research article potential immune modularly role of...

Hindawi Publishing CorporationClinical and Developmental ImmunologyVolume 2013 Article ID 808367 9 pageshttpdxdoiorg1011552013808367

Research ArticlePotential Immune Modularly Role of Glycine inOral Gingival Inflammation

Teresa Schaumann1 Dominik Kraus2 Jochen Winter3 Michael Wolf1

James Deschner4 and Andreas Jaumlger1

1 Department of Orthodontics Welschnonnenstraszlige 17 53111 Bonn Germany2Department of Prosthodontics Preclinical Education and Material Sciences Welschnonnenstraszlige 17 53111 Bonn Germany3Department of Periodontology Operative and Preventive Dentistry Welschnonnenstraszlige 17 53111 Bon Germany4 Experimental Dento-Maxillo-Facial Medicine (CRU 208) Welschnonnenstraszlige 17 53111 Bonn Germany

Correspondence should be addressed to Andreas Jager ajaegeruni-bonnde

Received 6 August 2013 Accepted 2 October 2013

Academic Editor Lenin Pavon

Copyright copy 2013 Teresa Schaumann et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Gingival epithelial cells (GECs) represent a physical barrier against bacteria and are involved in the processes of innate immunityRecently an anti-inflammatory and immune-modulatory effect of the amino acid glycine has been demonstrated However there isonly little information about the immune-modulatory effects of glycine in oral tissuesThis study aimed to investigate the existenceand role of the glycine receptor in gingival tissue analyzing tissuescells from extracted human molars via immunohistochemicalanalysis In vitro GECs were challenged by inflammatory conditions with IL-1120573 alone or in combination with glycine and analyzedfor cytokine expression of IL6IL8 via real-time PCR On protein level the effect of nuclear translocalization of NF120581B protein p65was analyzed using immunofluorescence analysis A distinct proof of the GlyR in oral gingival tissue and keratinocytes could bedemonstrated Isolated challenge of the keratinocytes with IL-1120573 as well as with glycine resulted in an upregulation of IL6 and IL8mRNA expression and activation of NF120581B pathway The presence of glycine in combination with the inflammatory stimulus led toa significant decrease in inflammatory parameters These results indicate a possible anti-inflammatory role of glycine in gingivalinflammation and encourage further research on the utility of glycine in the prevention or therapy of inflammatory periodontitis

1 Introduction

Periodontitis typically starts with inflammation of the gingivaand proceeds by spreading into the deeper structures ofthe periodontium leading to progressive destruction ofperiodontal tissues and the alveolar bone and to the lossof teeth [1] As a major part of the gingival tissue gingivalkeratinocytes represent a physical barrier to infections byperiodontal pathogens [2] While the epithelium was previ-ously thought to provide only a passive role in inflammationrecent articles demonstrated a new perspective to inflamma-tory conditions assigning an active role to the epithelium inthe host response to bacterial infections [3] According tothese authors the epithelium reacts to bacterial challengesby signaling host defense and integrating innate and acquiredimmune responses

Signaling pathways of gingival keratinocytes that aremodulated by bacteria such as Porphyromonas gingivalis inthe course of periodontal infection include changes in intra-cellular calcium ion (Ca2+) concentrations [4 5] Izutzu et al[4] demonstrated thatP gingivalis invasion and inflammatoryresponse in human gingival epithelial cells is related to arelease of Ca2+ from intracellular reservoirs and subsequentincrease in cytosolic Ca2+ Contact between P gingivalisand epithelial cells is shown to activate the host-cell Ca2+signaling system a further sign for its inflammatory impact

Similar inflammatory signaling is also reported for thecytokine interleukin 1120573 (IL-1120573) which is involved in thepathogenesis ofmany inflammatory diseases such as rheuma-toid arthritis inflammatory bowel disease atherosclero-sis and also periodontitis [6ndash8] In human keratinocytesinduction of IL-1120573 was demonstrated to take place upon

2 Clinical and Developmental Immunology

stimulation with bacterial lipopolysaccharide (LPS) phys-ical or thermal injury ultraviolet irradiation and a vari-ety of cytokines that is granulocyte-macrophage colony-stimulating factor (GM-CSF) tumor necrosis factor-120572 (TNF-120572) interleukin 6 (IL6) transforming growth factor-120573 (TGF-120572) and IL-1120573 [6 9] IL-1120573 is reported to trigger chemotaxisof neutrophil granulocytes as well as T and B cell activationIn addition it stimulates the expression of the early responsegenes multiple cytokines and inflammatory factors thatdrive extracellular matrix degradation [6 7]

For an anti-inflammatory purpose the use of glycine isreported to induce beneficial immune-modulatory and cyto-protective effects [10ndash12] L-glycine is the smallest nonessen-tial amino acid that consists of a methylene carbon moleculeattached to an amino- and a carboxyl group In nonner-vous tissue glycine is considered to be biologically neutralIn the last years however numerous investigations haverevealed significant effects of glycine on the activation ofcells belonging to the innate as well as the adaptive immunesystem including macrophages polymorphonuclear neu-trophils (PMNs) and lymphocytes [13ndash15] In patientrsquos treat-ment glycine has been reported to have several benefi-cial effects including protection against toxicity induced byanoxia oxidative stress and various toxic agents at the celltissue and whole body levels [10 12 16] For instance itwas demonstrated that a diet enriched with glycine protectedagainst LPS-induced lethality hypoxia-reperfusion injuryafter liver transplantation D-galactosamine-mediated liverinjury and experimental arthritis [10ndash12 17]

The glycine receptor (GlyR) is composed of four 48 kDa120572-subunits and a 58 kDa 120573-subunit and comprises a pen-tameric complex that forms a chloride-selective trans-mem-brane channel [18] In support of pharmacological evi-dence for the existence of GlyR in nonneuronal cells statedabove recent studies provide molecular evidence for theGlyR in nonneuronal cells A wide variety of cells suchas neutrophils alveolar macrophages endothelial cells andKupffer cells have been shown to contain glycine-gatedchloride channels [10ndash12 16] In addition Denda et al [19]demonstrated that the existence of the GlyR in epidermalkeratinocytes might play a crucial role in cutaneous barrierhomeostasis

