A three-dimensional model system to study the interactions between human leukocytes and endothelial cells

Leukocyte adhesion to endothelial cells and migration into the subendothelial matrix was studied with a three-dimensional model system, consisting of human endothelial cells cultured on a loose collagen matrix. We developed a new method to separate the endothelial cell monolayer and adhering leukocytes, from the subendothelial matrix, allowing simultaneous analysis of leukocyte adhesion and transendothelial migration. Monocytes adhered more avidly to untreated endothelial cells than did neutrophils (2.5 +/- 0.3 vs. 1.0 +/- 0.2 leukocytes per endothelial cell). Only a small fraction (10%-20%) of these leukocytes migrated into the subendothelium. Pretreatment of endothelial cells with interleukin 1 (IL 1) enhanced adhesion (20%), but not migration of monocytes. In contrast, neutrophil adhesion was markedly and in a time-dependent manner increased by IL 1 treatment (i.e. 200% after 6 h and 110% after 24 h of IL 1 treatment). Moreover, IL 1 pretreatment enhanced neutrophil migration twofold. Activation of leukocytes with formyl-methionyl-leucyl-phenylalanine (fMLP) enhanced both monocyte and neutrophil adhesion, but did not affect leukocyte migration. Under all conditions, monocyte adhesion was only partly (30%-40%) inhibited by monoclonal antibodies (mAb) against the common beta subunit of the leukocyte-cell adhesion molecules (LeuCAM: CD18) and 25%-30% by mAb against the alpha subunit of LFA-1 (CD11a). In contrast, mAb against the alpha subunits of Mac-1 (CD11b) and p150.95 (CD11c) were hardly effective. fMLP-mediated neutrophil adhesion was reduced to below baseline levels by anti-LeuCAM (CD18) mAb, whereas the LeuCAM contribution in IL 1-mediated neutrophil adhesion was less pronounced and varied in time. IL 1-mediated neutrophil migration, however, was completely blocked by anti-LeuCAM mAb. fMLP-mediated neutrophil adhesion was inhibited by mAb against the alpha subunits of Mac, while mAb against the alpha subunits of LFA-1 and Mac-1 both reduced IL 1-mediated adherence. In summary, we describe a novel leukocyte adhesion/migration method and demonstrate that the contribution of the LeuCAM complex in leukocyte-endothelium interaction varies depending on cell type and stimulus used.     

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.     

Cytohesin-1 regulates human blood neutrophil adhesion to endothelial cells through β2 integrin activation

Cytohesin-1 is a guanine nucleotide exchange factor for ADP ribosylation factor 6 (Arf6) in human blood neutrophils and differentiated PLB-985 neutrophil-like cells. Cytohesin-1 regulates adhesion and the transendothelial migration of monocytes, dendritic cells and T lymphocytes through activation of the β2 integrin LFA-1. In this study we investigated the role of cytohesin-1 in neutrophil and neutrophil-like cell adhesion to HUVECs, immobilized ICAM-1, and the α4β1 and α5β1 integrin extracellular matrix ligand fibronectin. We show that cytohesin-1 knockdown or inhibition with secinH3 inhibits fMLF-mediated cell adhesion to HUVECs and immobilized ICAM-1, whereas cytohesin-1 over-expression has the opposing effect. Binding of PLB-985 cells to HUVECs correlated with expression of the high-affinity β2 integrin epitope recognized by mAb24. Adhesion to HUVECs was inhibited by soluble ICAM-1, anti-ICAM-1, anti-CD11a and anti-CD18, but not anti-CD11b, blocking antibodies. We also demonstrate that cytohesin-1 knockdown promotes fMLF-mediated cell adhesion to fibronectin whereas cytohesin-1 over-expression has the opposing effect. Crosstalk between β1 and β2 integrins also exists since inhibition of β1 integrin functions with blocking antibodies enhanced adhesion of PLB-985 over-expressing cytohesin-1 to ICAM-1. We suggest that cytohesin-1 is a key regulator of neutrophil adhesion to endothelial cells and to components of extracellular matrix, which may influence cell emigration through its dual opposing effect on β2 and β1 integrin activation.     

