Monocyte migration to arthritis in the rat utilizes both CD11/CD18 and very late activation antigen 4 integrin mechanisms

In human and experimental models of arthritis, blood monocytes migrate into the inflamed synovium and joint space. The mechanisms required for monocyte migration across the vascular endothelium in joints is poorly defined. Radiolabeled rat blood monocytes were used to measure monocyte migration to the inflamed joints of rats with adjuvant arthritis, and the role of monocyte adhesion molecules was analyzed. Monocyte accumulation in the inflamed joints was maximal 14-21 d after immunization with adjuvant, when arthritis had fully developed. Blocking mAbs to lymphocyte function-associated antigen 1 (LFA-1), Mac- 1, and very late activation antigen 4 (VLA-4) were used to evaluate the role of these integrins in the migration. Migration to the joints was not inhibited by treatment of the animals with mAb to LFA-1, Mac-1, or VLA-4 alone, and was partially (50%) inhibited in only the most arthritic joint, the talar joint, by the combination of mAb to LFA-1 plus Mac-1. In contrast, this combination inhibited migration to dermal inflammation induced by C5ades Arg, endotoxin, tumor necrosis factor alpha, and polyinosine-cytosine by 60-70%. When mAbs to LFA-1 and VLA-4 were combined, migration to all the inflamed joints was strongly inhibited (80-98%, depending on the joint). Treatment with the combination of the three mAbs to LFA-1, Mac-1, and VLA-4 completely eliminated monocyte migration to all joints and dermal inflammation. The results show that 51Cr blood monocytes can be used to quantify monocyte migration to arthritic joints in the rat. LFA-1 alone or VLA-4 alone is sufficient to mediate most of this migration, and either LFA-1 or VLA-4 is required for monocyte migration to joint inflammation. These results indicate that both the VLA-4 and LFA-1 integrins should be therapeutic targets for suppression of monocyte infiltration of joints in arthritis.     

Identification of surface proteins mediating adherence of CD11/CD18-deficient lymphoblastoid cells to cultured human endothelium.

Patients with the severe form of leukocyte adhesion deficiency syndrome do not express the CD11/CD18 adhesion complex on any of their leukocytes. Nevertheless, their lymphocytes, unlike their phagocytes, emigrate to extravascular sites of inflammation, demonstrating that surface proteins other than CD11/CD18 can mediate lymphocyte adherence to endothelium. Using a B-lymphoblastoid cell line (B-LCL) established from a CD11/CD18-deficient patient and cultured human umbilical vein endothelial cells (HEC), we investigated the CD11/CD18-independent mechanism(s) of lymphocyte adherence to endothelium. Monoclonal antibodies directed to the alpha 4 polypeptide (CD49d) and the beta 1 polypeptide (CD29) of the lymphocyte VLA-4 integrin receptor (CD49d/CD29), and to vascular cell adhesion molecule-1 (VCAM-1) on the endothelial cell significantly inhibited the adherence of the CD11/CD18-deficient B-LCL to untreated HEC and to HEC treated with recombinant human tumor necrosis factor-alpha. We suggest that the interaction of the lymphocyte receptor VLA-4 with the endothelial ligand VCAM-1 induced by cytokines at sites of inflammation or immune reaction represents a CD11/CD18-independent pathway of lymphocyte emigration.     

Lymphocyte adhesion through very late antigen 4: evidence for a novel binding site in the alternatively spliced domain of vascular cell adhesion molecule 1 and an additional alpha 4 integrin counter-receptor on stimulated endothelium

