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PubMed1997

VLA-4-mediated interactions between normal human hematopoietic progenitors and stromal cells.

The alpha 4 beta 1 integrin very late activation antigen-4 (VLA-4) has been implicated to play a role in the adhesive interactions between hematopoietic progenitor cells (HPC) and bone marrow stromal cells which express the vascular cell adhesion molecule-1 (VCAM-1) or produce fibronectin (FN). Here, we summarize some of the recent advances made in the elucidation of the role of these particular adhesive interactions for the regulation of normal hematopoiesis. HPC bind to stroma mainly through VLA-4/VCAM-1 interactions. There is evidence which suggests that more primitive HPC constitutively express VLA-4 in a high-affinity state. In vitro studies in the mouse have shown that monoclonal antibodies (mAb) against VLA-4 partly block the development of lymphocytes, myelopoietic cells, and erythropoiesis, whereas in the human system outgrowth of TdT+ B cells is severely retarded by such mAb. In vivo studies revealed that VLA-4 is involved in erythropoietic development, and is particularly important for homing and lodgement of HPC in the bone marrow. Hematopoiesis in mice with deficient expression of alpha 4 integrin or VLA-4's ligand VCAM-1 appears to develop normally. However, chimeras developed from wild-type blastocysts and beta 1 -/- embryonic stem cells do not contain beta 1 -/- hematopoietic cells, although these are present as blood islands in the yolk sac. These beta 1 -/- hematopoietic cells are capable of forming colonies, indicating that beta 1-integrin is not involved in hematopoietic differentiation, but is primarily important for migration of hematopoietic cells into the fetal hematopoietic organs. In addition to the role of VLA-4 in migration, it may also have other regulatory functions. It has been demonstrated that ligation of VLA-4 induces phosphorylation of the protein tyrosine kinase (PTK) pp125FAK as well as other proteins which may be involved in the regulation of ligand affinity. Indeed, it has been shown that tyrosine kinase-dependent stimulation of CD34+ hematopoietic cell lines with c-kit ligand (KL), IL-3 or GM-CSF transiently activates the ability of VLA-4 to bind to VCAM-1 or FN. These events are most probably involved in the induction of quiescence in HPC which adhere to stromal cells. This claim was recently substantiated: when HPC were treated with Fab fragments of an anti-VLA-4 mAb, entry into S-phase of the cell cycle was prevented. Taken together, the present data point to a role for VLA-4 in HPC migration, cell cycle regulation, erythropoiesis and B-lymphopoiesis. Moreover, these insights may explain how defects in adhesive behavior of leukemic HPC through VLA-4 contribute to their dysregulated growth and provide a rationale for therapeutically correcting those defects.

Animals↗
PubMed1994

Activation dependent and independent VLA-4 binding sites on vascular cell adhesion molecule-1.

Vascular cell adhesion molecule-1 (VCAM) is a cytokine-inducible member of the immunoglobulin superfamily which binds to the integrin VLA-4. VCAM is expressed predominantly on the vascular endothelium where it is involved in the recruitment of mononuclear cells and lymphocytes to sites of inflammation. Two forms of VCAM containing six and seven Ig domains (VCAM-6d; VCAM-7d) are generated by alternative splicing but the physiological significance of this is unknown. We have utilised VCAM deletion mutants, VCAM-transfected cell lines and monoclonal antibodies to assess the functional importance of the individual VCAM domains. We have identified two binding sites on VCAM-7d located in domains 1 and 4 that are involved in the adhesion of the U937 human myelomonocytic cell line. Adhesion to domain 1 is temperature-independent, inhibited by the anti-VCAM mAbs 4B2 or lE10, and insensitive to PMA activation. In contrast, adhesion to domain 4 is temperature sensitive, unaffected by mAbs 4B2 or lE10 and augmented by PMA. Adhesion to both domains can be totally inhibited by the anti-VLA-4 mAb, 2B4. The anti-VCAM mAb 4B2 inhibits adhesion of U937 cells to stably transfected VCAM-7d-CHO cells at 4 degrees C, but, at 37 degrees C the effect of 4B2 on adhesion is modest with incubation times of less than 60 minutes duration. With longer incubation times, its effectiveness gradually increases, so that by 2 hours > 75% of the response can be blocked. Co-incubation with PMA prevents this time-dependent enhancement of 4B2 efficacy but has no significant effect on the inhibitory activity of the anti-VLA-4 mAb 2B4. These data can be explained by postulating a two stage ligand-receptor interaction that involves activation-induced changes in the avidity of VLA-4 for domain 4 of VCAM.

