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Of the many intracellular events that occur after mitogenic stimulation of cells, the phosphorylation of the retinoblastoma protein (RB) in early G1-phase appears to play a pivotal role in controlling cell-cycle progression. RB phosphorylation results in release from a proliferative block imposed by hypophosphorylated RB. Several investigators have presented evidence, using models produced in vitro, that the serine kinase p34CDC2 phosphorylates RB and is responsible for regulating RB phosphorylation. Using human T-cells as a model, we show that lectin treatment of resting T-cells results in detectable RB phosphorylation by 24 h after treatment. Further, using immunoprecipitation and immunoblotting, no detectable p34CDC2 could be seen until 48 h after lectin stimulation. Analysis of the relative histone H1 activity of p34CDC2, purified by immunoprecipitation, revealed that RB phosphorylation does not parallel increases in p34CDC2 activity as T-cells progress into S-phase, supporting the contention that p34CDC2 activation as a histone H1 kinase is not a critical regulator of RB phosphorylation. Further treatment of activated T-cells, arrested in G1-phase, with interleukin 2 results in a 95% increase in RB phosphorylation within 4 h with no detectable increase in the histone H1 kinase activity of p34CDC2. Together, these data suggest that p34CDC2 activation is not required for early cell-cycle phosphorylation of RB.

The mechanism of action of prolactin (PRL) has remained obscure despite the unveiling of the primary structure of PRL receptors. The present study demonstrates rapid PRL receptor-mediated tyrosine phosphorylation of at least three cellular proteins, designated p120, p97, and p40, in a rat T-lymphoma (Nb2-11C) as revealed by antiphosphotyrosine immunoblotting. One of the phosphotyrosyl proteins, p120, co-purified with activated PRL receptor complexes obtained using either anti-ligand or anti-receptor antibodies. Furthermore, in vitro incubation of affinity-purified PRL receptor complexes from PRL-stimulated cells with ATP in the presence of a tyrosine phosphatase inhibitor, resulted in a 10-15-fold increase in the phosphotyrosine content of p120, as revealed by antiphosphotyrosine immunoblotting. Parallel experiments utilizing [gamma-32P]ATP confirmed a rapid and time-dependent incorporation of phosphate into p120 in the same affinity-purified PRL receptor complexes. These data provide strong evidence for the involvement of a tyrosine kinase in PRL signal transduction and suggest the presence of a tyrosine kinase within the activated PRL receptor complex.

Results and conclusions concerning the ability of HIV glycoprotein (gp) 120 to stimulate monokine secretion have been equivocal, based on observations using natural gp120 derived from infected human cells and a Chinese hamster ovary (CHO) cell-derived recombinant fusion protein. Current studies were designed to determine whether differences in recombinant gp120 proteins could result in failure to trigger monokine production. We found that natural gp120 could stimulate monocytes to release TNF-alpha, IL-1 beta, IL-6, and granulocyte-macrophage-CSF, and this effect could be blocked with soluble CD4. Full-length rgp120 either expressed from an adenovirus vector and purified from infected human cells, or derived from CHO cells, could function similarly. In contrast, full-length recombinant envelope protein expressed in a baculovirus system and a CHO cell-derived recombinant fusion protein tested previously, consistently failed to stimulate monokine production. The stimulatory capacity of both natural and full-length CHO cell-derived gp120 was eliminated by heating at 100 degrees C, and could be blocked with excess CHO cell-derived gp120 fusion protein. Inasmuch as the baculovirus-expressed gp120 and the CHO cell-derived recombinant fusion protein can bind to CD4, these results suggest that HIV gp120 binding to CD4 on the monocyte surface may of itself be insufficient for stimulation of monokine secretion. Therefore, primary protein structure, as well as posttranslational protein modifications, may determine this activity.

Interleukin (IL) 4 is a potent immunomodulatory cytokine secreted by T-helper 2 (Th2) cells and Th2 mast cells that promotes the commitment of cells. However, unregulated production and release of IL-4 can exacerbate allergic reactions and increase susceptibility to infectious organisms and viruses. Here, we present evidence that AG-490, a Janus tyrosine kinase (JAK) 2-JAK3 inhibitor, effectively blocked IL-4 gene expression and secretion in the Th2 cell line D10 that was not occurring after anti-CD3 antibody stimulation, whereas AG-490 had no inhibitory effect on production of other Th2 cytokines or cytokines synthesized by the corresponding Th1 cell line clone 29. AG-490 potently inhibited IL-4-mediated proliferation of both D10 and the IL-4-dependent cell line CT.4S. Moreover, AG-490 markedly inhibited IL-4 activation of JAK3 and blocked the downstream activation of signal transducer and activator of transcription 6, as judged by tyrosine phosphorylation, DNA binding, and transcription assays. In contrast, AG-490 did not affect tumor necrosis factor alpha activation of NF-kappaB at similar concentrations of drug. These data suggest that tyrosine kinase inhibitors that inhibit JAK3 may have previously unrecognized and selective clinical potential as immunotherapeutic drugs to treat Th2-mediated diseases driven by IL-4. (Blood. 2000;95:3816-3822)

Interleukin 6 (IL-6) is a cytokine that regulates not only immune and inflammatory responses but also the growth of some tumors, including prostate carcinomas. IL-6 signals through Janus kinase, signal transducer and activator of transcription 3 (STAT3), and mitogen-activated protein kinase and is also able to induce androgen receptor (AR)-mediated gene activation in prostate cancer, which is an important process in prostate cancer androgen-independent progression. We now show that IL-6-induced AR-mediated gene activation requires the activation of STAT3 by IL-6 in LNCaP prostate cancer cells. In particular, STAT3 associates with AR in an androgen-independent but IL-6-dependent manner. Inhibition of STAT3 rather than mitogen-activated protein kinase results in inhibition of AR-mediated gene activation in response to IL-6. These findings not only identify STAT3 as an important signaling molecule required for IL-6-signaling to induce AR-mediated gene activation in prostate carcinoma cells but also reveal the importance of activated STAT3 in human tumor development and progression.

T lymphocyte activation is highlighted by the induction of interleukin-2 (IL-2) gene expression, which governs much of the early lymphocyte proliferation responses. Peroxisome proliferator-activated receptor-gamma (PPARgamma) is a member of the nuclear receptor superfamily of ligand-activated transcription factors. PPARgamma mRNA expression was found in human peripheral blood T lymphocytes, raising the possibility of PPARgamma involvement in the regulation of T cell function. Here we show that PPARgamma ligands, troglitazone and 15-deoxy-Delta(12,14) prostaglandin J(2), but not PPARalpha agonist Wy14643, inhibited IL-2 production and phytohemagglutinin-inducible proliferation in human peripheral blood T-cells in a dose-dependent manner. This inhibitory effect on IL-2 was restricted to the PPARgamma2-expressing, not the PPARgamma-lacking, subpopulation of transfected Jurkat cells. The activated PPARgamma physically associates with transcriptional factor NFAT regulating the IL-2 promoter, blocking NFAT DNA binding and transcriptional activity. This interaction with T-cell-specific transcription factors indicates an important immunomodulatory role for PPARgamma in T lymphocytes and could suggest a previously unrecognized clinical potential for PPARgamma ligands as immunotherapeutic drugs to treat T-cell-mediated diseases by targeting IL-2 gene expression.

