Selective Binding and Presentation of CCL5 by Discrete Tissue Microenvironments during Renal Inflammation
Stephan Segerer*,
Roghieh Djafarzadeh*,
Hermann-Josef Gröne,
Christian Weingart*,
Dontscho Kerjaschki,
Christian Weber,
Andreas J. Kungl||,
Heinz Regele,
Amanda E.I. Proudfoot¶ and
Peter J. Nelson*
* Medizinische Poliklinik-Innenstadt, University of Munich, Germany; Department of Cellular and Molecular Pathology, German Cancer Research Center, Heidelberg, Germany; Clinical Institute of Pathology, University of Vienna, Vienna, Austria; Institute for Molecular Cardiovascular Research, University of Aachen, Aachen, Germany; || University of Graz, Graz, Austria; and ¶ Serono Pharmaceutical Research Institute, Geneva, Switzerland
Address correspondence to: Dr. Stephan Segerer, Medizinische Poliklinik-Innenstadt, University of Munich, Pettenkoferstrasse 8a, 80336 Munich, Germany. Phone: +49-89-5160-3565; Fax: +49-89-5160-4439; E-mail: stephan.segerer{at}lrz.uni-muenchen.de
Received for publication August 9, 2006.
Accepted for publication March 29, 2007.
T cells are differentially recruited to the tubulointerstitiumduring renal inflammation. The selective presentation of chemokinesby surface structures may in part underlie this phenomenon.In an attempt to better characterize the presentation of chemokinesby tissue environments an exemplary chemokine with a well-definedstructure was selected, and a binding assay for the proteinon fixed archival tissue sections was developed. This articledescribes the selective binding of the chemokine CCL5 to renalstructures. CCL5 was shown to bind to endothelial regions, interstitialextracellular matrix, tubular epithelial cells, and tubularbasement membranes but rarely to glomerular structures in well-preservedkidneys. In contrast, binding of CCL5 to glomerular componentswas seen in renal biopsies with acute allograft glomerulitis(in which T cells accumulate in glomeruli). The N terminus mediatesreceptor binding, whereas two clusters of basic amino acid residues(44RKNR47 and 55KKWVR59) are involved in the presentation ofCCL5 by extracellular structures. Mutation of either loop abrogatedCCL5 binding to tissue sections. Variations of the N terminusand a mutation that prevents higher order oligomerization didnot change the binding pattern. The data suggest that renalcompartments differ in their capacity to present chemokines,which may help explain the differential recruitment of leukocytesduring allograft injury. Both clusters of basic residues inCCL5 are necessary for sufficient binding of CCL5 to tissuesections.
Underlying the recruitment of leukocytes to inflamed tissuesand allografts is a complex interaction between chemokines andtheir corresponding receptors, adhesion molecules, and ligands(1–4). Chemokines that are bound to endothelial surfacestrigger the firm adhesion of leukocytes by the activation ofintegrins and facilitate migration of cells through tissues(5,6).
Chemokines are thought to be bioactive in vivo when presentedby extracellular structures (e.g., glucosaminoglycan [GAG] structureson cell surfaces or on extracellular matrix [7,8]). This presentationof chemokines is thought to facilitate adhesion under flow andhaptotaxis through tissues (migration along a surface gradient).
The chemokine CCL5 acts as a ligand for the chemokine receptorsCCR1, CCR3, and CCR5 and helps to direct the migration of monocytes/macrophagesand T cells (2,9). CCL5 has two clusters of basic residues,a BBXB motif on the "40s loop" of the primary amino acid sequence(44RKNR47) and a BBXXB motif on the "50s loop" (54KKWVR59; Figure 1A).Both regions represent consensus domains for matrix-bindingsites, but only the 40s cluster has been shown to facilitatebinding to GAG (10). A mutation of the 40s loop 44AANA47 CCL5is unable to recruit cells in a model of peritoneal cell recruitment(7,10). We recently found that the 54KKWVR59 loop may be importantfor CCL5 binding to sulfatides (S.S. et al., manuscript in preparation).
