Activation of integrin receptors is required for growth factor-induced smooth muscle cell dysfunction

PURPOSE: Growth factors and cytokines such as platelet-derived growth factor (PDGF), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and transforming growth factor beta (TGF-beta) stimulate smooth muscle cell (SMC) proliferation and extracellular matrix (ECM) protein production by binding and activating their respective receptors. Recent investigations suggest that simultaneous activation of integrins, which are heterodimeric receptors for ECM, may also be required for growth factor and cytokine function. In this study, we tested the hypothesis that activation of two integrins, alpha v beta 3 and alpha 2 beta 1, both previously identified in vascular SMCs, is necessary for growth factor- and cytokine-induced vascular SMC dysfunction. METHODS: DNA synthesis was measured after stimulation of SMCs derived from human saphenous vein with the growth factors PDGF-BB, EGF, and bFGF. SMC fibronectin (Fn) production was measured (by means of Western blotting) in SMCs stimulated for 72 hours with TGF-beta1 or EGF. Both endpoints were measured in the presence and absence of antibodies that block the function of the alpha v beta 3 and alpha 2 beta 1 integrins as well as the alpha2 and beta1 subunits. RESULTS: The alpha v beta 3 integrin blocking antibody significantly inhibited PDGF-BB-, EGF-, and bFGF-induced SMC proliferation. The alpha v beta 3 integrin antibody also markedly inhibited TGF-1- and EGF-induced SMC Fn production. Neither the alpha 2 beta 1 integrin nor the alpha2 or the beta1 subunits inhibited either proliferation or matrix protein production in response to any of these agonists. CONCLUSION: The alpha v beta 3 integrin is required for growth factor- and cytokine-induced SMC proliferation and FN production, whereas alpha 2 beta 1 is not. Since activation of alpha v beta 3 is required for the activity of at least four distinct growth factors and cytokines, inhibition of this integrin might be used as a therapeutic tool for the prevention of intimal hyperplasia.     

Enhancement of fibronectin synthesis and fibrillogenesis by BMP-4 in cultured rat osteoblast.

The skeletal extracellular matrix produced by osteoblasts contains the glycoprotein fibronectin (Fn), which regulates the adhesion, differentiation, and function of osteoblasts. Fn fibrillogenesis is involved in the process of bone mineralization. Bone morphogenetic proteins (BMPs) can be isolated from organic bone matrix and are able to initiate de novo cartilage and bone formation. In this study, the effect of BMP-4 on Fn fibrillogenesis in cultured rat osteoblasts was examined. BMP-4 enhanced Fn synthesis and extracellular Fn assembly in primary cultured osteoblasts. In addition, the extracellular assembly of Fn from exogenously applied soluble human Fn was also increased by BMP-4. It has been reported that alpha5beta1 integrin is related to Fn fibrillogenesis. The synthesis of both alpha5 and beta1 integrins was upregulated by BMP-4. Immunocytochemistry showed that the clustering of alpha5 and beta1 integrins was also increased by BMP-4. BMP-4 increased fibril formation of Fn and the adhesion of osteoblasts onto Fn matrix, which was inhibited by disintegrin triflavin and Gly-Arg-Gly-Asp-Ser (GRGDS) peptide. Phosphorylation of extracellular signal-regulated kinase (ERK) and focal adhesion kinase (FAK) was increased by BMP-4. Enhancement of extracellular Fn fibrillogenesis and the mRNA expression of beta1 integrin by BMP-4 were inhibited by ERK kinase (MEK) inhibitor PD98059. These results suggest that the enhancement of extracellular Fn fibrillogenesis by BMP-4 in cultured osteoblasts is related to the increase of the synthesis of Fn and clustering of alpha5 and beta1 integrins. ERK is involved in the signaling pathway of BMP-4 in regulating Fn fibrillogenesis in osteoblasts.     

