MAB1962 Sigma-AldrichAnti-Integrin β2 Antibody, clone P4H9

Andes virus (ANDV) causes a fatal hantavirus pulmonary syndrome (HPS) in humans and Syrian hamsters. Human alpha(v)beta(3) integrins are receptors for several pathogenic hantaviruses, and the function of alpha(v)beta(3) integrins on endothelial cells suggests a role for alpha(v)beta(3) in hantavirus directed vascular permeability. We determined here that ANDV infection of human endothelial cells or Syrian hamster-derived BHK-21 cells was selectively inhibited by the high-affinity alpha(v)beta(3) integrin ligand vitronectin and by antibodies to alpha(v)beta(3) integrins. Further, antibodies to the beta(3) integrin PSI domain, as well as PSI domain polypeptides derived from human and Syrian hamster beta(3) subunits, but not murine or bovine beta(3), inhibited ANDV infection of both BHK-21 and human endothelial cells. These findings suggest that ANDV interacts with beta(3) subunits through PSI domain residues conserved in both Syrian hamster and human beta(3) integrins. Sequencing the Syrian hamster beta(3) integrin PSI domain revealed eight differences between Syrian hamster and human beta(3) integrins. Analysis of residues within the PSI domains of human, Syrian hamster, murine, and bovine beta(3) integrins identified unique proline substitutions at residues 32 and 33 of murine and bovine PSI domains that could determine ANDV recognition. Mutagenizing the human beta(3) PSI domain to contain the L33P substitution present in bovine beta(3) integrin abolished the ability of the PSI domain to inhibit ANDV infectivity. Conversely, mutagenizing either the bovine PSI domain, P33L, or the murine PSI domain, S32P, to the residue present human beta(3) permitted PSI mutants to inhibit ANDV infection. Similarly, CHO cells transfected with the full-length bovine beta(3) integrin containing the P33L mutation permitted infection by ANDV. These findings indicate that human and Syrian hamster alpha(v)beta(3) integrins are key receptors for ANDV and that specific residues within the beta(3) integrin PSI domain are required for ANDV infection. Since L33P is a naturally occurring human beta(3) polymorphism, these findings further suggest the importance of specific beta(3) integrin residues in hantavirus infection. These findings rationalize determining the role of beta(3) integrins in hantavirus pathogenesis in the Syrian hamster model.

Acute lung injury affects close to 200,000 people in the United States annually and leads to death in 40-50% of the affected patients. Chronic ethanol abuse is thought to contribute to up to 40-50% of subjects who develop acute lung injury. We previously demonstrated in a rat model that chronic ethanol ingestion promoted acute lung injury and associated with chronic oxidant stress, activated matrix metalloproteinases, increased release of transforming growth factor-beta, and increased expression and deposition of fibronectin, a matrix glycoprotein implicated in lung injury and repair. Because fibronectin can activate monocytes to increase pro-inflammatory cytokine expression, we hypothesized that generation of fibronectin-enriched matrices during chronic ethanol ingestion might contribute to the development of acute lung injury by stimulating unopposed inflammation. To test this hypothesis, we harvested alveolar type II cells from rats fed the Lieber-DeCarli diet (6 weeks; 36% of calories from ethanol). After 96h of culture, the matrices deposited ex vivo by the type II cells derived from ethanol-fed rats showed increased amounts of fibronectin protein as demonstrated by ELISA. When monocytic U937 cells were plated atop these matrices, there was increased expression of interleukin-1beta (IL-1beta). This stimulation was inhibited by antibodies against alpha5beta1, a receptor that mediates many of the biological effects of fibronectin. We then tested whether antioxidants ameliorated these effects. Dietary supplements of the antioxidants N-acetylcysteine and procysteine normalized matrix production by type II cells. Furthermore, the newly derived matrices did not stimulate IL-1beta expression over control cells. These studies suggest that chronic ethanol exposure induces oxidant stress and activates lung tissue remodeling characterized by increased expression of fibronectin by alveolar type II cells. The newly deposited fibronectin-enriched matrices may stimulate the expression of pro-inflammatory cytokines in monocytic cells recruited to the lung after injury thereby explaining the priming effects of ethanol.

