Stimulation of integrin-mediated adhesion of T lymphocytes and monocytes: two mechanisms with divergent biological consequences.

We show that the adhesion of T lymphoid cells to immobilized fibronectin can be increased by two distinct mechanisms. The first is by increasing the affinity of the fibronectin receptor/ligand interaction using the anti-beta 1 integrin monoclonal antibody 8A2. The second is by treating the cells with phorbol 12-myristate 13-acetate (PMA), which alters events that occur after receptor occupancy (e.g., cell spreading) without affecting receptor affinity. The effects of these two mechanisms on adhesion in the presence of physiological concentrations of soluble fibronectin suggest that they have different biological consequences. Under these conditions, the net effect of increasing the affinity of the fibronectin receptors is to decrease cell adhesion, whereas the increase in adhesion induced by PMA is unaffected. This suggests that the high affinity receptors are not primarily available for cell adhesion under these circumstances, and that they have an alternative function. We further show that high affinity binding of soluble fibronectin can be induced by either differentiation of the monocytic cell line THP-1 or by cross-linking the T cell receptor complexes on the T lymphoid cell line HUT-78. The differentiated monocytic cells express two populations of fibronectin receptors: a minority in a high affinity state, and the majority in a low affinity state. Thus they will both continue to adhere in the presence of physiological concentrations of soluble fibronectin and bind significant amounts of soluble fibronectin at the cell surface.

Lipid IVA inhibits synthesis and release of tumor necrosis factor induced by lipopolysaccharide in human whole blood ex vivo.

Tumor necrosis factor (TNF) released by lipopolysaccharide (LPS)-stimulated mononuclear phagocytes is a critical mediator of sepsis. We examined the capacities of rough mutant Salmonella typhimurium LPS (Rc) and LPS partial structures lipid A, monophosphoryl lipid A (MPLA), lipid IVA, and lipid X to induce production of TNF in whole blood. Rc LPS (0.0001-10 ng/ml) produced a dose-dependent release of TNF as determined by cytotoxicity of actinomycin D-sensitized L929 murine fibroblasts. Lipid A, MPLA, lipid IVA, and lipid X exhibited decreasing capacities to stimulate production of TNF in whole blood, respectively. Fractional deacylation of LPS by incubation with acyloxyacyl hydrolase isolated from human leukocytes produced a reduction in the capacity of LPS to induce TNF release in whole blood. Maximal enzymatic deacylation reduced activity of LPS by greater than 100-fold. Coincubation with lipid IVA inhibited TNF release induced by Rc LPS or lipid A, but not by phorbol ester. In contrast, MPLA, lipid X, and deacylated LPS failed to inhibit LPS-stimulated release of TNF. Corresponding to the inhibition of the release of TNF protein, lipid IVA also inhibited the accumulation of TNF mRNA in LPS-stimulated mononuclear cells. These results suggest that lipid IVA may act as a competitive antagonist of LPS, perhaps at the receptor level.

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

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

Activation-dependent recognition by hematopoietic cells of the LDV sequence in the V region of fibronectin.

It has been shown that the alpha 4 beta 1 integrin is the lymphocyte receptor for the carboxy terminal cell-binding domain of fibronectin which comprises adhesion sites in Hep 2 and a high affinity site, CS-1, in the type III connecting segment or V (for variable) region. In the present studies, using a series of peptides derived from CS-1, we identify the tripeptide leu-asp-val (LDV), as the minimal peptide capable of supporting stable lymphocyte or melanoma cell adhesion. However, only cells which expressed an active form of the alpha 4 beta 1 complex were capable of attaching to and spreading on LDV peptide. On a molar basis, LDV minimal peptides were either not active or 10-20 times less active than intact CS-1 in promoting the adhesion of lymphocytes expressing the resting form of the receptor. In cells which express the high avidity form of the receptor, LDV and CS-1 were equally effective in promoting cell adhesion and spreading. The avidity of the alpha 4 beta 1 complex could be altered with mAbs to beta 1 which specifically activate beta 1 dependent function. The high avidity form of the alpha 4 beta 1 complex could be induced on U937 cells, T, and B lymphoblastoid cell lines, or PHA-stimulated T cell blasts. Resting PBL could not be induced to bind LDV peptide conjugates by activating antibodies to beta 1 implying that two signals are required for LDV recognition by T cells. In conclusion, these data show clearly that the minimal peptide for the alpha 4 beta 1 complex in CS-1 is the LDV sequence. Although numerous cell populations can interact with intact CS-1 only cells which express an active alpha 4 beta 1 complex can bind the LDV sequence. This implies that cell interaction with the carboxy terminal cell-binding domain of fibronectin can be regulated at several levels: (a) alpha 4 beta 1 expression; (b) activation of the alpha 4 beta 1 complex; and (c) alternate splicing of CS-1 into V+ isoforms of fibronectin.

