J Am Soc Nephrol 9: 2283-2290, 1998

Hyaluronan Induces Monocyte Chemoattractant Protein-i

Expression in Renal Tubular Epithelial Cells

BEATRICE BECK�SCHIMMER,*t BEAT OERTLI,*� THOMAS PASCH,t and

RUDOLF P. WUTHRICH*�*physiological Institute, University of Zllrich-lrchel; �Department ofAnesthesiology and �Division of

Nephrology, Department of Medicine, University Hospital, Zurich, Switzerland.

Abstract. Hyaluronan (HA) is a nonsulfated glycosaminogly-

can that accumulates in the renal interstitium in immune-

mediated kidney diseases. The functional significance of such

HA deposition in the kidney has not been elucidated. Several

studies have suggested that HA may exhibit proinflammatory

effects. Since chemokines such as monocyte chemoattractant

protein- 1 (MCP- 1) play an important robe in the recruitment of

beukocytes in renal injury, this study tested whether HA and its

fragments could promote MCP- 1 production by renal paren-

chymal cells. Mouse cortical tubular cells were stimulated with

fragmented HA or with high molecular weight HA (Heabon) in

vitro and were examined for MCP-l expression. Fragmented

HA, but not Heabon, increased MCP-l mRNA within 30 mm

with a peak after 2 h. In addition, a 10-fold increase of MCP- 1

protein in the supernatant was found after a 6-h stimulation

with fragmented HA. The enhanced MCP-l mRNA and pro-

tein expression in response to HA was dose-dependent between

1 and 100 �sg/mb. Upregubation of MCP-l protein production

could be blocked by preincubation with actinomycin D or

cycboheximide, suggesting that MCP-l mRNA and protein

expression in response to HA are based on de novo synthesis.

The HA-stimulated MCP-l production was also inhibited with

anti-CD44 antibodies, suggesting that MCP-l is upregubated at

least in part by signaling through CD44. In summary, frag-

mented HA markedly stimulates renal tubular MCP-l produc-

tion by mechanisms that involve binding to the HA receptor

CD44. It is hypothesized that the accumulation of HA in

immune renal injury could participate in the recruitment and

activation of inflammatory cells in vivo through production of

MCP-1.

The synthesis and the degradation of components of the extra-

cellular matrix (ECM) represent important steps in chronic

inflammation. One of the constituents of the altered ECM in

inflammation is hyaluronan (HA), a nonsulfated gbycosamino-

glycan that is composed of repeating units of (f31-4)-o-glucu-

ronic acid-(�3b-3)-N-acetyl-D-glucosamine (1). Under normal

conditions, HA is synthesized as a high molecular weight

extracellubar pobysaccharide (2). In inflammatory conditions,

high molecular weight HA may undergo degradation into low

molecular weight components, presumably under the influence

of oxygen free radicals or inflammation-specific hyaluroni-

dases (3-5).

High and low molecular weight forms of hyaburonan exert

different biological effects on tissues and cells. In macro-

phages, for example, which express the main HA receptor

CD44 abundantly, high molecular weight HA has no prom-

flammatony action, but it acts as an inhibitor of phagocytosis

(6), whereas low molecular weight HA induces proinflamma-

tory genes, growth factors, and nitric oxide synthase (7-10).

Depending on the balance of synthesis and degradation, HA

Received February 24, 1998. Accepted June 1, 1998.Correspondence to Dr. Rudolf P. WUthnich, Division of Nephrobogy, Univer-

sity Hospital. Rhmistrasse 100. 8091 ZUrich, Switzerland.

1046-6673/0901 2-2283$03.00/0

Journal of the American Society of Nephrology

Copyright 0 1998 by the American Society of Nephrology

might therefore play an important role in macrophage activa-

tion in vivo.

Very little HA is present in the normal kidney. In inflam-

matory conditions, however, HA markedly accumulates in the

kidney cortex, particularly in crescentic gbomerubonephritis

( 1 1 ), tububointerstitial injury ( I 2, 1 3), and albograft rejection

(14,15). In immune renal injury, HA accumulation is most

prominent at sites where the HA receptor CD44 is also in-

duced, e.g. , in glomerubar crescents and particularly in the

peritubular interstitium adjacent to injured proximal tubular

epithebial cells (13).

The functional significance of HA accumulation in the kid-

ney cortex has not been elucidated. Given its proinflammatory

effects, we were interested in examining the effects of HA and

its fragments on the expression of chemokine genes in the

kidney. In a previous study, we have shown that fragments of

HA can upregulate the expression of the adhesion molecules

intercellular adhesion molecule- 1 (ICAM- 1 ) and vascular cell

adhesion molecule-b (VCAM-b) in cultured kidney tubular

epithebiab cells ( 16). These studies have suggested indirectly

that HA accumulation in immune-mediated renal injury could

play a robe in beukocyte recruitment and adhesion.

