Chemokines and renal disease

PHYSIOLOGY AND CELL BIOLOGY UPDATE

Chemokines and Renal Disease Ulrich 0. Wenzel, MD, and Hanna E. Abboud,

MD

0 Chemokines are low molecular weight inflammatory cytokines with chemoattractant properties as their major biologic effect. They are classified into at least two families. C-X-C chemokines (alpha subfamily) act primarily on neutrophils, while C-C chemokines act preferentially on monocytes. Chemokine receptors are C proteincoupled receptors that form a family of structurally and functionally related proteins. Chemokines are induced in cells and tissus in response to proinflammatory cytokines. They are produced by glomerular, tubular interstitial, and blood vessel cells. There is good evidence that chemokines contribute to nsutrophil and mononuclear cell infiltration in glomeruli and intersMum. Their expression is increased in renal disease, and neutralization studies using antibodies in vivo demonstrated a role for certain chemokines in mediating renal pathology and proteinurfa. Intedeukin-8, RANTES, and monocyte chemotactic peptide are the best-studied chemokines in the kidney. Development of specific anttbodiis and receptor antagonists shoukf help establish the precise role of these medietors in renal disease. Important therapeutic implications may result from this work. 0 lgS5 by the National Kidney Foundation, Inc. INDEX WORDS: Kidney; glometulonephritis; migration.

monocyte

chemoattractant

I

T HAS BEEN KNOWN since the pioneering work of Dutrochet, Cohnheim, Metchnikov, and Ehrlich in the 19th century that local tissue irritation causes the focal adherence of leukocytes to blood vessel wall, leukocyte transmigration, and accumulation in the tissue.“’ Leukocyte migration from the circulation into the tissue is an essential component of the immune-inflammatory response, and is, consequently, carefully regulated. The role of mononuclear leukocytes in the pathophysiology of kidney disease has recently been reviewed.‘-6 It is now well known that infiltration of monocytes/macrophages and lymphocytes into the glomerulus and the tubulointerstitium is also a common feature of renal disease. Migration of monocytes and lymphocytes may be important in acute and chronic renal disease since once in the tissue, monocyteslmacrophages represent a reservoir and a source of From the Division of Nephrology, Department of Medicine, The University of Texas Health Science Center at San Antonio, San Antonio, TX. Received January 10, 1995; accepted in revised form June 13, 1995. Supported by the Veterans Administration Medical Research Service and National Institutes of Health, Bethesda, MD, grants no. DK33465 and DK43988. Dr Wenzel is supported by the German Research Foundation. Address reprint requests to Ulrich 0. Wenzel, MD, Department of Medicine, Division of Nephrology, The University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Dr, San Antonio, TX 78284-7882. 0 1995 by the National Kidney Foundation, Inc. 0272~6386/95/2404-0015$3.00/0

982

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Journal

of Kidney

protein-l;

RANTES; intedeukinb;

cell

inflammatory mediators. Bioactive lipids, including arachidonic acid metabolites, reactive oxygen metabolites, proteolytic enzymes, and a variety of cytokines and growth factors, promote activation of resident renal somatic cells. Macrophage infiltration has been associated with proteinuria and declining renal function, in both humans and experimental animals. Numerous members of a superfamily of proinflammatory cytokines, referred to as chemokines, have been identified. The term “chemokine” was proposed at the Third International Symposium of Chemotactic Cytokines in Austria in 1992.7 The term is an abbreviation of chemotactic cytokine and reflects the early characterization of these cytokines as chemoattractants and modulators of inflammatory cells. Previous names used for the chemokine family are “intercrines,” SIS (for small inducible secreted), or chemotactic cytokines. It should be noted that not all cytokines that posses chemoattractant activity are considered chemokines since the term is based as much on structural as on functional considerations. The bewildering array of acronyms and codes ranges from the prosaic (PF4 platelet factor 4) to the prolix RANTES (for regulated on activation, normal T cell expressed and secreted). The following review is focused on certain aspects of the biology of chemokines and their potential role in renal disease. Detailed discussions of the biology of chemokines and their pathophysiologic role in other diseases have been extensively reviewed elsewhere.*-” Diseases,

Vol26,

No 6 (December),

1995:

pp 982-994

CHEMOKINES

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DISEASE

983

Fig 1. The chemokine superfamily. Abbreviations are defined in Tables 1 and 2. (Modified and reprinted with permission.2)

