Biological activity of soluble CD100. II. Soluble CD100, similarly to H-SemaIII, inhibits immune cell migration

CD100 is a human 150-kDa homodimer expressed at the surface of most hemopoietic cells, and its gene belongs to the Ig and semaphorin gene families. Semaphorin genes encode soluble and membrane-bound proteins, most of which have been shown to act as chemorepellents on growth cone guidance. CD100 is discrete, as it is a transmembrane leukocyte surface molecule that can also exist in a soluble form. While our previous studies using mAbs suggested that the transmembrane form of CD100 plays a role in lymphocyte activation, no function was shown for its soluble form. Here, we investigated the effect of soluble CD100 in a cell migration assay; both CD100 spontaneously shed from a stable transfectant and soluble recombinant CD100 inhibited spontaneous and chemokine-induced migration of human monocytes. Interestingly, only the dimeric form of CD100 exerted an effect. Moreover, soluble CD100 inhibited migration of cells from monocytic and B cell lineages. A similar inhibitory effect on migration was observed with H-SemaIII, but not H-SemaIV, semaphorins. In addition, both CD100 and H-SemaIII were recognized by two CD100 mAbs in an ELISA, and one of these mAb abolished the inhibitory effect of each of these semaphorins. We also provide evidence that CD100 and H-SemaIII act through the same receptor on immune cells, which is not neuropilin-1. Furthermore, we describe a function on immune cells for H-SemaIII, a semaphorin to date only studied in the nervous system.     

Prolidase activity in fibroblasts is regulated by interaction of extracellular matrix with cell surface integrin receptors

Prolidase (EC 3.4.13.9) is a ubiquitously distributed imidodipeptidase that catalyzes the hydrolysis of C-terminal proline or hydroxyproline containing dipeptides. The enzyme plays an important role in the recycling of proline for collagen synthesis and cell growth. An increase in enzyme activity is correlated with increased rates of collagen turnover indicative of extracellular matrix (ECM) remodeling, but the mechanism linking prolidase activity and ECM is poorly understood. Thus, the effect of ECM-cell interaction on intracellular prolidase activity is of special interest. In cultured human skin fibroblasts, the interaction with ECM and, more specifically, type I collagen mediated by the β₁ integrin receptor regulates cellular prolidase activity. Supporting evidence comes from the following observations: 1) in sparse cells with a low amount of ECM collagen or in confluent cells in which ECM collagen was removed by collagenase (but not by trypsin or elastase) treatment, prolidase activity was decreased; 2) this effect was reversed by the addition of type I collagen or β₁ integrin antibody (agonist for β₁ integrin receptor); 3) sparse cells (with typically low prolidase activity) showed increased prolidase activity when grown on plates coated with type I collagen or on type IV collagen and laminin, constituents of basement membrane; 4) the relative differences in prolidase activity due to collagenase treatment and subsequent recovery of the activity by β₁ integrin antibody or type I collagen treatment were accompanied by parallel differences in the amount of the enzyme protein recovered from these cells, as shown by Western immunoblot analysis. Thus, we conclude that prolidase activity responded to ECM metabolism (tissue remodeling) through signals mediated by the integrin receptor. J. Cell. Biochem. 67:166–175, 1997. Published 1997 Wiley-Liss, Inc.     

A soluble form of the human T cell differentiation antigen CD27 is released after triggering of the TCR/CD3 complex.

The human T cell Ag CD27 belongs to a recently defined family of cell surface receptors, including the nerve growth factor receptor, two distinct tumor necrosis factor receptors, and the B cell specific molecule CD40. On resting T cells, CD27 is a transmembrane homodimer with subunits of 50 to 55 kDa (p55). T cell activation via the TCR/CD3 complex causes a strong enhancement of p55 expression. Concomitantly, an alternative form of the CD27 molecule with a molecular mass of 28 to 32 kDa (p32) appears at the cell surface. With the use of ELISA, we here show that a soluble form of CD27 (sCD27) can be detected in the supernatant of T cells activated with anti-CD3 or combinations of anti-CD2 mAb. Moreover, sCD27 is found in both serum and urine from healthy donors. sCD27, purified from either culture supernatant or urine, has a molecular mass of 28 to 32 kDa and is, according to peptide mapping, structurally homologous to the p55 membrane form of CD27. Quantification of sCD27 levels may be used as a marker for T lymphocyte activation in vivo.     

