Plus Phorbol 12-myristate 13-acetate Research Articles

WNT5A Enhances LH-Mediated Expression of HAS2 in Granulosa Cells.

In adult ovary, WNT5A is involved in follicular responses to gonadotropins and necessary for ovarian follicle development. However, the mechanism by which gonadotropins regulate WNT5A and the role of WNT5A in modulating follicular responses to gonadotropins are unclear. In mice, we discovered that the expression of Wnt5a was increased in granulosa cells of mouse ovaries during ovulation, and regulated by gonadotropin-activated intra-ovarian cytokine interleukin 6 (IL6). Using human granulosa-like KGN cells, we confirmed that forskolin plus phorbol myristate acetate (PMA) which mimic the luteinizing hormone (LH) action induced the expression of WNT5A and cumulus expansion gene HAS2. However, this effect was suppressed by a NF-κB pathway inhibitor. Inhibition of NF-κB pathway also blocked the activation of WNT5A signaling components ROR2 and JNK. Moreover, exogenous WNT5A enhanced the expression of HAS2 in KGN cells through JNK and AKT signaling pathways. Knockdown of WNT5A expression by siRNA disrupted LH-mediated expression of HAS2. Our findings indicate that WNT5A could be a fine tuner for LH-induced HAS2 expression in ovarian granulosa cells.

Anti-inflammatory effects of novel barbituric acid derivatives in T lymphocytes

We have used a high throughput small molecule screen, using a fission yeast-based assay, to identify novel phosphodiesterase 7 (PDE7) inhibitors. One of the most effective hit compounds was BC12, a barbituric acid-based molecule that exhibits unusually potent immunosuppressive and immunomodulatory actions on T lymphocyte function, including inhibition of T cell proliferation and IL-2 cytokine production. BC12 treatment confers a >95% inhibition of IL-2 secretion in phytohaemagglutinin (PHA) plus phorbol-12-myristate-13-acetate (PMA) stimulated Jurkat T cells. The effect of BC12 on IL-2 secretion is not due to decreased cell viability; rather, BC12 blocks up-regulation of IL-2 transcription in activated T cells. BC12 also inhibits IL-2 secretion in human peripheral T lymphocytes stimulated in response to CD3/CD28 co-ligation or the combination of PMA and ionomycin, as well as the proliferation of primary murine T cells stimulated with PMA and ionomycin. A BC12 analog that lacks PDE7 inhibitory activity (BC12-4) displays similar biological activity, suggesting that BC12 does not act via PDE7 inhibition. To investigate the mechanism of inhibition of IL-2 production by BC12, we performed microarray analyses using unstimulated and stimulated Jurkat T cells in the presence or absence of BC12 or BC12-4. Our studies show these compounds affect the transcriptional response to stimulation and act via one or more shared targets to produce both anti-inflammatory and pro-stress effects. These results demonstrate potent immunomodulatory activity for BC12 and BC12-4 in T lymphocytes and suggest a potential clinical use as an immunotherapeutic to treat T lymphocyte-mediated diseases.

Stunned silence: gene expression programs in human cells infected with monkeypox or vaccinia virus.

Poxviruses use an arsenal of molecular weapons to evade detection and disarm host immune responses. We used DNA microarrays to investigate the gene expression responses to infection by monkeypox virus (MPV), an emerging human pathogen, and Vaccinia virus (VAC), a widely used model and vaccine organism, in primary human macrophages, primary human fibroblasts and HeLa cells. Even as the overwhelmingly infected cells approached their demise, with extensive cytopathic changes, their gene expression programs appeared almost oblivious to poxvirus infection. Although killed (gamma-irradiated) MPV potently induced a transcriptional program characteristic of the interferon response, no such response was observed during infection with either live MPV or VAC. Moreover, while the gene expression response of infected cells to stimulation with ionomycin plus phorbol 12-myristate 13-acetate (PMA), or poly (I-C) was largely unimpaired by infection with MPV, a cluster of pro-inflammatory genes were a notable exception. Poly(I-C) induction of genes involved in alerting the innate immune system to the infectious threat, including TNF-alpha, IL-1 alpha and beta, CCL5 and IL-6, were suppressed by infection with live MPV. Thus, MPV selectively inhibits expression of genes with critical roles in cell-signaling pathways that activate innate immune responses, as part of its strategy for stealthy infection.