Chemokines represent a family of structurally rebated che-

motactic cytokines produced by a variety of cells, including

kidney proximal tubular cells (17-19). Monocyte chemoattrac-

tant protein- 1 (MCP-l) is a particularly important chemokine

that is ovenexpressed in many immune-mediated renal diseases

and also in abbograft rejection (1 7). It has been suggested that

2284 Journal of the American Society of Nephrobogy J Am Soc Nephrol 9: 2283-2290, 1998

MCP-l may play a key role in the perpetuation of tububoin-

terstitial inflammation by attracting mainly monocytes and T

cells (20-22). Since HA fragments are known to induce prom-

flammatory genes, we have now examined whether HA in-

duces MCP- 1 upregubation in kidney tubular epithelial cells. In

this study, we demonstrate that fragmented HA is a potent

stimulatory factor for MCP- 1 expression by cultured tubular

cells. The HA-induced stimulation of MCP- 1 is mediated at

least in part by interaction with the CD44 molecule. Therefore,

HA-induced signaling through CD44 and subsequent MCP-l

gene transcription could be an important event in the genera-

tion and maintenance of a chemoattractant gradient in immune

renal injury.

Materials and MethodsChemicals and Reagents

Actinomycin D (ACTD), cycboheximide (CHX), and lipopoly-

sacchanide (LPS) were purchased from Sigma Chemical Co. (St.

Louis, MO). Fragmented HA derived from human umbilical cord

(molecular weight approximately 0.08 to 0.8 X 106 daltons) was

purchased from Fluka (Buchs, Switzerland). The HA samples were

checked for significant LPS contamination with a Limulus ame-

bocyte lysate assay (COATEST#{174} Endotoxin kit, Endosafe, Inc.,

Charleston, SC). LPS concentrations of all HA preparations were<20 pg/mb at the maximum concentration of HA used to stimulate

cells, a concentration that did not stimulate MCP-l per se. High

molecular weight HA (Heabon#{174}, >106 Da) was a generous gift

from Phanmacia (Uppsala, Sweden).

Cell Lines and Cell Culture

The simian virus 40-transformed mouse cortical tubular (MCT) cell

line was grown to confluence in Dulbecco’s modified Eagle’s medium

with Glutamax-l (DMEM) containing 10% fetal bovine serum (FBS),

10 mM Hepes, 100 U/mb penicillin, and 100 �.rg/ml streptomycin (all

from Life Technologies, Gaithensburg, MD) at 37#{176}Cwith 5% CO,

(23). The medium was then changed to DMEM containing 1% FBS

for 18 h to rest the cells. Cells were subsequently stimulated with HA

fragments or Heabon at different concentrations in the presence or

absence of inhibitors. HA fragments and Heabon were not toxic at

concentrations up to I mg/mb as assessed by morphology and trypan

blue exclusion. The KM8 1 hybridoma producing the rat anti-munine

CD44 IgG monocbonal antibody (mAb) was obtained from American

Type Culture Collection (Rockville, MD) (24). The hybridoma was

grown in RPMI containing 20% FBS. The control IgG mAb FF-l0targeting the Thy-l molecule has been described (25). Purified mAb

were generated from hybnidoma supernatants using affinity chnoma-tography with protein 0-Sepharose (Pharmacia).

RNA Extraction and Northern Blot Analysis

Total RNA was isolated from MCT cells using the Tnizob#{174}reagent

(Life Technologies) and phenol/chloroform extraction as described

previously (26,27). For Northern blot analysis, b0-p�g abiquots of total

RNA were size-fractionated by electrophoresis on 1 % aganose-form-

abdehyde gels and transferred to Zeta-Probe#{174} blotting membranes

(Bio-Rad, Hercules, CA). Methylene blue staining to detect the 18Sand 285 ribosomal RNA (rRNA) bands was performed to ensure

equal loading. After prehybridization overnight at 65#{176}Cin a solution

containing 1 mM ethybenediaminetetra-acetic acid, 7% sodium dode-

cyl sulfate in 0.5 M Na,HPO4, pH 7.2, the blots were hybridized

overnight with an MCP-b eDNA fragment previously labeled with

32P(dCTP). The MCP-l cDNA probe was generated by reverse tran-scniption-PCR, using the following primers: 5’-CCC AAT GAG TAO

OCT OGA GA-3’ (forward primer), 5’-AAA ATO GAT CCA CAC

CTT GC-3’ (reverse primer), yielding a 435-bp MCP-l eDNA fnag-ment (28). Blots were washed at 65#{176}Cin 40 mM sodium phosphate

buffer with decreasing concentrations of sodium dodecyb sulfate.

Autoradiography was performed using X-OMAT AR film (Kodak,

Rochester, NY). Blots were rehybnidized with an 800-bp eDNA probe

for the housekeeping gene �3-actin.

Enzyme-Linked Immunosorbent Assay for MCP-1

Protein

MCP- 1 protein production was examined in the supernatant ofstimulated MCT cells, using a standard sandwich enzyme-linked

immunosorbent assay (ELISA) technique as described elsewhere (29).

