CD28 costimulatory signals are required for lymphokine production and T cell proliferation. CD28 signaling recruits the intracellular proteins PI 3-kinase, ITK, and GRB-2/SOS. PI 3-kinase and GRB-2/SOS bind the CD28 cytoplasmic pYMNM motif via SH2 domains. We generated CD28 pYMNM mutants and found that Y191 mutation (Y191CD28F) disrupted both PI 3-kinase and GRB-2 binding, while M194 mutation (M194CD28C) disrupted only PI 3-kinase binding. Both mutants still bound ITK. We have assessed the ability of these selective mutants to support IL-2 production upon TCR zeta/CD3 ligation in the presence of CHO-CD86 (B7-2) cells. Both Y191CD28F and M194CD28C mutants failed to generate IL-2. These data directly implicate PI 3-kinase in CD28-mediated costimulation leading to IL-2 secretion. Wortmannin, an inhibitor of PI 3-kinase, induced cell apoptosis and as such was unsuitable for use in this study.

Thapsigargin (TG), a sesquiterpene lactone and non-phorbol 12-myristate 13-acetate tumor promoter, stimulates a rapid increase in intracellular free Ca2+ [( Ca2+]i) in human T lymphocytes clone P28. The [Ca2+]i response to TG is sustained in the presence of 1 mM extracellular Ca2+, while it becomes transient in Ca2(+)-free medium suggesting that TG activates both the release of Ca2+ from intracellular stores and the entry of Ca2+ from extracellular spaces. TG-induced Ca2+ influx is completely abolished after cell depolarization caused by increased extracellular concentrations of K+. The rise in [Ca2+]i stimulated by TG occurs in the absence of detectable production of inositol phosphates. Moreover, TG does not alter the early biochemical events of T cell activation triggered through the CD2 or the CD3 T cell antigens. Indeed, both inositol phosphate production and intracellular pH increase induced by specific monoclonal antibodies (mAb) remain unchanged after TG treatment. These data suggest that in human T lymphocytes TG releases Ca2+ from an intracellular pool by a mechanism which is independent of the phospholipase C metabolic pathway. Preincubation with TG of T cell clone P28 empties both the CD2 and the CD3-sensitive intracellular Ca2+ pool(s). Conversely, prestimulation of T cell clone P28 by CD3 or CD2-specific mAb inhibits the Ca2(+)-mobilizing effect of TG. Thus it appears that TG and CD2- or CD3-specific mAb mobilize Ca2+ from common Ca2+ pool(s). Taken together, these results demonstrate that Ca2+ influx in human T cells may be linked to mobilization of intracellular Ca2+ pools and by a mechanism independent of phosphoinositide hydrolysis. They further indicate that the release of intracellular Ca2+ pool(s) may play a major role in the opening of cell membrane Ca2+ channels observed during the CD2- or CD3-induced stimulation of human T lymphocytes.

The contribution of neuropeptide Y (NPY), deriving from adrenal medulla, to the adrenosympathetic tone is unknown. We found that in response to NPY, primary cultures of mouse adrenal chromaffin cells secreted catecholamine, and that this effect was abolished in cultures from NPY Y1 receptor knockout mice (Y1؊͞؊). Compared with wild-type mice (Y1؉͞؉), the adrenal content and constitutive release of catecholamine were increased in chromaffin cells from Y 1؊͞؊ mice. In resting animals, catecholamine plasma concentrations were higher in Y1؊͞؊ mice. Comparing the adrenal glands of both genotypes, no differences were observed in the area of the medulla, cortex, and X zone. The high turnover of adrenal catecholamine in Y 1؊͞؊ mice was explained by the enhancement of tyrosine hydroxylase (TH) activity, although no change in the affinity of the enzyme was observed. The molecular interaction between the Y 1 receptor and TH was demonstrated by the fact that NPY markedly inhibited the forskolin-induced luciferin activity in Y 1 receptor-expressing SK-N-MC cells transfected with a TH promoter sequence. We propose that NPY controls the release and synthesis of catecholamine from the adrenal medulla and consequently contributes to the sympathoadrenal tone. (3,4). NPY is an important neurotransmitter of the sympathetic function that potentiates the catecholamine vasoconstrictor activity through the Y 1 receptor and exerts prejunctional inhibitory effects on NE release from the sympathetic nerve endings of the heart through the Y 2 receptor (5). In addition, the nerve terminals of parasympathetic neurons in the mouse heart possess Y 2 receptors, which, when activated, reduce acetylcholine release, also causing an inhibition of the parasympathetic nervous system (6). We have shown that NPY Y 1 knockout mice (Y 1 Ϫ͞Ϫ) lose their ability to potentiate NE-induced vasoconstriction and have normal blood pressure, probably indicating a minor role of NPY in the maintenance of blood pressure homeostasis (7). Recently, these mice were investigated for their cardiac sympathovagal balance in baseline conditions and during an acute social challenge. Reduced somatomotor activity during nonsocial challenges, lower heart rate in baseline conditions, and larger heart rate responsiveness during social defeat were reported (8). Besides its presence in nerve endings, NPY is produced by chromaffin cells of adrenal medulla of different species, including human (9). The mouse has higher adrenal NPY content than rat, pig, or humans (10, 11). The effect of NPY on the adrenal medulla is controversial. NPY stimulated catecholamine release from intact rat adrenal capsular tissue (12), although an inhibitory effect of NPY on catecholamine secretion in rat adrenomedullary primary cell cultures was also observed (13). Moreover, there is a weak inhibitory effect of NPY on NE and epinephrine (EP) release from bovine chromaffin cells, evoked by addition of a cholinergic agonist (14, 15). However, depending on the experimental conditions, conflicting results wer...

