Expression and functional significance of an activation-dependent epitope of the β1 integrins in chronic inflammatory diseases

The avidity of VLA integrins for their ligands can be increased by their transition to an active conformational state. This conformational change can be detected with a novel monoclonal antibody (mAb), termed 15/7, that recognizes an activation-dependent conformational epitope on the common β1 polypeptide of different VLA αβ1 integrins. In an attempt to understand the possible role of the active conformational state of β1 integrins in vivo, we first investigated the expression of 15/7 epitope on T lymphocytes from patients with chronic inflammatory joint diseases. An enhanced expression of the 15/7 epitope was found in the synovial fluid (SF) T lymphocytes from these patients as compared to their peripheral blood (PB) T cells. The effect of different cytokines on the appearance of the 15/7 activation epitope in PB T lymphocytes was subsequently analyzed; interferon-γ, interleukin-2 and, to a lower extent, tumor necrosis factor-α were able to induce an increased expression of the 15/7 epitope. This enhanced 15/7 expression correlated with a higher binding ability to fibronectin of cytokine-activated T cells. The presence of this activation epitope was detected in a small proportion of T lymphocytes scattered within inflammatory foci of synovial membrane from rheumatoid arthritis and thyroid glands from Hashimoto's chronic thyroiditis. We then analyzed the possible role of 15/7 epitope expression on cell adhesion in vitro. Immunofluorescence studies showed that the 15/7 epitope displayed a spot-like distribution, selectively decorating adhesive contacts of U-937 myelomonocytic cells attached to the 80 kDa proteolytic fragment of fibronectin (FN80). Furthermore, the anti-β1 15/7 mAb was able to induce both T lymphocyte, Jurkat and U-937 cellular binding and spreading on FN80. Altogether these results indicate that an activated conformation of β1 integrins is detected in vivo in lymphocyte infiltrates from chronic inflammatory conditions. The active conformations of β1 integrins are regulated by physiologic mediators such as cytokines, play an important role in cellular attachment and spreading, and appear to be involved in the development of inflammatory processes.     

Functional relevance during lymphocyte migration and cellular localization of activated β1 integrins

The state of integrin activation can be assessed by monoclonal antibodies (mAb) that selectively recognize integrins in their active form. We demonstrate herein that the expression of the epitope recognized by mAb HUTS-21 is induced on T lymphoblasts upon binding of soluble vascular cell adhesion molecule (VCAM)-1 and an 80-kDa tryptic fragment of fibronectin (FN80) to the β1 integrins very late activation antigen (VLA)-4 and VLA-5, and that this effect is dependent on ligand concentration and is specific for β1 integrins. On T lymphoblasts adhering to immobilized fibronectin, the HUTS-21 epitope localized exclusively to sites of integrin binding to fibronectin. These results indicate that mAb HUTS-21 recognizes a ligand-induced binding site (LIBS) on the common β1 subunit of VLA proteins. Engagement of β1 integrins through this LIBS epitope inhibited T lym-phoblast movement on fibronectin, as determined by quantitative time-lapse video microscopy studies. Furthermore, the HUTS-21 mAb also prevented T lymphoblast-directed migration through gradients of substratum-immobilized β1 integrin ligands such as fibronectin or VCAM-1, whereas it did not affect migration on intercellular adhesion molecule (ICAM)-1. This anti-LIBS mAb stimulated cell adhesion through postreceptor events, without affecting receptor affinity for ligand, and appears to interfere with cell migration by a mechanism distinct from that of other anti-β1 activating antibodies.     

Functional role of alpha 2/beta 1 and alpha 4/beta 1 integrins in leukocyte intercellular adhesion induced through the common beta 1 subunit.