The role of alpha(4) and LFA-1 integrins in selectin-independent monocyte and neutrophil migration to joints of rats with adjuvant arthritis

Monocytes and neutrophils are chronically recruited to joints in rheumatoid arthritis. In the joints of rats with adjuvant arthritis, this is mediated, in part, by selectin-dependent and selectin-independent mechanisms. To define the selectin-independent mechanisms, (51)Cr-labeled blood monocytes, (111)In-labeled neutrophils and function blocking mAb to the selectins and integrins were utilized. Integrins contributed to the selectin-independent monocyte migration to arthritic joints with 58-70% inhibition of this recruitment by anti-alpha(4) or anti-LFA-1 mAb, relative to selectin blockade alone. alpha(4) plus P-selectin blockade was as effective as combined blockade of alpha(4), P-, E- and L-selectin, mediating approximately 83% of the overall monocyte migration to the joints. In contrast, LFA-1 was the predominant selectin-independent mechanism for neutrophil recruitment to the joints. LFA-1 together with P-selectin had essential roles in the talar joint. In dermal inflammation in the arthritic rats, LFA-1 accounted for most (69%) of the selectin-independent monocyte migration to the chemoattractant C5a(desArg) (zymosan-activated serum), whereas LFA-1 and Mac-1 both contributed to selectin-independent neutrophil recruitment to C5a(desArg). alpha(4) integrin and P-selectin in concert mediated monocyte recruitment to lipopolysaccharide and IFN-gamma lesions (81%). Thus: (1) either alpha(4) or LFA-1 can mediate monocyte migration to arthritic joints in the absence of selectin function and alpha(4) together with P-selectin is particularly important; (2) LFA-1 is the predominant mechanism of selectin-independent migration of neutrophils to inflamed joints; and (3) in arthritic rats, selectin-independent migration of monocytes and neutrophils to dermal inflammation is mediated by alpha(4) or LFA-1 or both LFA-1 and Mac-1, depending on the leukocyte type, and inflammatory stimulus.     

Enhancement of monocyte transendothelial migration by granulocyte-macrophage colony-stimulating factor: requirement for chemoattractant and CD11a/CD18 mechanisms

Granulocyte-macrophage colony-stimulating factor (GM-CSF) enhances and primes monocyte functions, but its role in monocyte migration is poorly understood. We examined monocyte migration across human umbilical vein endothelial cells (HUVEC) grown on filters. GM-CSF had no chemotactic or chemokinetic effect. However, GM-CSF enhanced monocyte transendothelial migration (TEM) through unstimulated and IL-1-activated (5 h) HUVEC in response to C5a or monocyte chemoattractant protein-1 in a dose-dependent fashion, increasing the migration from 28.7 +/- 5.3% to 41.8 +/- 6.2% (n = 8, p < 0.05) and from 34.8 +/- 6% to 50.3 +/- 3.1%, p < 0.05), respectively. The enhanced TEM was inhibited by monoclonal antibodies (mAb) to LFA-1, but not by mAb to Mac-1 or to VLA-4. Furthermore, GM-CSF up-regulated and activated LFA-1, as assessed by NKI-L16 neoepitope expression. The results indicate that: (1) GM-CSF can prime monocytes for increased TEM, (2) GM-CSF enhances LFA-1-mediated monocyte TEM and (3) this effect is in part mediated by increasing LFA-1 expression and activation. Thus, increased GM-CSF production may promote monocyte accumulation in inflammation not only by inducing monocytosis, but also enhancing migration.     