Recent studies demonstrate that alternative splicing of mRNA from a single gene can produce two forms of vascular cell adhesion molecule 1 (VCAM-1): a six-immunoglobulin (Ig) domain form (VCAM-6D) and a seven-Ig domain form (VCAM-7D). Using a COS cell transient expression assay, we investigated whether VCAM-6D and VCAM-7D differ functionally in adhesion to the integrin VLA-4 (CD49d/CD29) on lymphoid cells. Binding of lymphoid cell lines and peripheral blood lymphocytes was completely blocked by VLA-4 monoclonal antibody (mAb) and one VCAM-1 mAb (4B9) to both VCAM-6D and VCAM-7D, whereas one VCAM-1 mAb (E1/6) completely blocked binding to VCAM-6D but only partially inhibited binding to VCAM-7D. We conclude that there is one VLA-4 binding site in the six Ig domains shared between VCAM-6D and VCAM-7D, and that the alternatively spliced domain 4 present in VCAM-7D provides a second VLA-4 binding site that is blocked by 4B9 but not the E1/6 mAb. We compared the inhibitory effects of anti-VCAM-1 and anti-VLA-4 mAbs on lymphoid cell adhesion to cultured human umbilical vein endothelial cells (HUVEC). The anti-VCAM-1 mAb 4B9 blocked the binding of PBL and lymphoid tumor cells to stimulated HUVEC better than the anti-VCAM-1 mAb E1/6. Because VCAM-7D is the predominant form of VCAM-1 expressed by stimulated endothelial cells, this difference in VCAM-1 mAb inhibition is attributed to lymphoid cell binding to VCAM-7D on stimulated HUVEC. Although the anti-VLA-4 mAb and anti-VCAM-1 mAb 4B9 equally inhibited PBL binding to stimulated HUVEC, mAb 4B9 inhibited the binding of two lymphoid cell lines significantly less than anti-VLA-4 mAb. Combination of 4B9 mAb with function-blocking antiserum to human fibronectin, a second known ligand for VLA-4, also failed to inhibit as much as anti-VLA-4 mAb. These findings suggest that adhesion of lymphoid cell lines through VLA-4 or other alpha 4 integrins may involve inducible counter-receptor(s) on endothelium distinct from either VCAM-1 or fibronectin. Time course experiments indicate that the fraction of alpha 4 integrin-dependent binding that can be blocked by anti-VCAM-1 mAb E1/6 rises and peaks within 2 h of tumor necrosis factor (TNF) stimulation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Very late activation antigen 4-vascular cell adhesion molecule 1 interaction is involved in the formation of erythroblastic islands

Erythroblastic islands are anatomical units consisting of a central macrophage surrounded by erythroblasts. We studied the adhesion molecules involved in the formation of these structures. Central macrophages of erythroblastic islands isolated from the spleens of phlebotomized mice were clearly stained for vascular cell adhesion molecule 1 (VCAM-1). The surrounding erythroblasts of the erythroblastic islands strongly expressed the alpha 4 integrin of very late activation antigen 4 (VLA-4: alpha 4 beta 1 integrin), the counter receptor of VCAM-1, whereas most reticulocytes and erythrocytes did not. Both monoclonal antibodies (mAbs) against alpha 4 integrin and VCAM-1 disrupted the erythroblastic islands cultured in the presence of erythropoietin. Moreover, adhesion of splenic erythroblasts to tumor necrosis factor alpha-stimulated mouse splenic endothelial cells, which showed high expression of VCAM-1 but not intercellular adhesion molecule 1, was inhibited by the anti-VCAM-1 and anti-alpha 4 mAbs. These findings suggest that VLA-4-VCAM-1 interaction plays a crucial role in the formation of erythroblastic islands.     

The chemokine KC, but not monocyte chemoattractant protein-1, triggers monocyte arrest on early atherosclerotic endothelium

In a reconstituted flow chamber system, preincubation with chemokines can trigger the arrest of rolling monocytes, suggesting that this interaction could help recruit these cells to early atherosclerotic lesions. To date, however, the contribution of endothelium-derived chemokines found in these lesion to monocyte arrests has not been investigated. The endothelium of lesion-prone carotid arteries from apolipoprotein E-deficient (ApoE(-/-)) mice, but not control mice, presents the chemokines KC (mouse GRO-alpha) and JE (mouse monocyte chemoattractant protein-1 [MCP-1]). Arrest of a monocytic cell line or mouse blood monocytes perfused through carotid arteries of ApoE(-/-) mice was reduced by treating with either pertussis toxin, an antagonist of CXCR2, or an antibody to KC, but this process was insensitive to agents that blocked CCR-2 or JE. Conversely, monocyte accumulation more than doubled upon pre-perfusion of the carotid artery with KC but not with mouse MCP-1. Blockade of alpha(4)beta(1) integrin (VLA-4) or vascular cell adhesion molecule-1, but not CD18 or intercellular adhesion molecule-1, almost completely inhibited the arrest of monocytes. We conclude that when presented by early atherosclerotic lesions, KC but not murine MCP-1 triggers VLA-4-dependent monocyte recruitment.     

Characterization of adhesive interactions between human endothelial cells and megakaryocytes.

