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PubMed1994

Activation of the alpha 4 beta 1 integrin through the beta 1 subunit induces recognition of the RGDS sequence in fibronectin.

Lymphocyte attachment to fibronectin is mainly mediated by the interaction of alpha 5 beta 1 and alpha 4 beta 1 integrins with the RGD and CS-1/Hep II sites, respectively. We have recently shown that the anti-beta 1 mAb TS2/16 can convert the partly active alpha 4 beta 1 present on certain hemopoietic cells that recognizes CS-1 but not Hep II, to a high avidity form that binds both ligands. In this report we have studied whether mAb TS2/16 also affects alpha 4 beta 1 ligand specificity. Incubation of the B cell lines Ramos and Daudi (which lack alpha 5 beta 1) with mAb TS2/16 induced specific attachment to an 80-kD fragment which lacks CS-1 and Hep II and contains the RGD sequence. mAbs anti-alpha 4 and the synthetic peptides CS-1 and IDAPS inhibited adhesion to the 80-kD fragment thus implying alpha 4 beta 1 as the receptor for this fragment. Interestingly, the synthetic peptide GRGDSPC and a 15-kD peptic fibronectin fragment containing the RGD sequence also inhibited B cell adhesion to the 80-kD fragment. Because we have previously shown that RGD peptides do not affect the constitutive function of alpha 4 beta 1, we tested whether TS2/16-activated alpha 4 beta 1 acquired the capacity to recognize RGD. Indeed RGD peptides inhibited TS2/16-treated B cell adhesion to a 38-kD fragment containing CS-1 and Hep II but did not affect binding of untreated cells to this fragment. An anti-fibronectin mAb reactive with an epitope on or near the RGD sequence also efficiently inhibited cell adhesion to the 80-kD fragment, indicating that the RGD sequence is a novel adhesive ligand for activated alpha 4 beta 1. These results emphasize the role of alpha 4 beta 1 as a receptor with different ligand specificities according to the activation state, a fact that may be important for lymphocyte migration, localization, and function.

Amino Acid Sequence↗

PubMed1995

Changes in the fibronectin-specific integrin expression pattern modify the migratory behavior of sarcoma S180 cells in vitro and in the embryonic environment.

The molecules that mediate cell-matrix recognition, such as fibronectins (FN) and integrins, modulate cell behavior. We have previously demonstrated that FN and the beta 1-integrins are used during neural crest cell (NCC) migration in vitro as well as in vivo, and that the FN cell-binding domains I and II exhibit functional specificity in controlling either NCC attachment, spreading, or motility in vitro. In the present study, we have analyzed the effect of changes in the integrin expression patterns on migratory cell behavior in vivo. We have generated, after stable transfection, S180 cells expressing different levels of alpha 4 beta 1 or alpha 5 beta 1 integrins, two integrins that recognize distinct FN cell-binding domains. Murine S180 cells were chosen because they behave similarly to NCC after they are grafted into the NCC embryonic pathways in the chicken embryo. Thus, they provide a model system with which to investigate the mechanisms controlling in vitro and in vivo migratory cell behavior. We have observed that either the overexpression of alpha 5 beta 1 integrin or the induction of alpha 4 beta 1 expression in transfected S180 cells enhances their motility on FN in vitro. These genetically modified S180 cells also exhibit different migratory properties when grafted into the early trunk NCC migratory pathways. We observe that alpha 5 and low alpha 4 expressors migrate in both the ventral and dorsolateral paths simultaneously, in contrast to the parental S180 cells or the host NCC, which are delayed by 24 h in their invasion of the dorsolateral path. Moreover, the alpha 4 expressors exhibit different migratory properties according to their level of alpha 4 expression at the cell surface. Cells of the low alpha 4 expressor line invade both the ventral and dorsolateral pathways. In contrast, the high expressors remain as an aggregate at the graft site, possibly the result of alpha 4 beta 1-dependent homotypic aggregation. Thus, changes in the repertoire of FN-specific integrins enable the S180 cells to exploit different pathways in the embryo and regulate the speed with which they disperse in vivo and in culture. Our studies correlate well with known changes in integrin expression during neural crest morphogenesis and strongly suggest that neural crest cells that migrate into the dorsolateral path, i.e., melanoblasts, do so only after they have upregulated the expression of FN receptors.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1995

Cooperative signaling by alpha 5 beta 1 and alpha 4 beta 1 integrins regulates metalloproteinase gene expression in fibroblasts adhering to fibronectin.

