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At least 703 records · Page 39Linked to original sources
PubMed1996

Antigen-mediated IGE receptor aggregation and signaling: a window on cell surface structure and dynamics.

The high-affinity receptor for immunoglobulin E, Fc epsilon RI, serves as an archtype for multisubunit immunoreceptors that mediate cell activation in response to foreign antigens. Antigen-mediated aggregation of this receptor at the surface of mast cells and basophils initiates a biochemical cascade that uses nonreceptor tyrosine kinases as key participants in the earliest steps of this signal transduction process. Cross-linking of Fc epsilon RI with ligands of well-defined structure and valency has revealed detailed information about the fundamental requirements for functionally active receptor aggregates. Cross-linking-dependent changes in the interaction of these receptors with other cellular components have been characterized with biochemical and biophysical methods to develop a more complete view of signal initiation. Recent evidence suggests that this process involves the interaction of aggregated Fc epsilon RI with specialized plasma membrane domains that may localize important signaling molecules in the vicinity of aggregated receptors. Although these various studies were aimed toward understanding the operation of one cell surface receptor, they provide new insights into plasma membrane structure and dynamics that are generally relevant to the function of most nucleated mammalian cells.

Amino Acid Sequence↗
PubMed2001

Characterization of alpha 5-integrin-dependent mast cell adhesion following Fc epsilon RI aggregation.

BACKGROUND: Integrin receptors are engaged in the upregulation of mast cell adhesion to extracellular matrix components upon stimulation with cytokines and antigen. Fibronectin receptor containing the alpha 5-integrin subunit is critical for mast cell interaction with the extracellular matrix protein fibronectin (FN). METHODS: The murine MCP5/L mast cell line was employed to investigate the process of Fc epsilon RI-mediated mast cell adhesion to FN. RT-PCR and cytofluorimetric analysis were used to assess the expression of alpha 5 integrin in MCP5/L mast cells. Radiolabelled mast cells were sensitized with monoclonal IgE and used in adhesion assays. Anti-alpha 5-integrin antibody (Ab), monovalent hapten and metabolic inhibitors were used to characterize antigen-mediated mast cell adhesion to FN. RESULTS: Addition of antigen to IgE-sensitized cells resulted in transient upregulation of mast cell adhesion to FN with a maximum adhesion following 30 min of incubation. Mast cell adhesion was inhibited with anti-alpha 5-integrin monoclonal antibodies blocking FN receptor or with excess monovalent hapten preventing antigen-mediated IgE cross-linking. The presence of the protein kinase C (PKC) inhibitor staurosporine also inhibited mast cell adhesion in a dose-dependent fashion. The process of Fc epsilon RI-mediated upregulation of mast cell adhesion to FN was not associated with an increase in surface expression of mast cell FN receptors. CONCLUSION: The major FN receptor on MCP5/L mast cell surface, an integrin containing the alpha 5 subunit mediates a transient change in mast cell adhesiveness following IgE cross-linking. Fc epsilon RI-derived signals engage PKC and upregulate mast cell adhesion in a process which might involve changes in integrin avidity rather than integrin expression.

Animals↗
PubMed1986

Specific effect of age on lectin-induced mitogenesis of lymphocytes from diseased patients.

Using several models of multivariate analysis, it is found that age per se is a specific determinant on the in vitro lymphocyte response to phytohemagglutinin A in a random sample of 30 diseased patients. Depending upon the used model, sex and lymphocyte subsets appear as other significant variables. The multiple regression coefficients reach 0.85. A 2% fall per decade is expected for phytohemagglutinin A responsiveness expressed in 1n cpm.

Adolescent↗
PubMed2001

Secretion of interleukin-1 receptor antagonist from human mast cells after immunoglobulin E-mediated activation and after segmental antigen challenge.