Whereas all of the integrins in the VLA protein subfamily are involved in cell-extracellular matrix interactions, only VLA-4 (through the alpha 4 subunit) has been implicated in the triggering of intercellular adhesion. Here we describe that the VLA protein beta 1 subunit (CD29) is also involved in the induction of homotypic cell aggregation. We have obtained three novel anti-beta 1 monoclonal antibodies (mAb) with the ability to induce cell aggregation on different leukocyte cell types. These mAb recognize an antigenic site on the common beta 1 chain of VLA proteins which is topographically and/or functionally distinct from other epitopes previously defined by several prototype anti-beta 1 mAb. Induction of cell aggregation by anti-beta 1 mAb is epitope specific, isotype and Fc independent, and displays kinetics similar to alpha 4-mediated aggregation. This cell aggregation requires an intact cellular metabolism, the presence of divalent cations in the extracellular medium, and the integrity of the cytoskeleton. We also have found that the Na+/H+ antiporter may be essential for this process. For Ramos cells, which bear only the VLA alpha 4/beta 1 heterodimer, intercellular adhesion induced through the VLA-beta 1 chain could be selectively inhibited by other anti-beta 1 mAb as well as by anti-alpha 4 mAb. Interestingly, anti-beta 1 mAb which induced strong aggregation of VLA-alpha 2- or VLA-alpha 4-transfected K562 cells, had minimal effect on the alpha 2- alpha 4- alpha 5+ K562 cell line. Furthermore, the beta 1-mediated induction of cell aggregation on alpha 2-K562- and alpha 4-K562-transfected cells was blocked by preincubation with either anti-alpha 2 or anti-alpha 4 mAb, respectively, as well as by other anti-beta 1 mAb. Interestingly, parental K562 cells were able to interact with both alpha 2- and alpha 4-transfected K562 cells, thus suggesting that counter-receptors for both integrins (VLA-2 and VLA-4) might exist on these cells. Together these results provide strong evidence supporting the involvement of alpha 2/beta 1 and alpha 4/beta 1 heterodimers in intercellular interactions and underline the pivotal role of the common beta 1 chain of VLA proteins in the integrin-mediated induction of cell aggregation.     

VLA family in rheumatoid arthritis: evidence for in vivo regulated adhesion of synovial fluid T cells to fibronectin through VLA-5 integrin.

Adhesion of T cells to extracellular matrix (ECM) proteins through VLA integrin receptors is crucial for lymphocyte trafficking, tissue localization and inflammatory function. We have investigated the expression of different VLA integrins (VLA-1-5) on peripheral blood (PB) and synovial fluid (SF) T lymphocytes from patients with rheumatoid arthritis (RA). Their expression on different cell types from synovial membrane (SM) is also reported. The role of VLA-4 fibronectin (FN) receptors in the interaction of activated SF T cells from RA patients with a 38-kD fragment of FN has been previously demonstrated. Here we have focused functional studies on VLA-5 as an alternative FN receptor for RA T cells. A significant higher proportion of SF T cells were able to bind to an 80-kD fragment of FN, containing the Arg-Gly-Asp (RGD) cell binding site, compared with PB T cells. This attachment was almost completely inhibited by anti-VLA-5 MoAbs as well as by RGD peptides. This enhanced capability by SF T cells appears to be independent of the level of the surface expression of the receptor and correlates better with their activation state as determined by the expression of the activation molecule AIM (CD69). The evidence for the expression of VLA heterodimers on both SF and SM cells from RA patients suggests the possible implication of ECM proteins in mediating and perpetuating inflammation in vivo.     

CD9 antigen is an accessory subunit of the VLA integrin complexes

The CD9 antigen is a cell surface glycoprotein of unknown function which belongs to the tetraspans family. We demonstrate here, by precipitation, Western blotting and co-capping experiments, that this molecule is associated with a large fraction of β1 integrins in two cell lines, the pre-B cell line NALM-6 and the megakaryocytic cell line HEL. In HEL cells, CD9 antigen is only associated with VLA-4. In contrast, in NALM-6 cells, CD9 antigen is associated with both VLA-4 and VLA-5. On the other hand, only the β1 chain is co-precipitated with the CD9 antigen in transfected L cells. These data show that the CD9 antigen is associated with the β1 chain rather than with a particular integrin. CD9 monoclonal antibodies (mAb) did not modify the binding of HEL and NALM-6 cells to fibronectin, laminin or collagen. The association of CD9 antigen to VLA integrins is strengthened by the fact that both CD9 and anti-VLA mAb induce aggregation of the two cell lines and inhibit their migration in Transwell chambers. Because the aggregating effect, but not the inhibition of migration, is observed in CEM or CD9-transfected CEM cells, these two effects are likely to be mediated by different mechanisms.     

Very late antigen integrins are involved in the adhesive interaction of lymphoid cells to human gingival fibroblasts.