Cell-cell adhesion is essential for many immunological functions and is believed to be important in the regulation of hematopoiesis. Adhesive interactions between human endothelial cells and megakaryocytes were characterized in vitro using the CMK megakaryocytic cell line as well as marrow megakaryocytes. Although there was no adhesion between unactivated human umbilical vein endothelial cells (HUVEC) and megakaryocytes, treatment of HUVEC with inflammatory cytokines such as IL-1 beta, tumor necrosis factor alpha, INF-gamma, or the phorbol ester phorbol myristate acetate (PMA) resulted in a time- and dose-dependent increase in adhesion. Stimulation of marrow megakaryocytes or CMK cells with the cytokines IL-1 beta, GM-CSF, IL-6, IL-3, or PMA augmented their adhesion to endothelium. Monoclonal antibodies against the LFA-1 subunit of the leukocyte adherence complex CD18 inhibited the binding of marrow megakaryocytes or CMK cells to HUVEC. Adhesion blocking experiments also demonstrated that the VLA-4/VCAM-1 pathway was important for megakaryocyte attachment to HUVEC. Adhesion promoted maturation of megakaryocytic cells as measured by increased expression of glycoproteins GpIb and GpIIb/IIIa and by increased DNA content. These observations suggest that alterations in megakaryocyte adhesion may occur during inflammatory conditions, mediated by certain cytokines, resulting in augmented megakaryocyte maturation.     

Rat blood neutrophils express very late antigen 4 and it mediates migration to arthritic joint and dermal inflammation

Blood neutrophils contribute to joint injury in human and experimental models of arthritis. Neutrophil migration out of the blood in joint inflammation involves both the CD18 (beta2) integrins and a CD18 integrin-independent pathway. To investigate this migration, radiolabeled rat blood neutrophils were used to measure neutrophil accumulation in the inflamed joints of rats with adjuvant arthritis and the role of leukocyte integrins in migration to these joints and to dermal inflammation was determined. Neutrophils migrated rapidly (<2 h) to the inflamed joints 14-18 d after immunization with adjuvant. Blocking monoclonal antibodies (mAbs) to both LFA-1 and Mac-1 together, as well as a mAb to CD18, inhibited neutrophil accumulation in the inflamed joints by 50-75%. However, migration to dermal inflammation induced by C5a(des Arg)' tumor necrosis factor alpha, lipopolysaccharide, and poly-inosine:cytosine was inhibited by approximately 90%. Flow cytometry revealed the expression of low levels of very late antigen 4 (VLA-4) on nearly all rat blood neutrophils. Treatment with anti-VLA-4 plus anti-LFA-1 but neither mAb alone, strongly (60-75%) inhibited neutrophil accumulation in arthritic joints. This mAb combination also inhibited neutrophil migration to dermal inflammatory reactions by 30-70%. Blocking VLA-4 together with the CD18 integrins inhibited neutrophil accumulation by 95-99%, virtually abolishing neutrophil accumulation in cutaneous inflammation. A similar blockade of VLA-4 and CD18 decreased neutrophil accumulation in the inflamed joints by 70-83%, but a significant portion of the neutrophil accumulation to these joints still remained. In conclusion, rat blood neutrophils express functional VLA-4 that can mediate neutrophil migration to both inflamed joints and dermal inflammatory sites. VLA-4 appears to be able to substitute for LFA-1 in this migration and is particularly important for accumulation in inflamed joints. However, there exists an additional CD18- and VLA-4-independent pathway of neutrophil migration to arthritic joints that is not involved in acute dermal inflammation.     

Molecules and structures involved in the adhesion of natural killer cells to vascular endothelium

