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PubMed1996

VLA-5 in the plasma cell line, FR4ds, acts as a common regulator of VLA-4 and VLA-6 in spreading induced by fibronectin and laminin.

VLA-5 recognizes the GRGDSP sequence of fibronectin (FN) in the extracellular matrix (ECM). We examined the role of beta1 integrin in the spreading of the human plasma cell line, FR4ds, induced by FN and laminin (LN). We first examined the role of VLA-5 in the spreading induced by FN. Anti-alpha4 antibody induced 46.4% inhibition, whereas anti-alpha5 had no effect. A combination of anti-alpha4 and anti-alpha5 enhanced the inhibition of spreading significantly. Complementary inhibition was also demonstrated using the GRGDSP peptide plus anti-alpha4 and the GRGDNP peptide of LN plus anti-alpha4. The results suggested that VLA-5 is a regulator of VLA-4 and that it is involved in the recognition of GRGDNP. We then examined the role of VLA-5 in the spreading induced by LN. Anti-alpha6 induced 53.1% inhibition. Anti-alpha5 alone had no effect. A combination of alpha5 and anti-alpha6, however, significantly enhanced the inhibition of spreading. The combination of GRGDSP plus anti-alpha6 and GRGDNP plus anti-alpha6 resulted in complete inhibition. These results suggested that VLA-5 participates in the recognition of LN cooperatively with VLA-6 and that VLA-5 is a common regulator of VLA-4 and the LN receptor, VLA-6.

Cell Movement↗
PubMed1997

Expression of functional ICAM-1 and VCAM-1 adhesion molecules by an immortalized epithelial cell clone derived from the small intestine.

The role of small bowel-derived epithelial cells in regulating the accumulation of inflammatory cells within the inflamed gut epithelium is poorly understood because of the difficulties in culturing the epithelial cells in vitro. We have recently developed a cloned epithelial cell line (IEC-4.1) derived from the small intestine of BALB/c mice. In the present study, we examined whether IEC-4.1 cells could express adhesion molecules ICAM-1 and VCAM-1 and the molecular basis of macrophage adhesion to the epithelial cells. Northern blot analysis demonstrated that IEC-4.1 cells constitutively expressed ICAM-1 and VCAM-1 mRNA at low levels. Stimulation with LPS (12 microg/ml) or TNF-alpha (2.5 ng/ml) markedly upregulated ICAM-1 and VCAM-1 gene expression in IEC-4.1 cells. ICAM-1 mRNA started to increase 2 hr after LPS stimulation, peaked at 4 hr, and then decreased rapidly to the basal level at 8 hr. VCAM-1 mRNA had the similar pattern of upregulation but the increased VCAM-1 mRNA sustained over a longer period of time and did not return to the basal level until 24 hr after the stimulation. IEC-4.1 cells expressed very low basal levels of ICAM-1 and VCAM-1 on the cell surface as demonstrated by immunofluorescence staining and FACS analysis. Stimulation of IEC-4.1 cells with LPS or TNF-alpha markedly increased the surface expression of both ICAM-1 and VCAM-1, which correlated with the increased binding of macrophages to the stimulated IEC-4.1 cells. Adherence of macrophages to the IEC-4.1 cells was mediated by both LFA-1/ICAM-1 and VLA-4/VCAM-1 since blocking both adhesion pathways inhibited macrophage adhesion by about 90%. These findings suggest that small bowel-derived epithelial cells may be capable of expressing a defined set of functional adhesion molecules during mucosal inflammation.

Animals↗
PubMed1998

Interleukin-3 and Flt3-ligand induce adhesion of Baf3/Flt3 precursor B-lymphoid cells to fibronectin via activation of VLA-4 and VLA-5.

