Collagen XIII Induced in Vascular Endothelium Mediates α1β1 Integrin-Dependent Transmigration of Monocytes in Renal Fibrosis
2010; Elsevier BV; Volume: 177; Issue: 5 Linguagem: Inglês
10.2353/ajpath.2010.100017
ISSN1525-2191
AutoresJameel Dennis, Daniel T. Meehan, Duane Delimont, Marisa Zallocchi, Greg A. Perry, Stacie O'Brien, Hongmin Tu, Taina Pihlajaniemi, Dominic Cosgrove,
Tópico(s)Mast cells and histamine
ResumoAlport syndrome is a common hereditary basement membrane disorder caused by mutations in the collagen IV α3, α4, or α5 genes that results in progressive glomerular and interstitial renal disease. Interstitial monocytes that accumulate in the renal cortex from Alport mice are immunopositive for integrin α1β1, while only a small fraction of circulating monocytes are immunopositive for this integrin. We surmised that such a disparity might be due to the selective recruitment of α1β1-positive monocytes. In this study, we report the identification of collagen XIII as a ligand that facilitates this selective recruitment of α1β1 integrin-positive monocytes. Collagen XIII is absent in the vascular endothelium from normal renal cortex and abundant in Alport renal cortex. Neutralizing antibodies against the binding site in collagen XIII for α1β1 integrin selectively block VLA1-positive monocyte migration in transwell assays. Injection of these antibodies into Alport mice slows monocyte recruitment and protects against renal fibrosis. Thus, the induction of collagen XIII in endothelial cells of Alport kidneys mediates the selective recruitment of α1β1 integrin-positive monocytes and may potentially serve as a therapeutic target for inflammatory diseases in which lymphocyte/monocyte recruitment involves the interaction with α1β1 integrin. Alport syndrome is a common hereditary basement membrane disorder caused by mutations in the collagen IV α3, α4, or α5 genes that results in progressive glomerular and interstitial renal disease. Interstitial monocytes that accumulate in the renal cortex from Alport mice are immunopositive for integrin α1β1, while only a small fraction of circulating monocytes are immunopositive for this integrin. We surmised that such a disparity might be due to the selective recruitment of α1β1-positive monocytes. In this study, we report the identification of collagen XIII as a ligand that facilitates this selective recruitment of α1β1 integrin-positive monocytes. Collagen XIII is absent in the vascular endothelium from normal renal cortex and abundant in Alport renal cortex. Neutralizing antibodies against the binding site in collagen XIII for α1β1 integrin selectively block VLA1-positive monocyte migration in transwell assays. Injection of these antibodies into Alport mice slows monocyte recruitment and protects against renal fibrosis. Thus, the induction of collagen XIII in endothelial cells of Alport kidneys mediates the selective recruitment of α1β1 integrin-positive monocytes and may potentially serve as a therapeutic target for inflammatory diseases in which lymphocyte/monocyte recruitment involves the interaction with α1β1 integrin. Alport syndrome is a relatively common (1 in 5000) hereditary basement membrane disorder caused by mutations in the collagen IV α3, α4, or α5 genes.1Barker D Hostikka SL Zhou J Chow LT Oliphant AR Gerkin SC Gregory MC Skolnick MH Atkin CL Tryggvasson K Identification of mutations in the COL4A5 collagen gene in Alport syndrome.Science. 1990; 348: 1224-1227Crossref Scopus (656) Google Scholar, 2Lemmink HH Mochizuki T van den Heuvel LPWJ Schroder CH Barrientos A Monnens LAH van Oost BA Brunner HG Reeders ST Smeets HJM Mutations in the type IV collagen α3 (COL4A3) gene in autosomal recessive Alport syndrome.Hum Mol Gen. 1994; 3: 1269-1273Crossref PubMed Scopus (197) Google Scholar, 3Mochizuki T Lemmink HH Mariyama M Antignac C Gubler M-C Pirson Y Verellen-Dumoulin C Chan B Schroder CH Smeets HJ Reeders ST Identification of mutations in the α3(IV) and α4(IV) collagen genes in autosomal recessive Alport syndrome.Nat Genet. 