DNA microarray-based gene expression profiles of cytomegalovirus infection and acute rejection in liver transplants

An association between cytomegalovirus (CMV) infection and alloresponse has been suggested. CMV increases inflammation and adhesion molecule expression in graft, and induces cytokines and growth factors, linked with transplant vasculopathy and chronic rejection. We have investigated the gene expression of various inflammatory factors in the CMV-associated immune response and compared this with the immune response of acute rejection in liver transplants by using DNA microarray technology. Gene expression was studied at mRNA level in biopsies from liver transplant patients experiencing CMV infection or acute rejection. RNA extracted from liver grafts after reperfusion was used as control material. Among the strongly upregulated genes in the specimens obtained from liver transplants during CMV infection were IFN-gamma, caspases 1 and 3, granzymes A and B, TGF-beta receptors II and III, IL-10 receptor alpha, VCAM-1, TNF receptor, IL-4, TNF-alpha, IL-10, IL-2 receptor beta, IL-1beta, PDGF-receptor beta, vascular adhesion protein-1, TGF-beta2, and ICAM-1. In biopsies with acute liver allograft rejection, the most significantly upregulated genes were MHC class II, IFN-gamma, caspases 1 and 3, IL-2R beta and gamma, granzymes A and B, VLA-4, L-selectin, E-selectin, VCAM-1, and IL-1beta. Upregulated genes common for CMV and alloresponse were granzyme A and B, E-selection, IFN-gamma, VCAM-1, VLA-4, TNF, caspases 1, 3, and 8, and PDGF. Microarray analysis defined different entities in the immune responses of CMV infection and acute rejection. The differences and similarities of the gene expression profiles related to those in CMV infection and rejection may help to understand the intragraft immunologic events.     

Porcine mononuclear cells adhere to human fibronectin independently of very late antigen-5: implications for donor-specific tolerance induction in xenotransplantation

To combat the shortage of donor organs, transplantation across species barriers has been proposed. Induction of tolerance would overcome the substantial immunologic barriers to xenotransplantation and would avoid the chronic use of immunosuppressive agents. Successful transplantation of hematopoietic cells induces robust specific tolerance to donor antigens in allogeneic and xenogeneic models. The beta1 integrin class of adhesion molecules and their interactions with extracellular matrix components are thought to be integral to the engraftment and maturation of hematopoietic stem cells. We therefore examined the efficacy of porcine very late antigen-5 (VLA-5) and VLA-4 interactions with the human extracellular matrix (ECM) protein, fibronectin. Peripheral blood mononuclear cells (PBMCs) from humans and miniature swine were flourochrome labeled and adhesion to plates coated with whole human fibronectin (whFN) or its 120 KDa fragment containing the VLA-5 binding region was determined. Flow cytometry and immuno- precipitation were used to identify a monoclonal antibody that cross-reacted on porcine VLA-5. Human and pig PBMC adhesion to human fibronectin (hFN) or 120 kDa fragment-coated plates was assessed following incubation with control ab, anti-VLA-4, anti-VLA-5, or soluble fibronectin. Using rabbit complement, cells expressing VLA-5 were purged from PBMC preparations before performing the adhesion assay. Porcine and human PBMC both adhered to hFN in a divalent cation-dependent and activation-dependent manner. Adhesion to hFN of human but not pig PBMC was blocked by anti-VLA-5 monoclonal antibody SAM-1, although this mAb immunoprecipitated a heterodimeric cell surface molecule (155/135 kDa) resembling VLA-5 from pig PBMC. Complement-mediated depletion of VLA-5-expressing cells ablated specific binding of human but not porcine cells to hFN and its 120 kDa fragment. Addition of soluble fibronectin was capable of blocking adhesion of PBMC of both species to hFN. Anti-VLA-4 reduced the binding of PBMC from both species to hFN to a similar extent. Human and pig cells can specifically adhere to hFN and its 120 kDa fragment, suggesting that this critical cell-ECM interaction is preserved across species. While human cells exclusively use VLA-5 for binding to the 120 kDa fragment, porcine cells could not be shown to adhere to whFN or its 120 kDa fragment via VLA-5. However, porcine VLA-4 is capable of mediating adhesion to human FN. We conclude that disparities in the adhesive interactions of beta1 integrins may be a barrier to the use of porcine hematopoietic stem cell transplantation as a means of inducing donor-specific tolerance in the pig to human species combination.     

