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PubMed2000

Soluble vascular cell adhesion molecule 1 mediation of monocyte chemotaxis in rheumatoid arthritis.

OBJECTIVE: Rheumatoid arthritis (RA) is characterized by infiltration of leukocytes, including monocyte/ macrophages, into synovial tissue (ST), but factors mediating the ingress of these cells are poorly understood. Vascular cell adhesion molecule 1 (VCAM-1) plays an important role in adhesion of leukocytes to the vasculature. This study was undertaken to test the hypothesis that soluble VCAM-1 (sVCAM-1) might mediate chemotaxis of monocytes in RA. METHODS: Chemotaxis assays were performed using a modified Boyden chamber to determine the effects of sVCAM-1 on and the role of very late activation antigen 4 (VLA-4) in peripheral blood (PB) monocyte migration. Synovial fluids (SF) were immunodepleted of sVCAM-1 to identify a role for sVCAM-1 in RA. Immunohistochemistry and flow cytometry analyses were performed to show the expression of VLA-4 in ST, SF, and PB. Tyrosine phosphorylation was studied by Western blot analysis on PB monocyte lysates in the presence of signaling inhibitors. RESULTS: Soluble VCAM-1 induced monocyte migration in the nM range, in a concentration-dependent manner. Anti-VLA-4 significantly inhibited sVCAM-1-induced monocyte migration, suggesting that sVCAM-1 acts in part via a VLA-4-dependent mechanism. In RA SF, incubation with anti-VCAM-1 resulted in a reduction in the ability to induce monocyte migration (mean 28%). VLA-4 immunolocalized to RA ST, SF, or PB, monocytes, macrophages, and lymphocytes. Soluble VCAM-1 stimulated tyrosine phosphorylation in monocytes, and pertussis toxin, chelerythrine chloride, and staurosporine significantly reduced sVCAM-1-mediated monocyte chemotaxis, suggesting that signaling pathways via G proteins and protein kinase C are required for sVCAM-1-mediated monocyte migration. CONCLUSION: These results demonstrate a novel function for sVCAM-1 as a monocyte chemotactic agent in RA and suggest a new potential target for modulating monocyte ingress into inflamed RA ST.

Arthritis, Rheumatoid↗
PubMed1995

Mechanisms of VCAM-1 and fibronectin binding to integrin alpha 4 beta 1: implications for integrin function and rational drug design.

Integrin alpha 4 beta 1 can mediate both cell-cell and cell-extracellular matrix adhesion by binding to either fibronectin or vascular cell adhesion molecule 1 (VCAM-1). Both interactions are important for extravasation of leukocytes from the blood implying that rationally designed inhibitors of alpha 4 beta 1 function may be useful for treating a various inflammatory conditions. The mechanisms of ligand binding by alpha 4 beta 1 are complicated by the fact that alternative splicing can generate different isoforms of the receptor-binding domains in both fibronectin and VCAM-1. Therefore, in addition to developing alpha 4 beta 1 antagonists, we have also been interested in identifying isoform-specific functions. Recombinant ligand variants have been tested in adhesion and direct receptor-binding assays and each molecule was found to have a different inherent affinity for alpha 4 beta 1 that endows them with different adhesive activities. This suggests that alternative splicing may regulate alpha 4 beta 1-dependent motility in vivo. The initial strategy that we have adopted to develop alpha 4 beta 1 inhibitors has been to identify key amino acid residues and peptide sequences participating in the receptor-ligand binding event and to use this information to generate synthetic mimetics. Three active sites have been identified in fibronectin by testing truncated proteins, expressing recombinant fragments and screening synthetic peptides. Two of these sites employ versions of a novel integrin-binding motif, LDVP/IDAP. A key active site in VCAM-1 has been identified by similar approaches as the related sequence IDSP. Since IDSP-like sequences are probably used by other integrin-binding immunoglobulins, derivatives of these peptides may turn out to be the forerunners of a new generation of therapeutic agents with multiple applications.

Amino Acid Sequence↗
PubMed1994

Regulated expression of p150,95 (CD11c/CD18; alpha X/beta 2) and VLA-4 (CD49d/CD29; alpha 4/beta 1) integrins during myeloid cell differentiation.