The present study was designed to define molecules and structures involved in the interaction of natural killer (NK) cells with the vascular endothelium in vitro. Resting and interleukin 2 (IL-2)-activated NK cells were studied for their capacity to adhere to resting and IL-1-treated human umbilical vein endothelial cells (EC). In the absence of stimuli, NK cells showed appreciable adhesion to EC, with levels of binding intermediate between polymorphs and monocytes. The binding ability was increased by pretreatment of NK cells with IL-2. Using the appropriate monoclonal antibody, the beta 2 leukocyte integrin CD18/CD11a was identified as the major adhesion pathway of NK cells to unstimulated EC. Activation of EC with IL-1 increased the binding of NK cells. In addition to the CD18-CD11a/intercellular adhesion molecule pathway, the interaction of resting or IL-2-activated NK cells to IL-1-activated EC involved the VLA-4 (alpha 4 beta 1)-vascular cell adhesion molecule 1 receptor/counter-receptor pair. No evidence for appreciable involvement of endothelial-leukocyte adhesion molecule was obtained. Often, NK cells interacted either with the culture substrate or with the EC surface via dot-shaped adhesion structures (podosomes) protruding from the ventral surface and consisting of a core of F-actin surrounded by a ring of vinculin and talin. The identification of molecules and microanatomical structures involved in the interaction of NK cells with EC may provide a better understanding of the regulation of NK cell recruitment from blood, their extravasation, and their migration to tissues.     

Monocyte adhesion and transmigration induce tissue factor expression: role of the mitogen-activated protein kinases

The expression of tissue factor (TF) by monocytes that have transmigrated across the endothelium to sites of extravascular inflammation acts both to focus and amplify the inflammatory response. Because clustering of the integrins responsible for endothelial adhesion and transmigration induces tyrosine phosphorylation and activation of the mitogen-activated protein (MAP) kinases, we postulated that transmigration might lead to monocyte activation and TF production. Monocytes were migrated across TNFalpha-primed ECV304 cells grown on fibronectin-coated Transwell chambers in response to FMLP (10(-8) M). After transmigration, monocytes showed a time-dependent increase in surface TF expression and biological procoagulant activity. TF expression was dependent on monocyte adhesion to ECV304 cells. Specifically, TF was not induced by FMLP treatment of suspended monocytes, by migration across fibronectin alone, or by soluble factors induced during migration, whereas monocyte-ECV304 adhesion was sufficient to stimulate TF. Antibodies against CD29 (beta1 integrin), but not against CD18 (beta2 integrin) or CD31 (PECAM-1), inhibited TF expression. Monocyte adhesion to ECV304 cells induced tyrosine phosphorylation of cellular proteins and specifically of the ERK and p38 MAP kinases. Tyrosine kinase inhibition with genistein (10 microg/mL) blocked transmigration, whereas selective ERK inhibition with PD98059 (50 microM) or p38 inhibition with SB203580 (20 microM) did not. However, both ERK and p38 inhibition dose dependently abolished TF expression. These studies suggest that an extravascular focus of infection or inflammation can promote both intravascular thrombosis and extravascular fibrin deposition during the process of adhesion and transmigration across the endothelial barrier. The selective inhibition of the mitogen-activated protein kinases may offer a novel therapeutic means of modulating this inflammatory sequence.     

Freezing adhesion molecules in a state of high-avidity binding blocks eosinophil migration

Leukocyte extravasation is mediated by multiple interactions of adhesive surface structures with ligands on endothelial cells and matrix components. The functional role of beta 1 (CD29) integrins (or very late antigen [VLA] proteins) in eosinophil migration across polycarbonate filters was examined under several in vitro conditions. Eosinophil migration induced by the chemoattractant C5a or platelet- activating factor was fully inhibited by monoclonal antibody (mAb) 8A2, a recently characterized "activating" CD29 mAb. However, inhibition by mAb 8A2 was observed only under filter conditions that best reflected the in vivo situation, i.e., when the eosinophils migrated over filters preincubated with the extracellular matrix (ECM) protein fibronectin (FN), or when the filters were covered with confluent monolayers of cultured human umbilical vein endothelial cells (HUVEC). When bare untreated filters were used, mAb 8A2 had no effect, whereas the C5a- directed movement was prevented by CD18 mAb. Studies with alpha-subunit (CD49)-specific mAbs indicated that the integrins VLA-4 and -5 mediated migration across FN-preincubated filters, and VLA-2, -4, -5, and -6 were involved in eosinophil migration through filters covered with HUVEC. In contrast with the activating CD29 mAb 8A2, a combination of blocking CD49 mAbs or the nonactivating but blocking CD29 mAb AIIB2 failed to inhibit completely eosinophil migration over FN-preincubated or HUVEC-covered filters. mAb 8A2 stimulated binding to FN but not to HUVEC. Moreover, eosinophil migration over FN-preincubated or HUVEC- covered filters was significantly inhibited by anti-connecting segment 1 (CS-1) mAbs, as well as the soluble CS-1 peptide (unlike migration across bare untreated filters). Thus, inhibition of eosinophil migration by mAb 8A2 depended upon the presence of ECM proteins and not upon the presence of HUVEC per se. In conclusion, "freezing" adhesion receptors of the beta 1 integrin family into their high-avidity binding state by the activating CD29 mAb 8A2 results in a complete inhibition of eosinophil migration under physiological conditions. Hence, activation of beta 1 integrin-mediated cell adhesion may represent a new approach to prevent influx of inflammatory cells.     