The capturing antibody was a purified hamster anti-mouse MCP-l

IgG mAb (clone 2H5) and the detecting antibody was a biotinylated

hamster anti-mouse MCP-l IgO mAb (clone 4E2/MCP), both pun-

chased from Phanmingen (San Diego, CA). Recombinant mouse

MCP- 1 was used as a standard (Pharmingen).Antibodies, standards, and samples were diluted in phosphate-

buffered saline (PBS) containing 10% FBS and 0.05% Tween. All

washing steps were performed 3 times with 200 �tl, using PBS

containing 0.05% Tween. Then, 96-well plates (Costar) were coated

overnight with purified 2H5 anti-mouse-MCP- 1 mAb (2 p.g/ml, 50

�rl/well) in 0. 1 M Na2HPO4 binding buffer, pH 9.0. After a blocking

step with PBS/l0% FBS for 20 mm at 37#{176}C,standards and samples

(100 j.d per well) were incubated for I h at 37#{176}C,followed by the

incubation with the biotinylated 4E2/MCP hamster anti-mouse-

MCP-1 mAb (I j�g/ml, 100 pJ per well) for 45 mm at 37#{176}C.Strepta-vidin-conjugated horseradish penoxidase (Southern Biotechnology

Associates, Birmingham, AL) at a dilution of I :6000 was then added

to the plate. After washing, the plates were developed with 200 �rl of

an o-phenylenediamine dihydrochlonide solution (Sigma). The reac-

tion was stopped by adding 50 jrl of 95% sulfuric acid to each well.

Optical density was determined with an ELISA reader at 492 nm.

Statistical Analyses

All samples were run in triplicate, and the results (mean ± SEM)were calculated. Experiments were performed at beast three times, and

results from typical experiments are shown in the figures.

ResultsTime- and Dose-Dependent Upregulation of MCP-1

mRNA by HA

The effect of HA on MCP- 1 gene expression by MCT cells

was studied by Northern blot analysis. MCT cells were slim-

ulated with fragmented HA for various time periods, and total

RNA was extracted. Figure 1A shows that the steady-state

transcript levels for MCP- 1 increased markedly in response to

short-term stimulation with HA compared with unstimulated

cells that express MCP-l constitutively. MCP-l mRNA levels

peaked between 1 .5 and 3 h. Although MCP- I mRNA levels

also increased slightly in unstimulated MCT cells when exam-

med for up to 72 h, the HA-stimulated cells always displayed

higher steady-state mRNA bevels for MCP-1 when compared

with the corresponding control (Figure 1B).

To examine the effect of increasing HA doses on MCP- 1

mRNA expression, MCT cells were stimulated with HA at

f3-actin 2.2 kb

time(h) 0 1 1.5 2 3 6A

I control ,� HA

MCP-1

methylene blue

time (h) 24 48 72 24 48 72B lh 1.5h 2h 3h 6h

timeFigure 1. Steady-state mRNA bevels for monocyte chemoattractant

protein- 1 (MCP-b) are time dependently upregubated by fragmented

hyaburonan (HA) when compared with �-actin. Mouse cortical tubular

(MCT) cells were stimulated with HA (100 p.g/mb) for 0 to 6 h.Specific MCP-l mRNA is at a maximum level between 1.5 and 3 h

(A). Extending the stimulation oven 72 h demonstrates upregulation ofMCP-l mRNA at all time points oven control, with only a slight

increase in control cells (B). Methylene blue reveals undegraded total

RNA.

0

C00 HA

50

40

30

20

10

0

A

100

90

80

70‘I)

.� 60

(00

x

� A0

30

20

10

0

B

Figure 3. The MCP-l protein is time dependently upregulated in

response to HA. Supernatants from HA-stimulated (100 pg/mb) MCT

cells were collected, and MCP- 1 protein determination was performed

by enzyme-linked immunosonbent assay (ELISA). The amount of

MCP-l above control in the supernatants of stimulated cells at dif-fenent time points increased steadily within 6 h (A). Prolonged incu-

bation with HA (100 �g/ml) oven 72 h reveals that the rate of MCP-b

production is greatest in the first 6 h. The concentration of MCP-l is

always greater in HA-stimulated plates compared with unstimulated

plates. *p < 001 (comparisons made within each time point) (B). The

figure represents one of three similar experiments.

MCP-1 0.8 kb

0 0.1 1_�_10 100 1000 dose �.tg/ml

�-actin

Oh 6h 24h 48h 72h

time

112.2

Figure 2. MCP-l mRNA is dose dependently upregubated by HA.MCT cells were stimulated with various HA concentrations (0. 1 to1000 p�g/mb for 2 h), and RNA was extracted and analyzed by

Northern blotting for MCP-1 and (3-actin. Transcript levels for MCP-b

increase from 0.1 to 100 j.tg/ml, with a peak at 100 �.rg/ml. With an

HA concentration of 1000 p�g/ml, the mRNA bevel is decreased again.

J Am Soc Nephrol 9: 2283-2290, 1998 Hyaluronan Upregulates MCP-l 2285

concentrations between 0. 1 and 1000 j.tg/mb. Figure 2 shows

tflill M#{128}T-I mRNAupr�u1�tion wii� � �t’t’�’�n1 and 100 j.tg/mb, peaking at 100 �tg/ml. At 1000 pjg/mb, the

MCP- 1 transcript bevels were slightly lower compared with

100 j.sg/ml HA.

MCP-1

0C..

0Or

HA

0.8 kb

Time- and Dose-Dependent Upregulation of MCP-1

f3r�t(n(?yttuiWe then examined MCP-l protein synthesis in response to

HA by measuring MCP- 1 concentrations in the supernatant of

HA-stimulated MCT cells with a sensitive and specific ELISA.