Integrins are a family of cell surface heterodimers which mediate both cell-cell and cell-extracellular matrix interactions and affect cellular differentiation through their signal transduction capacity. Integrin expression is regulated during differentiation as well as by numerous growth factors and cytokines. We have analyzed the changes in p150,95 (CD11c/CD18 or alpha X/beta 2) and VLA-4 (CD49d/CD29 or alpha 4/beta 1) integrin subunits mRNA levels that take place during the myeloid differentiation of HL60 and U937 cells, and compared them to other integrins with similar functional activities. Northern blot analysis revealed that the monocytic differentiation of U937 and HL60 cells alters the alpha X and alpha 4 mRNA steady-state levels: alpha X mRNA is induced de novo whereas alpha 4 mRNA decreases to undetectable levels. Both changes were dependent on the activity of protein kinase C and were also observed upon granulocytic differentiation of HL60 cells. Parallel analysis of other integrin subunits mRNA (beta 1, alpha 5, beta 7) demonstrated that the mRNA levels for the alpha subunits of the fibronectin receptors alpha 4/beta 1 (VLA-4) and alpha 5/beta 1 (VLA-5) are differentially regulated during the monocytic differentiation of myeloid cell lines, and suggested that myeloid cells express a heterodimer formed by the association of beta 7 with an integrin alpha subunit distinct from alpha 4. Nuclear transcription assays and functional analysis of the alpha X and alpha 4 promoter regions demonstrated that the transcription rate of the alpha X gene is considerably elevated after phorbol 12-myristate 13-acetate treatment of U937 cells, while that of alpha 4 is almost unaffected, suggesting that post-transcriptional mechanisms are causing the extremely low alpha 4 mRNA levels observed in differentiated U937 cells.

Blotting, Northern↗
PubMed1996

Modulation of integrin-mediated intercellular adhesion during the interaction of thymocytes with stromal cells expressing VLA-4 and LFA-1 ligands.

Mature peripheral T cells closely regulate their intercellular interactions by modulating integrin adhesion functions. The ability of members of the integrin family to mediate intercellular adhesion is dependent on signals from within the cells (inside-out signaling) that increase the avidity of integrins for their ligands. These changes in avidity are independent of the quantitative changes on the number of receptors, and there is evidence to suggest that phosphorylation events play a predominant role in the regulation of the avidity state of the integrins. Whether such regulatory mechanisms are operative during T cell development had hitherto been an opened question. In the present work, we have used an in vitro adhesion assay between thymocytes and target cells expressing VLA-4 and LFA-1 counter ligands to determine how thymocytes can discriminate between integrin-specific signals during T cell development. Our findings are that VLA-4, but not LFA-1, is constitutively expressed in its high-avidity state during the early stages of T cell development, and that the high-avidity state of thymocytes for VCAM-1-expressing cells is closely regulated by signaling through protein kinase C and protein tyrosine kinase pathways. At later stages of development, mature thymocytes prior to leaving the thymus turn off both VLA-4 and LFA-1 adhesion functions. Our results show that the low-affinity state of integrins on peripheral mature T cells is established before mature thymocytes leave the thymus. Only when mature T cells recognize antigenic peptides in the context of major histocompatibility complex in the periphery will they turn on the adhesion function of VLA-4 and/or LFA-1 integrins.

Animals↗
PubMed1997

Functional relevance during lymphocyte migration and cellular localization of activated beta1 integrins.

The state of integrin activation can be assessed by monoclonal antibodies (mAb) that selectively recognize integrins in their active form. We demonstrate herein that the expression of the epitope recognized by mAb HUTS-21 is induced on T lymphoblasts upon binding of soluble vascular cell adhesion molecule (VCAM)-1 and an 80-kDa tryptic fragment of fibronectin (FN80) to the beta1 integrins very late activation antigen (VLA)-4 and VLA-5, and that this effect is dependent on ligand concentration and is specific for beta1 integrins. On T lymphoblasts adhering to immobilized fibronectin, the HUTS-21 epitope localized exclusively to sites of integrin binding to fibronectin. These results indicate that mAb HUTS-21 recognizes a ligand-induced binding site (LIBS) on the common beta1 subunit of VLA proteins. Engagement of beta1 integrins through this LIBS epitope inhibited T lymphoblast movement on fibronectin, as determined by quantitative time-lapse video microscopy studies. Furthermore, the HUTS-21 mAb also prevented T lymphoblast-directed migration through gradients of substratum-immobilized beta1 integrin ligands such as fibronectin or VCAM-1, whereas it did not affect migration on intercellular adhesion molecule (ICAM)-1. This anti-LIBS mAb stimulated cell adhesion through postreceptor events, without affecting receptor affinity for ligand, and appears to interfere with cell migration by a mechanism distinct from that of other anti-beta1 activating antibodies.

