Steinman, R. (1 984) Stimulation of lymphokine release from T lymphoblasts. Requirement for mRNA synthesis and inhibition by cyclosporin A. J Ekp Med l60:1792-1802.

Grynckiewicz, G., Poenie M. and Tsien, R.Y. (1985) A new generation of ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260: 3440-3450.

Hajnoczky, G., Robb-Gaspers, L.D., Seitz, M.B. and Thomas, A.P. (1 995) Decoding of cytosolic calcium oscillations in the mitochondria. Ce// 82: 41 5-424.

Hall ,WB. (1 969) ~ i l a n t i n ~ hyperplasia: a preventable lesion. J Periodontol Res: 4: 36-37.

Hall, B.K. and Squier, C.A. (1 982) Ultrastructural quantitation of connective tissue changes in phenytoin-induced gingival overgrowth in the ferret. J. Dent Res 61 :942-952.

Hancock, R.H. and Swan, R.H. (1992) Nifedipine-induced gingival overgrowth. Report of a case treated by controlling plaque. J Clin Periodontol19(1): 12-1 4.

Hanschumaker, R.E., Harding, M.W., Rice, J., Drugge, R.J., and Speicher, D.W. (1 984) Cyclophilin: a specific cytosolic binding protein for CsA. Science 22:544- 547.

Haworth, R.A. and Hunter, D.R. (1 979) The ca2+-induced membrane transition inmitochondria. II. Nature of the ca2' trigger site. Arch Biochem Biophys 195:460-467.

Hefti, A.F., Eshenaur, A.E., Hassell, T.M. and Stone, C. (1 994) Gingival overgrowth in cyclosporine A treated multiple sclerosis patients. J Periodontoi 65: 744-749.

Hofer, A.M., Landolfi, B., Debellis, L., Pouan, T. and Curci, S. (1 998) Free [ca2'] dynamics measured in agonist-sensitive stores of single living intact cells: a new look at the refilling process. EMBO J 1 7(7):1986-1995.

Hui, M.-& Tenen baum, H.C. and McCulloch, C.A.G. (1 997) Collagen phagocytosis and apoptosis are induced by high level alkaline phosphatase expression in rat fibro blasts. J Cell Physioi 172:323-333.

Ichas, F., Jouaville, L.S. and Mazat, J.P. (1 997) Mitochondria are excitable organelles capable of generating and conveying electrical and calcium signals. Celi 89: 1 1 45-53.

Ingman, T., Sorsa, T., Michaelis, J. and Konttinen, Y.T. (1 994) lmmunohistochemical study of neutrophil- and fibroblast tpe collagenases and stromelysin-1 in adult periodontitis. Scand J Dent Res 102(6):342-349.

Irvine, R.F. (1990) 'Quantal' calcium release and the control of calcium entry by inositol phosphate - a possible mechanism. FEBS Lett 263:5-9.

Isakov, N., Mally, M.I., Scholz, W. and Altman, A. (1 987) T lymphocyte activation: the role of protein kinase C and the bifurcating inositol phospholipid signal transduction pathway. lmmunol Rev. 95:89-111.

Isberg, R.R. and Tran Van Nhieu, G. (1995) The mechanism of phagocytic uptake promoted by invasin-integrin interaction. Trends Ce11 Bioi 5: 1 20-1 24.

Jaconi, M.E.E., Lew, D.P., Carpentier, J.-L., Mangnusçion, K.E., Sjogren, M. and Stendhal? 0. (1990) Cytosolic free calcium elevation mediates the phagosome- lysosome fusion during phagocytosis in human neutrophils. J Cell Bi01 110:1555- 1564.

Johnson, D.W., Saunders, H.J., Johnson, F.J., Huq, S.O., Field, M.J. and Pollock, C.A. (1 999) Fibrogenic effects of cyclosporin A on the tubulointerstitium: role of cytokines and growth factors. Exp Nephrol7:470-478.

Jones, C. (1986) Gingival hyperplasia associated with nifedipine. Br Dent J 16O:416-417.

Kapur, R.N., Grigis, S., Little, T.M. and Masotti, R.E. (1973) Diphenylhydantoin induced gingival hyperplasia: Its relation to dose and serum level. Dev Med Child Neurol15: 483-487.

Kay, J.E. (1 989) lnhibitory effects of CsA on lymphocyte activation. In: Cvclos~orin. Mode of action and clinical a~plication (ed._ A.W. Thomson) Lancaster, UK: Kluwer Academic Publisher, p 4.

Kay, J.E. and Benzie, C.R. (1986) Lymphocyte activation by OKT3: cyclosporine sensitivity and synergism with phorbol ester. lmmunology 57:195-199.

Kay, J.E. (1 987) The cyclosporin A-sensitive event in lymphocyte activation. Ann lnst Pasteur lmmunol138(4):622-625.