1994; 8: 77-81Crossref PubMed Scopus (436) Google Scholar The disease manifests with progressive renal disease associated with hearing loss and retinal flecks. There are several models for Alport's Syndrome including a collagen IV α3 knockout mouse.4Cosgrove D Meehan DT Grunkemeyer JA Kornak JM Hunter WJ Samuelson GC Collagen COL4A3 knockout: a mouse model for autosomal Alport syndrome.Genes Dev. 1996; 10: 2981-2992Crossref PubMed Scopus (296) Google Scholar, 5Miner JH Sanes JR Molecular and functional defects in kidneys of mice lacking collagen α3(IV): implications for Alport syndrome.J Cell Biol. 1996; 135: 1403-1413Crossref PubMed Scopus (254) Google Scholar In the 129 Sv Alport mouse model, animals develop glomerular and interstitial fibrosis followed by end stage renal failure between 8 and 9 weeks of age. Increased extracellular matrix deposition, mesangial matrix expansion, impaired glomerular filtration, scarring and tubular atrophy observed in this model correlate with Alport's syndrome pathogenesis reported in humans. In this model two biochemical pathways are known to contribute to disease progression. The first pathway requires transforming growth factor-β, while the second is α1-integrin dependent.6Cosgrove D Rodgers K Meehan D Miller C Bovard K Gilroy A Gardner H Kotelianski V Gotwasl P Amatucci A Kalluri R Integrin alpha1beta1 and transforming growth factor-beta1 play distinct roles in Alport glomerular pathogenesis and serve as dual targets for metabolic therapy.Am J Pathol. 2000; 157: 1649-1659Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar Monocytes express transforming growth factor-β which facilitates myofibroblast accumulation and matrix deposition in Alport mice. Monocytes also express matrix metalloproteinases and associated proteins capable of degrading tubular basement membranes and promoting tubular epithelial cell death.7Rodgers KD Rao V Meehan DT Fager N Gotwals P Ryan ST Koteliansky V Nemori R Cosgrove DE Monocytes may promote myofibroblast accumulation and apoptosis in Alport renal fibrosis.Kidney Int. 2003; 63: 1338-1355Crossref PubMed Scopus (46) Google Scholar These findings suggest that monocytes are of principal importance in promoting scarring and tubular atrophy in chronic renal fibrosis. This connection has been corroborated in other models of renal fibrosis.8Kitagawa K Wada T Furuichi K Hashimoto H Ishiwata Y Asano M Takeya M Kuziel WA Matsushima K Mukaida N Yokoyama H Blockade of CCR2 ameliorates progressive fibrosis in kidney.Am J Pathol. 2004; 165: 237-246Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar, 9Chow F Ozols E Nikolic-Paterson DJ Atkins RC Tesch GH Macrophages in mouse type 2 diabetic nephropathy: correlation with diabetic state and progressive renal injury.Kidney Int. 2004; 65: 116-128Crossref PubMed Scopus (433) Google Scholar Thus the cellular mechanisms that facilitate transmigration and proliferation of interstitial monocytes are important factors in promoting the progression of interstitial disease. In an earlier report, we showed that nearly all of the monocytes in Alport kidneys express α1β1 integrin.10Sampson NS Ryan ST Enke DA Cosgrove D Koteliansky V Gotwals P Identification of a role for alpha1 integrin in renal pathogenesis using global analysis of gene expression.J Biol Chem. 2001; 276: 34182-34188Crossref PubMed Scopus (54) Google Scholar We also have shown that integrin α1-null Alport mice live nearly twice as long as Alport mice, an observation that correlates well with a marked reduction in interstitial monocyte accumulation.6Cosgrove D Rodgers K Meehan D Miller C Bovard K Gilroy A Gardner H Kotelianski V Gotwasl P Amatucci A Kalluri R Integrin alpha1beta1 and transforming growth factor-beta1 play distinct roles in Alport glomerular pathogenesis and serve as dual targets for metabolic therapy.Am J Pathol. 