LPS is an outer-membrane glycolipid component of gram-negative bacteria known for its fervent ability to activate monocytic cells and for its potent proinflammatory capabilities. In addition, LPS triggers the release of cytokines and chemokines as well as cell-cell adhesion molecules. We postulate that LPS may also affect the expression of matrix-binding integrin receptors, thereby modulating cell-adhesive functions in monocytic cells. To test this hypothesis, we investigated the effects of LPS on the expression of the integrin alpha(5)beta(1), a fibronectin receptor, in a human monocytic cell line (U937) as well as in isolated human peripheral blood mononuclear cells (PBMCs). We found that LPS increased the expression of alpha(5)beta(1) receptors and enhanced the adherence of U937 cells and PBMCs to fibronectin-coated surfaces; this was blocked by anti-alpha(5)beta(1) antibodies. LPS increased alpha(5)-subunit mRNA accumulation in a dose- and time-dependent manner. The induction by LPS occurred, at least in part, at the level of gene transcription as indicated by experiments using alpha(5) intact and deletion promoter constructs. LPS-induced alpha(5) gene transcription was associated with rapid induction of conventional PKC-alpha protein and activity, was blocked by PKC inhibitors, and was mimicked by lipid A. Finally, we found that an anti-CD14 antibody was able to inhibit the LPS response. Overall, the data suggest that LPS stimulates alpha(5) gene transcription via CD14 and PKC-dependent signals to enhance the expression of functional alpha(5)beta(1) receptors in monocytic cells. This process may help stimulate monocytic cell activation and facilitate their migration into fibronectin-containing tissues during infection.

Adhesive interactions between monocytes and vascular smooth muscle cells (VSMC) may contribute to subendothelial monocyte-macrophage retention in atherosclerosis. We investigated the effects of angiotensin II (ANG II) and platelet-derived growth factor (PDGF)-BB on VSMC-monocyte interactions. Treatment of human aortic VSMC (HVSMC) with ANG II or PDGF-BB significantly increased binding to human monocytic THP-1 cells and to peripheral blood monocytes. This was inhibited by antibodies to monocyte beta(1)- and beta(2)-integrins. The binding was also attenuated by blocking VSMC arachidonic acid (AA) metabolism by inhibitors of 12/15-lipoxygenase (12/15-LO) or cyclooxygenase-2 (COX-2). Conversely, binding was enhanced by overexpression of 12/15-LO or COX-2. Direct treatment of HVSMC with AA or its metabolites also increased binding. Furthermore, VSMC derived from 12/15-LO knockout mice displayed reduced binding to mouse monocytic cells relative to genetic control mice. Using specific signal transduction inhibitors, we demonstrated the involvement of Src, phosphoinositide 3-kinase, and MAPKs in ANG II- or PDGF-BB-induced binding. Interestingly, after coculture with HVSMC, THP-1 cell surface expression of the scavenger receptor CD36 was increased. These results show for the first time that growth factors may play additional roles in atherosclerosis by increasing monocyte binding to VSMC via AA metabolism and key signaling pathways. This can lead to monocyte subendothelial retention, CD36 expression, and foam cell formation.

During cell migration, integrin attachments to the substratum provide the means to generate the traction and force necessary to achieve locomotion. Once the cell has moved over these attachments, however, it is equally important that integrins detach from the substratum. The fate of integrins after detachment may include release from the cell, lateral diffusion across the cell surface, or endocytosis and redelivery to the cell surface. Polymorphonuclear neutrophils (PMNs) become stuck on the extracellular matrix proteins fibronectin and vitronectin when their intracellular free calcium concentration ([Ca(++)]i) is buffered. Taking advantage of this feature of PMN migration, we investigated the fate of integrins to differentiate among various models of migration. We demonstrate that alpha5beta1, one of the fibronectin-binding integrins, is responsible for immobilization of [Ca(++)](i)-buffered PMNs on fibronectin. We find that alpha5 and beta1 are in endocytic vesicles in PMNs and that alpha5 colocalizes with a marker for an endocytic recycling compartment. When [Ca(++)](i) is buffered, alpha5 and beta1 become concentrated in clusters in the rear of the adherent cells, suggesting that [Ca(++)](i) transients are required for alpha5beta1 detachment from the substratum. Inhibition of alpha5beta1 detachment by buffering [Ca(++)](i) results in the depletion of alpha5 from both endocytic vesicles and the recycling compartment, providing compelling evidence that integrins are normally recycled by way of endocytosis and intracellular trafficking during cell migration. This model is further refined by our demonstration that the endocytic recycling compartment reorients to retain its localization just behind the leading lamella as PMNs migrate, indicating that membrane recycling during neutrophil migration has directionality. (Blood. 2000;95:2471-2480)