Kay, J.E., Meehan, R.T. and Benzie, CR. (1983) Activation of T lymphocytes by 12-O-tetradecanoylphorbol-1 Sacetate is resistant to inhibition by cyclosporin A. lmmunol Lett 7: 151 -1 56.

Kimball, 0. (1 939) The treatment of epilepsy with sodium diphenyl-hydantoinate. JAMA 112: 1244.

King, S. (1 988) An assessrnent of p hosphoinositide hydrolysis in antigenic signal transduction in lymphocytes. Immunology 65: 1 -7.

Kirchhofer, D., Languino, L.R., Ruoslahti, E., and Pierschbacher, MD. (1990) a2Bi integrins from different ceIl types show different binding specifities. J Bioi Chem 265:615-618.

Kobayashi, K., Takahashi, K. and Nagasawa, S. (1995) The role of tyrosine phosphorylation and ca2+ accumulation in Fcy receptor-mediated phagocytosis in neutrophils J Biochem 1 17: 1 156-1 161.

Kulkarni, G.V. and McCulloch, C.A.G. (1 995) Concanavalin A induced apoptosis in fibroblasts: The role of cell surface carbohydrates in lectin rnediated cytotoxicity. J Cell Physioll65: 1 1 9-1 33.

Kulkarni, G.V., Lee, W., Seth, A. and MCCUIIOC~,C.A.G. (1998) Role of mitochondrial membrane potential in concanavalin A-induced apoptosis in human fibroblasts. Exp CeIl Res 244: 170-1 78.

Kwiatkowska K, Sobota A. Signaling pathways in phagocytosis. (1 999) BioEssays 21 : 422-431 .

Lainson, P.A. (1 986) Gingival overgrowth in a patient treated with nifedipine (Procardia). Periodontal Case Rep 8: 1 4- 1 6.

Landolfi, B., Curci, S., Debellis, L., Pouan, T. and Hofer, A.M. ca2' homeostasis in the agonist-sensitive interna1 store: functional interactions between mitochondria and the ER measured in situ in intact cells. J Ceil Biol 142:1235- 1243.

Lederman, D., Lumerman, H., Reuben, S. and Freedman, P.D. (1 984) Gingival hyperplasia associated nifedipine treatment. Report of a case. Oral Surg Oral Med Oral Path 57(6):620-622.

Lee, W., Sodek, J. and McCulloch, C.A.G. (1996) Role of integrins in regulation of collagen phagocytosis by human fibroblasts. J Ce11 Physiol168:695-704.

LeGrue, S. J., Friedman, A.W., and Kahan, B.D. (1 983). Binding of cyclosporine by human lymphocytes and phospholipid vesicles. J lmmunol131:712-718.

Lindholm, A. and Kahan, B.D. (1 993) Influence of cyclosporine pharmacokinetics, trough concentrations, and AUC monitoring on outcorne after kidney transplantation. Clin Pharmacol Ther M(2): 205-21 8.

Liu, X., Wu, H., Byrne, M., Jeffrey, J., Krane, S. and Jaenisch, R. (1995) A targeted mutation at the known collagenase cleavage site in mouse type 1 collagen impairs tissue remodelling. J Ce11 Biol l30:227-237.

Lee, H., Theilade, E. and Jensen, S. (1965) Experimental gingivitis in man. J Periodontol36: 177-187.

Lucas, R.M., Howell, L.P. and Wall, B.A. (1 985) Nifedipine-induced gingival hyperplasia. A histochemical and ultrastructural study. J Periodontol56(4):211- 21 5.

Marusich, M.F., Robinson, B.H., Taanman, J.-W., Kim, S.J., Schillace, R., Smith, J.L. and Capaldi, RA.. (1 997) Expression of mtDNA and nDNA encoded respiratory chain proteins in chemically and genetically-derived Rh00 human fibroblasts: a cornparison of subunit proteins in normal fibroblasts treated with ethidium bromide and fibroblasts from a patient with mtDNA depletion syndrome.. Biochim Biophys Acta 1382: 145-1 59.

McCulloch, C.A.G. and Knowles, G.C. (1 993) Deficiencies in collagen phagocytosis by human fibroblasts in vitro: A mechanism for fibrosis? J Ce11 Physiol 1 55:461-471.

McGaw, W.T., Lam, S. and Coates, J. (1 987) Cyclosporine-induced gingival overgrowth: correlation with dental plaque scores, gingivitis scores and cyclosporine levels in serum and saliva. Oral Surg Oral Med Oral Pathol Oral Radio1 Endodo 64: 293-297.

McGaw, W.T. and Porter H. (1 988) Cyclosporine-induced gingival overgrowth: An ultrastructural stereologic study. Oral Surg Oral Med Oral Pathol65:186-90.