Inhibitory effects of chitooligosaccharides on degranulation and cytokine generation in rat basophilic leukemia RBL-2H3 cells

Chitooligosaccharides (COS) of three different molecular weight ranges (1–3kDa, 3–5kDa and 5–10kDa) were investigated for their abilities against allergic reactions in RBL-2H3 cells. At the high concentration of 1000μg/ml, COS with molecular weights of 1–3kDa greatly attenuated the histamine and β-hexosaminidase release and intracellular Ca2+ elevation induced by calcium ionophore A23187, at the rates of 32% and 34% release and 37% elevation, respectively. Furthermore, the inhibitory activities of COS on calcium ionophore A23187 plus phorbol 12-myristate 13-acetate (PMA)-induced production as well as mRNA expression of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-4, and IL-6 were evidenced. Notably, the suppressive effects on the phosphorylation of extracellular signal-regulated kinases (MEK/ERK) and p38 kinase by COS were confirmed. These results indicate that COS could be used as an inhibitor in regulating mast cell-mediated allergic inflammatory responses.

Inhibitory effects of blueberry root methanolic extract on degranulation in KU812F cells

The effect of blueberry (Vaccinium angustifolium) root methanolic extract (BRM) on the A23187 plus phorbol myristate acetate (PMA)-induced degranulation in human basophilic KU812F cells was investigated. The total phenolic content (TPC) was 170±1.9 mg gallic acid equivalents (GAE)/g BRM. BRM reduced levels of histamine and β-hexosaminidase released from stimulated KU812F cells. Intracellular calcium concentration ([Ca2+]i) were determined by spectrofluorometric analysis using Fura 2-AM was found to be reduced by BRM with dose-dependent manner. Moreover, Western blot analysis revealed that protein kinase C (PKC) translocation was inhibited by BRM with dose-dependent manner. These results indicated that BRM inhibited histamine and β-hexosaminidase through the suppression of Ca2+ influx and PKC translocation. Therefore, we suggest that BRM is potent inhibitor of degranulation in mast cells and basophils, and may be useful in preventing the allergic reactions.

A novel mechanism for neutrophil priming in trauma: Potential role of peritoneal fluid

We sought to determine the effect of peritoneal fluid from a novel animal model of abdominal compartment syndrome (ACS) on the proinflammatory status of polymorphonuclear leukocytes (PMNs) and monocytes. We hypothesize that peritoneal fluid is a potential priming and/or activating agent for PMNs/monocytes. ACS was induced in female Yorkshire swine, and peritoneal fluid was collected at the time of decompressive laparotomy. Naïve PMNs/monocytes were primed and/or activated with peritoneal fluid, phosphatidylcholine (PAF) plus peritoneal fluid, peritoneal fluid plus n-formyl-met-leu-phe (fMLP), and peritoneal fluid plus phorbol 12-myristate 13-acetate (PMA). Activation was determined by surface marker expression of integrins (CD11b an CD18) and selectins (CD62L). Additionally, proinflammatory cytokines in peritoneal fluid were analyzed. Peritoneal fluid did not activate PMNs but increased CD11b expression on monocytes. When used as a primer for fMLP- or PMA-induced activation, peritoneal fluid significantly increased CD11b and CD18 expression on PMNs and monocytes. Peritoneal fluid collected at 6 and 12 h post decompressive laparotomy had similar effects. Interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) levels were increased in peritoneal fluid. Peritoneal fluid represents a primer for PMNs/monocytes and seems to act through receptor-dependent and receptor-independent pathways. Strategies to reduce the amount of peritoneal fluid may decrease the locoregional and systemic inflammatory response by reducing priming and activation of neutrophils/monocytes.

Inhibitory Effect of Acteoside Isolated fromCistanche tubulosaon Chemical Mediator Release and Inflammatory Cytokine Production by RBL-2H3 and KU812 Cells

The immediate-type allergic reaction is involved in many allergic diseases such as asthma, allergic rhinitis, and sinusitis. In this study, we investigated the effect of acteoside extracted from CISTANCHE TUBULOSA (Schrenk) R. Wight on the basophilic cell-mediated allergic reaction. The effect of acteoside on β-hexosaminidase release and intracellular [Ca (2+)] I level from rat basophilic leukemia (RBL-2H3) cells was determined. Also, ELISA was used to determine the level of histamine, tumor necrosis factor (TNF)- α, and interleukin (IL)-4 on human basophilic (KU812) cells. The effect of acteoside on basophilic cell viability was determined using the 3-[4,5-dimethylthiazolyl]-2,5-diphenyltetrazolium bromide (MTT) assay. These results indicated that 0.1-10.0 µg/mL acteoside inhibits the release of β-hexosaminidase and [Ca (2+)] I influx from IgE-mediated RBL-2H3 cells. Moreover, acteoside inhibited histamine release, TNF- α, and IL-4 production in a dose-dependent manner from calcium ionophore A23187 plus phorbol 12-myristate 13-acetate (PMA) or compound 48/80-stimulated KU812 cells. Our findings provide evidence that acteoside inhibits basophilic cell-derived immediate-type and delayed-type allergic reactions. This is the first report describing antiallergic activity of acteoside extracted from CISTANCHE TUBULOSA on basophilic cells.