Cell surface CD4 interferes with the infectivity of HIV-1 particles released from T cells.

The CD4 protein is required for the entry of human immunodeficiency virus (HIV) into target cells. Upon expression of the viral genome, three HIV-1 gene products participate in the removal of the primary viral receptor from the cell surface. To investigate the role of surface-CD4 in HIV replication, we have created a set of Jurkat cell lines which constitutively express surface levels of CD4 comparable to those found in peripheral blood lymphocytes and monocytes. Expression of low levels of CD4 on the surface of producer cells exerted an inhibitory effect on the infectivity of HIV-1 particles, whereas no differences in the amount of cell-free p24 antigen were observed. Higher levels of cell surface CD4 exerted a stronger inhibitory effect on infectivity, and also affected the release of free virus in experiments where the viral genomes were delivered by electrotransfection. The CD4-mediated inhibition of HIV-1 infectivity was not observed in experiments where the vesicular stomatitis virus G protein was used to pseudotype viruses, suggesting that an interaction between CD4 and gp120 is required for interference. In contrast, inhibition of particle release by high levels of cell-surface CD4 was not overcome by pseudotyping HIV-1 with foreign envelope proteins. Protein analysis of viral particles released from HIV-infected Jurkat-T cells revealed a CD4-dependent reduction in the incorporation of gp120. These results demonstrate that physiological levels of cell-surface CD4 interfere with HIV-1 replication in T cells by a mechanism that inhibits envelope incorporation into viral membranes, and therefore provide an explanation for the need to down-modulate the viral receptor in infected cells. Our findings have important implications for the spread of HIV in vivo and suggest that the CD4 down-modulation function may be an alternative target for therapeutic intervention.     

The biologic effects of allogeneic effect factor on T lymphocytes. I. The mitogenic activity and the autonomous induction of cytotoxic T lymphocytes by AEF

Studies presented herein illustrate the capacity of the soluble mediator, allogeneic effect factor (AEF), which is derived from histoincompatible cell interactions, to induce the in vitro differentiation of normal murine splenic lymphocytes into mature cytotoxic cells capable of exerting activity on H-2-identical target cells. This process requires the presence of T lymphocytes during the sensitization phase, and the lytic activity on tumor cells is mediated by cytotoxic T lymphocytes (CTL). The capacity of AEF to induce differentiation of such CTL does not require the presence of stimulating target cells in the sensitization phase. The induction of CTL requires the presence of AEF at the initiation of culture, although exposure to AEF as brief as 1 hr is sufficient to induce fresh spleen cells to differentiate into CTL during the subsequent 5 days in culture. In addition to its ability to induce CTL, AEF is highly mitogenic for T lymphocytes. However, the mitogenic and the CTL-inducing activities of AEF can be experimentally dissociated, indicating that different subpopulations of T lymphocytes may be involved in the response to AEF. In contrast to similar soluble helper factors derived from allogeneic cell interactions, AEF appears to be unique in its ability to autonomously induce a primary CTL response in vitro.     

The laminin-binding activity of the alpha 7 integrin receptor is defined by developmentally regulated splicing in the extracellular domain.

The expression pattern of the laminin-binding alpha 7 beta 1 integrin is developmentally regulated in skeletal, cardiac, and smooth muscle. The X1/X2 alternative splicing in the extracellular domain of alpha 7 is found in the variable region between conserved alpha-chain homology repeat domains III and IV, a site implicated in ligand binding. To assess differences in X1/X2 isoform activity, we generated MCF-7 cell lines transfected with alpha 7-X1/X2 cDNAs. Transfectants expressing the alpha 7-X2 variant adhered rapidly to laminin 1, whereas those expressing alpha 7-X1 failed to attach. That alpha 7-X1 exists in an inactive state was established in assays using an activating beta 1 antibody that induced X1-dependent cell adhesion and spreading. Furthermore, the activation of alpha 7-X1 was cell type specific, and when expressed in HT1080 cells, the integrin was converted into a fully functional receptor capable of promoting adhesion. Thus, the expression of the alpha 7-X1/X2 integrin is a novel mechanism that regulates receptor affinity states in a cell-specific context and may modulate integrin-dependent events during muscle development and repair.     