HISTORICAL BACKGROUND, PROPERTIES, AND RECEPTORS FOR CHEMOKINES

Chemokines constitute a large family of structurally and functionally related proteins (Fig 1; Tables 1 and 2) with highly conserved amino acid sequences, indicating that they arose from a single ancestral gene. Most of the members of the chemokine family have a protein molecular weight in the 8 to 10 kd range and arc basic hepatin-binding polypcptides. Structurally, chemokines form a superfamily of at least 22 known proteins characterized by a four cysteine motif. The superfamily can be divided into two subfamilies, alpha and beta, based on the chromosomal location of the gene, the overall sequence homology, and the disposition of the first two of the four conserved cysteine residues. In the C-X-C or alpha subfamily, the first two cysteines are separated by a single intervening residue; in the C-C or beta subfamily, they are adjacent. C-X-C chemokines cluster on human chromosome 4 and act primarily on neutrophils. C-C chemokines, on the other hand, act preferentially on monocytes and are clustered on human chromosome 17. The murine C-C cluster lies on chromosome 11. A new class of chemokines, the C subfamily, has been recently proposed by Schall* based on the cloning and characterization of lymphotactin, a lymphocyte chemoattractant that lacks .the first and third cysteine in the four-cysteine chemokine motif.” The chemokine family was originally little more than a collection of cDNAs encoding small structurally related proteins of unknown function. The introduction of the chemotaxis chamber by Boyden in 1962, which allows in vitro quantitation of leukocyte migration in defined gradients of soluble chemoattractants,‘8 was a prerequisite

for the functional description of the chemokines 30 years later. Whereas many of the C-X-C chemokines were identified first by protein chemistry through purification, most of the C-C chemokines were discovered as a result of molecular cloning techniques. Investigators identified novel

Table 1. C-X-C Chemokirtes C-X-C chemokines GCP-2 SDF-1 alpha/beta PBP Beta-TG CTAP-III NAP-2 ENA-

MGSAIGRO

IL-8 C-X-C chemokines IP-10 MIG PF-4

that contain the ELR motif’ Granulocyte chemotactic protein-2 Stromal cell-derived factors-l alpha/beta Platelet basic protein. Precursor of Beta thromboglobulin Connective tissue-activating protein Ill Neutrophil-activating peptide 2 Epithelial-derived neutrophil attractant 78 (mature protein is 78 amino acids long) Melanoma growth-stimulating activity/growth-related oncogen; KC is murine and CINC (cytokine-induced neutrophil chemoattractant) is rat homologue of GRO alpha; MIP2 alpha and beta are rat counterparts of GRO beta and gamma Interleukin-8 (= NAP-l) that lack the ERL motif* Gamma interferon-inducible protein 10 Monokine induced by interferongamma Platelet factor 4

The ELR motii represents the three amino acids GluLeu-Arg that immediately precede the first cysteine amino acid residue of the proteins. This motif is important in receptor/ligand interaction on neutrophils. l

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984 Table MCP-1,

2, 3

Cl0 I-309

MIP-1 alphal beta BANTES Eotaxin FIC

2. C-C

Chemokines

Monocyte chemoattractant protein-l, -2, and -3; MCP-2 is identical to HC14 Murine chemokine without human homologue Human analog of TCA3 (T-cell activation gene 3); P500 is a splice variant of TCA3 Macrophage inflammatory protein-l alpha/beta Regulated on activation, normal T cell expressed and secreted Eosinophil-active chemokine Munne chemokine with 57% homology to MCP-146

genes that are expressed in a cell-specific or activation state-specific fashion. Three common properties of chemokines are apparent. First, chemokines attract one or more myeloid cell types in vitro. Second, their production and/or secretion is markedly induced by proinflammatory stimuli, such as lipopolysaccharide (LPS), tumor necrosis factor-a (TNF-cr), or interleukin-1 (IL-l). Finally, all those chemokines that have been tested induce inflammatory infiltrates when injected intradermally into animals. In addition to their chemotactic activity, most of the chemokines also activate several functions of leukocytes, such as degranulation and oxidant burst. Other biologic activities of chemokines include modulation of leukocyte adhesiveness, vasoconstriction, increased vascular permeability, stimulation or inhibition of proliferation, and angiogenesis. To gain access to the interstitial tissue compartment, circulating leukocytes must first adhere to endothelial cells that line the vessel wall, migrate through endothelial cell junctions, and penetrate the basement membrane. The so-called adhesion cascade has been reviewed and involves a three-step process.‘9,20 The first step is rolling and loose binding or “tethering” of leukocytes to the endothelium via one of the selectin family of molecules. This is followed by triggering, in which a signal transduced to the leukocyte converts the inactive integrin molecules to an active adhesive configuration. The last step is strong adhesion mediated by binding of leukocyte integrins to their ligands on endothelial cells. Migration into

