Evidence that TNF-induced respiratory burst of adherent PMN is mediated by integrin alpha(L)beta(2) (original) (raw)
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Evidence that TNF-induced respiratory burst of adherent PMN is mediated by integrin αLβ2
Journal of Leukocyte Biology, 2002
Polymorphonuclear leukocytes (PMN) respond to tumor necrosis factor (TNF) with a respiratory burst (RB) only after adherence to surfaces coated with extracellular matrix proteins such as fibronectin and fibrinogen (permissive substrates) but not with others such as laminin or collagen (nonpermissive substrates). As PMN adherence to both types of surfaces is dependent on  2 integrins, we investigated the molecular basis of the different metabolic response to TNF. In particular, we evaluated the relative role of each  2 integrin (␣ L  2 , ␣ M  2 , and ␣ X  2) in adherence and O 2 ؊ production of PMN residing on fibronectinand laminin-coated surfaces, which were considered as models of permissive and nonpermissive surfaces, respectively. By using ␣ chain-specific monoclonal antibodies (mAb), we show that ␣ M  2 and ␣ X  2 mediate adherence to fibronectin and laminin; ␣ L  2 is not involved in adherence to laminin and has only a minimal contribution in adherence to fibronectin. Furthermore, production of O 2 ؊ in response to TNF was induced by immobilized anti-␣ L  2 but not anti-␣ M  2 or anti-␣ X  2 mAb. A strong correlation was also found between expression of ␣ L  2 and TNF-induced RB on fibronectin. Lastly, PMN responded to TNF on laminin with a RB after the inclusion of ␣ L-specific mAb in the laminin coat. Thus, we conclude that TNFinduced RB by PMN residing on fibronectin is mediated by ␣ L  2 and that ␣ M  2 and ␣ X  2 are likely to play an ancillary role to the signaling activity of ␣ L  2 by facilitating its recruitment to sites of adherence. The nonpermissiveness of laminin appears to be a consequence of its inability to act as a ligand for ␣ L  2 .
Evidence that TNF-induced respiratory burst of adherent PMN is mediated by integrin L2
Polymorphonuclear leukocytes (PMN) respond to tumor necrosis factor (TNF) with a respiratory burst (RB) only after adherence to surfaces coated with extracellular matrix proteins such as fibronectin and fibrinogen (permissive substrates) but not with others such as laminin or collagen (nonpermissive substrates). As PMN adherence to both types of surfaces is dependent on  2 integrins, we investigated the molecular basis of the different metabolic response to TNF. In particular, we evaluated the relative role of each  2 integrin (␣ L  2 , ␣ M  2 , and ␣ X  2 ) in adherence and O 2 ؊ production of PMN residing on fibronectinand laminin-coated surfaces, which were considered as models of permissive and nonpermissive surfaces, respectively. By using ␣ chain-specific monoclonal antibodies (mAb), we show that ␣ M  2 and ␣ X  2 mediate adherence to fibronectin and laminin; ␣ L  2 is not involved in adherence to laminin and has only a minimal contribution in adherence to fibronectin. Furthermore, production of O 2 ؊ in response to TNF was induced by immobilized anti-␣ L  2 but not anti-␣ M  2 or anti-␣ X  2 mAb. A strong correlation was also found between expression of ␣ L  2 and TNF-induced RB on fibronectin. Lastly, PMN responded to TNF on laminin with a RB after the inclusion of ␣ L -specific mAb in the laminin coat. Thus, we conclude that TNFinduced RB by PMN residing on fibronectin is mediated by ␣ L  2 and that ␣ M  2 and ␣ X  2 are likely to play an ancillary role to the signaling activity of ␣ L  2 by facilitating its recruitment to sites of adherence. The nonpermissiveness of laminin appears to be a consequence of its inability to act as a ligand for ␣ L  2 . J. Leukoc. Biol. 72: 718 -726; 2002.
Journal of Experimental Medicine, 1990
We used mAbs against polymorphonuclear leukocyte (PMN) surface proteins to investigate the mechanisms by which stimulated human neutrophils (PMNs) adhere in vitro to laminin, the major glycoprotein of mammalian basement membrane. mAb IB4, which is directed against the common beta 2 chain of the CD11/CD18, only partially inhibited the adherence of PMA-stimulated PMNs to both laminin and to subendothelial matrices. In contrast, IB4 completely inhibited PMA-stimulated PMN adherence to gelatin, fibronectin, collagen IV, and endothelial cell monolayers. PMA-stimulated PMNs from a patient with severe congenital CD11/CD18 deficiency also adhered to laminin, but not to gelatin or endothelial cell monolayers. Therefore, PMA-stimulated PMNs adhere to laminin by both CD11/CD18-dependent and CD11/CD18-independent mechanisms. Expression of CD11/CD18-independent adherence to laminin was agonist dependent, occurring after stimulation with the calcium ionophore A23187 and recombinant TNF-alpha, but...
