Bharathi Vayuvegula

The induction of intercellular adhesion molecule 1 (ICAM-1) expression on human fetal astrocytes by interferon-λ, tumor necrosis factor α, lymphotoxin, and interleukin-1: relevance to intracerebral antigen presentation

Antigen presentation reactions are dependent upon the expression of the class II major histocompatibility antigens (MHC), the T-cell receptor, and the presented antigen. Recent studies demonstrate that such processes also require the presence of adhesion molecules such as lymphocyte functional antigen 1 (LFA-1) and its cell surface ligand, intercellular adhesion molecule 1 (ICAM-1). It has been suggested that the brain astrocyte can function as a facultative antigen presenting cell (APC). This hypothesis is based upon the ability to induce the expression of the class II MHC antigens on astrocytes, and on their ability to present myelin basic protein to encephalitogenic T-cells in vitro. The best in vivo data showing that astrocytes serve as intracerebral APCs is the finding that astrocytes in multiple sclerosis plaques are DR+ (class II MHC in human). However, it still remains to be resolved whether the in vivo expression of the MHC antigens in disease states is instrumental to antigen presentation mechanisms or whether these cell surface glycoproteins are expressed secondary to brain immune responses. If astrocytes function as immunocompetent APCs within the brain, it would seem that they would also be able to express molecules important for intercellular adhesion. Here, we present the first data that indicates that human astrocytes are capable of expressing ICAM-1 in response to cytokines that either induce or upregulate the expression of DR. In essence, cytokines derived from different cell types seem to exert similar pleiotropic effects on the modulation of MHC and ICAM-1 expression on astrocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Norepinephrine inhibits gamma-interferon-induced MHC class II (Ia) antigen expression on cultured brain astrocytes

Recent evidence that astrocytes can be induced to express the class II major histocompatability (MHC) antigens suggests that these cells may be involved in the development of intracerebral immune responses. The principal inducer of MHC class II antigen (Ia) expression is a soluble lymphokine, gamma-interferon (gamma-IFN). Normally astrocytes do not express significant levels of Ia antigens despite the fact that agents such as gamma-IFN may be present in the central nervous system (CNS). Here we report that a major neurotransmitter, norepinephrine (NE), inhibits, in a dose-response fashion, the ability of gamma-IFN to induce Ia antigen expression on cultured astrocytes derived from newborn BALB/c mice. This finding may indicate that the brain contains inhibitory modulators that serve to prevent the up-regulation of intracerebral immune responsiveness.

Human immunodeficiency virus-associated changes in signal transduction

It is well established that the activation of T lymphocytes by mitogen/antigen is accompanied by a rise in intracellular free calcium ([Ca2+]i), changes in membrane potential, metabolism of inositol phospholipid, and activation of protein kinase C. Theseearly events of signal transduction culminate inlate events of lymphocyte activation, namely, DNA synthesis, lymphokine production, and cellular proliferation. In this study we examined the effect of human immunodeficiency virus (HIV) on changes in membrane potential and [Ca2+]i levels. The membrane potentials were markedly decreased (depolarized) in T cell lines infected with HIV (H9/HTLV IIIb) and did not respond normally to phytohemagglutinin (PHA) or anti-T3 (anti-CD3) monoclonal antibody compared to uninfected H9 cell line. The basal [Ca2+]i levels in H9/HTLV IIIb cells were increased in comparison to those in H9 cells; however, there was very little further increase in [Ca2+]i in H9/HTLV IIIb cells following activation with PHA or anti-T3 monoclonal antibody. This is in contrast to a significant rise in [Ca2+]i in H9 cells following similar stimulation. These data demonstrate abnormalities in the plasma membrane potential and [Ca2+]i levels in chronically infected T cells with HIV. These abnormalities in signal transduction of the T-cell activation pathway could be responsible for T-cell dysfunction in patients with HIV infection.