Several reports showed that glycine suppresses formationof inflammatory cytokines [10ndash12] As stated above the pro-duction of proinflammatory mediators induced by bacterialLPS depends on the increase in intracellular Ca2+ an effectthat was demonstrated to be blunted after treatment withglycine The exact mechanism of how increased intracellularcalcium levels are blocked by glycine is not yet completelyunderstood

In recent investigations Breivik and coworkers [20]pointed to the anti-inflammatory potential of glycine in oraltissues In their animal experiments these authors wereable to demonstrate a significant reduction of artificiallyinduced periodontal infection following a specific glycinediet Substitution of glycine to animalrsquos food preventedanimals from severe periodontal breakdown However infor-mation is missing whether glycine and its receptors maybe able to modulate the development of inflammatory oral

gingivitis and if they are able to protect oral tissues fromsevere periodontal disease

In this context the first aim of the present study wasexpression analysis of glycine receptors in gingival tissue andfurther analysis of the glycine signaling role in oral gingivalkeratinocytesWehypothesized that glycine receptors are alsoexpressed within the oral gingival tissue and that glycineapplication might be able to modulate the inflammatoryresponse of gingival keratinocytes in inflammatory condi-tions

2 Material and Methods

All experimental protocols were reviewed and approved bythe Ethics Committee of the University of Bonn

21 Human Gingival Tissue Tissue samples from humangingiva were obtained during routine extraction treatmentof third molars Human gingiva samples were collected fromthree different human donors aged between 12 and 14 yearsshowing no clinical signs of gingivitis and periodontitis Theteeth had been extracted for orthodontic reasons and withwritten parental consent Following extraction human teethincluding gingiva were perfused with phosphate-bufferedsaline supplemented with 4 paraformaldehyde for fixationpurposes Afterwards the gingiva of each tooth was dissectedand prepared for light microscopical examination as recentlydescribed [21 22]

22 Histology For histological analyses all specimens wereprocessed for paraffin histology To perform histology 5ndash7 120583m serial sagittal sections of each specimen were preparedFor orientation purposes selected sections were stained withhematoxylin and eosin

23 Immunohistochemistry To analyze glycine receptor ex-pression tissue sections were processed for immunohisto-chemical detection of glycine receptor expression accordingto previously established protocols [22 23] In brief sectionswere incubated with primary antibody of rabbit origin raisedagainst a peptide mapping at the carboxy terminus of theprotein (anti-glycine receptor SYSY Gottingen Germany)in a 1 250 working solution of TBSBSA at 4∘C overnightin a humidified chamber A 1 100 dilution of a goat anti-rabbit immunoglobulin (DakoAS Denmark) was incubatedas secondary antibody for 30min Following further rinsinga PAP complex (1 150 in TBSBSA Dako AS Denmark) wasadministered for 30min prior to the visualization of anti-body bindingwith 331015840-diaminobenzidine (SigmaChemicalsUSA) solution for about 5min Thereafter specimens werecounterstained with Mayerrsquos hematoxylin dehydrated andcover-slipped for light microscopical analysis

In order to prove the specificity of the immunoreactionsnegative controls were carried out by (a) omitting the primaryantibody or using nonimmune IgG instead and (b) omittingboth the primary and secondary antibody and using TBSBSA instead

Clinical and Developmental Immunology 3

Preadsorption controls were used to exclude unspecificbinding of the antibodies to unrelated antigensThe antibodywas combined with a twofold excess of blocking peptideand incubated at 4∘C overnight Following neutralization theantibodypeptide mixture was diluted into the appropriateworking solution Afterwards immunohistochemistry wascarried out as described above

24 Explantation of Gingival Keratinocytes Gingival ker-atinocytesepithelial cells (GECs) were cultured from gingivaof three periodontal healthy patients as described aboveThe cells were isolated by collagenase digestion and sub-sequent mechanical isolation of the epithelial layer fromthe connective tissue as described previously [24] Follow-ing explantation cells were seeded on 60mm dishes andcultured in KGM-Medium (Keratinocyte Growth MediumPromoCell Heidelberg Germany) supplemented with CaCl

2

(015mM) and 05 antibiotics (diluted from a stock solutioncontaining 5000UmL penicillin and 5000UmL strepto-mycin Biochrom AG Germany) under a saturated humid-ified atmosphere containing 5 CO

2at 37∘C The medium

was changed every other day Prior to experimental usegingiva keratinocytes were characterized as described previ-ously [24] For cell culture experiments cells were used inpassage 4

25 Induction of Gingival Inflammation In Vitro To mimicinflammatory impact on gingival keratinocytes similar to theenvironment in gingivitis cells were cultured in presenceof the amount of 5 ngmL IL-1120573 (Promokine HeidelbergGermany) as measured in patients suffering from gingivalinfection and proven to be effective to induce inflam-matory response [24ndash26] For experiments in vitro cellswere grown to a confluent state (90) One hour prior tostimulation cells were adapted to a glycine-free medium(SMEM Supplemented Eaglersquos Minimum Essential MediumGIBCOInvitrogen Darmstadt Germany) For stimulationexperiments cells were treated with 5 ngmL IL-1120573 in thepresence or absence of glycine (5mM glycine) Following astimulation time of 30 or 60 minutes cells were analyzedfor changes in gene expression of proinflammatory markersIL6 and IL8 Preliminary experiments demonstrated a 30minstimulation time for IL6 and 60min stimulation time for IL8to be most effective for inflammatory response induction

26 Glycine Stimulation Experiments To analyze the possibleanti-inflammatory effect of glycine on gingiva keratinocytesin addition to the IL-1120573 conditioned medium cells wereadditionally stimulated with 5mM glycine (Sigma-AldrichTaufkirchen Germany) as demonstrated to be effective pre-viously by Vardar-Sengul et al [26] For negative controlglycine was administered alone

