Subendothelial Heparan Sulfate Proteoglycans Become Major L-Selectin and Monocyte Chemoattractant Protein- Ligands upon Renal Ischemia/Reperfusion

【摘要】  Leukocyte infiltration into inflamed tissues is considered to involve sequential steps of rolling over the endothelium, adhesion, and transmigration. In this model, the leukocyte adhesion molecule L-selectin and its ligands expressed on inflamed endothelial cells are involved in leukocyte rolling. We show that upon experimental and human renal ischemia/reperfusion, associated with severe endothelial damage, microvascular basement membrane (BM) heparan sulfate proteoglycans (HSPGs) are modified to bind L-selectin and monocyte chemoattractant protein-1. In an in vitro rolling and adhesion assay, L-selectin-binding HSPGs in artificial BM induced monocytic cell adhesion under reduced flow. We examined the in vivo relevance of BM HSPGs in renal ischemia/reperfusion using mice mutated for BM HSPGs perlecan (Hspg23/3), collagen type XVIII (Col18a1C/C), or both (cross-bred Hspg23/3xCol18a1C/C) and found that early monocyte/macrophage influx was impaired in Hspg23/3xCol18a1C/C mice. Finally, we confirmed our observations in human renal allograft biopsies, showing that loss of endothelial expression of the extracellular endosulfatase HSulf-1 may be a likely mechanism underlying the induction of L-selectin- and monocyte chemoattractant protein-1-binding HSPGs associated with peritubular capillaries in human renal allograft rejection. Our results provide evidence for the concept that not only endothelial but also (microvascular) BM HSPGs can influence inflammatory responses.
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Upon tissue damage, leukocytes are recruited to the site of inflammation to exert their functions. This process is generally considered to involve an initial phase of tethering of leukocytes to activated endothelium, mediated by leukocyte-expressed L-selectin and endothelium-expressed E- and P-selectin and their ligands, followed by activation and firm adhesion, mediated by chemokines and integrins, respectively.1,2 Finally, leukocytes transmigrate through the endothelium and vascular basement membrane (BM) to the site of inflammation.
Although this multistep model of leukocyte extravasation is well established, there are some clinically important situations in which additional factors may come into play. For example, it is well known that the endothelium of the heart and kidney is severely damaged as a result of prolonged ischemia and subsequent reperfusion (I/R),3-8 which is particularly important in transplantation settings. Upon renal I/R, endothelial cell swelling, loss of endothelial cell-cell attachment, and even complete loss of endothelial lining has been reported in peritubular capillaries as early as 24 hours after reperfusion.4,5 In this environment, leukocytes encounter a damaged endothelial layer with exposed vascular BM, over which they will transmigrate.
Vascular BM is composed of various matrix components including laminins, collagens, and proteoglycans.9,10 Proteoglycans are glycoconjugates consisting of extended linear carbohydrate side chains (glycosaminoglycans; GAGs) linked to a protein core.11 A specific subset of proteoglycans, heparan sulfate proteoglycans (HSPGs), is especially well known for its ability to bind a number of proteins important for the inflammatory response, including the leukocyte-expressed adhesion molecule L-selectin, and various chemokines.12 Binding of these proteins to HSPGs is dependent on the presence of specific binding domains on the HS GAG chain.13,14 We and others have previously shown that for L-selectin binding to HSPGs a number of determinants, including 6-O-sulfation of the GAG chain, are crucial.15,16 Because the presence or absence of these domains on HS GAG chains determines whether HSPGs will bind molecules like L-selectin and chemokines, active regulation of these domains, eg, during biosynthesis and/or by later modification, could clearly affect the inflammatory response. Interestingly, extracellular endosulfatases that specifically cleave 6-O-linked sulfate residues of HSPGs have been identified in humans (HSulf-1 and HSulf-2),17 and although a change in expression pattern of these enzymes has not been reported in inflammatory settings to date, differential expression of HSulf-1 has been described in carcinomas.18,19
In the normal kidney, HSPGs are abundantly present in glomeruli, tubular BM (TBM), microvascular BM, vasa recta bundles, papilla, and on epithelial cells. However, only a subset of HSPGs, present in medullary TBMs, vasa recta bundles, and papilla, are able to bind L-selectin under normal conditions.15 In this study, we examine the expression and relevance of HSPGs that bind L-selectin and chemokine upon renal I/R, a model used to study the early inflammatory processes involved in kidney transplantation. Because we are interested in HSPG alterations that can have functional consequences for the inflammatory response, we chose to directly detect binding of L-selectin and monocyte chemoattractant protein-1 (MCP-1) to HSPGs in tissue sections,20 rather than using antibodies directed against structurally well defined, but not necessarily functional, HS epitopes. We show that upon renal I/R, microvascular BM HSPGs in the renal interstitium are modified to bind L-selectin and MCP-1. Using an in vitro model for leukocyte rolling and adhesion, we provide evidence for a functional role of L-selectin-binding HSPGs in monocytic cell adhesion, and we show that in vivo monocyte influx is impaired in kidneys of mice that lack functional BM HSPGs perlecan and collagen type XVIII. Finally, we extrapolate our findings to human renal transplant biopsies and show that down-regulation of endothelial-expressed HSulf-1 could be a mechanism for the observed HSPG alterations.