Therapy of recurrent high grade gliomas with surgery, and autologous mitogen activated IL-2 stimulated killer (MAK) Lymphocytes: I. Enhancement of MAK lytic activity and cytokine production by PHA and clinical use of PHA

Nineteen patients with recurrent high grade gliomas were treated in a phase I/II trial with aggressive debulking of the tumor, mitogen activated IL-2 stimulated peripheral blood lymphocytes (MAK cells), and rIL-2. Phytohemagglutin (PHA) was introduced into the tumor site in 16 patients prior to implanting MAK cells and IL-2 in an attempt to trigger more effective lysis of the tumorin vivo. In vitro both TNF bioactivity and cytolytic activity of long term cultured MAK (LMAK) cells were dramatically enhanced by adding PHA to the cultures of these activated PBL. Three of eleven patients (27%) had a decrease in size of the enhancing lesion on CT and/or MRI. Seven (37%) patients clinically improved. Median survival after therapy was 30 weeks. PHA was shown to be safein vivo and more effective than IL-2 triggering enhanced effector functionin vitro.

Disseminated cryptococcal infection in a patient with hypogammaglobulinemia and normal T cell functions

The immunologic status of a patient with disseminated cryptococcosis and no underlying predisposing factor was evaluated. The proportions and numbers of T cells, T cell subsets, B cells and proliferative responses to mitogens and soluble antigens, including cryptococcal antigen, were comparable to those in healthy control subjects; however, the patient had moderately severe hypogammaglobulinemia. The patient continues to do well clinically with intravenous gammaglobulin and antifungal therapy. This study suggests that antibodies might also play a role in the defense against cryptococcal antigen.

Vasoactive intestinal polypeptide inhibits the expression of the MHC class II antigens on astrocytes

The brain has been traditionally viewed as an immunologically privileged site. However, recent findings suggest that the brain is in fact equipped with its own immune circuitry. Astrocytes and microglia have been considered the most likely candidates to assume the role of intracerebral antigen presenting cells (APC). Using the techniques of immunofluorescence cytochemistry and flow cytometric analysis, we observed that vasoactive intestinal polypeptide (VIP) can significantly inhibit gamma-interferon (IFN-gamma)-induced Ia expression on astrocytes derived from newborn Lewis rats. Further, we analyzed a number of neuropeptides and transmitters for their ability to exert a similar inhibitory modulation on IFN-gamma induced Ia expression or for the ability to induce or augment Ia expression on rat astrocytes. Our results showed that only norepinephrine (NE), a major brain neurotransmitter, and VIP, a ubiquitous brain peptide, have the ability to inhibit Ia expression on Lewis rat astrocyte cultures. Alternately, we report that cholecystokinin (CCK), a brain/gut peptide, has the ability to induce Ia on about 5-10% of the cells analyzed. These findings suggest that endogenous brain substances have the ability to modulate intracerebral immune responses by regulating the expression of Ia on astrocytes.

Potentiation of immunosuppressive effects of cyclosporin A by 1α,25-dihydroxyvitamin D3

Both cyclosporin A (CsA) and 1 alpha,25-dihydroxyvitamin D3 (vitamin D3) inhibit T-cell functions. Because CsA has a dose-related systemic toxicity, there is a need for approaches by which the dosage of CsA could be reduced while maintaining the required immunosuppression. Therefore, we examined for any potentiating effect of vitamin D3 on CsA-induced suppression of T cell functions in vitro. Peripheral blood mononuclear cells were stimulated with phytohemagglutinin in the presence or absence of various concentrations of CsA (1.6-13.2 micrograms/ml) or vitamin D3 (10(-10)-10(-7) M) or both together and [3H]thymidine incorporation (TdR), interleukin 2 (IL-2) production, IL-2 receptor (IL-2R) expression, and the response to exogenous recombinant IL-2 (rIL-2) on TdR were measured. IL-2 production was measured on CTLL cell line and IL-2R expression with anti-Tac monoclonal antibody using FACScan. There was a dose-dependent inhibition of TdR, IL-2 production, and IL-2R expression by CsA. Vitamin D3 inhibited TdR and IL-2 production in a dose-dependent manner but had no significant (P greater than 0.1) effect on IL-2R expression. Recombinant IL-2 had no effect on CsA-induced inhibition of TdR, whereas rIL-2 completely reversed the vitamin D3-induced inhibition of TdR. A significantly (P less than 0.05) increased inhibition of TdR and IL-2 production was observed when two agents were used together as compared to expected inhibition by the addition of each agent separately. A consistent synergism was observed between all concentrations of CsA used and vitamin D3 (10(-8) and 10(-7) M). This study shows that the potentiation of immunosuppressive effects of CsA by vitamin D3 could be used as an approach to reduce the dosage of CsA in clinical use without compromising immunosuppression, thus preventing or minimizing the dose-related toxic effects of CsA.