Rabbit synovial fibroblasts (RSF) express basal levels of the metalloproteinases (MMP) collagenase, stromelysin-1 and 92-kD gelatinase when plated on intact fibronectin (FN), but elevated levels when plated on either the central RGD-containing cell-binding region of FN (120FN) or antibody against the alpha 5 beta 1 integrin, suggesting that domains outside 120FN may suppress the induction of MMP (Werb, Z., P. M. Tremble, O. Behrendtsen, E. Crowley, and C.H. Damsky. 1989. J. Cell Biol. 109:877-889). We therefore attempted to reconstitute the basal signaling of intact FN by plating RSF on 120FN together with domains of FN outside this region. Large COOH-terminal fragments containing both the heparin-binding and HICS domains suppressed MMP when combined with 120FN. To map the active sequences, peptides from this region and larger fragments that did, or did not, include the CS-1 portion of IIICS were tested. Only CS-1 peptide, or larger fragments containing CS-1, suppressed MMP expression induced by 120FN. In contrast, peptide V from the heparin-binding region, shown previously to stimulate focal contact formation, further enhanced MMP expression by RSF when present on the substrate with 120FN. RSF expressed alpha 4 beta 1 integrin, the receptor for CS-1, and the anti-alpha 4 mAb blocked the ability of CS-1 to suppress MMP induction by 120FN. These results show that signals modulating MMP expression and focal contact assembly are regulated independently, and that cooperative signaling by alpha 5 beta 1 and alpha 4 beta 1 integrins plays a dominant role in regulating expression of these extracellular matrix-remodeling genes in response to FN. This work demonstrates directly the modular way in which information in the extracellular matrix is detected and processed by cell surface receptors.

Amino Acid Sequence↗

PubMed1996

Sequential regulation of alpha 4 beta 1 and alpha 5 beta 1 integrin avidity by CC chemokines in monocytes: implications for transendothelial chemotaxis.

Leukocyte emigration possibly requires dynamic regulation of integrin adhesiveness for endothelial and extracellular matrix ligands. Adhesion assays on purified vascular cell adhension molecule (VCAM)-1, fibronectin, and fibronectin fragments revealed distinct kinetic patterns for the regulation of very late antigen (VLA)-4 (alpha 4 beta 1) and VLA-5 (alpha 5 beta 1) avidity by the CC chemokines monocyte inflammatory protein (MIP)-1 alpha, RANTES (regulated on activation, normal T expressed and secreted), or monocyte chemoattractant protein (MCP)-1 in monocytes. CC chemokines induced early activation and subsequent deactivation of VLA-4, whereas upregulation of VLA-5 avidity occurred later and persisted. Controlled detachment assays in shear flow suggested that adhesive strength of VLA-4 for VCAM-1 or the 40-kD fragment of fibronectin (FN40) is more rapidly increased and subsequently reduced by MCP-1 than by MIP-1 alpha, and confirmed late and sustained activation of the adhesive strength of VLA-5 for the 120-kD fragment of fibronectin (FN120). Mn2+ or the stimulating beta 1 mAb TS2/16 strongly and stably enhanced monocyte binding to VCAM-1 or fibronectin, and locked beta 1 integrins in a high avidity state, which was not further modulated by CC chemokines. Mn2+ and mAb TS2/16 inhibited CC chemokine-induced transendothelial migration, particularly chemotaxis across stimulated endothelium that involved VLA-4 and VCAM-1. VLA-4 on Jurkat cells is of constitutively high avidity and interfered with migration across barriers expressing VCAM-1. Low but not high site densities of VCAM-1 or FN40 promoted, while FN120 impaired, beta 1 integrin-dependent monocyte chemotaxis to MCP-1 across filters coated with these substrates. Thus, we show that CC chemokines can differentially and selectively regulate avidity of integrins sharing common beta subunits. Transient activation and deactivation of VLA-4 may serve to facilitate transendothelial diapedesis, whereas late and prolonged activation of VLA-5 may mediate subsequent interactions with the basement membrane and extracellular matrix.