Contribution of interleukin 17 to human cartilage degradation and synovial inflammation in osteoarthritis

OBJECTIVE: To compare the effect of interleukin (IL)-17, IL-1beta and TNF-alpha on chemokine production by human chondrocytes and synovial fibroblasts isolated from patients with osteoarthritis (OA). The expression of IL-1beta mRNA by OA chondrocytes was also assessed, as well as the presence and expression of IL-17 receptor (IL-17R) in OA chondrocytes and synovial fibroblasts after stimulation with IL-17, IL-1beta and TNF-alpha. DESIGN: Synovial fibroblasts and chondrocytes isolated from patients with OA were stimulated in vitro with IL-17, IL-1beta or TNF-alpha. Supernatants were collected and immunoassayed for the presence of IL-8, GRO-alpha (CXC chemokines) and MCP-1, RANTES (CC chemokines). The cells were used to detect the presence of IL-17R and the expression of IL-17R mRNA. Stimulated chondrocytes were also used to detect IL-1beta production and mRNA expression. RESULTS: IL-17 upregulated the release of IL-8 and GRO-alpha both by synovial fibroblasts and chondrocytes, and the release of MCP-1 only by chondrocytes. IL-17 was a weaker stimulator than IL-1beta and TNF-alpha, except for GRO-alpha release which was maximally upregulated by IL-1beta, less by IL-17 and minimally by TNF-alpha. When compared to IL-1beta, IL-17 was more active on chondrocytes than on fibroblasts. In chondrocytes the expression of IL-1beta mRNA was enhanced by IL-17 and TNF-alpha, with a maximum level reached by IL-1beta. IL-17 and TNF-alpha stimulated IL-1beta release in few subjects. Neither IL-17, IL-1beta nor TNF-alpha modulated the presence of IL-17R and the expression of IL-17R mRNA. CONCLUSIONS: These data suggest that IL-17 could contribute to cartilage breakdown and synovial infiltration in OA by inducing both the release of chemokines by chondrocytes and synovial fibroblasts and, in a less extent, the synthesis of IL-1beta by chondrocytes.     

Integrin-mediated laminin-1 adhesion upregulates CXCR4 and IL-8 expression in pancreatic cancer cells

BACKGROUND: We have shown recently that alpha(2)beta(1) integrin-mediated type I collagen adhesion promotes a more malignant phenotype in pancreatic cancer cell lines than other extracellular matrix (ECM) proteins. MiaPaCa-2 cells, by contrast, do not express collagen-binding integrins, but are metastatic in our orthotopic mouse model and migrate maximally on laminin-1 (Ln-1). It has also been shown that CXCR4 and IL-8 expression correlates directly with metastasis in pancreatic cancer in vivo. We therefore examined the potential of the ECM to regulate CXCR4 and IL-8 expression in pancreatic cancer cells. METHODS: We cultured 8 pancreatic cancer cell lines on fibronectin (Fn), types I and IV collagen, Ln-1 and vitronectin (Vn), and examined cell lysates for CXCR4 by immunoblotting and media for IL-8 by ELISA. We also conducted cell migration assays with stromal-derived factor-1 (SDF-1) as the chemoattractant to examine integrin-binding specificity and CXCR4 function. RESULTS: All cell lines expressed CXCR4 protein. MiaPaCa-2 cell growth on Ln-1 increased significantly CXCR4 and IL-8 expression relative to other ECM proteins. Migration inhibition studies showed that both the alpha(6)beta(1) and alpha(3)beta(1) integrins mediate MiaPaCa-2 migration on Ln-1. Growth studies showed further that CXCR4 expression on Ln-1 was mediated by the alpha(6)beta(1) integrin whereas IL-8 expression was mediated by both the alpha(6)beta(1) and alpha(3)beta(1) integrins. The expression of functional CXCR4 was also shown in migration assays, where SDF-1 significantly increased pancreatic cancer cell chemotaxis on Ln-1. CONCLUSIONS: These data indicate that integrin-mediated Ln-1 adhesion upregulates CXCR4 and IL-8 expression and may play a mechanistic role in pancreatic cancer metastases.     

