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Infusion of a simianized anti-α 4 β 7 mAb (Rh-α 4 β 7 ) just before and following SIV infection protected rhesus macaques from developing AIDS and partially from vaginal SIV acquisition. Recently, short-term treatment with Rh-α 4 β 7 in combination with cART was found to lead to prolonged viral suppression after withdrawal of all therapeutic interventions. The humanized form of Rh-α 4 β 7 , vedolizumab, is a highly effective treatment for inflammatory bowel disease. To clarify the mechanism of action of Rh-α 4 β 7 , naive macaques were infused with Rh-α 4 β 7 and sampled in blood and tissues before and after treatment to monitor several immune cell subsets. In blood, Rh-α 4 β 7 increased the CD4 + and CD8 + T cell counts, but not B cell counts, and preferentially increased CCR6 + subsets while decreasing CD103 + and CD69 + lymphocytes. In mucosal tissues, surprisingly, Rh-α 4 β 7 did not impact integrin α 4 + cells, but decreased the frequencies of CCR6 + and CD69 + CD4 + T cells and, in the gut, Rh-α 4 β 7 transiently decreased the frequency of memory and IgA + B cells. In summary, even in the absence of inflammation, Rh-α 4 β 7 impacted selected immune cell subsets in different tissues. These data provide new insights into the mechanisms by which Rh-α 4 β 7 may mediate its effect in SIV-infected macaques with implications for understanding the effect of treatment with vedolizumab in patients with inflammatory bowel disease., (Copyright © 2018 by The American Association of Immunologists, Inc.)

Intravenous administration of a novel recombinant rhesus mAb against the α4β7 gut-homing integrin (mAb) into rhesus macaques just prior to and during acute SIV infection resulted in significant decrease in plasma and gastrointestinal (GI) tissue viral load and a marked reduction in GI tissue proviral DNA load as compared with control SIV-infected rhesus macaques. This mAb administration was associated with increases in peripheral blood naive and central memory CD4(+) T cells and maintenance of a high frequency of CCR5(+)CD4(+) T cells. Additionally, such mAb administration inhibited the mobilization of NK cells and plasmacytoid dendritic cells characteristically seen in the control animals during acute infection accompanied by the inhibition of the synthesis of MIP-3α by the gut tissues. These data in concert suggest that blocking of GI trafficking CD4(+) T cells and inhibiting the mobilization of cell lineages of the innate immune system may be a powerful new tool to protect GI tissues and modulate acute lentiviral infection.

CD4+ T cells are essential for development and perpetuation of Crohn's disease, a chronic immune-mediated condition that affects primarily the small intestine. Using novel models of Crohn's disease-like ileitis (i.e., SAMP1/YitFc and CD4+ T cell transfer models), we have begun to understand the adhesive pathways that mediate lymphocyte trafficking to the chronically inflamed small bowel. Expansion of the CD4/beta7+ population and increased mucosal addressin cell adhesion molecule-1 (MAdCAM-1) expression were observed within the intestinal lamina propria with disease progression. However, Ab blockade of the beta7 integrin, the alpha4beta7 heterodimer, MAdCAM-1, or L-selectin did not attenuate inflammation. Blockade of two pathways (L-selectin and MAdCAM-1 or alpha4 integrins) was required to improve ileitis. Further analyses showed that 55 +/- 7% of the mesenteric lymph node alpha4beta7+CD4 expressed L-selectin. These L-selectin+ T cells were the main producers of TNF-alpha and the predominant ileitis-inducing subpopulation. Mechanistically, combined blockade of L-selectin and MAdCAM-1 depleted the intestinal lamina propria of CD4+ T cells that aberrantly coexpressed alpha4beta7 and alpha4beta1 integrins, markedly decreasing local production of TNF-alpha and IFN-gamma. Thus, pathogenic CD4+ T cells not only use the physiologic alpha4beta7/MAdCAM-1 pathway, but alternatively engage alpha4beta1 and L-selectin to recirculate to the chronically inflamed small intestine.

The Ig-like receptor family member, PIR-B, has been shown to play an inhibitory role in receptor signaling within B cells, mast cells, and dendritic cells. As it has been implicated in integrin-mediated responses, we investigated the effect of loss of the PIR-B protein on integrin-mediated signaling in primary murine myeloid cells. The pir-b-/- neutrophils displayed enhanced respiratory burst, secondary granule release, and a hyperadhesive phenotype when plated on surfaces coated with either extracellular matrix proteins or cellular adhesion molecules in the presence or absence of the soluble inflammatory agonist TNF-alpha. The pir-b-/- and wild-type cells responded equivalently when stimulated with TNF-alpha in suspension, indicating that the hyperresponsive phenotype of the pir-b-/- cells during adhesion was due to enhanced integrin signaling. Both wild-type and pir-b-/- neutrophils expressed similar levels of integrin subunits. Primary bone marrow-derived macrophages from pir-b-/- mice were also hyperadhesive and spread more rapidly than wild-type cells following plating on surfaces that cross-linked cellular beta2 integrins. Biochemical analysis of macrophages from pir-b-/- mice revealed enhanced phosphorylation and activation of proteins involved in integrin signaling. These observations point to a nonredundant role for PIR-B in the regulation of leukocyte integrin signaling.