The role of E- and P-selectin in neutrophil and monocyte migration in adjuvant-induced arthritis in the rat

The role of the endothelial adhesion molecules E- and P-selectin in leukocyte accumulation in arthritis is not known. We investigated this role in rat adjuvant arthritis by employing adhesion function-blocking monoclonal antibodies (mAb) to rat P- and E-selectin. The acute migration (2 h) of radiolabeled rat blood neutrophils and monocytes to joints and skin was determined. Anti-P-selectin mAb significantly reduced accumulation of monocytes (by 50%) and neutrophils (by 40%) in the talar joint, and of neutrophils in tail joints (by 90%). Anti-E-selectin mAb alone did not attenuate leukocyte migration, but when combined with anti-P-selectin mAb, it enhanced inhibition of neutrophil accumulation in the talar and carpal joints. In the same animals, anti-P-selectin mAb significantly inhibited neutrophil and monocyte migration to dermal inflammatory reactions induced by zymosan-activated rat serum (ZAS) containing the chemotactic factor C5a_{des Arg}, endotoxin (LPS), interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α). In contrast, anti-E-selectin mAb alone had no effect on monocyte or neutrophil accumulation in inflamed skin of arthritic animals, but again enhanced the inhibition when combined with mAb to P-selectin. The addition of anti-L-selectin mAb to anti-P- and E-selectin mAb did not further suppress monocyte or neutrophil migration to inflamed skin or joints. These results demonstrate that optimal leukocyte migration to arthritic joints and inflamed skin is P-selectin dependent, and E-selectin is not essential. However, E-selectin contributes to migration when P-selectin mechanisms are not operative. L-selectin does not play a role in E- and P-selectin-independent leukocyte migration to joints or skin inflammation in arthritic rats. However, it is likely that additional selectin-independent pathways also mediate neutrophil and monocyte migration to joint and skin inflammation.     

Role of p150,95 in adhesion, migration, chemotaxis and phagocytosis of human monocytes

The leukocyte function-associated antigen-1 (LFA-1), the C3bi receptor (CR3) and the p150,95 antigen belong to a family of leukocyte surface molecules consisting of bimolecular complexes with alpha chains of 170 kDa, 165 kDa and 150 kDa, respectively, and a common beta subunit with a mol. mass of 95 kDa. In order to determine the function of the p150,95 antigen on human monocytes and U937 cells, and to study the functional relationship between this antigen and LFA-1 or CR3, we investigated the influence of monoclonal antibodies (mAb) directed against these cell surface molecules on the adhesive properties of these cells. The observation that anti-beta chain mAb strongly inhibited migration, chemotaxis, adhesion and phagocytosis of monocytic cells indicates a major role for LFA-1 family antigens in monocyte functions. Detailed analysis with a panel of anti-alpha chain antibodies demonstrated that both p150,95 and LFA-1 mediate random migration whereas in contrast, p150,95 and CR3 were shown to be involved in the directed migration of monocytes to f-Met-Leu-Phe. Furthermore, adhesion of monocytes to plastic surfaces or monolayers of endothelial cells as well as phagocytosis of latex particles was mediated by p150,95. The results demonstrate that, in spite of its relative low expression, the p150,95 glycoprotein is a major adhesion-associated molecule expressed by human monocytic cells.     

Prostaglandin E2 and monoclonal antibody to lymphocyte function-associated antigen-1 differentially inhibit migration of T lymphocytes across microvascular retinal endothelial cells in rat.