Figure 3A represents a time course over 6 h, showing a marked

increase of MCP-l protein within 6 h. Compared with the

mRNA bevels for MCP- 1 , the MCP- 1 protein concentrations

increased with a 2- to 3-h delay, suggesting that the de novo

transcription precedes MCP-l mRNA translation. Stimulation

with HA over 72 h showed that MCP- 1 levels increased the

most within the first 6 h, with very little further increase after

U)

a,

U

0.8kb ‘�‘C

40

30

U)

U

‘Do 20‘C

10

0

Acontrol HA ACTD+ HA CHX+ HA

MCP-1

methylene blue

4- x

0� 4 +� I a

0C)4

4I+

IC.)

28S

18S

50

40

30

20

Cl)

4)U

0‘C

a)0.

10

0

2286 Journal of the American Society of Nephrology J Am Soc Nephrol 9: 2283-2290, 1998

this time point (Figure 3B). Control plates that were not stim-

ulated with HA showed increasing constitutive MCP- 1 protein

bevels, particularly between 24 and 72 h. MCP- 1 concentra-

tions in plates stimulated with HA always exceeded the con-

centrations measured in control plates.

Figure 4 shows that with increasing HA concentrations

(from 0. 1 to 100 p�gImb), MCP- 1 protein expression was en-

hanced dose dependently with a maximum expression at 100

pg/mb HA. With 1000 �g/ml HA, however, the MCP- 1 protein

concentration was bower again. These data correlate very well

with the MCP-l mRNA levels.

Mechanisms of HA-Induced MCP-1 Synthesis

To determine whether MCP-l mRNA and protein are syn-

thesized de novo in HA-stimulated MCT cells, we then exam-

med the effect of transcriptional and translational blockade,

using the transcriptional blocker ACTD or the protein synthesis

inhibitor CHX. Thirty minutes before HA stimulation was

started in MCT cells, ACTD or CHX were added. Figure SA

shows that the HA-stimulated MCP-b protein expression was

completely prevented with ACTD or CHX. Figure SB shows

that the increase in steady-state MCP- 1 mRNA levels in HA-

stimulated MCT cells was also prevented in the presence of

ACTD, whereas CHX did not inhibit the stimulation of MCP-l

mRNA in response to HA. These data suggest that de novo

transcription and translation are both involved in the HA-

mediated upregubation of MCP-l in MCT cells.

involvement of the CD44 Receptor in HA-inducedMCP-1 Expression

We have previously shown that MCT cells express the main

HA receptor CD44 abundantly (30). To determine whether the

0 0.1 1 10

HA dose (jsglml)

Figure 4. HA (0.1 to 1000 �tg/ml) upregulates MCP-b protein dose

dependently. MCT cells were stimulated with HA at various concen-

trations, and supernatants were collected after 6 h and assayed for

MCP- 1 content by ELISA. Data are mean ± SEM above control. A

peak is reached with bOO j.tg/ml, decreasing again at 1000 jrg/ml HA.

The figure represents one of three similar experiments.

B

Eu0.8 kb

Figure 5. MCP-l expression in response to HA after blocking withactinomycin D (ACTD) or cycloheximide (CHX). Cells were prein-

cubated with 0.5 pg/mb ACTD or 5 jtg/mb CHX for 1 h before adding

100 �tg/ml HA. RNA was extracted after 2 h, and supennatants were

collected after 6 h. MCP-l protein expression is totally blocked with

both inhibitors (A). At the transcriptional level, the HA-stimulated

increase in MCP-l mRNA is prevented with ACTD but not with CHX

(B).

effects of HA on MCP- 1 are mediated by CD44, we performed

blocking studies with the anti-CD44 antibody KM81, which

recognizes the HA binding site on CD44. MCT cells were

preincubated for 1 h with KM8 1 antibody on the irrelevant

control antibody FF- 10 (10 j�g/ml), followed by an incubation

with HA (10 .tg/ml). As shown in Figure 6A, MCP-l protein

expression could be inhibited by approximately 40% with

KM8 1 but not with FF- 10. Similar results were obtained at the

mRNA bevel, where KM81 but not FF-l0 also partially inhib-

ited the upregubation of MCP-l (Figure 6B). When given

alone, the KM81 and the FF-b0 mAb only marginally in-

creased MCP-l mRNA and protein production.

We also determined whether the high molecular weight HA100 1000 preparation Heabon could inhibit the upregulation of MCP-b

induced by fragmented HA. MCT cells were preincubated for

1 h with Heabon before adding fragmented HA. As seen in

Figure 7A, Heabon blocked the HA-stimulated MCP- 1 protein

expression by approximately 45%. Heabon itself downregu-

bated the constitutive MCP- 1 protein expression slightly. At the

mRNA bevel, Heabon also inhibited the HA-stimulated rise in

MCP-l expression (Figure 7B).

40

30

30

10

0

A

0

Co KM KM+ HA FF10 FFIO+ HAHA

A

MCP1

Co Healon

MCP-1

0.8 kb

HA

HA

0.8 kb

<

<

�

���u-L1-

�

<x

0‘-

LI.

Z

.�

��..