McGaw, W.T. and Ten-Cate, A.R. (1983) A role for collagen phagocytosis by fibroblasts in scar remodelling: An ultrastructural sterologic study. J lnvest Dermat0181 :375-78.

McKeown, M., Knowles, G. and McCulloch, C.A.G. (1 990) Role of the cellular attachment domain of fibronectin in the phagocytosis of beads by human gingival fibroblasts in vitro. Ce11 Tissue Res 262:523-530.

Melcher, A.H. and Chan, J. (1 981 ) P hagocytosis and digestion of collagen by gingival fibroblasts in vivo: A study of serial sections. J Ultrastruct Res 77:l-36.

Mignatti, P., Rifkin, D.B., Welgus, HG. and Parks, W.C. (1996) Proteinases and tissue rernodeling. In The Molecular and Cellular Bioloav of Wound Re~air. Second Edition. (ed. R.A.F.Clark)Plenurn Press, New York:pp 427-474.

Mochly-Rosen, D. and Gordon, A.S. (1 998) Anchoring proteins for protein kinase C: a means for isozyme selectivity. FASEB J 12:35-45.

Mutsaers, S.E., Bishop, J.E., McGrouther, G. and Laurent, G.J. (1997) Mechanism of tissue repair: from wound healing to fibrosis. lnt J Biochem Cell Bi01 29:5-17.

Nabel, G.J. (1 999) A transformed view of cyclosporin. Nature 397(6719):47l- 472.

Narayanan, A.S. and Page, R.C. (1 983) Connective tissues of the periodontium: A summary of current work. Collagen Re/ Res 3:33-64.

Newell, J. and Irwin, C R . (1 997) Comparative effects of cyclosporin on glycosaminoglycan synthesis by gingival fibroblasts. J Periodontol68(5):443-47.

Nicolli, A., Basso, E., Petronilli, V., Wenter, R.M. and Bernardi, P. (1996) Interactions of cyclophlin with the rnitochondrial inner membrane and regulation of the permeability transition pore, a cyclosporin A-sensitive channel. J Bid Chem 271 :2185-2192,

Panuska, H.J., Gorlin, R.J., Bearman, J.E. and Mitchell, D.F. (1961) The effect of anticonvulsant drugs upon the gingiva. A series of 1048 patients. II. J Periodontol 32: 15-28.

Park, K.S., Kim, T.K. and Kim, D.H. (1 999) Cyclosporin A treatment alter characte ristics of ca2'-release channel in sarcoplasmic reticulum. Am J Physiol 276 (Heart Circ Physiol45):H865-H872.

Pender, N. and McCulloch, C.A.G. (1 991) Quantitation of actin polymerization in two human fibroblast subtypes responding to mechanical stretching. J Ce// Sci 100: 1 87-1 93.

Putney, J.W. Jr. (1 986) A model for receptor-regulated calcium entry. Ce// Calcium 7: 1 -1 2.

Rabinovitch, M. (1 995) Professional and non-professional phagocytes: an introduction. Trends Celi Bio/5:85-87,

Raeste, A.M ., Collan, Y. and Kilpinen, E. (1 978) Hereditary fibrous hyperplasia of the gingiva with varying penetrance and expressivity. Scand J Dent Res 86: 357- 365.

Rateitschak-PIiiss, E.M., Hefti, A., Lortscher, R. and Thiel, G. (1983) Initial observation that cyclosporine A induces gingival enlargement in man. J Clin Periodontol 10: 237-246.

Riuuto R., Brini M., Murgia, M. and Pouan T. (1993) Microdomains with high ca2+ close to IP3-sensitive channels that are sensed by neighbouring mitochondria. Science 262:744-747.

Rossrnann, J.A., Ingles, E. and Brown, R.S. (1 994) Multirnodal treatment of drug- induced gingival hyperplasia in a kidney transplant patient. Compendium Contin Educ Dent 15: 1266-1 274.

Savage, M.K., Jones, D.P. and Reed D.J. (1 991 )Calcium- and phosphate- dependent release and loading of glutathione by liver mitochondria. Arch Biochem Biophys 290(1):51-6.

Schwartz, M.A. (1 993)Spreading of human endothelial cells on fibronectin or vitronectin triggers elevation of intracellular free calcium. J Ce11 Bi01 120(4):1003- 1010

Sells, R. Long-term cyclosporin: Delivering effective therapeutic levels. London, England: Royal Society of Medicine Press Ltd., 1 994: 1 -3, 56.

Seymour, R.A., Smith, D.G. and Rogers, S.R. (1 987) The comparative effects of azathioprine and cyclosporine on some gingival health parameters of renal treansplant patients. A longitudinal study. J Clin Periodontol14: 61 0-61 3.