Lymphocyte-transendothelial cell migration is a complex event, and although much is known about the receptor-ligand interactions involved, little is understood about the intracellular events which accompany these interactions, or their regulation by inflammatory mediators. In this study we have shown that activation of T lymphocytes increased the proportion of cells migrating across monolayers of cultured retinal microvascular endothelial cells by both lymphocyte function-associated antigen-1 (LFA-1)-dependent and LFA-1-independent mechanisms. In preliminary experiments, it was found that activation of T cells with mitogens such as concanavalin (Con A) increased significantly T-cell migration across the endothelial monolayers. In contrast, activation of the endothelial monolayer with interferon-gamma (IFN-gamma) (96 hr) had no effect on transendothelial migration. Investigation of adhesion molecule requirements for transendothelial migration indicated that LFA-1 was necessary (P < 0.02) but that intracellular adhesion molecule-1 did not appear to be involved. Investigation of the role of prostaglandins in transendothelial migration revealed that, while prostaglandin E2 (PGE2) did not affect adhesion molecule expression on endothelial cells or T cells, treatment of either cell significantly blocked transendothelial migration (P < 0.05). Pretreatment with PGE2 combined with LFA-1 blockade had an additive effect on inhibition of T-cell transendothelial migration, indicating that two independent mechanisms were operative. PGE2 also had a direct inhibitory effect on T-cell adhesion to the endothelium. These results highlight the importance of considering non-adhesion receptor-mediated mechanisms, perhaps involving cytoskeletal and/or motility, in the migration of T cells across endothelial monolayers.     

Characterization of the rat leukocyte integrin, CD11/CD18, by the use of LFA-1 subunit-specific monoclonal antibodies.

We have attempted to characterize the rat leukocyte integrin, CD11/CD18, by the use of newly generated monoclonal antibodies (mAb) WT.1 (anti-CD11a) and WT.3 (anti-CD18) in conjunction with an mAb, OX42, reactive with a rat integrin-like molecule, with respect to the biochemistry, cellular distribution and function. The conclusion that the mAb WT.1 and WT.3 specifically recognize the rat CD11a and CD18, respectively, was based on: (a) their ability to inhibit homotypic aggregation of splenic concanavalin A (Con A) blasts; (b) sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the antigens recognized; (c) their ability to inhibit binding of Con A blasts to the purified ligand, namely the ICAM-1 antigen and (d) their blocking abilities in mixed leukocyte reaction. In the rat, CD18 has an apparent molecular mass of 95-100 kDa and can associate with at least three distinct alpha subunits of 160-170 kDa (CD11a), 140-150 kDa and 120-130 kDa. The latter two are precipitated by OX42 from M phi but not from unstimulated lymphocytes. They presumably represent the rat CD11b and CD11c, respectively. Rat thymocytes, PBL, thoracic duct lymphocytes, monocytes and neutrophils expressed differential levels of CD11a and CD18. Peritoneal M phi showed virtually no CD11a expression, although CD18 was expressed at levels similar to those seen on blood monocytes, showing an interesting pattern of LFA-1 expression regulation in this cell lineage. Both WT.1 (anti-CD11a) and WT.3 (anti-CD18) apparently recognize a "low-affinity" as well as a "high-affinity" form of LFA-1 and do not discriminate between the two.     

T cell interaction with ICAM-1-deficient endothelium in vitro: transendothelial migration of different T cell populations is mediated by endothelial ICAM-1 and ICAM-2.

The trafficking of T lymphocytes is carefully regulated by adhesive interactions with the vascular endothelium. Depending on their maturation and activation stage, T lymphocytes exhibit distinctive patterns of homing and recirculation, which is at least partly due to the selective expression of cell adhesion molecules (CAM) on the T cell surface. In order to define whether the differential usage of CAM during the steps of transendothelial migration is involved in organ-specific recirculation of different T cell subsets we compared the interaction of three different T cell populations with mouse endothelioma cell lines in vitro. Using a novel approach, where we directly compared T cell interaction with ICAM-1-deficient endothelium to wild-type endothelium, we recently demonstrated that endothelial ICAM-1 and ICAM-2 play a key role in mediating the transendothelial migration of CD4(+) memory T cells. Here we show that endothelial ICAM-1 and ICAM-2 are equally required for the transendothelial migration of other T cell populations such as thymocytes and T lymphoma cells, which differ from CD4(+) memory T cells in their maturation and activation stage, as well as in their surface expression of adhesion molecules. Our data therefore demonstrate that transendothelial migration of different T cell populations is mediated by the same endothelial CAM, i.e. ICAM-1 and ICAM-2, and thus subset-specific interaction of T cells with endothelial cells must be regulated prior to transendothelial migration.     