2000; 157: 1649-1659Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, 10Sampson NS Ryan ST Enke DA Cosgrove D Koteliansky V Gotwals P Identification of a role for alpha1 integrin in renal pathogenesis using global analysis of gene expression.J Biol Chem. 2001; 276: 34182-34188Crossref PubMed Scopus (54) Google Scholar Alpha1beta1 integrin (also known as VLA-1, or very late antigen 1) mediates collagen dependent cell proliferation and adhesion.11Gardner H Kreidberg J Koteliansky V Jaenisch R Deletion of integrin α1 by homologous recombination permits normal murine development but gives rise to a specific deficit in cell adhesion.Dev Biol. 1996; 175: 301-313Crossref PubMed Scopus (226) Google Scholar, 12Pozzi A Wary KK Giancotti FG Gardner HA Integrin α1β1 mediates a unique collagen-dependent proliferation pathway in vivo.J Cell Biol. 1998; 142: 587-594Crossref PubMed Scopus (247) Google Scholar However, a role for α1β1 integrin in transmigration of inflammatory cells across the microvascular barrier into the interstitial spaces has not been directly demonstrated. Monocyte and lymphocyte transmigration into the interstitial space is a principal event underlying both acute and chronic inflammatory response mechanisms.13Hogg N Henderson R Leitinger B McDowall A Porter J Stanley P Mechanisms contributing to the activity of integrins on leukocytes.Immunol Rev. 2002; 186: 164-171Crossref PubMed Scopus (144) Google Scholar Many aspects of the cellular events underlying the initiation and progression of monocyte efflux have been elaborated in recent years, as these pathways are central to pathobiology of many inflammatory diseases. The initiation of the inflammatory response involves cellular expression of chemokines and inflammatory cytokines, which have profound effects on adjacent cells. The vascular and capillary endothelial cells respond by up-regulating expression of selectins and intercellular adhesion molecules.14Springer TA Traffic signals on endothelium for lymphocyte recirculation and leukocyte emigration.Annu Rev Physiol. 1995; 57: 827-872Crossref PubMed Scopus (1387) Google Scholar, 15Jung U Norman KE Scharffetter-Kochanek K Beaudet AL Ley K Transit time of leukocytes rolling through venules controls cytokine-induced inflammatory cell recruitment in vivo.J Clin Invest. 1998; 102: 1526-1533Crossref PubMed Scopus (230) Google Scholar The selectins loosely adhere to lymphocytes and monocytes resulting in a “slow rolling” effect that can be visualized directly using intravital microscopy.16Jones TR Nozomu S Adams AB Pearson TC Larsen CP Intravital microscopy identifies selectins that regulate T cell traffic into allografts.J Clin Invest. 2003; 112: 1714-1723Crossref PubMed Scopus (21) Google Scholar Intercellular adhesion molecules and related inducible endothelial cell surface ligands provide the substrate for firm adhesion through interactions with the integrin family of heterodimeric receptors on the surface of the monocytes.13Hogg N Henderson R Leitinger B McDowall A Porter J Stanley P Mechanisms contributing to the activity of integrins on leukocytes.Immunol Rev. 2002; 186: 164-171Crossref PubMed Scopus (144) Google Scholar Firm adhesion results in monocyte activation, inducing the expression of proteins needed to degrade the capillary basal lamina, allowing invasion into the interstitial space.17Correale J Bassani Molinas ML Temporal variations of adhesion molecules and matrix metalloproteinases in the course of MS.J Neuroimmunol. 