We explore the binding sites for mAbs to the alpha I domain of the integrin alphaLbeta2 that can competitively inhibit, allosterically inhibit, or activate binding to the ligand ICAM-1. Ten mAbs, some of them clinically important, were mapped to species-specific residues. The results are interpreted with independent structures of the alphaL I domain determined in seven different crystal lattices and in solution, and which are present in three conformational states that differ in affinity for ligand. Six mAbs bind to adjacent regions of the beta1-alpha1 and alpha3-alpha4 loops, which show only small (mean, 0.8 angstroms; maximum, 1.8 angstroms) displacements among the eight I domain structures. Proximity to the ligand binding site and to noncontacting portions of the ICAM-1 molecule explains competitive inhibition by these mAbs. Three mAbs bind to a segment of seven residues in the beta5-alpha6 loop and alpha6 helix, in similar proximity to the ligand binding site, but on the side opposite from the beta1-alpha1/alpha3-alpha4 epitopes, and far from noncontacting portions of ICAM-1. These residues show large displacements among the eight structures in response to lattice contacts (mean, 3.6 angstroms; maximum, 9.4 angstroms), and movement of a buried Phe in the beta5-alpha6 loop is partially correlated with affinity change at the ligand binding site. Together with a lack of proximity to noncontacting portions of ICAM-1, these observations explain variation among this group of mAbs, which can either act as competitive or allosteric antagonists. One agonistic mAb binds distant from the ligand binding site of the I domain, to residues that show little movement (mean, 0.5 angstroms; maximum, 1.0 angstroms). Agonism by this mAb is thus likely to result from altering the orientation of the I domain with respect to other domains within an intact integrin alphaLbeta2 heterodimer., (Copyright 2004 The American Association of Immunologists, Inc.)

The Src family kinase Lyn has been shown to play both stimulatory and inhibitory roles within several hemopoietic cell types. In this study, we investigated the role played by Lyn in neutrophil integrin signaling. Loss of Lyn resulted in a hyperresponsive phenotype on engagement of surface integrins at low valency. Lyn(-/-) neutrophils displayed enhanced respiratory burst, secondary granule release, and a hyperadhesive phenotype when adherent to surfaces coated with either cellular counterreceptors or extracellular matrix proteins. In contrast, Lyn-deficient and wild-type cells expressed similar levels of surface integrins and responded equivalently to activating agents in suspension, indicating that the enhanced responses of lyn(-/-) cells was specific to the integrin signaling pathways. Lyn-deficient macrophages also displayed a hyperadhesive phenotype. Biochemical analysis of macrophages from lyn(-/-) mice revealed that Lyn plays an essential role in the adhesion-dependent phosphorylation of the immunoreceptor tyrosine-based inhibitory motif of the inhibitory receptors SIRP1alpha and PIR-B, which in turn recruit the phosphatase SHP-1. These observations suggest that reduced mobilization of SHP-1 to the membrane in lyn(-/-) neutrophils results in a hyperadhesive and hyperactive phenotype. This hypothesis is further supported by the fact that neutrophils from me(v)/me(v) mice, which have significantly reduced SHP-1 activity, are also hyperresponsive following integrin engagement. This is the first direct evidence using primary leukocytes from lyn(-/-) mice that this kinase functions as a negative regulator in integrin signaling.

Microglia are the primary immune effector cells resident within the CNS, whose activation into migratory, phagocytic cells is associated with increased expression of cell adhesion molecules of the integrin family. To determine which specific factors are important regulators of microglial activation and integrin expression, we have examined the influence of individual cytokines and extracellular matrix (ECM) substrates by quantifying cell surface expression of MHC and individual integrins by flow cytometry. We found that the proinflammatory cytokines TNF and IFN-alpha promoted microglial activation, as assessed by amoeboid morphology and increased expression of MHC class I, and also increased expression of the alpha(4)beta(1) and Mac-1 integrins. In contrast, TGF-beta1 had the opposite effect and was dominant over the other cytokines. Furthermore, the ECM substrates fibronectin and vitronectin, but not laminin, also promoted microglial activation and increased expression of the alpha(4)beta(1), alpha(5)beta(1) and Mac-1 integrins, but significantly, the influence of fibronectin and vitronectin was not diminished by TGF-beta1. Taken together, this work suggests that, in addition to cytokines, the ECM represents an important regulatory influence on microglial activity. Specifically, it implies that increases in the local availability of fibronectin or vitronectin, as a result of blood-brain barrier breakdown or increased expression in different pathological states of the CNS, could induce microglial activation and increased expression of integrins.