Affinity modulation of integrin alpha 5 beta 1: regulation of the functional response by soluble fibronectin.

We report that a beta 1 integrin (alpha 5 beta 1) can exist in different affinity states for its soluble ligand, fibronectin. The alpha 5 beta 1 expressed by the erythroleukemic cell line K562 binds soluble fibronectin with low affinity (Kd > 1 microM), but is induced to bind it with 20-fold higher affinity (Kd-54 nM) in the presence of the anti-beta 1 mAb 8A2. This activation seems to be due to direct antibody-induced change in the receptor that does not require intracellular signaling, and is a plausible basis for the 8A2-induced enhancement of beta 1-dependent adhesion to fibronectin and other immobilized ligands (Kovach, N. L., T. M. Carlos, E. Yee, and J. M. Harlan. 1992. J. Cell Biol. 116: 499-509). Fab fragments of 8A2 bind with higher affinity to alpha 5 beta 1 receptor that is occupied by the GRG-DSP peptide ligand suggesting that the antibody functions by stabilizing a high affinity (occupied) conformer of the receptor. A functional consequence of the affinity modulation is that soluble fibronectin (at physiological concentrations) occupies the high affinity receptors, and so becomes an effective inhibitor of adhesion to immobilized fibronectin. In contrast, the majority of low affinity receptors remain unoccupied and are still to mediate cellular adhesion.

A monoclonal antibody to beta 1 integrin (CD29) stimulates VLA-dependent adherence of leukocytes to human umbilical vein endothelial cells and matrix components.

The leukocyte beta 1 integrin receptor very late activation antigen-4 (VLA-4) (alpha 4 beta 1, CD49d/CD29) binds to vascular cell adhesion molecule-1 (VCAM-1) expressed on cytokine-activated endothelium. A mAb designated 8A2 was identified that stimulated the binding of U937 cells to CHO cells transfected with VCAM-1 cDNA but not endothelial-leukocyte adhesion molecule or CD4 cDNA. mAb 8A2 also rapidly stimulated the adherence of peripheral blood lymphocytes (PBLs) to VCAM-1-transfected CHO cells or recombinant human tumor necrosis factor-treated human umbilical vein endothelial cells. mAb 8A2-stimulated binding of PBL was inhibited by mAbs to VLA-4 or VCAM-1. Surface expression of VLA-4 was not altered by mAb 8A2 treatment and monovalent Fab fragments of mAb 8A2 were active. Immunoprecipitation studies reveal that mAb 8A2 recognizes beta 1-subunit (CD29) of integrin receptors. In contrast to mAbs directed to VLA-4 alpha-subunit (alpha 4, CD49d), mAb 8A2 did not induce homotypic aggregation of PBL. Additionally, mAb 8A2 stimulated adherence of PBL and hematopoietic cell lines to purified matrix components laminin and fibronectin. This binding was blocked by mAbs to the VLA alpha-subunits alpha 6 (CD49f), or alpha 5 (CD49e) and alpha 4 (CD49d), respectively. We conclude that mAb 8A2 modulates the affinity of VLA-4 and other leukocyte beta 1 integrins, and should prove useful in studying the regulation of beta 1 integrin function.