Alternative Splicing↗
PubMed1994

MAb 18D3 triggering of integrin beta 1 will prevent but not terminate proliferation of human T cells.

Triggering of integrins can deliver signals that will regulate T cell activation and proliferation when coupled with TCR/CD3 signaling. While co-activation stimuli can be achieved either with immobilized natural ligands or immobilized monoclonal antibodies specific for various integrin subunits, counterposing effects can be delivered by ligation of the integrin beta 1 chain (CD29) resulting in the downregulation of T cell proliferation. Thus, integrins may play a pivotal role in cell activation and are involved in both positive and negative regulatory pathways. In this report, anti-beta 1 mAb 18D3 was used to investigate the role of beta 1 in the negative regulation of T cell proliferation. T lymphocytes were stimulated to proliferate when activated with immobilized mAb to CD3 in conjunction with all of a panel of immobilized mAb to different alpha 4 (CD49d) and beta 1 epitopes, except the anti-beta 1.1 mAb 18D3. In soluble form, mAb 18D3 inhibited the induction of DNA synthesis dependent on costimulation of CD3 and the integrin alpha 4 subunit by a mechanism independent of anti-adhesive properties. In kinetic experiments, the addition of mAb 18D3 effectively inhibited the ultimate induction of DNA synthesis at all time points until the time coinciding with the onset of T cell proliferation, indicating that triggering the beta 1.1 epitope may only act to quench activation events prior to cellular commitment to synthesize DNA. MAb 18D3 did not induce cell death nor render cells incompetent for restimulation, but appeared to selectively inhibit IL-2 synthesis with little effect on the induction of IL-2 receptor expression.

Antibodies, Monoclonal↗
PubMed1995

Adhesion of lymphoma cells to fibronectin: differential use of alpha 4 beta 1 and alpha 5 beta 1 integrins and stimulation by the 9EG7 mAb against the murine beta 1 integrin subunit.

Murine ESb and MDAY-D2 lymphoma cells are highly metastatic, in particular to the liver, and are highly invasive in hepatocyte cultures. This may involve adhesion to hepatocyte surface-associated fibronectin (Kemperman et al., 1994, Cell Adh. and Communic. 2:45). Both ESb and MDAY-D2 cells express the fibronectin receptor alpha 4 beta 1, and MDAY-D2 cells in addition also alpha 5 beta 1. Yet, adhesion of ESb cells to fibronectin was low, and MDAY-D2 cells did not adhere at all, but adhesion of both cells was stimulated by phorbol myristate acetate (PMA) and Mn2+. In ESb cells, this adhesion was mediated by alpha 4 beta 1. In MDAY-D2 cells, however, only alpha 5 beta 1 was involved, despite alpha 4 beta 1 levels similar to ESb cells. The alpha 4 beta 1 integrin was functional since it mediated adhesion of MDAY-D2 cells to VCAM-1. An alpha 5 beta 1-negative variant of MDAY-D2 adhered to fibronectin and this was mediated by alpha 4 beta 1. These results indicate that alpha 4 beta 1 function in these cells is suppressed in the presence of alpha 5 beta 1. Adhesion of ESb cells to hepatocytes was inhibited by anti-alpha 4 antibody, but only by 30%, and fibronectin adhesion was found to have no role in the interaction of MDAY-D2 cells with hepatocytes. This suggests that alpha 4 beta 1 and alpha 5 beta 1 function is not activated during this interaction. The 9EG7 antibody against mouse beta 1 integrin was described to inhibit beta 1 integrins (Lenter et al., 1993, Proc. Natl. Acad. Sci. USA, 90, 9051). In contrast, we observed that 9EG7 stimulated beta 1-integrin function: Adhesion of ESb and MDAY-D2 cells not only to fibronectin, but also to laminin was induced or enhanced.