27 Analyses of NF120581B Pathway Activation To further char-acterize pro- or anti-inflammatory effects of IL-1120573 with orwithout glycine activation of NF120581B pathway was investi-gated According to a protocol published previously [24]gingival keratinocytes were stimulated for 6 h in combination

with IL-1120573with and without glycine as described above Afterincubation cells were fixed and further analyzed for nucleartranslocation of p65 immune reactions using immune cyto-chemistry with a primary antibody against NF120581B (BioLe-gend Uithoorn Netherlands) in a 1 100 concentration [7]The amount of cells showing positive immunoreactionsover the cell nucleus was assessed semiquantitatively by amodification of the grading system published previously[21] The grading system was set up as follows 0 = noimmune reaction in cell nucleus 1 = 13 of the cell nucleusdemonstrated immune reactions 2 = 23 of the cell nucleusdemonstrated immune reactions 3 = immune reaction allover the cell nucleus and the cytoplasm were visible 4 =immune reactivity was only located in cell nucleus Cells werecounted in relation to total cell number at the investigatedregion using AxioVision software (Carl Zeiss Germany) Toensure reproducibility all counts were performed on fourindependent experiments per stimulation group

28 RNA Extraction and cDNA Synthesis RNA was iso-lated using the ldquoRNeasy Protect Mini Kitrdquo (Qiagen HildenGermany) and quantified using the NanoDrop ND-1000Spectrophotometer (NanoDrop Technologies WilmingtonUSA) 1 120583g total RNAwas reversely transcribed using ldquoiScriptSelect cDNASynthesis Kitrdquo (BioRadMunichGermany)witholigo(dT)-primers [24]

29 Quantitative Real-Time PCR Gene expression of 120573-actin IL6 and IL8 was analyzed by real-time PCR withthe iCycler Thermal Cycler (BioRad) SYBR Green servedas fluorophore for online monitoring of generated PCRproducts as described previously [24] In brief all primerswere synthesized according to the highest quality standards(Metabion Martinsried Germany) and verified by computeranalysis for specification (BLAST) Primer sequences arepresented in Table 1 Amplification for the detection of 120573-actin was performed under the following conditions 95∘Cfor 5min followed by 40 cycles of 95∘C for 15 s 69∘C for30 s and 72∘C for 30 s IL6 and IL8 were amplified underthe same conditions except for the annealing temperaturewhich was set at 68∘C 50 ng cDNA was added to a mastermix containing primers and IQ SYBR Green Supermix(BioRad) The reference gene 120573-actin was used as standardfor normalizing the crossing point Cloned PCR productsderived from the specific primers served as positive controlsfor the PCR while water was used as the negative controlPCR was performed for all samples individually Resultinggene expressions were averaged Relative differential geneexpression was calculated using the method described byPfaffl [27] PCR efficiencies were determined with dilutionseries as listed above (Table 2)

210 Immunofluorescence andCytochemistry GECswere cul-tured on sterile coverslips and grown to 90 of conflu-ency The cells were washed with phosphate-buffered saline(PBS PAA Laboratories Colbe Germany) fixed with 4paraformaldehyde for 15min at room temperature washedwith PBS and permeabilized thereafter in 01 Triton X-100


Table 1 Primer sequences for real-time PCR for 120573-actin IL-6 andIL-8

Gene Primer sequence

120573-actin Sense 51015840-CATGGATGATGATATCGCCGCG-31015840

Antisense 51015840-ACATGATCTGGGTCATCTTCTCG-31015840

IL-6 Sense 51015840-ATGAACTCCTTCTCCACAAGC-31015840

Antisense 51015840-CTACATTTGCCGAAGAGCCC-31015840

IL-8 Sense 51015840-ATGACTTCCAAGCTGGCCGTGG-31015840

Antisense 51015840-TGAATTCTCAGCCCTCTTCAAAAAC-31015840

Table 2 Primer efficiencies and corresponding annealing tempera-tures for 120573-actin IL-6 and IL-8

Gene Efficiency Temperature (∘C)120573-actin 184 69IL-6 212 68IL-8 202 68

in PBS for 10min After washing with PBS nonspecific bind-ing was blocked by a 60min treatment in a 1 bovine serumalbumin solution (BSA Sigma-Aldrich) Incubation with theprimary anti-glycine receptor antibody (SYSY GottingenGermany 1250) was performed overnight at room temper-ature Following extensive washing a Cy-3-conjugated anti-rabbit IgG secondary antibody (Dianova Hamburg Ger-many) was applied for 50min at room temperature Finallycells were washed three times with PBS Nuclear staining wasachieved by incubating the cells in DAPI for 8min followedby washing twice with Aqua dest Cells were mountedwith MowiolDABCO (Roth Karlsruhe Germany) for flu-orescence microscopic imaging using a Zeiss fluorescencemicroscope (AXIO ImagerA1 Carl ZeissMicroImaging) Forcytochemistry a horseradish-peroxidase- (HRP-) conjugatedsecondary antibody (Dako Invision) was used instead andcells were counterstained with hemalaun as described above

211 Statistical Analysis The PCR data are presented asmeans plusmn SD of 10 independent experiments Statisticalsignificant differences in means were assessed using one-way analysis of variances (ANOVA) in GraphPad Prismversion 403 for Windows (Graph Pad Software San DiegoCalifornia USA httpwwwgraphpadcom) A value of 119875 lt005 was considered statistically significant

3 Results

31 Presence of the Glycine Receptor inHumanGingival TissueExplants of human gingival tissue were analyzed for glycinereceptor expression by immunohistochemistry to investigatewhether glycine receptor is expressed in human gingivaltissue The immune staining of healthy human gingivaltissue sections revealed a strong glycine receptor expressionwithin the epithelial layers of the gingiva tissue Within theinvestigated samples glycine receptor immune reactivity wasmainly concentrated to gingival keratinocytes (Figures 1(a)

and 1(b)) At cellular level positive immune reactions weremainly located at both cell membranes and in the cytoplasmof the cells The underlying basal membrane of the gingivatissue as well as the other connective tissues showed nopositive immune reactions for the glycine receptor