The transcription factor, signal transducer and activator of transcription (Stat) 6, regulates T(H)2-lymphocyte activity by controlling the expression and responsiveness to interleukin (IL)-4, which plays a key role in numerous allergic maladies. Therefore, we sought to use a phosphorothiolate cis-element decoy to target disruption of Stat6 transcriptional activity. Here we showed that the Stat6 decoy potently ablated the messenger RNA expression and production of IL-4, but not of several other cytokines. The Stat6 decoy functionally disrupted IL-4-inducible cell proliferation of murine T(H)2 cells and primary human CD4(+) T lymphocytes. Specificity of the decoy was demonstrated by its ability to directly block Stat6 binding to a cis-element probe and transactivation, but not affect Stat6 tyrosine phosphorylation or expression of the IL-4 receptor chains. Moreover, the decoy failed to inhibit non-Stat6-dependent signaling pathways since IL-2 was competent to induce cell proliferation and activation of Stats 1, 3, and 5a/b. With the use of laser scanning confocal microscopy, fluorescently tagged Stat6 decoy was detectable in the cytoplasm and nucleus; however, greater levels of oligonucleotide were present in the latter following IL-4 treatment. Taken together, these data suggest that IL-4-driven T(H)2 cell activity can be preferentially restricted via targeted disruption of Stat6 by a novel and specific decoy strategy that may possess gene therapeutic potential. (Blood. 2000;95:1249-1257)

AG-490 is a member of the tyrphostin family of tyrosine kinase inhibitors. While AG-490 has been considered to be a Janus kinase (JAK)2-specific inhibitor, these conclusions were primarily drawn from acute lymphoblastic leukemia cells that lack readily detectable levels of JAK3. In the present study, evidence is provided that clearly demonstrates AG-490 potently suppresses IL-2-induced T cell proliferation, a non-JAK2-dependent signal, in a dose-dependent manner in T cell lines D10 and CTLL-2. AG-490 blocked JAK3 activation and phosphorylation of its downstream counterpart substrates, STATs. Inhibition of JAK3 by AG-490 also compromised the Shc/Ras/Raf/mitogen-activated protein kinase (MAPK) signaling pathways as measured by phosphorylation of Shc and extracellular signal-related kinase 1 and 2 (ERK1/2). AG-490 effectively inhibited tyrosine phosphorylation and DNA binding activities of several transcription factors including STAT1, -3, -5a, and -5b and activating protein-1 (AP-1) as judged by Western blot analysis and electrophoretic mobility shift assay. These data suggest that AG-490 is a potent inhibitor of the JAK3/STAT, JAK3/AP-1, and JAK3/MAPK pathways and their cellular consequences. Taken together, these findings support the notion that AG-490 possesses previously unrecognized clinical potential as an immunotherapeutic drug due to its inhibitory effects on T cell-derived signaling pathways.

Kaposi's sarcoma-associated herpesvirus (KSHV)/human herpesvirus 8, a virus that appears to be involved in the pathogenesis of Kaposi's sarcoma and primary effusion lymphomas, encodes a G protein-coupled receptor (KSHV-GPCR) that exhibits constitutive signaling. In this report, we show that two chemokines, interleukin 8 (IL-8) and growth-related protein-alpha, activate KSHV-GPCR over constitutive levels. Moreover, as with human receptors, the integrity of the ELR motif of these chemokines is required for activation of KSHV-GPCR. Other residues that are required for IL-8 binding to human chemokine receptors CXCR1 and CXCR2 are important for KSHV-GPCR activation also. Thus, it appears that the ELR binding site and other key domains of ELR chemokine activation have been preserved in the virus KSHV-GPCR. The results suggest that KSHV-GPCR originated from CXCR1 or CXCR2 and that activation of KSHV-GPCR by endogenous chemokines may affect the pathobiology of KSHV infection in humans.

Microinjection of expression plasmids encoding either JAK2 or hyperactive (Ndelta661)rJAK2 into serum-starved NIH 3T3 cells resulted in 20-30-fold induction of DNA synthesis. Control microinjections of buffer or parental pcDNA3 vector resulted in only 3-5-fold induction of DNA synthesis. Induction of DNA synthesis was blocked when plasmid encoding JAK2 was microinjected in the presence of the JAK2-selective inhibitor AG-490, whereas AG-490 did not block DNA synthesis induced by microinjected plasmid encoding (Ndelta661)rJAK2. The ability of JAK2 to initiate the G(o)/S cell cycle transition is comparable to that of other proto-oncogenes, and supports a mechanistic role for overexpressed Janus kinases in carcinogenesis.

The tyrosine kinases JAK1 and JAK3 have been shown to undergo tyrosine phosphorylation in response to interleukin-2 (IL), IL4, IL7, and IL9, cytokines which share the common IL2 receptor gamma-chain (IL2R gamma), and evidence has been found for a preferential coupling of JAK3 to IL2R gamma and JAK1 to IL2R beta. Here we show, using human premyeloid TF-1 cells, that IL4 stimulates JAK3 to a larger extent than JAK1, based upon three different evaluation criteria. These include a more vigorous tyrosine phosphorylation of JAK3 as measured by anti-phosphotyrosine immunoblotting, a more marked activation of JAK3 as determined by in vitro tyrosine kinase assays and a more manifest presence of JAK3 in activated IL4-receptor complexes. These observations suggest that IL4 receptor signal transduction does not depend on equimolar heterodimerization of JAK1 and JAK3 following IL4-induced heterodimerization of IL4R alpha and IL2R gamma. Indeed, when human IL4R alpha was stably expressed in mouse BA/F3 cells, robust IL4-induced proliferation and JAK3 activation occurred without detectable involvement of JAK1, JAK2, or TYK2. The present study suggests that JAK1 plays a subordinate role in IL4 receptor signaling, and that in certain cells exclusive JAK3 activation may mediate IL4-induced cell growth. Moreover, mutational analysis of human IL4R alpha showed that a membrane-proximal cytoplasmic region was critical for JAK3 activation, while the I4R motif was not, which is compatible with a role of JAK3 upstream of the recruitment of the insulin receptor substrate-1/4PS signaling proteins by IL4 receptors.

Pervanadate has been shown to rapidly increase the level of tyrosine phosphorylation in intact cells. Because one of the most rapidly detectable events following treatment of human T cells with interleukin-2 (IL-2) is tyrosine kinase activation, we were interested to determine whether pervanadate could act to induce IL-2-associated events. We show here that pervanadate does act to induce IL-2 signal transduction pathways as determined by induction of mitogenesis and interferon gamma production in normal human T cells and the factor independent T cell line YT. Analysis of signal transduction events shows that pervanadate induces the activity of the src family of tyrosine kinases lck and fyn and the tyrosine phosphorylation of a major IL-2 responsive protein of 97 kDa. Pervanadate does not, however, induce the activity of tyrosine kinases associated with the IL-2 receptor or the phosphorylation of a major IL-2 responsive protein of 116 kDa (Jak-3). Together these data suggest that src family kinase activation is a down stream event following IL-2 stimulation and is not directly associated with the activation of the IL-2 receptor-associated tyrosine kinase. The data also imply that tyrosine phosphorylation of p116/Jak-3 is strictly associated with activation of tyrosine kinases associated with the IL-2 receptor. With the use of pervanadate as a tool, we have established a dissociation of src family kinases with IL-2 receptor activation and imply the involvement of two distinct tyrosine kinase pathways, a receptor-associated pathway closely coupled with Jak-3 phosphorylation and a downstream pathway involving src family kinase activation.