Animals↗
PubMed1996

The alpha 4 beta 1 fibronectin ligands CS-1, Hep II, and RGD induce different intracellular events in B lymphoid cells. Comparison with the effects of the endothelial ligand VCAM-1.

The lymphocyte integrin alpha 4 beta 1 is the receptor for the Hep II domain and CS-1 site in fibronectin (Fn) as well as for VCAM-1. We recently showed that upon activation with anti-beta 1 mAb TS2/16, alpha 4 beta 1 also recognizes the RGD Fn sequence. To determine the physiological role of these multiple interactions, we have now studied some intracellular events induced by "resting" and activated alpha 4 beta 1 binding to its different ligands. Analyses of actin and tubulin reorganization upon adhesion of B lymphoid cells to Fn fragments or VCAM-1 showed that VCAM-1, a 38 kDa fragment (Hep II+CS-1), and the CS-1 synthetic peptide induced formation of transient cytoplasmic projections; however, cells attached to a 58 kDa (Hep II) or 80 kDa (RGD) fragments remained rounded. Using transfilter assays, we showed that VCAM-1, 38 kDa and CS-1 also induced dose-dependent B cell migration mediated by alpha 4 beta 1. Furthermore, these three ligands, but not the 80 kDa fragment or a synthetic peptide (H1) containing a sequence from Hep II shown to bind alpha 4 beta 1, induced tyrosine phosphorylation of a 110 kDa protein. Activation of alpha 4 beta 1 with TS2/16 inhibited the cytoplasmic protrusions and cell migration but did not affect the pattern of phosphorylation. Our results indicate that the various alpha 4 beta 1 ligands induce different cellular responses. Most importantly they show that alpha 4 beta 1 interaction with CS-1 is sufficient to trigger intracellular events in B cells. Furthermore, they suggest a regulation by the activation form of the receptor as well as by the ligand in events involving lymphocyte adhesion and migration.

Amino Acid Sequence↗
PubMed1996

The type III connecting segment of fibronectin contains an aspartic acid residue that regulates the rate of binding to integrin alpha 4 beta 1.

The type III connecting segment (IIICS) within fibronectin is the major binding site for the integrin alpha 4 beta 1. Most integrin ligands have an essential acidic residue within their integrin binding site, in IIICS this residue is hypothesized to be the aspartic acid at position 21. Alanine scanning mutagenesis was used to determine the amino acid residues within the intact IIICS domain required for interaction with alpha 4 beta 1. IIICS was cloned and expressed as a fusion protein with glutathione S-transferase. This recombinant form of IIICS supports the adhesion of CHO cells that express human alpha 4 beta 1 in a cation dependent manner. Alanine scanning mutagenesis of the EILDVP sequence in recombinant IIICS demonstrated that only two of these residues are critical for adhesion of alpha 4 beta 1 expressing cells. Mutations of leucine at position 20 and aspartic acid at position 21 to alanine significantly reduced cell adhesion. Conservative mutations of aspartic acid at position 21 to asparagine or glutamic acid also reduced the ability of the recombinant protein to support cell adhesion, although not to the same extent as the corresponding alanine replacement. Most importantly, we show that although the mutation of asp 21 impairs cell adhesion, an examination of cell adhesion as a function of time demonstrated that asp 21 is not necessary for cell adhesion through alpha 4 beta 1. In comparison to wild type IIICS, the asp 21 to ala mutant supported minimal adhesion at early time points (10-30 min.), but was equivalent to wild type IIICS in supporting adhesion over one hour.

Amino Acids↗
PubMed1998

Dual control by divalent cations and mitogenic cytokines of alpha 4 beta 1 and alpha 5 beta 1 integrin avidity expressed by human hemopoietic cells.