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the tissue follows. Chemokines seem to play a role in each of these steps.‘y-2’ All the chemokines are basic and will bind to heparin and hence, presumably, to extracellular matrix components. This property led to the suggestion that they act by haptotaxis, the movement of target cells up a gradient of an attractant that is immobilized rather than free in the extracellular fluid. Soluble cytokines would be rapidly washed away from the area of production by blood flow. Proteoglycans are good candidates to present proadhesive and chemotactic cytokines, and therefore trigger leukocyte migration.20-22 Haptotactic properties have been demonstrated for IL-8.23 Receptors for chemokines are members of the G protein-coupled receptor family. The chemokine receptors, like their ligands, form a family of structurally and functionally related proteins that can be divided into four groups: specific receptors that bind only one chemokine; shared receptors specific for alpha or beta chemokines, but not both; herpesvirus homologues of the leukocyte alpha or beta chemokine receptors; and the Duffy erythrocyte antigen, which binds promiscuously several alpha and beta chemokines. Detailed overviews have been published recently.‘*24,25The Duffy antigen/chemokine receptor localizes in the kidney to endothelial cells lining postcapillary venules.26 Postcapillary venules represent a dynamic interface that may be an important site for leukocyte transmigration from the vascular space into the tissue compartment during inflammation. The receptor could act as a docking protein to concentrate ligands at the cell surface for presentation to specific receptors on the appropriate target cells. Altematively, it might act as an intravascular physiologic “sink,” which could bind and inactivate circulating chemokines. This would presumably generate a chemokine gradient with a higher concentration of chemokines found in the subendothelial matrix facilitating extravasation and migration of leukocytes. The physiologic and pathophysiologic relevance of the Duffy antigen/chemokine receptor in the kidney clearly needs further investigation. C-X-C

CHEMOKINES

The C-X-C group of chemokines claims a longer and larger history of experimental investigation, with its prototype PF4 being first de-

CHEMOKINES Table

NAP-l NAF NAP GCP MDNCF MONAP

AND 3. Former for

RENAL

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DISEASE

and Alternative Interleukin-8

Names

Neutrophil-activating peptide-l Neutrophil-activating factor Neutrophil-activating protein Granulocyte chemotactic peptide Monocyte-derived neutrophil chemotactic factor Monocyte-derived neutrophil-activating peptide

scribed in 1955.” PF4 was originally identified for its ability to bind to heparin, leading to inactivation of heparin’s anticoagulant function. While possessing little chemotactic activity for inflammatory cells, PF4 is a potent inhibitor of angiogenesis. This activity led to the evaluation of its antitumor properties. Phase I and II clinical trials are currently under way to test its efficacy.2x Growth-related oncogen (GRO)-alpha, -beta, and -gamma are closely related C-X-C chemokines, with GRO-alpha originally being described as melanoma growth stimulatory activity. NH2-terminal truncated forms of platelet basic protein include connective tissue-activating protein III, beta-thromboglobulin, and neutrophilactivating protein-2. The NH2-terminal truncated forms are generated when platelet basic protein is released from platelet alpha granules and undergoes proteolytic cleavage by monocyte-derived proteases. The most extensively studied chemokine of this family is IL-S (Table 3), forprotein 1” merly called ‘ ‘neutrophil-activating (Table 4). It was the discovery of IL-8 in 1987 that revealed the existence of a novel class of small cytokines, now called “chemokine~.“‘~*~~ Similar to MCP-1 of the C-C family, IL-8 is produced by an array of immune and nonimmune cells. In addition to being a potent chemoattractant and activator of neutrophils, it recently has been characterized as a potent stimulant of endothelial cell chemotaxis and angiogenesis as well as mitogenesis of vascular smooth muscle cells.“‘,“2 The C-X-C chemokines can be further divided into ELR containing or lacking proteins. The ELR motif represents the three amino acids Glu-Leu-Arg that immediately precede the first cysteine amino acid residue of the proteins. This motif is important in receptor/ligand interaction on neutrophils. ELR motif lacking chemokines

like PF4, IP-10, and MIG are only weak chemoattractants for neutrophils (Table 1). However, insertion of the ELR motif at the N terminus of PF4 converts it to a potent neutrophil activator and attractant that binds to the IL-8 receptor.“” C-X-C CHEMOKINES AND THE KIDNEY