Minimally modified low-density lipoprotein induces monocyte adhesion to endothelial connecting segment-1 by activating beta1 integrin.

We have shown previously that treatment of human aortic endothelial cells (HAECs) with minimally modified low-density lipoprotein (MM-LDL) induces monocyte but not neutrophil binding. This monocyte binding was not mediated by endothelial E-selectin, P-selectin, vascular cell adhesion molecule-I, or intercellular adhesion molecule-I, suggesting an alternative monocyte-specific adhesion molecule. We now show that moncytic alpha4beta1 integrins mediate binding to MM-LDL-treated endothelial cells. We present data suggesting that the expression of the connecting segment-1 (CS-1) domain of fibronectin (FN) is induced on the apical surface of HAEC by MM-LDL and is the endothelial alpha4beta1 ligand in MM-LDL-treated cells. Although the levels of CS-1 mRNA and protein were not increased, we show that MM-LDL treatment causes deposition of FN on the apical surface by activation of beta1integrins, particularly those associated with alpha5 integrins. Activation of beta1 by antibody 8A2 also induced CS-1-mediated monocyte binding. Confocal microscopy demonstrated the activated beta1 and CS-1colocalize in concentrated filamentous patches on the apical surface of HAEC. Both anti-CS-1 and an antibody to activated beta1 showed increased staining on the luminal endothelium of human coronary lesions with active monocyte entry. These results suggest the importance of these integrin ligand interactions in human atherosclerosis.

Activation of beta1 integrins on CML progenitors reveals cooperation between beta1 integrins and CD44 in the regulation of adhesion and proliferation.

Adhesion of normal colony-forming cells (CFC) to bone marrow (BM) stroma and the extracellular matrix (ECM) component fibronectin (FN) depends at least in part on the alpha4beta1 and alpha5beta1 integrins and the CD44 receptor. Aside from anchoring progenitors in the marrow microenvironment, beta1 integrin-dependent adhesion of normal CFC is associated with inhibition of their proliferation. In contrast to normal CFC, chronic myelogenous leukemia (CML) Ph+ CFC adhere significantly less to either stroma or FN. CML Ph+ CFC proliferation is also not inhibited by coculture with stroma or FN. However, equal numbers of alpha4, alpha5, and beta1 integrins and CD44 are present on CML and normal CD34+ cells. We have previously demonstrated that beta1-dependent adhesion to and subsequent proliferation inhibition by FN can be restored when CML Ph+ CFC are incubated with the beta1 integrin activating antibody, 8A2, and demonstrated a role for the alpha5beta1 integrin in this phenomenon. Since the integrin alpha4beta1 and the proteoglycan form of CD44 may cooperate in establishing normal CFC adhesion to FN, we examined if treatment of CML Ph+ CFC with 8A2 also restores the cooperativity between beta1 integrins and CD44. We demonstrate that 8A2 induces adhesion of CML Ph+ CFC not only to intact FN but also to alpha4beta1, alpha5beta1, and proteoglycan binding fragments of FN. 8A2-induced adhesion to these fragments and peptides also results in a significant inhibition of the proliferation of CML Ph+ CFC. Addition of antibodies to either the alpha5, alpha4, or beta1 integrins, antibodies against the CD44 receptor, or removal of chondroitin sulfate glycosaminoglycans from the surface of CML CD34+ HLA-DR+ cells significantly reduced the 8A2-induced adhesion to and adhesion-mediated inhibition of proliferation by FN. These studies demonstrate that activation of beta1 integrins on CML Ph+ CFC not only results in upregulation of beta1 integrin-dependent adhesion and adhesion-mediated inhibition of proliferation, but also in the restoration of cooperation between beta1 integrins and CD44. These studies suggest that decreased beta1 integrin avidity may also affect the function of the proteoglycan adhesion receptor CD44, both of which may contribute to the abnormal circulation and expansion of malignant progenitors in CML.