Figure 1. Scheme of the binding assay and CCL5 binding to lymphatic tissue. (A) Scheme of the CCL5 primary structure. (B) Scheme detailing the in situ CCL5 binding assay. The anti-human CCL5 antibody does not bind to endogenous CCL5 under these conditions. Sections of human tonsils were exposed to wild-type (WT)-CCL5 (C), CCL5 E26A (D), aminooxypentane (AOP)-RANTES (E), and PBS (G) and stained for CD34-positive endothelial cells (F).(C and D) Prominent binding of WT-CCL5 and E26A (a CCL5 mutant that does not form higher order multimers) to structures of the extracellular matrix and to cells within a vessel is seen. Note that there is no apparent difference between the staining patterns, which illustrates that larger CCL5 multimers are not necessary in this assay. (E and F) Consecutive sections were stained using binding of AOP-RANTES (E) and of CD34 (F), which demonstrates binding to a high endothelial venule (arrowhead in E). AOP-RANTES demonstrated the same staining pattern as WT-RANTES. The vehicle control is negative (G). Magnification, x200.
During inflammatory kidney diseases, CCR5-positive T cells preferentiallylocalize to the tubulointerstitial compartment, whereas monocytes/macrophagesare found both in the tubulointerstitium and in glomerular tufts(11,12). One exception to this observation is the acute glomerularinflammatory response that is seen in renal allografts (acuteallograft glomerulitis), whereby CCR5-positive T cells are oftendetectable within glomerular tufts (13). We sought to determinewhether differences in chemokine binding between glomeruli andthe tubulointerstitium may help to explain this observation.With the use of CCL5 as an exemplary proinflammatory chemokine,potential chemokine binding/presentation structures in normalkidneys and allografts were characterized. The relative importanceof the 40s and 50s loops in the CCL5 protein for presentationwas studied.
CCL5 Protein and Mutations
The wild-type and mutated CCL5 proteins were expressed in Escherichiacoli and purified as described previously (7,10). The bindingsites in CCL5 are illustrated in Figure 1A.
Tissue Specimens
Human tonsils and renal nephrectomies (allografts or normalareas of tumor nephrectomies) were used for establishment ofconsecutive and double-antibody labeling. Renal allograft biopsiesfrom 15 patients with acute transplant glomerulitis were comparedwith 10 biopsies without glomerulitis. Patients without glomerulitisdemonstrated no significant lesions (n = 5), vascular allograftrejection (Banff 2, n = 4), and tubular injury (n = 1). Diagnosesin patients with glomerulitis were humoral allograft rejection(n = 4), borderline lesion (n = 1), vascular allograft rejection(Banff 2, n = 5), interstitial rejection (Banff 1, n = 1), tubularinjury (n = 1), and thrombotic microangiopathy (n = 3). Materialswere fixed in 4% buffered formalin and embedded in paraffinfollowing protocols for routine procedures.
CCL5 Binding Assay
The binding assay (Figure 1B) was based on a modified immunohistochemistryprotocol (14). Sections were deparaffinized in xylene and rehydratedin a graded series of ethanol. Endogenous peroxidase was blockedby incubation with 3% hydrogen peroxide in methanol. Antigenretrieval was performed as described previously (14). Endogenousbiotin was blocked using the Avidin/Biotin blocking Kit (VectorLaboratories, Burlingame, CA). Slides were incubated with CCL5for 1 h. As controls, CCL5 was replaced by vehicle (PBS), thecorresponding concentration of bovine albumin, or CCL5 was preabsorbedby exposure to heparin. The anti-CCL5 antibody (VL2) does notdetect CCL5 in formalin-fixed, paraffin-embedded tissue underthe conditions used, as controls did not demonstrate stainingafter omission of CCL5. Only exogenously added CCL5 proteincould be detected on the tissue sections. For detection, eitherFITC-labeled streptavidin (Vector Laboratories) or the ABC reagent(Vector Laboratories) with diaminobenzidine and metal enhancementwas used (15). Immunohistochemistry for inflammatory cell markersand extracellular matrix was performed as described previously(16).