Anti-CD44-mediated blockade of leukocyte migration in skin-associated immune diseases

CD44 plays an important role in leukocyte extravasation, which is fortified in autoimmune diseases and delayed-type hypersensitivity (DTH) reactions. There is additional evidence that distinct CD44 isoforms interfere with the extravasation of selective leukocyte subsets. We wanted to explore this question in alopecia areata (AA), a hair-follicle centric autoimmune disease, and in a chronic eczema. The question became of interest because AA is treated efficiently by topical application of a contact sensitizer, such that a mild DTH reaction is maintained persistently. Aiming to support the therapeutic efficacy of a chronic eczema in AA by anti-CD44 treatment, it became essential to control whether a blockade of migration, preferentially of AA effector cells, could be achieved by CD44 isoform-specific antibodies. Anti-panCD44 and anti-CD44 variant 10 isoform (CD44v10) inhibited in vitro migration of leukocytes from untreated and allergen-treated, control and AA mice. In vivo, both antibodies interfered with T cell and monocyte extravasation into the skin; only anti-panCD44 prevented T cell homing into lymph nodes. Contributing factors are disease-dependent alterations in chemokine/chemokine receptor expression and a blockade of CD44 on endothelial cells and leukocytes. It is important that CD44 can associate with several integrins and ICAM-1. Associations depend on CD44 activation and vary with CD44 isoforms and leukocyte subpopulations. CD44 standard isoform preferentially associates with CD49d in T cells and CD44v10 with CD11b in monocytes. Accordingly, anti-panCD44 and anti-CD49d inhibit T cell, anti-CD11b, and anti-CD44v10 macrophage migration most efficiently. Thus, allergen treatment of AA likely can be supported by targeting AA T cells selectively via a panCD44-CD49d-bispecific antibody.     

Roles of alpha(4) integrins/VCAM-1 and LFA-1/ICAM-1 in the binding and transendothelial migration of T lymphocytes and T lymphoblasts across high endothelial venules

Several cell adhesion molecules that mediate the binding of lymphocytes to high endothelial venules (HEV) from flowing blood have been identified but the regulation of lymphocyte migration across the HEV wall into the lymph node (LN) is far from understood. In this study we have used an in vitro model of lymphocyte migration across HEV, and analysed the roles of two integrins in the binding and transendothelial migration of T lymphocytes and T lymphoblasts. The adhesion of T lymphocytes to high endothelial cells (HEC) cultured from rat LN HEV differed from that of T lymphoblasts since the percentage of T lymphoblasts that adhered and transmigrated was higher and was not increased by IFN-gamma pretreatment of HEC. Antibodies to alpha(4) integrins, VCAM-1 or LFA-1 maximally inhibited T lymphocyte adhesion by 40-50%, whereas antibodies to ICAM-1 were less effective (<20% inhibition). The effects of alpha(4) integrin and LFA-1 antibodies were additive, giving >90% inhibition. T lymphocytes which adhered in the presence of LFA-1 antibody showed reduced levels of transmigration and, in the presence of alpha(4) integrin antibody, slightly increased transmigration. Antibodies to alpha(4) integrins, VCAM-1, LFA-1 or ICAM-1 had little effect on T lymphoblast adhesion (maxima of 10-30% inhibition) and T lymphoblasts transmigrated normally in the presence of either alpha(4) integrin or LFA-1 antibodies. However, the effects of alpha(4) integrin and LFA-1 antibodies on T lymphoblast adhesion were synergistic, giving >90% inhibition of adhesion. These results suggest that the majority of T lymphoblasts use either alpha(4) integrins or LFA-1 to bind and transmigrate HEV, and the roles of these integrins on activated T cells are overlapping and redundant. In contrast, either integrin supports half-maximal binding of unactivated T lymphocytes to the surface of HEV and LFA-1 makes a larger contribution than alpha(4) integrins to transendothelial migration.     