2003; 140: 198-209Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 18Matias-Roman S Galvez BG Genis L Yanez-Mo M de la Rosa G Sanchez-Mateos P Sanchez-Madrid F Arroya AG Membrane type 1-matrix metalloproteinase is involved in migration of human monocytes and is regulated through their interaction with fibronectin or endothelium.Blood. 2005; 105: 3956-3964Crossref PubMed Scopus (102) Google Scholar The activated monocyte produces additional chemokines and cytokines, which further accelerate monocyte recruitment and the progression of the inflammatory response. Research aimed at defining the specific cellular mechanisms underlying monocyte and lymphocyte recruitment has been prolific. The discovery of integrins, a vastly important family of cell surface receptors that mediate adhesion, cell migration and signal transduction, resulted from studies aiming to identify the adhesion receptors on peripheral blood monocytes and lymphocytes, as well as their cognate ligands on activated vascular endothelium.19Hynes RO Integrins: versatility, modulation, and signaling in cell adhesion.Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (9002) Google Scholar Monoclonal antibodies that block the interaction of these cells with endothelial cell surface receptors have emerged as potentially effective therapeutic approaches for treating chronic inflammatory diseases such as multiple scleroses and psoriasis. A large number of such agents are currently in various stages of preclinical and clinical trials.20Harlan JM Winn RK Leukocyte-endothelial interactions: clinical trials of anti-adhesion therapy.Crit Care Med. 2002; 30: S214-S219Crossref PubMed Scopus (166) Google Scholar, 21Szekanecz Z Koch AE Therapeutic inhibition of leukocyte recruitment in inflammatory diseases.Curr Opin Pharmacol. 2004; 19: 423-428Crossref Scopus (42) Google Scholar The α1-integrin heterodimerizes only with β1-integrin. The heterodimer is found in the plasma membrane of a variety of cell types, and is widely viewed as a collagen binding integrin, although binding to other matrix molecules has been demonstrated.22Dickeson SK Mathis NL Rahman M Bergelson JM Santoro SA Determinants of ligand binding specificity of the alpha(1)beta(1) and alpha(2)beta(1) integrins.J Biol Chem. 1999; 274: 32182-32191Crossref PubMed Scopus (72) Google Scholar, 23Jokinen J Dadu E Nykvist P Kapyla J White DJ Ivaska J Vehvilainen P Reunanen H Larjava H Hakkinen L Heino J Integrin-mediated cell adhesion to type I collagen fibrils.J Biol Chem. 2004; 279: 31956-31963Crossref PubMed Scopus (282) Google Scholar We used a monocyte-specific cell trafficking assay to determine whether selective transmigration of α1β1 integrin-positive monocytes contributes to the accumulation of these cells in the Alport mouse kidneys. Our results suggest that α1β1 integrin-positive monocytes are indeed selectively recruited to the interstitium and the rate of transendothelial migration increases over time. We used Phage display and biopanning strategies to identify the α1β1 integrin ligand involved in selective recruitment as collagen XIII (a membrane bound collagen). Collagen XIII mRNA and protein are induced in the vascular endothelium of Alport mice. Monoclonal antibodies raised against the binding site on collagen XIII for α1β1 integrin block monocyte adhesion to collagen XIII on embryonic fibroblasts, and when administered systemically to Alport mice, markedly decrease monocyte efflux into the tubulointerstitial space. Matrix accumulation and tubulointerstitial damage are also markedly reduced. Collectively, these data suggest that collagen XIII is an inducible endothelial cell ligand for α1β1 integrin on peripheral blood monocytes, and mediates monocyte