To date, it is still unclear how the trafficking and retention of activated lymphocytes in periodontal lesions are regulated. In this study, we investigated the molecular basis for the adhesive interactions between lymphocytes and human gingival fibroblasts (HGF). Peripheral blood T lymphocytes (PBT) exhibited binding ability, but only when the calls were activated with phorbol 12-myristate 13-acetate (PMA). Among several human cell lines tested, PMA-stimulated Molt-4, a human T-cell leukaemia line, also displayed significant binding ability to HGF. In order to clarify the molecule(s) involved in this cell-cell interaction, a panel of monoclonal antibodies (mAb) was prepared to PMA-activated Molt-4 and one clone, 4-145, was selected on the basis of its ability to block the binding of PMA-activated Molt-4 to HGF. Moreover, 4-145 inhibited the binding of not only activated Molt-4 but also activated PBT and other cell types to HGF. Biochemical and flow cytometric analyses revealed that 4-145 probably recognizes the beta 1 chain of very late antigen (VLA) integrins. Blocking experiments using mAb specific for the alpha-chain of VLA integrins demonstrated the involvement of alpha 4 (VLA-4) and, to a lesser extent, alpha 5 (VLA-5) chains in the adhesive interactions between T cells and HGF. Despite the significant involvement of VLA integrins in the adhesive interaction between PBT and HGF, the binding of PBT to human dermal fibroblasts (HDF) was not abrogated by 4-145, suggesting that HGF and HDF differ in their requirement of VLA integrins for adhesion to activated PBT. Furthermore, the fact that vascular cell adhesion molecule-1 (VCAM-1), one of the ligands of VLA-4, was not detected on HGF by flow cytometry and anti-fibronectin (FN) Ab did not block the adhesive interaction to HGF suggests that not-yet-identified ligand(s) for VLA-4 might be present on HGF.     

VLA-4-dependent adhesion activities of U937 cells and guinea pig bronchoalveolar lavage leukocytes

VLA-4-dependent binding to fibronectin (FN) and to a human vascular cell adhesion molecule (hVCAM-1)-transfected murine cell line was measured using U937 cells and guinea pig (GP) bronchoalveolar lavage (BAL) cells. A species cross-reactive, blocking monoclonal antibody directed against human VLA-4 (TY 21.6) inhibited U937/FN binding by 71±7%. The presence of TY 21.6 inhibited the stimulated binding of U937 cells to hVCAM-1 by 84%. However, TY 21.6 was unable to inhibit the BAL/FN binding. With the addition of TY 21.6, the binding of PMA-stimulated BAL cells to hVCAM-1 was inhibited by 57±5%. In summary, human and guinea-pig leukocytes express binding activity to both FN and hVCAM-1. A specific VLA-4 blocking monoclonal antibody, TY 21.6, inhibited U937 and BAL cell binding to hVCAM-1, but only inhibited FN binding with U937 cells.In memory of Robert Sturm, January 1993.     

Differential expression of VLA-4 integrin by resident and peripheral blood B lymphocytes. Acquisition of functionally active α4β1-fibronectin receptors upon B cell activation

Very-late antigen (VLA)-4 (CD49d/CD29) constitutes the only member of the β1 integrin family that plays a role in the interaction of lymphoid cells with both extracellular matrix and endothelial cells through two identified ligands: fibronectin (FN) and VCAM-1, respectively. The expression and functional activity of VLA-4 has been studied in different maturation and activation stages of B cells from several cellular compartments. Resident B lymphocytes of different lymphoid organs were almost negative for VLA-4 as detected by both immunoperoxidase staining and flow cytometry analysis. However, a high expression of both chains of this heterodimer was observed when tonsillar B cells were activated in vitro with different stimuli, such as phorbol esters or Staphylococcus aureus Cowan I (SAC). Both nonactivated and in vitro activated B cells from peripheral blood constitutively expressed high levels of this surface antigen. The induced expression of VLA-4 after activation of tonsillar B lymphocytes was accompanied by the acquisition of the capacity to bind to a 38-kDa proteolytic fragment, containing the connecting segment I domain, of FN. Interestingly, nonactivated peripheral blood B cells were unable to attach to this FN fragment, in spite of their constitutive expression of VLA-4, and only acquired this functional capacity after cell activation with phorbol esters and SAC. This FN-binding acquisition was not affected by preincubation with inhibitors of protein and RNA synthesis. These results underline that the FN-binding activity of VLA-4 is dependent on processes affecting cellular activation as described for other members of the integrin family. By contrast, VLA-4-mediated homotypic aggregation of peripheral blood B cells could be triggered by anti-α4 monoclonal antibodies independently of the cell activation state.     