Adhesion of hematopoietic cells to extracellular matrix components is important for blood cell development. However, little is known regarding the potential influence of IL-3 on this process for precursor B cells and Flt3-ligand has not yet been implicated in induction of adhesion of any blood cell types to extracellular matrix components. Therefore, we examined the characteristics of cytokine-induced cell adhesion to fibronectin (FN), using as a model the murine precursor B cell line, Baf3, a factor-dependent cell line requiring IL-3 for both growth and survival. Since factor-dependent hematopoietic cell lines expressing Flt3 receptor are extremely rare, we also studied Baf3/Flt3, a subline of Baf3 transduced with the Flt3 receptor gene. IL-3 induced adhesion of Baf3 and Baf3/Flt3 cells to FN, while Flt3-ligand induced adhesion of Baf3/Flt3 cells only. Whereas both Baf3 and Baf3/Flt3 cells expressed VLA-4 and -5 integrins as FN receptors, expression levels of VLA-4 and -5 were not affected by IL-3 or Flt3-ligand treatment. However, blocking experiments using anti-integrin antibodies showed that cytokine-induced adhesion of cells depended on both VLA-4 and -5 suggesting that IL-3 and Flt3-ligand activated these integrins. PI-3 kinase inhibitor wortmannin, PKC inhibitor H-7, or PKA inhibitor HA1004 did not suppress adhesion induced by IL-3 or Flt3-ligand; in contrast, PLC inhibitor U-73122 did suppress adhesion, suggesting the possibility that PLC, but not PI-3 kinase, PKC, or PKA, may be involved in this process. Since it is known that IL-3 and Flt3-ligand receptors are expressed on precursor B cells, and these receptors are downregulated during B cell maturation of primary cells, the induction of precursor B cell adhesion to FN by IL-3 and Flt3-ligand may contribute a mechanism by which precursor B cells adhere to bone marrow stroma, thereby influencing their development.

Animals↗
PubMed1998

Prevention of experimental allergic encephalomyelitis by an antibody to CD45RB.

CD45 is involved in the regulation of lymphocyte activation, and it has been demonstrated that ligation of CD45 induces apoptosis of T and B lymphocytes. Recently anti-CD45RB antibody therapy was shown to block acute allograft rejection in a mouse model of transplantation. Therefore, we wanted to examine the effects of anti-CD45RB antibody treatment on the course of an autoimmune disorder, experimental allergic encephalomyelitis (EAE), a Th1-mediated process. Mice immunized with myelin basic protein and treated with anti-CD45RB antibody did not develop EAE. Histologically, there was no evidence of lymphocytic infiltrates in the central nervous system. T cell proliferation and TNF-alpha production were significantly decreased in anti-CD45RB-treated mice. Furthermore, there was a significant reduction in the production of other Th1 cytokines including interferon-gamma and IL-2, but not IL-4 or IL-6. However, levels of a number of adhesion markers or markers of activation such as VLA-4 and LFA-1 on T cells were no different in treated versus control animals. Thus, anti-CD45RB can prevent EAE and appears to do so by altering T cell proliferation and cytokine production.

Animals↗
PubMed1999

Characterization of B lymphocytes rescued from apoptosis by platelet-activating factor.

B lymphocyte development is characterized by deletion via apoptosis of immature cells that are insufficiently stimulated. We have previously demonstrated that crosslinking of the B cell receptor (BCR) using anti-IgM antibodies (alphaIgM) (2 microg/ml) in Ramos B lymphoblastoid cells causes deletion of 30-40% of cells by apoptosis in 24 h. Addition of the potent lipid mediator platelet-activating factor (10(-7) M) to alphaIgM stimulated Ramos cells significantly decreases the number of apoptotic cells as measured by annexin V labeling. We have characterized the phenotype of Ramos cells that have not become apoptotic following BCR stimulation. In these cells, there is a significant decrease in the surface expression of the VLA-4 adhesion molecule (31% of control expression) and surface IgM expression (sIgM) (53% of control expression). Significantly fewer cells co-incubated with platelet-activating factor (PAF) underwent apoptosis, and the remaining cells maintained control levels of VLA-4 (104% of control expression) and sIgM expression (104% of control). All of these protective effects were inhibited by the specific PAF receptor antagonist, WEB 2170. The action of PAF on alphaIgM induced apoptosis was not inhibited by either cycloheximide or cytochalasin B, suggesting that de novo protein synthesis and F-actin polymerization were not implicated in the rescue of Ramos cells by PAF. In contrast, the ability of PAF to maintain sIgM and VLA-4 expression at control levels was inhibited by cycloheximide (7. 5 microg/ml). Cytochalasin B (5 microg/ml) had no effect on sIgM expression but blocked the decrease in VLA-4 expression mediated by alphaIgM. These data indicate that PAF's effect on rescuing and maintaining alphaIgM stimulated Ramos B cells is mediated via at least two pathways. Abrogation of apoptosis does not require de novo protein synthesis, while maintenance of sIgM and VLA-4 expression requires protein synthesis.