Mast cells produce substances with antiinflammatory properties in addition to their capacity to release proinflammatory mediators. To further probe the antiinflammatory aspect of mast-cell function we investigated the ability of human mast cells (huMCs) to produce interleukin (IL)-1 receptor antagonist (IL-1ra) in response to high-affinity Fc receptor for immunoglobulin E (Fcalpha RI) aggregation, and examined IL-1ra in bronchoalveolar lavage fluid (BALF) to determine whether it might be of mast-cell origin. Using a ribonuclease protection assay, flow cytometry, and enzyme-linked immunosorbent assay (ELISA), IL-1ra message and protein were found to be constitutively expressed in cultured huMCs. Upon stimulation through Fcalpha RI, IL-1ra message was upregulated in huMCs and IL-1ra protein secreted from cultured huMCs and isolated human lung mast cells. By immunoblot analysis, huMCs were found to produce the 17-kD form of IL-1ra and the presence of IL-1ra in human lung mast cells was confirmed by immunohistochemistry. In BALF obtained from allergic asthmatic subjects, IL-1ra production increased after specific antigen challenge, with the 17-kD isoform of IL-1ra predominating. These findings demonstrate that huMCs produce and release IL-1ra after Fcalpha RI aggregation, which may contribute to a local inhibition of IL-1-dependent effects on inflammation in the lung.

Allergens↗
PubMed1984

Severe deficiency of B lymphocytes in peripheral blood from multiple myeloma patients.

A major problem in the assessment of circulating B lymphocytes in multiple myeloma is the extent to which cells with passively absorbed Ig contribute to the assay. We have analyzed peripheral blood B cell numbers in 51 patients in various treatment categories by using an assay that is not subject to artifacts involving cytophilic Ig. We have defined a B lymphocyte by three different criteria (a) expression of a high surface density of Ig (b) expression of a high density of HLA.DR and (c) expression of a marker exclusive to surface Ig+ B cells. By these criteria, normal individuals have an average of 6% B cells. In multiple myeloma patients, B cell levels in purified mononuclear cell preparations are severely reduced. Untreated patients and the majority of patients on intermittent chemotherapy have 20-600-fold fewer B cells than do normal donors (average = 0.3%). This decrease was even greater in whole blood of patients as compared with normal donors (100-1,000-fold fewer B cells). The number of B cells did not correlate with disease status or paraprotein concentration. We found no evidence to support the idea that B lymphocytes in patients include a substantial monoclonal subset.

B-Lymphocytes↗
PubMed1996

Integrin-dependent induction of functional urokinase receptors in primary T lymphocytes.

In order to reach the sites of inflammation, lymphocytes leave the bloodstream and migrate into peripheral tissues, in a process involving integrin-mediated adhesion to the vascular endothelium, followed by transmigration across the endothelial barrier and through the underlying interstitial matrix. We have investigated the role of the plasminogen activator/plasmin system in normal T cell migration. Receptors for urokinase plasminogen activator (uPAR) were not expressed in resting T lymphocytes, but could be efficiently induced at the mRNA and protein level by coclustering of the antigen receptor complex and beta1 or beta2 integrins, through a signalling pathway involving both protein kinase C activation and an increase in intracellular cyclic AMP. Catalytic activation of plasminogen by uPAR-expressing T cells promoted their migration through an extracellular matrix in vitro. Plasmin-induced invasion was inhibited by plasmin-and urokinase inhibitors and by anti-uPAR antibodies. Finally, cytofluorimetric and immunohistochemical analysis of primary human tumor specimens showed the presence of uPAR positive infiltrating T cells in vivo. Collectively, these findings suggest that plasminogen activation may play a role in lymphocyte migration in vivo, and that integrin-dependent expression of membrane-associated endopeptidases could represent an additional step in the regulated process of leukocyte transmigration.

Adenylyl Cyclases↗
PubMed1996

GPI-anchored proteins, glycosphingolipids, and sphingomyelin are sequestered to caveolae only after crosslinking.