Reduced prostacyclin survival after fasting-induced elevation of plasma free fatty acids.

The anti-thrombotic effect of prostacyclin (PGI2) may be determined not only by its synthetic rate but also by its subsequent survival in blood. After its release from the vascular wall, prostacyclin binds to plasma albumin which stabilizes the molecule and prolongs its inhibitory effects on platelets. In vitro studies have shown that free fatty acids compete for the same albumin binding sites and may therefore displace PGI2 and substantially shorten its survival. To see if this competition could also occur in vivo, we produced a three-fold rise of plasma free fatty acid concentrations in ten normal volunteers by four days of fasting, which led to a significant reduction in prostacyclin survival as measured by a functional assay based on inhibition of ADP-induced platelet aggregation. The shortening of prostacyclin survival was associated with evidence of increased platelet reactivity as measured by the circulating platelet aggregate ratio test. Diseases that produce marked elevations of free fatty acids such as acute myocardial infarction may also lead to shortened PGI2 survival with potentiation of platelet mediated thrombosis.

Functional down-regulation of alpha 5 beta 1 integrin in keratinocytes is reversible but commitment to terminal differentiation is not.

Extracellular matrix receptors of the integrin family have a dual role in the epidermis, regulating both adhesion and differentiation. Loss of contact with the extracellular matrix causes keratinocytes to become committed to terminal differentiation, and results in a decrease in the ability of the alpha 5 beta 1 integrin to bind fibronectin. We have investigated whether the decrease in ligand-binding ability is reversible and, if so, whether commitment to terminal differentiation can also be reversed. Keratinocytes that had been placed in suspension for 5 hours to induce commitment were compared with the starting population (0 hour cells) in the presence or absence of 8A2, an activating anti-beta 1 antibody. 8A2 IgG or FAb fragments increased the amount of alpha 5 beta 1 in cell extracts that bound to fibronectin-Sepharose and in the presence of 8A2 the amount of bound alpha 5 beta 1 in 0 hour and 5 hour extracts was equal. 8A2 also restored alpha 5 beta 1 function in adhesion assays of intact 5 hour cells. Ca2+, Mg2+ and Mn2+ alone, at concentrations of up to 1 mM, did not increase the adhesiveness of 5 hour cells relative to 0 hour cells; however, the effect of 8A2 on keratinocytes was dependent on Ca2+. Although 8A2 restored alpha 5 beta 1 ligand-binding ability it did not prevent committed cells from withdrawing from the cell cycle and expressing involucrin, a differentiation marker.(ABSTRACT TRUNCATED AT 250 WORDS)

Stem cell factor modulates avidity of alpha 4 beta 1 and alpha 5 beta 1 integrins expressed on hematopoietic cell lines.

Interactions between hematopoietic cells and bone marrow (BM) stroma, composed of extracellular matrix and stromal cells, are crucial for hematopoiesis. Integrins facilitate these interactions by mediating adherence of hematopoiesis. Integrins facilitate these interactions by mediating adherence of hematopoietic cells to both the extracellular matrix and stromal cells. Marrow stromal cells secrete a variety of growth factors, including stem cell factor (SCF). Because treatment with SCF in vivo mobilizes primitive hematopoietic cells from the BM, we investigated the effect of the growth factor SCF of hematopoietic cell adhesion. These studies show that SCF modulates adhesive function in a dose- and time-dependent manner, but does not modulate expression of the integrins alpha 4 beta 1 and alpha 5 beta 1 in the SCF-responsive cell line MO7E. Treatment of MO7E cells with SCF (200 ng/mL) produced a transient increase in adherence to cytokine-activated human umbilical vein endothelial cells (HUVECs) or to vascular cell adhesion molecule 1 (VCAM-1)-transfected Chinese hamster ovary (CHO) cells with peak adhesion at 30 minutes and return to baseline by 60 to 90 minutes. This increase in adhesion was paralleled by increased binding of the beta 1 activation-dependent monoclonal antibody (MoAb) 15/7, as determined by flow cytometry. However, prolonged incubation of MO7E with SCF induced a marked decrease in integrin-mediated adherence, with maximal inhibition by 24 hours. No change in expression of integrins, as determined by flow cytometry, was observed with short- or long-term incubation with SCF. SCF-treated cells were still able to respond to phorbol esters and to the activating beta 1 MoAb 8A2 with increased adherence, but not to the level seen in control cells. This suggests that a subpopulation of expressed alpha 4 beta 1 and alpha 5 beta 1 integrins is disengaged by prolonged incubation with SCF.