Induction of homotypic T cell adhesion by triggering of leukocyte function-associated antigen-1 alpha (CD11a): differential effects on resting and activated T cells.

The leukocyte integrin LFA-1 (CD11a/CD18) plays a key role in many adhesive interactions involving cells of the immune system. Recently, it has been shown that LFA-1 is not only involved in cell adhesion, but that stimulation of LFA-1 can also contribute to cell activation. We now demonstrate that triggering of LFA-1 on T lymphocytes by monoclonal antibodies (mAb) against the LFA-1 alpha chain, but not against the LFA-1 beta chain, promotes cell adhesion. Induction of homotypic adhesion was only observed in T cells that had been pre-activated with anti-CD3 and not in resting peripheral blood T lymphocytes. The induced homotypic adhesion is mediated by LFA-itself, because it was inhibited by anti-LFA-1 beta mAb. This notion is supported by the temperature and divalent cation dependence which is characteristic of LFA-1-mediated adhesion. mAb against ICAM-1 (CD54) did not block LFA-1 alpha-induced adhesion. The sensitivity of LFA-1 alpha-induced adhesion to H7, which prevents the activation of protein kinase C and protein kinase A, and to cytochalasin B, which inhibits microfilament formation, suggests that the activation of the LFA-1 pathway through the LFA-1 alpha chain involves cell activation and requires an intact cytoskeleton.     

Biochemical and functional characteristics of the human leukocyte membrane antigen family LFA-1, Mo-1 and p150,95

The human leukocyte function-associated (LFA-1) antigen, the monocyte differentiation antigen Mo-1 which is characterized as the C3bi receptor and the glycoprotein p150,95 are characterized biochemically. Immunoprecipitations carried out with 6 different monoclonal antibodies (mAb) against LFA-1 indicated that four mAb (SPV-L1, SPV-L5, SPV-L7 and SPV-L11) were directed against the alpha chain, whereas mAb CLB54 and MHM-23 were found to react with the common beta chain of LFA-1, Mo-1 and p150,95. LFA-1 and Mo-1 expressed on KG-1 cells or lymphocytes, monocytes and granulocytes from one donor were homogeneous. Interestingly the alpha chain of p150,95 showed heterogeneity. The molecular weight of the alpha chain expressed on monocytes was consistently higher than that of the alpha chain on granulocytes. The beta subunits of LFA-1 and Mo-1 (as detected by mAb Bear-1) are not only similar in molecular weight and isoelectric focusing patterns, but it is demonstrated here that they are also identically glycosylated and have similar protein backbones as judged by tryptic peptide mapping. In spite of their structural similarities. LFA-1 and Mo-1 differ completely in some of their biological functions. Anti-LFA-1 mAb strongly inhibited monocyte-dependent T cell proliferation induced by tetanus toxoid or Helix pomatia hemocyanin and pokeweed mitogen-driven specific antibody production in vitro, whereas the anti-Mo-1 antibody Bear-1 was ineffective. These results suggest that the differences in these biological functions of LFA-1 and Mo-1 may be related to their different alpha subunits, which may recognize specific counter structures.     

ICAM-3, the third LFA-1 counterreceptor,is a co-stimulatory molecule for both resting and activated T lymphocytes