Antibodies, Monoclonal↗

PubMed1996

Altered secondary myogenesis in transgenic animals expressing the neural cell adhesion molecule under the control of a skeletal muscle alpha-actin promoter.

The majority of skeletal muscle fibers are generated through the process of secondary myogenesis. Cell adhesion molecules such as NCAM are thought to be intricately involved in the cell-cell interactions between developing secondary and primary myotubes. During secondary myogenesis, the expression of NCAM in skeletal muscle is under strict spatial and temporal control. To investigate the role of NCAM in the regulation of primary-secondary myotube interactions and muscle fusion in vivo, we have examined muscle development in transgenic mice expressing the 125-kD muscle-specific, glycosylphosphatidylinositol-anchored isoform of human NCAM, under the control of a human skeletal muscle alpha-actin promoter that is active from about embryonic day 15 onward. Analysis of developing muscle from transgenic animals revealed a significantly lower number of myofibers encased by basal lamina at postnatal day 1 compared with nontransgenic littermates, although the total number of developing myofibers was similar. An increase in muscle fiber size and decreased numbers of VCAM-1-positive secondary myoblasts at postnatal day 1 was also found, indicating enhanced secondary myoblast fusion in the transgenic animals. There was also a significant decrease in myofiber number but no increase in overall muscle size in adult transgenic animals; other measurements such as the number of nuclei per fiber and the size of individual muscle fibers were significantly increased, again suggesting increased secondary myoblast fusion. Thus the level of NCAM in the sarcolemma is a key regulator of cell-cell interactions occurring during secondary myogenesis in vivo and fulfills the prediction derived from transfection studies in vitro that the 125-kD NCAM isoform can enhance myoblast fusion.

Actins↗

PubMed1998

Endothelial cell E- and P-selectin and vascular cell adhesion molecule-1 function as signaling receptors.

Previous studies have shown that polymorphonuclear leukocyte (PMN) adherence to endothelial cells (EC) induces transient increases in EC cytosolic free calcium concentration ([Ca2+]i) that are required for PMN transit across the EC barrier (Huang, A.J., J.E. Manning, T. M. Bandak, M.C. Ratau, K.R. Hanser, and S.C. Silverstein. 1993. J. Cell Biol. 120:1371-1380). To determine whether stimulation of [Ca2+]i changes in EC by leukocytes was induced by the same molecules that mediate leukocyte adherence to EC, [Ca2+]i was measured in Fura2-loaded human EC monolayers. Expression of adhesion molecules by EC was induced by a pretreatment of the cells with histamine or with Escherichia coli lipopolysaccharide (LPS), and [Ca2+]i was measured in single EC after the addition of mAbs directed against the EC adhesion proteins P-selectin, E-selectin, intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), or platelet/endothelial cell adhesion molecule-1 (PECAM-1). Both anti-P- and anti-E-selectin mAb, as well as anti-VCAM-1 mAb, induced transient increases in EC [Ca2+]i that were comparable to those induced by 200 microM histamine. In contrast, no effect was obtained by mAbs directed against the endothelial ICAM-1 or PECAM-1. PMN adherence directly stimulated increases in [Ca2+]i in histamine- or LPS-treated EC. mAbs directed against leukocyte CD18 or PECAM-1, the leukocyte counter-receptors for endothelial ICAM-1 and PECAM-1, respectively, did not inhibit PMN-induced EC activation. In contrast, mAb directed against sialyl Lewis x (sLex), a PMN ligand for endothelial P- and E-selectin, completely inhibited EC stimulation by adherent PMN. Changes in EC [Ca2+]i were also observed after adherence of peripheral blood monocytes to EC treated with LPS for 5 or 24 h. In these experiments, the combined addition of mAbs to sLex and VLA-4, the leukocyte counter-receptor for endothelial VCAM-1, inhibited [Ca2+]i changes in the 5 h-treated EC, whereas the anti-VLA-4 mAb alone was sufficient to inhibit [Ca2+]i changes in the 24 h-treated EC. Again, no inhibitory effect was observed with an anti-CD18 or anti-PECAM-1 mAb. Of note, the conditions that induced changes in EC [Ca2+]i, i.e. , mAbs directed against endothelial selectins or VCAM-1, and PMN or monocyte adhesion to EC via selectins or VCAM-1, but not via ICAM-1 or PECAM-1, also induced a rearrangement of EC cytoskeletal microfilaments from a circumferential ring to stress fibers. We conclude that, in addition to their role as adhesion receptors, endothelial selectins and VCAM-1 mediate endothelial stimulation by adhering leukocytes.

