Integrin-dependent tyrosine phosphorylation and growth regulation by Vav.

The proto-oncogene product p95Vav (Vav) undergoes rapid phosphorylation on tyrosine following stimulation of the T or B cell antigen receptor, and in response to a variety of other cell surface stimuli. Vav contains, among other, a guanine nucleotide exchange factor domain with homology to the Rho/Rac/CDC42 exchange protein Db1. It has been recently shown that Vav is functionally linked to small GTPases of the Rho family, suggesting that it is an activator of Rho GTPases and may participate in regulation of cytoskeletal organization. The present study shows that cell adhesion to fibronectin triggers rapid phosphorylation of Vav on tyrosine in Vav-transfected CHO cells and in Jurkat T cells. Vav phosphorylation is strongly dependent on adhesion and is mediated by beta 1 integrins. Furthermore, Vav overexpression enhances the adhesion-dependent increase in the rate and extent of phosphorylation on focal adhesion kinase and paxillin, and the formation of stress fibers and lamellipodia. In addition, there is a marked increase in the amount of Vav localized to the triton-insoluble fraction following 1 h of incubation on FN. Finally, Vav increases the growth rate of the cells in an adhesion-dependent manner. Our results strongly implicate Vav as a mediator of integrin signal transduction.

Reactivity patterns of antiphospholipid antibodies and endothelial cells: effect of antiendothelial antibodies on cell migration.

Antiphospholipid syndrome (APS) is characterized by the presence of a heterogeneous class of antibodies directed against phospholipids and associated with high occurrence of thrombotic complications. Antiendothelial cell antibodies (AECAs) have been identified in various autoimmune disorders including APS, but their reactivity patterns remain unclear. We used eluted endothelial membrane-bound antibodies (EC eluates) to investigate possible cross-reactivity of AECAs and their pathogenic effects on endothelial cell integrity. The heterogeneous and nonspecific nature of AECAs was confirmed by our finding that they cross-react with fibroblasts and platelets and bind to cardiolipin. In addition, platelet-bound antibodies from sera of patients with APS reacted with endothelial cells. A dose-dependent binding of human monoclonal anticardiolipin antibody was demonstrated, but this antibody did not compete with AECAs in EC eluates, indicating that only small portion of AECAs are directed against cardiolipin. Although sera from APS patients prolonged coagulation tests, EC eluates did not affect coagulation, suggesting that AECAs may belong to antiphospholipid antibodies subsets that does not interfere with coagulation. Vascular damage is a common feature of autoimmune disorders associated with AECAs. Possible effects of AECAs on vascular perturbance were investigated by cytotoxicity, attachment, and migration assays. Although AECAs were not shown to be cytotoxic or to affect cell attachment, sera from APS patients caused reduced cellular migration (by 30%), and EC eluates caused even more significant inhibition (by 50%). These findings suggest possible interference of AECAs in vascular repair mechanisms and provide an explanation for the thrombotic complications frequently seen in APS patients.

Vav: function and regulation in hematopoietic cell signaling.

Vav, a 95 kDa proto-oncogene product expressed specifically in hematopoietic cells, was originally isolated as a transforming human oncogene. Vav contains an array of functional domains that are involved in interactions with other proteins and, possibly, with lipids. These include, among others, a putative guanine nucleotide exchange domain, a cysteine-rich region similar to the phorbol ester/diacylglycerol-binding domain of protein kinase C, a pleckstrin-homology domain, and Src-homology 2 and 3 (SH2 and SH3, respectively) domains. The presence of these domains, the transforming activity of the vav oncogene, and the rapid increase in tyrosine phosphorylation of Vav induced by triggering of diverse receptors indicate that it plays an important role in hematopoietic cell signaling pathways. Such a role is supported by recent studies using "knockout" mice and transiently transfected T cells, in which Vav deletion or overexpression, respectively, had marked effects on lymphocyte development or activation. The presence of a putative guanine nucleotide exchange domain, the prototype of which is found in the dbl oncogene product, implies that Vav functions as a guanine nucleotide exchange factor (GEF) for one (or more) members of the Ras-like family of small GTP-binding proteins. In support of such a role, Vav preparations were found in some (but not other) studies to mediate in vitro-specific GEF activity for Ras. Additional studies are required to identify the physiological regulators and targets of Vav, and its exact role in hematopoietic cell development and signaling.