32 Demonstration of Glycine Receptor in Isolated GingivaKeratinocytes In a second step the aim was to verify theobserved in vivo findings of glycine receptor expression inhuman gingival tissue sections in isolated human gingivalkeratinocytes in order to provide the basis for furtherexperiments on those cells Similar to the in vivo findingspositive immune reactions for glycine receptor were alsofound in cell cultures of isolated gingival keratinocytes Inour experiments fourth passage gingival keratinocytes fromhealthy humane donors demonstrated a significant amountof immune reactions for glycine receptor expression mainlylocated on the outer cell membrane and in the cell cytoplasm(Figures 2(a) 2(b) and 2(c))

33 Application of Isolated Challenge with IL-1120573 To mimican inflammatory impact on gingival keratinocytes similar toconditions in oral gingivitis in patients cells were culturedin presence of the amount of IL-1120573 which was measured ingingival inflamed patients previously [26] Following IL-1120573stimulation an impact on mRNA expression of proinflam-matorymarkers interleukin 6 and interleukin 8was observedCompared to untreated controls themRNAexpression of IL6was increased by 23-fold (Figure 3(a)) and IL8 expressionwasupregulated by 35-fold (Figure 3(b)) Changes in IL6 and IL8were demonstrated to be significant to control cultures

34 Effect of Glycine on IL6 and IL8 mRNA Expression Inaddition to the IL-1120573 administration cells were additionallystimulated with glycine to analyze the anti-inflammatorypotential of glycine on induced gingival inflammatory con-ditions Following glycine administration the expression ofthe analyzed proinflammatory markers was demonstratedto be downregulated The observed inflammatory effect onIL6 expression was significantly reduced compared to theisolated IL-1120573 stimulation group (08- instead of 23-fold ofcontrol Figure 3(a)) The effect on IL8 mRNA expressionwas also significantly reduced when glycine was added tothe conditioned cell culture medium (24- instead of 35-foldof control (Figure 3(b)) Interestingly isolated application ofglycine seems also to induce a slight upregulation of IL6 inmRNA expression (Figure 3(a)) In combination with IL-1120573 adownregulation was observed

35 Effect of Glycine on NF120581B Pathway-Mediated Inflam-matory Response As a second approach the immune-modulatory effect of glycine on inflammatory signalingpathway NF120581B using the NF120581B related protein p65 wasanalyzed In this context changes were addressed for p65protein expression which is localized in the cytoplasm innoninflammatory conditions In the present experimentsp65 protein was demonstrated to be located in the cell cyto-plasm of untreated human gingival keratinocytes (Figure 4)


(a) (b)

Figure 1 Demonstration of the glycine receptor (GlyR) in gingival tissue Overview (a) of oral tissue (magnification times10) isolatedfrom extracted upper third molars showing different region of the gingival tissue (OG oral gingival tissue st subepithelial tissue) (b)Representative gingival tissue section (magnification times200) assessed by immunohistochemistry using an anti-glycine receptor antibody(SYSY Gottingen Germany) at 4∘C overnight and counterstaining with DAB (brown color) There was strong immune reaction in theepithelial layers of the gingiva namely in gingival keratinocytes The underlying basal membrane as well as the connective tissue showednegative immune staining

(a) (b) (c)

Figure 2 Cytochemical ((a) negative control (b) anti-glycine receptor) and immunofluorescence ((c) anti-glycine receptor) staining forGlycine receptor in cultured human gingival keratinocytes (red color black arrow) For nuclear staining cells were treated with DAPI (bluecolor (c))

GlycineIL-1120573

IL6 m RNA expression

00

05

10

15

20

25

30

35

40

45

minus

lowast

minus

minus

minus+

+ +

+

mRN

A ex

pres

sion

(fold

of c

ontro

l)

(a)


lowast

lowast

00

05

10

15

20

25

30

35

40

45

GlycineIL-1120573

minus

minus

minus

minus+

+ +

+

mRN

A ex

pres

sion

(fold

of c

ontro

l)

(b)

Figure 3 Effect of IL-1120573 and glycine treatment on the mRNA expression of the proinflammatory cytokines IL6 (a) and IL8 (b) Confluentcultures of gingival keratinocytes were treated with IL-1120573 either alone or in combination with glycine to induce inflammatory conditions invitro Glycine administration to inflammatory challenged gingival keratinocytes resulted in a decreased proinflammatory cytokine mRNAexpression of both IL6 (a) and IL8 (b) Results are presented as means plusmn SD (119899 = 10) lowast119875 lt 005 difference between groups following ANOVAanalysis


Control 6h Glycine 6h

IL-1120573 6h IL-1120573 + glycine 6h

A B

C D

(a)

lowast

lowast

of p65

tran

sloca

tion

Con

trol

Gly

cine

IL-1120573

IL-1120573

+ gl

ycin

e

0

1

2

3

4

Sem

iqua

ntita

tive a

sses

smen

t

NF-120581B activation p65

(b)

Figure 4 Effects of IL-1120573 andor glycine on NF120581B signaling (a) Immune fluorescence staining of NF120581B signaling protein p65 of cells (A)glycine (B) IL-1120573 (C) and IL-1120573 + glycine (D) treated human gingival keratinocytes The photographs demonstrate a localization of p65protein primarily in the cell cytoplasm in untreated cells Untreated controls (A) and glycine treated cells (B) did not affect p65 expressionFollowing the induction of inflammation by IL-1120573 administration NF120581B pathway was activated as demonstrated by translocation of p65protein to the cell nucleus (C)The addition of glycine to the inflammatory cell culturemedium reduced the observed p65 protein translocation(D) (b) Semiquantitative assessment of p65 protein translocation in human gingival keratinocytes following administration of IL-1120573 incombination with and without glycine lowast119875 lt 005 difference between treated and control groups following ANOVA analysis

119875 lt 005difference between IL-1120573 and IL-1120573 + glycine treated groups