Apoptosis↗
PubMed2000

CD8(+) T cells secreting type 2 lymphokines are defective in protection against viral infection.

Effector T cells secreting type 1 and/or type 2 lymphokines (Tc1, Tc0, Tc2) were generated in vitro from CD8(+) T cells of mice with a transgenic TCR recognizing lymphocytic choriomeningitis virus (LCMV) glycoprotein to compare their effector function in vitro and in vivo. Tc1, Tc2, and Tc0 showed similar Fas- and perforin-mediated cytotoxicity in vitro. Upon adoptive transfer, Tc2 and Tc0 effectors were less efficient than Tc1 at controlling LCMV or recombinant vaccinia virus expressing the LCMV glycoprotein in vivo. Tc2 and Tc0 had decreased surface VLA-4 density and deficient activation-induced LFA-1/ICAM-1-dependent homotypic adhesion in vitro. Therefore, the reduced antiviral activity in vivo of Tc2 and Tc0 compared with Tc1 is not due to reduced cytotoxic activity or IFN-gamma secretion but may be explained by defective homing to the target organ due to decreased expression and/or lower activity of adhesion molecules.

Adoptive Transfer↗
PubMed2001

Adhesion of neutrophils to fibronectin: role of the cd66 antigens.

Adhesion of neutrophils to substrate is initiated by receptor-ligand interactions that induce outside-in signaling. Inside-out signals and lateral interactions between surface molecules further fine tune the response. This study investigates the role of CD66 in adhesion of neutrophils to fibronectin, using domain-mapped monoclonal antibodies to CD66. Neutrophils express CD66a, CD66b, and CD66c on their surface. The neutrophil surface molecules that bind to fibronectin are the alpha(4)beta(1) and alpha(5)beta(1) integrins. Our results show that the monoclonal antibody Kat4c, which recognizes the AB domain of CD66a, b, and c and the polyclonal anti-CD66 (anti-carcinoembryonic antigen), augments neutrophil adhesion to fibronectin, while monoclonal antibodies to the individual CD66 antigens, the Fab fragment of Kat4c, and a mixture of the individual antibodies to CD66 antigens were unable to affect the adhesion. Thus heterodimerization of CD66a, b, and c is required for promoting neutrophil adhesion to fibronectin. The increased adhesion in presence of Kat4c was inhibited by antibodies to the beta(1) and beta(2) integrins. Antibody ligation of CD66 antigens causes their clustering and concomitant coclustering of the alpha(M) subunit of the beta(2) integrin, thereby activating the integrin. The sugar alpha-methyl mannoside inhibited anti-CD66-mediated clustering, indicating that a carbohydrate-lectin interaction may exist between CD66 and alpha(M) integrin. It also reduced the increased adhesion of neutrophils to fibronectin, suggesting that beta(2) integrin activation precedes beta(1) integrin activation. Further, the anti-CD66-mediated adhesion to fibronectin is accompanied by increased localization of Src family kinases (lyn and hck) to the cytoskeleton and an increase in their kinase activity. These results suggest that crosslinking of CD66a, CD66b, and CD66c promotes activation of the beta(2) integrin and in turn an alteration in the affinity of the beta(1) integrin, which enhances the adhesion of neutrophils to fibronectin.

Antibodies, Monoclonal↗
PubMed1997

T-lymphocytes from individuals with filarial inflammatory disease have increased transendothelial migration in vitro.

The in vitro transendothelial migration of circulating filarial antigen-specific T-cells was examined in Wuchereria banerofti infection. Circulating T-cells from individuals with filaria-induced lymphatic pathology (LP) had significantly greater migration through unstimulated HUVEC monolayers than did T-cells from asymptomatic infected (MF) individuals (P = 0.04). In contrast to the MF individuals where no effect was seen, transendothelial migration of 48-hr filarial antigen stimulated T-cells from LP individuals was significantly (P = 0.01) greater than migration of 48-hr media-stimulated T-cells. In six of seven patients examined, inhibition of the VLA-4/VCAM-1 pathway resulted in greater than 50% inhibition of transendothelial migration of T-cells.