There is in vitro and in vivo evidence that this family of chemokines may be involved in mediating renal pathology. Rat mesangial cells are induced to express KC, the murine analog of GRO-alpha, in response to IL- 1. Dexamethasone inhibits transcription of KC.“4 Tumor necrosis factor-a induces the expression of IL-8 in human glomerular epithelial’5 and in proximal tubular epithelial cells.“’ Human mesangial cells in culture, when stimulated by TNF-(I! and IL-l, also express IL-8 mRNA and protein that associates with both cell and extracellular matrix. Dexamethasone treatment partially inhibits the release of extracellular IL-8, while IL-8 mRNA and cellassociated IL-8 are not significantly altered.““’ Interstitial inflammatory cell infiltrate is a prominent component of renal allograft failure. Both mononuclear and polymorphonuclear cells infiltrate the renal interstitium in acute rejection. The mechanisms by which cells are attracted into the tubulointerstitial compartment remain to be fully elucidated. Human renal cortical epithelial cells express and secrete IL-8 in response to IL1, LPS, or TNF-a in both a time- and dose-dependent manner.76 Proximal and distal epithelial cells in renal biopsy specimens from patients with acute allograft rejection stain strongly for IL-8.“9 Cyclosporine exerts its immunosuppressive activities by binding to immunoregulatory proteins like cyclophilins. Interleukin-8 can bind specifically to cyclosporine. Putative binding sites for cyclosporine on IL-8 can be identified on the basis of structural similarities between IL8 and cyclosporine. 40The biologic importance of this binding needs to be investigated. An increased urinary excretion of IL-8 in patients with

Table NAP-l NAP-2 NAP-3 NAP-4

4. Nsutrophil-Activating Interleukin-8 Neutrophil-activating MGSAlGRO alpha Lacks C-X-C motif

Peptides

peptide

2

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several forms of proliferative glomerulonephritis was found by Wada et a14’ The elevated urinary IL-8 levels were associated with the immunohistologic detection of IL-8 in diseased glomeruli.4’ These studies suggest that IL-8 may contribute to the infiltration of polymorphonuclear cells (PMNs) in acute allograft rejection and proliferative forms of glomerulonephritis. Serum and urine levels of IL-8 are elevated in patients with acute pyelonephritis. Patients with high concentrations of IL-8 in urine during acute pyelonephritis have a lower glomerular filtration rate at follow-up compared with patients with lower urinary levels of IL-8. This suggests that the secretion of IL-8 from renal cells may participate in the initiation and maintenance of renal inflammation and perhaps the decline in glomerular filtration rate.42 There is a close temporal relationship between chemokine expression and neutrophil or monocyte infiltration?’ There is also good evidence, using neutralizing antibodies, that C-X-C chemokines contribute to the influx of leukocytes and development of renal pathology in experimental models of glomerulonephritis. A rapid influx of neutrophils into the glomerulus occurs within the first hours after the administration of anti-glomerular basement membrane antibody in the rat. Wu et al4 recently demonstrated that mRNA for cytokine-induced neutrophil chemoattractant, the rat counterpart of the human GRO-alpha and the murine KC, was induced in the kidney. The corresponding protein was also elaborated by isolated glomeruli after induction of nephritis. Moreover, in vivo administration of an anti-cytokine-induced neutrophil chemoattractant antibody selectively attenuated the influx of neutrophils into the glomerulus by 60% and commensurately diminished proteinuria, strongly indicating a role for cytokine-induced neutrophil chemoattractant in the early events that accompany immune complex-mediated glomerulonephritis.& Feng et al found increased expression of another chemokine, MIP-2, in a similar model of nephritis.45 The administration of a neutralizing antibody against MIP-2 decreased the neutrophi1 influx by 40% and diminished proteinuria, indicating that MIP-2, a neutrophil chemoattractant, contributes to the influx of neutrophils in anti-glomerular basement membrane antibody disease.45 It is noteworthy that in both studies,

Table 5. Alternative EDCF GDCF LDCF MCAF SMC-CF TDCF JE MCP-1

alpha/

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ABBOUD

Names and Clones for MCP-1 Endothelial cell-derived chemotactic factor Glioma-derived chemotactic factor Lymphocyte-derived chemotactic factor Monocyte chemotactic and activating factor Smooth muscle cell-derived chemotactic factor Tumor-derived chemotactic factor Murine MCP-1 homologue Monocyte chemoattractant protein-l

the influx of PMNs was reduced but not abolished. These data suggest that more than one chemokine is likely responsible for the infiltration of the glomerulus by neutrophils in this model of glomerular injury. The release of multiple chemokines at a single inflammatory site may indeed be the rule rather than the exception, suggesting that more than one chemokine or a common receptor needs to be neutralized to prevent or attenuate inflammatory cell infiltration. C-C CHEMOKINES