Production of human tumor necrosis factor from whole blood ex vivo.

Since monocytes are the major source of tumor necrosis factor-alpha (TNF) in whole blood, we have developed a new method for stimulating TNF production using heparinized human whole blood instead of isolated monocytes. Test agents were dissolved in endotoxin-free buffer and 25 microliters aliquots were added directly to 225 microliters of whole blood. Following incubation at 37 degrees C, TNF levels were measured directly from diluted plasma (10%) by enzyme-linked immunoassay or L929 bioassay. Unstimulated whole blood released no detectable TNF (less than 150 pg/ml; less than 40 U/ml) over a 24 hour incubation period, but significant TNF release could be detected following a 6 hour incubation with 10 ng/ml of LPS (2163 pg/ml; 390 +/- 240 U/ml). In contrast to methods using isolated monocytes, the measurement of TNF production in whole blood ex vivo avoids monocyte activation by an adherence step, reduces the risk of contamination by endotoxin during isolation, and eliminates potentially confounding exogenous serum factors. More importantly, this method examines monocyte TNF release in response to stimuli in the relevant physiologic milieu.

MAP kinase activation by flow in endothelial cells. Role of beta 1 integrins and tyrosine kinases.

Local alterations in the hemodynamic environment regulate endothelial cell function, but the signal-transduction mechanisms involved in this process remain unclear. We previously demonstrated that mitogen-activated protein (MAP) kinase is rapidly stimulated by flow in bovine aortic endothelial cells. Integrin receptors may act as mechanotransducers, as suggested by rapid remodeling of focal adhesion complexes in response to flow. To study the role of integrins in flow-mediated MAP kinase activation, we compared the effects of beta 1 integrin activation (with 8A2 antibody) and flow in cultured human umbilical vein endothelial cells (HUVECs). Both 8A2 (3 micrograms/mL) and flow (shear stress, 12 dynes/cm2) stimulated MAP kinase, although the flow response was faster and greater. To characterize flow-activated tyrosine kinases, tyrosine-phosphorylated proteins were immunoprecipitated and identified by Western blot. There was a time-dependent increase in phosphotyrosine content in 60- to 80-kD, 110-kD, 125- to 150-kD, and 180- to 190-kD proteins. A 125-kD protein was identified as focal adhesion kinase (FAK), suggesting that flow activates integrins. In comparison with flow, 8A2 caused less tyrosine phosphorylation of fewer proteins, although FAK was tyrosine phosphorylated. Concurrent stimulation of HUVECs with 8A2 and flow caused additive increases in MAP kinase. Antibody 8A2 increased binding of the beta 1 affinity-sensitive antibody, 15/7, while flow failed to increase binding of 15/7. In summary, both a beta 1-activating antibody and flow stimulate tyrosine kinases, leading to activation of FAK and MAP kinase signal-transduction pathways. However, the cellular responses elicited by 8A2 represent only a portion of those stimulated by flow, suggesting that "costimulatory" events such as calcium mobilization, in addition to integrin activation, mediate the HUVEC response to fluid shear stress.

Activation-dependent alpha5beta1 integrin-mediated adhesion to fibronectin decreases proliferation of chronic myelogenous leukemia progenitors and K562 cells.