Actin Cytoskeleton↗
PubMed1997

Human T lymphocytes bind to germinal centers of human tonsils via integrin alpha4/VCAM-1 and LFA-1/ICAM-1 and -2.

Binding of T lymphocytes within the different compartments of the secondary lymphoid organs is crucial for the function of the cellular and the humoral immune response. It is still not known which adhesion molecules guide T cells to the distinct areas of the lymphoid microenvironment. In the current study an in situ adhesion assay was used to define the receptors for binding of T cells to human tonsils. The T cell lines Jurkat and MOLT-4 and normal, activated T cells were found to bind exclusively to germinal centers. Jurkat cells used the receptor pair integrin-alpha4 (VLA-4alpha)/VCAM-1, whereas activated MOLT-4 cells and normal T cells bound via both adhesion pathways, namely via integrin-alpha4/VCAM-1 and LFA-1/ICAM-1 and -2. It is suggested that these adhesion mechanisms are involved in the migration of T cells into the germinal centers of secondary lymphoid organs and that they influence the selection of B cells by apoptosis.

Antigens, CD↗
PubMed1997

Association between receptor density, cellular activation, and transformation of adhesive behavior of flowing lymphocytes binding to VCAM-1.

We investigated the ability of purified vascular cell adhesion molecule-1 (VCAM-1), adsorbed on plastic, to capture and immobilize flowing lymphocytes, and the dependence of adhesive behavior on activation of the counter-receptor, alpha 4 beta 1 integrin. This integrin/immunoglobulin interaction bound lymphocytes at a wall shear stress at which the beta 2-integrin family has previously been found ineffective (> 0.1 Pa), and whereas lymphocytes rolled on lower concentrations of VCAM-1 (10 micrograms/ml), they were stationary at high concentrations (100 micrograms/ml). Activation of alpha 4 beta 1 integrin by Mn2+ or by antibody 12G10 or treatment of lymphocytes with phorbol ester caused transformation to stationary adhesion, and increased binding significantly only at the lower concentrations of VCAM-1. We thus hypothesized that formation of a high density of ligand between VCAM-1 and alpha 4 beta 1 integrin actively transformed lymphocyte behavior. This concept was supported by the finding that the proportion of lymphocytes rolling on the higher concentrations of VCAM-1 increased if cells were pretreated with azide to block energy-dependent responses, or if intracellular Ca2+ was chelated. However, not all activation responses were equivalent: only phorbol ester induced marked spreading of immobilized cells, and if pretreatment was prolonged, this agent even reduced the efficiency of initial attachment of flowing lymphocytes. Azide treatment had no effect on transformation to stationary adhesion caused by Mn2+ or activating antibody. Thus, different forms of lymphocyte activation were identifiable: external modification of integrin converted rolling to stationary attachment, did not require ATP, and was reversible; high-density ligand binding induced an energy-dependent signal for conversion from rolling to stationary attachment, but not spreading; and protein kinase C activation promoted stationary attachment and spreading, but not necessarily capture. VCAM-1 is thus a versatile adhesion receptor capable of supporting all stages of leukocyte attachment, i.e. rolling, stationary, and spreading, and of ligand-induced transformation of adhesion, although an additional signal appears necessary to promote lymphocyte spreading and migration.

Animals↗
PubMed1997

Clustering the adhesion molecules VLA-4 (CD49d/CD29) in Jurkat T cells or VCAM-1 (CD106) in endothelial (ECV 304) cells activates the phosphoinositide pathway and triggers Ca2+ mobilization.