Janus tyrosine kinase (JAK) has recently been linked to signal transduction by cytokine receptors of the hematopoietin family. We have recently described a 116-kDa tyrosine kinase (p116) present in interleukin-2 (IL-2) receptor complexes in human YT cells that showed functional characteristics of a JAK kinase. These included receptor association, rapid and transient tyrosine phosphorylation kinetics in response to ligand, and in vitro autophosphorylating tyrosine kinase activity (Kirken, R. A., Rui, H., Evans, G. A., and Farrar, W. L. (1993) J. Biol. Chem. 268, 22765-22770). Here we extend these observations by demonstrating structural homologies between IL-2-modulated p116 and prolactin-modulated JAK2 in the rat T cell line Nb2. These include similar net charge as determined by nonequilibrium pH gradient electrofocusing and related primary structure based upon phosphopeptide mapping of V8 protease-digested hyperphosphorylated proteins. This putative JAK kinase underwent marked tyrosine phosphorylation in response to IL-2, IL-4, and IL-7, lymphoid growth factors that use the common IL-2 receptor gamma-chain, but not in response to prolactin. Furthermore, polyclonal antisera to JAK1, JAK2, or tyrosine kinase 2 did not recognize either rat or human p116. However, we identified the IL-2-modulated p116 as the recently cloned novel leukocyte Janus kinase, L-JAK, using an antiserum to a peptide corresponding to the COOH terminus of human L-JAK.

Prolactin (PRL) has recently been demonstrated to induce tyrosine phosphorylation and activation of the cytoplasmic tyrosine kinase JAK2 in PRL-dependent Nb2 lymphoma cells. The present study represents an initial effort to identify cytoplasmic regions of the PRL receptor (PRLR) that are critical to growth signal generation and JAK2 activation. Variably truncated rat PRLRs were stably expressed in the murine 32D cell line. PRL-induced proliferation was mediated by the full-length receptor and the mutant G328, which contains the membrane-proximal Homology Boxes 1 and 2 characteristic of hematopoietin receptors. In contrast, mutant receptors lacking either Box 2 or both Boxes 1 and 2 were not capable of transmitting a growth signal. The mitogenic capacity of the PRLR variants correlated with JAK2 association and activation, as well as with induction of mRNA levels for the growth-related gene ornithine decarboxylase. We conclude that mitogenic competence of rat PRLRs resides within the first 94 amino acids of the cytoplasmic domain. The data suggest that Homology Box 1/Box 2 region is critical to JAK2 phosphorylation and association with the PRLR. These findings will serve as a basis for more detailed molecular characterization of PRLR domains essential for JAK2 interaction and growth signal generation.

JAK family tyrosine kinases have recently been implicated in intracellular signal transduction by transmembrane cytokine receptors of the interferon (IFN) and hematopoietin receptor families. Using the prolactin (PRL)-dependent rat pre-T cell line Nb2, a PRL receptor-associated, candidate tyrosine kinase of 120-130 kDa was recently characterized (1). In the present work this protein is identified as JAK2, based upon reciprocal anti-JAK2 and anti-phosphotyrosine immunoprecipitation and immunoblotting. JAK2 underwent rapid and transient tyrosine phosphorylation in response to receptor activation, reaching peak levels within 5 min of exposure to 100 nM PRL at 37 degrees C. In vitro tyrosine kinase assays using either [gamma-32P]ATP and autoradiography or unlabeled ATP combined with anti-phosphotyrosine immunoblotting, demonstrated that the activity of JAK2 was stimulated by PRL. Phosphoamino acid analysis of JAK2 after in vitro tyrosine kinase assay revealed that the majority of phosphate was incorporated into tyrosine residues. Furthermore, JAK2 was associated with PRL receptors to a comparable extent before and after PRL binding, as demonstrated by anti-receptor immunoprecipitation and subsequent anti-JAK2 immunoblotting. We propose that binding of ligand to the PRL receptor activates preassociated JAK2, and that this enzyme generates the initial signal in the intracellular communication cascade.

In this paper we have extended previous results on interleukin-2 receptor (IL2-R) signal transduction and focused on the interleukin-2 (IL-2)-stimulated tyrosine phosphorylation of a 116-kDa protein (p116) observed in IL-2 responsive cells. This protein exhibited rapid and transient phosphorylation kinetics in both human T-lymphocytes and the YT cell line, attaining maximum tyrosine phosphorylation within 5 min of stimulation with IL-2. Tyrosine phosphorylated p116 co-purified with activated IL-2 receptor beta-chain (IL2-R beta) when IL2-R complexes were covalently stabilized with the membrane-permeable cleavable cross-linking agent dithiobis(succimidyl propionate) prior to detergent cell lysis and immunoprecipitation with monoclonal anti-IL2-R beta antibodies. Under these conditions comparable amounts of tyrosine-phosphorylated p116 were immunoprecipitated with either anti-IL2-R beta antibodies or anti-phosphotyrosine antibodies, suggesting that a major portion of tyrosine phosphorylated p116 is associated with the IL2-R beta subunit. Furthermore, unphosphorylated p116 was also associated with unactivated IL2-R beta, based on the observation that p116 from unstimulated YT cells underwent tyrosine phosphorylation in IL2-R beta immune-complex tyrosine kinase assay as demonstrated by anti-phosphotyrosine immunoblotting. The presence of tyrosine kinase activity in affinity-purified IL2-R beta complexes supports the notion of a preformed receptor-kinase complex. The co-association of both p116 and tyrosine kinase activity with the IL2-R beta supports the critical role of the beta-chain in IL2-R signal transduction and suggests that p116 may have a role in the dynamics of IL2-R activation.

The mechanism of action of prolactin (PRL) has remained obscure despite the unveiling of the primary structure of PRL receptors. The present study demonstrates rapid PRL receptor-mediated tyrosine phosphorylation of at least three cellular proteins, designated p120, p97, and p40, in a rat T-lymphoma (Nb2-11C) as revealed by antiphosphotyrosine immunoblotting. One of the phosphotyrosyl proteins, p120, co-purified with activated PRL receptor complexes obtained using either anti-ligand or anti-receptor antibodies. Furthermore, in vitro incubation of affinity-purified PRL receptor complexes from PRL-stimulated cells with ATP in the presence of a tyrosine phosphatase inhibitor, resulted in a 10-15-fold increase in the phosphotyrosine content of p120, as revealed by antiphosphotyrosine immunoblotting. Parallel experiments utilizing [gamma-32P]ATP confirmed a rapid and time-dependent incorporation of phosphate into p120 in the same affinity-purified PRL receptor complexes. These data provide strong evidence for the involvement of a tyrosine kinase in PRL signal transduction and suggest the presence of a tyrosine kinase within the activated PRL receptor complex.