Following the induction of inflammation by stimulationwith IL-1120573 a pronounced accumulation of NF120581B in the cellnucleus was observed When glycine was added to the condi-tioned cell culturemedium the inflammatory translocation ofp65 NF120581B pathway protein was almost completely abolishedSimilar to control experiments p65 immune reactions weremainly located in the cytoplasm of the cells and did nottranslocate to the cell nucleus in glycine treated cell cultureexperiments

4 DiscussionTo our knowledge this study is the first which analyzesthe existence of the glycine receptor in gingival tissue andgingival derived keratinocytes Both tissue sections of humangingival tissue and human gingival keratinocytes demon-strated a ubiquitous expression of the glycine receptor in thegingival tissuewhichwasmainly located at the cellmembraneand in the cytoplasmic region Similar to present findingsother authors documented similar findings on the location ofthe glycine binding receptor (GlyR) in different cell systemsFor example Webb and Lynch demonstrated in their studieson neural cells that the glycine receptor was located on thecell membrane which acts as a receptor with transmembranedomains and extracellular binding sites as well as chloride-sensitive transmembrane channels [28] Similar to this reportZhong et al [11] also demonstrated the GlyR as a membranereceptor in ldquoKupfferrdquo cells On the other hand Bechade[29] observed both a membrane and a more cytoplasmaticlocalization of the GlyR in oligodendrocytes and in glia cellsFrom the present findings the expression of GlyR both inthe membrane and within the cell cytoplasm in gingivalkeratinocytes can be suggested

To mimic the clinical situation of gingival inflammationsuch as in conditions of oral gingival inflammation in thepresent in vitro experiments isolated gingival keratinocyteswere challenged with IL-1120573 at the amount being found tobe expressed in patients suffering from gingivitis [30] Theused amount of interleukin 1120573 application has been provento be effective in the present findings as shown by theupregulation of cytokine mRNA expression levels of IL6and IL8 and the induction of NF120581B activation in treatedgingival keratinocytes Steinberg et al [7] and McKay andCidlowski [31] also reported similar effects of IL-1120573-inducedinflammation in their experiments on gingival keratinocytesafter stimulation with IL-1120573 Also the inflammatory responseof oral cells to cytokine application on protein level ofthe investigated inflammatory parameters was proven inprevious experiments suggesting the effectiveness of thepresent protocol [24 32 33] A significant inflammatoryresponse of treated specimens was observed in this studyand also in reports from other authors Corresponding to thepresented regulation of IL6 and IL8 mRNA expression alsoKraus and coworkers [24] presented in a recent work thatbacterial LPS stimulation was able to show similar and evenstronger inflammatory responses of gingival keratinocytesas documented in the present study Again the present dataon IL-1120573 induced inflammation underline the efficiency andcapability of IL-120573 to induce inflammatory reactions

The addition of glycine to the cell culture mediumof inflammatory challenged gingival keratinocytes demon-strated the ability to attenuate the observed inflammatoryresponse of gingival keratinocytes Since the standard Ker-atinocyte Growth Medium (KGM) contains different aminoacids including glycine we therefore analyzed its impact oncell culture conditions by using also glycine-free medium inpreliminary unpublished experiments Induction of differentinflammatory cytokines in keratinocytes was demonstratedto be nearly identical compared to the used standardmediumin these experiments Therefore the described standard cellculture medium was used in present experiments

Interestingly an initial upregulation of the mRNA expres-sion of the investigated early inflammatory marker IL6 butnot IL8 after 30min of stimulation with glycine alone wasobserved A possible explanation for this regulation might bedue to an early cellular response to changes within the cellculture medium by the application of glycine Interestinglyin combination with IL-1120573 protein this upregulation wasinhibited The regulation of IL8 expression which is knownas a more stable inflammatory marker was not affected byglycine application After adding glycine to the cytokine con-taminated medium a significant inhibition of the immediateupregulation of the cytokines IL6 and IL8 at the transcrip-tional level and a decrease in p65 nuclear translocation wereobserved This glycine-induced anti-inflammatory responsegoes hand in hand with reports of others on observations inseveral different cell and organ systems In his recent reviewarticle Zhong and coworkers [11] pointed to the observedanti-inflammatory potential of glycine They reported thatglycine is found to be protective under several inflammatoryconditions such as shock endotoxin and sepsis and toprevent ischemia-reperfusion injury to a variety of tissues andorgans including liver kidney heart intestine and skeletalmuscle Furthermore glycine also protected against pepti-doglycan polysaccharide-induced arthritis and protects thegastricmucosa against chemical and stress-induced ulcers Intheir review these authors concluded that glycine appears toexert several protective effects including anti-inflammatoryimmune-modulatory and direct cytoprotective actions [11]Furthermore it has been suggested that glycine is also able toact on inflammatory cells such as macrophages to suppressactivation of inflammatory transcription factors and theformation of free radicals and inflammatory cytokines Inthe light of the findings in the present investigation and thereported effects of glycine in the literature it can be suggestedthat also gingival tissue with special regard to gingivalkeratinocytes is able to perform a glycine-mediated anti-inflammatory response under inflammatory circumstances

5 Conclusions

In conclusion the present data demonstrate at first that theglycine receptor is expressed in gingival tissue and indicatesan immune-regulatory role for glycine in the response ofgingival keratinocytes to inflammatory conditions Togetherwith recent reports about glycine physiology the modula-tory potential of glycine within the inflammatory process


in gingival tissue becomes obvious The mapping of inter-cellular cytokine signaling networks and its modulatingreagents with special regard to glycine that functionallycouples gingival tissues and its activation to induce immuneresponse may also provide information for future potentialclinical targets This could include the prevention of severegingivitis and the further development of periodontal diseasewhich is discussed to be the result of long lasting gingivalinflammation Furthermore the immune-modulatory roleof glycine in the course of gingival inflammation extendsthe well accepted knowledge of anti-inflammatory effects ofglycine in the oral gingival tissue that again points to glycineas a promising treatment agent in inflammatory conditions