Ligation of very late antigen (VLA)-4 (alpha 4 beta 1 integrin) with a cross-linked anti-alpha 4 subunit monoclonal antibody (mAb) triggered a biphasic Ca2+ response in Jurkat cell populations and in peripheral human lymphocytes. Cross-linking vascular cell adhesion molecule (VCAM)-1 (the counter-receptor of VLA-4) in ECV 304 endothelial cells generated a biphasic Ca2+ response. Tumor necrosis factor-alpha-primed human umbilical cord vascular endothelial cells also responded to the cross-linked mAb with a biphasic Ca2+ profile. Ligated VLA-4 (Jurkat cells) or VCAM-1 (ECV 304) stimulated the production of myo-inositol 1,4,5-trisphosphate. ECV 304 cells induced a biphasic Ca2+ response in Fura2-loaded Jurkat cells, whereas a transient response was observed when Jurkat cells were added to Fura2-loaded ECV 304 cells. The Ca2+ responses in these experiments involved VLA-4/VCAM-1 interactions since they were significantly reduced (approximately 80%) by prior treatment of the target cells with the relevant noncross-linked mAb. Close contact between the cells triggered mutual Ca2+ signaling as shown by spectrofluorimetric and confocal microscopy time-dependent recordings. Fibronectin and its CS-1 fragment (V25) triggered a sustained Ca2+ response in Jurkat cells (confocal microscopy). Our results suggest that the VLA-4 and VCAM-1 adhesion molecules can transduce a signal that involves activation of the phosphoinositide pathway and the mobilization of Ca2+.

Calcium↗
PubMed1997

A role for the VLA-4 integrin in the activation of human memory B cells.

It is generally recognized that activation through membrane effector molecules such as CD40 or the B cell receptor (BCR) is mandatory to allow B cells to proliferate and differentiate into antibody (Ab)-secreting cells in response to cytokines. We show here that purified tonsillar B cells can be stimulated directly by a cytokine combination to proliferate and secrete immunoglobulins when cultures are performed at high cell density. The contact-mediated activation of B cells in this experimental system is strongly inhibited both by anti-very late antigen (VLA)-4 monoclonal Ab and by a peptide containing the LDV sequence specifically recognized by the alpha 4 integrin binding site. These reagents also significantly suppressed the B cell responses elicited by engagement of the BCR or CD40. Our data reveal that memory B cells but not virgin or germinal center B cells are sensitive to the direct stimulatory effect of cytokines in high-density cultures. Finally, we found that the dual expression of the alpha and beta chains of VLA-4 is a distinctive feature of the memory B cell population. Collectively, our findings support the notion that VLA-4-dependent homotypic B cell interactions can mediate a co-stimulatory signal to human memory B cells and might participate in the B cell activation triggered through the BCR and CD40.

B-Lymphocytes↗
PubMed2003

The HIV protease inhibitor indinavir reduces immature dendritic cell transendothelial migration.

Indinavir (IDV) is a protease inhibitor that successfully suppresses HIV-1 replication as part of anti-retroviral therapy. There is evidence to suggest that IDV may also act non-specifically upon host proteases. In this study we investigated whether IDV could modulate protease-dependent molecules involved in dendritic cell (DC) migration - a pivotal process in immunoregulation. Human monocyte-derived DC were exposed to IDV (IDV-DC) and transendothelial migration (TEM) to inflammatory chemokines was determined. TEM of IDV-DC was significantly impaired compared to non-treated DC (p<0.01). Phenotypic analysis revealed that IDV-DC had reduced DC-SIGN expression, correlating with reduced adhesion to immobilized ICAM-2. Nevertheless, the reduction in migration following exposure to IDV could not be fully attributable to DC-SIGN interactions alone. Investigation of IDV-DC interactions with the underlying matrix protein, fibronectin, demonstrated that IDV significantly impaired DC binding to immobilized fibronectin (p<0.01). IDV appeared to act upon VLA-4 and VLA-5 since addition of antagonist monoclonal antibodies (mAb) similarly reduced adhesion of non-treated DC to fibronectin. Combined blockade of DC using anti-VLA-4, VLA-5 and anti-DC-SIGN mAb inhibited TEM to a similar extent as IDV. Our results strongly suggest that IDV inhibits host proteases necessary for DC migration and may, therefore, affect DC immunoregulation in HIV-1-infected patients.

Antigens, CD↗
PubMed2004

Defective up-regulation of CD49d in final maturation of NOD mouse macrophages.

Macrophages are potent regulators of both innate and adaptive immunity. They play a central role in the development of autoimmune diabetes and are among the first cells to appear in peri-islet infiltrates of NOD mice that spontaneously develop diabetes. Since efficient adhesion and migration are crucial for proper macrophage trafficking, we examined the migration and fibronectin (FN) adhesion capacity of NOD macrophages, as well as the regulation and expression of the FN receptors alpha4beta1 and alpha5beta1. When compared to macrophages from control strains, resident NOD macrophages showed a reduced ability to adhere to and migrate on FN, a delayed clearance following peritoneal inflammation, and substantially lower expression levels of the alpha4beta1 integrin alpha chain, CD49d. NOD bone marrow-derived macrophages were specifically defective in the LPS-induced increase in CD49d expression. Moreover, the mitogen-activated protein kinase extracellular signal-regulated kinase-1/2 negatively regulated macrophage CD49d expression and strongly suppressed its expression in NOD macrophages. The data presented herein indicate that the LPS-activated signaling cascade plays a critical role in CD49d expression of macrophages. Mature NOD macrophages are characterized by decreased CD49d expression and show defective CD49d-mediated adhesion to FN.