Adult↗
PubMed1997

Characterization of adhesion molecule expression in the pathogenesis of homologous collagen-induced arthritis in the DA rat.

We have investigated the inflammatory cell infiltrates and adhesion molecule expression in the synovial fluid (SF) and synovial membranes (SM) of rats with homologous collagen-induced arthritis. Immunohistochemical staining revealed that the majority of the cells in the SF were granulocytes, expressing CD11b and CD11c. In SM, the majority of the cells were monocytes/macrophages. CD49d and CD49f were expressed mainly in the erosion zone in SM, and ICAM-1 was expressed in the lining layer, in the capillaries, and in the erosion zone. In SF 7% of the cells were ICAM-1 positive. CD2 was more abundant in SM than in SF. These findings might explain the difference in granulocyte counts between SF and SM. CD49d and CD49f expression might retain lymphocytes and monocytes in SM, while granulocytes not expressing CD49d and CD49f are not retained.

Animals↗
PubMed1996

The novel recognition site in the C-terminal heparin-binding domain of fibronectin by integrin alpha 4 beta 1 receptor on HL-60 cells.

The hematopoietic cell recognition sites of human fibronectin (FN) are the Arg-Gly-Asp-Ser (RGDS) sequence recognized by widely distributed integrin receptor alpha 5 beta 1 and the type III connecting segment (III CS) containing two cell-binding sites, designated CS1 and CS5, that are recognized by the alpha 4 beta 1 receptor. The C-terminal heparin-binding domain of FN (Hep II) has recently been demonstrated to support adhesion of alpha 4 beta 1-dependent melanoma cells [A. P. Mould and M. J. Humphries (1991) EMBO J. 10, 4089-4095]. Previously we demonstrated that this region of FN mediated binding of FN to HL-60 cells (acute promyelocytic leukemia cell line) by direct interaction independently of RGD and CS1 [H. Fujita et al., (1995) Exp. Cell Res. 217, 484-488]. In this study we have characterized a novel site in the Hep II region for binding to HL-60 cells. alpha 4 beta 1 and alpha 5 beta 1 were expressed on HL-60 cells, while alpha 2 beta 1 and alpha 3 beta 1 were not present, as shown by flow cytometry using monoclonal antibodies specific for the different integrins. Anti-alpha 4 beta 1 (P4C2) and anti-beta 1 (JB1a) antibodies inhibited binding of a 29-kDa dispase-digestive fragment of FN to HL-60 cells. This fragment contains the C-terminal heparin-binding domain of FN but lacks CS1 and CS5. Only the peptide representing the sequence from Val1866 to Arg1880, designated E1, inhibited the binding of the 29-kDa fragment to HL-60 cells. The active region of this peptide was a sequence of Thr-Asp-Ile-Asp-Ala-Pro-Ser (TAI-DAPS), which is homologous to Leu-Asp-Val-Pro-Ser (LDVPS) derived from the active site of CS1. Furthermore, labeled E1 peptide directly bound to HL-60 cells. The anti-alpha 4 beta 1 antibody (P4C2) inhibited this interaction. These results indicate that the site of binding to hematopoietic cells is present in the Hep II region of FN and the definition of the chemical structure of FN clarifies a fundamental mechanism of cell invasion of the extracellular matrix.

Amino Acid Sequence↗
PubMed1998

The disintegrin eristostatin interferes with integrin alpha 4 beta 1 function and with experimental metastasis of human melanoma cells.