Experimental autoimmune encephalomyelitis (EAE) is mediated by inflammatory cells recruited from the circulation to the CNS. We used intravital microscopy to investigate the mechanisms of this recruitment. No leukocyte rolling and very little adhesion was observed in healthy control mice. In contrast, both rolling and adhesion was observed in brain postcapillary venules before onset of physical symptoms of EAE. Rolling and adhesion remained elevated for 2 wk and returned to near normal levels by 5 wk postsymptom onset. Consistent with a role for P-selectin in recruitment to the CNS, P-selectin protein was detected in the brains and spinal cords of EAE mice. Expression was highest before symptom onset and decreased over the next 2 wk. The importance of alpha(4) integrin increased with time as anti-alpha(4) integrin blocked approximately 20, 50, and 60% of leukocyte rolling 2 days before disease onset, 5 days and 2 wk postonset of symptoms, respectively, and 85% of rolling 5 wk postsymptoms. Addition of anti-P-selectin to alpha(4) integrin Ab-treated mice blocked all remaining rolling at each time point. Interestingly, however, alpha(4) integrin-mediated rolling appeared to be entirely dependent on P-selectin as anti-P-selectin alone was able to completely block all leukocyte rolling. In the absence of rolling (with P-selectin Ab), a 70% reduction in adhesion was noted. A very similar reduction was seen when mice were treated with alpha(4) integrin-blocking Ab. In conclusion, we describe increased leukocyte trafficking in the brains of EAE mice with important overlapping roles for both P-selectin and alpha(4) integrin in mediating leukocyte-endothelial cell interactions.

Previous studies have shown that the CXC chemokine, IFN-gamma-inducible T cell alpha chemoattractant (I-TAC), was chemotactic for IL-2-activated human T lymphocytes, which express abundant CXCR3. However, because most memory T lymphocytes are also CXCR3(+), the ability of I-TAC to promote the migration of normal human blood T cells across HUVEC monolayers in Transwell chambers was examined. I-TAC induced a marked (4- to 6-fold) increase in transendothelial migration (TEM) of T cells across unstimulated HUVEC from 5.6 to 28% of input T cells and was substantially more active than IFN-gamma-inducible protein-10, another CXCR3 ligand. I-TAC significantly enhanced TEM of T cells across TNF-alpha, but not across IFN-gamma or IFN-gamma plus TNF-alpha-activated HUVEC. IFN-gamma or IFN-gamma plus TNF-alpha-activated HUVEC produced substantial amounts of I-TAC, in contrast to TNF-alpha-treated EC. Both CD4(+) and CD8(+) T cells migrated in response to I-TAC to a similar extent, while memory T cells migrated several fold better than naive T cells. Blockade of LFA-1 strongly inhibited I-TAC-induced T cell TEM across unstimulated HUVEC, and approximately 50-60% of the TEM across cytokine-activated HUVEC. However, blocking both LFA-1 and very late Ag-4 abolished I-TAC induced T cell TEM. In vivo significant levels of I-TAC were detected in arthritic synovial fluid. Thus, I-TAC is one of the most potent chemoattractants of normal human blood CD4 and CD8 T cell TEM and is likely a major mediator of blood memory T lymphocyte migration to inflammation.

Activation of naive T and B cells occurs only within the context of organized lymphoid tissue. Thus, the continuous recirculation of mature lymphocytes is crucial for the development of primary immune response to foreign Ags. We have previously shown that low levels of IFN-gamma inhibit homing of B cells to the secondary lymphoid organs. In this study, we demonstrate that similarly low doses of IFN-gamma down-regulate integrin-mediated adhesion and migration of naive T and Th2 cells, and have a profound effect on the in vivo homing of naive T cells to the lymph nodes. Moreover, we show that these low doses of IFN-gamma have anti-inflammatory effects in an in vivo asthma model. Thus, in contrast to the proinflammatory effects of IFN-gamma at relatively high concentrations, low dose IFN-gamma appears to exert global suppressory effects on T cell trafficking and may have clinical application as an anti-inflammatory agent.