The activity of the extracellular signal-regulated kinase 2 is regulated by differential phosphorylation in the activation loop

The mitogen-activated protein kinases (MAP kinases) play a central role in signaling pathways initiated by extracellular stimuli such as growth factors, cytokines, and various forms of environmental stress. Full activation of the MAP kinases requires dual phosphorylation of the Thr and Tyr residues in the TXY motif of the activation loop by MAP kinase kinases. Interestingly, down-regulation of MAP kinase activity can be initiated by multiple Ser/Thr phosphatases, Tyr-specific phosphatases, and dual-specificity phosphatases. This would inevitable lead to the formation of monophosphorylated MAP kinases. However, in much of the literature investigating MAP kinase signaling, there has been the implicit assumption that the monophosphorylated forms are inactive. Thus, the significance for the need of multiple phosphatases in regulating MAP kinase activity is not clear, and the biological functions of these monophosphorylated MAP kinases are currently unknown. We have prepared extracellular signal-regulated protein kinase 2 (ERK2) in all phosphorylated forms and kinetically characterized them using two proteins (the myelin basic protein and Elk-1) and ATP as substrates. Our results revealed that a single phosphorylation in the activation loop of ERK2 produces an intermediate activity state. Thus, the catalytic efficiencies of the monophosphorylated ERK2/pY and ERK2/pT (ERK2 phosphorylated on Tyr-185 and Thr-183, respectively) are approximately 2-3 orders of magnitude higher than that of the unphosphorylated ERK2 and are only 1-2 orders of magnitude lower than that of the fully active bisphosphorylated ERK2/pTpY. This raises the possibility that the monophosphorylated ERK2s may have distinct biological roles in vivo. Different phosphorylation states in the activation loop could be linked to graded effects on a single ERK2 function. Alternatively, they could be linked to distinct ERK2 functions. Although less active than the bisphosphorylated species, the monophosphorylated ERK2s may differentially phosphorylate pathway components.     

The extracellular domain of p185/neu is released from the surface of human breast carcinoma cells, SK-BR-3.

The human breast carcinoma cell line SK-BR-3, expresses the neu oncogene product, p185, which is a receptor tyrosine kinase. Using a double monoclonal antibody capture enzyme-linked immunosorbent assay for p185, activity was detected in conditioned media from cultures of SK-BR-3 cells. Two monoclonal antibodies specific for the extracellular domain of p185/neu immunoprecipitated a protein with a molecular mass of approximately 105 kDa. p105 was further shown to compete with p185 for binding to monoclonal antibodies and pulse-chase experiments indicate that it was generated by post-translational processing. Peptide maps showed that p105 and p185 are related polypeptides. Since p105 is close to the predicted size for the extracellular domain of p185/neu, we propose that SK-BR-3 cells specifically process and release this portion of the receptor into the medium. The release of the extracellular domain may have implications in oncogenesis and its detection could prove useful as a cancer diagnostic.     

Generation of CD8 T cell memory is regulated by IL-12

Various signals during infection influence CD8 T cell memory generation, but these factors have yet to be fully defined. IL-12 is a proinflammatory cytokine that has been shown to enhance IFN-gamma-producing T cell responses and has been widely tested as a vaccine adjuvant. In this study, we show that IL-12-deficient mice generate a weaker primary CD8 T cell response and are more susceptible to Listeria monocytogenes infection, but have substantially more memory CD8 T cells and greater protective immunity against reinfection. Kinetic analyses show that in the absence of IL-12 there is a reduced contraction of Ag-specific CD8 T cells and a gradual increase in memory CD8 T cells as a result of increased homeostatic renewal. By signaling directly through its receptor on CD8 T cells, IL-12 influences their differentiation to favor the generation of fully activated effectors, but hinders the formation of CD8 T cell memory precursors and differentiation of long-term CD8 T cell memory(.) These results have implications for understanding memory T cell development and enhancing vaccine efficacy, and offer new insight into the role of IL-12 in coordinating the innate and adaptive immune response.     

IL-1 alpha is produced by T lymphocytes activated via the CD2 plus CD28 pathways.