Immunofluorescence
Double fluorescence was used to further define the CCL5 bindingstructures. When two primary antibodies of the same specieswere combined, another heat treatment in a microwave oven wasperformed between the incubation steps. In each series of doublelabeling, each antibody was replaced by isotype-matched Ig (15).
Immunohistochemistry
Five allograft nephrectomies with acute vascular and interstitialrejection were used. Frozen sections from allograft nephrectomieswere fixed in acetone for 2 min. Immunohistochemistry was performedwith the mAb VL2 (17,18) against CCL5, using an amplificationsystem (CSA; Dako, Glostrup, Denmark) according to the protocolprovided by the manufacturer.
Antibodies
The following antibodies were used: Anti-CD68 for monocytes/macrophages(clone PG-M1; DAKO), anti-CD3 for T cells (clone CD3–12,rat anti-human; Serotec, Oxford, UK), anti-CD34 for endothelialcells, anti–D2–40 lymphatic endothelium (D2–40;Signet Laboratories, Dedham, MA), anti–smooth muscle antigen(SMA; clone 1A4; Dako), anti–human collagen I (providedby Larry Fischer, National Institutes of Health, Bethesda, MD),MC5 against CCR5 (monoclonal mouse antibody; provided by MatthiasMack), and 1C3 against human DARC (provided by Yves Collins,Paris, France) (19). As secondary reagents, biotinylated antibodies(Vector Laboratories), Streptavidin/FITC complex (Vector Laboratories),Cy3 labeled anti-rat antibody (Jackson Immunoresearch Laboratories,West Grove, PA), Texas red, and FITC-labeled anti-mouse IgG(Vector Laboratories) were used.
Localization of CCL5 Binding Structures in Tissue
A CCL5 binding assay based on a modified immunohistochemistryprotocol using formalin-fixed, paraffin-embedded tissue wasestablished to study the selective binding of chemokines totissue compartments (Figure 1B). The assay showed a reproducibleCCL5 binding pattern on human tonsils (Figure 1C). CCL5 boundto endothelial cells of high endothelial venules that showeda focal, granular deposition (Figure 1, E and F), on the extracellularmatrix that surrounded larger vessels and in a matrix patternthat surrounded follicles (Figure 1, C and D). Some circulatingcells in larger vessels demonstrated CCL5 binding (Figure 1C).
T cells were embedded in a matrix that bound CCL5. The stainingof extracellular matrix was similar but not overlapping withthe pattern of collagen I (data not shown). Comparison of thestaining with CD34 (an endothelial marker), D2–40 (a markerof lymphatic endothelium), and the chemokine- binding proteinDARC demonstrated that the majority of the binding structureswere not on lymphatic or vascular endothelial cells but associatedwith the extracellular matrix. Controls with replacement ofCCL5 protein by diluent (PBS; Figure 1G), bovine albumin (Figure 3A),or with preincubation with heparin resulted in absence of staining(Figure 2). Modifications of the N-terminal region of CCL5 (i.e.,aminooxypentane-RANTES and methionylated RANTES) did not changethe binding pattern (data not shown). Finally, CCL5 bindingto CD3-positive T cells was not detectable, suggesting thatCCL5 binding to T cell–expressed chemokine receptors (e.g.,CCR1, CCR5) cannot be detected in this assay.