Chronic myelogenous leukemia (CML) is a malignant disease of the hematopoietic stem cell characterized by abnormal circulation and proliferation of malignant progenitors. In contrast to their normal counterparts, CML progenitors adhere poorly to bone marrow stroma or fibronectin (FN). Aside from anchoring progenitors in the marrow microenvironment, beta1 integrin-dependent adhesion of normal progenitors is also associated with inhibition of their proliferation. As the beta1 integrin expression on CML progenitors is normal, we hypothesized that decreased integrin affinity may underlie the abnormal adhesive and proliferative characteristics of CML progenitors. We examined the effect of affinity modulation by the activating antibody 8A2 on the adhesion and proliferation of CML progenitors and the CML cell line, K562. 8A2 induced alpha5Beta1-dependent adhesion of Philadelphia chromosome-positive (Ph+) CD34+/HLA-DR+ cells and K562 cells to FN. Increased adhesion was 8A2- and FN concentration-dependent, time-dependent, and energy-dependent. Further, 8A2-induced adhesion to FN significantly inhibited the proliferation of malignant CML progenitors as well as K562 cells independent of cell differentiation, necrosis, or apoptosis. These studies demonstrate that affinity modulation of the alpha5Beta1 integrin on CML progenitors and K562 cells by 8A2 results in increased adhesion to FN with subsequent decreased proliferation, suggesting that decreased beta1 integrin affinity contributes to the abnormal circulation and proliferation of malignant progenitors in CML.

Role of protein synthesis and CD11/CD18 adhesion complex in neutrophil emigration into the lung.

The mechanism of neutrophil (PMN) emigration into the lung may be stimulus-dependent. This study examined PMN emigration in the lung induced by intratracheal instillation of lipopolysaccharide (LPS), Streptococcus pneumoniae (S. pneu) organisms, supernatant from S. pneu incubated with alveolar macrophages (AM phi), Escherichia coli (E. coli) organisms, or phorbol myristate acetate (PMA). Rabbits were pretreated with either the CD18 monoclonal antibody (MAb) 60.3, the protein synthesis inhibitor cycloheximide (Cx), or, in one case, both. Animals were then given one of the above stimuli to elicit PMN emigration. Four hours after the stimulus was instilled, animals were killed and total and differential cell counts were performed on bronchoalveolar lavage (BAL) fluid. PMN emigration in response to PMA was virtually abolished by MAb 60.3, but was not significantly inhibited by Cx. Emigration induced by LPS was inhibited by 80% by either MAb 60.3 or Cx, and greater than 94% when MAb 60.3 and Cx were given simultaneously. Emigration in response to E. coli organisms was 80% inhibited by MAb 60.3. Emigration induced by S. pneu was approximately 50% inhibited by MAb 60.3, but was greater than 90% blocked by Cx. The MAb 60.3 had approximately the same effect on PMN emigration toward the supernatant from co-incubation of AM phi with S. pneu as it did toward live S. pneu. It is concluded that the mechanism of PMN emigration into the lung is stimulus-dependent. The CD18-dependent mechanism is responsible for the majority of the emigration in response to PMA, E. coli LPS, and E. coli organisms. S. pneu and supernatant from S. pneu + AM phi produce a CD18-independent pathway. These data suggest the requirement for de novo protein synthesis for PMN emigration in response to LPS and S. pneu, but not for PMA-induced emigration.

A biochemical characterization of the binding of osteopontin to integrins alpha v beta 1 and alpha v beta 5.

