L-selectin and its ligands mediate infiltration of mononuclear cells into kidney interstitium after ureteric obstruction

J. Pathol. 188: 93–99 (1999)

L-SELECTIN AND ITS LIGANDS MEDIATEINFILTRATION OF MONONUCLEAR CELLS INTO

KIDNEY INTERSTITIUM AFTER URETERICOBSTRUCTION

1*, 2, 1, 1, 1, 3, 2, 2, 1 3

1Department of Medicine III, Okayama University Medical School, Okayama 700-8558, Japan2Department of Biochemistry, University of Shizuoka School of Pharmaceutical Science, Shizuoka 422-8526, Japan

3Department of Bioregulation, Biomedical Research Center, Osaka University Medical School, Osaka 565-0871, Japan

SUMMARY

It was previously reported that the L-selectin ligands detected by a rat L-selectin and human IgG chimeric molecule (rLEC–IgG) areexpressed in the distal tubules of the kidney, where no leukocyte traffic is seen under physiological conditions. In the present study, therole of L-selectin ligands in leukocyte infiltration into the kidney interstitium was investigated using a rat ureteric obstruction model.After ligation of the ureter, ligands for L-selectin rapidly disappeared from tubular epithelial cells and were relocated to the interstitiumand peritubular capillary walls, where infiltration of monocytes and CD8+ T cells subsequently occurred. Mononuclear cell infiltrationwas significantly inhibited by intravenous injection of a neutralizing monoclonal antibody (MAb) against L-selectin, indicating thepossible involvement of an L-selectin-mediated pathway. Interestingly, immunohistochemical studies with a MAb against sulphatideshowed that the distribution of sulphatide, known to be one of the candidates of L-selectin ligand, was almost indistinguishable from thestaining pattern of rLEC–IgG in both normal and ureteric obstructed kidneys, suggesting that sulphatide and/or related molecule(s)relocated to the renal interstitium were recognized by leukocyte L-selectin, leading to interstitial leukocyte infiltration. In line with thisnotion, intravenous injection of sulphatide markedly inhibited leukocyte infiltration, suggesting that L-selectin–sulphatide interactionmay play a pivotal role in interstitial leukocyte infiltration in the kidney following ureteric obstruction. Copyright ? 1999 John Wiley& Sons, Ltd.

KEY WORDS—sulphatide; L-selectin; inflammation; obstructive nephropathy

INTRODUCTION

Using a soluble fusion protein of rat L-selectin andhuman IgG (rat L-selectin–IgG chimera rLEC–IgG), wehave recently demonstrated that ligands for L-selectinare present in the kidney and localized exclusively in theepithelial cells of the distal tubules, although theirphysiological significance was not fully understood.1,2

Furthermore, the observation that the reactivity ofrLEC–IgG in frozen sections was completely abolishedby treatment with organic solvents (chloroform andmethanol) suggested that the ligands for L-selectin in thedistal tubules were possibly glycolipids.2 A sulphatedglycolipid, sulphatide, is a major constituent of glycoli-pids in the kidney.3 We and others have demonstratedthat sulphatide binds specifically to L-selectin throughthe sulphated sugar chain.4,5 Alon et al.6 also reportedthat sulphatide interacts with L-selectin and can contrib-ute to leukocyte tethering under physiological flow con-ditions, although with low efficiency. Thus, it is possiblethat sulphatide is involved in L-selectin binding to

CCC 0022–3417/99/060093–07$17.50Copyright ? 1999 John Wiley & Sons, Ltd.

the distal tubules, although the exact localization ofsulphatide in the kidney is unknown at present.

Experimental unilateral ureteric obstruction is amodel for human renal diseases such as acute uretericobstruction, vesico-ureteric reflux, and pyelonephritis.7–9

Ureteric obstruction induces extensive mononuclearcell infiltration into the kidney interstitium, which even-tually results in acute tubular injury. In the presentstudy, we evaluated the contribution of L-selectin andits ligands to mononuclear cell infiltration induced byureteric obstruction and examined whether sulphatidecontributes to this as a ligand for L-selectin. Our resultsindicated that the interaction between L-selectin andsulphatide or related molecule(s) plays an important rolein mononuclear cell infiltration into the interstitium andthat a therapeutic strategy based on blocking L-selectin–ligand interactions might be an effective new treatmentfor this pathological entity.