Authorsrsquo Contribution

Teresa Schaumann and Dominik Kraus contributed equallyto this work

Acknowledgments

The authors thank the German Research Foundation (DFG)as well as the Medical Faculty of the University of Bonn forproviding a research grant (KFO 208 TP8 LO-11812-2) Theauthors declare that they have no conflict of interests

References

[1] K S Kornman ldquoMapping the pathogenesis of periodontitis anew lookrdquo Journal of Periodontology vol 79 supplement 8 pp1560ndash1568 2008

[2] J Sandros C Karlsson D F Lappin P M Madianos D FKinane and P M Papapanou ldquoCytokine responses of oralepithelial cells to Porphyromonas gingivalis infectionrdquo Journalof Dental Research vol 79 no 10 pp 1808ndash1814 2000

[3] B A Dale ldquoPeriodontal epithelium a newly recognized role inhealth and diseaserdquo Periodontology 2000 vol 30 no 1 pp 70ndash78 2002

[4] K T Izutsu C M Belton A Chan et al ldquoInvolvement ofcalcium in interactions between gingival epithelial cells andPorphyromonas gingivalisrdquo FEMS Microbiology Letters vol144 no 2-3 pp 145ndash150 1996

[5] C M Belton P C Goodwin S Fatherazi M M Schubert RJ Lamont and K T Izutsu ldquoCalcium oscillations in gingi-val epithelial cells infected with Porphyromonas gingivalisrdquoMicrobes and Infection vol 6 no 5 pp 440ndash447 2004

[6] C A Dinarello ldquoBiologic basis for interleukin-1 in diseaserdquoBlood vol 87 no 6 pp 2095ndash2147 1996

[7] T Steinberg B Dannewitz P Tomakidi et al ldquoAnalysis ofinterleukin-1120573-modulated mRNA gene transcription in humangingival keratinocytes by epithelia-specific cDNAmicroarraysrdquoJournal of Periodontal Research vol 41 no 5 pp 426ndash446 2006

[8] O H Ryu S J Choi A M G Linares et al ldquoGingival epithe-lial cell expression of macrophage inflammatory pProtein-1120572induced by interleukin-1120573 and lipopolysacchariderdquo Journal ofPeriodontology vol 78 no 8 pp 1627ndash1634 2007

[9] J Ansel P Perry J Brown et al ldquoCytokine modulation ofkeratinocyte cytokinesrdquo Journal of Investigative Dermatologyvol 94 supplement 6 pp 101Sndash107S 1990

[10] M D Wheeler K Ikejema N Enomoto et al ldquoGlycine a newanti-inflammatory immunonutrientrdquo Cellular and MolecularLife Sciences vol 56 no 9-10 pp 843ndash856 1999

[11] Z Zhong M D Wheeler X Li et al ldquoL-Glycine a novel anti-inflammatory immunomodulatory and cytoprotective agentrdquoCurrent Opinion in Clinical Nutrition amp Metabolic Care vol 6no 2 pp 229ndash240 2003

[12] R Y Gundersen P Vaagenes T Breivik F Fonnum and PK Opstad ldquoGlycinemdashan important neurotransmitter and cyto-protective agentrdquo Acta Anaesthesiologica Scandinavica vol 49no 8 pp 1108ndash1116 2005

[13] A Spittler C M Reissner R Oehler et al ldquoImmunomodula-tory effects of glycine on LPS-treatedmonocytes reduced TNF-120572 production and accelerated IL-10 expressionrdquo FASEB Journalvol 13 no 3 pp 563ndash571 1999

[14] R F Stachlewitz X Li S Smith H Bunzendahl L M Gravesand R GThurman ldquoGlycine inhibits growth of T lymphocytesby an IL-2-independent mechanismrdquo Journal of Immunologyvol 164 no 1 pp 176ndash182 2000

[15] M Froh R G Thurman and M D Wheeler ldquoMolecular evi-dence for a glycine-gated chloride channel in macrophages andleukocytesrdquo American Journal of PhysiologymdashGastrointestinaland Liver Physiology vol 283 no 4 pp G856ndashG863 2002

[16] R Bruck J Wardi H Aeed et al ldquoGlycine modulates cytokinesecretion inhibits hepatic damage and improves survival in amodel of endotoxemia in micerdquo Liver International vol 23 no4 pp 276ndash282 2003

[17] X Li B U Bradford M DWheeler et al ldquoDietary glycine pre-vents peptidoglycan polysaccharide-induced reactive arthritisin the rat role for glycine-gated chloride channelrdquo Infection andImmunity vol 69 no 9 pp 5883ndash5891 2001

[18] S Rajendra J W Lynch and P R Schofield ldquoThe glycinereceptorrdquo Pharmacology andTherapeutics vol 73 no 2 pp 121ndash146 1997

[19] M Denda S Fuziwara and K Inoue ldquoInflux of calcium andchloride ions into epidermal keratinocytes regulates exocytosisof epidermal lamellar bodies and skin permeability barrierhomeostasisrdquo Journal of Investigative Dermatology vol 121 no2 pp 362ndash367 2003

[20] T Breivik Y Gundersen F Fonnum P Vaagenes and P KOpstad ldquoChronic glycine treatment inhibits ligature-inducedperiodontal disease in Wistar ratsrdquo Journal of PeriodontalResearch vol 40 no 1 pp 43ndash47 2005

[21] M Wolf S Lossdorfer N Abuduwali et al ldquoEffect of inter-mittent PTH(1-34) on human periodontal ligament cells trans-planted into immunocompromised micerdquo Tissue EngineeringPart A vol 18 no 17-18 pp 1849ndash1856 2012

[22] W Gotz T Gerber B Michel S Lossdorfer K O Henkeland F Heinemann ldquoImmunohistochemical characterization ofnanocrystalline hydroxyapatite silica gel (NanoBone) osteoge-nesis a study on biopsies from human jawsrdquo Clinical OralImplants Research vol 19 no 10 pp 1016ndash1026 2008

[23] M Wolf S Lossdorfer N Abuduwali and A Jage ldquoPotentialrole of high mobility group box protein 1 and intermittent PTH(1-34) in periodontal tissue repair following orthodontic toothmovement in ratsrdquo Clinical Oral Investigations vol 17 no 3 pp989ndash997 2013