Animals↗
PubMed1997

Sinusoidal endothelium release of hydrogen peroxide enhances very late antigen-4-mediated melanoma cell adherence and tumor cytotoxicity during interleukin-1 promotion of hepatic melanoma metastasis in mice.

The hepatic sinusoidal endothelium (HSE) releases large amounts of reactive oxygen species (ROS) in response to endotoxins and interleukin-1 (IL-1). Such pro-inflammatory mediators have been shown to promote hepatic metastasis. We have investigated the involvement of ROS released by IL-1-stimulated HSE in this promoting effect. Recombinant human interleukin-1 beta (rHuIL-1 beta) (5 micrograms/kg) was intravenously injected into C57BL/6J mice, and the hepatic metastasizing ability of B16 melanoma cells following intrasplenic injection was studied in the presence of ROS scavengers. rHuIL-1 beta-promoted hepatic metastases were significantly (P < .01) reduced by catalase (1 mg/kg) and enhanced by recombinant human superoxide dismutase (rHuSOD) (5 mg/kg). rHuIL-1 beta-stimulated HSE-conditioned medium (HSE-CM) significantly (P < .01) enhanced B16 melanoma cell adhesion to HSE compared with unstimulated HSE-CM, which in turn also significantly (P < .01) increased with melanoma cell adherence compared with basal medium. The addition of catalase completely abrogated proadhesive effects induced by rHuIL-1 beta-stimulated HSE-CM with respect to unstimulated HSE-CM, but did not affect the proadhesive effects induced by unstimulated HSE-CM over basal medium. The rat monoclonal antibody to mouse vascular cell adhesion molecule-1 (VCAM-1) significantly (P < .01) inhibited the enhanced melanoma cell adherence effects of both unstimulated and rHuIL-1 beta-stimulated HSE-CM, indicating that adherence was very late antigen-4 (VLA-4)-mediated. Not surprisingly, the percentage of VLA-4 expressing B16 melanoma cells significantly (P < .05) increased in response to unstimulated (21% of controls) and rHuIL-1 beta-stimulated (32% of controls) HSE-CM. Catalase addition abrogated these effects of rHuIL-1 beta-stimulated-HSE-CM. Melanoma cell damage was observed from the second hour of adhesion to HSE and significantly (P < .01) increased when the cells adhered to rHuIL-1 beta-stimulated HSE. This increase was abrogated by catalase. Cytolysis of the HSE was not observed during melanoma cell adhesion. Neither was the enhancement of B16 melanoma hydrogen peroxide production observed in response to rHuIL-1 beta. Thus, the effects of IL-1 in the liver may consist of a balance between the prometastatic effect of enhanced adherence to the HSE and the antimetastatic effect of H2O2-mediated cytotoxicity. Our results suggest that the enhancement of H2O2 production by the rHuIL-1 beta-stimulated HSE may contribute to the hepatic metastasis progression of ROS-resistant melanoma cells. Results in vitro indicate that this progression is associated with a H2O2-mediated increase in melanoma cell adhesion to HSE.

Animals↗
PubMed1994

Loss of polarity and de novo expression of the beta 1 family of integrins in thyroid tumors.

The expression and cell-membrane distribution of the beta 1 family of integrins (very-late-activation antigens, VLA) were investigated in benign and malignant human thyroid tumors. We compared tissue samples of normal glands, nodular goiters, adenomas and carcinomas. We also examined 3 thyroid-carcinoma cell lines cultured in vitro. The expression of subunits of the beta 1 family of integrins was assessed by flow cytometry and specific antibodies in dispersed single-cell suspensions and by immunofluorescence on frozen tissue sections. In contrast to the heterogeneity of the expression of beta 1 integrins observed in other tumors, thyroid neoplastic lesions showed a remarkably constant VLA profile. In all tumors, benign as well as malignant, and in carcinoma cell lines, all sub-units of beta 1 integrins were expressed at high levels. While sub-units alpha 1, alpha 3, alpha 5, alpha 6 and occasionally alpha 2 were also present in a cell sub-set of normal glands and nodular goiters, expression of alpha 4 was restricted to neoplastic lesions; this integrin can be therefore considered an antigen associated with thyroid tumors. It has been reported that in normal glands and in nodular goiters, the expression of beta 1 integrins is restricted to the basal-cell membrane. Immunofluorescence on tissue sections showed instead that, in adenomas and carcinomas, the polarized distribution of these integrins on the cell membrane is lost.