MATERIALS AND METHODS

*Correspondence to: Kenichi Shikata, MD, PhD, Department ofMedicine III, Okayama University Medical School, 2-5-1, Shikata-cho, Okayama, 700-8558, Japan.

Contract/grant sponsor: Ministry of Education, Science andCulture, Japan; Contract grant numbers: C06671141, C08671287.

Animals

Female Wistar rats weighing approximately 100 g(Charles River Co., Yokohama, Japan) were used in thepresent experiments.

Received 22 April 1998Revised 7 June 1998

Accepted 8 December 1998

94 K. SHIKATA ET AL.

Fig. 1—Staining with the fusion protein of rat L-selectin and human IgG (rLEC–IgG) (a, d),anti-sulphatide monoclonal antibody (GS-5) (b, e), and anti-Tamm Horsfall protein antibody (c, f) onnormal rat kidney section (serial sections). rLEC–IgG, anti-sulphatide monoclonal antibody, andanti-Tamm Horsfall protein antibody reacted exclusively with the distal tubules in normal rat kidney.(a–c) #50; (d–f) #400

Antibodies and fusion protein

Neutralizing anti-rat L-selectin MAb (HRL-3) andnon-neutralizing anti-rat L-selectin MAb (HRL-2) wereprepared and characterized as described previously.10

Preparation of the soluble fusion protein of ratL-selectin and human IgG (rLEC–IgG) has beendescribed previously by Tamatani et al.2

Anti-sulphatide MAb (GS5) (mouse IgM) was estab-lished by a procedure described previously by ourlaboratory,11 using liposomes containing sulphatide

Copyright ? 1999 John Wiley & Sons, Ltd.

(SO3H-3GalB1-1* ceramide) purified from the bovinebrain, dipalmitoylphosphatidylcholine, cholesterol, andlipopolysaccharides. The reactivity of GS5 to varioussulphated glycosphingolipids was determined by thethin-layer chromatography (TLC)/enzyme immuno-staining method11 and found to be strictly restricted tosulphatide and seminolipid (SO3H-3GalB1-alkylacyl-glycerol). GS5 showed a negative reaction to therelated sulphated glycosphinoglipids found in therat kidney, such as SM3 (SO3H-3GalB1-4GlcB1-1*ceramide), SM2 [GalNAcB1-4(SO H-3)GalB1-4GlcB1-
3
J. Pathol. 188: 93–99 (1999)

95L-SELECTIN IN LEUKOCYTE INFILTRATION INTO KIDNEY

1*ceramide], SB2 [SO3H-3GalNAcB1-4(SO3H-3)-GalB1-4GlcB1-1*ceramide], and SB1a [SO3H-3GalB1-3GalNAcB1-4(SO3H-3)GalB1-4GlcB1-1*ceramide]. How-ever, seminolipid is apparently absent from the ratkidney, and only sulphatide was detected to be reactivewith this MAb (GS5) as assessed by a TLC/enzymeimmunostaining method (Fig. 3).

Polyclonal anti-human Tamm Horsfall protein anti-body, which also reacts with rat Tamm Horsfall protein,was purchased from Chemicon (CA, U.S.A.). Anti-ratleukocyte common antigen MAb (OX1), anti-rat CD4MAb (W3/25), and anti-rat CD8 MAb (OX8) werepurchased from Serotec (Oxford, U.K.). Anti-ratmonocytes/macrophages MAb (ED1) was purchasedfrom Chemicon (CA, U.S.A.). The secondary antibody,biotinylated anti-mouse IgG antibody, was purchasedfrom Jackson Immunoresearch Laboratories (PA,U.S.A.). Biotinylated anti-human IgG antibody,biotinylated anti-sheep IgG, and FITC-conjugatedanti-human IgG antibody were purchased from EYLaboratories (CA, U.S.A.). Biotinylated and FITC-conjugated anti-mouse IgM antibodies were fromZymed Laboratories (CA, U.S.A.).