We have examined the signal transduction pathways of a number of cytokines that interact with receptors that are members of the hematopoietin receptor superfamily. A 97-kDa protein was phosphorylated on tyrosine in response to stimulation of appropriate target cells with interleukin (IL)-2, IL-3, granulocyte-macrophage colony-stimulating factor (CSF), granulocyte-CSF, or erythropoietin. These data suggest that a 97-kDa phosphotyrosylprotein represents a point of convergence for signal transduction by a number of growth factor receptors that do not have homology with any known protein tyrosine kinase. To address the possibility that p97 may represent a tyrosine kinase involved in multiple signal transduction pathways, we tested the capacity of this protein to bind a tyrosine kinase substrate or ATP. Indeed, a 97-kDa phosphotyrosylprotein purified from IL-2-stimulated lymphoid cells as well as granulocyte-macrophage-CSF-stimulated myeloid cells bound to a polymer of glutamic acid and tyrosine which is a tyrosine kinase substrate. Further, a 97-kDa phosphotyrosylprotein present in both lineages also bound 8-azido-ATP. These data indicate that a 97-kDa phosphotyrosylprotein with properties consistent with those of a protein tyrosine kinase is involved in the signal transduction pathways of certain members of the newly identified hematopoietin receptor superfamily and may represent an early point of convergence in the stimulus-response coupling of multiple cytokine receptors.

The intracellular events which are involved in controlling the G1 to S phase transition during the eucaryotic cell cycle are important to define in order to understand the mechanisms by which mitogenic and growth arrest-inducing agents control cell growth. Because a change in protein kinase activity is associated with the initial response of cells to mitogenic stimulants and growth factors, we used a kinase renaturation assay to identify specific protein kinases which are modulated as human T cells make the G1 to S phase transition after mitogenic stimulation with lectin. We identified four protein serine/threonine kinases of 180, 97, 85, and 38 kilodaltons which are increased in activity as these cells enter S phase. A-55 kDa serine/threonine kinase (PK55) was shown to have maximal activity during G0 and its activity was reduced by 95% upon movement into S phase. PK55 is inducible in human T cells by removal of interleukin 2 and low serum incubation which arrests cells in G1 phase, indicating that it is closely associated with G1 phase growth arrest. Furthermore, a similar PK55 activity was induced upon growth arrest in HL-60 cells treated with dimethyl sulfoxide and in Daudi cells treated with interferon alpha. Because the cAMP-dependent protein kinase (PK-A) family has been shown to be antiproliferative to lectin stimulated T cells, we were interested in determining whether PK55 was in fact an isozyme of PK-A. Comparative analysis using a specific peptide inhibitor of PK-A activity revealed that PK55 is catalytically distinct from PK-A. This data suggest that increases in PK55 may be associated with the growth-arrested state and further that PK55 is distinct from PK-A.

In an effort to identify unique tyrosine kinases found in human leukemia cell lines, we utilized polymerase chain reaction (PCR) technology and degenerate oligonucleotide primers to produce a cDNA library of kinase catalytic domains found in the human monocytic cell line AML-193. This search yielded a member of the class 3 tyrosine kinases closely related to the murine kinase FD-22. Previous work has identified this kinase as JAK1. This class of tyrosine kinases is characterized by being ubiquitously expressed, lacking both a ligand-binding domain and a SH2 domain, while containing a second domain similar to a degenerate kinase domain. Our studies focused on the further characterization of this class 3 tyrosine kinase using Northern blot analysis to demonstrate an increase in steady-state mRNA by interferon-gamma in human monocytes. A human-hamster somatic cell hybrid panel and linkage mapping was used to assign JAK1 (aml-116) to human chromosome 1.

Results and conclusions concerning the ability of HIV glycoprotein (gp) 120 to stimulate monokine secretion have been equivocal, based on observations using natural gp120 derived from infected human cells and a Chinese hamster ovary (CHO) cell-derived recombinant fusion protein. Current studies were designed to determine whether differences in recombinant gp120 proteins could result in failure to trigger monokine production. We found that natural gp120 could stimulate monocytes to release TNF-alpha, IL-1 beta, IL-6, and granulocyte-macrophage-CSF, and this effect could be blocked with soluble CD4. Full-length rgp120 either expressed from an adenovirus vector and purified from infected human cells, or derived from CHO cells, could function similarly. In contrast, full-length recombinant envelope protein expressed in a baculovirus system and a CHO cell-derived recombinant fusion protein tested previously, consistently failed to stimulate monokine production. The stimulatory capacity of both natural and full-length CHO cell-derived gp120 was eliminated by heating at 100 degrees C, and could be blocked with excess CHO cell-derived gp120 fusion protein. Inasmuch as the baculovirus-expressed gp120 and the CHO cell-derived recombinant fusion protein can bind to CD4, these results suggest that HIV gp120 binding to CD4 on the monocyte surface may of itself be insufficient for stimulation of monokine secretion. Therefore, primary protein structure, as well as posttranslational protein modifications, may determine this activity.

We have investigated whether PK-C-regulated events are independent of those biochemical events related to IL-3-induced tyrosine kinase activation by 32Dcl cells. The depletion of functional PK-C isoform activity by prolonged PMA treatment reduced the proliferative response to IL-3 by half that of untreated control cells. PK-C-deficient 32Dcl cells were unable to respond to PMA for the induction of c-myc and ODC mRNA accumulation. PK-C down-regulation did not affect IL-3-induced tyrosine phosphorylation and inhibited IL-3-regulated c-myc and ODC mRNA expression by only 30%. However, PK-C down-regulation had a pronounced inhibitory effect on IL-3 regulation of ODC enzymatic activity. While a PK-C-dependent and -independent pathway for the regulation of c-myc and ODC mRNA expression could be demonstrated, the regulation of ODC enzymatic activity appeared to require an intact PK-C system. The data suggest that the optimum biological and biochemical responses to IL-3 requires both pathways intact, however, tyrosine kinase activation and significant increases in gene products associated with proliferation can be achieved in the absence of a functional PK-C system.

The levels or diadenosine 5', 5'''-p1, p4, tetraphosphate (Ap4A), a putative signal molecule associated with DNA synthesis, has been measured in murine T lymphocytes. The level or Ap4A detected correlated with the stimulation of DNA synthesis in murine T lymphocytes. In interleukin-2 (IL-2) dependent cells previously deprived of IL-2, new DNA synthesis can be induced by adding IL-2; the synthesis of DNA is preceded by an increase in Ap4A levels. A significant increase in DNA synthesis was observed after the Ap4A concentration exceeded the Kd of DNA polymerase alpha for Ap4A. Similarly, in cells blocked from synthesizing DNA by hydroxyurea, the levels or Ap4A are maintained only in the presence of IL-2. Once IL-2 is removed, the potential to synthesize DNA decreases and is preceded by decreases in the level or Ap4A. The DNA synthesis potential decreases rapidly after the Ap4A concentration fell below the Kd of DNA polymerase alpha for Ap4A. It is possible that Ap4A is a second messenger molecule required for the proliferation of lymphocytes and that the production of Ap4A in IL-2 dependent murine T lymphocytes is regulated by the homologous growth factor.