adhesion and transmigration. Blocking collagen XIII may provide a novel therapeutic target for chronic inflammatory diseases where α1β1 integrin-positive interstitial monocytes (or T-cells) play a role. All mice were in the 129 Sv genetic background. Alport mice, integrin α1-null mice and double knock-out (DKO) mice have all been described previously.4Cosgrove D Meehan DT Grunkemeyer JA Kornak JM Hunter WJ Samuelson GC Collagen COL4A3 knockout: a mouse model for autosomal Alport syndrome.Genes Dev. 1996; 10: 2981-2992Crossref PubMed Scopus (296) Google Scholar, 7Rodgers KD Rao V Meehan DT Fager N Gotwals P Ryan ST Koteliansky V Nemori R Cosgrove DE Monocytes may promote myofibroblast accumulation and apoptosis in Alport renal fibrosis.Kidney Int. 2003; 63: 1338-1355Crossref PubMed Scopus (46) Google Scholar, 11Gardner H Kreidberg J Koteliansky V Jaenisch R Deletion of integrin α1 by homologous recombination permits normal murine development but gives rise to a specific deficit in cell adhesion.Dev Biol. 1996; 175: 301-313Crossref PubMed Scopus (226) Google Scholar All mice were used under a protocol approved by the Institutional Animal Care and Use Committee in accordance with the Guide for Care and Use of Laboratory Animals, and every effort was made to minimize pain and discomfort. Murine bone marrow was isolated by flushing the marrow cavities of the femur with Dulbecco's modified Eagle's medium (DMEM) supplemented with 2% fetal calf serum (FCS), and 1% pen/strep. Marrow cell clusters were passed through 1-ml syringe several times to dissociate cells. Cell suspension was washed 2× in Hanks' balanced salt solution (HBSS) (Gibco BRL). Pelleted cells were resuspended in ammonium chloride solution (20 mmol/L Tris, and 100 mmol/L NH4Cl, pH 7.2) and incubated for 5 minutes to remove red blood cells. Murine primary bone marrow derived monocytes were washed in DMEM with 2% FCS, then a final wash with HBSS. Cells were cultured in DMEM supplemented (Gibco, BRL) with 2% FCS. Endothelial cells were isolated from murine kidneys using a previously described method24Dong QG Bernasconi S Lostaglio S De Calmanovici RW Martin-Padura I Breviario F Garlanda C Ramponi S Mantovani A Vecchi A A general strategy for isolation of endothelial cells from murine tissues. Characterization of two endothelial cell lines from the murine lung and subcutaneous sponge implants.Arterioscler Thromb Vasc Biol. 1997; 17: 1599-1604Crossref PubMed Scopus (209) Google Scholar with some modifications. Four 10-week-old DKO mice were anesthetized and perfused with ice-cold PBS. Kidneys were harvested and immediately minced on ice in HBSS, then digested in 20 ml of 1 mg/ml collagenase A (Roche Diagnostics Corp., Indianapolis, IN) prepared in HBSS solution at 37°C for 45 minutes with gentle agitation. Digested material was filtered through a 70-μm nylon mesh and collected. Cells were collected by centrifugation and washed 2× in PBS. One ml of cell suspension was combined with 1 × 107 anti-CD31 coated metallic beads and mixed on a nutator for 30 minutes at 4°C. Rosetted cells were washed 4× in PBS with 0.1% bovine serum albumin using a metallic chamber. The metallic beads were liberated from the isolated endothelial cells with 0.5% trypsin (Gibco). Murine primary renal endothelial cells were cultured in DMEM-F12 supplemented with 50 μg/ml endothelial mitogen (Biomedical Technologies Inc., Stoughton, MA), 1% penicillin/streptomycin (Gibco, BRL), 20 mmol/L l-glutamine, 1U/ml heparin, and 20% FCS. The cells were immunopositive for CD-31 and formed microtubules in Matrigel after 72 hours in culture. Mouse embryonic fibroblasts were isolated from day 14 post copulation embryos. Skin was removed with forceps and treated with 0.25% trypsin/EDTA for 5 minutes at 37°C. Monoclonal