Correction of altered plasma membrane potentials

SummaryWe have recently shown that cyclosporin A (CsA) reverses pleiotropic drug resistance in human acute lymphatic leukemia in vitro and daunorubicin resistance in Ehlrich ascites tumor in vivo. In the present study we examined the mechanisms by which CsA might reverse pleiotropic drug resistance relative to changes in cellular plasma membrane potentials and intracellular calcium ([Ca2+]i). Membrane potentials were measured with DIOC5 dye flowcytometrically and [Ca2+]i levels with Quin 2 dye spectrofluorimetrically. All pleiotropic (PDR) drug-resistant tumor sublines had decreased membrane potentials (membrane depolarized) compared with their corresponding drug-sensitive parental tumors. In comparison, the membrane potentials of a control antimetaboliteresistant acute leukemia cell line were unchanged. The basal levels of [Ca2+]i in the PDR sublines were variable compared with those of parental drug-sensitive cell lines. Incubation of all PDR tumor sublines with CsA or verapamil resulted in the restoration of membrane potentials to that characteristic of the corresponding drug-sensitive parental tumor. Cyclosporin A produced variable changes in the levels of [Ca2+]i. These data suggest that alteration of membrane potentials is one of the mechanisms responsible for pleiotropic drug resistance in malignancy and show that this alteration is corrected by CsA and verapamil.

Lipopolysaccharide-induced murine B cell proliferation: a role of protein kinase C

Lipopolysaccharide (LPS)-induced murine B cell proliferation was blocked by 1-(5-isoquinoline-sulfonyl)-2-methylpiperazine dihydrochloride (H7), an inhibitor of protein kinase C (PKC), in a dose- and time-dependent manner. The maximum inhibition of B cell proliferation was observed when H7 was added at the initiation of cultures. H7-induced inhibition was prolonged and irreversible. Furthermore, pretreatment of B cells with phorbol myristate acetate ester, a process that degrades membrane-associated PKC, rendered them unresponsive to LPS. These data strongly suggest that the activation of PKC is one of the mechanisms of LPS-induced murine B cell proliferation.

T cell activation via the T cell receptor: A comparision between WT31 (defining αβ TcR)-induced and anti-CD3-induced activation of human T lymphocytes

Abstract The T cell receptor (TcR) heterodimer of α β glycoprotein is noncovalently associated with CD3 glycoprotein forming TcR/CD3 complex. The TcR have been shown to recognize antigen, and CD3 antigen is responsible for signal transduction. In this study we compared the effects of WT31 (defining α β TcR) monoclonal antibody (MoAb) and anti-CD3 MoAb on various steps of human T cell activation. Both antibodies depolarized plasma membranes, increased cell volume, induced IL-2 production and the expression of IL-2 receptors (CD25 antigen) and induced DNA synthesis. Furthermore, the two antibodies showed no synergistic effect on any of these parameters. However, both MoAb showed synergism with phorbol ester (PMA). WT31 -induced T cell activation was Ca2+-dependent because the addition of EGTA to the medium inhibited DNA synthesis and CD25 antigen expression. The blockers of protein kinase C (PKC), 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H7) and staurosporin, in a dose-dependent manner inhibited WT31-induced DNA synthesis. Cholera toxin but not the pertussis toxin inhibited WT31-induced T cell activation, suggesting involvement of G protein in WT31-induced T cell activation. These data indicate that WT31 antibody activates human T cells by a pathway that is similar to that of anti-CD3-induced T cell activation.