Beta-1 integrins have essential functions in hemopoietic and immune systems by controlling phenomenons such as cell homing and cell activation. The function alpha 4 beta 1 and alpha 5 beta 1 integrins is regulated by divalent cations and, as demonstrated more recently, by mitogenic cytokines which activate them by "inside-out" mechanisms. Using the adhesive interaction of a cytokine-dependent human hemopoietic cell line to immobilized fibronectin, we have analyzed the requirements in divalent cations Mn2+, Mg2+ and Ca2+ for alpha 4 beta 1 and alpha 5 beta 1 activation by "inside-out" mechanisms triggered by cytokines such as granulocyte-macrophage colony stimulating factor or KIT ligand, or by external conformational constraints with the function-activating anti-beta 1 integrin monoclonal antibody 8A2. The intrinsic difference between these two modes of beta 1 integrin activation was revealed by their different requirements in divalent cations. We found that in the absence of any divalent cations, alpha 4 beta 1 and alpha 5 beta 1 were non-functional even after further stimulation by cytokines or 8A2. However, whilst either Ca2+, Mg2+ or Mn2+ were able to restore adhesive functions of alpha 4 beta 1 and alpha 5 beta 1 when activated by 8A2, only Mg2+ and Mn2+ were able to support activation of alpha 4 beta 1 and alpha 5 beta 1 by cytokines. Furthermore, high concentrations of Ca2+ exceeding 20 mM dramatically inhibited cell adhesion to fibronectin induced by Mn2+ and cytokines but not by 8A2. On the contrary, in the presence of both Ca2+ and Mg2+, Mn2+ had an additive effect on the activation of alpha 4 beta 1 and alpha 5 beta 1 by mitogenic cytokines. The presence of the absence of these divalent cations did not inhibit early tyrosine phosphorylation induced by the binding of KIT ligand to its tyrosine-kinase receptor KIT. Therefore, we propose that in hemopoietic cells, Ca2+, Mg2+ and Mn2+ may modulate in vivo alpha 4 beta 1 and alpha 5 beta 1 regulation by mitogenic cytokines, a phenomenon involved in the regulation of hemopoietic progenitor cell homing within the bone marrow.

Anti-Allergic Agents↗
PubMed2001

Cell cycling status of human cord blood CD34+ cells during ex vivo expansion is related to the level of very late antigen expression.

Very late antigen-4 (VLA-4), which binds to the extracellular matrix protein fibronectin, is an integrin molecule known to be modulated during mobilization of CD34+ cells, and to be involved in signaling the mobilization stimuli. On the hypothesis that cell cycling status might be different depending on the level of VLA-4 expression, we investigated the DNA contents of human cord blood CD34+ cells during ex vivo expansion by recombinant human thrombopoietin and flt3-ligand with simultaneous measurement of surface VLA-4 at the 1st and 4th week. During this ex vivo expansion, expression of VLA-4 increased and almost all cells became VLA-4+ until the 4th day of culture. Expression of VLA-4 was maintained in the major population of the cultured cells until the 4th week. The cells in S/G2/M phase were greater in number in VLA-4 high fraction than in VLA-4 low fraction (n=4, p<.001). Furthermore, the fraction of cells in S/G2/M phase increased as the expression of VLA-4 became higher. These results suggest that cord blood CD34+ cells expressing high levels of VLA-4 have more proliferative activities.

Antigens, CD34↗
PubMed2004

Stem cells expressing homing receptors could be expanded from cryopreserved and unselected cord blood.

We assessed the cytokine combinations that are best for ex vivo expansion of cord blood (CB) and the increment for cell numbers of nucleated cells, as well as stem cells expressing homing receptors, by an ex vivo expansion of cryopreserved and unselected CB. Frozen leukocyte concentrates (LC) from CB were thawed and cultured at a concentration of 1 x 10(5)/mL in media supplemented with a combination of SCF (20 ng/mL)+TPO (50 ng/mL)+FL (50 ng/mL)+/-IL-6 (20 ng/mL)+/-G-CSF (20 ng/mL). After culturing for 14 days, the expansion folds of cell numbers were as follows: TNC 22.3+/-7.8 approximately 26.3+/-4.9, CFU-GM 4.7+/-5.1 approximately 11.7+/-2.6, CD34+CD38- cell 214.0+/-251.9 approximately 464.1+/-566.1, CD34+CXCR4+ cell 4384.5+/-1664.7 approximately 7087.2+/-4669.3, CD34+VLA4+ cell 1444.3+/-1264.0 approximately 2074.9+/-1537.0, CD34+VLA5+ cell 86.2+/-50.9 approximately 113.2+/-57.1. These results revealed that the number of stem cells expressing homing receptors could be increased by an ex vivo expansion of cryopreserved and unselected CB using 3 cytokines (SCF, TPO, FL) only. Further in vivo studies regarding the engraftment after expansion of the nucleated cells, as well as the stem cells expressing homing receptors will be required.