Analysis of T cell stimulation by superantigen plus major histocompatibility complex class II molecules or by CD3 monoclonal antibody: costimulation by purified adhesion ligands VCAM-1, ICAM-1, but not ELAM-1.

Many ligands of adhesion molecules mediate costimulation of T cell activation. The generality of this emerging concept is best determined by using model systems which exploit physiologically relevant ligands. We developed such an "antigen-specific" model system for stimulation of resting CD4+ human T cells using the following purified ligands: (a) major histocompatibility complex class II plus the superantigen Staphylococcus enterotoxin A, to engage the T cell receptor (TCR); (b) adhesion proteins vascular cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1), and endothelial leukocyte adhesion molecule 1 (ELAM-1), to provide potential cell surface costimulatory signals; and (c) recombinant interleukin 1 beta (rIL-1 beta)/rIL-6 as costimulatory cytokines. In this biochemically defined system, we find that resting CD4+ T cells require costimulation in order to respond to TCR engagement. This costimulation can be provided by VCAM-1 or ICAM-1; however adhesion alone is not sufficient since ELAM-1 mediates adhesion but not costimulation. The cytokines IL-1 beta and IL-6 by themselves cannot mediate costimulation, but augment the adhesion ligand-mediated costimulation. Direct comparison with the model of TCR/CD3 engagement by CD3 monoclonal antibody demonstrated comparable costimulatory requirements in both systems, thereby authenticating the commonly used CD3 model. The costimulation mediated by the activation-dependent interaction of the VLA-4 and LFA-1 integrins with their respective ligands VCAM-1 and ICAM-1 leads to increased IL-2R alpha (CD25) expression and proliferation in both CD45RA+ CD4+ and CD45RO+ CD4+ T cells. The integrins also regulate the secretion of IL-2, IL-4, and granulocyte/macrophage colony-stimulating factor. In contrast the activation-independent adhesion of CD4+ T cell to ELAM-1 molecules does not lead to T cell stimulation as measured by proliferation, IL-2R alpha expression, or cytokine release. These findings imply that adhesion per se is not sufficient for costimulation, but rather that the costimulation conferred by the VLA-4/VCAM-1 and LFA-1/ICAM-1 interactions reflects specialized accessory functions of these integrin pathways. The new finding that VLA-4/VCAM-1 mediates costimulation adds significance to observations that VCAM-1 is expressed on a unique set of potential antigen-presenting cells in vivo.     

Role of beta 1 and beta 2 integrins in the adhesion of human CD34hi stem cells to bone marrow stroma.

Hematopoietic stem cell interaction with elements of the underlying stroma is essential for sustained normal hematopoiesis. Here we have determined that adhesion receptors in the integrin family play a role in promoting adhesion of human hematopoietic stem cells to cultured human marrow stromal cells. Enriched CD34hi progenitor cells expressed VLA-4, VLA-5, and at least one or more beta 2 integrins. Homogeneous marrow stromal cell monolayers capable of supporting proliferation of cocultivated CD34hi cells expressed VCAM-1 and fibronectin (ligands for VLA-4 and VLA-5) as well as ICAM-1 (ligand for LFA-1 and Mac-1). Adhesion-blocking experiments indicated that VLA-4/VCAM-1, VLA-5/fibronectin, and beta 2-integrin/ICAM-1 pathways all are important for CD34hi cell attachment to stromal cells. Consistent with this suggestion, IL-1 stimulation of stromal cells caused both increased VCAM-1 and ICAM-1 expression and increased attachment by CD34hi bone marrow cells. In addition, CD34hi cells utilized VLA-4 to adhere to purified VCAM-1 and employed VLA-5 (and to a lesser extent VLA-4) to adhere to purified fibronectin. Together these results suggest that CD34hi stem cells may utilize multiple integrin-mediated adhesion pathways to localize within specialized microenvironmental niches created by marrow stromal cells.     