GPI-anchored proteins, glycosphingolipids, and sphingomyelin are all enriched in the detergent-insoluble complex which has been suggested to be purified caveolae. I studied the relationship of the molecules with caveolae in cultured cells by immunocytochemical methods. In cells reacted with antibodies to various membrane proteins and lipids on ice and fixed before applying secondary antibodies, labeling did not show concentration in caveolae. In contrast, when cells were incubated with the primary and secondary antibodies on ice and then transferred to 37 degrees C without fixation, labeled Thy-1.2, beta 2-microglobulin, lactosyl ceramide, ceramide tetrahexose, Forssman antigen, and sphingomyelin became concentrated in caveolae, whereas labeled transferrin receptor did not. Thy-1.2 and sphingolipids formed common patches and were sequestered in the same caveolae when crosslinked with two primary antibodies simultaneously. On the other hand, when either Thy-1.2 alone or lactosyl ceramide alone was crosslinked and sequestered to caveolae, the other antigen remained evenly distributed. Caveolar sequestration of the antigens occurred in the presence of cytochalasin D, nocodazole, or a mixture of the two reagents. The results show that not only GPI-anchored proteins but also glycosphingolipids and sphingomyelin are sequestered in caveolae only after crosslinking, and that the sequestration does not require the intact cytoskeleton.

Animals↗
PubMed2003

Selective blockade of CD28 and not CTLA-4 with a single-chain Fv-alpha1-antitrypsin fusion antibody.

B7-1 and B7-2 are costimulatory molecules expressed on antigen-presenting cells. The CD28/B7 costimulation pathway is critical for T-cell activation, proliferation, and Th polarization. Blocking both cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) and CD28 interactions with a CTLA-4/Ig fusion protein inhibits various immune-mediated processes in vivo, such as allograft rejection and autoimmunity. However, selective blockade of CD28 may represent a better strategy for immunosuppression than B7 blockade, because CTLA-4/B7 interactions have been shown to participate in the extinction of the T-cell receptor-mediated activation signal and to be required for the induction of immunologic tolerance. In addition, selective CD28 inhibition specifically decreases the activation of alloreactive and autoreactive T cells, but not the activation of T cells stimulated by exogenous antigens presented in the context of self major histocompatibility complex (MHC) molecules. CD28 blockade cannot be obtained with anti-CD28 dimeric antibodies, which cluster their target and promote T-cell costimulation, whereas monovalent Fab fragments can block CD28 and reduce alloreactivity. In this study, we report the construction of a monovalent single-chain Fv antibody fragment from a high-affinity antihuman CD28 antibody (CD28.3) that blocked adhesion of T cells to cells expressing the CD28 receptor CD80. Genetic fusion with the long-lived serum protein alpha1-antitrypsin led to an extended half-life without altering its binding characteristics. The anti-CD28 fusion molecule showed biologic activity as an immuno-suppressant by inhibiting T-cell activation and proliferation in a mixed lymphocyte reaction.

Abatacept↗
PubMed2003

Membrane cholesterol regulates LFA-1 function and lipid raft heterogeneity.

Many surface receptors and signaling molecules are thought to associate with unique membrane microdomains termed lipid rafts. We examined the involvement of lipid rafts in the activation of leukocyte function-associated antigen-1 (LFA-1). Depletion or sequestration of cholesterol with methyl-beta-cyclodextrin (MCD) or filipin, respectively, strongly inhibited LFA-1-mediated adhesion of T-cell lines and primary T cells. This inhibition was reversed by cholesterol reconstitution. LFA-1 on T-cell lines was detected in cold Triton X-100-insoluble lipid rafts, which were disrupted by MCD or filipin treatment. However, no LFA-1 on primary T cells was detected in lipid rafts isolated by the same procedures, and these rafts were resistant to cholesterol depletion or sequestration. Association of LFA-1 with lipid rafts of primary T cells could be detected only when they were isolated with another nonionic detergent, Brij 35. Upon treatment with MCD, LFA-1 in Brij 35-insoluble lipid rafts partially shifted to nonraft fractions. T-cell lines were found to have a high level of cholesterol and a low level of ganglioside GM1, a common marker for lipid rafts, whereas primary T cells have a much lower level of cholesterol and a very high amount of GM1. Cross-linking of LFA-1 on primary T cells induced cocapping of cholesterol but not GM1. These results suggest that lipid rafts of T cells are heterogenous, and LFA-1 associates with a subset of lipid rafts containing a high level of cholesterol. This association seems to regulate LFA-1 functions, possibly by facilitating LFA-1 clustering.

Animals↗
PubMed2006

Long-lasting CCR5 internalization by antibodies in a subset of long-term nonprogressors: a possible protective effect against disease progression.