Peptides containing the integrin recognition sequence, RGD, can inhibit experimental metastasis of mouse melanoma cells, but the integrin(s) affected in these experiments is unknown. Besides "classical" RGD-binding integrins such as alpha 5 beta 1 and alpha v beta 3, RGD has been reported to bind alpha 4 beta 1, and mAbs to alpha 4 beta 1 can inhibit melanoma metastasis. We investigated the mode of action of the disintegrin eristostatin, an RGD-containing peptide isolated from snake venom, in a human melanoma experimental metastasis model. Lung colonization following i.v. injection of MV3 cells in nude mice was strongly inhibited by eristostatin. MV3 cells bound FITC-eristostatin and adhered to eristostatin-coated wells. This adhesion was partially inhibited by a GRGDSP peptide and by alpha 4 mAb. Binding of FITC-eristostatin to Jurkat cells and adhesion of Jurkat (but not K562) cells to eristostatin-coated wells further suggested that eristostatin binds alpha 4 beta 1, even though, again, alpha 4 mAb only partially inhibited adhesion. Expression of alpha 4 beta 1 was enhanced in metastatic melanoma cells compared to normal melanocytes and nonmetastatic melanoma cells. Finally, eristostatin inhibited adhesion of both MV3 and CHO alpha 4 cells to the alpha 4 beta 1-ligand VCAM-1, while adhesion to other ligands via other integrins was not affected. These findings demonstrate that inhibition of melanoma cell metastasis by RGD-containing peptides such as eristostatin, may be due to interference with alpha 4 beta 1-VCAM binding, in addition to inhibition of the classical RGD-binding integrins.

Animals↗
PubMed2000

Differential induction of gelatinase B (MMP-9) and gelatinase A (MMP-2) in T lymphocytes upon alpha(4)beta(1)-mediated adhesion to VCAM-1 and the CS-1 peptide of fibronectin.

Integrin alpha(4)beta(1) on the surface of T lymphocytes interacts with vascular cell adhesion molecule-1 (VCAM-1) and fibronectin during migration of lymphocytes from the blood to sites of inflammation. Migrating lymphocytes actively modify their environment through a number of mechanisms including proteolysis of the extracellular matrix by matrix metalloproteinases (MMP) synthesized by the cells. In this study, expression of MMP upon alpha(4)beta(1)-mediated adhesion of leukocytes to two major ligands, the IIICS-1 domain of fibronectin and VCAM-1, has been examined. Adhesion of T lymphoblastoid Jurkat cells to the CS-1 peptide induced expression of mRNA for two MMPs, gelatinase A (MMP-2) and gelatinase B (MMP-9). As evaluated by relative RT-PCR and Northern blot analyses, the level of mRNA was upregulated about 4- to 5-fold for both MMPs compared to control cells maintained in suspension. With time, both enzymes were detected in conditioned media and inside the cells, and their identities were verified by Western blotting and gelatin zymography. Adhesion of Jurkat cells to the second major alpha(4)beta(1) ligand, VCAM-1, upregulated mRNA for MMP-2 (3.5-fold) and failed to induce expression of mRNA for MMP-9. Accordingly, only MMP-2 protein was detected in conditioned media of cells adherent to VCAM-1. Occupancy of alpha(4)beta(1) on the surface of suspended cells with soluble CS-1 peptide or VCAM-1 did not upregulate synthesis and release of MMPs. A similar pattern of induction of MMPs after adhesion to CS-1 and VCAM-1 was observed in T lymphocytes isolated from human blood. These results demonstrate that adhesion of T lymphocytes through alpha(4)beta(1) to different ligands, which bind to similar or overlapping sites in the integrin, induces intracellular events leading to distinct patterns of MMPs biosynthesis.

Amino Acid Sequence↗
PubMed2002

Adenosine suppresses alpha(4)beta(7) integrin-mediated adhesion of T lymphocytes to colon adenocarcinoma cells.

The interaction of T lymphocytes with tumor cells, a key step in the antitumor immune response, is suppressed by adenosine, a nucleoside produced at increased levels within the hypoxic tumor environment. We have explored the mechanism by which adenosine interferes with the lymphocyte:tumor cell interaction. The adhesion of anti-CD3-stimulated T cells to syngeneic MCA-38 mouse colon adenocarcinoma cells did not involve LFA-1 (alpha(L)beta(2)) or VLA-5 (alpha(5)beta(1)). However, antibodies against either lymphocyte alpha(4) or beta(7) (but not beta(1)) integrin subunits, or against VCAM-1 on the tumor cells, significantly suppressed adhesion, showing that the recognition of MCA-38 cells by T cells is strongly dependent upon the association of alpha(4)beta(7) on the effector cells with VCAM-1 on the tumor targets. This association is modulated by adenosine: The ability of adenosine to suppress T cell adhesion to MCA-38 cells was lost if alpha(4)beta(7) was functionally blocked with anti-alpha(4) antibodies (i) prior to or (ii) during the adhesion assay or if (iii) alpha(+)(4) cells were depleted from the T lymphocyte population. The binding of T cells to fibronectin through alpha(4)beta(1) was not suppressed by adenosine. We conclude that adenosine partially inhibits the interaction of T lymphocytes with tumor cells by blocking the function of integrin alpha(4)beta(7).