Interleukin-17: A new bone acting cytokine in vitro.

Interleukin-17 (IL-17) is a recently cloned cytokine that is exclusively produced by activated T cells, but its receptor has been found on several cells and tissues. Like other proinflammatory cytokines produced by activated T cells, IL-17 may affect osteoclastic resorption and thereby mediate bone destruction accompanying some inflammatory diseases. In the present study, we investigated whether osteogenic cells possess the receptor for IL-17 (IL-17R) and whether IL-17 affects osteoclastic resorption. We found that IL-17R mRNA is expressed both in mouse MC3T3-E1 osteoblastic cells and fetal mouse long bones, suggesting that osteogenic cells may be responsive to IL-17. In fetal mouse long bones, IL-17 had no effect on basal and IL-1beta-stimulated osteoclastic bone resorption, but when given together with tumor necrosis factor-alpha (TNF-alpha) it increased bone resorption dose dependently in serum-free conditions. In addition, IL-17 increased TNF-alpha-induced IL-1alpha, IL-1beta, and IL-6 mRNA expression in fetal mouse metatarsals and IL-1alpha and IL-6 mRNA expression in MC3T3-E1 cells. In conclusion, IL-17R mRNA was expressed by mouse osteoblastic cells and fetal mouse long bones, and IL-17 in combination with TNF-alpha, but not IL-1beta, increased osteoclastic resorption in vitro. IL-17 may therefore affect bone metabolism in pathological conditions characterized by the presence of activated T cells and TNF-alpha production such as rheumatoid arthritis and loosening of bone implants.     

LPS-stimulated PMN activation and proinflammatory mediator synthesis is downregulated by phosphodiesterase inhibition: role of pentoxifylline

BACKGROUND: Excessive production of reactive oxygen species by PMN is associated with tissue damage during inflammation. LPS interacts with the cell surface receptor CD14, which generates transmembrane signals through Toll-like protein 4 leading to mitogen activated protein kinase (MAPK) p38 activation, cytokine synthesis, PMN beta2-integrin expression and oxidative burst. Phosphodiesterase inhibition decreases proinflammatory cytokine production and tissue injury after LPS challenge. Its effects on PMN function after LPS stimulation, however, have not been fully investigated. We hypothesized that LPS-induced TNF-alpha synthesis and subsequent PMN beta2-integrin expression and oxidative burst are downregulated by concomitant treatment with the non-specific phosphodiesterase inhibitor pentoxifylline (PTX). METHODS: Whole blood was incubated with HBSS (control), LPS (100 microg/mL), fMLP (1 micromol/L), LPS+PTX (2 mmol/L) and fMLP+PTX for different time intervals at 37C. Oxidative burst, CD14, and CD-11b expression were measured by flow cytometry. Serum TNF-alpha levels were measured by ELISA. In an attempt to localize the site of action of PTX (proximal or distal to PKC) cell surface receptors were bypassed by PMA stimulation (1 microg/mL) and oxidative burst was measured with and without PTX. RESULTS: Up-regulation of CD14 expression was similar in LPS and LPS+PTX groups. LPS stimulation caused a significant increase in PMN oxidative burst, CD11b expression, and TNF-alpha serum levels. In addition, PMA and fMLP stimulation also caused significant increase in oxidative burst compared with controls. Concomitant addition of PTX to LPS led to a significant decrease in PMN oxidative burst (65%; p < 0.0001), PMN CD11b expression (20%; p = 0.012), and TNF-alpha levels (93%; p < 0.0001). Also, PMA- and fMLP-induced PMN oxidative burst were significantly decreased by PTX [77.5% (p < 0.0001) and 50% (p < 0.01), respectively]. CONCLUSIONS: These results suggest that PTX-inhibition of oxidative burst occurs distal to PKC and may be either due to direct inhibition of NADPH oxidase or inhibition of MAPK phosphorylation, leading to decreased adhesion molecule expression and TNF-alpha synthesis. Its use in clinical scenarios in which PMN are primed may be of clinical relevance.     