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The interference of the recombinant HIV-1 glycoproteins gp160 and gp120 with the CD3/T cell antigen receptor (TcR)-mediated activation process has been investigated in the CD4+ diphtheria toxoid-specific human P28D T cell clone. Both glycoproteins clearly inhibit the T cell proliferation induced in an antigen-presenting cell (APC)-free system by various cross-linked monoclonal antibodies specific for the CD3 Introduction HIV-1 infection in humans results in quantitative and qualitative defects in the CD4 helper/inducer T cell function (1). The selective infection of CD4+ T cells by HIV-1 is governed through the high affinity interaction between the viral envelope glycoprotein gp 120 and T cell surface expressed determinants of the CD4 molecule. Different works showed that soluble gp 120 itself could exert a suppressive effect on T cell responses. Thus, gp 120 could alter the antigen-specific proliferation of human uninfected CD4+ T cells (2-4) thereby

Lymph nodes with Hodgkin disease (HD)harbor few neoplastic cells in a marked leukocytic infiltrate. Since chemokines are likely to be involved in the recruitment of these leukocytes, the expression of potentially relevant chemokines and chemokine receptors were studied in lymph nodes from 24 patients with HD and in 5 control lymph nodes. The expression of regulated on activation, normal T cell expressed and secreted (RANTES), monocyte chemotactic protein (MCP)-1, macrophage inflammatory protein (MIP)-1␣, and MIP-1␤ was analyzed by in situ hybridization and that of CCR3 and CCR5 by immunohistochemistry and flow cytometry. It was found that, overall, the expression of all 4 chemokines was markedly enhanced, but the cellular source was different. RANTES was expressed almost exclusively by T cells whereas the expression of MCP-1, MIP-1␣, and MIP-1␤ was confined largely to macrophages. In control lymph nodes, chemokine expression was low, with the exception of MIP-1␣ in macrophages. CCR3 and CCR5 were highly expressed in T cells of HD involved but not of control lymph nodes. CCR3 was equally distributed in CD4 ؉ and CD8 ؉ cells, but CCR5 was associated largely with CD4 ؉ cells. In HD lymph nodes, CCR3 and CCR5 were also expressed in B cells, which normally do not express these receptors. All these chemokines and receptors studied, by contrast, were absent in the neoplastic cells. It was concluded that chemokines are involved in the formation of the HD nonneoplastic leukocytic infiltrate. Expression of CCR3 and CCR5 appears to be characteristic of HD, but the roles of these receptors' up-regulation for the disease process remain unclear. IntroductionHodgkin disease (HD) is characterized by the presence of few neoplastic mononuclear Hodgkin cells and polynuclear ReedSternberg (H-RS) cells surrounded by a dense nonneoplastic leukocytic infiltrate consisting of lymphocytes, plasma cells, granulocytes, and macrophages. 1 In the nodular sclerosis (NS) and mixed cellularity (MC) subtypes, CD4 ϩ T cells are particularly frequent in this infiltrate. They almost exclusively have the CD45RO ϩ and CD45RB ϩ phenotype and are in a state of activation that, based on their cytokine expression, appears to be related to a T-helper 2 (T H 2)-mediated immune response. [2][3][4] H-RS cells express various cell surface molecules, including CD58 (intercellular adhesion molecule 1), CD40, CD30, and CD80 (B7-1) as well as major histocompatibility complex class II molecules, 5,4 and produce numerous cytokines, such as interleukin (IL)-1, IL-5, IL-6, IL-9, macrophage-colony stimulating factor, tumor necrosis factor (TNF)-␣, 6 and IL-13, 7 that may have multiple effects on the surrounding leukocytes.Few studies have addressed the role of chemokines in HD. For instance, the IL-8 gene expressed by reactive cells in lymph nodes involved by HD is related to the presence of neutrophilic granulocytes. 8 Studies based mainly on reverse-transcription polymerase chain reaction technology show that HD tissues also express higher levels of inducible prot...

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CD80 (B7-1) and CD86 (B7-2) ligation of CD28 provide co-stimulatory signals required for optimal lymphokine production in response to TCR zeta-CD3 ligation. CD28 binds to several intracellular proteins including phosphatidylinositol 3-kinase (Pl3-kinase), the tyrosine kinase ITK and the growth factor receptor-bound protein/Son of Sevenless (GRB-2/SOS) complex. Previously, we showed that TCR zeta-CD3 and CD28 co-stimulation required Pl3-kinase binding to the pYMNM motif of the cytoplasmic domain of the co-receptor. In this study, we have investigated whether CD28-associated Pl3-kinase is required for CD80 and CD86 co-stimulation, as well as in co-signaling that involves different primary signals (i.e. TCR zeta-CD3 versus phorbol ester/lonomycin). In the presence of anti-CD3, ligation of CD28 by both CD80 and CD86 was found to induce Pl3-kinase recruitment and IL-2 production. Furthermore, mutations at Y-191 and M-194 within the pYMNM motif blocked the ability of both ligands to induce IL-2. CD80 and CD86 therefore share a common signaling pathway leading to IL-2 production. By contrast, CD28 mediated co-stimulation involving receptor ligation plus phorbol ester/lonomycin induced IL-2 independent of Pl3-kinase binding to CD28. These data indicate that TCR zeta-CD3-dependent CD80 and CD86 co-signaling requires Pl3-kinase binding to the CD28pYMNM motif, while phorbol ester and lonomycin can bypass this requirement in CD28 co-stimulation.