antibodies were raised against the peptide sequence GEKGAEGSPGL. The peptide was conjugated to KLH and mouse monoclonal antibodies raised at the University of Nebraska monoclonal core facility. Polyclonal antibodies were raised against the custom synthesized peptide sequence (GELGAPGPGTVALAEQ) from the NC1 domain of collagen XIII and produced by Invitrogen, Inc. (Carlsbad CA). Antibodies were qualified by enzyme-linked immunosorbent assay and immunostaining of collagen XIII Chinese hamster ovary (CHO) cells versus wild type cells (see below). All Alexa-fluorochrome conjugated secondary antibodies were purchased from Molecular Probes (Seattle, WA). Peroxidase conjugated secondary antibodies were purchased from Sigma (St. Louis, MO). Monocyte/macrophage specific marker α-CD11b was purchased from Cedar Lane Laboratories (Hornby, Ontario). Endothelial marker α-CD31 was purchased from Abcam (Cambridge, UK) and anti-α1β1 integrin antibodies were generously provided by Biogen Corp (Cambridge, MA), and described earlier.25Mendrick DL Kelly DM duMont SS Sandstrom DJ Glomerular epithelial and mesangial cells differentially modulate the binding specificities of VLA-1 and VLA-2.Lab Invest. 1995; 72: 367-375PubMed Google Scholar Anti-CD68 antibodies were from AbD Serotec (Raleigh, NC). The qualification of the AB2 antibody specificity was demonstrated using CHO cells either Mock-transfected, or transfected to a construct encoding the human collagen XIII linked to the mCherry fluorescent marker protein. Cloning of collagen XIII-mCherry chimera is based on two constructs, pRSET-B-mCherry26Shaner NC Campbell RE Steinbach PA Giepmans BN Palmer AE Tsien RY Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. Red fluorescent protein.Nature Biotechnol. 2004; 22: 1524-1525Crossref Scopus (3482) Google Scholar and hCOLXIII/pcDNA3.1 (Hashimoto T. unpublished data). Human collagen XIII cDNA was amplified by PCR from a human brain cDNA library using primers: HindIII-COLXIII fwd: 5′-GGATAGAAGCTTTTGGCAGCGGCTGTCGC-3′. XhoI-COLXIII rev: 5′-AATACTCGAGGTACAAACACACACACAGGC-3′, and then inserted following standard procedures into the HindIII and XhoI sites of pcDNA3.1(+)/hygromycin expression vector (Invitrogen). hCOLXIII/pcDNA3.1 was digested by HindIII and XhoI to form a HindIII-COLXIII-XhoI (2kb) and a HindIII-vector-XhoI (5.6 kb). HindIII-COLXIII-XhoI (2kb) was further digested by MfeI to form a HindIII-COLXIII(1.8kb)-MfeI and a MfeI-COLXIII(0.2kb)-XhoI. Collagen XIII with a linker to mCherry was amplified from MfeI-COLXIII(0.2kb)-XhoI using primers: XIII-MfeI fwd: 5′-GTATTCCAGGACCAATTGGAGTTC-3′. XIII-mCherry rev: 5′-TCCTCGCCCTTGCTCACCATCTTGTTCCAGCAGCC-TTGGACT-3′. mCherry with a linker to collagen XIII was amplified from pRSET-B-mCherry using primers: XIII-mCherry fwd: 5′-AGTCCAAGGCTGCTGGAACAAGATGGTGAGCAAGGGCGAGGA-3′. mCherry-XhoI rev: 5′-TCTAGACTCGAGTTACTTGTACAGCTCGTCCAT-3′. The two PCR products from steps 2 and 3 were used as templates for overlapping PCR with primers XIII-MfeI fwd and mCherry-XhoI rev. The product is MfeI-COLXIII (0.2kb)-mCherry-XhoI. The fragment from step 4 together with HindIII-COLXIII (1.8kb)-MfeI was then ligated into HindIII-vector-XhoI (5.6 kb) using T4 ligase (Fermentas) to form a transfection plasmid COLXIII-mChery/pcDNA3.1. CHO-K1 cells (ATCC) was transfected using FuGene HD (Roche) reagents. The stable transfected clones were selected by 200 μg/ml hygromycin. Cells grown on cytology slides (VWR, Batavia, IL) were rinsed with PBS, fixed with −20°C acetone for 5 minutes and dried for 2 hours at 25°C. Slides were rehydrated with PBS for 5 minutes, permeabilized with 0.3% Triton X-100 (Sigma) in PBS for 10 minutes, followed by three successive PBS washes and then incubated with Image-iT FX signal enhancer (Invitrogen, Grand Island, NY) for 30 minutes, humidified at 25°C. The slides were rinsed with PBS and blocked with 2% FCS, 0.2% fish gelatin (Sigma) in PBS for 2 hours. The slides were incubated in blocking solution with 0.3 μg/ml mouse anti-collagen XIII antibody overnight, humidified at 4°C. After three PBS washes, slides were incubated in blocking solution with a 1:1500 dilution of Alexa Fluor 488 goat ∞ mouse IgM (Invitrogen) for 2 hours, humidified at 25°C. After three PBS washes, slides were cover-slipped with Vectashield mounting medium with 4,6-diamidino-2-phenylindole (Vector, Burlingame, CA) and confocal images captured under a Zeiss AxioPlan 2IF MOT microscope interfaced with a LSM510 META confocal imaging system. Final images were assembled using Adobe Photoshop and Illustrator software (Adobe Systems, San Jose, CA). Collagen XIII IP Western: 50 μg of total cellular or glomerular lysate and 5 μg of AB-2 monoclonal Ab were incubated overnight at 4°C. 20 μl of Protein L Sepharose (Pierce, Rockford, Ill) was added to each sample and rocked for 2 hours at 4°C. Samples were spun at 13,200 rpm at 4°C, supernatant removed and pellet washed with 1 ml of 1 mol/L NaCl in PBS. Pellets were spun and washed an additional five times in 1 ml of PBS. 25 μl of SDS reducing buffer was added to pellets, boiled 3 minutes and centrifuged. 20 μl of each samples was loaded on a 7.5% SDS-polyacrylamide electrophoresis gel and fractionated for 45 minutes at 200 volts. Transfer to polyvinylidene difluoride membrane (Bio Rad, Hercules, CA) was completed in 1 hour at 100 volts in 25 mmol/L Tris, 192 mmol/L glycine buffer. The membrane was blocked in 1% bovine serum albumin (BSA), PBS plus 0.1% Tween for 2 hours at room temperature. An anti-rabbit polyclonal Ab against Collagen XIII was used to probe the membrane at 1:20,000 in blocking buffer overnight at 4°C and subsequently washed three times with PBS plus 0.1% Tween for 15 minutes. The membrane was incubated with anti-rabbit IgG (Sigma) 1:50,000 in blocking buffer and washed as before. The membrane was developed using enhanced chemiluminescence solution (Pierce) and exposed to film. Controls included no lysate plus AB-2/beads and lysate plus IgM/beads. Fluorescent dextrans were prepared according to the methods described by Luby-Phelps27Luby-Phelps K Preparation of fluorescently labeled dextrans and ficolls.Methods Cell Biol. 1989; 29: 59-73Crossref PubMed Scopus (26) Google Scholar with some modifications. Briefly, 1 mg of Alexa 568 (Molecular Probes, Inc. Eugene, OR) was combined with 39 mg of dextran (mol. wt ∼144,000) in the presence of pyridine, dimethyl sulfoxide, and tin dilaurate (Sigma-Aldrich Co.). Labeled dextran (ADC568) was precipitated with 95% ethanol, dialyzed in glass-distilled water and lyophilized. The dried product was then stored in 500 μg aliquots at {minus]20°C in a desiccator, protected from light. The collagen α3 (IV) knockout (Alport) mice were described previously.4Cosgrove D Meehan DT Grunkemeyer JA Kornak JM Hunter WJ Samuelson GC Collagen COL4A3 knockout: a mouse model for autosomal Alport syndrome.Genes Dev. 1996; 10: 2981-2992Crossref PubMed Scopus (296) Google Scholar Development and characterization of integrin α1-null and collagen α3 (IV)-null DKO mice was also described previously.6Cosgrove D Rodgers K Meehan D Miller C Bovard K Gilroy A Gardner H Kotelianski V Gotwasl P Amatucci A Kalluri R Integrin alpha1beta1 and transforming growth factor-beta1 play distinct roles in Alport glomerular pathogenesis and serve as dual targets for metabolic therapy.Am J Pathol. 