We have examined the effect and potential mechanism of Cyclosporin A (CsA) on the Interleukin-2-receptor alpha chain (IL-2R alpha) expression in human T-lymphocytes. CsA pretreatment of PHA-activated T-cells led to 30-50% decrease in Tac antigen surface expression and a concomitant decrease in the steady state IL-2R alpha mRNA levels. Transacting factors which recognize a kB-like sequence present in the IL-2R alpha chain regulatory region have been suggested to participate in the transcriptional regulation of the IL-2R alpha gene. Using oligonucleotides corresponding to the 5' regulatory region of the IL-2R alpha gene (i.e. 245 to 291 bp upstream of the start codon) and nuclear extract from resting T lymphocytes, we detected two specific bands by gel mobility shift assay. One of these bands is specifically increased after stimulation with phytohemagglutinin (PHA) and it is inhibited by CsA pretreatment. The same pattern of binding activity has been observed with the tandem repeat of NF-kB binding site present in the enhancer element of the human immunodeficiency virus long terminal repeat (HIV-1 LTR). These data suggest that CsA affects IL-2R receptor alpha chain expression by inhibiting the interaction of transacting factors to kB-like sequences after PHA activation. These findings may be of some relevance for the understanding of the immunosuppressive effects of CsA in normal human T lymphocytes.

Hemopoietic cells have an absolute requirement for survival and proliferation for specific growth factors. The growth factors maintain the critical vitality of the cells by stimulating adenosine triphosphate (ATP) synthesis and hexose transport. Intracellular alkalinization also occurs rapidly through the stimulation of the Na+/H+ antiporter. These immediate metabolic events, not initiated by serum components, appear to be necessary for the integrity of cellular viability (Fig. 6). Interleukin-3 has been shown to induce the activation of PK-C through a mechanism(s) not requiring the hydrolysis of phosphoinositol 4,5 bisphosphate. A role for Ca2+ influx or intracellular release in the action of CSFs or interleukins has not been shown. Although downregulation of cAMP has been reported in response to IL-2, the signal transduction process of CSFs and IL-2 appears not to be mediated by upregulation of cyclic nucleotide metabolism or "classical" phospholipid degradative pathways. Protein phosphorylation is clearly modulated by the hemopoietic cytokines, yet only the CSF-1 receptor has any known intrinsic kinase activity. Instead, the IL-3, GM-CSF receptors, and perhaps G-CSF appear to be coupling to kinases of both tyrosine and serine specificities. This may be a direct allosteric interaction with membrane-associated kinases or transduced through an intermediate protein such as those using GTP. Such is the case for many hormone receptors that couple to amplifying "second messenger" enzyme systems (i.e., adenylate cyclase, phospholipase C) or members of the insulin growth factor family that couple to tyrosine kinases in proximity to the receptors (IGF-II). One of the kinase systems that IL-2, IL-3, and other CSFs stimulate appears to have some characteristics similar to PK-C. Direct activators of PK-C stimulate some similar serine-threonine phosphorylation and perhaps even tyrosine phosphorylation. The hemopoietic growth factors, however, stimulate tyrosine phosphorylation of some proteins that are not phosphorylated in response to PK-C activators, suggesting that these kinase systems are independently regulated. Although phorbol esters stimulate many of the same metabolic activities (ATP synthesis in myeloid and lymphoid cell lines), growth-factor abrogation is clearly associated with the action of tyrosine kinase oncogenes or the nuclear oncogene effectors such as v-myc. It is likely, therefore, that tyrosine kinases are playing a critical role in the control of proliferation although the dominant amount of cellular protein phosphorylations are on serine. Both classes of kinases are apparently required for growth-factor action. All the hemopoietic growth factors examined thus far stimulate the steady-state accumulation of the nuclear protooncogenes.(ABSTRACT TRUNCATED AT 400 WORDS)

Haemopoietic growth factors stimulate a number of common biochemical and molecular events despite the high specificity of individual ligand-receptor interactions. Analysis of three distinct colony-stimulating factors (CSFs), interleukin 3 (IL-3), granulocyte-CSF and granulocyte macrophage-CSF, and the lymphocytotropic growth factor IL-2 revealed remarkably similar distal subcellular biochemical signals, although the mode of initial membrane signal transduction may differ significantly. Both early progenitor cell growth factors, such as IL-3, and late-acting factors, such as CSF-1, stimulate tyrosine and serine/threonine substrate phosphorylations. One substrate (p68) is phosphorylated in response to many CSFs and to IL-2, suggesting that it plays a highly conserved role in the signal transduction processes of many different receptor(s). The proliferative CSFs and IL-2 also stimulate the expression of many of the same genes, including protooncogenes, the ornithine decarboxylase gene, and members of the phylogenetically ancient family of stress response genes. Thus although initial membrane events may differ among the proliferative stimulants, the biochemical and molecular convergence of signalling pathways on highly conserved cellular substrates and on the programme of gene expression is seen.

14552 Background: Resistance to transforming growth factor-beta (TGF-beta) is common in solid tumors. Alterations of TGF-beta biology in prostate cancer are complex, with a downregulation of this cytokine reported in early disease and upregulation of TGF-beta in advanced disease. Mechanisms of TGF-beta resistance in early disease are not well understood. We have investigated the function of TGF-beta receptor 3 (TGFBR3) to determine its role in prostate cancer. Methods: We undertook an analysis of the data in seven published DNA microarray studies of nonneoplastic prostate tissue versus prostate cancer specimens using oncomine. Of the genes we identified as known to be directly involved in the TGF-beta pathway, loss of TGFBR3 was most significantly associated with prostate cancer. We determined TGFBR3 expression in prostate cancer cell lines and the immortalized prostate epithelial cell line RWPE-1. To assess the consequences of loss of TGFBR3 expression, we created an shRNA construct and stably infected RWPE-1 cells. We analyzed gene expression changes by DNA microarray analysis and confirmed changes by Western Blot. We then searched TGFBR3 regulated genes based on our knockdown studies in the data set from the seven previously published studies. Furthermore, we evaluated the phenotypic consequences of TGFBR3 knockdown. Results: TGFBR3 expression was significantly lost in 6 of 7 microarray studies. TGFBR3 was downregulated in 3 of 4 of prostate cancer cell lines, compared with RWPE-1. DNA microarray analysis of TGFBR3 knockdown in RWPE-1 cells showed that expression of DPYSL3, Vimentin, COCH, SERPINF1, PMP22, LTBP1 and BMP4 were decreased both with TGFBR3 knockdown and in the transition to prostate cancer. Of these genes, vimentin expression was most dramatically downregulated in TGFBR3 knockdown cells and we showed that vimentin protein expression was completely lost. The phenotypic consequence of TGFBR3 knockdown in RWPE-1 cells was formation of colonies that appear to reflect loss of contact inhibition. Conclusions: TGFBR3 is lost in prostate cancer. TGFBR3 knockdown leads to colony formation by immortalized prostate epithelial cells. Our data suggests that loss of TGFBR3 contributes to malignant transformation of the prostate. No significant financial relationships to disclose.

Tac antigen (as a measure of the IL 2 receptor) acquisition and regulation by IL 2, an antigen-receptor agonist (anti-T3), phorbol esters, and phytohemagglutinin (PHA) were studied. Phorbol esters stimulated de novo acquisition of Tac antigen, which was associated with the subcellular redistribution of protein kinase C (PK-C) from cytosol to particulate membranes of human T lymphocytes. PHA and anti-T3 (alpha-T3) antibody also stimulated a transient redistribution and activation of PK-C that reached a maximum within 20 min after stimulation. Both phorbol esters and alpha-T3 could increase Tac expression and stimulate PK-C translocation on 5 and 12 day activated T cells that were at the G0/G1 stage of the cell cycle due to IL 2 deprivation. Tac antigen-specific mRNA was seen in the nucleus within 2 hr after stimulation. In contrast, IL 2 alone could only increase Tac expression and stimulate PK-C translocation on day 5 but not day 12 activated T cells. IL 2 synergizes with alpha-T3 and phorbol ester for the regulation of Tac expression. Although IL 2 increased expression of Tac, the majority if not all of these receptors possessed low affinity for IL 2. These data suggest that the activation of PK-C is a common transmembrane signal shared by IL 2 and antigen stimulation. The results also imply that PK-C activation is necessary for the regulation of Tac antigen expression.