[24] D Kraus J Winter S Jepsen A Jager R Meyer and JDeschner ldquoInteractions of adiponectin and lipopolysaccharidefrom Porphyromonas gingivalis on human oral epithelial cellsrdquoPLoS ONE vol 7 no 2 Article ID e30716 2012


[25] M Nokhbehsaim B Deschner J Winter et al ldquoInteractions ofregenerative inflammatory and biomechanical signals on bonemorphogenetic protein-2 in periodontal ligament cellsrdquo Journalof Periodontal Research vol 46 no 3 pp 374ndash381 2011

[26] S Vardar-Sengul S Arora H Baylas and D MercolaldquoExpression profile of human gingival fibroblasts induced byinterleukin-1 120573 reveals central role of nuclear factor-kappa bin stabilizing human gingival fibroblasts during inflammationrdquoJournal of Periodontology vol 80 no 5 pp 833ndash849 2009

[27] M W Pfaffl ldquoA new mathematical model for relative quantifi-cation in real-time RT-PCRrdquo Nucleic acids research vol 29 no9 article e45 2001

[28] T I Webb and J W Lynch ldquoMolecular pharmacology ofthe glycine receptor chloride channelrdquo Current PharmaceuticalDesign vol 13 no 23 pp 2350ndash2367 2007

[29] C Bechade ldquoExpression of glycine receptor a subunits andgephyrin in cultured spinal neuronsrdquo European Journal ofNeuroscience vol 8 no 2 pp 429ndash435 1996

[30] A Rawlinson M H N Dalati S Rahman T F Walsh andA L Fairclough ldquoInterleukin-1 and IL-1 receptor antagonist ingingival crevicular fluidrdquo Journal of Clinical Periodontology vol27 no 10 pp 738ndash743 2000

[31] L I McKay and J A Cidlowski ldquoCross-talk between nuclearfactor-120581B and the steroid hormone receptors mechanisms ofmutual antagonismrdquoMolecular Endocrinology vol 12 no 1 pp45ndash56 1998

[32] M Nokhbehsaim S Eick A V Nogueira et al ldquoStimulationof MMP-1 and CCL2 by NAMPT in PDL cellsrdquo Mediators ofInflammation vol 2013 Article ID 437123 12 pages 2013

[33] M Nokhbehsaim B Deschner J Winter et al ldquoAnti-inflam-matory effects of EMD in the presence of biomechanical loadingand interleukin-1120573 in vitrordquo Clinical Oral Investigations vol 16no 1 pp 275ndash283 2012

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


stimulation with bacterial lipopolysaccharide (LPS) phys-ical or thermal injury ultraviolet irradiation and a vari-ety of cytokines that is granulocyte-macrophage colony-stimulating factor (GM-CSF) tumor necrosis factor-120572 (TNF-120572) interleukin 6 (IL6) transforming growth factor-120573 (TGF-120572) and IL-1120573 [6 9] IL-1120573 is reported to trigger chemotaxisof neutrophil granulocytes as well as T and B cell activationIn addition it stimulates the expression of the early responsegenes multiple cytokines and inflammatory factors thatdrive extracellular matrix degradation [6 7]

For an anti-inflammatory purpose the use of glycine isreported to induce beneficial immune-modulatory and cyto-protective effects [10ndash12] L-glycine is the smallest nonessen-tial amino acid that consists of a methylene carbon moleculeattached to an amino- and a carboxyl group In nonner-vous tissue glycine is considered to be biologically neutralIn the last years however numerous investigations haverevealed significant effects of glycine on the activation ofcells belonging to the innate as well as the adaptive immunesystem including macrophages polymorphonuclear neu-trophils (PMNs) and lymphocytes [13ndash15] In patientrsquos treat-ment glycine has been reported to have several benefi-cial effects including protection against toxicity induced byanoxia oxidative stress and various toxic agents at the celltissue and whole body levels [10 12 16] For instance itwas demonstrated that a diet enriched with glycine protectedagainst LPS-induced lethality hypoxia-reperfusion injuryafter liver transplantation D-galactosamine-mediated liverinjury and experimental arthritis [10ndash12 17]

The glycine receptor (GlyR) is composed of four 48 kDa120572-subunits and a 58 kDa 120573-subunit and comprises a pen-tameric complex that forms a chloride-selective trans-mem-brane channel [18] In support of pharmacological evi-dence for the existence of GlyR in nonneuronal cells statedabove recent studies provide molecular evidence for theGlyR in nonneuronal cells A wide variety of cells suchas neutrophils alveolar macrophages endothelial cells andKupffer cells have been shown to contain glycine-gatedchloride channels [10ndash12 16] In addition Denda et al [19]demonstrated that the existence of the GlyR in epidermalkeratinocytes might play a crucial role in cutaneous barrierhomeostasis

Several reports showed that glycine suppresses formationof inflammatory cytokines [10ndash12] As stated above the pro-duction of proinflammatory mediators induced by bacterialLPS depends on the increase in intracellular Ca2+ an effectthat was demonstrated to be blunted after treatment withglycine The exact mechanism of how increased intracellularcalcium levels are blocked by glycine is not yet completelyunderstood

In recent investigations Breivik and coworkers [20]pointed to the anti-inflammatory potential of glycine in oraltissues In their animal experiments these authors wereable to demonstrate a significant reduction of artificiallyinduced periodontal infection following a specific glycinediet Substitution of glycine to animalrsquos food preventedanimals from severe periodontal breakdown However infor-mation is missing whether glycine and its receptors maybe able to modulate the development of inflammatory oral

gingivitis and if they are able to protect oral tissues fromsevere periodontal disease

In this context the first aim of the present study wasexpression analysis of glycine receptors in gingival tissue andfurther analysis of the glycine signaling role in oral gingivalkeratinocytesWehypothesized that glycine receptors are alsoexpressed within the oral gingival tissue and that glycineapplication might be able to modulate the inflammatoryresponse of gingival keratinocytes in inflammatory condi-tions