【关键词】  subendothelial proteoglycans l-selectin monocyte chemoattractant protein- ischemia/reperfusion

Materials and Methods

Animals and Renal I/R

Adult male Wistar rats (300 to 350 g) were obtained from Harlan CPB (Zeist, The Netherlands). Adult male wild-type mice, Hspg23/3 mutant mice,21 Col18a1C/C mice,22 and crossbred Hspg23/3xCol18a1C/C mutant mice21 were all on C57BL/6 background and ranging from 10 to 18 weeks old. Unilateral renal warm I/R was performed by clamping left renal pedicle (mice) or renal artery (rats) of isoflurane-anesthetized animals for 45 minutes while keeping the abdominal cavity moist with saline, after which the clamp was released, reperfusion of the kidney was visually checked, and wounds were closed. During and after the operation, animals were kept warm until they regained consciousness, after which they had free access to food and water. Animals were sacrificed at t = 24 hours or t = 48 hours after reperfusion; both contralateral and I/R kidneys were removed and either snap-frozen or formalin-fixed and paraffin-embedded according to routine histology protocol. Animal housing and experiments were approved by local animal experimentation ethics committees.

Proteins, Enzymes, and Antibodies

L-selectin-IgM chimeric protein, consisting of the extracellular domain of human L-selectin linked to an IgM Fc-tail, was produced as described.20,23 Heparitinase I from Flavobacterium heparinum (EC4.2.2.8) was from Seikagaku Corp., Tokyo, Japan. Anti-rat CD31, anti-rat CD68 (ED1), and anti-mouse F4/80 were from Serotec, Oxford, UK. Anti-rat perlecan (10B2) was kindly provided by Dr. Couchman, Division of Biomedical Sciences, Imperial College, London, UK. Anti-rat agrin (GR14) and anti-human MCP-1 (5D3-F7) were previously described.24,25 Recombinant human MCP-1 was from Peprotech, London, UK. Anti-collagen XVIII NC11 was kindly provided by Dr. T. Sasaki, Max-Planck-Institut f?r Biochemie, Martinsried, Germany. DREG-56, MECA-79, and HECA-452 were from BD PharMingen (Erembodegem, Belgium); and anti-human CD31, CD34, and von Willebrand factor were from DAKO (Heverlee, Belgium). Alexa Fluor-labeled anti-human IgM, anti-mouse IgG, anti-rabbit IgG, anti-sheep IgG, anti-rat IgG, and streptavidin were from Molecular Probes (Invitrogen, Breda, The Netherlands) and biotinylated anti-rat IgG + IgM from Jackson ImmunoResearch (Cambridgeshire, UK).

Immunofluorescence

In situ L-selectin binding was performed as described on either formalin-fixed cryostat tissue sections or formalin-fixed, paraffin-embedded tissue sections.20 MCP-1 binding was performed accordingly on formalin-fixed, paraffin-embedded sections, incubating MCP-1 (2.5 µg/ml) overnight at 4??C. Specific digestion of HS GAG chains was performed by preincubation of tissue sections with 0.05 U/ml heparitinase I in acetate buffer (50 mmol/L C2H3O2Na, 5 mmol/L CaCl2?

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作者单位：From the Departments of Molecular Cell Biology and Immunology* and Hematology, Vrije Universiteit University Medical Center, Amsterdam, The Netherlands; the Department of Pathology, Academic Medical Center, Amsterdam, The Netherlands; and the Department of Medical Biochemistry and Molecular Biology,