Detection of immunoglobulin A anticardiolipin antibodies in cervical mucus from in vitro fertilization patients and fertile women.

OBJECTIVE: To identify and quantify antiphospholipid autoantibodies of the immunoglobulin (Ig) A isotype in cervical mucus obtained from IVF patients and fertile controls. DESIGN: The study was performed prospectively. Blood and cervical mucus samples were obtained from patients undergoing IVF treatment (n = 27) at the time of expected E2 peak, before administering hCG. Control samples were taken from fertile women (n = 16) around the time of ovulation during a spontaneous nonstimulated menstrual cycle. Anticardiolipin activity was tested using ELISA. SETTING: Infertility and IVF unit of an academic tertiary referral medical center and university-based basic research laboratory. RESULTS: Forty-eight percent (13/27) of the IVF patients and 43.8% (7/16) of the fertile controls exhibited anticardiolipin IgA activity in aspirated cervical mucus. The mean activity measured for the positive cases was similar in both groups. This activity was higher than that measured in peripheral blood of the women studied. No difference was noted between infertile patients undergoing IVF treatment and fertile women in this respect. CONCLUSIONS: In this preliminary work, we demonstrated for the first time anticardiolipin IgA activity in human cervical mucus. These observations have to be substantiated by larger scaled studies to assess their possible clinical significance.

A human monoclonal IgA autoantibody--185/12--behaves like an autoimmune antiphospholipid antibody.

A human monoclonal anticardiolipin autoantibody (ACA) of the IgA-k isotype, designated 185/12, is described. The antibody was prepared from peripheral B cells, obtained from a patient with a history of habitual abortion, by immortalization with Epstein-Barr virus (EBV). The antibody displays a strong binding activity to cardiolipin and phosphatidyl L-serine, but not to phosphatidylcholine, phosphatidylinositol, ssDNA and dsDNA. It binds to cardiolipin in a concentration-related and saturable manner (Kd = 3.0 x 10(-8) M). This reaction is dependent upon the presence of bovine serum, and is fully inhibited by cardiolipin vesicles. The 185/12 antibody exhibits different binding patterns to the solid-phase bound cardiolipin-serum complex and to its individual components (cardiolipin and bovine serum). The Bmax of 185/12 binding to the complex (0.968 OD units) is higher than the sum of the Bmax values calculated for each one of the complex components (0.352 + 0.179 = 0.531 OD units). Bovine serum as well as purified beta 2-glycoprotein I (beta 2-GPI) in suspension inhibit the binding of 185/12 to the complex. 185/12 binding capacity increases in direct relation to the rising concentration of beta 2-GPI. Collectively, these data may be interpreted to suggest that 185/12 antibody, which is an IgA isotype, exhibits characteristics usually attributed only to antiphospholipid autoantibodies (APA) of the IgG isotype, that are associated with the clinical spectrum of APA syndrome (APA-S). It is, therefore, possible that autoantibodies of the IgA isotype could play a pathogenic role, which may be different from that of the IgG isotype, in the development of autoimmune phenomena.

The relationship between in vitro fertilization and naturally occurring antibodies: evidence for increased production of antiphospholipid autoantibodies.