Adenocarcinoma↗
PubMed1997

Specific immunosuppression of corneal allograft rejection by combination of anti-VLA-4 and anti-LFA-1 monoclonal antibodies in mice.

It has been reported that allograft rejection is mediated by a variety of adhesion molecules. Using a corneal allograft model in mice, we studied the role of very late antigen (VLA)-4 and leukocyte function-associated antigen (LFA)-1 adhesion molecules in corneal allograft rejection and the effects of monoclonal antibodies (mAbs) to them in suppressing corneal rejection. C3H/He donor corneas were transplanted into BALB/c corneal beds. The allografted mice were treated with a control mAb (M18/2), mAbs to VLA-4, or LFA-1 or their combination by i.p. injection until day 7. The expression of VLA-4, LFA-1, major histocompatibility complex (MHC) class II antigens, interleukin (IL)-2, IL-2 receptor and interferon gamma (IFNgamma) in the grafted cornea were studied immunohistochemically. Cytotoxic T lymphocyte (CTL) responses to donor alloantigens were assessed. The skins from a syngeneic donor or a third-part strain were transplanted 8 weeks after the initial keratoplasty onto the mice treated with anti-LFA-1 plus anti-VLA-4 mAbs. Fourteen of 16 allografts in non-treated mice and control mAb-treated mice became opaque by 2 weeks after transplantation. At 2 weeks, non-treated allografts showed expression of MHC class II antigens on keratocytes and mononuclear cells at the host-graft junction. Also, mononuclear cells expressing VLA-4, LFA-1, IL-2, IL-2 receptor and IFNgammawere present in the stroma at the host-graft junction. The allografts treated with either anti-VLA-4 or anti-LFA-1 alone, or anti-VLA-4 plus anti-LFA-1 remained transparent for more than 2 weeks, and the survival rates at 14 weeks was 0%, 16.7%, and 75.0%, respectively. The combined use of anti-VLA-4 and anti-LFA-1 mAbs prolonged graft survival significantly (P<0.05) at 14 weeks as compared with anti-LFA-1 mAb alone. At 3 weeks, CTL responses to donor alloantigens were depressed in mice treated with either anti-LFA-1 alone or anti-LFA-1 plus anti-VLA-4. Specific prolongation of donor-syngeneic skin was observed after treatment with the combination of these two mAbs. These results indicate that VLA-4 and LFA-1 have important roles in rejection process of corneal allografts, and that the combined use of mAbs to these molecules has remarkable effects on inducing alloantigen-specific immunosuppression in corneal transplantation.

Animals↗
PubMed1998

Combined treatment of acute EAE in Lewis rats with TNF-binding protein and interleukin-1 receptor antagonist.

Experimental autoimmune encephalomyelitis (EAE) is a term given to describe a collection of animal models representing the human disease multiple sclerosis (MS). Although not fully understood, the involvement of cytokines and the immune system in either EAE or human MS is well established. Past efforts have shown that inhibition of proinflammatory cytokines tumor necrosis factor (TNF-alpha) or interleukin-1 (IL-1) result in amelioration of acute EAE in Lewis rats. The present study examined this model for the effect of concomitant inhibition of both TNF-alpha and IL-1, which resulted in a modest but significant therapeutic effect that was superior to inhibition of either single agent alone with respect to four of the five variables used to follow the progression of disease in this model, i.e., clinical severity, frequency of disease, loss of body weight, and day of onset. These results are in accordance with the idea that combination treatments are likely to prove superior to single agent therapy in the treatment of autoimmune inflammatory disease.

Animals↗
PubMed2001

Effect of lipopolysaccharide on the morphology and integrin immunoreactivity of ramified microglia in the mouse brain and in cell culture.