0C)

C

.i0Q)

I

<�

+

0#{149}�

.I

I Am Soc Nephrol 9: 2283-2290. 1998 Hyaluronan Upregulates MCP- I 2287

Cl)

4)U

IDo 20

‘C

C)

0.

methylene blue

B

28 S

185

Figure 6. Inhibition of HA-stimulated MCP-b expression with the

anti-CD44 monocbonal antibody (mAb) KM8I but not with the irreb-

evant anti-Thy-l mAb FF10. Purified KM81 or FF10 antibody (10

p�g/mb) was added to confluent MCT cells and incubated for I h,

followed by an incubation with 10 p.g/mb HA for another 6 h. KM81

inhibits MCP-l protein expression by approximately 40% (A). *P <

0.01. Using Northern blot analysis, the partial inhibition of MCP-l

mRNA upregulation can also be demonstrated with KM8 1 . When

given alone, KM8I and FF10 upregulated MCP-l slightly (B).

DiscussionIn this study, we demonstrate that HA, a matrix glycosami-

nogbycan that accumulates in the kidney cortex in immune-

mediated renal injury, potently upregulates MCP- 1 in cultured

tubular epitheliab cells. Numerous studies have documented

that MCP-l is strongly upregubated by proximal tubular cells in

experimental inflammatory renal diseases (31-34) and in hu-

mans with gbomerubonephritides and albognaft rejection (35-

40). It has been suggested that a correlation exists between the

enhanced expression of MCP- 1 and the degree of tububointen-

stitial infiltration with mononuclear cells (reviewed in refer-

ence 17). Since HA accumulation is a prominent feature of

20Cl)

4,U

0

‘C

C)0. io

methylene blue

B

28S

18S

Figure 7. Heabon blocks the expression of MCP-l induced withfragmented HA. MCT cells were incubated with 1000 p.g/ml Healon

for 1 h. HA (100 j.�g/mb) was then added. and cells were incubated for

another 6 h. MCP-l protein expression is downregulated by approx-

imately 45% by adding Heabon (*� < 0.01 ). Healon itself decreases

baseline MCP-l protein expression (A). At the mRNA level, Northern

blot analysis demonstrates that the stimulated increase in MCP-l

mRNA is inhibited with Healon (B).

inflammatory renal diseases, it is tempting to speculate that

HA, in addition to other factors such as proinfiammatory

cytokines, could promote MCP- I production in vivo.

Kidney proximal tubular epithebiab cells are one of the main

sites of MCP- 1 production in immune renal injury in vivo. The

stimuli that increase MCP- 1 in vivo remain to be defined but

could involve proinflammatony cytokines on could be caused

by the proteinunia of gbomerubar diseases. In cultured tubular

epithelial cells, MCP-l is upnegubated in response to prom-

flammatory cytokines (tumor necrosis factor-a, interbeukin- 1,

and interferon-y), LPS, and by urinary proteins such as albu-

mm and transfenrin (37,41,42). Our results show that extracel-

2288 Journal of the American Society of Nephrology J Am Soc Nephrol 9: 2283-2290. 1998

bubar matrix components such as HA have to be included in the

list of factors capable of promoting MCP- 1 production in

kidney tubular cells. Our data are in agreement with experi-

mental research in macrophages demonstrating that HA pro-

motes the synthesis of cytokines, chemokines, and growth

factors in these cells (7,8,43,44).

The effects of HA on MCP- 1 expression were dose-depen-

dent between 1 and 100 �g/ml. These concentrations could be

pathophysiobogicabby relevant, as the renal cortical accumula-

tion of HA in inflammatory renal diseases can be impressive.

In rejecting rat renal abbografts, for example, 300 to 400 p�g

HA/g dry weight can be detected in the kidney (45), and

concentrations > 1 jtg/mb can be found in the serum or in the

peritoneab diabysate of patients with end-stage renal disease

(46,47). There is no obvious explanation why higher concen-

trations of HA (up to 1 mg/mb) stimulated MCP-l productionless than bower doses (100 p�g/ml). Possible mechanisms could

include less efficient interaction with cell surface receptors for

HA or a downregulating effect of high HA doses on HAreceptor-mediated signaling events.

Earlier work has suggested that high molecular weight HA

inhibits inflammatory responses (6), and it has only recently

been recognized that fragments of HA display different effects

which are proinflammatory (48). In cultured MCT cells,

MCP-b was only induced with fragmented HA but not with

high molecular weight HA (Healon). In fact, high molecular

weight HA inhibited the effect of low molecular weight HA,

suggesting that the HA fragments were competed away with

high molecular weight preparations of HA. This is further

evidence of specificity of the HA-mediated effect on MCP-l

expression.

Our studies also demonstrate that the HA receptor CD44 is

involved in MCP- 1 production by cultured tubular cells, be-

cause anti-CD44 mAb inhibited the stimulation with HA. It

was not possible, however, to block the HA-stimulated MCP- 1

production completely. This could be due to several reasons.

First, relatively high concentrations of HA had to be used to

stimulate MCP- I expression in MCT cells; to compete away a

barge amount of HA, one needs to use also large amounts of

antibody. It is possible that the high antibody concentration

could have a partial agonist activity that negates the blocking

effect. Similar considerations were made previously by other

groups (43,44). Second, it may be intrinsically problematic to

block the binding of disaccharide moieties (HA) to protein

(CD44) with the use of protein (anti-CD44 antibodies). Third,

we cannot exclude that additional HA receptors could be

involved in the HA-stimulated generation of MCP-l by tubular

epitheliab cells. CD44 is very abundant on MCT cells, but

additional, less well characterized HA binding proteins such as

RHAMM could also be present (49,50). Additional studies will

be required to examine in detail whether HA promotes MCP-l

production solely via CD44 or whether additional HA recep-

tons are also involved.