OBJECTIVE: Assessment of possible effects of ovarian stimulation during in vitro fertilization (IVF) treatment cycles on circulating levels of antiphospholipid and antinuclear autoantibodies. DESIGN: The study was performed prospectively. Sera were obtained at three time points along IVF treatment cycle. Levels of autoantibodies directed against nuclear components, mitochondrial antigens, and phospholipids were determined using enzyme-linked immunosorbent assay. PATIENTS: Thirty-five patients, who underwent at least one previous IVF attempt, and 36 age- and sex-matched controls were analyzed. All participants were randomly selected. RESULTS: The mean levels of antiphospholipid (but not antinuclear) autoantibodies in sera from IVF-treated patients were found to be significantly higher than the corresponding values of the control group (for immunoglobulin [Ig]M isotype: anticardiolipin, antiphosphatidyl L-serine; for IgG isotype: anticardiolipin, antiphosphatidyl L-serine, and antiphosphatidylcholine; P less than 0.0001, assessed by Mann-Whitney test). The autoantibody levels remained more or less constant at different time points along the treatment cycle. No correlation with age and number of previous IVF cycles was demonstrated. CONCLUSIONS: Serum levels of antiphospholipid (but not antinuclear) autoantibodies increase after IVF treatment. Based on these preliminary data, it is not yet possible to estimate if the observed changes in autoantibody levels might have any future clinical influence on infertile patients undergoing IVF treatment.

CD5+ B cells and naturally occurring autoantibodies in cancer patients.

We have determined the percentage of CD5+ B lymphocytes in the peripheral blood of cancer patients and healthy controls, using antibodies directed at the CD5 and CD19 (pan-B) markers. The frequencies of CD5+ B cells, expressed as a percentage of total B cells, ranged from 14.3 to 57.5% in the controls and from 14.8 to 82.8% in the patient population. One-third of the cancer patients had frequencies greater than 2 s.d. above the mean of the control population. The CD5+ B cell fraction expressed as a percentage of total lymphocytes was also significantly elevated in this group of cancer patients. These results suggest that the CD5+ B cell compartment may be affected by the malignant process or by the therapy modality employed. The plasma levels of several naturally occurring autoantibodies, the products of the CD5+ B cells, were also assessed in cancer patients and controls. No significant differences were observed when reactivity to several autoantigens was measured. These included nuclear components and phospholipids.

Effect of sex hormones on human T cell activation by concanavalin A.

The effect of sex hormones on concanavalin A (Con A)-activated human T cells was studied. We show that neither 17 beta-estradiol (E2) nor progesterone, in concentrations of up to 10(-6) M, alters the proliferative response of peripheral-blood mononuclear cells (PBMC) of healthy postmenopausal women. Furthermore, the hormones had no effect on the composition of T cell populations and on the expression of activation markers. We extended our study to a unique T cell population that is characterized by the ability to form rosettes with human erythrocytes, following Con A activation (designated autorosette-forming cells; ARFC) and known to manifest suppressive activity. Indeed, the in vitro addition of E2 (neither progesterone nor testosterone) to Con A-stimulated PBMC brought an about 2- to 4-fold increase in the frequency of ARFC. Tamoxifen, an antiestrogen drug, reduced the frequency of estrogen-stimulated ARFC to the original low level. Furthermore, the inhibitory effect of growth medium from ARFC cultures originally stimulated with Con A + E2 was found to be higher than that of ARFC cultures originally stimulated with Con A alone.

Frequency and activity of autorosette forming cells in Con-A activated PBL from women with hyperplasia or carcinoma of the endometrium--possible role of estrogens.

In a previous study we showed that endometrial carcinoma (EC) patients have a T cell deficiency manifested in a reduced ability to be stimulated in vitro by PHA and to produce IL-2. In an attempt to understand the mechanism responsible for this alteration we present in this paper a study on T cells characterized by the ability to form rosettes, with human erythrocytes, following Con-A activation (designated auto-rosette forming cells--ARFC). These cells are also known to manifest suppressive activity. We show that the frequency of ARFC in con-A activated peripheral blood leukocytes (PBMC) of EC patients is significantly (2-5 fold) higher than that of healthy age-matched controls or that of patients with stage--I colon or vaginal cancer. Endometrial carcinoma is known to be associated with long term exposure to estrogens unopposed by progestins. Examining the possible role of estrogens in increasing the frequency of ARFC from EC patients, we found that in vitro addition of estradiol to Con-A stimulated PBMC from healthy donors increased the frequency of ARFC to levels found in EC patients. Tamoxifen, an anti estrogen drug, reduced the frequency of the estrogen stimulated ARFC to the original low level. Our results suggest a dual role for estrogen in carcinogenesis as well as in immunomodulation.