Actins↗

PubMed2000

The small GTPase, Rap1, mediates CD31-induced integrin adhesion.

Integrin-mediated leukocyte adhesion is a critical aspect of leukocyte function that is tightly regulated by diverse stimuli, including chemokines, antigen receptors, and adhesion receptors. How cellular signals from CD31 and other adhesion amplifiers are integrated with those from classical mitogenic stimuli to regulate leukocyte function remains poorly understood. Here, we show that the cytoplasmic tail of CD31, an important integrin adhesion amplifier, propagates signals that induce T cell adhesion via beta1 (VLA-4) and beta2 (LFA-1) integrins. We identify the small GTPase, Rap1, as a critical mediator of this effect. Importantly, CD31 selectively activated the small Ras-related GTPase, Rap1, but not Ras, R-Ras, or Rap2. An activated Rap1 mutant stimulated T lymphocyte adhesion to intercellular adhesion molecule (ICAM) and vascular cell adhesion molecule (VCAM), as did the Rap1 guanine nucleotide exchange factor C3G and a catalytically inactive mutant of RapGAP. Conversely, negative regulators of Rap1 signaling blocked CD31-dependent adhesion. These findings identify a novel important role for Rap1 in regulating ligand-induced cell adhesion and suggest that Rap1 may play a more general role in coordinating adhesion-dependent signals during leukocyte migration and extravasation. Our findings also suggest an alternative mechanism, distinct from interference with Ras-proximal signaling, by which Rap1 might mediate transformation reversion.

Antigens, CD↗

PubMed1994

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.

Animals↗

PubMed1994

Constitutive activation of integrin alpha 4 beta 1 defines a unique stage of human thymocyte development.

Our understanding of thymocyte development and of the positive and negative selection events involved in shaping the repertoire of mature T lymphocytes has been greatly facilitated by the use of transgenic and gene knockout animals. Much less is known about the factors that control the homing and population of the thymus by T cell precursors and the subsequent migration of developing thymocytes through the thymic architecture. As the integrins represent a candidate group of cell surface receptors that may regulate thymocyte development, we have analyzed the expression and function of alpha 4 beta 1 and alpha 5 beta 1 on human thymocytes. A major portion of double positive (CD4+ CD8+) human thymocytes express alpha 4 beta 1 in a constitutively active form and adhere to fibronectin and vascular cell adhesion molecule 1. alpha 4 beta 1 expression is similar on adherent and nonadherent populations, thus, activity reflects the receptor state and not simple expression. The adherent cells are immature, expressing high levels of CD4/CD8 and low levels of CD3 and CD69. In contrast, nonadherent cells possess the phenotype of thymocytes after positive selection, expressing intermediate levels of CD4 and/or CD8 and high levels of CD3 and CD69. The adherent population fails to respond to activation with anti-CD3 and fibronectin, whereas nonadherents exhibit an alpha 5 beta 1-dependent proliferation. Differential regulation of alpha 4 beta 1 and alpha 5 beta 1 receptors may provide a mechanism controlling cellular traffic, differentiation, and positive selection of thymocytes.