Our in vitro study raises important new questions regarding

the interaction of matrix components with kidney tubular epi-

thebial cells in interstitial renal diseases. In a normal situation,

the tubular basement membrane separates the proximal tubules

from the penitubular interstitial space, shielding anatomically

the proximal tubule. The situation is different, however, in

renal interstitial inflammation, where the tubular basement

membrane can be disrupted, as, for example, in albograft re-

jection. In such an inflammatory situation, HA accumulates in

the renal cortex, and it could interact with the HA receptor

CD44 on tubular cells. This interaction, and perhaps the bind-

ing of additional matrix breakdown products, could then trig-

ger proximal tubules to express proinflammatory genes such as

chemokines, which participate in the recruitment of leukocytes.

HA needs to bind to its receptors before it can produce

proinflammatory effects in proximal tubular cells. It is known

that the main HA receptor CD44 must be in an activated state

to bind HA (S I). In a previous study, we have shown that the

CD44 molecule is in this activated state in MCT cells, binding

the HA molecule abundantly (30). Not all cells that express

CD44 are capable of binding HA constitutively, however. T

cell lines, for example, are strongly CD44-positive but they

vary markedly in their capacity to bind HA, and certain lines

do not bind HA at all (52). Previously, we have shown that

MCT cells, but not primary cultures of mouse tubular epithebiab

cells, can be stimulated with HA fragments to express adhesion

molecules, presumably because of limited HA-binding by

CD44 in primary cultures (16). Primary cultures were also only

marginally responsive to HA in stimulating MCP-l (unpub-

lished observation). It is not excluded that CD44-positive prox-

imal tubular cells are in an active binding state in vivo in

inflammatory renal diseases, but this needs to be examined

further.

In summary, we have shown that the ECM component HA

markedly stimulates the expression of the chemokine MCP- 1

by cultured kidney tubular epithelial cells. HA needs to be in

fragmented form to promote MCP- 1 production since a high

molecular weight HA preparation did not stimulate MCP-l.

The stimulatory effect of fragmented HA is transduced at least

in part by the CD44 cell surface receptor on tubular epithebiab

cells. De novo transcription and translation are involved in the

HA-mediated upregubation of MCP- 1 production. We specu-

late that fragmented HA, in concert with other more short-lived

stimuli such as proinflammatory cytokines, could produce sus-

tamed expression of MCP-l and therefore contribute to the

activation and recruitment of mononuclear cells in the kidney

in immune renal injury.

AcknowledgmentsThis study was supported by the Swiss National Science Founda-

tion (Grants 32-40390.94 and 32-5072 1 .97 to Dr. WUthnich), the

Olga-Mayenfisch Foundation, the Hartmann-MUller Foundation, and

the Research Foundation of the University of ZUrich. Dr. Beck-

Schimmer is the recipient of a grant by the Federal Foundation for the

Advancement of Young Scientists and Scholars. Dr. Oentli is the

recipient of a Postgraduate Fellowship from the University of Zurich

and is supported by the Swiss National Science Foundation and the

Maurice E. Muller Foundation. Dr. W0thnich is the recipient of a

Physician Scientist Award (Grant 32-38821.93) from the Swiss Na-

tional Science Foundation. We thank C. Gasser for the illustrations.

J Am Soc Nephrol 9: 2283-2290, 1998 Hyaluronan Upregulates MCP-1 2289

References1 . Laurent TC, Fraser IRE: Hyaluronan. FASEB J 6: 2397-2404,

1992

2. Prehm P: Hyaluronate is synthesized at plasma membranes.

Biochem J 220: 597-600, 1984

3. Saani H: Oxygen derived free radicals and synovial fluid hyal-

uronate. Ann Rheum Dis 50: 389-392, 1991

4. Schenck P, Schneider 5, Miehlke R, Prehm P: Synthesis and

degradation of hyaluronate by synovia from patients with rheu-

matoid arthritis. J Rheu,natol 22: 400-405, 1995

5. Kreil 0: Hyaluronidases: A group of neglected enzymes. Protein

Sci 4: 1666-1669, 1995

6. Forrester IV, Babazs EA: Inhibition of phagocytosis by high

molecular weight hyalunonate. mmniuizolog�’ 40: 435-446, 1980

7. Noble PW, Lake FR, Henson PM, Riches DW: Hyabunonate

activation of CD44 induces insulin-bike growth factor-b expres-

sion by a tumor necrosis factor-a-dependent mechanism in mu-

nine macrophages. J Cliii mns’est 91 : 2368-2377, 1993

8. Lake FR, Noble PW, Henson PM, Riches DW: Functionalswitching of macnophage responses to tumor necrosis factor-a

(TNF-a) by interfenons: Implications for the pbeiotropic activities

of TNF-a. J C/in Ins’est 93: 1661-1669, 1994

9. McKee CM, Lowenstein CI, Horton MR. Wu J, Bao C, Chin BY,

Choi AMK, Noble PW: Hyaluronan fragments induce nitric-

oxide synthase in murine macrophages through a nuclear factor

RB-dependent mechanism. J Biol Chein 272: 8013-8018, 1997

10. Rockey DC, Chung II, McKee CM, Noble PW: Stimulation of

inducible nitric oxide synthase in rat liven by hyaburonan frag-

ments. Hepatology 27: 86-92, 1998

1 1. Nishikawa K, Andres 0, Bhan AK, McCluskey RT, Collins AB,

Stow IL, Stamenkovic I: Hyaburonate is a component of cres-cents in rat autoimmune gbomerubonephnitis. Lob m�ivest 68: 146-