Thin-layer chromatography (TLC)/enzymeimmunostaining

TLC/enzyme immunostaining was performed asdescribed previously.11 Briefly, standard bovine brain


sulphatide (1 nmol) and rat kidney alkali-stableglycolipid fraction (30 µg) were developed on asilica gel thin-layer plate (Nagel Sil G) using a solventmixture of chloroform–methanol–acetone–acetic acid–water (6:2:4:2:1, by vol.). The plates were stained withGS5 to detect sulphatide or with orcinol–H2SO4 reagentto detect sugar.

Fig. 2—Immunohistochemical analyses of the kidneys of sham-operated rats and rats with unilateral ureteric obstruction using the fusion proteinof rat L-selectin and human IgG (rLEC–IgG) and anti-sulphatide MAb (GS5). (a, b) rLEC–IgG (a) and GS5 (b) reacted exclusively with the distaltubular epithelial cells in the kidney of a sham-operated rat. Glomeruli, peritubular capillaries, and arteries were not reactive. (c–f). The reactivitieswith rLEC–IgG (c, d) and GS5 (e, f) were markedly diminished in the distal tubules but became strongly reactive in the interstitium andperitubular capillaries (arrows) in rats with ureteric obstruction. T=tubular lumen. (a–f) #400

Experimental design

To examine the distribution of ligands for L-selectinand also sulphatide in normal and surgically manipu-lated kidneys, ten female Wistar rats were divided intotwo groups (N=5): sham-operated rats and rats withunilateral ureteric ligation. Under ether anaesthesia, therats underwent a sham operation or right uretericligation using the method described previously byDziukas et al.7 The right kidneys were harvested24 h after surgery. Four-micrometre-thick cryostatsections including the entire inner and outer medulla andcortex were processed for indirect immunoperoxidasestaining.

To study the in vivo preventive effect of neutralizingMAb against L-selectin and sulphatide, 20 femaleWistar rats, each weighing approximately 100 g, weredivided into four treatment groups (N=5): rats givenF(ab)2 fragment of neutralizing anti-rat L-selectinMAb HRL3,10 F(ab)2 fragment of non-neutralizinganti-rat L-selectin MAb HRL2,10 native sulphatide, or

J. Pathol. 188: 93–99 (1999)


galactosyl ceramide (Gal-Cer), a desulphated sulphatide.Native sulphatide was purified from the bovine brain asdescribed previously.4 The right ureter was ligated ineach rat as described above. Gal-Cer was also preparedas described previously.4 Each rat received HRL3 (5 mg/kg body weight), HRL2 (5 mg/kg body weight), sul-phatide (10 mg/kg body weight), or Gal-Cer (10 mg/kgbody weight) in 1 ml of PBS intravenously 10 min afterthe operation. After harvesting, all kidneys were pro-cessed for morphological examination by immuno-peroxidase staining and light microscopy. Cells positivefor OX1, ED1, and CD8 in the interstitium werecounted and statistically analysed as described below.F(ab)2 fragments of HRL2 and HRL3, native sul-phatide, and Gal-Cer did not induce leukopenia wheninjected into rats in this study. For each animal group,the data were expressed as the mean (&SEM) numberof cells/mm2.

l

,,

.


scopic fields in the cortex in each section. The totalnumber of each cell type was expressed as cells/mm2

using the method described by Saito and Atkins.12

Immunofluorescence staining was performed as de-scribed previously.13 Briefly, 4-micrometre-thick freshfrozen sections were incubated with GS5 and then withFITC anti-mouse IgM. The sections were examinedusing a fluorescence microscope (Olympus, Tokyo,Japan).

Immunostaining with rLEC–IgG was performed aspreviously described2 using biotinylated anti-humanIgG antibody or FITC anti-human IgG antibody.

Statistical analyses

Differences between groups were tested for statisticalsignificance by Wilcoxon’s test and the Welch t-test.p values less than 0·01 were considered significant.