Pyk2 is differentially regulated by β 1 integrin- and CD28-mediated co-stimulation in human CD4+ T lymphocytes

β₁ integrins can provide T cell co-stimulation, but little is known concerning their downstream signaling pathways. We found that Pyk2, a focal adhesion kinase-related tyrosine kinase, is regulated by β₁ integrin signaling in human T cells. Stimulation of Jurkat T cells with the α₄β₁ integrin ligand VCAM-1 results in Pyk2 tyrosine phosphorylation, and combined stimulation with VCAM-1 and anti-CD3 mAb induces rapid and sustained synergistic Pyk2 phosphorylation. Studies with mAb suggest that in synergistic CD3- and α₄β₁ integrin-mediated Pyk2 tyrosine phosphorylation, a major contribution of CD3-derived signals is independent of their effects on regulating integrin adhesion. Analysis of resting human CD4⁺ T cells confirmed the ability of CD3-derived signals to synergize with β₁ integrin-dependent signals in the induction of Pyk2 tyrosine phosphorylation. In addition, although CD28-mediated co-stimulatory signals were able to synergize with CD3-mediated signals in inducing ERK and JNK activation and secretion of IL-2 in the primary T cells, they did not contribute to the induction of Pyk2 phosphorylation. Taken together, these results indicate a potential role for Pyk2 in T cell costimulation mediated specifically by β₁ integrins.     

Involvement of β1 integrins in the binding and entry of Trypanosoma cruzi into human macrophages

We have investigated the role of integrin molecules in the binding and entry of Trypanosoma cruzi into human macrophages, with the help of monoclonal antibodies (mAb). Addition of Lia 1/2 mAb and in lesser extent of Lia 1/5 mAb, which are both specific for the β₁ subunit of the VLA integrin family, to human macrophages blocked T. cruzi uptake and subsequent replication inside the macrophages. This inhibition correlated with their respective ability to block Fibronectin (Fn) binding to macrophages. Furthermore, another anti-β₁ mAb, Alex 1/4, which binds to a different epitope on the β₁ molecule and was unable to block Fn binding, did not affect T. cruzi invasion. The inhibition by Lia 1/2 and Lia 1/5 was dose dependent and clearly observable with doses as low as 1 μg/ml. Moreover, this inhibition was T. cruzi specific since the Lia 1/2 and Lia 1/5 were unable to block uptake of Leishmania pifanoi or Escherichia coli by human macrophages. In contrast, the TS 1/18 mAb, which blocks ligand binding to β₂ integrin, inhibited entry of L. pifanoi but not of T. cruzi. Finally, mAb specific for the a₄ and a₅ subunits of the two major Fn binding molecules of macrophages (VLA-4 and VLA-5, respectively), either alone or in combination, were poor inhibitors of T. cruzi uptake, suggesting that several members of the VLA family, including VLA-4 and VLA-5, are involved in binding and entry of T. cruzi into macrophages.     

T lymphocyte adhesion to the fibronectin and laminin components of the extracellular matrix is regulated by the CD4 molecule.

The adhesion of T cells to components of the extracellular matrix (ECM) is mediated by the beta 1 subfamily of integrin receptors, designated VLA. It has been recently demonstrated that the binding of VLA receptors to protein components of the ECM is rapidly augmented by the activation of the T cells without, however, any actual change in the level of expression of the VLA receptors for fibronectin (FN) or laminin (LN). Thus, it is likely that activation of existing VLA receptors is required for binding. The activation must be regulated by T cell surface molecules capable of transducing signals into the cell. We studied the role of the CD4 molecule in the binding of rat CD4+ T cells to the FN and LN components of the ECM. We now report that the CD4 molecule appears to play a major role in regulating T cell interactions with ECM. This conclusion is based on the following observations: (a) monoclonal antibodies directed against the CD4 molecule inhibited T cell adhesion to both FN and LN; (b) down-regulation of the CD4 molecule resulted in partial loss of the ability of CD4+ T cells to adhere to FN and LN; (c) a CD4+ T cell clone adhered to both FN and LN while a CD4-CD8- clone expressing an identical T cell receptor bound weakly to both proteins and (d) treatment of the CD4+ T cells with an inhibitor of the CD4-associated tyrosine protein kinase activity inhibited T cell adhesion to both ECM proteins.     

The beta 1 integrin, very late activation antigen-4 on human neutrophils can contribute to neutrophil migration through connective tissue fibroblast barriers.