Carcinoma, Papillary, Follicular↗
PubMed1995

Adhesion molecules involved in the binding of murine myeloma cells to bone marrow stromal elements.

In previous work, we reported the development of the B9/BMI syngeneic murine bone marrow metastasis model. Interleukin (IL)-6-dependent, IL-I-producing B9/BMI cells, which preferentially home to and colonise the vertebral and femoral marrow after i.v. injection, exhibit striking similarity in cell surface phenotype to human myeloma cells, especially the expression of 3 adhesion molecules, CD44, VLA-4 and ICAM-I. Because the haematopoietic microenvironment consists of different cell types, such as endothelial cells, fibroblasts, adipocytes and macrophages, we investigated the functional significance of these adhesion molecules in heterotypic binding assays between B9/BMI cells and a newly established bone marrow-derived endothelial cell line (BMEC), a fibroblastoid pre-adipocyte cell line (BMS2.2) and primary bone marrow-derived macrophages. B9/BMI cells adhered well to all stromal elements: a combination of monoclonal antibodies (MAbs) against CD44 and VLA-4 significantly inhibited the adherence of B9/BMI cells to BMEC and BMS2.2 cells, whereas binding of B9/BMI cells to macrophages was partially blocked with an anti-ICAM-I MAb. Our results implicate multiple recognition mechanisms, including those involving CD44, VLA-4 and ICAM-I, in the retention of B9/BMI cells in the bone marrow.

Animals↗
PubMed1991

Identification of a novel recognition sequence for the integrin alpha 4 beta 1 in the COOH-terminal heparin-binding domain of fibronectin.

The type III connecting segment of fibronectin contains two cell binding sites, represented by the peptides CS1 and CS5, that are recognized by the integrin receptor alpha 4 beta 1. Using assays measuring the spreading of A375-SM human melanoma cells, we now report that the adhesion promoting activity of a 29 kDa protease fragment of fibronectin containing the COOH-terminal heparin-binding domain (HepII), but lacking CS1 and CS5, is completely sensitive to anti-alpha 4 and anti-beta 1 antibodies, suggesting that HepII contains a third alpha 4 beta 1-binding sequence. Examination of the primary structure of HepII revealed a sequence with homology to CS1. A 19mer peptide spanning this region (designated H1) was found to support cell spreading to the same level as the 29 kDa fragment. H1-dependent adhesion was completely sensitive to anti-alpha 4 and anti-beta 1 antibodies. When soluble peptides were tested for their ability to block cell spreading on the 29 kDa fragment, a 13mer peptide comprising the central core of H1 was found to be completely inhibitory. The active region of H1 was localized to the pentapeptide IDAPS, which is homologous to LDVPS from the active site of CS1. Taken together, these results identify a novel peptide sequence in the HepII region of fibronectin that supports alpha 4 beta 1-dependent cell adhesion.

Amino Acid Sequence↗
PubMed1995

Critical amino acid residues for ligand binding are clustered in a predicted beta-turn of the third N-terminal repeat in the integrin alpha 4 and alpha 5 subunits.

Integrin alpha 4 beta 1 is a receptor for vascular cell adhesion molecule (VCAM)-1 and fibronectin (CS-1). The alpha 4 beta 1-ligand interaction is involved in the pathogenesis of diseases and is, therefore, a therapeutic target. Here, we identified critical residues of alpha 4 for ligand binding using alanine-scanning mutagenesis of the previously localized putative ligand binding sites (residues 108-268). Among 43 mutations tested, mutations of Tyr187, Trp188 and Gly190 significantly inhibited cell adhesion to both VCAM-1 and CS-1. This inhibition was not due to any gross structural changes of alpha 4 beta 1. These critical residues are clustered in a predicted beta-turn structure (residues 181-190) of the third N-terminal repeat in alpha 4. The repeat does not contain divalent cation binding motifs. Notably, the mutations within the corresponding region of alpha 5 significantly reduced fibronectin-alpha 5 beta 1 interaction. These findings suggest that the predicted beta-turn structure could be ubiquitously involved in ligand binding of non-I domain integrins.