RESULTS

Distribution of L-selectin ligand in the kidney afterureteric obstruction

The L-selectin–Ig chimera, rLEC–IgG, reacted exclu-sively with the distal tubules in normal and sham-operated kidneys (Fig. 1). The tubules reactive withrLEC–IgG also stained positively with antibody againstTamm Horsfall protein (Fig. 1) that localizes selectivelyin distal tubules,7 confirming the restricted expression ofL-selectin ligands in the distal tubules. However,glomeruli, peritubular capillaries, and arteries were notreactive. After ureteric ligation, the reactivity ofrLEC–IgG against the distal tubules was markedlydiminished and rapidly showed a staining pattern similarto peritubular capillaries in interstitium (Fig. 2) whereextensive infiltration of leukocytes was observedwithin 24 h after ureteric ligation. The infiltratingleukocytes consisted mainly of monocytes and CD8+ Tcells (Fig. 4). Other recombinant Ig chimeras tested,such as RVR–Ig2 or CD44–Ig,14 showed no positivebinding to the distal tubules in normal and obstructedkidneys (not shown), showing the specificity of bindingof rLEC–IgG.

Distribution of sulphatide in normal kidney and afterureteric obstruction

Among the ligands for L-selectin reported hitherto,sulphatide is known to be abundantly present in thekidney. Immunohistochemical studies using a mono-clonal antibody to sulphatide (GS5) showed that itsdistribution in the kidney was strikingly similar to thatof L-selectin ligands. Specifically, GS5 reacted exclu-sively with the distal tubular epithelial cells in bothnormal and sham-operated rat kidneys (Fig. 1). Uretericligation caused a marked reduction in the staining onthe distal tubules and shifted the reactivities of GS5 tothe peritubular capillary walls and interstitium (Fig. 2).TLC/enzyme immunostaining showed that GS5reacted only with sulphatide in rat kidney alkali-stableglycolipid fraction (Fig. 3).

Fig. 3—Reactivity of GS5 to rat kidney alkali-stable glycolipid frac-tion on thin-layer chromatography (TLC). GS5 reacted only withsulphatide (arrow). This band was also positive for orcinol–H2SO4

(data not shown). Lane 1: bovine brain sulphatide; lane 2: rat kidneyalkali-stable glycolipid

Immunohistochemistry

Immunoperoxidase staining was performed using theABC kit (Vector Laboratories, CA, U.S.A.). The first setof antibodies consisted of goat anti-Tamm Horsfalprotein polyclonal antibody and MAbs against sul-phatide (GS5), rat leukocyte common antigen (OX1)rat monocytes/macrophages (ED1), rat CD4 (W3/25)rat CD8 (OX8), and rat L-selectin (HRL-2). Bioti-nylated anti-sheep goat IgG, biotinylated anti-mouseIgG, biotinylated anti-mouse IgM or biotinylated anti-hamster IgG antibody was used as the second antibodyCells positive for OX1, ED1, CD4 and CD8 presentin the interstitium were counted using a micrometer(Olympus, Tokyo, Japan) by examining 20 randomlyselected microscopic fields in the medulla and 20 micro-

J. Pathol. 188: 93–99 (1999)


Inhibitory effect of anti-L-selectin MAb on mononuclearcell infiltration induced by ureteric ligation

In order to examine the contribution of L-selectin tomononuclear cell infiltration in the interstitium, weadministered either a neutralizing or a non-neutralizinganti-rat L-selectin MAb into rats with ureteric obstruc-tion. The neutralizing MAb against rat L-selectin(HRL3) significantly reduced leukocyte infiltration intothe renal interstitium, whereas the non-neutralizingMAb HRL2 did not (the per cent reduction of totalleukocytes, monocytes, and CD8+ T cells with HRL3was 62, 58 and 57 per cent, respectively, compared withthe values of rats treated with HRL2, Fig. 5a), indicatingthat L-selectin is involved in this inflammatory process.