Polymorphonuclear leucocyte (PMNL) accumulation in extravascular tissues and inflammatory exudates is dependent on their migration through blood vessel endothelium and then through connective tissue. Previously we utilized a barrier of human synovial and dermal fibroblasts (HSF or HDF) grown on microporous filters, as a model of PMNL migration through connective tissue. Those studies showed that beta 2 (CD18) and the beta 1 integrins, very late activation antigen-5 (VLA-5) and VLA-6, in part mediate this PMNL migration. Here we report that VLA-4, which can also be expressed at low levels on activated PMNL, is also involved in PMNL migration induced by C5a through fibroblast (HSF and HDF) barriers, because monoclonal antibody (mAb) to VLA-4 significantly inhibited (by 20-30%) PMNL migration. Blocking the function of CD18, VLA-5 or VLA-6 was not required for detection of the VLA-4-mediated migration. Combination treatment with mAb to VLA-4 and with mAb to VLA-5 or to VLA-6 further inhibited PMNL migration, irrespective of whether CD11/CD18 mechanisms were blocked with anti-CD18 mAb or not. Treatment of PMNL with a peptide based on the VLA-4-binding domain in the CS-1 fragment of fibronectin, but not a control peptide, inhibited PMNL migration to a comparable extent to treatment with mAb to VLA-4. A low level of VLA-4 was expressed on C5a-activated PMNL, detected by immunofluorescence flow cytometry. These results suggest that VLA-4 can be mobilized by human peripheral blood PMNL and can, in addition to VLA-5, VLA-6 and CD11/CD18 integrins, mediate PMNL migration through connective tissue. This is in marked contrast to PMNL transendothelial migration, where beta 1 integrins appear to play no significant role.     

Cell type determines the relative proportions of (-) and (+) strand RNA during poliovirus replication.

To examine the influence of the host cell type on poliovirus RNA synthesis we compared the levels of (-) and (+) strand poliovirus RNA during infection of epithelial (HeLa and HEp-2), leukocytic (U-937, HL-60 and K-562) and nerve (IMR-32) cells. The levels of (-) strand RNA were higher in IMR-32, U-937, K-562 or HL-60 cells than those in HeLa or HEp-2 cells. By contrast, (+) strand RNA content was greater in HeLa or HEp-2 cells. Although significant levels of (+) strand RNA were detected in U-937, K-562 and HL-60 cells, no viral protein synthesis was detected by polyacrylamide gel electrophoretic analysis of metabolically labelled proteins. The molar ratio of poliovirus (-) and (+) RNAs was 2-3 fold higher in IMR-32, U-937 and K-562 cells than in HeLa or HL-60 cells and 5-6 fold higher than in HEp-2 cells. Differentiation of HL-60 cells with a variety of inducers produced differential effects on poliovirus (-) and (+) RNA content and modified the molar ratio of (-)/(+) strand RNAs. These findings indicate that host cell components play a critical role in the regulation of the amount of poliovirus (-) and (+) strand RNAs synthesized during infection.     

LPAM-1 (integrin α4β7)-ligand binding: overlapping binding sites recognizing VCAM-1, MAdCAM-1 and CS-1 are blocked by fibrinogen,a fibronectin-like polymer and RGD-like cyclic peptides

The α4 integrin LPAM-1 (α4β7) mediates lymphocyte attachment within the extracellular matrix (ECM) by adhering to the connecting segment (CS)-1 site of fibronectin (FN). Here we reveal that very late antigen (VLA)-4⁻ LPAM-1⁺ T cell lymphoma TK-1 cells bind via LPAM-1 to multiple copies of the RGD sequence engineered within an FN-like polymer. Further, the small conformationally restrained RGD-like cyclic peptides 1-adamantaneacetyl-Cys-Gly-Arg-Gly-Asp-Ser-Pro-Cys and Arg-Cys-Asp-thioproline-Cys inhibit the adhesion of TK-1 cells to immobilized CS-1 peptide, and to endothelial counterreceptors for LPAM-1, namely mucosal addressin cell adhesion molecule (MAdCAM)-1 and vascular cell adhesion molecule (VCAM)-1. Spontaneous adhesion of the VLA-4⁻ LPAM-1⁺ B lymphoma cell line RPMI 8866 to CS-1 was likewise inhibited, confirming a previously undocumented ability of LPAM-1 to recognize the RGD tripeptide. The RGD-binding site in LPAM-1 either overlaps or is identical to sites required for interaction with MAdCAM-1, VCAM-1, and the CS-1. The binding of LPAM-1 and VLA-4 to RGD-containing ligands may have relevance in vivo given that fibrinogen at physiological concentrations is able to partially block the binding of TK-1 cells to MAdCAM-1. Hence fibrinogen and other vascular RGD-containing proteins may have mild anti-inflammatory activity required for maintaining effective homeostasis, analogous to the anti-thrombogenic activity of the vascular endothelium.     