Inhibition of murine cytotoxic T cell responses by progesterone.

In the present study we evaluated the effect of sex hormone on the generation of murine cytotoxic T cell responses. We show that the in vitro CTL response was strongly inhibited by progesterone but not by E1, E2 or testosterone. Our experiments attempting to understand the mechanism of the hormone action on CTL development have revealed that the ability of the cells to generate helper signals was not affected. This was demonstrated by the fact that neither IL-2 production nor IL-2 receptor expression was altered by the hormone. Rather, it appears that the capacity of the cells to respond to the signals and to become cytotoxic was modified. Furthermore, we show that the hormone mediated an inhibition of CTL development in thymocyte cultures externally supplemented with all the required helper factors. These results strongly suggest that progesterone has a direct effect on the differentiation of cytotoxic effector cells.

Comparison of NK activity in mouse spleen and peripheral blood lymphocytes.

Natural killer (NK) cells originating in mouse peripheral blood were studied with regard to their lytic activity against YAC-1 target cells and to their expression of asialo-GM1 marker on their surface. In Balb/c, CBA/LAK and A/J mice, PBL were found to be approximately twice as effective as splenocytes. Splenic and peripheral NK cells were shown by flow cytometry to have similar lytic potential per cell; the difference in NK activity found in the spleen and in PBL was solely due to the differences in the size of the NK cell population found in the two sites. Strain distribution of NK activity in PBL followed the same pattern observed in splenocytes. The difference in NK activity between CBA and Balb/c mice was shown to be due to the fact that the lytic potential per NK cell was approximately twice as high in the former.

Miniaturization of the standard 51Cr release assay for long term follow-up of NK activity of individual mice.

Two modifications for miniaturizing the standard 51chromium release assay have been described. Using peripheral blood lymphocytes (PBL) as effectors and very few target cells, this permits longitudinal studies on NK cell activity of individual mice to be performed. The modifications are based on methods to increase the chromium uptake by the target cell. Firstly, chromium uptake by the YAC-1 target cells was enhanced to such an extent that appreciable activity could be detected against as little as 750 cells/well at an E/T ratio of 50:1. Secondly, the uptake was increased even further by labelling the target cells in the presence of isotonic sucrose, thus permitting the use of only 125 target cells/well at an E/T of 25:1. The long term pattern of NK activity in individual BALB/c mice was studied by repeated bleedings over a period of 8 months. Considerable fluctuations with time were observed in the pattern of NK activity of individual mice. However, the NK activity averaged over a large number of mice did not show a significant decrease with age. These methods may allow the study of the long term pattern of NK activity in individual mice and the response of this activity to physiological and environmental factors.

The immune system during the pre-cancer and the early cancer period. IL-2 production by PBL from post-menopausal women with and without endometrial carcinoma.

We report on alterations in IL-2 production and cell proliferation following PHA stimulation of peripheral blood lymphocytes (PBL) from stage-I endometrial carcinoma (EC) patients, and on mechanisms involved in these alterations. Our study includes 3 groups: EC patients, post-menopausal women at high risk of developing EC, and age-matched healthy women. IL-2 production was markedly lower in most EC patients than in healthy controls. Varying levels of IL-2 were produced by PBL from women in the high-risk group. The proliferative response of PBL to PHA appeared to correlate with levels of IL-2 production. Our results suggest that macrophages are involved, in part, in the modulation of T-cell functions of EC patients.

The analysis of measurements of murine natural killer cell activity.