Adhesion of human basophils, eosinophils, and neutrophils to interleukin 1-activated human vascular endothelial cells: contributions of endothelial cell adhesion molecules

Cytokines such as interleukin 1 (IL-1) promote adhesiveness in human umbilical vein endothelial cells for leukocytes including basophils, eosinophils, and neutrophils, and induce expression of adherence molecules including ICAM-1 (intercellular adhesion molecule-1), ELAM-1 (endothelial-leukocyte adhesion molecule-1), and VCAM-1 (vascular cell adhesion molecule-1). In the present study, blocking monoclonal antibodies (mAb) recognizing ICAM-1, ELAM-1, and VCAM-1 have been used to compare their roles in IL-1-induced adhesion of human basophils, eosinophils, and neutrophils. IL-1 treatment of endothelial cell monolayers for 4 hours induced a four- to eight-fold increase in adhesion for each cell type. Treatment of endothelial cells with either anti-ICAM-1 or anti-ELAM-1 mAb inhibited IL-1-induced adherence of each cell type. In contrast, treatment with anti-VCAM-1 mAb inhibited basophil and eosinophil (but not neutrophil) adhesion, and was especially effective in blocking eosinophil adhesion. The effects of these mAb were at least additive. Indirect immunofluorescence and flow cytometry demonstrated expression of VLA-4 alpha (very late activation antigen-4 alpha, a counter-receptor for VCAM-1) on eosinophils and basophils but not on neutrophils. These data document distinct roles for ICAM-1, ELAM-1, and VCAM-1 during basophil, eosinophil, and neutrophil adhesion in vitro, and suggest a novel mechanism for the recruitment of eosinophils and basophils to sites of inflammation in vivo.     

Integrin mediated adhesion of mononuclear cells from patients with familial hypercholesterolemia

BACKGROUND: Low-density lipoproteins (LDL) can induce the adhesion of monocytes to endothelial cells. Monocytes of patients with familial hypercholesterolemia (FH) are exposed to high concentrations of LDL, and it has been reported that adhesiveness of these cells in hypercholesterolemic patients is enhanced. We investigated whether LFA-1 or VLA-4 mediated adhesion is altered in FH patients and whether HMG-CoA reductase inhibitors influence this adhesion. PATIENTS AND METHODS: LFA-1 and VLA-4 mediated adhesion to ICAM-1 and VCAM-1 coated beads was investigated using freshly isolated monocytes and T-lymphocytes from patients with homozygous FH, heterozygous FH (before and after cholesterol lowering treatment), and from controls. In addition, the expression of beta1- and beta2-integrins on these cells was determined. RESULTS: Both LFA-1 and VLA-4 mediated adhesion and integrin expression of monocytes and CD3+ cells from patients with homozygous FH and heterozygous FH was similar to that of monocytes from a control population. Treatment with HMG-CoA reductase inhibitors did not affect the adherence to ICAM-1 or VCAM-1, and did not influence the expression of integrins. CONCLUSIONS: In contrast to studies by others, we demonstrated in the present study that the actual LFA-1 and VLA-4 mediated adhesion of T-lymphocytes and monocytes is not altered in patients with FH.     

Role of vascular cell adhesion molecule 1/very late activation antigen 4 and intercellular adhesion molecule 1/lymphocyte function-associated antigen 1 interactions in antigen-induced eosinophil and T cell recruitment into the tissue