Fibrinogen, with or without its conversion to fibrin, in the extravascular spaces of injured and inflamed lung tissues is thought to promote inflammatory responses that can eventually lead to pulmonary fibrosis. One of these responses likely involves the elaboration of the proinflammatory cytokine interleukin (IL)- 1beta. We reported that both fibrinogen and fibrin stimulated production of IL-1beta message and protein by binding to CD18 integrin receptors on normal human monocytes (J. Immunol., 1995;154:1879-1887). The purpose of the current work was to extend our previous observations by characterizing the transcriptional regulation of fibrinogen-induced IL-1beta expression. Our model was the human monocytic cell line U937 transfected with the human IL-1beta promoter connected to reporter genes. We found that fibrinogen induced the IL-1beta promoter and that induction could be blocked by anti-CD18 antibody. Transfection with deletion constructs of the promoter and DNA electrophoresis mobility gel shift assays suggested that sequences containing activator protein (AP)-1, cyclic adenosine monophosphate response element (CRE), and nuclear factor (NF)-kappaB cis-acting motifs regulate IL-1beta gene expression by fibrinogen. In combination with competitive cotransfection studies using consensus oligonucleotides mimicking these motifs, we conclude that transactivation of an NF-kappaB-like sequence is necessary for induction of the IL-1beta gene, that activation of CRE may repress induction of the gene, and that AP-1 potentially modulates induction and repression of the gene induced by fibrinogen. This study begins to define the molecular mechanisms by which fibrin(ogen) promotes and regulates expression of the IL-1beta gene and further substantiates a role for fibrin(ogen) in tissue injury and inflammation.

Extracellular matrices (ECMs) can stimulate human monocytic cells to express interleukin-1beta (IL-1beta), a proinflammatory cytokine implicated in the regulation of tissue inflammation. In this study, we explored the intracellular mechanisms responsible for ECM induction of IL-1beta using human promonocytic U937 cells transfected with the full-length human IL-1beta gene promoter connected to a reporter gene. Using this system, we demonstrated that the ECM molecules fibronectin (FN), type I collagen (Coll), fibrin (Fb) and laminin (Lm) induced transcription of the IL-1beta gene (which was associated with a modest increase in IL-1beta protein secretion) in suspended cells, when used in their soluble monomeric form. This effect was mimicked or blocked by anti-integrin monoclonal antibodies (mAbs) and was dependent on the activation of protein kinase C (PKC). Three of the ECMs tested (FN, Coll and Fb) induced the activation of mitogen-activated protein kinases (MAPKs), whereas Lm had no effect. FN, Coll and Fb (but not Lm) also induced DNA binding of the transcription factor activator protein-1 (AP-1), but not that of nuclear factor-kappaB. Co-transfection of U937 cells with a competing AP-1 oligomer blocked the IL-1beta response induced by FN, but not that induced by the other ECMs. By inhibiting AP-1 translocation, glucocorticoids also blocked the FN-induced response, but not that of the other ECMs. These studies suggest that the signalling pathways which mediate ECM induction of IL-1beta expression in human monocytic cells converge at the level of PKC activation. However, they diverge in other aspects, as demonstrated by the differential activation of MAPKs and the need for diverse transcription factors.

Fluorescence confocal microscopy was used to obtain three-dimensional (3-D) images of human neutrophils migrating through a 3-D matrix of amniotic membrane with a temporal resolution of 30-60 s and a spatial resolution of approximately 2 microm in the z-dimension. Neutrophils migrating in response to a chemoattractant gradient within a 3-D matrix were apparently able to generate traction by use of lateral pseudopods inserted into footholds in the matrix as evidenced by matrix distortion. Similar anchored pseudopods were seen in cells migrating across polycarbonate membranes with 0.8-microm pores; the presence of these pores increased cell polarization and migration compared with cells on membranes without pores. Expansion of pseudopods distal to narrow constrictions in the matrix and porous filters was observed and appeared to be used to pull cells through the openings. Neutrophils deformed parts of the elastic amnion matrix during migration without permanently altering the substrate. Contact guidance of neutrophils crawling along matrix fibrils was also observed. These observations show that neutrophils migrating in 3-D are able to utilize mechanical structures in the matrix, not present on 2-D surfaces, to generate traction for locomotion.