Several methods of analysing murine NK response measurements have been compared in order to select a quantitative objective measure of NK activity . The fitting of data from 51chromium release experiments to the formula y=A(1-e-kx)((termed the "k method" and shown by Pross et al. (J. Clin. Immunol. (1981) 1,51) to be beneficial in analysing human the NK response) has been particularly evaluated. Computer simulated curves as well as experimental NK dose response curves were analysed testing data in which a plateau level of chromium release had not been reached. Results obtained by the "k method" were very dependent on both the portion of the curve used for the analysis and on small deviations of the data from the theoretical form of the equation. In the analysis of murine NK response the "k method" has no clear advantage over other methods.

Contribution of activated C3 to lymphocyte-mediated target lysis: complement-dependent cellular cytotoxicity.

Lysis of Raji cells by human lymphocytes was found to be enhanced if human serum was added to the assay. This was not due to the contribution of antibodies because hypogammaglobulinemic serum also augmented cytotoxicity. The results suggest that the mechanism of the enhancement was due to activation of C3 by the Raji cells. We assume that the cleavage products are deposited on cell surfaces in such a way that they contribute to contact between effector and target. Previous reports in two antibody-dependent cellular cytotoxicity systems provided other examples for the existence of this phenomenon.

Complement-dependent cellular cytotoxicity due to alternative pathway C3 activation by the target cell membrane.

The cytotoxic activity of human blood lymphocytes toward Raji cells was strongly elevated when human serum (HS) was included in the cytotoxicity assay. This phenomenon also occurred when the effector cells were activated by interferon (IFN). Hypogammaglobulinemic serum (HyS) and heat-inactivated serum could also augment cytotoxicity, but C3-depleted serum was inefficient. IFN treatment of Raji cells decreased their sensitivity to lysis and this effect was counteracted by addition of HS to the system. It is likely that C3 activation by, and deposition on, Raji cells when used as targets for cytotoxicity facilitate their recognition and lysis by lymphocytes. These events may represent one mechanism operating in the natural killing phenomenon.

Lymphokine-activated killer cells: lysis of fresh syngeneic natural killer-resistant murine tumor cells by lymphocytes cultured in interleukin 2.

Normal splenocytes that are cultured in the lymphokine, interleukin 2 (IL-2), for as short as 2 days develop lytic activity for fresh syngeneic natural killer-resistant tumor cells as well as natural killer-sensitive YAC cells in a 4-hr 51Cr release assay. Lymphokine-activated killer (LAK) cells do not lyse syngeneic fresh lymphocytes but do lyse syngeneic concanavalin A-induced lymphocyte blasts. Lysis is not due to the presence of lectin or xenogeneic serum and appears to be an intrinsic property of lymphocytes activated in IL-2. The activation appears universal in that lymphocytes from all strains of mice activated in this manner exhibited similar patterns of lysis for fresh tumor target cells. To characterize the cells responsible for this lysis, we analyzed the phenotypic expression of surface markers on these cells with depletion techniques using monoclonal antibody and complement. These studies indicate that the precursor of the LAK cell is Thy-1+ and nonadherent to plastic or nylon wool. Lysis of syngeneic tumor was inhibited when LAK cells were treated with an anti-Thy-1.2, or anti-Lyt-2.2 monoclonal antibody and complement but not with anti-Lyt-1.2 monoclonal antibody and complement, indicating that the observed lytic activity was due to a Thy-1+ Lyt-1-2+ cell. Furthermore, LAK cell-mediated lysis could be inhibited by the addition of anti-Lyt-2 or LFA-1 monoclonal antibody to cytotoxicity assays. Cold target inhibition analysis revealed that the syngeneic tumor cells were lysed by recognition of a determinant not present on normal lymphocytes or lymphocyte blasts. This lysis of fresh solid tumor cells by lymphoid cells grown in IL-2 may be of value in the study of tumor-host immunological interactions. The biological significance of tumor lysis by IL-2-activated cells requires further study.