Expression of functionally active α4β1 integrin by thymic epithelial cells

We have investigated the expression and function of the VLA-4 heterodimer α₄β₁, a member of the β₁ integrin subfamily, on human thymic epithelial cells (TEC) derived from cortical epithelium. The expression of the α₄ integrin chain was studied in four different cloned TEC lines derived from either fetal or post-natal human thymus by both flow cytometry and immunoprecipitation techniques with anti-α₄ MoAbs. All different cell lines assayed expressed significant levels of α₄, as revealed by their reactivity with MoAbs specific for distinct α₄ epitopes. The α₄ subunit expressed by TEC was associated to β₁ but not to β₇ chain, and displayed the characteristic 80/70 kD pattern of proteolytic cleavage. The VLA-4 integrin in these cells was constitutively active in terms of adhesiveness to both fibronectin and vascular cell adhesion molecule-1 (VCAM-1). In addition, this heterodimer localized to punctate regions of the cell in the area of contact with the substratum, named point contacts assessed by staining with the anti-β₁ activation epitope 15/7 MoAb. According to the cortical origin of the TEC lines expressing VLA-4, human thymus sections stained with different anti-α₄ antibodies revealed the presence of cortical, and in smaller numbers medullary epithelial cells bearing α₄ integrin. The expression of α₄ in the thymus was also found in both adult and fetal rats, in which epithelial cells were also specifically stained. Altogether, our data show that VLA-4 is an additional component of the integrin repertoire of TEC, and suggest that it could have an important role in thymus epithelial cell–thymocyte interactions.     

CSF-1 (M-CSF) enhances the inflammatory response of fibronectin-primed macrophages: pathways involved in activation of the cytokine network

We have previously reported that the priming of thioglycollate-elicited peritoneal macrophages (PMphi), as a representative population of mononuclear phagocytes (MNP), by macrophage-colony-stimulating factor (M-CSF or CSF-1) rendered these cells more susceptible to secondary stimulation by extracellular matrix (ECM) proteins, in particular fibronectin (FN), and that at least two beta1 integrins, VLA 4 (alpha4beta1 or CD49d) and VLA 5 (alpha5beta1 or CD49e), regulate IL-6 gene expression when PMphi come into contact with FN. In this report, we focused our attention on resident PMphi, as a more mature/differentiated MNP subpopulation. By using granulocyte-macrophage colony-stimulating factor (GM-CSF)- and IL-6-knockout (null) mice, we demonstrated that the cooperative effect between CSF-1 and FN in IL-6 release was a result of a sequential stimulation of the GM-CSF, but not the TNF-alpha, gene via interaction with VLA 5. We also showed that regardless of the presence or absence of CSF-1 or FN, IL-6 inhibits GM-CSF and TNF-alpha gene expression in an autocrine manner. The observed effects were specific because CSF-1 enhanced VLA 5 expression and blocking FN-treated resident PMphi in vitro with VLA 5 monoclonal antibodies inhibited the IL-6 response. We found that treatment of resident PMphi with the protein kinase C inhibitor, staurosporine, and the activator, phorbol myristate acetate (PMA), resulted in marked modulation of either FN- or FN/CSF-1-induced cytokine release. An increased level of VLA 5 expression was observed in PMA-treated resident PMphi. We concluded that in inflammatory processes, CSF-1 drives a number of pathways involved in the regulation of the expression of several genes and renders MNP highly susceptible to stimulation by ECM proteins that transform the MNP into secretory inflammatory cells.     