Amino Acid Sequence↗
PubMed2003

Monocyte/macrophage interactions with base and linear- and star-like PEG-modified PEG-poly(acrylic acid) co-polymers.

Poly(ethylene glycol):poly(acrylate) PEG-g-PA co-polymers were made that inhibited nonspecific protein and cellular adhesion. PEG-g-PA co-polymers were then covalently modified with either cell adhesion peptides or fragments of antibodies to monocyte/macrophage integrin receptors (anti-VLA4, anti-beta(1), anti-beta(2), and anti-CD64) known to enhance macrophage adhesion and, perhaps, modulate their activation. Peptides were either directly conjugated to the base material or linked by way of PEO-star tethers. Fragments of the antibody region containing the antigen-binding site (Fab' fragments) were coupled to other PEG-g-PA samples using the sulhydryl end groups on Fab' fragments to amine-bearing PEO stars. Macrophage adhesion rates, phagocytic response (oxidative burst), and cytokine expression were determined for each PEG-g-PA material. Luminol-enhanced chemiluminescence was used as a semiquantitative indication of monocyte-macrophage phagocytic activation (oxidative burst). Macrophage cytokine expression in response to control, base, and modified materials was determined by ELISAs for TNF-alpha, IL-1 beta, IL-6, and IL-8. Tissue culture poly(styrene) (TCPS)-mediated the greatest number of adherent monocyte/macrophage cells relative to PEG-g-PA materials. Both YRGDS and YEILDV peptides, whether directly or indirectly (via StarPEO) conjugated to PEG-g-PA, increased adhesion versus controls. Fab' fragments of all four antibodies also promoted enhanced adhesion versus controls. Fab'StarPEO materials presented two orders of magnitude fewer ligands per surface unit area than peptide star materials (10(8) vs. 10(10)), but were able to adhere similar numbers of cells. For surfaces presenting Fab'(VLA-4) or YEILDV, both of which may both bind to a cell's VLA-4 receptor, the Star:VLA4 surface showed a greater number of adherent monocyte/macrophages. This result suggests that the Fab' had a higher affinity to the cell receptor than a corresponding minimal peptide binding sequence. All materials exhibited low oxidative burst (luminescence counts per minute, LCPM) per cell DNA without the addition of exogenous stimuli (LCPM/DNA < 100). Directly conjugated peptide materials, poly(propylene) (PP), and TCPS showed the lowest levels of LCPM/DNA without the addition of exogenous stimulus (LCPM/DNA < 20). There was no correlation between LCPM/DNA ratios, with and without added LPS stimulus, versus the individual substrates. Monocyte/macrophages adherent to TCPS substrata showed the overall highest stimulatory potential in cytokine expression response to exogenous LPS, followed by PP > PEG-g-PA > StarPEO. Cells adherent to peptide-modified materials and Fab'-modified materials were overall less stimulated. The method of presenting the peptides (i.e., directly or via Star PEO) influenced the level of cytokine secreted by the adherent macrophage.

Acrylates↗
PubMed1993

Homotypic leukocyte aggregation triggered by a monoclonal antibody specific for a novel epitope expressed by the integrin beta 1 subunit: conversion of nonresponsive cells by transfecting human integrin alpha 4 subunit cDNA.

The monoclonal antibody 33B6 was found to be specific for the beta 1 integrin subunit. Treatment of leukocytes with this antibody induced a vigorous homotypic aggregation that had similar physiologic conditions as aggregation induced by a monoclonal antibody specific for the alpha 4 subunit. Expression of a beta 1 subunit on the cell surface was not sufficient for mAb 33B6-mediated aggregation to occur, since cells of the K562 erythroleukemia line failed to respond even though they expressed the beta 1 subunit and the 33B6 epitope. However, after transfection with cDNA encoding the alpha 4 subunit, K562 cells acquired the ability to aggregate in response to mAb 33B6 binding. By contrast, mAb 33B6 blocked cell binding to the endothelial surface protein vascular cell adhesion molecule-1 and the extracellular matrix protein fibronectin. These results suggest that the beta 1 epitope defined by mAb 33B6 may play a novel role in regulating leukocyte adhesive interactions.

Animals↗