To determine the role of vascular cell adhesion molecule 1 (VCAM- 1)/very late activation antigen 4 (VLA-4) and intercellular adhesion molecule 1 (ICAM-1)/lymphocyte function-associated antigen 1 (LFA-1) interactions in causing antigen-induced eosinophil and T cell recruitment into the tissue, we studied the effect of the in vivo blocking of VCAM-1, ICAM-1, VLA-4, and LFA-1 by pretreatment with monoclonal antibodies (mAb) to these four adhesion molecules on the eosinophil and T cell infiltration of the trachea induced by antigen inhalation in mice. The in vivo blocking of VCAM-1 and VLA-4, but not of ICAM-1 and LFA-1, prevented antigen-induced eosinophil infiltration into the mouse trachea. On the contrary, the in vivo blocking of VCAM-1 and VLA-4, but not of ICAM-1 and LFA-1, increased blood eosinophil counts after antigen challenge, but did not affect blood eosinophil counts without antigen challenge in sensitized mice. Furthermore, the expression of VCAM-1 but not ICAM-1 was strongly induced on the endothelium of the trachea after antigen challenge. In addition, pretreatment with anti-IL-4 mAb decreased the antigen-induced VCAM-1 expression only by 27% and had no significant effect on antigen-induced eosinophil infiltration into the trachea. The in vivo blocking of VCAM- 1 and VLA-4 inhibited antigen-induced CD4+ and CD8+ T cell infiltration into the trachea more potently than that of ICAM-1 and LFA-1. In contrast, regardless of antigen challenge, the in vivo blocking of LFA- 1, but not of ICAM-1, increased blood lymphocyte counts more than that of VCAM-1 and VLA-4. These results indicate that VCAM-1/VLA-4 interaction plays a predominant role in controlling antigen-induced eosinophil and T cell recruitment into the tissue and that the induction of VCAM-1 expression on the endothelium at the site of allergic inflammation regulates this eosinophil and T cell recruitment.     

Fractalkine preferentially mediates arrest and migration of CD16+ monocytes

CD16+ monocytes represent 5-10% of peripheral blood monocytes in normal individuals and are dramatically expanded in several pathological conditions including sepsis, human immunodeficiency virus 1 infection, and cancer. CD16+ monocytes produce high levels of proinflammatory cytokines and may represent dendritic cell precursors in vivo. The mechanisms that mediate the recruitment of CD16+ monocytes into tissues remain unknown. Here we investigate molecular mechanisms of CD16+ monocyte trafficking and show that migration of CD16+ and CD16- monocytes is mediated by distinct combinations of adhesion molecules and chemokine receptors. In contrast to CD16- monocytes, CD16+ monocytes expressed high CX3CR1 and CXCR4 but low CCR2 and CD62L levels and underwent efficient transendothelial migration in response to fractalkine (FKN; FKN/CX3CL1) and stromal-derived factor 1 alpha (CXCL12) but not monocyte chemoattractant protein 1 (CCL2). CD16+ monocytes arrested on cell surface-expressed FKN under flow with higher frequency compared with CD16- monocytes. These results demonstrate that FKN preferentially mediates arrest and migration of CD16+ monocytes and suggest that recruitment of this proinflammatory monocyte subset to vessel walls via the CX3CR1-FKN pathway may contribute to vascular and tissue injury during pathological conditions.     

The chemokine SDF-1 stimulates integrin-mediated arrest of CD34+ cells on vascular endothelium under shear flow

The chemokine SDF-1 plays a central role in the repopulation of the bone marrow (BM) by circulating CD34(+) progenitors, but the mechanisms of its action remain obscure. To extravasate to target tissue, a blood-borne cell must arrest firmly on vascular endothelium. Murine hematopoietic progenitors were recently shown in vivo to roll along BM microvessels that display selectins and integrins. We now show that SDF-1 is constitutively expressed by human BM endothelium. In vitro, human CD34(+) cells establish efficient rolling on P-selectin, E-selectin, and the CD44 ligand hyaluronic acid under physiological shear flow. ICAM-1 alone did not tether CD34(+) cells under flow, but, in the presence of surface-bound SDF-1, CD34(+) progenitors rolling on endothelial selectin rapidly developed firm adhesion to the endothelial surface, mediated by an interaction between ICAM-1 and its integrin ligand, which coimmobilized with SDF-1. Human CD34(+) cells accumulated efficiently on TNF-activated human umbilical cord endothelial cells in the absence of SDF-1, but they required immobilized SDF-1 to develop firm integrin-mediated adhesion and spreading. In the absence of selectins, SDF-1 also promoted VLA-4-mediated, Gi protein-dependent tethering and firm adhesion to VCAM-1 under shear flow. To our knowledge, this is the first demonstration that SDF-1 expressed on vascular endothelium is crucial for translating rolling adhesion of CD34(+) progenitors into firm adhesion by increasing the adhesiveness of the integrins VLA-4 and LFA-1 to their respective endothelial ligands, VCAM-1 and ICAM-1.     