Monoclonal antibodies to the integrin α−4 subunit inhibit the murine contact hypersensitivity response

Lymphocyte-endothelial cell recognition is an active multistep process central to the pathophysiology of inflammation. In vitro models of lymphocyte adhesion predict that the β1 integrin very late antigen?4 (VLA?4), an activation-dependent adhesion receptor, can mediate the firm sustained attachment required for the extravasation of memory lymphocytes. We have used murine contact hypersensitivity as an in vivo model in which to evaluate the role of α?4 integrins in an evolving inflammatory response. We demonstrate that the intravenous administration of 75μg of the anti-α?4 specific monoclonal antibodies Rl-2 or PS/2 4–6 h prior to challenge significantly inhibits the efferent response of 2,4 dinitrofluorobenzene, or oxazalone-sensitized mice. The disease-modifying effect of anti-α4 treatment was evident as a 50–60% reduction in the ear swelling response. By histological analysis treated animals scored lower for edema, number of epidermal lesions and degree of leukocyte infiltration. Antibody-treated animals have elevated numbers of circulating mononuclear leukocytes present in the same relative ratio as untreated control animals, suggesting that the inhibitory effect was not due to antibody-dependent cellular depletion of effector lymphocytes. These data are consistent with a central role for VLA-4 in the pathophysiologic process of inflammation.     

Contribution of CD11a/CD18, CD11b/CD18, ICAM-1 (CD54) and −2 (CD102) to human monocyte migration through endothelium and connective tissue fibroblast barriers

Recently we reported that monocyte migration through a barrier of human synovial fibroblasts (HSF) is mediated by the CD11/CD18 (β₂) integrins, and the β₁ integrins VLA-4 and VLA-5 on monocytes. Here we investigated in parallel the role of β₂ integrin family members, LFA-1 (CD11a/CD18) and Mac-1 (CD11b/CD18) on monocytes, and the immunoglobulin supergene family members, ICAM-1 and ICAM-2 on HSF and on human umbilical vein endothelial cells (HUVEC), in monocyte migration through HSF and HUVEC monolayers. Using function blocking monoclonal antibodies (mAb), when both VLA-4 and VLA-5 on monocytes were blocked, treatment of monocytes with mAb to both LFA-1 and to Mac-1 completely inhibited monocyte migration across HSF barriers, although blocking either of these β₂ integrins alone had no effect on migration, even when VLA-4 and VLA-5 were blocked. This indicates that optimal β₂ integrin-dependent monocyte migration in synovial connective tissue may be mediated by either LFA-1 or Mac-1. Both ICAM-1 and ICAM-2 were constitutively expressed on HSF and on HUVEC, although ICAM-2 was only minimally expressed on HSF. Based on results of mAb blockade, ICAM-1 appeared to be the major ligand for LFA-1-dependent migration through the HSF. In contrast, both ICAM-1 and ICAM-2 mediated LFA-1-dependent monocyte migration through HUVEC. However, neither ICAM-1 nor ICAM-2 was required for Mac-1-dependent monocyte migration through either cell barrier, indicating that Mac-1 can utilize ligands distinct from ICAM-1 and ICAM-2 on HSF and on HUVEC during monocyte transmigration.     

Activation of beta1 integrins on blood eosinophils by P-selectin

Activation of β(1) integrins of blood eosinophils, assessed by mAb N29, correlates inversely with FEV(1) in two paradigms for studying control of human asthma. We asked whether P-selectin causes eosinophil β(1) integrin activation and results in increased adhesivity. By dual-label flow cytometry, eosinophils with high levels of surface-associated P-selectin had higher reactivity with the activation-sensitive anti-β(1) mAbs N29, 8E3, and 9EG7 than eosinophils with no or with a low-level of surface-associated P-selectin. Among patients with nonsevere asthma, surface P-selectin correlated with N29, 8E3, and 9EG7 signals. By immunofluorescence microscopy, surface-associated P-selectin was present in patches on eosinophils, some of which stained for the platelet marker thrombospondin-1. Activated β(1) and P-selectin partially colocalized on eosinophils. Soluble P-selectin added to whole blood enhanced activation of eosinophil β(1), but not β(2), integrins. In contrast, IL-5 activated eosinophil β(2), but not β(1), integrins. Eosinophils that did not attach to vascular cell adhesion molecule-1 (VCAM-1) in a static adhesion assay had a lower N29 signal than the original population. Soluble P-selectin added to whole blood enhanced eosinophil adhesion to VCAM-1. These findings are compatible with a scenario whereby P-selectin, on eosinophil-associated activated platelets or acquired from plasma or from prior interactions with endothelial cells or platelets, activates eosinophil α(4)β(1) integrin and stimulates eosinophils to adhere to VCAM-1 and move to the airway in asthma.