2000; 157: 1649-1659Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar Male wild type 129SV and 129SV/J mice along with Alport and DKO mice were tail vein injected with 50 μg of Alexa 568 labeled dextran reconstituted in 100 μl HBSS (pH 7.2). Animals were given a single injection at weekly intervals ranging from 4 to 12 weeks (WT), 5 to 8 weeks (Alport), and 8 to 12 weeks (DKO). The high molecular weight dextrans do not cross the vascular endothelium, but are phagocytized by circulating monocytes which become labeled. Under conditions of inflammation, the labeled cells will cross the endothelium into the interstitial spaces at the inflammatory site. Three days post injection animals were anesthetized with Avertin (0.55 mg/g body weight; i.p.) and perfused with ice cold PBS. Kidneys were removed, immersed in 30% ice-cold sucrose for 3 hours, embedded in Tissue Tek optimal cutting temperature mounting medium (Sakura Finetek USA, Inc., Torrance, CA) and stored at −80°C. Fresh frozen kidney sections (4 μm) were fixed in 2% paraformaldehyde. CD11b positive cells were labeled in three sections at least 100 μm apart then coverslipped with mounting medium (0.1g N-propyl-Galate, 5 ml PBS, 5 ml glycerol). Approximately ten pictures were taken of each section using an Olympus BH2-RFCA microscope. Images were recorded (number of red/green cells per 200× field) using a Spot RT digital camera and analyzed with Image Pro Plus software (Media Cybernetics, Inc. Silver Spring, MD). Endothelial cells were isolated from 7-week-old Alport kidneys as described above. Poly A+ RNA was isolated and cDNA was generated with HIND III random primers and methylated dNTPs provided in the Orient Express T7 Select Phage expression system (Novagen, Inc., Madison WI). EcoRI/HIND III linkers were ligated to the cDNA, followed by digestion with HINDIII and EcoRI restriction enzymes. The digested product was filtered through a size fractionation column (Novagen, Inc., Madison WI). cDNA larger than 300bp was collected then ligated to T7 select vector arms for preparation of the phage library using T7 select phage packaging extract (Novagen, Inc., Madison WI). The number of recombinants was determined by plaque assay using bacterial strain BLT5403 (Novagen, Inc., Madison WI). Immulon 2HB 96 well plates (Fisher Scientific) were coated with human α1β1 integrin purified from placenta (α1β1 integrin, Chemicon Intl., Temecula, CA) at 5 μg/ml in coating buffer (0.035 mol/L NaHCO3, 0.015 mol/L Na2CO3) overnight at 4°C. After coating with α1β1 integrin, the wells were washed 3× with 1× Tris-buffered saline, blocked with 5% nonfat milk Tris-buffered saline buffer then washed 5× with distilled water. Phage preps (titer ∼5.9 × 108/ml) were added to α1β1 integrin coated wells in 200 μl biopanning buffer (10 mmol/L Tris-HCl, pH 8.0, 0.15 mol/L NaCl, 0.1% Tween-20, 1 mmol/L MgCl2, 1 mmol/L CaCl2) and kept at room temperature for 45 minutes. Wells were washed 5× with biopanning buffer and bound phage were eluted with elution buffer (20 mmol/L Tris, neutral pH, 1.0% SDS) for 20 minutes. BLT5403 bacterial cells were then added to the coated wells to recover high affinity phage that may not have been collected in the eluate. 90% of the eluted phage were combined with 50 ml bacterial cell culture at OD600 = 0.5 and amplified for 3 hours at 37°C with shaking. The remaining 10% was used to determine the number of phage recovered from each round of biopanning. Amplified phage from each round of biopanning was titered by plaque assay. The biopanning procedure was repeated 3× with 1 × 108 phage/α1β1 integrin-coated well for a total of four rounds of biopanning. Amplified phage collected after the fourth round of biopanning were diluted and individual plaques collected for sequence analysis. One ml phage extraction buffer (20 mmol/L Tris-HCl, pH 8.0, 100 mmol/L NaCl, 6 mmol/L MgSO4) was added to each plug and stored at 4°C. Plaques were dispersed in 100 μl o
Referência(s)