Antibodies↗

PubMed1995

P-selectin and vascular cell adhesion molecule 1 mediate rolling and arrest, respectively, of CD4+ T lymphocytes on tumor necrosis factor alpha-activated vascular endothelium under flow.

This report examines the adhesive interactions of human CD4+ T lymphocytes with tumor necrosis factor alpha-activated human endothelial cell monolayers in an in vitro model that mimics microcirculatory flow conditions. Resting CD4+ T cell interactions with activated endothelium consisted of initial attachment followed by rolling, stable arrest, and then spreading and transendothelial migration. P-selectin, but not E-, or L-selectin, mediated most of this initial contact and rolling, whereas beta 1-integrins (alpha 4 beta 1), interacting with endothelial-expressed vascular cell adhesion molecule 1, participated in rolling and mediated stable arrest. In contrast, beta 2-integrins were primarily involved in spreading and transmigration. These findings highlight an important role for P-selectin and suggest discrete functions for beta 1- and beta 2-integrins during lymphocyte recruitment to sites of immune-mediated inflammation.

Animals↗

PubMed1995

Engaging CD19 or target of an antiproliferative antibody 1 on human B lymphocytes induces binding of B cells to the interfollicular stroma of human tonsils via integrin alpha 4/beta 1 and fibronectin.

Adhesion of B lymphocytes within the different compartments of secondary lymphoid organs is essential for the function of the humoral immune response. It is not currently known how the temporary immobilization of B cells in distinct areas of this complex microenvironment is regulated. The present study aimed at defining B cell antigens that initiate binding of B cells to human tonsil sections in situ. Engaging the B cell antigens CD19 and target of an antiproliferative antibody 1 (TAPA-1) with monoclonal antibodies induced adhesion of these B cells to the interfollicular stroma. This binding occurred through the integrin alpha 4 beta 1 on the B cell surface and via the extracellular matrix protein fibronectin expressed in the interfollicular compartment of the tonsil. Signaling through either antigen, CD19 or TAPA-1, depended on tyrosine kinases. Binding induced by engaging CD19 required an intact cytoskeleton, whereas TAPA-1-transmitted adhesion did not. We suggest that CD19 and TAPA-1 have a novel and unique function by regulating an alpha 4 beta 1/fibronectin-mediated binding of B cells to the interfollicular stroma of lymphoid tissues.

Antibodies, Monoclonal↗

PubMed1996

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

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

Animals↗

PubMed1996

Cell adhesion and migration are regulated at distinct stages of thymic T cell development: the roles of fibronectin, VLA4, and VLA5.

T cell development in the thymus requires the establishment of stable interactions with cell-selecting elements such as the cortical epithelium followed by a regulated movement of selected progenitors to the medulla. Cell adhesion and migration are mediated by integrins in a number of biological systems though little is known regarding their function in the thymus. We demonstrated previously that immature CD3loCD69lo double positive human thymocytes adhere avidly to FN via the integrin, VLA4. We now demonstrate that the interaction of mature CD3hiCD69hi thymic subsets with FN triggers migration rather than firm adhesion. Migration requires the engagement of VLA4 in cooperation with VLA5 and both receptors regulate the persistence and directionality of movement. While migration capability is linked to maturation state, ligand concentration determines the efficiency of migration. In fact, FN and the alternatively spliced CS1 site are predominant in the thymic medulla, suggesting an instructive role of this ECM protein in vivo. Our studies identify a novel VLA4 and VLA5/FN-mediated pathway likely to be involved in regulating cell traffic between the cortex and medulla of the thymus. Moreover, the data provides evidence that VLA4 exists in at least two functional states at distinct stages of T cell development. While different states of VLA4 activation have been described on cell lines, this represents the first evidence supporting a biological significance for this integrin property.