Nine different C-C chemokines have been identified. These include MCP-1, 2, and 3; MIP1 alpha and beta; RANTES; I 309, FIC,& and Cl0 (Fig 1; Table 2). A new C-C chemokine, eotaxin, has been recently discovered.47 A number of cDNAs encoding MCP-1 and MIP-18.48 proteins and their human homologues have been isolated independently by different groups using a variety of methods (Table 5). RANTES is the only member of this family whose transcription is not rapidly induced in hematopoietic cells. The lack of rapid induction may explain why RANTES is unusual in this family in not having been independently isolated by a number of groups. Studies pertinent to renal pathology have focused on the potential role of RANTHS and MCP-1. Human RANTES was first identified by its differential expression in selected cDNA libraries, in which its cDNA was expressed at higher levels in T cells than in B cells.4g RANTPS is chemoattractant for monocytes, basophils, eosinophils, and T cells. The murine cDNA also has been cloned from renal tubular epithelium5’ and macrophages5’ The history of

CHEMOKINES

AND RENAL DISEASE

MCP-1 can be traced to the discovery of murine JE by Co&ran et al in 198352 and its subsequent cloning and characterization by Rollins and colleagues in 1988.5”,54JE was discovered as a transcript rapidly induced by platelet-derived growth factor in fibroblasts. In 1984, Valente et al purified a chemotactic protein from baboon vascular smooth muscle cells and called it smooth musclederived chemotactic factor; this protein was later identified as MCP-1.55 The purification of a human monocyte-chemoattractant protein from a glioma cell line (glioma-derived chemotactic factor [GDCFI) and from a monocytic cell line (monocyte chemotactic and activating factor [MCAF]) by two different groups was achieved in 1989.56,57Subsequent cloning of the cDNA for MCP-1 showed it to be highly similar to murine JE.54The MCP-1 protein is 62% identical to murine JE in a region of 68 shared N-terminal residues, but murine JE extends an additional 49 residues in the carboxy portion of the molecule. Although it is widely accepted that JE and MCP1 are species homologues, the significance of the predicted protein differences is not clear. MCP1 also shares 21% homology at the amino acid level with IL-8. The rat JE gene is 82% homologous to the murine JE. Human MCP-1 is a peptide of 76 residues. MCP-1 resolves on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) as multiple species of molecular masses ranging between 8 to 18 kd. Leonard and Yoshimura reported that a glioma cell line secretes 15 and 13-kd species, which were designated MCP- 1-alpha and MCP- 1-beta. I4 The size difference is not due to different amino acid composition, but rather to posttranslational modification. Rollins et al reported the secretion of three major forms of recombinant protein in COS cells even in the presence of tunicamycin, an inhibitor of N-linked glycosylation.54 Data from Jiang et al, on the other hand, suggest that differences in the processing of O-linked carbohydrates account for the heterogeneity of MCP-1 produced by different cell lines.58 The sequence organization of the human MCP- 1 gene has been described.54.59Three exons and two introns of the gene span approximately 1,900 base pairs of DNA. Several regulatory DNA sequences have been described in the 595 base pairs of the 5’ flanking region. Ueda et al recently cloned human MCP- 1 promoter and de-

scribed an upstream NFkB element essential for the induction of MCP- 1 by IL- 1, TNF, and LPS.@-’ We have isolated an 1,874 base pair clone of the 5’ flanking region. Transfection of luciferase constructs into vascular smooth muscle cells indicates transcriptional regulation of MCP-1 by thrombin.“’ cDNAs that encode two MCP-lspecific receptors with alternatively spliced carboxy1 tails have been cloned recently.62.6” Signaling by the MCP- 1 receptor involves mobilization of intracellular calcium and inhibition of adenyl cyclase by pertussis toxin-sensitive G proteins.@ Recent reports by Van Damme et al have revealed the existence of MCP-2 and MCP-3 with 62% and 73% amino acid identity to MCP-1.65 Most cells or tissues examined produce MCP-1 on activation. However, the targets of MCP-1 appear to be limited to monocytes, basophils, and, as reported recently, lymphocytes.66,67 MCP1 activates freshly isolated monocytes and induces the release of the granule-associated enzyme N-acetyl beta-D-glucosaminidase and production of superoxide anions.68 Rollins et al also demonstrated that MCP-1 stimulates H202 production in monocytes,69 whereas Leonard and Yoshimura found that monocytes release little or no superoxide in response to MCP-1 .I4 MCP-1 also stimulates the release of histamine from human basophils primed with IL-3.” Ikeda et al recently reported that leukocytes are not the only target for MCP-1. These investigators found a small but significant inhibitory effect of MCP-1 on proliferation of rat vascular smooth muscle cells?’ This is the first report demonstrating a biologic effect of MCP-1 on somatic cells. RANTES