The Journal of Immunology
Certain inflammatory cytokines and growth factors have been previously shown to interact with glycosaminoglycan moieties of the extracellular matrix (ECM). We have examined the association of the pleiotropic cytokine TNF-alpha with glycoprotein constituents of ECM. TNF-alpha interacted with fibronectin (FN) and laminin, and to a lesser degree with collagen. The major binding site for TNF-alpha on FN was localized to its 30-kDa N-terminal fragment (FN-N') with a Ki in the sub-nM range. The binding of 125I-labeled TNF-alpha to immobilized FN or FN-N' persisted for at least 24 h, and was specifically inhibited by antibodies to FN, mAb directed against the FN-N' domain, unlabeled TNF-alpha, and by the truncated forms of TNF-alpha receptors. Once bound to immobilized FN or FN-N', the cytokine could not be released by the soluble TNF-alpha-receptors, although it could be released by anti-TNF-alpha Ab. TNF-alpha was also found to interact with soluble FN, although with a lo...
Journal of cell science, 2000
Tumor necrosis factor (alpha) (TNF-(alpha) can change the interaction of lung endothelial cell monolayers with their extracellular matrix in association with an increase in endothelial monolayer protein permeability. Using immunofluorescence microscopy and flow cytometry, we determined if exposure of calf pulmonary artery endothelial monolayers to TNF-(alpha) may influence cell-matrix interactions by altering the clustering as well as internalization of the (&agr;)5(beta)1 integrins (or fibronectin receptors) on the surface of endothelial cells. Immunofluorescence microscopy revealed that TNF-(alpha) caused an increase in the intracellular staining of (alpha)5(alpha)1 integrins within structures similar to endocytic vesicles as well as an increase in antibody-induced clustering of the integrins at the cell periphery. Flow cytometric analysis of endothelial cells incubated at 37 degrees C after antibody-labeling of their surface (alpha)5(beta)1 integrins at 4 degrees C confirmed an i...
Increased recycling of α5β1 integrins by lung endothelial cells in response to tumor necrosis factor
2000
Tumor necrosis factor α (TNF-α) can change the interaction of lung endothelial cell monolayers with their extracellular matrix in association with an increase in endothelial monolayer protein permeability. Using immunofluorescence microscopy and flow cytometry, we determined if exposure of calf pulmonary artery endothelial monolayers to TNF-α may influence cell-matrix interactions by altering the clustering as well as internalization of the α5β1 integrins (or fibronectin receptors) on the surface of endothelial cells. Immunofluorescence microscopy revealed that TNF-α caused an increase in the intracellular staining of α5β1 integrins within structures similar to endocytic vesicles as well as an increase in antibody-induced clustering of the integrins at the cell periphery. Flow cytometric analysis of endothelial cells incubated at 37°C after antibody-labeling of their surface α5β1 integrins at 4°C confirmed an increase in the rate of α5β1 integrin internalization which was at least 3...
β 1 integrin activation on human neutrophils promotes β 2 integrin-mediated adhesion to fibronectin
European Journal of Immunology, 2001
Although the importance of g 1 integrin-mediated binding to adhesion molecules and extracellular matrix (ECM) molecules is well established for most types of leukocytes, the expression patterns and functional importance of g 1 integrins on neutrophils have remained controversial. Using flow cytometry, we found that human neutrophils express the § 4 , § 5 , § 9 and g 1 integrin subunits. To examine whether the integrins VLA-4 ( § 4 / g 1 ) and VLA-5 ( § 5 / g 1 ) have a functional role on neutrophils, we studied adhesion to their ligand fibronectin. Treatment of neutrophils with antibody 8A2, which specifically binds and activates g 1 integrins, resulted in increased binding to fibronectin. However, addition of blocking mAb revealed that 8A2induced adhesion did not depend on g 1 integrins, but on the g 2 integrin CD11b/CD18. Similarly, activation of g 1 integrins by 8A2 resulted in CD11b-dependent binding of neutrophils to fibrinogen. 8A2 treatment increased expression of an activation epitope of CD11b/CD18, which depended on phosphoinositide 3-OH kinase activity and an adequate concentration of intracellular free Ca 2+ . These data suggest that engagement of g 1 integrins on neutrophils results in a cross-talk signal that leads to activation of the g 2 integrin CD11b/CD18, followed by CD11b-mediated adhesion. As transmigrated neutrophils are surrounded by both g 1 and g 2 ligands in the ECM, this integrin cross-talk could play a role in modifying migration and cellular activation in inflamed tissues.