Functional antibodies to leukocyte adhesion molecules in antithymocyte globulins

BACKGROUND: Polyclonal antithymocyte globulins (ATG) induce T-cell depletion and functional impairment of nondeleted lymphocytes. Interference of ATG with the main leukocyte surface molecules involved in cellular adhesion and leukocyte-endothelium interaction was investigated in the present study. METHODS: In three rabbit ATG, the authors measured antibodies to integrins, beta2-integrin ligands, and chemokine receptors by flow cytometry; chemotactic responses; and down-modulation of cell surface expression on lymphocytes, monocytes, and neutrophils. RESULTS: Antibodies to CD11a/CD18 (leukocyte function-associated antigen-1 [LFA-1]) present in ATG induced a dose-dependent down-modulation of cell surface expression of this beta2 integrin on lymphocytes, monocytes, and neutrophils. In contrast, anti-LFA-1 monoclonal antibodies did not induce LFA-1 modulation unless cross-linked by a second antibody. ATG also contained functional antibodies to the beta1 integrin CD49d/CD29 (VLA-4), the alpha4beta7 integrin, CD50, CD54, and CD102 but not to CD62L. ATG were shown to bind to CXCR4 and CCR7 on lymphocytes, CXCR4, and CCR5 on monocytes; to down-modulate cell surface expression of CCR7; and to decrease monocyte chemotactic response to CCL5 (RANTES) and lymphocyte chemotactic response to CCL19 (MIP-3beta). CONCLUSION: These results show that ATG may interfere with leukocyte responses to chemotactic signals but mostly inhibit the expression of integrins required for firm cellular adhesion. The latter property of inhibition is not shared by monoclonal antibodies, and it may contribute to decreasing graft cellular infiltration during acute rejection and possibly after postischemic reperfusion.     

Role for beta1 integrin and its associated alpha3, alpha5, and alpha6 subunits in development of the human fetal pancreas

The integrin receptors play a major role in tissue morphogenesis and homeostasis by regulating cell interactions with extracellular matrix proteins. We have examined the expression pattern of integrin subunits in the human fetal pancreas (8-20 weeks fetal age) and the relevance of beta1 integrin function for insulin gene expression and islet cell survival. Its subunits alpha3, alpha5, and alpha6 beta1 integrins are expressed in ductal cells at 8 weeks, before glucagon- and insulin-immunoreactive cells bud off; their levels gradually increase in both ductal cells and islet clusters up to 20 weeks. Colocalization of alpha3, alpha5 and alpha6 beta1 integrins with endocrine cell markers was frequently observed in 8- to 20-week fetal pancreatic cells. When the beta1 integrin receptor was functionally blocked in cultured islet-epithelial clusters with a beta1 immunoneutralizing antibody or following transient beta1 integrin small interfering RNA treatment, there was inhibition of cell adhesion to extracellular matrices, decreased expression of insulin, and increased cell apoptosis. These data offer evidence for dynamic and cell-specific changes in integrin expression during human pancreatic islet neogenesis. They also provide an initial insight into a molecular basis for cell-matrix interactions during islet development and suggest that beta1 integrin plays a vital role in regulating islet cell adhesion, gene expression, and survival.     

Monocyte adhesion to mesangial matrix modulates cytokine and metalloproteinase production