Optimal activation of human T cells mediated by ligation of CD3/T cell receptor (TcR) complex requires co-stimulatory signals. These can be provided by the adhesive interaction between receptor molecules on T cells and their counter-receptors on antigen-presenting cells. Soluble ICAM-3, anti-ICAM-3 and anti-CD3 mAb were utilized to address the role of the ICAM-3/LFA-1 pathway in TcR/CD3-dependent or -independent T cell activation. Immunoaffinity-purified ICAM-3 co-immobilized with suboptimal concentrations of anti-CD3 monoclonal antibody (mAb) stimulated T lymphocytes as monitored by the expression of the lymphocyte activation antigens CD25 and CD69. The mechanism underlaying this activation appear to involve the interaction of ICAM-3 with a β2 integrin, likely to be LFA-1, since mAb to the CD18 chain completely inhibited T cell activation. Similar experiments demonstrated that anti-ICAM-3 mAb were able to co-stimulate both resting (cord blood) and activated (T cell clones) T lymphocytes. On the contrary, anti-ICAM-1 mAb were only co-stimulatory for CD25 expression on activated but not on resting T cells. In addition, we have found that some γδ T cell clones bearing the Vδ1 segment were activated by direct mAb engagement of ICAM-3 in the absence of TcR/CD3 occupancy. Furthermore, immobilized anti-ICAM-3 mAb also induced development of dentritic processes. In conclusion, our data suggest that ICAM-3 on the surface of both T cells and antigen-presenting cells plays an essential role in the initiation of the immune response.     

CD13 is a novel mediator of monocytic/endothelial cell adhesion

During inflammation, cell surface adhesion molecules guide the adhesion and migration of circulating leukocytes across the endothelial cells lining the blood vessels to access the site of injury. The transmembrane molecule CD13 is expressed on monocytes and endothelial cells and has been shown to mediate homotypic cell adhesion, which may imply a role for CD13 in inflammatory monocyte trafficking. Here, we show that ligation and clustering of CD13 by mAb or viral ligands potently induce myeloid cell/endothelial adhesion in a signal transduction-dependent manner involving monocytic cytoskeletal rearrangement and filopodia formation. Treatment with soluble recombinant (r)CD13 blocks this CD13-dependent adhesion, and CD13 molecules from monocytic and endothelial cells are present in the same immunocomplex, suggesting a direct participation of CD13 in the adhesive interaction. This concept is strengthened by the fact that activated monocytic cells adhere to immobilized recombinant CD13. Furthermore, treatment with anti-CD13 antibodies in a murine model of peritonitis results in a decrease in leukocyte infiltration into the peritoneum, suggesting a potential role for CD13 in leukocyte trafficking in vivo. Therefore, this work supports a new direction for CD13 biology, where these cell surface molecules act as true molecular interfaces that induce and participate in critical inflammatory cell interactions.     

Role of the adhesion molecule lymphocyte function associated antigen 1 in toxic shock syndrome toxin 1-induced tumor necrosis factor alpha and interleukin-1 beta secretion by human monocytes.

We previously demonstrated that the induction by staphylococcal toxic shock syndrome toxin 1 (TSST-1) of tumor necrosis factor alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) secretion by human monocytes requires direct T cell-monocyte contact. In the present study, a role for the adhesion molecule lymphocyte function associated antigen 1 (LFA-1) in TSST-1-induced cytokine secretion by human monocytes among 12 normal healthy donors was investigated. Monoclonal antibodies to the alpha chain (anti-CD11a) and to the beta chain (anti-CD18) of LFA-1 significantly inhibited TSST-1-induced TNF-alpha and IL-1 beta secretion (P < 0.025; Wilcoxon signed-rank test, two tailed), while a control monoclonal antibody directed against the monocyte CD14 antigen had no effect. These results suggest that LFA-1 may play an important role in the secretion of TNF-alpha and IL-1 beta by TSST-1-stimulated human monocytes, likely by promoting cell-cell adhesion between monocytes and lymphocytes.     