Microglial cells form the first line of defense in brain infection. They are related to monocytes and macrophages and can be readily activated by cell wall components of bacteria such as lipopolysaccharides (LPS). In the present study, we explored the effect of this endotoxin in mouse on the morphology of microglia and their immunoreactivity for the integrin family of cell adhesion molecules in vitro and in vivo. Subcutaneous injection of LPS led to a dose-dependent activation of alpha M beta 2-positive microglia, with a saturating effect at 1 microg LPS in the blood-brain barrier deficient area postrema, at 10 microg in the directly adjacent tissue, and at 100 microg throughout the brainstem and cerebellum. Morphologically, this activation was characterized by the swelling of the microglial cell body, a thickening of the proximal processes, and a reduction in distal ramification. Microglial immunoreactivity for the integrins alpha 4 beta 1, alpha 5 beta 1, alpha 6 beta 1, and alpha M beta 2 was strongly increased. In vitro, ramified microglia were obtained using a coculture on top of a confluent astrocyte monolayer. Two days exposure to LPS resulted in a morphological activation of the cultured cells with an increase of the integrin immunoreactivity for alpha 5 (5.7-fold), alpha 4 (3.1-fold), beta 1 (2.3-fold), and alpha M (1.5-fold), and a decrease in the alpha 6-staining intensity by 39%. Even a sublethal dose of LPS (3 mg in vivo and 500 microg/ml in vitro, respectively) did not induce the phagocyte-associated integrin alpha X beta 2 (CD11c/CD18, p150,95) and did not lead to a morphological transformation of the ramified microglia into phagocytes.

Animals↗
PubMed2002

Developmental regulation of beta1 integrins during angiogenesis in the central nervous system.

We examined beta1 integrin expression during angiogenesis in the developing mouse CNS. Maturation of blood vessels was accompanied by three main events: (1) marked upregulation of beta1 integrin expression; (2) a switch in beta1 integrin expression, from alpha4 and alpha5 at postnatal day 1 to alpha1 and alpha6 in the adult; and (3) downregulation of fibronectin and upregulation of laminin expression. Thus, blood vessel maturation was associated with a developmental switch, from fibronectin-mediated signaling early in angiogenesis, to laminin-mediated signaling at later stages. To investigate the potential role of alpha4 and alpha5 integrins in angiogenesis, we developed a novel approach to purify endothelial cells from the CNS by selective adhesion to fibronectin. BrdU incorporation showed that fibronectin induced more endothelial cell proliferation than laminin, and antibody-blocking studies revealed that fibronectin mediated its effect via both alpha4 and alpha5 integrins. This work provides the first evidence that there is a developmental switch in the use of beta1 integrins during angiogenesis. Furthermore, it suggests that regulation of beta1 integrin and ECM expression by endothelial cells are important factors influencing vascular development in the CNS.

Aging↗
PubMed1997

Activated eosinophils stimulate endothelin-1 release from airway epithelial cells by direct adherence via adhesion molecules.

Endothelin-1 (ET-1), synthesized in airway epithelial cells, has a potent constrictive action on airway smooth muscle. In this study, we investigated the effect of eosinophils on ET-1 release from guinea pig cultured tracheal epithelial cells. Eosinophils with or without the activation were directly co-cultured with tracheal epithelial cells. Eosinophils activated by GM/CSF or IL-5 potentiated ET-1 release, but non-activated ones did not. Treatment of activated eosinophils with antibodies against macrophage-1 (Mac-1) and/or very late antigen-4 (VLA-4) suppressed the potentiated ET-1 release. However, inhibition of lipid mediators derived from activated eosinophils could not suppress the potentiated ET-1 release. Moreover, separated co-culture of activated eosinophils with tracheal epithelial cells using Millicell-CM had no effect on ET-1 release. These observations suggest that adherence of activated eosinophils to epithelial cells via adhesion molecules such as Mac-1 and VLA-4 was essential for potentiation of ET-1 release. Since presence of eosinophils in the epithelial layer has been commonly demonstrated in bronchial biopsies and autopsy specimens from patients with asthma, epithelial cells would be activated by adherence of eosinophils via adhesion molecules and potentiate ET-1 release in vivo.

Animals↗