BACKGROUND: Monocytes migrate into the glomerular mesangium during acute inflammatory renal disease, differentiate into macrophages, and may play a key role in the development and progression of glomerular scarring. Treatment strategies that inhibit monocyte infiltration ameliorate glomerular injury in animal models. Mesangial matrix contains several potential monocyte-binding domains that may contribute to monocyte entrapment and modulate cell activation. METHODS: Adhesion of peripheral blood-derived monocytes to matrix synthesized by human mesangial cells and to individual matrix proteins was assessed by colorimetry of nuclear staining with crystal violet. Monoclonal antibodies were used to identify the cell-surface integrins and matrix ligands involved. Monocyte proliferation was assessed by 3H-thymidine incorporation and cytokine production using enzyme-linked immunosorbent assay (ELISA). Secretion of metalloproteinases and their inhibitors was determined by zymography and ELISA, respectively. RESULTS: Monocytes bound to matrix synthesized by mesangial cells. Prestimulation of mesangial cells with tumor necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta (TGF-beta) enhanced matrix fibronectin content (P < 0.001) and monocyte binding (P < 0.001). Blocking antibodies to fibronectin, as well as to the integrins very late antigen-4 (VLA-4) and VLA-5, reduced monocyte adhesion to mesangial matrix by approximately 50%. Incubation of monocytes with matrix, fibronectin, laminin and collagen IV enhanced production of interleukin-1beta (IL-1beta), interleukin-6 (IL-6), TNF-alpha and metalloproteinase-9 (MMP-9) when compared to cells incubated in plastic wells. However, there was no apparent difference in proliferation rate and no change in production of metalloproteinase inhibitors. CONCLUSION: Monocyte activation within the glomerulus may be mediated by binding to mesangial matrix components, particularly fibronectin. Matrix-mediated activation enhances production of inflammatory cytokines and matrix-degrading enzymes.     

Polymorphonuclear leukocyte rigidity is defective in patients with chronic renal failure.

BACKGROUND: The purpose of the study was to investigate the rigidity of polymorphonuclear leukocytes (PMNs) in non-dialysed chronic renal failure (CRF) and haemodialysis (HD) patients. METHODS: PMN rigidity as well as tumour necrosis factor alpha (TNF-alpha) and interleukin 1beta (IL-1beta) plasma levels were assessed in 10 early-stage CRF, 10 late-stage non-HD, and 10 HD patients, before and during dialysis. In HD patients both cellulose acetate and polysulphone membranes were used. Ten healthy subjects served as controls. Rigidity was tested by counting the deformability in morphologically passive PMNs by the micropipette method. Cytokine levels were measured by enzyme-linked immunosorbent assay. RESULTS: PMN rigidity was significantly increased in end-stage CRF patients regardless of HD but not in early-stage CRF. In HD patients PMN rigidity increased significantly 60 min after initiation of HD. There was an increase of TNF-alpha and IL-1beta levels in end-stage non-HD and HD patients and a further increase at 60 min after initiation of HD. The percentage of morphologically activated PMNs was increased only during dialysis. The nature of the HD membrane had no influence on rigidity, PMN activation, or cytokine production. CONCLUSIONS: The results indicate that PMN rigidity is defective in end-stage chronic CRF patients and is further increased 60 min after initiation of HD, regardless of the nature of the HD membrane used. PMN activation, increased TNF-alpha and IL-1beta levels, or a direct PMN impairment may cause the observed cell rigidity.     

Identification of integrin cell-substratum adhesion receptors on cultured rat bone cells.

The interactions of bone cells with their extracellular matrix is of major importance in bone development, repair, and disease. We examined the ability of rat calvarial bone cells to adhere to various matrix proteins and to define the role of integrin cell-substrate adhesion receptors in these interactions. Isolated newborn rat calvarial bone cells prelabeled with 3H-thymidine and plated on plastic wells that had been precoated with serial dilutions of various substrates showed typical dose-response adherence curves to fibronectin, fibrinogen, laminin, vitronectin, and collagen I and IV. Cell adherence to poly-D-lysine, a nonspecific cell adherent, was high at all substrate concentrations > 0.0001 micrograms/ml. A polyclonal anti-rat integrin antibody blocked cell adhesion to all substrates tested except poly-D-lysine. Isolated rat calvarial bone cells were surface labeled with 125I, extracted, and immunoprecipitated with polyclonal antibodies made against the rat integrin complex and peptides derived from the cytoplasmic domains of the alpha 2, alpha 3, and alpha 5 subunits. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (nonreduced) identified four bands representing a mixture of integrins including the alpha 1 beta 1 laminin/collagen receptor, the alpha 5 beta 1 fibronectin receptor, and the alpha V beta 3 (or possibly alpha V beta 5) vitronectin receptor. These experiments show that bone cells adhere to a wide variety of extracellular matrix proteins via specific integrins. Increased knowledge about the regulation of these receptors and the mechanisms by which they transmit information to the cell will be important for a more complete understanding of bone physiology and pathophysiology.     