In addition to activation via the TCR complex, resting purified T cells can be activated to proliferate by mAb directed against the two surface molecules, CD2 and CD28. We demonstrate here that only the CD2 plus CD28 combined activation induces the expression and secretion of IL-1 alpha, a cytokine classically considered as a monokine. In contrast, neither IL-1 beta nor IL-6 were produced. A second monokine, TNF-alpha was transiently expressed by T cells activated with either CD2 or CD28 mAb, but was expressed to higher levels and with a prolonged kinetics in cells activated by the CD2 plus CD28 combination. The prolonged expression of the IL-1 alpha gene could account, at least in part, for the monocyte-independent and long lasting T cell proliferation induced by the CD2 plus CD28 co-stimulation. Secretion of monokines, such as IL-1 alpha, by activated T cells, could play a regulatory role in immune responses, as well as contribute to autoimmune processes.     

Macrophage--T cell interactions. I. The uptake by T cells of Fc receptors released from macrophages.

Using a rosette assay for the detection of cells carrying Fc receptors (Fc+) we have been able to show that in nylon wool (NWC), separated spleen cells from different strains of mice 12 to 18% are Fc+.Within 4 hr of culture in vitro at 37 °C, 75 to 85% of the Fc+ cells lose their Fc receptors and remain Fc receptor negative even after culture for 24 hr. However the addition of 5 to 10% syngeneic (but not allogeneic) peritoneal macrophages in the NWC, resulted in the preservation of the Fc receptors on 75 to 85% of the Fc+ cells originally present.Brief exposure of NWC which have been cultured in vitro for 4 hr (lost their Fc receptors) to supernates from 3 hr cultures of peritoneal macrophages reconstituted the fc+ cells by 75 to 85%. Only the supernates from syngeneic, but not allogeneic macrophages are active. Evidence is presented which indicates that these supernates contain Fc receptor molecules of small molecular weight. These molecules can be removed by antisera directed against the I region of the major histocompatibility complex.     

CD39 is highly involved in mediating the suppression activity of tumor-infiltrating CD8+ T regulatory lymphocytes

CD39 is an ectoenzyme, present on different immune cell subsets, which mediates immunosuppressive functions catalyzing ATP degradation. It is not known whether CD39 is expressed and implicated in the activity of CD8+ regulatory T lymphocytes (Treg). In this study, CD39 expression and function was analyzed in both CD8+ and CD4+CD25^hi Treg from the peripheral blood of healthy donors as well as from tumor specimens. CD39 was found expressed by both CD8+ (from the majority of healthy donors and tumor patients) and CD4+CD25^hi Treg, and CD39 expression correlated with suppression activity mediated by CD8+ Treg. Importantly, CD39 counteraction remarkably inhibited the suppression activity of CD8+ Treg (both from peripheral blood and tumor microenvironment) suggesting that CD39-mediated inhibition constitutes a prevalent hallmark of their function. Collectively, these findings, unveiling a new mechanism of action for CD8+ Treg, provide new knowledge on intratumoral molecular pathways related to tumor immune escape, which could be exploited in the future for designing new biological tools for anticancer immune intervention.     

MLC-specific suppressor T lymphocytes in man. I. Their induction in vitro by soluble HLA-DR antigens

Human T lymphocytes precultured for 36 hr in the presence of soluble HLA-DR antigens suppress the MLR response of autologous peripheral blood lymphocytes to allogeneic stimulating cells. The suppression is DR antigen-specific in that it appears that the MLR stimulating cell donor and the soluble suppressor-inducing antigen must share DR specificities. The soluble DR antigens were fractionated from the sera of normal donors using QAE-Sephadex chromatography and CNBr-activated Sepharose immunoadsorption. Similarly prepared HLA-A and -B antigens failed to induce suppressive activity. The suppressive activity of DR-antigen cultured T cells is resistant to mitomycin C treatment and, further, the antigen specificity is maintained with or without mitomycin C treatment. The kinetics of suppressor cell induction as well as the kinetics of suppression in the test MLR cultures are presented. The implications of these results are discussed.     

Novel salmon cardiac peptide hormone is released from the ventricle by regulated secretory pathway

We used the secretion of the novel salmon cardiac peptide (sCP) as a model to examine the mechanisms of ventricular hormone release. Mechanical load increased dose dependently the secretion of immunoreactive sCP from isolated perfused salmon ventricle, with 3. 3-fold increase when a load of 13 cmH(2)O was applied. Endothelin-1 (5 nmol/l) was also able to rapidly increase the secretion of sCP. The released peptide corresponded to the biologically active sCP-29, whereas the large ventricular storage consisted of pro-sCP-sized material. With the use of immunoelectron microscopy, a large number of granules containing immunoreactive sCP could be detected in salmon ventricle. As judged by RNA blot analysis, there was very active basal expression of the sCP gene in the ventricle, which was not increased by mechanical load of up to 2-h duration. Our results show that the ventricle actively expresses the gene of sCP, stores the prohormone in secretory granules, and releases the peptide in response to mechanical load and endothelin-1. Thus the salmon ventricle uses the regulated pathway to produce and release a hormone structurally related to the mammalian natriuretic peptides.     