Seymour, R. and Heasman, P. (1 988) Drugs and the periodontium. J Clin Periodontol 1 5: 1 - 1 6,

Silverstein, L.H., Koch, J.P., Lefkove, M.D., Garnick, J.J., Singh, B. and Steflik, D.E. (1 995) Nifedipine-induced gingival enlargement around dental implants: a clinical report. J Oral implant01 21 : 1 1 6-1 20.

Slavin, J. and Taylor, J. (1 987) Cyclosporine, nifedipine, and gingival hyperplasia. Lancet 2(8561): 739.

Srniley, S.T., Reers, M., Mottola-Hartshorn, C., Lin, M., Chen, A., Smith, T.W., Steele, G.D. Jr. and Chen, L.B. (1 991 ) lntracellular heterogeneity in mitochondrial membrane potentials revealed by a J-aggregate-forming lipophilic cation JC-1. Proc Nat1 Acad Sci USA 88:3671-3675.

Sodek, J. and Overall, C. (1988) Matrix degradation in hard and soft connective tissues. ln The Bioloaical Mechanisms of Tooth Eru~tion and Root Resorption (ed. 2. Davidovitch) Birmingham, AL:EBSCO Media:303-311.

Staple, P.H., Reed, M.J., Mashimo, P.A., Sedransk, N. and Umemoto, T. (1 978) Diphenylhydantoin gingival hyperplasia in Macaca arctoides. Prevention by inhibition of dental plaque deposition. J Periodontol49: 31 0-325.

Stendahl, O., Krause, K.-H., Krischer, J., Jerstrdm, P., Theler, J.M., Clark, RA., Carpentier, J.L. and Lew, D.P. (1994) Redistribution of intracellular calcium stores during phagocytosis in human neutrophils. Science 265: 1439-1 441 .

Sullivan, P.G., Thompson, M.B. and Scheff, W. (1 999) Cyclosporin A attenuates acute rnitochondiral dysfunciton following traumatic brain injury. Experimental Neurology 1 60: 226-234.

Svoboda, E.L.A., Shiga, A. and Deporter, D.A. (1 981) A stereologic analysis of collagen phagocytosis by fibroblasts in three soft connective tissues with differing rates of collagen turnover. Anat Rec 199:473-480.

Thiru, S. Pathological effects of cyclosporin A in clinical practice. ln Cvclos~orin Mode of Action and Clinical A~plication (ed. AW Thomson) Lancaster. UK: Kluwer Academic Publisher, 1989: 324-359.

Thomason, J.M., Sloan, P. and Seymour, R.A. (1 998) lmmunolocalization of collagenase (MMP-1) and stromelysin (MMP-3) in the gingival tissues of organ transplant patients medicated with cyclosporin. J Clin Periodontol25:554-560.

Tipton, D.A., Stricklin, G.P. and Dabbous, M.K. (1 991) Fibroblast heterogeneity in collagenolytic response to cyclosporine. J Ce11 Biochern 46(2): 152-1 65.

Van Der Zee, E. Everts, V., Hoeben, K. and Beertsen, W. (1995) Cytokines rnodulate phagocytosis and intracellular digestion of collagen fibrils in rabbit periosteal explants. Inverse effects on procollagenase production and collagen phagocytosis. J Ce11 Sci 108: 3307-331 5.

Van Rossum, DB., Patterson, R.L., Ma, H.T. and Gill, D.L. (2000) Calcium entry mediated by store-depletion, S-nitrosylation and TRP3 channels: cornparison of coupling and function. J Bi01 Chem epub ahead of print.

Wilson TG, Kornman KS. Fundarnentals of periodontics. Carol Stream, IL: Quintessence Publishing Co., 1 996: 263-265.

Young, J.D., Ko, S.S. and Cohn, Z.A. (1 984) The increase in intracellular free calcium associated with IgG gamma Pblgamma 1 Fc receptor-ligand interactions: role in phagocytosis. Proc Nati Acad Sei USA 81 (1 7): 5430-5434.

Zamzami, N., Marchetti, P., Castedo, M., Decaudin, D., Mach, A., Hirsch, T., Susin, S.A., Petit, X.A., Mignotte, B. and Droemer, G. (1995) Sequential reduction of mitochondrial transmembrane potential and generation of reactive oxygen species in early programmed ceIl death. J Exp Med 182:367-377.

Zilberman, Y., Howe, L.R., Moore, J.P., Hesketh, T.R. and Metcalfe, J.C. (1987) Calcium regulates inositol 1,3,4,5-tetrabisphosphate production in lysed thymocytes and in intact cells stimulated with concanavalin A. EMBO J6:957- 962.

Zoratti, M. and Szabo, 1. (1 995) The mitochondrial permeability transition. Biochim Biophys Acta 1241 : 1 39-1 76.