AND THE KIDNEY

RANTES is expressed in whole kidney tissue, tubular epithelium, and mesangial cells.” Tumor necrosis factor-a, and to a lower extent IL-l and LPS, increase RANTES mRNA levels and stimulate protein secretion in murine mesangial cells.72 The induction of RANTES in mesangial cells involves reactive oxygen intermediates and is attenuated by forskolin.” RANTES mRNA expression is increased in glomeruli harvested from rats following renal in vivo perfusion with TNF-~x.‘~ Increased expression of RANTES also is seen in rats with accelerated nephrotoxic serum nephritis74 and in rats with anti-Thy-l nephritis (R.A.K. Stahl, personal communication, 1994).

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0

12

24

36

48 hours

CON

CM

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ABBOUD

CM+ABCM+NRS

Fig 2. (A) Production of monocyte chemotactic activity by human mesangial cells in culture. The medium was conditioned by mesangial cells for dint time periods, and human monocyte chemotactic activity was determined in chemotaxis chambers. (B) Inhibition of mesangial cell-derived monocyte chemotactic activity by MCP1 antibodies. Medium condiiioned for 48 hours by mesangial cells was incubated with or without a 1:loO dilution of rabbit antiserum to MCP-1 and assayed for monocyte chemotactic activity. CON, control; CM, condiined medium; CM + AB, conditioned medium preincubated with anti-baboon MCP-1 antibody; CM + NRS, condiioned medium preincubated wlth normal rabblt serum.

RANTES is not expressed in routine human renal biopsy specimens taken 1 hour after transplantation or in native renal biopsy specimens from patients with cyclosporine nephrotoxicity, but is upregulated during cell-mediated transplant rejection. In these studies, RANTES mRNA was detected in infiltrating mononuclear cells, and renal tubular epithelium and FWNTES protein was localized to mononuclear cells, tubular epithelium, and vascular endothelium. RANTES therefore may play a role in allograft rejection.75376 Disruption of RANTES by homologous recombination may provide a useful model to directly assess the role of RANTES in inflammatory renal disease.77 MCP-1

AND THE KIDNEY

There is good in vitro and in vivo evidence for a potential role of MCP-1 in mediating infiltration of inflammatory cells in renal disease. Human mesangial cells release monocyte chemotactic activity in culture (Fig 2A). This activity is neutralized approximately 85% by a specific MCP-1 antibody (Fig 2B). Thus, MCP-1 is responsible for the bulk of the monocyte chemotac-

tic activity produced by cultured human mesangial cells, suggesting that other members of the C-C chemokine family play a minimal role in the monocyte chemotactic activity released by these cells.‘* On SDS-PAGE and immunoblotting of mesangial cell-conditioned medium, three protein bands with molecular weights of 11, 15, and 16 kd are identified.78 MCP-1 is upregulated in mesangial cells in response to stimulation by cytokines and growth factors. Interferon-gamma, IL-l-alpha and -beta, and TNF-a are the most potent inducers of MCP- 1 mRNA expression and protein secretion.78-8’ Moreover, LPS, immunoglobulin G immune complexes, LDL, and thrombin induce MCP-1 expression in mesangial cells.82~8hLipopolysaccharide also induces MCP1 in the isolated perfused rat kidney. Expression was found in peritubular capillary endothelial cells.87 Interleukin- 1, TNF-(Y, interferon-gamma, and thrombin regulate MCP-1 gene expression in bovine glomerular endothelial cells.88 There is evidence that oxygen radicals are second messengers that enhance the expression of MCP-1 in mouse mesangial cells.89 Agents that increase cyclic adenosine monophosphate levels, such as

CHEMOKINES

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Table 6. MCP-1 in Renal Disease Model

lschemia Hydronephrosis Anti-Thy-l nephritis Anti-GEM nephritis (rat) Streptozotocin-induced diabetes Renovascular hypertension PAN nephrosis Anti-GBM nephritis (mouse)

Localization Thick ascending limbs and distal nephrong’ Cortical tubulesg3 Mesangiumw GlomerulusaG Mesangiumg8 NW Tubuleslo ND”“’

Abbreviations: GBM, glomerular basement membrane; ND, not determined; PAN, puromycin aminonucleoside.