Exposure to HIV-1 does not necessarily result in infection and progression toward disease, thus suggesting that the control of viral infection may be achieved. Antibodies to CCR5 have been detected in HIV-exposed but uninfected subjects (ESNs); thus, these antibodies could be involved in HIV protection. To assess whether anti-CCR5 antibodies may also contribute to slow HIV disease progression, we searched for anti-CCR5 antibodies in 497 subjects, including 85 long-term nonprogressors (LTNPs), 70 progressors, 135 HIV(+) patients treated with highly active antiretroviral therapy (HAART), and 207 seronegative donors. We found anti-CCR5 antibodies in a fraction of the LTNPs(23.5%) but not in the other populations studied (P < .001). These antibodies recognized a conformational epitope within the first extramembrane loop of CCR5, and they induced a stable and long-lasting downregulation of CCR5 on the surface of T lymphocytes, which inhibited HIV entry. In addition, CD4(+) lymphocytes from LTNPs having anti-CCR5 antibodies are resistance to R5 strains of HIV-1. Follow-up studies showed that the loss of anti-CCR5 antibodies occurred in some subjects, and this loss was significantly associated with a progression toward disease, whereas subjects who retained anti-CCR5 Abs maintained their LTNP status. Induction of anti-CCR5 Abs could be relevant to vaccine design and therapeutics.

AIDS Vaccines↗
PubMed2006

CD157 plays a pivotal role in neutrophil transendothelial migration.

Paracellular diapedesis, a key step in leukocyte recruitment to the site of inflammation, occurs at endothelial junctions and is regulated by highly coordinated interactions between leukocytes and endothelium. We found that CD157, a glycosylphosphatidylinositol-anchored ectoenzyme belonging to the NADase/ADP-ribosyl cyclase family, plays a crucial role for neutrophil diapedesis, because its ligation with specific monoclonal antibodies (both on neutrophils or endothelial cells) results in altered neutrophil movement on the apical surface of endothelium and, ultimately, in loss of diapedesis. Real-time microscopy revealed that CD157 behaves as a sort of compass during the interaction between neutrophils and endothelial cells; indeed, following CD157 ligation, neutrophils appear disoriented, meandering toward junctions where they eventually stop without transmigrating. These findings are relevant in vivo because CD157-deficient neutrophils obtained from patients with paroxysmal nocturnal hemoglobinuria are characterized by a severely impaired diapedesis.

ADP-ribosyl Cyclase↗
PubMed2002

Tyrosine phosphorylation of B-cell adaptor for phosphoinositide 3-kinase is required for Akt activation in response to CD19 engagement.

CD19 is a coreceptor that amplifies signaling initiated by antigen cross-linking of the B-cell antigen receptor (BCR). CD19 can also signal independently of BCR coligation. This study shows that B-cell adaptor for phosphoinositide 3-kinase (BCAP), previously characterized as a substrate of the tyrosine kinases upon BCR engagement, is phosphorylated by cross-linking of CD19. Tyrosine phosphorylation of BCAP, mediated by Lyn, provides binding site(s) for phosphoinositide 3-kinase (PI3K), thereby participating in Akt activation. Thus, these results provide evidence that BCAP serves as an adaptor molecule for CD19 to activate the PI3K pathway in B cells.

Amino Acid Motifs↗
PubMed2003

Selective inhibition of T cell activation via CD147 through novel modulation of lipid rafts.

The plasma membrane is compartmentalized into microdomains and the association/dissociation of receptors and signaling molecules with/from these membrane domains is a major principle for regulation of signal transduction. By following the reorganization of microdomains on living cells and performing biochemical studies, we show that Ab targeting of the T cell activation-associated Ag CD147 prevents TCR stimulation-dependent reorganization and clustering of microdomains. Triggering CD147 induces a displacement of the GPI-anchored coreceptors CD48 and CD59 from microdomains in human T lymphocytes. This perturbation of microdomains is accompanied by a selective inhibition of TCR-mediated T cell proliferation. The CD147-inhibited cells secret normal levels of IL-2 but acquire reduced amounts of the IL-2 receptor alpha-chain CD25. These results indicate that negative regulating signals can modulate microdomains and suggest a general mechanism for inhibition of receptor signaling.