Binding Sites↗

PubMed1997

Human peripheral blood eosinophils express a functional c-kit receptor for stem cell factor that stimulates very late antigen 4 (VLA-4)-mediated cell adhesion to fibronectin and vascular cell adhesion molecule 1 (VCAM-1).

We evaluated mature peripheral blood eosinophils for their expression of the surface tyrosine kinase, c-kit, the receptor for the stromal cell-derived cytokine, stem cell factor (SCF). Cytofluorographic analysis revealed that c-kit was expressed on the purified peripheral blood eosinophils from 8 of 8 donors (4 nonatopic and 4 atopic) (mean channel fluorescence intensity 2.0- 3. 6-fold, average 2.8 +/- 0.6-fold, greater than the negative control). The uniform and selective expression of c-kit by eosinophils was confirmed by immunohistochemical analysis of peripheral blood buffy coats. The functional integrity of c-kit was demonstrated by the capacity of 100 ng/ml (5 nM) of recombinant human (rh) SCF to increase eosinophil adhesion to 3, 10, and 30 microg/ml of immobilized FN40, a 40-kD chymotryptic fragment of plasma fibronectin, in 15 min by 7.7 +/- 1.4-, 5.3 +/- 3.3-, and 5.4 +/- 0. 2-fold, respectively, and their adhesion to 0.1, 0.5, and 1.0 microg/ml vascular cell adhesion molecule-1 (VCAM-1), by 12.7 +/- 9. 2-, 3.8 +/- 2.5-, and 1.7 +/- 0.6-fold, respectively. The SCF-stimulated adhesion occurred without concomitant changes in surface integrin expression, thereby indicating an avidity-based mechanism. rhSCF (100 ng/ml, 5 nM) was comparable to rh eotaxin (200 ng/ml, 24 nM) in stimulating adhesion. Cell adhesion to FN40 was completely inhibited with antibodies against the alpha4 and beta1 integrin subunits, revealing that the SCF/c-kit adhesion effect was mediated by a single integrin heterodimer, very late antigen 4 (VLA-4). Thus, SCF represents a newly recognized stromal ligand for the activation of eosinophils for VLA-4-mediated adhesion, which could contribute to the exit of these cells from the blood, their tissue localization, and their prominence in inflammatory lesions.

Cell Adhesion↗

PubMed2000

Human endothelial cells regulate survival and proliferation of human mast cells.

Mast cells (MCs) are immunoregulatory and inflammatory tissue cells preferentially located around blood vessels. Since endothelial cells have been suggested to regulate MC functions, we analyzed MC-endothelial cell interactions in vitro by performing coculture experiments with purified human intestinal MCs and human umbilical vein endothelial cells (HUVECs). We found that HUVECs provide signals allowing MCs to survive for at least 3 wk and to proliferate without addition of cytokines; otherwise all MCs died. HUVEC-dependent MC proliferation was more pronounced than that induced by stem cell factor (SCF), known to act as an MC growth factor both in vitro and in vivo. After coculture with HUVECs, most MCs were of the tryptase and chymase double-positive phenotype (MC(TC)). Transwell experiments suggested that the HUVECs' effects on MCs are not mediated by soluble factors. HUVEC-dependent MC adhesion and proliferation were inhibited by neutralizing antibodies directed against SCF and vascular cell adhesion molecule (VCAM)-1 expressed on HUVECs, and c-kit and very late antigen 4 (VLA-4) on MCs. The data suggest that two mechanisms (membrane-bound SCF/c-kit and VCAM-1/VLA-4) are involved in human MC-endothelial cell interactions. In conclusion, our study provides evidence that endothelial cells regulate MC survival and preferentially support human MC(TC) development.