forskolin or prostaglandin E2, inhibit the production of MCP-1 in mesangial cells.” The normal adult kidney expresses relatively small amounts of MCP-1. MCP-1 expression is increased in diverse models of renal disease ranging from ischemia, hydronephrosis, glomerulonephritis, puromycin aminonucleoside nephrosis, diabetic nephropathy, and renovascular hypertension’” (Table 6). Safirstein et a19’found increased expression of MCP-1 in rats up to 96 hours after the induction of ischemic acute renal failure. MCP- 1 protein localized immunocytochemically to the apical regions of the cortical and medullary thick ascending limbs as well as to the lumen of the distal nephron. The chemokine KC, the rat counterpart of GRO-alpha, also was upregulated in the same model of ischemia.” MCP-1 and KC, by attracting leukocytes into the ischemic kidney, may participate in tubular cell injury or, alternatively, may promote renal regenerative response. Treatment of rats with methylprednisolone blocked the constitutive expression and inhibited ischemia-induced elevation of MCP-1 mRNA levels in the kidney.92 Renal cortical monocytesl macrophage infiltration may play a role in the late development of interstitial fibrosis after experimental hydronephrosis. Diamond et al recently described a temporal relationship between the expression of MCP-1 and transforming growth factor-/? and the appearance of monocytes in experimental hydronephrosis.” Similar to the findings in the ischemic kidney, MCP1 protein expression was detected in the apical segments of cortical tubules. In cell culture, MCP-1 protein is rapidly secreted after its

mRNA is induced. The immunolocalization of a chemoattractant protein on the luminal surface of tubular epithelium, in both hydronephrosis and the renal ischemia models, does not necessarily indicate that these cells are the source of the chemokine. It remains unclear how the apical location of a chemoattractant may be responsible for the presence of monocytes along the basement membrane of the kidney tubules. MCP-1 is likely produced and released at different sites before it gains access to the apical aspect of the tubular epithelium. Thus, passive trapping or binding to apical cell membranes may explain such localization. Combined in situ hybridization studies together with immunohistochemical staining for specific cell markers should clarify the site of production of MCP- 1. Monocytes allegedly rapidly lose binding sites for MCP-1 after they migrate into the tissue. Therefore, it is also important to determine the expression of MCP-1 receptors on monocytes and other potential target cells in the inflamed tissue. In rats with anti-Thy-l nephritis, a model of proliferative glomerulonephritis, Stahl et al reported increased expression of MCP-1 in mesangial areas.% MCP-1 mRNA and protein expression increased as early as 30 minutes after induction of nephritis followed by reduction at 24 hours. Increased MCP-1 expression was found again after 5 and 21 days. The chronology of MCP-1 expression correlated with mesangial immune complex formation (30 minutes), mesangiolysis (24 hours), and development of proliferative glomerulonephritis (5 and 21 days). Induction of nephritis in the isolated perfused kidney also resulted in increased expression of MCP-1 n&NAY5 An increase in MCP-1 mRNA levels together with monocyte infiltration also was found in the glomerular mesangium of rats with nephrotoxic nephritis. Irradiation prior to induction of the nephritis prevented monocyte infiltration and decreased the induction of MCPl.% An increased number of monocytes in mesangial areas and in the interstitium associated with increased MCP-1 protein expression in the mesangium was also found in streptozotocin-induced diabetes in the rat. Insulin treatment prevented monocyte infiltration and decreased MCP- 1 staining?7’98 Infiltration of the glomerular mesangium and cortical interstitium by macrophages and lymphocytes also has been reported

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in rats with renovascular hypertension.99-‘0’ Increased expression of MCP-1 mRNA was found at an early time point in the clipped kidney of two-kidney, one-clip renovascular hypertensive rats and in the contralateral kidney at later time points.“’ Increased glomerular expression of RANTES mRNA and protein is also seen in the clipped kidney of renovascular hypertensive rats (unpublished observation). Eddy recently reported increased renal MCP-1 mRNA levels and de novo appearance of the MCP-1 protein in the tubules of rats with experimental nephrosis.“” Neugarten et al reported increased expression of MCP-1 and RANTES mRNA in mice with antiglomerular basement membrane glomerulonephlitiS.‘w Of interest in this study is the suggestion that proteinuria and glomerular pathology are not dependent on monocyte/macrophage infiltration in the colony-stimulating factor- 1 -deficient mice studied. Preliminary data also suggest that urinary MCP-1 levels are increased in patients with lupus nephritis.“’ Taken together, the results of these studies indicate that MCP-1 is a ubiquitous chemokine that may be induced in diverse forms of renal injury and that is responsible for leukocyte infiltration in renal disease. CONCLUSION