Figure 3. CCL5 binding to allograft nephrectomies. Consecutive tissue sections from an allograft nephrectomy were exposed to albumin (A; control), [54AAWVA59]-CCL5 (B), WT-CCL5 (C), and an antibody against CD34 (D). Mutation of the 54AAWVA59 residues completely abolishes tissue binding of CCL5 in this assay. WT-CCL5 binds to extracellular matrix (C) within the severe interstitial infiltrate but does not bind to the glomerular tuft (C). The pattern is not consistent with endothelial cells (D). Double immunofluorescence was performed for CCR5 (red; E) and WT-CCL5 (green; F), and an overlay was performed with a nuclear counterstain (G). Note that the glomerulus in the center does not contain CCR5-positive cells and demonstrates very little CCL5 binding, but the interstitial CCR5-positive infiltrate is embedded in structures that bind CCL5. Magnification, x200
Figure 2. CCL5 binding to preserved renal tissue and collecting ducts in allografts. Tissue sections were taken from the noninvolved part of a tumor nephrectomy that was exposed to CCL5-E26A (C and D) or to diluent (A and B). Note the prominent binding of CCL5-E26A to the basolateral side of tubular epithelial cells, weak binding to extracellular matrix of the interstitium, but very little binding to glomerular structures. Consecutive tissue sections from an allograft nephrectomy were exposed to WT-CCL5 (E) and methionylated (Met)-RANTES (F). This resulted in the same binding pattern with prominent binding to collecting duct cells and interstitial matrix. Magnification, x400.
CCL5 Binding to Normal Renal Tissue and Allograft Nephrectomies
Normal tissue from tumor nephrectomies was used to test CCL5binding. The tubulointerstitium was the principal site of CCL5binding (Figure 2, C and D). CCL5 bound to tubular epithelialcells at the basolateral surface and in a granular pattern inthe cytoplasm (Figure 2, C and D). A strong staining of thebasolateral area was also found on collecting ducts (Figure 2,E and F). Binding to tubular basement membrane was commonlyobserved, whereas glomerular regions did not demonstrate significantstaining (Figure 2C). Binding was pronounced in areas of focalaccumulation of extracellular matrix in the interstitium inthe control tissue. Positive staining of glomerular structureswas rare, but in these instances, a staining of glomerular endothelialcells was found (Figure 2C). Binding of CCL5 to peritubularcapillaries in controls was uncommon, and, when present, a granulardeposition was seen.
After CCL5 binding to control renal tissue was established,binding to diseased tissue was evaluated. Inflammatory cellsin the interstitium of renal allografts were found to be surroundedby CCL5 binding structures (Figures 3C and 4). Areas of interstitialfibrosis demonstrated binding of CCL5 (Figure 3C). CCR5-positivecells that were found predominantly in the tubulointerstitiumwere surrounded by a matrix that bound CCL5 (Figure 3, C throughG). CCL5 binding to peritubular capillaries was more prominentthan that seen in controls (Figure 4F).
Figure 4. CCL5 binding to a transplant nephrectomy. Consecutive sections of a transplant nephrectomy were exposed to WT-CCL5 (A and F), Met-RANTES (B), and [54AAWVA59]-CCL5 (C) and stained for collagen I (D) or CD34 (E). At a high magnification, binding of WT-CCL5 to circulating leukocytes and to the endothelial surface in a granular pattern can be demonstrated in a peritubular vessel (F). Frozen sections from an allograft nephrectomy were stained with an isotype control Ig (G) and an anti-CCL5 antibody (VL2; H), and binding of WT-CCL5 was localized (I). Note the prominent signal on infiltrating cells (H) for CCL5 protein and the binding to structures of the interstitial matrix (I) but not to glomeruli.
Variations in the amino terminus of CCL5 (i.e., aminooxypentane-RANTESand methionylated RANTES) did not affect the binding pattern(Figure 2, E and F). Mutation of amino acid 26 in the primarysequence of CCL5 from a glutamic acid to alanine (E26A) leadsto the preferential formation of CCL5 tetramers as apposed tohigher order oligomers (20). This modification did not changethe general distribution of CCL5 binding, suggesting that oligomerizationis not needed for CCL5 binding to renal structures.