153, 1993

12. Wu I, Fan 0, Kitazawa K, Sugisaki T: The relationship of

adhesion molecules and leukocyte infiltration in chronic tububo-

interstitial nephnitis induced by puromycin aminonucbeoside in

Wistar rats. C/in hn,nunol mtnmiinopathol 79: 229-235, 1996

13. Sibalic V, Fan X, Loffing I, Wuthrich RP: Upregulated renal

tubular CD44, hyaluronan and osteopontin in kdkd mice with

interstitial nephnitis. Nephrol Dial Transplant 12: 1344-1353,

1997

14. Wells AF, Lansson E, Tengblad A, Fellstrom B, Tufveson 0,

Kbareskog L, Laurent TC: The localization of hyalunonan in

normal and rejected human kidneys. Transplantation 50: 240-

243, 1990

15. Wells A, Larsson E, Hanas E, Laurent T, Hhllgnen R, Tufveson

0: Increased hyaluronan in acutely rejecting human kidney

grafts. Transplantation 55: 1346-1349, 1993

16. Oentli B, Beck-Schimmer B, Fan X, Wuthrich RP: Mechanisms

of hyaluronan-induced upregulation of ICAM-l and VCAM-l

expression by murine kidney tubular epithelial cells: Hyaluronan

triggers cell adhesion molecule expression through a mechanism

involving activation of nuclear facton-[K]B and activating pro-

tein-!. J mmnzunol 161: 3431-3437, 1998

17. Schl#{246}ndorffD, Nelson P1, Luckow B, Banas B: Chemokines andrenal disease. Kidney mnt 51: 610-621, 1997

18. Adams DH, Lloyd AR: Chemokines: Leucocyte recruitment and

activation cytokines. Loncet 349: 490-495, 1997

19. Luster AD: Chemokines: Chemotactic cytokines that mediate

inflammation. N Engi J Med 338: 436-445, 1998

20. Eddy AA: Experimental insights into the tububointerstitial dis-

ease accompanying primary gbomerulan lesions. J Am Soc Neph-

rol 5: 1273-1287, 1994

2 1 . Schl#{246}ndorff D: The robe of chemokines in the initiation andprogression of renal disease. Kidney mnt Suppi 49: 544-547,

1995

22. Banas B, Wenzeb U, Stahl RA, Schl#{246}ndorff D: Role of chemo-

kines in glomerular diseases. Kidney Blood Press Res 19: 270-

280, 1996

23. Haverty TP, Kelly CI, Hines WH, Amenta PS, Watanabe M,Harper RA, Kefalides NA, Neilson EG: Characterization of a

renal tubular epitheliab cell line which secretes the autobogous

target antigen of autoimmune experimental interstitial nephnitis.

J Cell Biol 107: 1359-1368, 1988

24. Miyake K, Medina KL, Hayashi 5, Ono 5, Hamaoka T, Kincade

PW: Monocbonal antibodies to Pgp-l/CD44 block lympho-he-mopoiesis in long-term bone marrow cultures. J Exp Med I 7 1:

477-488, 1990

25. Fan X, Wuthrich RP: ICAM- I -, -2- and LFA- 1-independent

homotypic T cell aggregation induced by a novel activating

monoclonal antibody targeting the murine Thy-I molecule. mn-flammation 20: 401-41 1, 1996

26. Chomczynski P, Sacchi N: Single-step method of RNA isolation

by acid guanidinium thiocyanate-phenol-chborofonm extraction.

AnalBiochem 162: 156-159, 1987

27. Benz PS, Fan XH, W0thnich RP: Enhanced tubular epithelial

CD44 expression in MRL-lpr lupus nephnitis. Kidney mnt 50:

156-163, 1996

28. Rollins BI, Morrison ED, Stiles CD: Cloning and expression of

JE, a gene inducible by platelet-derived growth factor and whose

product has cytokine-like properties. Proc Nail Acad Sci USA 85:

3738-3742, 1988

29. Beck-Schimmen B, Schimmen RC, Warner RL, Schmal H, Nor-

dbbom 0, Fbory CM, Lesch ME, Friedb HP, Schnien Di, Ward PA:

Expression of lung vascular and airway ICAM- 1 after exposure

to bacterial bipopolysaccharide. Am J Respir Cell Mo! Biol 17:

344-352, 1997

30. Oentbi B, Fan X, Wuthnich RP: Charactenisation of CD44-medi-

ated hyalunonan binding by renal tubular epithelial cells. Nephro!

Dial Transplant 13: 271-278, 1998

3 1 . Stahl RAK, Thaiss F, Disser M, Hebmchen U, Hona K, Schl#{246}n-

dorff D: Increased expression of monocyte chemoattractant pro-

tein- I in anti-thymocyte antibody-induced gbomerulonephnitis.