The neuroendocrine antidiuretic hormone arginine vasopressin (AVP) was capable of replacing the interleukin 2 (IL 2) requirement for gamma-interferon (IFN gamma) production by Lyt-2+ cells from C57BL/6 mouse spleen cells. The AVP replacement did not stimulate DNA synthesis in the target lymphocytes. This suggested that AVP was capable of replacing an IL 2 function that did not involve stimulation of cellular proliferation or DNA synthesis. This was confirmed by the demonstration that mitomycin C inhibition of IFN gamma production was reversed by IL 2 or AVP without concomitant reversal of blockage of DNA synthesis. Oxytocin, which is structurally related to AVP, was also capable of replacing IL 2 requirement for IFN gamma production, whereas insulin was ineffective. The data show that the neuroendocrine hormones AVP and oxytocin are capable of lymphokine-like activity. This activity may involve the induction of a second messenger such as cyclic GMP.

Interleukin 2 (IL 2) induces specific mRNA synthesis and secretion of an important immunoregulatory molecule, interferon-gamma (IFN-gamma). We have observed that treatment of an IL 2 independent murine T cell line, BUD-27, with IL 2, calcium ionophore A23187, or agents that activate phospholipid/Ca2+-dependent protein kinase C results in increased IFN-gamma mRNA transcription and release of anti-viral activity. These same agents each induced the subcellular redistribution of protein kinase C from cytosol to plasma membrane in both the BUD-27 cell line and its IL 2-dependent parent, CT6. Ionophore concentrations greater than 1 micron exhibited the most significant induction of IFN-gamma mRNA, which also correlated with the dose of ionophore, inducing translocation of protein kinase C. This correlation between increased mRNA levels and protein kinase C translocation suggests that a calcium-dependent event is involved in induction of IFN-gamma mRNA synthesis. Furthermore, the magnitude of the translocation of protein kinase C from cytosol to plasma membrane corresponded to the physiologic IL 2 dose-response for IFN-gamma secretion. The data suggest that the activation of protein kinase C and/or coordinate elevation of intracellular calcium may provide at least one mechanism of signal transduction for the regulation of IFN-gamma gene transcription.

The stimulation of activated human T lymphocytes with IL-2 results in increased tyrosine kinase activity. IL-2 treatment of Tac+ T cells stimulates the rapid phosphorylation of multiple protein substrates at M of 116, 100, 92, 70 to 75, 60, 56, 55, 33, and 32 kDa. Phosphorylation on tyrosine residues was detected by immunoaffinity purification of protein substrates with Sepharose linked antiphosphotyrosine mAb, 1G2. Although phorbol ester stimulated serine phosphorylation of the IL-2R alpha (p55) subunit recognized by alpha TAC mAb, IL-2 did not stimulate any detectable phosphorylation of IL-2R alpha or associated coimmune precipitated proteins. In fact, the tyrosine phosphorylated proteins did not coprecipitate with alpha Tac antibody and similar phosphoproteins were stimulated by IL-2 in IL-2R alpha- human large granular lymphocytes which express only the 70 to 75 kDa IL-2R beta subunit of the high affinity IL-2R. Anti-Tac mAb could inhibit IL-2-stimulated tyrosine phosphorylation in activated T cells, which express both IL-2R subunits that together form the high affinity receptor complex, but not in large granular lymphocytes expressing only the IL-2R beta subunit. The data suggest that IL-2 stimulation of tyrosine kinase activities requires only the IL-2R beta subunit.

Interleukin 2 (IL-2) stimulated the differentiation of human peripheral blood leukocytes into lymphokine-activated killer cells, as well as DNA synthesis of human T lymphocytes. Both effects of IL-2 could be inhibited by prostaglandin E2, a potent stimulator of adenylate cyclase; however, the inhibitory effect of prostaglandin E2 could be overcome by increased concentrations of IL-2. The opposite effects of IL-2 and prostaglandin E2 were paralleled by their respective abilities to inhibit and stimulate cAMP production in intact cells. Other agents, which inhibit adenylate cyclase directly (somatostatin, beta-endorphin, UK 14.3041) or indirectly by activation of protein kinase C (phenylephrine), could stimulate both differentiation and proliferation. None of these agents alone or in combination were as effective as maximal concentrations of IL-2. However, all agents potentiated differentiation and proliferation induced by submaximal and maximal concentrations of IL-2. Additionally, combinations of agents which stimulated protein kinase C with those that inhibited adenylate cyclase were additive in the potentiation of IL-2-induced differentiation. Neither inhibition nor potentiation of IL-2-induced lymphokine-activated killer cell differentiation was accompanied by changes in Tac expression or gamma-interferon production. The data indicate that the stimulation of lymphokine-activated killer cell differentiation and lymphocyte proliferation in human cells share a common initial biochemical signal. Although the inhibition of adenylate cyclase is not sufficient to maximally stimulate either process and cannot bypass the requirement for IL-2, modulation of this enzyme complex, positively or negatively, can regulate the ultimate physiologic response to IL-2.

Proliferation of NSF-60.8 cells, a CSF-dependent murine myeloid cell line, is strongly inhibited by murine IFN-gamma. Northern analysis of growth arrested, NSF-60.8 cells activated by addition of CSF in the presence or absence of IFN-gamma indicated that IFN-gamma inhibited the induction of c-myc mRNA steady state level by CSF. The effect was observed as early as 30 min after induction and the inhibition was complete after 20 h. IFN-gamma did not impair the transcriptional activation of c-myc gene, and it had only a slight destabilizing effect on the mature c-myc message. Study of the processing steps of c-myc mRNA precursor indicated that in the presence of IFN-gamma, a putative 3.6-kb splice intermediate accumulated instead of the mature message, suggesting that IFN-gamma inhibits the splicing of c-myc precursor.

The activation of alloantigen-specific cytotoxic T lymphocyte precursors is dependent upon the presence of both macrophages and helper T cells or regulatory molecules derived from these facilitative cells. Three biochemically distinct helper factors have been identified: interleukin 1 (macrophage-derived), Interleukin 2 (T cell derived), and immune interferon. All 3 factors are found in supernatants of mixed lymphocyte cultures (MLC), however, the removal of macrophages from these cultures completely ablates the production of these factors as well as the induction of cytotoxic T lymphocytes (CTL). The addition of IL 2 to these macrophage-depleted MLC restores the ability of responder T cells to: 1) bypass the requirement for macrophage soluble function, 2) produce immune interferon, and 3) generate CTL. The kinetics and dose response of immune interferon production in response to IL 2 correlates with the generation of CTL. The production of immune interferon as well as the generation of CTL requires T cells, alloantigen, and IL2. Furthermore, the induction of CTL by IL2 was neutralized by the addition of anti-immune interferon. These data suggest that: 1) the regulation of immune interferon production is based on a T to T cell interaction mediated by IL 2, and 2) immune interferon production may be required for IL 2 induction of CTL. These findings are consistent with the hypothesis that the induction of CTL involves a linear cell-factor interaction in which IL 1 (macrophage-derived) stimulates T cells to produce IL 2, which in turn stimulates other T cells to produce immune interferon and become cytotoxic.