The matrix binding domains that were previously studied in theCCL5 protein (10) include a BBXB binding motif found in a loopof amino acids that include R44, K45, and R47 (44AANA47 40sloop) and a BBXXB motif that includes K55, K56, and R59 (54AAWVA5950s loop). Mutation of either the 50s loop or the 40s loop abolishedCCL5 binding to renal structures (Figures 3, A and B, and 4C),suggesting that both loops are necessary for efficient bindingof CCL5 to presentation structures in renal tissue.
For localization of CCL5 in human allografts, immunohistochemistryfor CCL5 was performed on frozen sections of allograft nephrectomies(n = 5; Figure 4). CCL5 was mainly localized to the cytoplasmof infiltrating cells in the tubulointerstitium and infiltratingarterial walls in vascular rejection (Figure 4, F through H).Even on frozen materials, only intracellular CCL5 was detectableby immunohistochemistry (17,18). Consecutively, binding of CCL5was found on structures of the extracellular matrix as in theformalin-fixed, paraffin-embedded materials in the tubulointerstitium.Therefore, the main site of CCL5 expression corresponded tothe main site of CCL5 binding structures. CCL5 expression wasonly occasionally seen in glomerular tufts, by infiltratingcells.
Binding of CCL5 to Renal Allograft Biopsies
A total of 25 renal allograft biopsies were studied. Fifteenof the biopsies were classified as showing acute allograft glomerulitis.In biopsies without glomerulitis, little or no binding of CCL5was seen in glomerular structures (eight of 10 biopsies). Bycontrast, in biopsies that showed glomerulitis, a prominentbinding of CCL5 to glomeruli was found in 11 of the 15 biopsies(Figure 5, C through F). Here, CCL5 binding to infiltratingcells in the glomerular tuft, peripheral glomerular walls (Figure 5,E, F, and H), glomerular endothelial cells, and extracellularmatrix in the glomerular tuft was documented (Figure 5H).
Figure 5. CCL5 binding to renal allograft biopsies. Binding of human WT-CCL5 was studied on biopsies without allograft glomerulitis (A, B, and G) and with allograft glomerulitis (C through F and H). Note that biopsies with and without glomerulitis do not differ in the tubulointerstitial binding of CCL5 (e.g., G, H), but there is prominent binding to the glomerular tuft in biopsies with glomerulitis (C through F). Magnifications: x200 in A through D; x400 in E through H.
CCL5 was found to bind predominantly to the tubulointerstitiumin all biopsy samples (Figure 5). The binding to the basolateralsurface of tubular epithelial cells described previously wasunchanged between the various disease entities studied (Figure 5G).In tubular interstitial areas that showed leukocyte accumulation,the infiltrates were embedded within a network of CCL5 bindingstructures. Tubular basement membranes bound CCL5, whereas glomerularbasement membrane did not.
The formation of microenvironments in lymphatic tissue is aprerequisite for immune surveillance and effector cell accumulation.Recent studies have shown that similar processes lead to inflammatorycell accumulation in specific tissue compartments (19,21). Chemokinesfulfill several basic functions. One is integrin activation(leading to induced firm adhesion), a second is the directedmigration of leukocytes to specific tissue environments. Forboth actions, chemokines need to be presented by extracellularstructures. To date, the biology of chemokine presentation innormal or diseased tissues is poorly understood (22–24).