Kidneylnt44: 1036-1047, 1993

32. Tang WW, Qi M, Warren IS: Monocyte chemoattnactant protein1 mediates gbomerular macrophage infiltration in anti-GBM Ab

ON. Kidney hit 50: 665-671, 1996

33. Zoja C, Liu XH, Donadelli R, Abbate M, Testa D, Corna D,

Tanaboletti 0, Vecchi A, Dong QG, Rollins BI, Bertani T,

Remuzzi 0: Renal expression of monocyte chemoattnactant pro-

tein-l in lupus autoimmune mice. JAm Soc Nephrol 8: 720-729,

1997

34. Tang WW, Qi M, Warren IS, Van GY: Chemokine expression in

experimental tubulointerstitiab nephritis. J mm,nu,zol 159: 870-

876, 1997

35. Rovin BH, Rumancik M, Tan L, Dickerson I: Olomenulan ex-

pression of monocyte chemoattractant protein- 1 in expenimental

and human glomenulonephritis. Lob mnvest 7 1 : 536-542, 1994

36. Prodjosudjadi W, Gemtsma IS, van Es LA, Daha MR. Bnuijn

IA: Monocyte chemoattnactant protein- I in normal and diseasedhuman kidneys: An immunohistochemical analysis. C!i,z Neph-

rol44: 148-155, 1995

37. Prodjosudjadi W, Gemtsma IS, KIar-Mohamad N, Oemtsen AF,

2290 Journal of the American Society of Nephrology I Am Soc Nephrol 9: 2283-2290, 1998

Bruijn JA, Daha MR. van Es LA: Production and cytokine-mediated regulation of monocyte chemoattractant protein- I by

human proximal tubular epithelial cells. Kidney mnt 48: 1477-

1486, 199538. Prodjosudjadi W, Daha MR. Gerritsma IS, Flonijn KW, Baren-

dregt IN, Bruijn IA, van der Woude Fl, van Es LA: Increasedurinary excretion of monocyte chemoattnactant protein- 1 during

acute renal allograft rejection. Nephrol Dial Transplant 1 1:

1096-1103, 1996

39. Gesualdo L, Grandaliano 0, Ranieri E, Monno R, Montinaro V.

Manno C, Schena FP: Monocyte recruitment in cryogbobubine-mic membranoprolifenative glomenulonephritis: A pathogenetic

role for monocyte chemotactic peptide-b. Kidney mnt 51 : 155-

163, 1997

40. Grandaliano 0, Gesualdo L, Ranieri E, Monno R, Stallone 0,

Schena FP: Monocyte chemotactic peptide- 1 expression and

monocyte infiltration in acute renal transplant rejection. Trans-

plantation 63: 414-420, 1997

41. Schmouder RL, Stnieter RM, Kunkeb SL: Interfenon-y regulation

of human renal cortical epithebiab cell-derived monocyte chemo-

tactic peptide-l. Kidney mnt 44: 43-49, 1993

42. Wang Y, Chen I, Chen L, Tay YC, Rangan OK, Harris DC:

Induction of monocyte chemoattractant protein- 1 in proximaltubule cells by urinary protein. JAm Soc Nephrol 8: 1537-1 545,

1997

43. McKee CM, Penno MB, Cowman M, Burdick MD, Strieten RM,

Bao C, Noble PW: Hyaluronan (HA) fragments induce chemo-kine expression in alveolar macrophages: The robe of HA size

and CD44. J Clin mnvest 98: 2403-2413, 1996

44. Hodge-Dufour J, Noble PW, Horton MR, Bao C, Wysoka M,

Burdick MD, Strieter RM, Tninchieri 0, Pure E: Induction of

IL- I 2 and chemokines by hyalunonan requires adhesion-depen-

dent priming of resident but not elicited macrophages. J Immunol

159: 2492-2500, 1997

45. Hallgren R, Gerdin B, Tufveson 0: Hyaluronic acid accumula-

tion and redistribution in rejecting rat kidney graft: Relationship

to the transplantation edema. J Exp Med 17 1 : 2063-2076, 1990

46. Turney IH, Davison AM, Forbes MA, Cooper EH: Hyaluronic

acid in end-stage renal failure treated by haemodiabysis: Clinical

correlates and implications. Nephro! Dial Transplant 6: 566-570, 1991

47. Lipkin OW, Forbes MA, Cooper EH, Turney IH: Hyaluronicacid metabolism and its clinical significance in patients treated

by continuous ambulatory peritoneal dialysis. Nephrol Dial

Transplant 8: 357-360, 1993

48. Aruffo A: CD44: One ligand, two functions. J Clin Invest 98:2191-2192, 1996

49. Yang B, Yang BL, Savani RC, Turley EA: Identification of a

common hyalunonan binding motif in the hyaluronan bindingproteins RHAMM, CD44 and link protein. EMBO J 13: 286-

296, 1994

50. Entwistbe J, Hall CL, Turbey EA: HA receptors: Regulators of

signalling to the cytoskeleton. J Cell Biochem 61 : 569-577, 1996

51. Lesley I, Hyman R, Kincade PW: CD44 and its interaction with

extracebbular matrix. Adv Immunol 54: 271-335, 1993

52. Lesley I, Schulte R, Hyman R: Binding of hyaburonic acid tolymphoid cell lines is inhibited by monocbonal antibodies againstPgp-l. Exp Cell Res 187: 224-233, 1990