ADP-ribosyl Cyclase↗
PubMedDate not supplied

Obstructive jaundice facilitates hepatic metastasis of B16F1 mouse melanoma cells: participation of increased VCAM-1 expression in the liver.

Obstructive jaundice facilitates experimental liver metastasis in the rat model, but the detailed mechanisms of this facilitation remain unclear. This study aimed to evaluate the contribution of vascular cell adhesive molecules-1 (VCAM-1) to augmented hepatic metastasis in cases of obstructive jaundice. Obstructive jaundice was induced in male C57BL/6 mice by common bile duct obstruction for 5 days using a Surgiclip. For the biliary decompression, obstructive jaundice was induced for 5 days, followed by removal of the Surgiclip. Liver specimens and blood samples were obtained from animals 5 days after biliary obstruction (OJ5) or sham operation and 2, 5, 11, 14 days after biliary decompression. The expression of VCAM-1 mRNA was increased in the livers from the OJ5 group. Western blot analysis demonstrated increased expression of VCAM-1 protein in the livers of the OJ5 group, in contrast with low VCAM-1 expression in the sham group. The expression of VCAM-1 protein was sustained at high levels at 2 days and decreased at 5 days after biliary decompression (BD5). For the induction of experimental hepatic metastasis, male C57BL/6 mice were randomized to three groups (sham, OJ5, BD5) of six animals each. B16F1 melanoma cells were introduced into the animals by an intraportal injection. Metastatic colonies in the livers were investigated 13 days after inoculation. The mean number of metastatic colonies was significantly increased in the OJ5 group (70.5+/-51.2) compared to that of the sham group (7.2+/-7.9) (p<0.05). This augmentation of hepatic metastasis was abrogated in the BD5 group (16.8+/-20.3). In conclusion, our results suggest that augmented hepatic metastasis in cases of obstructive jaundice are partly mediated through VCAM-1/VLA-4 interaction.

Animals↗
PubMed2000

Alpha 4 beta 1 integrin/VCAM-1 interaction activates alpha L beta 2 integrin-mediated adhesion to ICAM-1 in human T cells.

Modulation of integrin affinity and/or avidity provides a regulatory mechanism by which leukocyte adhesion to endothelium is strengthened or weakened at different stages of emigration. In this study, we demonstrate that binding of high-affinity alpha 4 beta 1 integrins to VCAM-1 strengthens alpha L beta 2 integrin-mediated adhesion. The strength of adhesion of Jurkat cells, a human leukemia T cell line, or MnCl2-treated peripheral blood T cells to immobilized chimeric human VCAM-1/Fc, ICAM-1/Fc, or both was quantified using parallel plate flow chamber leukocyte detachment assays in which shear stress was increased incrementally (0.5-30 dynes/cm2). The strength of adhesion to VCAM-1 plus ICAM-1, or to a 40-kDa fragment of fibronectin containing the CS-1 exon plus ICAM-1, was greater than the sum of adhesion to each molecule alone. Treatment of Jurkat or blood T cells with soluble cross-linked VCAM-1/Fc or HP2/1, a mAb to alpha 4, significantly increased adhesion to ICAM-1. These treatments induced clustering of alpha L beta 2 integrins, but not the high-affinity beta 2 integrin epitope recognized by mAb 24. Up-regulated adhesion to ICAM-1 was abolished by cytochalasin D, an inhibitor of cytoskeletal rearrangement. Taken together, our data suggest that the binding of VCAM-1 or fibronectin to alpha 4 beta 1 integrins initiates a signaling pathway that increases beta 2 integrin avidity but not affinity. A role for the cytoskeleton is implicated in this process.