T lymphocyte infiltration into inflamed tissues is thought to involve lymphocyte rolling on vascular endothelial cells. Because both selectin and alpha(4) integrin adhesion molecules can mediate leukocyte rolling, the contribution of these receptors to lymphocyte migration to inflammation was examined. The recruitment of (111)In-labeled spleen T cells to intradermal sites injected with IFN-gamma, TNF-alpha, LPS, poly inosine-cytosine, and Con A was measured in the rat, and the effect of blocking mAbs to E-selectin, P-selectin, very late activation Ag-4 (VLA-4), and LFA-1 was determined on this T cell migration in vivo. Anti-E-selectin and anti-P-selectin mAbs each inhibited 10-40 and 20-48%, respectively, of the T lymphocyte migration to the inflammatory sites, depending on the stimulus. Blocking VLA-4 inhibited 50% of the migration to all of the lesions except Con A. Treatment with both anti-VLA-4 and anti-E-selectin mAbs inhibited up to 85% of the lymphocyte accumulation, while P-selectin and VLA-4 blockade in combination was not more effective than VLA-4 blockade alone in TNF-alpha, IFN-gamma, LPS, and poly inosine-cytosine lesions. Inhibiting E-selectin, P-selectin, and VLA-4 together nearly abolished lymphocyte migration to all inflammatory sites. Anti-LFA-1 mAb strongly inhibited lymphocyte accumulation by itself, and this inhibition was not significantly further reduced by E- or P-selectin blockade. Thus, T cell migration to dermal inflammation is dependent on E-selectin, P-selectin, and VLA-4, likely because these three receptors are required for rolling of memory T lymphocytes, but VLA-4 and E-selectin are especially important for lymphocyte infiltration in these tissues.

Integrin binding to physiologic ligands requires divalent cations and an inside-out-driven switch of the integrin to a high-affinity state. Divalent cations at the metal ion-dependent adhesion site (MIDAS) face of the alpha subunit-derived A domain provide a direct bridge between ligands and the integrin, and it has been proposed that activation dependency is caused by reorientation of the surrounding residues relative to the metal ion, forming an optimal binding interface. To gain more insight into the functional significance of the protein movements on the MIDAS face, we raised and characterized a murine mAb 107 directed against the MIDAS face of the A domain from integrin CD11b. We find that mAb 107 behaves as a ligand mimic. It binds in a divalent-cation-dependent manner to solvent-exposed residues on the MIDAS face of CD11b, blocks interaction of 11bA or the holoreceptor with ligands, and inhibits spreading and phagocytosis by human neutrophils. However, in contrast to physiologic ligands, mAb 107 preferentially binds to the inactive low-affinity form of the integrin, suggesting that its antagonistic effects are exerted in part by stabilizing the receptor in the low-affinity state. These data support a functional relevance of the protein movements on the MIDAS face and suggest that stabilizing the A domain in the low-affinity state may have therapeutic benefit.

LFA-1 exists in a low avidity state on resting leukocytes and is believed to adopt a high avidity state when the cells are exposed to a stimulus. Current evidence supports both aggregation of LFA-1 on the cell surface and conformational changes in the reversible acquisition of a high avidity state. We studied this regulation by selecting a Jurkat T cell clone, J-lo1.3, that expresses LFA-1 yet fails to bind to purified ICAM-1 despite treatment of the cells with PMA or Mn2+. Several lines of evidence demonstrated the absence of any changes within LFA-1 itself. LFA-1 protein purified from the J-lo1.3 clone and the wild-type Jurkat clone, Jn.9, were found to be functionally equivalent. The cDNA sequences encoding the LFA-1 alpha- and beta-chains from J-lo1.3 were identical with the published sequences except for nine base pairs. However, these differences were also found in a Jurkat mutant with a constitutively avid phenotype, J+hi1.19 or the wild-type Jn.9 genomic or cDNA. Fusion of J-lo1.3 with Jn.9 yielded hybrids that exhibited the J-lo1.3 adhesion phenotype, which indicated a dominant mutation in J-lo1.3. This phenotype was relatively specific for LFA-1 among all integrins expressed by Jurkat. Interestingly, the J-lo1.3 cells had a 1.2-fold faster doubling time than did the Jn.9 cells. Reversion of J-lo1.3 to the wild-type adhesion phenotype by mutagenesis and selection also decreased the growth rate. These data support a connection between cellular growth and cellular adhesion in lymphocytes.

T cells express a variety of surface proteins as they develop to maturity in the thymus. In addition to the TCR-CD3 complex and the two major coreceptors, CD4 and CD8, other surface proteins expressed include receptors for cytokines, growth factors, counterreceptors, and extracellular matrix molecules. To determine the role of integrin adhesion receptors in T cell development, we have expressed a trans-dominant inhibitor of integrin function in the thymus. This inhibitor leads to a block of adhesion to fibronectin due to reduced activation of integrin receptors. This reduced adhesion leads to a partial block in differentiation from CD4-CD8- cells to CD4+CD8+ cells, after the CD25+ stage, suggesting that integrins are important during Lck-mediated differentiation. Furthermore, the overall production of CD4+ cells is reduced compared with that of CD8+ cells without changes in negative selection, suggesting that integrins may be involved in the determination of the fate of the cell as well. These results demonstrate that integrin receptor function is required for proper thymocyte development in vivo.