Antibodies, Monoclonal↗
PubMed2004

Relevance of CD6-mediated interactions in T cell activation and proliferation.

CD6 is a cell surface receptor expressed on immature thymocytes and mature T and B1a lymphocytes. The ultimate function of CD6 has not been deciphered yet, but much evidence supports a role for CD6 in T cell activation and differentiation. In this study, we show that a fraction of CD6 molecules physically associates with the TCR/CD3 complex by coimmunoprecipitation, cocapping, and fluorescence resonance energy transfer experiments. Image analysis of Ag-specific T-APC conjugates demonstrated that CD6 and its ligand, activated leukocyte cell adhesion molecule (CD166), colocalize with TCR/CD3 at the center of the immunological synapse, the so-called central supramolecular activation cluster. The addition of a soluble rCD6 form significantly reduced the number of mature Ag-specific T-APC conjugates, indicating that CD6 mediates early cell-cell interactions needed for immunological synapse maturation to proceed. This was in agreement with the dose-dependent inhibition of CD3-mediated T cell proliferation induced by soluble rCD6. Taken together, our data illustrate the important role played by the intra- and intercellular molecular interactions mediated by CD6 during T cell activation and proliferation processes.

Activated-Leukocyte Cell Adhesion Molecule↗
PubMed1999

Regulation of apoptosis in mature alphabeta+CD4-CD8- antigen-specific suppressor T cell clones.

The regulation of apoptosis in mature CD4+ or CD8+ alphabeta+ T cells has been well studied. How the survival and death is regulated in peripheral CD4-CD8- (double negative, DN) alphabeta+ T cells remains unknown. Recent studies suggest that peripheral DN T cells may play an important role in the regulation of the immune responses mediated by CD4+ or CD8+ T cells. Here, we used immunosuppressive DN T cell clones to elucidate the mechanisms involved in the regulation of death and survival of alphabeta+ DN T cells. The DN T cell clones were generated from the spleen cells of 2C transgenic mice, which express the transgenic TCR specific for Ld and permanently accepted Ld+ skin allografts after pretransplant infusion of Ld+ lymphocytes. We report that 1) the mature DN T cells are highly resistant to TCR cross-linking-induced apoptosis in the presence of exogenous IL-4; 2) Fas/Fas-ligand and TNF-alpha/TNFR pathways do not play an apparent role in regulating apoptosis in DN T cells; 3) the DN T cells constitutively express a high level of Bcl-xL, but not Bcl-2; 4) both Bcl-xL and Bcl-2 are up-regulated following TCR-cross-linking; and 5) IL-4 stimulation significantly up-regulates Bcl-xL and c-Jun expression and leads to mitogen-activated protein kinase phosphorylation in DN T cells, which may contribute to the resistance to apoptosis in these T cells. Taken together, these results provide us with an insight into how mature DN T cells resist activation-induced apoptosis to provide a long-term suppressor function in vivo.

Animals↗
PubMed1999

Glucocorticoid suppresses autocrine survival of mast cells by inhibiting IL-4 production and ICAM-1 expression.

When mast cells are activated through their high affinity IgE receptors (FcepsilonRI), release of chemical mediators is followed by secretion of multiple cytokines. In this work, we report that IL-3-dependent mast cell line MC9 undergoes apoptosis when IL-3 is withdrawn. However, cross-linking of FcepsilonRI prevents apoptosis of MC9 by an autocrine mechanism, producing IL-3, IL-4, and GM-CSF. Although stimulated MC9 synthesizes mRNAs and proteins of these cytokines, secretion of endogenous IL-3 and GM-CSF is not enough for cell survival, whereas IL-4 itself does not have survival effect on MC9, but it induces cell aggregation by expressing LFA-1 and makes it reactive to endogenous growth factors. Addition of dexamethazone (DXM) to MC9 results in significant down-regulation of IL-4 mRNA in activated MC9. However, mRNA levels of IL-3 and GM-CSF are not changed by DXM. DXM also directly down-regulates the expression of ICAM-1 that is the high affinity ligand of LFA-1, by which the self-aggregation of MC9 is inhibited. Thus, glucocorticoids suppress autocrine survival of mast cells by inhibiting IL-4 production and ICAM-1 expression.

Animals↗