Elevation of intracellular cyclic adenosine 3':5' monophosphate (cAMP) inhibits interleukin 2 (IL-2)-stimulated proliferation of a murine cytotoxic cell clone, CT6. The effects of antiproliferative dosages of stable cAMP-derivative, 8-bromoadenosine 3':5'-monophosphate (8-Br-cAMP), on steady state mRNA expression stimulated by IL-2 was examined. IL-2 stimulated mRNA accumulation of three nuclear proto-oncogenes c-fos, c-myc, and c-myb. 8-Br-cAMP alone stimulated c-fos, c-myb, and IL-2 receptor mRNA accumulation as determined by Northern blot analysis. 8-Br-cAMP, however, markedly inhibited c-myc expression stimulated by IL-2. Furthermore, although c-fos and IL-2 receptor mRNA expression was potentiated by 8-Br-cAMP, suppression of protein synthesis was seen. We show that antiproliferative cAMP stimulates similar mRNA expression as does IL-2, with the exception of c-myc. Although a comparative stimulation of steady state mRNA accumulation of some genes occurs, cAMP may profoundly effect protein synthesis. cAMP, therefore, acts on multiple targets involved in the macromolecular events stimulated by IL-2.

Cell-mediated immune responses contributing to macrophage activation were compared in mice that demonstrated partial resistance to challenge Schistosoma mansoni infection as a result of vaccination with radiation-attenuated cercariae or of ongoing low-grade primary infection. Vaccinated mice developed significant delayed hypersensitivity reactions to soluble schistosome antigens in vivo. Splenocytes from vaccinated animals responded to in vitro culture with various specific antigens (soluble adult worm extract, living or disrupted schistosomula) by proliferation and production of macrophage-activating lymphokines as did lymphocytes from S. mansoni-infected animals. Macrophage-activating factors produced by spleen cells from vaccinated mice upon specific antigen stimulation eluted as a single peak on Sephadex G-100 with a molecular weight of approximately 50,000 and contained gamma interferon activity. Moreover, peritoneal macrophages with larvicidal and tumoricidal activity were recovered from vaccinated mice after intraperitoneal challenge with soluble schistosome antigens, a procedure also observed to elicit activated macrophages in S. mansoni-infected animals. These observations demonstrate that vaccination with irradiated cercariae stimulates many of the same cellular responses observed after primary S. mansoni infection, and suggest that lymphokine-activated macrophages may participate in the effector mechanism of vaccine-induced and concomitant immunity to challenge schistosome infection. This is the first demonstration of a potential immune effector mechanism in the irradiated vaccine model.

Acquired immunodeficiency syndrome is associated with a viral (HTLV-III/LAV)-mediated progressive depletion of a helper/inducer T4+ T cell subset, whereas acute T cell leukemia is associated with a viral (HTLV-I)-mediated growth of the same T cell subset. Because large granular lymphocytes (LGL) with natural killer (NK) activity have been shown to spontaneously lyse several virus-infected target cells, the ability of NK cells to lyse both HTLV-I- and HTLV-III/LAV-infected lymphoid cell lines and fresh lymphocytes was explored. Normal lymphocytes (T cells and LGL), with and without pretreatment with recombinant interleukin 2 (IL 2), as well as monocytes, with and without pretreatment with interferon-gamma were employed as effectors. Both IL 2-activated T cells and NK cells were cytolytic for HTLV-I-infected targets. However, only LGL demonstrated significant spontaneous activity against HTLV-I-infected targets. Similarly, LGL showed spontaneous cytolytic activity against HTLV-III/LAV-infected targets, and this cytotoxicity was considerably augmented by IL 2. In contrast, T cells and monocytes were unable to lyse HTLV-III/LAV targets, and only minimal activity was induced by activation. LGL cells, B cells, and monocytes were infectible in vitro by high titers of HTLV-III/LAV. However, levels of reverse transcriptase found in these cultures were significantly lower than the levels in T cell cultures. In contrast, only T cells were susceptible to infection by HTLV-I. Experiments with the use of cell cocultures showed that LGL afforded T cells protection from infection by HTLV-I (as indicated by lack of transformation and viral protein expression) but not from infection by HTLV-III/LAV. Collectively, these results indicate that NK cells may play a role in protecting cells against HTLV infection.

The cloned murine cytotoxic T cell line CT6 solely requires interleukin 2 (IL 2) for viability and cell cycle progression. Treatment of G arrested cultures of CT6 cells with recombinant IL 2 induces the rapid sequential expression of the nuclear proto-oncogenes c-fos, c-myc, and c-myb but does not affect the expression of several cytosolic or membrane-associated proto-oncogenes. A comparison of early genes induced by growth factor treatment of quiescent NIH/3T3 fibroblasts and CT6 cells demonstrated that only c-fos and c-myc induction is shared in the two different lineages. Factor-independent lines derived from CT6 cells show no mitogenic response to IL 2, yet binding of IL 2 with its receptor in the cells was capable of inducing the expression of c-fos and c-myc. In factor-independent cell lines, c-myc was uniformly expressed at high constitutive levels, suggesting that c-myc abrogates growth factor requirements of these cells. The levels of c-myc expression in the factor-independent lines was not due to an autocrine production of IL 2 but may be a consequence of constitutively activated IL 2 receptors.

Several investigations have suggested that products of arachidonic acid metabolism have modulatory effects on the development of cellular immunity. In this report we have studied the role of arachidonic acid metabolism in the specific effects of interleukin 1 (IL 1) induction of interleukin 2 (IL 2), and also IL 2 stimulation of proliferation and interferon-gamma (IFN-gamma) production. Utilizing cell lines that are specifically responsive to IL 1 or IL 2, it was found that both interleukins stimulate lipoxygenation of arachidonic acid in their respective target cell. The ability of each interleukin to induce monohydroxyeicosatetraenoic acid (HETE) correlated with the induction of secondary lymphokine secretion. Utilizing selective and partially selective pharmacologic inhibitors of arachidonic acid metabolism, the data suggest that the participation of lipoxygenase activity is required for both IL 1 induction of IL 2 production and IL 2 regulation of proliferation and IFN-gamma secretion. The same requirement for lipoxygenase activity was seen when phorbol myristate acetate (PMA) was used as a secretory stimulant, suggesting a similar mode of action for stimulation-secretory activity between PMA and interleukins. Studies performed with an endogenous inhibitor of 5-lipoxygenase (15-HETE) demonstrated the requirement of this enzyme system for IL 2-dependent proliferation and IFN-gamma production. Although leukotrienes could replace IL 2 for IFN-gamma secretion, they had no effect on IL 2 growth promotion. The results suggest that both IL 1 and IL 2, and PMA, may share the lipoxygenase pathway of arachidonic acid metabolism which is a component of the intracellular signal transduction process that regulates secretory activity and/or cellular proliferation.