Neutrophil migration to lung alveoli is a characteristic of lung diseases and is thought to occur primarily via capillaries rather than postcapillary venules. The role of adhesion molecules CD18 and CD29 on this migration in a mouse model of lung inflammation has been investigated. The number of neutrophils present in bronchoalveolar lavage fluid was determined 4 h after intratracheal instillation of LPS (0.1-1 microg) or murine recombinant KC (CXC chemokine, 0.03-0.3 microg). Both stimuli produced a dose-related increase in neutrophil accumulation. Intravenous anti-mouse CD18 mAb, 2E6 (0.5 mg/mouse), significantly (p < 0.001) attenuated LPS (0.3 microg)- but not KC (0.3 microg)-induced neutrophil accumulation. The anti-mouse CD29 mAb, HM beta 1-1 (0.02 mg/mouse), significantly (p < 0.05) inhibited both LPS (0.3 microg)- and KC (0.3 microg)-induced neutrophil migration. A second mAb to CD18 (GAME-46) and both F(ab')(2) and Fab of HM beta 1-1 produced similar results to those above, while coadministration of mAbs did not result in greater inhibition. Electron microscopy studies showed that CD29 was involved in the movement of neutrophils from the interstitium into alveoli. The effect of mAbs to CD49 (alpha integrin) subunits of CD29 was also examined. mAbs to CD49e and CD49f inhibited both responses, while anti-CD49b and CD49d significantly inhibited responses to KC only. These data suggest that CD29 plays a critical role in neutrophil migration in pulmonary inflammation and that CD49b and CD49d mediate CD18-independent neutrophil accumulation.

Dendritic cells (DC) are highly specialized APC that are critical for the initiation of T cell-dependent immune responses. DC exert a sentinel function while immature and, after activation by inflammatory stimuli or infectious agents, mature and migrate into lymphoid organs to prime T cells. We have analyzed integrin expression on monocyte-derived DC (MDDC) and found that expression of CD49d integrins (CD49d/CD29 and CD49d/beta7) was induced/up-regulated during TNF-alpha- or LPS-initiated MDDC maturation, reflecting the induction/up-regulation of CD49d and beta7 mRNA. CD49d mRNA steady-state level increased more than 10 times during maturation, with the highest levels observed 24 h after TNF-alpha treatment. CD49d integrin expression conferred mature MDDC with an elevated capacity to adhere to the CS-1 fragment of fibronectin, and also mediated transendothelial migration of mature MDDC. Up-regulation of CD49d integrin expression closely paralleled that of the mature DC marker CD83. CD49d integrin expression was dependent on cell maturation, as its induction was abrogated by N:-acetylcysteine, which inhibits NF-kappaB activation and the functional and phenotypic maturation of MDDC. Moreover, CD49d integrin up-regulation and MDDC maturation were prevented by SB203580, a specific inhibitor of p38 mitogen-activated protein kinase, but were almost unaffected by the mitogen-activated protein/extracellular signal-related kinase kinase 1/2 inhibitor PD98059. Our results support the existence of a link between functional and phenotypic maturation of MDDC and CD49d integrin expression, thus establishing CD49d as a maturation marker for MDDC. The differential expression of CD49d on immature and mature MDDC might contribute to their distinct motility capabilities and mediate mature DC migration into lymphoid organs.

Selective eosinophil recruitment is the result of orchestrated events involving cell adhesion molecules, chemokines, and their receptors. The mechanisms by which chemokines regulate eosinophil adhesion and migration via integrins are not fully understood. In our study, we examined the effect of CCR3-active chemokines on eosinophil adhesion to VCAM-1 and BSA under both static and flow conditions. When eotaxin-2 or other CCR3-active chemokines were added to adherent eosinophils, it induced rapid and sustained eosinophil detachment from VCAM-1 in a concentration-dependent manner. Adhesion was detectably reduced within 3 min and was further reduced at 10-60 min. Simultaneously, eotaxin-2 enhanced eosinophil adhesion to BSA. Preincubation of eosinophils with the CCR3-blocking mAb 7B11 completely prevented chemokine-induced changes in adhesion to VCAM-1 and BSA. Using a different protocol, pretreatment of eosinophils with chemokines for 0-30 min before their use in adhesion assays resulted in inhibition of VCAM-1 adhesion and enhancement of BSA adhesion. By flow cytometry, expression of alpha4 integrins and a beta1 integrin activation epitope on eosinophils was decreased by eotaxin-2. In a flow-based adhesion assay, eotaxin-2 reduced eosinophil accumulation and the strength of attachment to VCAM-1. These results show that eotaxin-2 rapidly reduced alpha4 integrin function while increasing beta2 integrin function. These findings suggest that chemokines facilitate migration of eosinophils by shifting usage away from beta1 integrins toward beta2 integrins.