Osteopontin (OPN) is an extracellular matrix protein that binds to integrin alpha v beta 3. Here we demonstrate that two other integrins, alpha v beta 1 and alpha v beta 5, are also receptors for OPN. Human embryonic kidney 293 cells adhere to human recombinant osteopontin (glutathione S-transferase-osteopontin; GST-OPN) using integrin alpha v beta 1. When the 293 cells are transfected with the beta 5 subunit, they can also adhere to GST-OPN using integrin alpha v beta 5. Divalent cations regulate the binding of GST-OPN to both alpha v beta 1 and alpha v beta 5. Mg2+ and Mn2+ support the binding of GST-OPN to these integrins but Ca2+ does not. The highest affinity is observed in Mn2+. In the presence of this ion, the affinity of GST-OPN for alpha v beta 1 is 18 nM and the affinity for alpha v beta 5 is 48 nM. The antibody 8A2, which is an agonist for beta 1, promotes the adhesion of 293 cells to GST-OPN even when Ca2+ is present. This observation suggests that cellular events could modulate the affinity of alpha v beta 1 for OPN. Collectively, these findings prove that integrins alpha v beta 1, alpha v beta 3, and alpha v beta 5 have similar affinity for OPN. Therefore, all three integrins must be considered when evaluating the biological affects of OPN.

Regulation of alpha 4 integrin-mediated adhesion of human eosinophils to fibronectin and vascular cell adhesion molecule-1.

BACKGROUND: Eosinophils selectively accumulate at sites of allergic inflammation. Their recruitment is dependent on both the expression and functional activity of cell adhesion molecules. How the functional activity of cell adhesion molecules on eosinophils is regulated is poorly understood. OBJECTIVE: Our objective was to examine the functional activity of alpha 4 integrins on human eosinophils and its regulation by various agents. METHODS: Function of alpha 4 integrins on human eosinophils was examined by testing adhesion to immobilized fibronection and vascular cell adhesion molecule-1 (VCAM-1) in the presence or absence of a monoclonal antibody (mAb) (8A2) that activates beta 1 integrin function. RESULTS: Spontaneous eosinophil adhesion to VCAM-1 was enhanced by 8A2, but adhesion to fibronectin could only be detected in the presence of 8A2. Concentrations of 8A2 that were approximately 100-fold less than saturating induced maximal eosinophil adhesion. Adhesion to VCAM-1 in the presence of 8A2 was effectively inhibited by alpha 4 and beta 1 integrin mAbs: beta 7 mAb had partial inhibitory activity. Connecting segment-1 peptide and alpha 4 mAb blocked 8A2-dependent fibronectin binding: beta 1, beta 2, and beta 7 integrin mAbs had partial inhibitory activity. Eosinophils obtained from bronchoalveolar lavage fluids and blood eosinophils stimulated with IL-5, platelet-activating factor, or RANTES displayed increased beta 2 integrin-dependent, not alpha 4 integrin-dependent, attachment. Spontaneous adhesion of eosinophils to VCAM-1 was significantly reduced by the tyrosine kinase inhibitor tyrphostin B46 (inhibitory concentration of 50% approximately equal to 20 mumol/L); this effect was reversed by 8A2. CONCLUSIONS: The functional activity of integrins on eosinophils can be positively and negatively regulated. Altered integrin avidity may influence eosinophil recruitment in vivo.

Clinical relevance of parvovirus B19 as a cause of anemia in patients with human immunodeficiency virus infection.

Parvovirus B19 (B19) DNA was detected by dot blot hybridization in sera from 5 (17%) of 30 human immunodeficiency virus (HIV)-infected patients with hematocrits (HCT) of < or =24 and 4 (31%) of 13 HRV-infected patients with HCT of < or =20, suggesting that B19 is a reasonably common cause of severe anemia in HIV infection. The anemia promptly remitted after immunoglobulin therapy in 3 of 4 treated patients. The presence of IgM to B19, the clinical circumstance in which anemia developed, and the marrow morphology were poor predictors of chronic B19 infection. DNA hybridization studies of sera from 191 HIV-infected and 117 HIV-seronegative homosexual males attending a clinic in the Seattle area revealed that 1 (0.5%) and 2 (2%) samples, respectively, from the 2 groups contained B19. However, when assayed by polymerase chain reaction (PCR), 5% of the serum samples from HIV-infected persons and 9% from uninfected persons contained B19, although each had an HCT of > or =40. The data argue that anemia results from chronic high-titer B19 infection. Although a negative PCR assay excludes this diagnosis, DNA hybridization may be the more specific serum test.