Expression patterns of integrin receptors and extracellular matrix proteins in chronic rejection of human liver allografts

The beta1-integrin family of adhesion molecules is supposed to mediate cell-to-matrix interactions involved in a variety of immune reactions, especially in those associated with tissue remodelling. In an attempt to determine the role of beta1-integrins in the initiation and maintenance of fibrotic deposition observed in chronic rejection after liver transplantation, we immunohistochemically analysed the expression of different extracellular matrix components and the very late antigen (VLA) family of beta1-integrins in 11 samples of chronically rejected human liver allografts and compared results to findings in acutely rejected transplants and nontransplanted chronic inflammatory livers. In contrast to normal liver specimens, chronically rejected human liver allografts displayed a general overexpression of matrix components along sinusoids throughout the tissue and an additional characteristic accumulation in pericentral areas. Accordingly, VLA-1, -5 and -6 demonstrated a linear upregulation or de novo expression on sinusoidal lining cells, VLA-1 and VLA-4 additionally displayed concentration within pericentral fibrotic deposits. VLA-2 and -3 were only sporadically found. In accordance with findings in chronic rejection, chronic inflammatory livers showed overexpression of VLA-1, -5 and -6 within sinusoids and accumulation of VLA-1 and -4 in fibrotic septa. In contrast, acutely rejected allografts displayed slight overexpression of ECM components without characteristic accumulates, hence beta1-integrins were seen to be equally distributed throughout the parenchyma. Altogether, our analysis showed an upregulation of integrin receptors which corresponded to the extent of ECM deposition and thus suggested an important role for these molecules in the iniation of fibrosis observed in these specimens. Individual integrins showed different expression patterns within sinusoids and fibrotic areas, indicating distinct roles in differential stages of matrix accumulation, but induction patterns were generally similar in chronic inflammatory and chronically rejected livers, suggesting independence of the underlying disease.     

Leukocyte integrin expression in patients undergoing cardiopulmonary bypass

BACKGROUND: The recruitment of leukocytes to vascular endothelium is controlled by adhesion events mediated through the beta2 integrins, whereas the response of extravasated leukocytes within the tissues is controlled through the beta1 integrins. Although cardiopulmonary bypass (CPB) has been shown to be associated with a systemic inflammatory response and elevated levels of beta2 integrins on leukocytes, its effect on the beta1 integrins is not known. This study investigated the effect of the protease inhibitor aprotinin on the expression of the beta1 and beta2 integrins on circulating leukocytes in patients undergoing CPB. METHODS: Patients undergoing primary elective coronary artery bypass grafting were randomized into full-dose aprotinin or placebo groups. Blood samples were obtained at nine time points preoperatively, intraoperatively, and up to 6 days postoperatively. The surface expression of the beta1 integrins VLA-1, -3, -4, -5, and -6 and of the beta2 integrins CD11a/CD18, CD11b/CD18, and CD11c/CD18 was measured by flow cytometry on gated neutrophil and monocyte subpopulations in whole blood. RESULTS: Expression of the beta1 integrins was not significantly altered during the study period and, therefore, aprotinin had no effect on the expression of these molecules. Of the beta2 integrins, CD11b/CD18 expression was significantly increased on neutrophils at 15 minutes after onset of CPB in the placebo group (p < 0.01) but not in the aprotinin group. CONCLUSIONS: This study showed that expression of the beta1 integrins on neutrophils and monocytes did not alter during the first 6 days after CPB. Expression of the beta2 integrin CD11b/CD18 increased significantly on neutrophils during CPB in control patients but not in patients treated with full-dose aprotinin.