Recent studies suggest that IgE-independent mechanisms of airway inflammation contribute significantly to the pathophysiology of allergic airway inflammatory diseases such as asthma. Such mechanisms may involve direct interactions between inhaled allergens and cells of the respiratory tract such as macrophages, dendritic cells, and epithelial cells. In this study, we investigated receptor-mediated interactions occurring between alveolar macrophages and allergen-containing pollen starch granules (PSG). We report here that PSG are released from a range of grass species and are rapidly bound and phagocytosed by alveolar macrophages. Human monocyte-derived dendritic cells also bound PSG but no internalization was observed. Phagocytosis of PSG was dependent on Mg2+ and Ca2+ and was inhibited by neo-glycoproteins such as galactose-BSA and N-acetylgalactose-BSA. Partial inhibition of phagocytosis was also seen with the Arg-Gly-Asp-Ser (RGDS) motif and with an anti-CD18 mAb (OX42). The combination of both neo-glycoprotein and anti-CD18 achieved the greatest degree of inhibition (>90%). Together, these data suggest a role for both C-type lectins and beta2-integrins in the binding and internalization of PSG. The consequences of this interaction included a rapid up-regulation of inducible NO synthase mRNA and subsequent release of NO by alveolar macrophages. Thus, receptor-mediated recognition of inhaled allergenic particles by alveolar macrophages may represent a potential mechanism for modulating the inflammatory response associated with allergic airway diseases such as asthma.

In acute lung inflammation, blood neutrophils and monocytes migrate into the lung parenchyma and bronchoalveolar space. The infiltration of the inflamed lung by monocytes is poorly understood because of difficulties in quantifying these cells in the presence of resident macrophages. Radiolabeled monocytes were used to study monocyte migration into the inflamed rat lung. Monocytes and neutrophils were purified from blood, labeled with 51Cr and (111)In, respectively, and injected i.v. into rats given an intratracheal injection of LPS. The accumulation of 51Cr-labeled monocytes increased > 10-fold in the lung parenchyma and 170-fold in the bronchoalveolar lavage (BAL) 18 h after LPS. (111)In-labeled neutrophils increased > 30-fold in the lung tissue and 500-fold in the BAL. Treatment of rats with a blocking anti-CD18 mAb inhibited monocyte accumulation in the lung and BAL by about 30%, whereas blocking very late activation Ag-4 (VLA-4) had no effect. Combined blockade of VLA-4 and CD18 inhibited approximately 30% of the migration to the lung parenchyma, but decreased the BAL by 80%. Monocyte migration to cutaneous inflammation was completely abolished by the combined mAb treatment. Neutrophil accumulation in the lung and BAL was not decreased by blocking either CD18 or VLA-4 and was only partially reduced by blocking CD18 plus VLA-4. Thus, monocyte migration to the LPS inflamed lung is substantially CD11/CD18 and VLA-4 independent, but accumulation in BAL is mediated by CD18 and VLA-4. Monocytes as well as neutrophils may use a previously unrecognized endothelial adhesion and migration pathway in the lung.

Integrins play an important role in lymphocyte adhesion to cellular and extracellular components of their microenvironment. The regulation of such adhesion often involves changes in the functional state of the integrins rather than alterations in their expression levels. Although the functional basis for such transitions is unknown, a possible role for disulfide exchange might be postulated based on the observations that integrin function can be activated by bifunctional reducing agents or by Abs that react with areas adjacent to predicted long-range disulfide bonds in integrins. Recently, it has been reported that enzymes that catalyze disulfide exchanges such as protein disulfide isomerase (PDI) are present on the surface of lymphoid cells, raising the possibility that such enzymes might be involved in the control of lymphocyte adhesion. A number of inhibitors of PDI function were examined for their effects on integrin-mediated adherence of T cells. The results did not support role for PDI in the regulation of integrin function, as the inhibitors somatostatin A, tocinoic acid, dithiobisnitrobenzoic acid, and anti-PDI mAb did not interfere with adherence. However, one of the PDI inhibitors, bacitracin, selectively interfered with the beta1 integrin-mediated adherence of lymphoid cells to collagen, fibronectin, laminin, and VCAM-1, and with alpha4beta7-dependent adherence to fibronectin and to VCAM-1. In contrast, alpha(v)beta3- and alpha(L)beta2-mediated adherence were not inhibited. Thus, it appears that bacitracin may be a selective inhibitor of beta1 and beta7 integrin functions by an as yet unknown mechanism.