Journal of Biological Chemistry, 2001
Regulated adhesion of leukocytes to the extracellular matrix is essential for transmigration of blood vessels and subsequent migration into the stroma of inflamed tissues. Although  2-integrins play an indisputable role in adhesion of polymorphonuclear granulocytes (PMN) to endothelium, we show here that  1-and  3-integrins but not  2-integrin are essential for the adhesion to and migration on extracellular matrix molecules of the endothelial cell basement membrane and subjacent interstitial matrix. Mouse wild type and  2-integrin null PMN and the progranulocytic cell line 32DC13 were employed in in vitro adhesion and migration assays using extracellular matrix molecules expressed at sites of extravasation in vivo, in particular the endothelial cell laminins 8 and 10. Wild type and  2-integrin null PMN showed the same pattern of ECM binding, indicating that  2-integrins do not mediate specific adhesion of PMN to the extracellular matrix molecules tested; binding was observed to the interstitial matrix molecules, fibronectin and vitronectin, via integrins ␣ 5  1 and ␣ v  3 , respectively; to laminin 10 via ␣ 6  1 ; but not to laminins 1, 2, and 8, collagen type I and IV, perlecan, or tenascin-C. PMN binding to laminins 1, 2, and 8 could not be induced despite surface expression of functionally active integrin ␣ 6  1 , a major laminin receptor, demonstrating that expression of ␣ 6  1 alone is insufficient for ligand binding and suggesting the involvement of accessory factors. Nevertheless, laminins 1, 8, and 10 supported PMN migration, indicating that differential cellular signaling via laminins is independent of the extent of adhesion. The data demonstrate that adhesive and nonadhesive interactions with components of the endothelial cell basement membrane and subjacent interstitium play decisive roles in controlling PMN movement into sites of inflammation and illustrate that  2-integrins are not essential for such interactions.
Inhibition of β2 Integrin–mediated Leukocyte Cell Adhesion
2001
Many integrins mediate cell attachment to the extracellular matrix by recognizing short tripeptide sequences such as arginine-glycine-aspartic acid and leucine-aspartate-valine. Using phage display, we have now found that the leukocyte-specific  2 integrins bind sequences containing a leucine-leucine-glycine (LLG) tripeptide motif. An LLG motif is present on intercellular adhesion molecule (ICAM)-1, the major  2 integrin ligand, but also on several matrix proteins, including von Willebrand factor. We developed a novel  2 integrin antagonist peptide CPCFLLGCC (called LLG-C4), the structure of which was determined by nuclear magnetic resonance. The LLG-C4 peptide inhibited leukocyte adhesion to ICAM-1, and, interestingly, also to von Willebrand factor. When immobilized on plastic, the LLG-C4 sequence supported the  2 integrin-mediated leukocyte adhesion, but not  1 or  3 integrin-mediated cell adhesion. These results suggest that LLG sequences exposed on ICAM-1 and on von Willebrand factor at sites of vascular injury play a role in the binding of leukocytes, and LLG-C4 and peptidomimetics derived from it could provide a therapeutic approach to inflammatory reactions.
Journal of leukocyte biology, 2017
Although essential for inflammatory responses, leukocyte recruitment to blood vessel walls in response to inflammatory stimuli, such as TNF-α, can contribute to vascular occlusion in inflammatory diseases, including atherosclerosis. We aimed to further characterize the mechanisms by which TNF stimulates adhesive and morphologic alterations in neutrophils. Microfluidic and intravital assays confirmed the potent effect that TNF has on human and murine neutrophil adhesion and recruitment in vitro and in vivo, respectively. Inhibition of actin polymerization by cytochalasin D significantly diminished TNF-induced human neutrophil adhesion in vitro and abolished TNF-induced membrane alterations and cell spreading. In contrast, TNF-induced increases in β2-integrin (Mac-1 and LFA-1) expression was not significantly altered by actin polymerization inhibition. Consistent with a role for cytoskeletal rearrangements in TNF-induced adhesion, TNF augmented the activity of the Rho GTPase, RhoA, in...