Fibronectin fragments modulate monocyte VLA-5 expression and monocyte migration

To identify the mechanisms that cause monocyte localization in infarcted myocardium, we studied the impact of ischemia-reperfusion injury on the surface expression and function of the monocyte fibronectin (FN) receptor VLA-5 (alpha(5)beta(1) integrin, CD49e/CD29). Myocardial infarction was associated with the release of FN fragments into cardiac extracellular fluids. Incubating monocytes with postreperfusion cardiac lymph that contained these FN fragments selectively reduced expression of VLA-5, an effect suppressed by specific immunoadsorption of the fragments. Treating monocytes with purified, 120-kDa cell-binding FN fragments (FN120) likewise decreased VLA-5 expression, and did so by inducing a serine proteinase-dependent proteolysis of this beta(1) integrin. We postulated that changes in VLA-5 expression, which were induced by interactions with cell-binding FN fragments, may alter monocyte migration into tissue FN, a prominent component of the cardiac extracellular matrix. Support for this hypothesis came from experiments showing that FN120 treatment significantly reduced both spontaneous and MCP-1-induced monocyte migration on an FN-impregnated collagen matrix. In vivo, it is likely that contact with cell-binding FN fragments also modulates VLA-5/FN adhesive interactions, and this causes monocytes to accumulate at sites where the fragment concentration is sufficient to ensure proteolytic degradation of VLA-5.     

Surface expression of alpha 4 integrin by CD4 T cells is required for their entry into brain parenchyma

Cloned CD4 T cell lines that recognize the Ac1-16 peptide of myelin basic protein bound to I-Au were isolated and used to analyze the immunopathogenesis of experimental autoimmune encephalomyelitis (EAE). T helper type 1 (Th1) clones induced disease, while Th2 clones did not. Using variants of a single cloned Th1 line, the surface expression of alpha 4 integrins (very late antigen 4 [VLA-4]) was identified as a major pathogenic factor. Encephalitogenic clones and nonencephalitogenic variants differ by 10-fold in their level of surface expression of alpha 4 integrin and in their ability to bind to endothelial cells and recombinant vascular cell adhesion molecule 1 (VCAM-1). The alpha 4 integrin-high, disease-inducing cloned Th1 T cells enter brain parenchyma in abundance, while alpha 4 integrin-low, nonencephalitogenic Th1 cells do not. Moreover, antibodies to alpha 4 integrin, its ligand VCAM-1, and intercellular adhesion molecule 1 all influence the pathogenicity of this encephalitogenic clone in vivo. The importance of the expression of VLA-4 for encephalitogenicity is not unique to cloned T cell lines, as similar results were obtained using myelin basic protein-primed lymph node T cells. alpha 4 integrin levels did not affect antigen responsiveness or production of the Th1 cytokines interleukin 2, interferon gamma, and lymphotoxin/tumor necrosis factor beta; and antibodies against alpha 4 integrin did not block antigen recognition in vitro. Thus, we conclude that surface expression of alpha 4 integrin is important in CD4 T cell entry into brain parenchyma. A general conclusion of these studies is that alpha 4 integrins may be crucial in allowing activated effector T cells to leave blood and enter the brain and other tissues to clear infections.     

LFA-1和VLA-4参与人高增殖潜能内皮祖细胞与血管内皮的黏附和跨内皮迁移

为了了解淋巴细胞功能相关抗原1（lymphocyte function—associated antigen1，LFA-1）和极迟反应抗原4（very lateantigen 4，VLA-4）在高增殖潜能内皮祖细胞（high proliferative potential endothelial progenitor cells，HPP—EPCs）归巢过程中与血管内皮的黏附和跨内皮迁移中的作用，利用流式细胞术检测HPP—EPC中整合蛋白B1和B2的表达以及小鼠骨髓内皮细胞相应的受体的表达。利用体外黏附和迁移实验研究经过功能级别的中和抗体阻断VLA-4和LFA-1后HPP—EPC黏附和迁移细胞数目的变化。结果表明，HPP—EPC表达整合蛋白B1和B2，活化后小鼠骨髓内皮细胞表达细胞间黏附分子1（intercellular adhesion molecule1，ICAM-1）和血管细胞黏附分子1（vascular cell adhesion molecule1，VCAM-1）；加CDlla抗体组黏附细胞或CD49d抗体组黏附和迁移细胞均较同型对照抗体组少，而且加CDlla和CD49d两种抗体联用组黏附和迁移细胞明显减少，其细胞数较任何单一抗体组少。结论：LFA-1和VLA-4在HPP—EPC与血管内皮的黏附和跨内皮迁移中发挥了重要的作用。