Cell surface expression of CD147/EMMPRIN is regulated by cyclophilin 60

CD147, also known as extracellular matrix metalloproteinase inducer, is a regulator of matrix metalloproteinase production and also serves as a signaling receptor for extracellular cyclophilins. Previously, we demonstrated that cell surface expression of CD147 is sensitive to cyclophilin-binding drug cyclosporin A, suggesting involvement of a cyclophilin in the regulation of intracellular transport of CD147. In this report, we identify this cyclophilin as cyclophilin 60 (Cyp60), a distinct member of the cyclophilin family of proteins. CD147 co-immunoprecipitated with Cyp60, and confocal immunofluorescent microscopy revealed intracellular co-localization of Cyp60 and CD147. This interaction with Cyp60 involved proline 211 of CD147, which was shown previously to be critical for interaction between CD147 and another cyclophilin, cyclophilin A, in solution. Mutation of this proline residue abrogated co-immunoprecipitation of CD147 and Cyp60 and reduced surface expression of CD147 on the plasma membrane. Suppression of Cyp60 expression using RNA interference had an effect similar to that of cyclosporin A: reduction of cell surface expression of CD147. These results suggest that Cyp60 plays an important role in the translocation of CD147 to the cell surface. Therefore, Cyp60 may present a novel target for therapeutic interventions in diseases where CD147 functions as a pathogenic factor, such as cancer, human immunodeficiency virus infection, or rheumatoid arthritis.     

Cutting edge: homeostatic proliferation of peripheral T lymphocytes is regulated by clonal competition

Homeostatic proliferation functions to maintain peripheral T cell numbers and is regulated by cytokines. In this study, we provide evidence that T cell homeostasis is also regulated by clonal competition. Naive polyclonal T cells divided when transferred to TCR transgenic hosts, as did monoclonal T cells when transferred to TCR transgenic hosts of differing clonotype. However, T cells did not divide in hosts of identical clono-type. Transgenic T cell proliferation was inhibited in irradiated hosts of the same clonotype, while cotransferred nontransgenic T cells proliferated extensively. These results show that clonal competition is a component of homeostasis that may contribute to selection of the peripheral T cell repertoire.     

Adhesion of T lymphocytes to human endothelial cells is regulated by the LFA-1 membrane molecule

Human T lymphocyte adhesion to human endothelial cells is the initial event in T cell migration to areas of extravascular inflammation. The molecular basis for T cell-endothelial cell adhesion was investigated using two different cell-cell adhesion assays: a) a fluorescein cell-cell adhesion assay using nonadherent endothelial cells and fluorescein-labeled T lymphocytes, and b) a radionuclide cell-cell adhesion assay using adherent endothelial cells and 51Cr-labelled T cells. Both assay systems demonstrated comparable quantitative assessment of cell-cell adhesions. The assays were performed at 22 degrees C and adhesions were maximal at 30 min. The results of these adhesion assays confirmed previous reports that T cells adhere to endothelial cells. In addition, we have shown that T cells adhere only marginally to foreskin fibroblasts or bone marrow derived fibroblasts. T cell-endothelial cell adhesions were significantly stronger than either monocytes or B lymphoblastoid cells adhesion to endothelial cells. To demonstrate the molecular mechanisms involved in regulating T cell-endothelial cell adhesions, a panel of function-associated monoclonal antibodies (MAb) were tested for their ability to inhibit T cell adhesion. MAb reactive with the leukocyte surface glycoprotein LFA-1 significantly inhibited T cell-endothelial cell adhesions in both assay systems. In contrast, MAb directed at other surface antigens did not inhibit T cell adhesion. The involvement of the LFA-1 glycoprotein in T lymphocyte adhesion to endothelial cells suggest that the LFA-1 molecule may be important in the regulation of leukocyte interactions.