CCL5 is a proinflammatory chemokine that is rapidly inducedunder various forms of tissue injury under the control of NF-B(25). Immune effector cells such as T cells and monocyte/macrophagescan be a rich source of CCL5 (26). Intrinsic renal cells, includingmesangial cells, podocytes, tubular epithelial cells, and interstitialfibroblasts, also release CCL5 after stimulation in vitro (4).In previous studies, we showed that microvascular endothelialcells upregulate an unknown CCL5 presentation antigen in concertwith intercellular adhesion molecule, vascular cellular adhesionmolecule, and E-selectin after activation with proinflammatorycytokines (27). During acute allograft rejection, CCL5 was localizedin frozen material to infiltrating cells (in glomeruli and thetubulointerstitium), tubular epithelial cells, and endothelialcells of peritubular capillaries (18,28). In diabetic nephropathyand in membranous nephropathy, CCL5 expression was found tobe predominantly localized to the tubulointerstitium (29,30).A systematic evaluation of CCL5 on a large number of biopsieshas not been performed to date. Cohen et al. (31) describedCCL5 protein and mRNA expression in glomeruli from renal allografts.As was previously found (17,18), intracellular CCL5 proteinwas localized in frozen materials predominantly to infiltratingcells in the tubulointerstitium in renal allograft nephrectomies.Binding of CCL5 to extracellular matrix could not be localizedsufficiently by immunohistochemistry alone, which is most likelya concentration problem. Therefore, consecutive binding studiesin tissue add information.
Here, exogenously added CCL5 was used as a probe to study theselective or regulated presentation on tissues (32–35).We hypothesized that the tubulointerstitium and the glomerulartuft differ in their CCL5 binding capacity, because these regionsshow a differential inflammatory infiltrate. An "in situ" assaywas developed to study this question. In this assay, bindingof CCL5 seemed not to be dependent on interactions with thechemokine binding protein DARC or other chemokine receptors.Binding of CCL5 could be prevented by preincubation of the proteinwith heparin or by mutating either the 40s or the 50s basicloop residues in CCL5. Lymphocytes in the tonsil, as well astubulointerstitial leukocytes in renal allografts, were foundto be embedded in a network of CCL5 binding structures. In thekidney, a prominent compartmentalization was detectable withstrong binding of CCL5 seen in the tubulointerstitium. In contrast,binding to structures of the glomerular tuft was weak or absentin well-preserved tissue. Therefore, CCL5 bound predominantlyto the renal compartment in which T cells, expressing the correspondingreceptor CCR5, accumulate (13). Expression of CCL5 has alsobeen predominantly localized to the tubulointerstitium evenin glomerular diseases (29). Unfortunately, we could not demonstrateendogenous CCL5 protein because we did not succeed in establishinga reliable immunohistochemistry protocol on archival, formalin-fixed,and paraffin-embedded tissue yet. Therefore, we could not directlycompare endogenous protein expression and CCL5 binding.
In a recent study on frozen sections of human renal allografts,the authors described binding of CCL5 to be restricted to thetubular basement membrane. The CCL5 binding was found to begenerally increased during rejection (36), but only focal bindingto tubular basement membrane was described in the rejectingallografts (36). This is in marked contrast to the results presentedhere, where use of well-preserved fixed renal tissue alloweddemonstration of binding of CCL5 to tubular epithelial cells,tubular basement membrane, and endothelial cells on some peritubularcapillaries.
It could be hypothesized that binding of CCL5 in the tubulointerstitiummight perpetuate the ongoing inflammatory response. Bindingof chemokines to tubular epithelial cells, tubular basementmembrane, and areas of extracellular matrix accumulation duringacute release might result in prolonged CCL5 gradients, attractingCCR5-positive cells to the tubulointerstitium.
Importantly, CCL5 binding within the glomerular tuft was foundto change in allografts during acute allograft glomerulitis.The change in binding of CCL5 was not a general phenomenon butspecifically presented within the glomerular tuft. The increasedbinding of CCL5 to glomerular structures seen is consistentwith the glomerular influx of CCR5-positive T cells that wasseen in this allograft pathology (13).