Inhibitory effect of sulphatide on mononuclear cellinfiltration induced by ureteric ligation

To examine further if sulphatide is the responsibleligand for L-selectin in interstitial mononuclear cellinfiltration, we injected sulphatide intravenously.Administration of sulphatide markedly reduced the infil-


tration of mononuclear cells following ureteric ligation.In contrast, administration of galactosyl ceramide didnot influence mononuclear cell infiltration, suggestingthat the inhibitory effect of sulphatide is dependent onsulphation (the per cent reduction of total leukocytes,monocytes, and CD8+ T cells was 54, 45, and 71 percent, respectively, compared with the values of ratstreated with galactosyl ceramide, Figs 5a–5c).

Fig. 4—Leukocyte infiltration into the renal interstitium following ligation of the ureter. (a) Light microscopy revealscell infiltration (arrows) in the interstitium in the kidney of the rat with unilateral ureteric obstruction (periodic acidSchiff staining; #400, reduced to 80 per cent in printing). (b) Immunoperoxidase staining showing infiltration ofnumerous leukocytes (OX1+ cells) (#400, reduced to 80 per cent in printing). (c) Analysis of infiltrating mononuclearcells showed that the majority were monocytes (positive for ED1) and CD8+ T cells (*p<0·01). Closed column:sham-operated group (n=5). Open column: ureteric obstruction group (n=5)

DISCUSSION

In the present study we investigated the role of ligandsfor L-selectin expressed in the renal distal tubules in theinfiltration of mononuclear cells into the kidney afterureteric ligation. Relocation of L-selectin ligands fromthe distal tubules to the interstitium where extensiveinfiltration of leukocytes subsequently occurs is in linewith the notion that the inflammatory cell traffic is underthe influence of adhesive interactions between leukocyteL-selectin and its ligands. In direct support of thisconclusion, administration of the F(ab*)2 fractionof a blocking monoclonal antibody against L-selectin

J. Pathol. 188: 93–99 (1999)


Fig. 5—Inhibitory effects of anti-rat L-selectin MAbs and sulphatide on leukocyte infiltration in the interstitium of thekidneys of rats with unilateral ureteric obstruction. (a) Total leukocytes (OX1+ cells), monocytes (ED1+ cells), andCD8+ T cells were significantly reduced in number in rats treated with HRL3 or native sulphatide compared with ratsadministered HRL2 or galactosyl ceramide (Gal-Cer) (*p<0·01). (b, c) Immunoperoxidase staining of the kidneys of ratstreated with native sulphatide (c) and of rats treated with Gal-Cer (b) using the MAb against rat leukocytes (OX1+ cells).The number of leukocytes in the kidneys of rats treated with native sulphatide is dramatically reduced compared withrats treated with Gal-Cer. (#200, reduced to 80 per cent in printing)

significantly inhibited the infiltration of mononuclearcells.

Studies from our laboratory and others showed thatsulphatide, a major constituent of glycolipids in thekidney,3 binds to L-selectin in vitro.4 Interestingly, thestaining obtained with an anti-sulphatide monoclonalantibody (GS5) was almost indistinguishable from thatobtained with soluble L-selectin as demonstrated in thepresent study, suggesting that sulphatide is one of themajor ligands for L-selectin in the kidney. It is of note inthis regard that GS5 raised against bovine brain sul-phatide also reacts with other sulphated glycolipids suchas seminolipid.11 However, our TLC studies indicatedthat in the kidney sulphatide is the predominant glyco-lipid reactive with GS5 and that seminolipid is absent. Itis also possible that GS5 reacts with certain glycopro-teins modified with particular sulphated carbohydrates,although it does not recognize high endothelial venulesin lymph nodes (M. Miyasaka, unpublished obser-vation) that express mucin-type sulphated glycoproteins(reviewed by Rosen et al.15). Unless the GS5-reactivematerial is purified from the kidney and submitted fordetailed biochemical analysis, it cannot be finally con-cluded that sulphatide is the ligand for L-selectin


responsible for leukocyte infiltration in the kidney.However, based on immunohistochemical as well as onin vivo analyses, it is strongly suggested that sulphatideand/or related molecule(s) selectively expressed in thedistal tubules and normally excluded from the systemiccirculation are potential ligands for L-selectin in thekidney and that they become a target for leukocyteL-selectin once redistributed to the renal interstitium inresponse to mechanical stimuli such as ureteric ligation.Monocytes and CD8+ T cells express L-selectin, whichmay also be utilized as a tissue retention mechanismby recognizing its ligand(s) in the intersitium afterextravasation.