Leukocyte transmigration is modulated by chemokine‐mediated PI3Kγ‐dependent phosphorylation of vimentin

Phosphoinositide 3‐kinase γ (PI3Kγ) plays a fundamental role in mediating leukocyte migration to inflammation sites. However, the downstream cytoplasmic events triggered by its signaling activity are still largely obscure. To address this issue, tyrosine and serine/threonine phosphorylated proteins of chemokine‐stimulated WT or PI3Kγ‐null macrophages were investigated. Among the proteins analyzed, the intermediate filament vimentin was found as a downstream effector of the PI3Kγ signaling pathway. Specific analysis of the phosphorylation state of vimentin in macrophages showed that this protein becomes rapidly phosphorylated in both tyrosine and serine residues upon chemokine stimulation. In the absence of PI3Kγ or the kinase activity of PI3Kγ (PI3Kγ^KD/KD), phosphorylation of vimentin was reduced. PI3Kγ‐null macrophages displayed impaired chemokine‐driven vimentin fiber disassembly as well as reduced ability to transmigrate across endothelial cells. While WT macrophages infected with a vimentin mutant resistant to N‐terminal serine phosphorylation showed a reduction in transendothelial migration, infection of PI3Kγ‐null macrophages with a vimentin mutant mimicking serine phosphorylation of N‐terminal residues rescued the transendothelial migration defect. These results define vimentin N‐terminal phosphorylation and fiber reorganization as a target of chemokine‐dependent PI3Kγ signaling in leukocytes.     

Transendothelial migration of lymphocytes mediated by intraendothelial vesicle stores rather than by extracellular chemokine depots

Chemokines presented by the endothelium are critical for integrin-dependent adhesion and transendothelial migration of naive and memory lymphocytes. Here we found that effector lymphocytes of the type 1 helper T cell (T(H)1 cell) and type 1 cytotoxic T cell (T(C)1 cell) subtypes expressed adhesive integrins that bypassed chemokine signals and established firm arrests on variably inflamed endothelial barriers. Nevertheless, the transendothelial migration of these lymphocytes strictly depended on signals from guanine nucleotide-binding proteins of the G(i) type and was promoted by multiple endothelium-derived inflammatory chemokines, even without outer endothelial surface exposure. Instead, transendothelial migration-promoting endothelial chemokines were stored in vesicles docked on actin fibers beneath the plasma membranes and were locally released within tight lymphocyte-endothelial synapses. Thus, effector T lymphocytes can cross inflamed barriers through contact-guided consumption of intraendothelial chemokines without surface-deposited chemokines or extraendothelial chemokine gradients.     

ICAM-1/LFA-1 interactions in T-lymphocyte activation and adhesion to cells of the blood-retina barrier in the rat.

To identify the signals involved in the adhesion and subsequent migration of lymphocytes across the endothelium (REC) and pigment epithelium (RPE) of the blood-retina barrier we have studied the effects of monoclonal antibodies (mAb) to rat adhesion/accessory molecules on the binding of normal and concanavalin A (Con A)-activated rat spleen lymphocytes to cultured unstimulated and interferon-gamma (IFN-gamma)-stimulated RPE and REC. Forty to 48% of unactivated T cells were found to bind to normal REC or RPE by leucocyte function-associated antigen-1/intercellular adhesion molecule-1 (LFA-1/ICAM-1)-independent mechanisms, despite constitutive expression of ICAM-1 by the RPE cells and LFA-1 by the T cells. Con A-activated lymphocytes showed an enhanced adhesion to both RPE and REC. However, IFN-gamma-stimulated RPE and REC did not demonstrate a significant increase in adhesiveness for normal lymphocytes highlighting the importance of lymphocyte integrin activation from low-affinity to high-affinity state. Activated lymphocyte adhesion to unstimulated RPE and REC was significantly blocked by LFA-1 mAb (35%, P < 0.0001) and ICAM-1 mAb (20%, P < 0.001). Inhibition of adhesion by antibody to CD2 was not significant. Both ICAM-1 and LFA-1 mAb also significantly (P < 0.05) blocked antigen presentation following retinal extract stimulation of lymphocytes from immunized rats in proliferation assay. These data suggest that the ICAM-1/LFA-1 system is important in lymphocyte trafficking into the eye only after lymphocyte activation.