Cell Adhesion↗
PubMed2000

The accumulation of dendritic cells in the lung is impaired in CD18-/- but not in ICAM-1-/- mutant mice.

Bone marrow-derived dendritic cell (DC) precursors migrate via the blood stream to peripheral tissues to adopt their sentinel function. To identify factors facilitating their emigration to the lung, mutant mice deficient in E-selectin, P-selectin, E/P-selectin, ICAM-1, or CD18 and their respective controls were examined. DCs and monocytes/macrophages were immunolabeled with M5/114 and MOMA-2 mAbs, respectively, and quantified morphometrically. Of these genotypes, the numbers of DC and MOMA-2+ cells were significantly less only in the lungs of CD18-/- mice by 68 and 35% in alveolar walls and by 28 and 26% in venous walls, respectively. DCs were reduced by 30 and 41% around large and small airways, respectively, but the number of MOMA-2+ cells in these locations was not significantly different from controls. Ablation of a single gene may be associated with augmented expression of other, related gene products. Therefore, we examined the expression of VCAM-1. Increased numbers of arteries exhibited continuous luminal VCAM-1 staining in both CD18-/- and ICAM-1-/- mutants. VCAM-1 expression was absent in pulmonary capillaries and unchanged in veins. These data suggest that under nonperturbing conditions, CD18-mediated adhesion is required for the full complement of DC precursors to accumulate in the lungs. However, the defect in CD18-/- mice is partial, suggesting that CD18-independent adhesion occurs. The alternative pathway may involve VLA-4/VCAM-1 in arteries and venules but not in capillaries. The smaller defect in ICAM-1-/- mice suggests that the CD11/CD18 complex recognizes ligands other than ICAM-1 at some sites.

Animals↗
PubMed2000

Alpha 4 integrin-dependent leukocyte recruitment does not require VCAM-1 in a chronic model of inflammation.

Rats immunized with Mycobacterium butyricum in Freund's adjuvant develop a chronic vasculitis, with large increases in leukocyte rolling and adhesion in mesenteric postcapillary venules that are significantly inhibited with an alpha 4 integrin Ab. Using intravital microscopy to visualize chronically inflamed microvessels, we demonstrated that alpha 4 integrin-dependent leukocyte rolling and adhesion was inhibited with a beta 1 integrin, but not a beta 7 integrin Ab. To date, VCAM-1 has been presumed to be the primary ligand for alpha 4 beta 1 integrin in the vasculature. However, alpha 4 beta 1 integrin-dependent interactions were not reduced by monoclonal or polyclonal VCAM-1 Abs or a VCAM-1 antisense oligonucleotide despite increased VCAM-1 expression in the mesenteric vasculature. To ensure that the VCAM-1 Abs were functional and used at saturating concentrations, blood from Ab-treated rats was perfused over monolayers of CHO cells transfected with rat VCAM-1. Sufficient alpha 4 integrin or VCAM-1 Ab was present to inhibit leukocyte interactions with rat VCAM-1 by 95-100%. Under in vitro flow conditions, only mononuclear leukocytes were recruited from blood of control rats onto purified VCAM-1. However, neutrophils were also recruited onto VCAM-1 from whole blood of adjuvant-immunized animals via alpha 4 integrin. Another ligand for alpha 4 beta 1 integrin is the connecting segment-1 (CS-1) region of fibronectin. An Ab to the CS-1 portion of fibronectin, which did not reduce rolling and adhesion in adjuvant arthritis animals, completely inhibited leukocyte adhesion to CS-1 under static conditions. These findings provide the first evidence that alpha 4 beta 1 integrin-dependent leukocyte rolling and adhesion can occur in vivo via a mechanism other than VCAM-1.

Adjuvants, Immunologic↗
PubMed2000

Migration of eosinophils across endothelial cell monolayers: interactions among IL-5, endothelial-activating cytokines, and C-C chemokines.