The alpha 4 integrins mediate leukocyte adhesion to specific counter-receptors, including vascular cell adhesion molecule-1 (VCAM-1), the fibronectin splice variant containing connecting segment 1 (CS1), and mucosal addressin cell adhesion molecule-1. A series of cyclized peptides based on the LDV sequence of CS1 were synthesized and assayed for inhibition of alpha 4 integrin binding. The most potent peptide, C*WLDVC* (where * indicates disulfide-linked residues), inhibited alpha 4 beta 1-dependent binding of lymphocytes to VCAM-1 and CS1 with half-maximal inhibition achieved at 1 to 3 microM of peptide. The peptide proved more potent when the lymphocytes were activated with 1 mM MnCl2; half-maximal inhibition was reached at 0.4 and 0.05 microM for VCAM-1 and CS1, respectively. This represents a 100- to 800-fold increase in potency over a linear CS1 peptide in these same assays. C*WLDVC* also inhibited alpha 4 beta 7-dependent lymphocyte binding to the ligands mucosal addressin cell adhesion molecule-1, VCAM-1 and CS1. Immunoprecipitation of radiolabeled integrin indicated that the peptide could bind alpha 4 beta 1 and alpha 4 beta 7 directly and elute alpha 4 beta 1 from a CS1-conjugated agarose resin. The peptide showed selectivity for alpha 4 integrins in that it effectively inhibited alpha 4 beta 1-dependent, but not alpha 5 beta 1-dependent, binding of cells to intact fibronectin. Due to its small size and potency, C*WLDVC* may serve as a useful tool for the study of alpha 4 integrin biology and the development of small molecule therapeutics.

TGF-beta 1 regulates cell growth, differentiation, and adhesion and is a potent immunosuppressant, in part through its well-recognized growth-inhibitory effects. However, certain T cell subsets, particularly of naive phenotype, can instead be costimulated to proliferate by TGF-beta 1. We have previously demonstrated that naive murine CD8+ T cells, TCR activated by platebound anti-CD3 Ab or SEB superantigen, are growth stimulated by TGF-beta 1, acquire a memory phenotype, express elevated IL-10 and TGF-beta 1, and cause T cell growth inhibition as effector CD8+ T cells. TGF-beta 1 causes growth among certain nonlymphoid cells in part by inducing or mimicking integrin activation. The present studies thus addressed mediation of TGF-beta 1-dependent growth and survival of anti-CD3-triggered CD8+ T cells via beta 1 integrins. TGF-beta 1 reduced anti-CD3-activated alpha 4 beta 1 integrin expression and constitutive adhesion to fibronectin, while initial alpha 5 beta 1 expression was heightened and adhesive function sustained. Fibronectin-based RGD peptides that bind alpha 5 beta 1 integrins and alpha 5 or beta 1 integrin chain-specific Abs blocked TGF-beta 1-dependent proliferation, while connecting segment-1 peptide that binds alpha 4 beta 1 integrin and alpha 4 chain-specific Abs had no effect. Cross-linked alpha 5- but not alpha 4-specific Ab mimicked TGF-beta 1 function by costimulating CD8+ T cell growth. TGF-beta 1 also caused RGD peptide-sensitive CD8+ T cell aggregation. Additionally, TGF-beta 1-costimulated proliferation correlated with TGF-beta 1 protection of CD8+ T cells from anti-CD3-induced apoptosis. RGD peptides and alpha 5 integrin-specific Ab abolished TGF-beta 1 prevention of activation-induced apoptosis. Therefore, TGF-beta 1 costimulates CD8+ T cell growth via activation of the alpha 5 beta 1 integrin and/or its ligand and supports sustained growth at least in part by alpha 5 beta 1-mediated protection from activation-induced apoptosis.

The leukocyte integrin receptor, alpha 4 beta 7, and the mucosal addressin cell adhesion molecule-1 (MAdCAM-1) are postulated to be important in regulating lymphocyte trafficking to normal intestine. Here we provide the first description of MAdCAM-1 expression in inflamed intestine. Using mouse models of experimentally induced colitis, we show a concordant increase in MAdCAM-1 expression associated with increased cellular infiltrates in areas of intestinal inflammation. To understand more of the molecular nature of the interactions between MAdCAM-1 and its leukocyte ligand, the alpha 4 beta 7 integrin receptor, we have analyzed the structural and functional properties of chimeric recombinant MAdCAM-1 proteins in vitro. Using site-directed mutagenesis and molecular modeling, we demarcate the alpha 4 beta 7 binding motif as three linear residues within the C-D loop in the first domain of MAdCAM-1. Mutation of residue L40, D41, or T42 in the first domain completely abrogates alpha 4 beta 7+ cell binding and cellular activation. Mutagenesis of other residues in the first domain do not impact these functions. We have modeled peptides based on the predicted structure of the alpha 4 beta 7 integrin binding motif on MAdCAM-1 and are able to show specific and selective blocking of cell binding. These observations suggest that the amino acid residues LDT on MAdCAM-1 play a role in the interaction with alpha 4 beta 7 in cell adherence and cell activation.