The factor(s) derived from fibrosarcoma-induced suppressor T cells was sensitive to pronase and neuraminidase, but not to trypsin, beta-galactosidase, DNase, or RNase. Protein and RNA, but not DNA, synthesis were required to mediate suppression. Suppressor T cell-derived factor(s) could be precipitated by a 50% saturated ammonium sulfate (SAS) solution. The 50% SAS fraction inhibited both in vitro and in vivo spleen cell blastogenesis, whereas the 80% and unprecipitated fractions had no inhibitory activity. Using Sephadex G-200 chromatography, the 2nd protein fraction (fraction II) contained an inhibitor of both DNA polymerases (IDP) and DNA synthesis (IDS) activity, which possessed no cytotoxic activity. In vitro DNA polymerase alpha activity was suppressed by fraction II, whereas DNA polymerase beta and gamma activities remained unchanged. Molecular weight of IDP/IDS, as determined by Sephadex G-200 gel filtration chromatography, was approximately 14,500. Attempts to separate IDP/IDS activities found in fraction II by anion-exchange chromatography and slab gel electrophoresis were not successful, which suggested that the 2 activities were the same or very similar molecules.

The initiation of murine alloantigen-specific cytotoxic T cell responses is macrophage dependent. The requirement for macrophages can be replaced in macrophage-depleted cytotoxic T lymphocyte assay by a soluble factor released from phorbol myristic acetate-stimulated mouse peritoneal adherent cells or P388D1 macrophage tumor cells. On the basis of the cell of origin, m.w. (15,000), and elution profiles on DEAE cellulose and polyacrylamide gels, this macrophage-replacing factor for T cell-mediated cytotoxicity was identical to lymphocyte activating factor (LAF). The stimulatory effect of LAF was totally dependent on the presence of the priming alloantigen. The results presented in this report support the hypothesis that macrophage-mediated antigen-induced T cell activation is dependent on two signals, antigen and LAF.

Peripheral blood monocytes from AIDS patients exhibit defective migratory responses to chemotactic stimuli in vitro and to inflammatory sites in vivo. In studies presented here, normal monocytes were infected with the HIV-1/HTLV-IIIBa-L isolate in vitro and evaluated for chemotactic responsiveness. Within 2 days after viral exposure, but before evidence of virus production in the monocytes, chemotactic activity was significantly impaired. Decreased chemotactic activity was associated with modulation of receptors for the chemotactic ligands, C5a and FMLP, on the monocyte cell surface. In addition to HIV-1, monocytes treated with purified HIV-1 envelope glycoprotein gp120 demonstrated a comparable modulation of chemotactic ligand receptors and migratory function. In addition, the HIV-1 or HIV-1 gp120-treated monocytes were induced to undergo differentiation as monitored by HLA-DR expression. Immunoprecipitation of the gp120 with a specific antibody reversed its effects on monocyte chemotaxis and HLA-DR expression. Taken together, these data indicate that the initial interaction of HIV-1 with the monocyte is not passive, but that the binding of HIV-1 and/or HIV-1 gp120 to the CD4R on monocytes transduces a signal leading to transient monocyte activation.

Interleukin 2 (IL-2) and the synthetic diacylglycerol, 1-oleoyl-2-acetylglycerol (OAG), a direct activator of protein kinase C, induce phosphorylation of the ribosomal S6 protein in a murine IL-2-dependent lymphocyte clone. The phosphorylation of S6 protein was correlated with increased protein synthesis in this cell line. Using cell-free assay systems, two unique kinases capable of phosphorylating the S6 protein were identified, namely, a calcium/phospholipid-dependent phosphotransferase, protein kinase C, and a second phospholipid-independent kinase detected in crude cytosolic fractions. Peptide mapping of the S6 protein demonstrated that the degree of S6 phosphorylation stimulated by IL-2 and OAG was similar to that achieved using the second (calcium/phospholipid-independent) kinase but not to the level of phosphorylation achieved with protein kinase C. The kinase responsible for phosphorylating S6 was soluble in stimulated cells and was induced in a time-dependent manner by either IL-2 or diacylglycerol treatment of intact cells. These data support the notion that, although protein kinase C is activated by IL-2 or OAG, subsequent events such as S6 phosphorylation may be the result of the activation of secondary phosphotransferase systems regulated by protein kinase C.

Phorbol-12-myristate-13-acetate stimulates a subline of mouse EL-4 thymoma cells to produce, in vitro, in very high titer, T cell growth factor (Interleukin 2, IL 2). The EL-4-derived IL 2 has the same m.w. (30,000) and isoelectric point heterogeneity (pI 3.8-4.4) as the IL 2 produced by Con A-stimulated spleen cells. In addition, the thymoma-derived IL 2 exhibits the same spectrum of biologic activities as has been reported for spleen cell-derived IL 2.

T helper cells have recently been divided into two subsets. The Th1 subset secretes and responds to IL-2 in an autocrine manner. The Th2 subset upon mitogen or antigen stimulation releases IL-4. Here we describe a novel technology that allowed us to confirm this distinction. We have used synthetic oligonucleotides complementary to the 5' end of mouse IL-2 and IL-4 to specifically block the biosynthesis of IL-2 or IL-4 in two murine helper T cell clones from the Th1 or Th2 subset. We show that the antisense IL-2 oligonucleotide inhibited the proliferation of the Th1 clone and had no effect on the Th2 clone. In parallel experiments, the antisense IL-4 oligonucleotide blocked the proliferation of the Th2 clone and not the proliferation of the Th1 clone. The inhibition was significantly reversed in both cases by the addition of the relevant lymphokine (IL-2 in the case of the Th1 clone, IL-4 in the case of the Th2 clone). Northern analysis, using cDNA probes specific for the two lymphokines, showed a decrease in the steady-state level of the relevant lymphokine mRNA, suggesting the specific degradation of the mRNA by an RNase H-like enzymatic activity. This strategy, which allows the specific blockade of the biosynthesis of a lymphokine, could be useful for future studies on the role of each T helper subset in physiological immune responses.

A Lyt-2+ lymphocyte is responsible for immune interferon (IFN gamma) production in mitogen-stimulated mouse spleen cell cultures. A Lyt-1+ helper cell is required for the induction of IFN gamma. Interleukin 2 (IL 2) can specifically replace the Lyt 1 cell requirement. IL 2 will also abrogate suppressor cell inhibition of IFN gamma production. IFN gamma production appears to be regulated by a dynamic interaction between helper cells (source of IL 2), suppressor cells (absorption of IL 2?) and IFN gammna-producer cells.

C-myc protein expression in human T cells was specifically inhibited by a 15-mer deoxy-oligonucleotide complementary to the 5' end of the human c-myc gene second exon. The oligonucleotide penetrates the cells without any treatment, with a plateau of cell association reached in 2 h. The oligonucleotide specifically blocked the de novo synthesis of c-myc protein, induced by PHA in human resting peripheral T cells, without impairing the overall synthesis of other proteins, as shown by two-dimensional analysis of [35S]methionine pulse-labeled proteins. The specific inhibition of c-myc protein synthesis prevented the entry into S phase of resting T cells, induced to proliferate by PHA, or IL-2-dependent T cells induced by IL-2, as shown by [3H]thymidine incorporation. The inhibition of proliferation was specific since it was not observed with the corresponding sense-oligonucleotide and was reversed by preincubation of the cells with an excess of sense oligonucleotide. These results clearly support a role for c-myc protein in the proliferation process and show that inducible protein expression can be blocked by means of synthetic oligonucleotides complementary to a coding exon.