AND PERSPECTIVE

The discovery and characterization of the chemokine superfamily has defined a new area in immunobiology. The distinct patterns of their biologic activity suggest that chemokines are expressed selectively in pathologic conditions and that their activation is of importance in the regulation of inflammatory cell recruitment and perhaps activation. Much of the evidence obtained is consistent with the idea that the C-X-C chemokines might play a role in acute inflammation mediated through neutrophils, while C-C chemokines are involved in less acute or chronic inflammation mediated through monocytes and lymphocytes. Many chemokines share receptors and exert similar biologic functions. However, overlapping but distinct sets of targets allow for a fine-tuning of the immune response by chemokines.2*48It also is likely that these proteins exert additional functions that have not yet been discovered. The properties of PF4 and IL-8 make it clear that we ought to not restrict our view of chemokines solely to the immune system. It

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should also be noted that a number of other inflammatory cytokines have chemotactic properties. Differential expression of inflammatory cytokines therefore could result in the recruitment of the appropriate combination of inflammatory cell types. Dramatic differences in chemoattractant potency also must be considered as biologically relevant. For example, transforming growth factor-p is optimally active as a monocyte chemoattractant at approximately 10-l’ mol/L, whereas the optimal concentration of MCP-1 is about 10m9 mol/L. Therefore, if present at levels well in excess of its optimal concentration, a chemotactic agonist is ineffective at promoting directed migration. I5 Thus, a highly potent chemokine may be an effective chemoattractant at some distance from an inflammatory focus, but it may be ineffective locally where high concentrations are achieved in the immediate vicinity of its production or release. Conversely, a less potent chemokine produced at the same site might be an effective chemoattractant for leukocytes that are nearby, but not for those located at greater distances from the inflammatory site.” The potential involvement of chemokines in kidney disease is currently the subject of intense investigation. A close temporal relationship between chemokine expression and biologic response, such as neutrophil, monocyte, or lymphocyte infiltration, has been established in certain forms of renal injury. In the context of cytokine network, chemokines appear to be involved at a very early stage of renal injury. They are induced in renal somatic cells by diverse mechanisms, including hemodynamic, hypoxic, and immune-mediated.‘060’07 This rapid induction and release of chemokines results in infiltration by inflammatory cells that are likely involved in mediating the acute injury followed by either healing or progression. One remarkable feature of certain renal diseases is the fact that the structural and functional abnormalities may be totally reversible. This is a feature of some experimental models of glomerular injury, such as anti-Thy-nephritis, and of certain human forms of glomerulonephritis, such as poststreptococcal glomerulonephritis. To what extent chemokines contribute to injury or repair remains to be defined. PMNs, monocytes, and lymphocytes may contribute to acute renal cell injury and/or to progression of glomerular

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disease. On the other hand, by promoting migration and proliferation of glomerular cells, monocytes may help to repopulate the glomerulus after cell lysis or necrosis. Secreted collagenases and proteases likely participate in this process and may be responsible for matrix reabsorption and remodeling, thereby restoring glomerular integrity. A constructive role for chemokines is suggested by studies demonstrating that infiltrating monocytes also appear to have a role in clearing the mesangium of deposits of immune complexes.“’ The temporal association of chemokines and renal pathology does not establish a casual relationship. In vivo intervention studies to block the biologic activity with antibodies or other antagoniststW will be needed to evaluate the significance of chemokine expression in renal disease. Neutralizing studies in acute models of renal disease are feasible and the early results are promising. The challenge is to extend these studies to additional animal models with acute progressive and chronic lesions relevant to human disease, such as diabetic nephropathy or progressive forms of nephrosclerosis. Immunohistochemical and in situ hybridization techniques should be applied to renal biopsy specimens to establish temporal relationships in human disease.95 Transgenic animals that overexpress chemokines or induction of disease in knockout animals that lack a particular chemokine provide an additional tool for studying the role of these mediators in renal disease.“.’ ‘O.’’ ’ Delineating a precise role for chemokines in mediating pathologic changes in the kidney is an area of fruitful investigation. ACKNOWLEDGMENT The authors thank Dr Antbony Valente for critical reading of the manuscript, and Leah Sankey and Olga German for typing the manuscript. REFERENCES 1. Ahuja SK, Gao JL, Murphy PM: Chemokine receptors and molecular mimicry. Immunol Today 15:320-326, 1994 2. Schall TJ: Chemokine Receptor-Ligand and Intracellular Signaling Interactions, in Chemotactic Cytokines. Targets for Therapeutic Development. IBC Conference, Washington, DC, September 19-20, 1994 3. Cattell V: Macrophages in acute glomerular intlammation. Kidney Int 45:945-952, 1994 4. Klahr S: Interstitial macrophages. Semin Nephrol 13:488-502. 1993

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