TNF-alpha inhibits collagen synthesis and at high concentrations stimulates collagenase synthesis in fibroblasts. As fluid from chronic inflammatory lesions contains significant levels of TNF-alpha, it is puzzling why these lesions exhibit dense accumulations of disorganized collagen. In this study we determined if low concentrations of TNF-alpha may inhibit the collagen phagocytic pathway in fibroblasts and thereby contribute to fibrosis. Collagen phagocytosis was measured by flow cytometric assessment of internalized, fluorescent collagen beads. TNF-alpha induced a dose-dependent reduction (optimal dose: 40% at 10 ng/ml; p<0.001) in the proportion of phagocytic cells and a twofold reduction of the number of internalized beads per cell but did not alter the total number of vital cells. TNF-alpha reduced by twofold the degradation of collagen films. Fluid flow shear-force assays demonstrated that TNF-alpha caused a 72% reduction (p < 0.05) in strong binding of collagen-coated beads to cells indicating that TNF-alpha may inactivate receptors and inhibit collagen binding. Furthermore, TNF-alpha reduced cell contact area with collagen substrates by threefold and inhibited reattachment of trypsinized cells by fourfold. Although levels of collagen receptors were increased by TNF-alpha (53% increase in alpha(2) (beta)1 integrin; p<0.001, 20% increase in alpha(1)beta(1)), the receptors were inactivated by the cytokine. The reduced phagocytic activity of TNF-alpha-treated cells was restored to control levels by treatment with the integrin-activating Abs A16G6 and JBS2. TNF-alpha inhibited focal adhesion formation and phosphotyrosine staining in focal adhesions. These effects were replicated by the tyrosine kinase inhibitor genistein, which also inhibited phagocytosis. Collectively, these data indicate that TNF-alpha inhibits adherence and phagocytosis of collagen. These effects are mediated by a reduction in the strength of alpha(2)beta(1) integrin binding to collagen, possibly through tyrosine kinases in focal adhesions. At low concentrations of TNF-alpha (10 ng/ml) that are found in the periphery of chronic inflammatory lesions, we suggest that inhibition of the collagen phagocytic pathway may contribute to fibrosis.

Video microscopy and digital imaging were used to quantitatively analyze lymphocyte adhesion and formation of pseudopodia on the extracellular matrix protein fibronectin (FN). A morphology kinetics assay comparing pseudopodial extension values over a 24-h period showed that HPB-ALL T leukemic cells undergo a wave of morphologic change, returning to a round shape after 8 h. Using anti-alpha 4 and anti-alpha 5 mAbs and a panel of cell types that are single or double positive for expression of the alpha 4/beta 1 and alpha 5/beta 1 FN binding integrins, it was determined that cell adhesion to FN was influenced by both beta 1-integrins, whereas alpha 4/beta 1 was found to be the major FN receptor mediating pseudopodia extension. The protein kinase inhibitor staurosporine, the protein kinase C inhibitors calphostin C and chelerythrine, and the protein tyrosine kinase inhibitor herbimycin A blocked pseudopodial extension in HPB-ALL cells. In contrast, two cAMP-dependent protein kinase inhibitors H8 and H89 did not inhibit. Inhibitors of phospholipase A2, lipoxygenases, and cyclooxygenases could block formation of pseudopodia, yet had little or no effect on cell adhesion to FN. The preincubation of cells with arachidonic acid could prevent the inhibition mediated by the reversible phospholipase A2 inhibitor cibacron blue. We conclude that the formation of lymphocyte pseudopodia in response to FN can utilize the adhesive and signaling activities of the alpha 4/beta 1-integrin and the enzymatic activities of protein kinases and phospholipases.

alpha M beta 2, a beta 2 integrin expressed on cells of myelomonocytic differentiation, functions as a receptor for factor X (X) and fibrinogen (Fg) and participates in leukocyte adhesion to vascular endothelium. Acquisition of high affinity ligand binding has been suggested to result from a conformational change of alpha M beta 2 in response to agonist-induced leukocyte stimulation. We now describe mAb 7A10, which preferentially binds to alpha M beta 2 on activated cells and reports the functional activation of this integrin. Agonist-stimulated monocytes and monocytic cells, but not resting cells, maximally expressed the 7A10 neoepitope, whereas expression of other selected alpha M beta 2 epitopes remained unchanged. The neoepitope was elicited equally by exposure of cells to either ADP or FMLP and did not require divalent cations for expression. Saturation of the 7A10 neoepitope by this Ab on stimulated THP-1 cells inhibited both X and Fg binding and abolished the alpha M beta 2-driven cellular coagulant response. Stimulated monocytic cells, which bound X and/or Fg, exhibited a sustained adhesion to unstimulated endothelial cell monolayers and Ab 7A10 inhibited this sustained adhesion. We conclude that activated conformers of alpha M beta 2 mediate X and Fg binding, that assembly of either X or Fg or both on alpha M beta 2 mediates leukocyte adhesion to unstimulated endothelium through sparse ICAM-1, and that mAb 7A10 can report and functionally inhibit this pathway of leukocyte adhesion.