2 Material and Methods

All experimental protocols were reviewed and approved bythe Ethics Committee of the University of Bonn

21 Human Gingival Tissue Tissue samples from humangingiva were obtained during routine extraction treatmentof third molars Human gingiva samples were collected fromthree different human donors aged between 12 and 14 yearsshowing no clinical signs of gingivitis and periodontitis Theteeth had been extracted for orthodontic reasons and withwritten parental consent Following extraction human teethincluding gingiva were perfused with phosphate-bufferedsaline supplemented with 4 paraformaldehyde for fixationpurposes Afterwards the gingiva of each tooth was dissectedand prepared for light microscopical examination as recentlydescribed [21 22]

22 Histology For histological analyses all specimens wereprocessed for paraffin histology To perform histology 5ndash7 120583m serial sagittal sections of each specimen were preparedFor orientation purposes selected sections were stained withhematoxylin and eosin

23 Immunohistochemistry To analyze glycine receptor ex-pression tissue sections were processed for immunohisto-chemical detection of glycine receptor expression accordingto previously established protocols [22 23] In brief sectionswere incubated with primary antibody of rabbit origin raisedagainst a peptide mapping at the carboxy terminus of theprotein (anti-glycine receptor SYSY Gottingen Germany)in a 1 250 working solution of TBSBSA at 4∘C overnightin a humidified chamber A 1 100 dilution of a goat anti-rabbit immunoglobulin (DakoAS Denmark) was incubatedas secondary antibody for 30min Following further rinsinga PAP complex (1 150 in TBSBSA Dako AS Denmark) wasadministered for 30min prior to the visualization of anti-body bindingwith 331015840-diaminobenzidine (SigmaChemicalsUSA) solution for about 5min Thereafter specimens werecounterstained with Mayerrsquos hematoxylin dehydrated andcover-slipped for light microscopical analysis

In order to prove the specificity of the immunoreactionsnegative controls were carried out by (a) omitting the primaryantibody or using nonimmune IgG instead and (b) omittingboth the primary and secondary antibody and using TBSBSA instead




2
























3 Results








(a) (b)


(a) (b) (c)


GlycineIL-1120573


00

05

10

15

20

25

30

35

40

45

minus

lowast

minus

minus

minus+

+ +

+

mRN

A ex

pres

sion

(fold

of c

ontro

l)

(a)


lowast

lowast

00

05

10

15

20

25

30

35

40

45

GlycineIL-1120573

minus

minus

minus

minus+

+ +

+

mRN

A ex

pres

sion

(fold

of c

ontro

l)

(b)




IL-1120573 6h IL-1120573 + glycine 6h

A B

C D

(a)

lowast

lowast

of p65

tran

sloca

tion

Con

trol

Gly

cine

IL-1120573

IL-1120573

+ gl

ycin

e

0

1

2

3

4

Sem

iqua

ntita

tive a

sses

smen

t


(b)









5 Conclusions






Acknowledgments


References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















2
























3 Results








(a) (b)


(a) (b) (c)


GlycineIL-1120573


00

05

10

15

20

25

30

35

40

45

minus

lowast

minus

minus

minus+

+ +

+

mRN

A ex

pres

sion

(fold

of c

ontro

l)

(a)


lowast

lowast

00

05

10

15

20

25

30

35

40

45

GlycineIL-1120573

minus

minus

minus

minus+

+ +

+

mRN

A ex

pres

sion

(fold

of c

ontro

l)

(b)




IL-1120573 6h IL-1120573 + glycine 6h

A B

C D

(a)

lowast

lowast

of p65

tran

sloca

tion

Con

trol

Gly

cine

IL-1120573

IL-1120573

+ gl

ycin

e

0

1

2

3

4

Sem

iqua

ntita

tive a

sses

smen

t


(b)









5 Conclusions






Acknowledgments


References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





























3 Results








(a) (b)


(a) (b) (c)


GlycineIL-1120573


00

05

10

15

20

25

30

35

40

45

minus

lowast

minus

minus

minus+

+ +

+

mRN

A ex

pres

sion

(fold

of c

ontro

l)

(a)


lowast

lowast

00

05

10

15

20

25

30

35

40

45

GlycineIL-1120573

minus

minus

minus

minus+

+ +

+

mRN

A ex

pres

sion

(fold

of c

ontro

l)

(b)




IL-1120573 6h IL-1120573 + glycine 6h

A B

C D

(a)

lowast

lowast

of p65

tran

sloca

tion

Con

trol

Gly

cine

IL-1120573

IL-1120573

+ gl

ycin

e

0

1

2

3

4

Sem

iqua

ntita

tive a

sses

smen

t


(b)









5 Conclusions






Acknowledgments


References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a) (b)


(a) (b) (c)


GlycineIL-1120573


00

05

10

15

20

25

30

35

40

45

minus

lowast

minus

minus

minus+

+ +

+

mRN

A ex

pres

sion

(fold

of c

ontro

l)

(a)


lowast

lowast

00

05

10

15

20

25

30

35

40

45

GlycineIL-1120573

minus

minus

minus

minus+

+ +

+

mRN

A ex

pres

sion

(fold

of c

ontro

l)

(b)




IL-1120573 6h IL-1120573 + glycine 6h

A B

C D

(a)

lowast

lowast

of p65

tran

sloca

tion

Con

trol

Gly

cine

IL-1120573

IL-1120573

+ gl

ycin

e

0

1

2

3

4

Sem

iqua

ntita

tive a

sses

smen

t


(b)









5 Conclusions






Acknowledgments


References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















IL-1120573 6h IL-1120573 + glycine 6h

A B

C D

(a)

lowast

lowast

of p65

tran

sloca

tion

Con

trol

Gly

cine

IL-1120573

IL-1120573

+ gl

ycin

e

0

1

2

3

4

Sem

iqua

ntita

tive a

sses

smen

t


(b)









5 Conclusions






Acknowledgments


References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















5 Conclusions






Acknowledgments


References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















Acknowledgments


References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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