Human umbilical vein endothelial cells display high-affinity c-kit receptors and produce a soluble form of the c-kit receptor.

Stem cell factor (SCF) is a hematopoietic growth factor produced by fibroblasts and endothelial cells that stimulates the growth of primitive hematopoietic cells. SCF triggers cell growth by binding to the c-kit receptor. Because endothelial cells can respond to certain hematopoietic growth factors, we tested human umbilical vein endothelial cells for display of the c-kit receptor and examined the effect of SCF on endothelial cell proliferation, adhesion molecule expression, and production of tissue factor. Quantitative binding experiments with 125I-SCF showed both high-affinity (Kd = 42 pmol/L) and low-affinity (Kd = 1.7 nmol/L) c-kit receptors. There were approximately 1,100 high-affinity c-kit receptors, and 5,400 low-affinity c-kit receptors per endothelial cell. Enzyme immunoassays showed that endothelial cells released soluble c-kit receptor and SCF. The transmembrane form of SCF was detected by indirect immunofluorescence analysis using monoclonal or polyclonal anti-SCF receptor antibodies. The addition of SCF (100 ng/mL) did not alter endothelial cell proliferation over a 7-day period. Similarly, there was no change in the release of tissue factor or expression of inducible endothelial adhesion molecules (intercellular adhesion molecule-1, endothelial-leukocyte adhesion molecule-1, and vascular cell adhesion molecule-1) measured by enzyme-linked immunosorbant assay at 4 and 24 hours after SCF addition. The neutralizing anti-c-kit receptor monoclonal antibody SR-1 blocked binding of 125I-SCF to the c-kit receptor by 98% but did not alter endothelial cell proliferation or adhesion-molecule expression. c-kit receptors were also detected on adult endothelial cells lining small blood vessels in normal human lymph nodes. These data indicate that normal human endothelial cells produce SCF and show high-affinity c-kit receptors that have the capacity to dimerize. The lack of response to exogenous SCF may be because of intracellular activation of the c-kit receptor via autocrine production of SCF. Alternatively, SCF and c-kit may play a role other than stimulation of proliferation, adhesion-molecule display, or tissue factor production by endothelial cells. The production of soluble c-kit receptors by normal human endothelial cells may serve to regulate the bioactivity of SCF within the bone marrow microenvironment.

Pentoxifylline inhibits integrin-mediated adherence of interleukin-2- activated human peripheral blood lymphocytes to human umbilical vein endothelial cells, matrix components, and cultured tumor cells.

Peripheral blood lymphocytes (PBLs) cultured in the presence of recombinant human interleukin-2 (rhIL-2) develop a natural killer (NK) cell phenotype (CD16+, CD56+, CD3-) and are referred to as lymphokine-activated killer cells (LAK). In developing the LAK phenotype, enhanced adherence to matrix components and endothelial cells have been described. In this report we investigated the functional behavior of adhesion receptors in rhIL-2-activated PBLs by in vitro adhesion assay and by flow cytometry. Compared to PBLs, IL-2-activated PBLs had increased integrin-mediated adherence to: (1) fibronectin (FN), (2) human umbilical vein endothelial (HUVE) cells, and (3) cultured melanoma and pancreatic tumor cell lines. This increase in adherence was mediated by increased surface expression of members of the beta 1 and beta 2 integrin subfamilies, as determined by flow cytometric analysis. No induction of an activation-dependent beta 1 (CD29) epitope was detected. We also investigated the effects of the methylxanthine derivative pentoxifylline (PTX) on PBLs and rhIL-2-activated PBL adhesion. PBLs co-cultivated in the presence of rhIL-2 (1,000 U/mL) and PTX exhibited reduced adherence to FN, HUVE and cultured tumor cell lines. This inhibition by PTX was concentration- and time-dependent. The increased expression of integrins induced by rhIL-2 was only in part inhibited by PTX, suggesting that PTX induced a subpopulation of integrins that are expressed but functionally inactive.