Antibodies↗

PubMed2002

Adhesion molecule-dependent mechanisms regulate the rate of macrophage clearance during the resolution of peritoneal inflammation.

Macrophage clearance is essential for the resolution of inflammation. Much is known about how monocytes enter the inflammatory site but little is known about how resultant macro-phages are cleared. We have previously demonstrated that macrophage clearance from resolving peritonitis occurs by emigration into draining lymphatics rather than local apoptosis. We now examine mechanisms for this process, in particular by evaluating the hypothesis that modulation of adhesion interactions between macrophages and cells lining the lymphatics regulates the rate of macrophage clearance. We demonstrate in vivo that macrophages adhere specifically to mesothelium overlying draining lymphatics and that their emigration rate is regulated by the state of macrophage activation. We observed that macrophage-mesothelial adhesion is Arg-Gly-Asp (RGD) sensitive and partially mediated by very late antigen (VLA)-4 and VLA-5 but not alpha(v) or beta(2) integrins. Moreover, macrophage clearance into lymphatics can be blocked in vivo by RGD peptides and VLA-4 and VLA-5 but not beta(2) blocking antibodies. This is the first evidence that macrophage emigration from the inflamed site is controlled and demonstrates that this is exerted through specific adhesion molecule regulation of macrophage-mesothelial interactions. It highlights the importance of adhesion molecules governing entry of cells into the lymphatic circulation, thus opening a new avenue for manipulating the resolution of inflammation.

Animals↗

PubMed2003

Integrin-dependence of lymphocyte entry into the splenic white pulp.

The steps involved in lymphocyte homing to the white pulp cords of the spleen are poorly understood. We demonstrate here that the integrins lymphocyte function associated (LFA)-1 and alpha 4 beta 1 make essential and mostly overlapping contributions necessary for B cell migration into white pulp cords. T cell entry to the white pulp is also reduced by blockade of LFA-1 and alpha 4 beta 1. The LFA-1 ligand, intercellular adhesion molecule 1 is critical for lymphocyte entry and both hematopoietic cells and radiation-resistant cells contribute to this requirement. Vascular cell adhesion molecule 1 contributes to the alpha 4 beta 1 ligand requirement and a second ligand, possibly fibronectin, also plays a role. By contrast with the entry requirements, antigen-induced movement of B cells from follicles to the outer T zone is not prevented by integrin blocking antibodies. Comparison of the distribution of integrin-blocked B cells and B cells treated with the G alpha i inhibitor, pertussis toxin, early after transfer reveals in both cases reduced accumulation in the inner marginal zone. These observations suggest that chemokine receptor signaling and the integrins LFA-1 and alpha 4 beta 1 function together to promote lymphocyte transit from the marginal zone into white pulp cords.

Adoptive Transfer↗

PubMed1999

Phenotypically activated gammadelta T lymphocytes in the peripheral blood of patients with tuberculosis.

Surface molecules with the potential relevance for resistance against Mycobacterium tuberculosis were investigated. The expression of lymphocyte function antigen-1, very late antigen (VLA)-4, l-selectin, intercellular adhesion molecule (ICAM)-1, major histocompatibility complex class II, Fas, and CD40 on alphabeta T cells, gammadelta T cells, NK cells, and monocytes of healthy donors and patients with tuberculosis were analyzed. A high activation status of gammadelta T cells and increased levels of soluble ICAM-1 in plasma of patients with tuberculosis versus healthy individuals was detected. Tuberculosis patients with and without an underlying systemic disease could be segregated by differential expression of VLA-4 and ICAM-1 on gammadelta T cells and on monocytes. The composition of peripheral blood mononuclear cells varied slightly, whereas the proportion of monocytes decreased significantly in patients with tuberculosis, compared with healthy controls. The activation phenotype of peripheral gammadelta T cells in patients with tuberculosis emphasizes the role of these T cells in controlling the inflammatory process during tuberculosis and perhaps other microbial infections.

Adolescent↗