There are two possible mechanisms that may accountfor changes in the distribution of L-selectin ligands afterureteric obstruction. One is that L-selectin ligands areshed from injured tubular epithelial cells and move intothe interstitium and peritubular capillary walls. A simi-lar event has been observed from Tamm Horsfall pro-tein, which is also expressed selectively in the distaltubule and released into the interstitium upon an inflam-matory stimulus.7 Another possible mechanism is thatL-selectin ligands are newly synthesized in the inter-stitium as well as peritubular capillary walls following

J. Pathol. 188: 93–99 (1999)


ureteric ligation. In either case, since leukocytes rarelyenter the renal interstitium under normal conditions,involvement of other factors, such as increased vas-cular permeability and/or deposition of translocatedL-selectin ligands to capillaries, may also occur subse-quent to ureteric ligation. Selective infiltration ofmonocytes and CD8+ T cells into the interstitium asobserved in the present study must be explained by afactor(s) not related to L-selectin, since both types ofcells express L-selectin equally well. A mechanism(s)that allows lineage-specific extravasation of inflamma-tory cells into the renal interstitium remains to bedetermined.

Systemic administration of sulphatide inhibited theinfiltration of mononuclear cells into the renal inter-stitium induced by ureteric obstruction. Recently,sulphatide was reported to activate human neutrophilsin vitro.16 However, the anti-inflammatory effect ofsulphatide in vivo observed in this study is not likely tobe due to systemic activation of leukocytes, sinceadministration of sulphatide did not alter the numberof blood leukocytes, which is a sensitive parameterreflecting luekocyte activation.17 It is possible that thesystemically administered sulphatide directly inhibitedthe binding of L-selectin to its ligands without acti-vation of leukocytes. Given the ability of sulphatide tobind P-selectin, it is conceivable that sulphatide alsoinhibited the P-selectin-dependent adhesion pathway,although the extent of the contribution of P-selectin tothe inflammatory process was not assessed in thisexperimental model.

Recently, Mulligan et al. showed the preventive effectsof oligosaccharides and sulphatide in P-selectin-dependent lung injury.18,19 Interstitial infiltration ofmononuclear cells is observed not only in obstructivenephropathy such as is seen following ureteric ligation,but also in tubulo-interstitial nephritis, severe glomeru-lonephrities, lupus nephritis, focal glomerulosclerosis,and rejection of transplanted kidney.20 Infiltratinginflammatory cells are likely to contribute to the pro-gression of renal tissue injury.12,21 Thus, a therapeuticstrategy based on blocking L-selectin–ligand inter-actions might be an effective new treatment forrenal inflammatory diseases. Since renal ischaemic-reperfusion injury was not ameliorated in L-selectin-deficient mice,22 the significance of the L-selectinpathway in various pathological states in kidney diseaseremains to be further investigated.

Note added to proof

After the submission of this manuscript, we foundthat chondroitin sulfate and heparan sulfate proteo-glycans in the kidney distal tubules react with L-selectin(Li Y-F, Kawashima H, Watanabe N and Miyasaka M.Identification and characterization of ligands forL-selectin in the kidney. II. Expression of chondroitinsulfate and heparan sulfate proteoglycans reactive withL-selectin. FEBS Letters, in press).


ACKNOWLEDGEMENTS

We thank Dr Yasukazu Ohmoto, Cellular Tech-nology Institute, Otsuka Pharmaceutical Co. Ltd.,Japan, for his technical support and Dr Ziqiang Ding ofthe Department of Bioregulation, Biomedical ResearchCenter, Osaka University Medical School for stimulat-ing discussion. This work was supported in part by aGrant-in-Aid for Scientific Research from the Ministryof Education, Science and Culture, Japan (C06671141,C08671287).

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L-selectin and its ligands mediate infiltration of mononuclear cells into kidney interstitium after ureteric obstruction