Eosinophils are the predominant cell type recruited in inflammatory reactions in response to allergen challenge. The mechanisms of selective eosinophil recruitment in allergic reactions are not fully elucidated. In this study, the ability of several C-C chemokines to induce transendothelial migration (TEM) of eosinophils in vitro was assessed. Eotaxin, eotaxin-2, monocyte chemotactic protein (MCP)-4, and RANTES induced eosinophil TEM across unstimulated human umbilical vein endothelial cells (HUVEC) in a concentration-dependent manner with the following rank order of potency: eotaxin approximately eotaxin-2 > MCP-4 approximately RANTES. The maximal response induced by eotaxin or eotaxin-2 exceeded that of RANTES or MCP-4. Preincubation of eosinophils with anti-CCR3 Ab (7B11) completely blocked eosinophil TEM induced by eotaxin, MCP-4, and RANTES. Activation of endothelial cells with IL-1beta or TNF-alpha induced concentration-dependent migration of eosinophils, which was enhanced synergistically in the presence of eotaxin and RANTES. Anti-CCR3 also inhibited eotaxin-induced eosinophil TEM across TNF-alpha-stimulated HUVEC. The ability of eosinophil-active cytokines to potentiate eosinophil TEM was assessed by investigating eotaxin or RANTES-induced eosinophil TEM across resting and IL-1beta-stimulated HUVEC in the presence or absence of IL-5. The results showed synergy between IL-5 and the chemokines but not between IL-5 and the endothelial activator IL-1beta. Our data suggest that eotaxin, eotaxin-2, MCP-4, and RANTES induce eosinophil TEM via CCR3 with varied potency and efficacy. Activation of HUVEC by IL-1beta or TNF-alpha or priming of eosinophils by IL-5 both promote CCR3-dependent migration of eosinophils from the vasculature in conjunction with CCR3-active chemokines.

Antibodies, Monoclonal↗
PubMed2000

Up-regulation of VCAM-1 and differential expansion of beta integrin-expressing T lymphocytes are associated with immunity to pulmonary Mycobacterium tuberculosis infection.

Immune responses rely on an intricate system of adhesion molecules to coordinate the homing and retention of lymphocytes in both secondary lymphoid tissues and at sites of infection. To define the events associated with pulmonary immune responses, the expression of endothelial addressins and integrins on T cells was analyzed during Mycobacterium tuberculosis infection. In infected lung, expression of endothelial VCAM-1, but not mucosal addressin cell adhesion molecule-1, was up-regulated from 4 wk postinfection and persisted to at least 12 wk. Subsequent analysis of the corresponding integrins expressed on lung CD4+ and CD8+ T cells revealed an accumulation of beta1high/beta7-/low, and to a lesser extent beta7high, integrin-expressing T cells during infection. Examination of integrin heterodimers showed that while alpha4 integrin was predominantly expressed on beta1high/beta7-/low cells, alphaE integrin was primarily associated with beta7high. The majority of activated/memory T cells recruited during infection expressed high levels of beta1 integrin and undetectable or low levels of beta7 integrin. These T cells were capable of producing IFN-gamma, a cytokine crucial for controlling M. tuberculosis infection. Rapid expansion of beta1high, beta7-, and beta7high T cell populations in the lung upon secondary mycobacterial infection indicates the participation of these populations in the acquired immune response to the infection. Furthermore, treatment of infected mice with mAb to alpha4 or alpha4beta7 integrin led to a reduction in lymphocytes and increase in granulocytes in the pulmonary infiltrate. These results reveal a crucial role for adhesion molecules in the generation of an effective pulmonary immune response to M. tuberculosis infection.

Animals↗
PubMed2000

Multiple defects in Fc epsilon RI signaling in Syk-deficient nonreleaser basophils and IL-3-induced recovery of Syk expression and secretion.