The binding of CCL5 to extracellular matrix structures in thetubulointerstitium, particularly at sites of inflammation andfibrosis, was generally more prominent than the binding thatcould be demonstrated to endothelial surfaces. The predominantinterstitial binding could be important for a rapid generationof chemokine surface gradients within tissues or for sequesteringleukocytes in interstitial areas where chemokines are generated.GAG and sulfatides both can act as chemokine binding molecules.The BBXB cluster of basic amino acids in the 44RKNR47 loop promotesCCL5 binding to GAG (7,10). It was previously demonstrated that[44AANA47]-CCL5 retains high-affinity binding to CCR5 but hasa reduced CCR1 binding (10). The [44AANA47]-CCL5 mutation isstill able to induce chemotaxis in Boyden chamber assays (10).We have found that the 50s loop may play an additional rolein the efficient binding of CCL5 to extracellular structuresto sulfatides, suggesting that chemokine binding in vivo mayinvolve a more complex biology than previously suggested (S.S.,manuscript in preparation). Here, both loops with clusters ofbasic residues had to be intact to promote detectable bindingto tissue sections. Mutation of the basic residues of eitherbasic loop abolished CCL5 binding to renal tissue sections.Efficient presentation of CCL5 could strengthen the interactionbetween leukocyte and endothelium, bringing the N-terminus ofthe chemokine in contact with the corresponding chemokine receptoron the leukocyte. An additional possible mode of action couldinvolve the "hand-off" of chemokine on the endothelial surfaceto the leukocyte. However, the demonstration that CCL5 bindingto tissue sections was lost by mutation of either site wouldbe more consistent with a model in which both sites interactto increase the binding strength of CCL5.
CCL5 binding structures were localized to tubular epithelialcells, basement membrane, and some endothelial cells under basalconditions. The tubulointerstitium was the predominant siteof CCL5 binding. Binding to glomerular structures was uncommonbut was found in renal biopsies with allograft glomerulitis(consistent with our hypothesis). Binding to tissue compartmentsmay be important for the in vivo function of CCL5. Differencesbetween renal compartments, as well as changes in binding structuresduring inflammation, may help to explain the differences thatwere seen in the composition of inflammatory cell subsets thatwere recruited to inflamed renal tissues. Chemokine presentationadds another level to the complexity of chemokine biology (37).In situ protein binding studies might help interpretation offindings using in situ hybridization or immunohistochemistry.
The work was supported by a grant of the Else Kröner-FreseniusStiftung (Bad Homburg an der Höhe, Germany), grants fromthe Deutsche Forschungsgemeinschaft (SE 888/4-1 to S.S.; SFB571 C2 and TR-SFB 36 B6 to P.J.N.), the EU (Network of Excellence"MAIN" FP6-502935 to S.S. and P.J.N.), and P6 "INNOCHEM" toP.J.N, H.J.G., and A.E.I.P.
We thank Dan Draganovici for technical support and MatthiasMack for providing the CCR5 antibody.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
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J. Am. Soc. Nephrol. 2007 18: 1617-1618.
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Abstract
Introduction
B (25). Immune effector cells such as T cells and monocyte/macrophages can be a rich source of CCL5 (26). Intrinsic renal cells, including mesangial cells, podocytes, tubular epithelial cells, and interstitial fibroblasts, also release CCL5 after stimulation in vitro (4). In previous studies, we showed that microvascular endothelial cells upregulate an unknown CCL5 presentation antigen in concert with intercellular adhesion molecule, vascular cellular adhesion molecule, and E-selectin after activation with proinflammatory cytokines (27). During acute allograft rejection, CCL5 was localized in frozen material to infiltrating cells (in glomeruli and the tubulointerstitium), tubular epithelial cells, and endothelial cells of peritubular capillaries (18,28). In diabetic nephropathy and in membranous nephropathy, CCL5 expression was found to be predominantly localized to the tubulointerstitium (29,30). A systematic evaluation of CCL5 on a large number of biopsies has not been performed to date. Cohen et al. (31) described CCL5 protein and mRNA expression in glomeruli from renal allografts. As was previously found (17,18), intracellular CCL5 protein was localized in frozen materials predominantly to infiltrating cells in the tubulointerstitium in renal allograft nephrectomies. Binding of CCL5 to extracellular matrix could not be localized sufficiently by immunohistochemistry alone, which is most likely a concentration problem. Therefore, consecutive binding studies in tissue add information. 