Human basophils respond to Ag-induced cross-linking of their high affinity IgE receptor, FcepsilonRI, by releasing histamine and other mediators from granules, producing IL-4 and other cytokines and, as shown in this study, by forming membrane ruffles and showing increased very late Ag-4 (VLA-4)-mediated adhesion to VCAM-1-expressing target cells. We have identified five blood donors whose basophils lack detectable levels of the FcepsilonRI-associated protein tyrosine kinase, Syk. Despite showing no obvious ultrastructural differences from normal basophils, nonreleaser basophils fail to form membrane ruffles, to show increased VLA-4-mediated adhesive activity, or to produce IL-4 in response to FcepsilonRI cross-linking. Although Syk protein levels are suppressed in basophils from all five donors, Syk mRNA is consistently present. Furthermore, culturing nonreleaser basophils for 4 days with IL-3 restores Syk protein expression and FcepsilonRI-mediated histamine release. Understanding the reversible suppression of Syk protein expression in nonreleaser basophils, and learning to replicate this property in patients with allergic inflammation could be a powerful and specific way to limit symptomatic disease.

Antibodies, Anti-Idiotypic↗
PubMed2000

Annexin 1 binds to U937 monocytic cells and inhibits their adhesion to microvascular endothelium: involvement of the alpha 4 beta 1 integrin.

Annexin 1 (ANX1), a calcium-binding protein, participates in the regulation of early inflammatory responses. Whereas some of its effects depend on intracellular interactions, a growing number of observations indicate that ANX1 may also act via autocrine/paracrine functions following externalization to the outer side of the plasma membrane. We studied the effects of ANX1 on leukocyte adhesion to endothelial cells using as a model system the monocytic cell line U937 and human bone marrow microvascular endothelial cells. Exogenous rANX1, as well as endogenous ANX1 externalized by U937 differentiated in vitro, inhibited monocyte firm adhesion to vascular endothelium. Both binding of ANX1 to U937 cells and ANX1-mediated inhibition of cell adhesion involved the short N-terminal domain of the ANX1 molecule. Under experimental conditions in which ANX1 inhibited U937 adhesion to human bone marrow microvascular endothelial cells, this protein specifically colocalized with the alpha 4 integrin, and a direct interaction between ANX1 and the alpha 4 integrin could be documented by immunoprecipitation experiments. Moreover, ANX1 competed with the endothelial integrin counterreceptor, VCAM-1, for binding to alpha 4 integrin. These results indicate that ANX1 plays an important physiological role in modulating monocyte firm adhesion to the endothelium.

Annexin A1↗
PubMed2001

Homing of in vitro-generated donor antigen-reactive CD4+ T lymphocytes to renal allografts is alpha 4 beta 1 but not alpha L beta 2 integrin dependent.

The extravasation and sequestration of Ag-reactive T lymphocytes into vascularized organ allografts depend on a cascade of complex interactions among circulating lymphocytes, endothelial cells, and extracellular matrix proteins. Ag-activated donor-specific CD4 T cells are major initiators and effectors in the allograft rejection response. Interfering with the intragraft homing of activated CD4 T cells may represent a novel therapeutic approach in transplant recipients. We have developed a FACS-based short-term homing assay that allows tracing in vitro-generated Ag-reactive CD4 T cells after adoptive transfer in test rat recipients. Allospecific cell lines were preincubated with anti-alpha(4)beta(1) or anti-alpha(L)beta(2) mAb, because of enhanced expression of both integrin receptors after alloactivation. The pretreated Lewis(BN) lymphocytes were carboxyfluorescein diacetate succinimidyl ester labeled and adoptively transferred into Lewis rat recipients of Brown Norway kidney allografts. The injection of equal numbers of PKH-26-labeled untreated cells allowed quantitative comparison of both populations in the same animal. Ex vivo treatment with anti-alpha(4)beta(1) mAb diminished intragraft infiltration of adoptively transferred T cells by 85% in a donor-specific fashion. In contrast, treatment with anti-alpha(L)beta(2) mAb did not affect intragraft cell sequestration. Hence, blocking alpha(4)beta(1) integrin interactions represents a novel strategy in preventing local intragraft recruitment of Ag-reactive CD4 T cells in transplant recipients.

Adoptive Transfer↗