Inhibitory effect of various Tunisian olive oils on chemical mediator release and cytokine production by basophilic cells

Tunisian olive oils have been traditionally used as a medicinal food for chronic inflammation. To investigate the antiallergic effect of virgin olive oil samples from five principal olive varieties grown in various regions of Tunisia, we used the type I allergy reaction model using rat basophilic leukemia (RBL-2H3) cells and different dilutions of olive oil samples to determine β-hexosaminidase release inhibition at two different response stages. Results showed that the Sayali olive oil significantly inhibited β-hexosaminidase release by the IgE antibody-sensitized, BSA antigen-stimulated RBL-2H3 cells at the antibody-antigen binding stage. The result of our experiment shows that the anti-allergic effect of olive oil at this binding stage may be dependent on their flavone content. The Zarrazi olive oil significantly inhibited β-hexosaminidase release at the antigen-receptor binding stage. Moreover, we investigated the effect of olive oil samples on histamine release and production of cytokines by activated human basophilic (KU812) cells. Different dilutions of Sayali olive oil dose-dependently inhibited the production of tumor necrosis factor-α (TNF-α) and interleukin-4 (IL-4), and different dilutions of Zarrazi olive oil dose-dependently inhibited histamine release and IL-4 production by calcium ionophore A23187 plus phorbol 12-myristate 13-acetate (PMA)-stimulated KU812 cells.

Defective integration of activating signals derived from the T cell receptor (TCR) and costimulatory molecules in both CD4+ and CD8+ T lymphocytes of common variable immunodeficiency (CVID) patients.

CVID is characterized by hypogammaglobulinaemia and impaired antibody production. Previous studies demonstrated defects at the T cell level. In the present study the response of purified CD4+ and CD8+ T lymphocytes to stimulation with anti-TCR monoclonal antibody (the first signal) in combination with anti-CD4 or anti-CD8, anti-CD2 and anti-CD28 MoAbs (the costimulatory signals) was investigated. Both CD4+ and CD8+ T cells from the patients showed significantly reduced IL-2 release following stimulation via TCR and costimulation via CD4 or CD8 and CD2, respectively. However, normal IL-2 production following TCR plus phorbol myristate acetate (PMA) costimulation and normal expression of an early activation marker, CD69, after TCR+CD28 stimulation indicated that TCR was able to transduce a signal. Furthermore, both IL-2 and IL-4 release were impaired in CD4+ lymphocytes following TCR+CD28 stimulation. In addition, stimulation via TCR+CD28 resulted in significantly decreased expression of CD40 ligand in the patients. These results suggest that the integration of activating signals derived from the TCR and costimulatory molecules is defective in CVID patients; the defect is not confined to costimulation via a single molecule, or restricted to cells producing Th1-type cytokines such as IL-2, and is expressed in both CD4+ and CD8+ T cell subsets.

Cryptotanshinone inhibits cyclooxygenase-2 enzyme activity but not its expression

Cyclooxygenase-2 (COX-2) is a key enzyme that catalyzes the biosynthesis of prostaglandins from arachidonic acid and plays a critical role in some pathologies including inflammation, neurodegenerative diseases and cancer. Cryptotanshinone is a major constituent of tanshinones, which are extracted from the medicinal herb Salvia miltiorrhiza Bunge, and has well-documented antioxidative and anti-inflammatory effects. This study confirmed the remarkable anti-inflammatory effect of cryptotanshinone in the carrageenan-induced rat paw edema model. Since the action of cryptotanshinone on COX-2 has not been previously described, in the present study, we further examined the effect of cryptotanshinone on cyclooxygenase activity in the exogenous arachidonic acid-stimulated insect sf-9 cells, which highly express human COX-2 or human COX-1, and on cyclooxygenases expression in human U937 promonocytes stimulated by lipopolysaccharide (LPS) plus phorbolmyristate acetate (PMA). Cryptotanshinone reduced prostaglandin E 2 synthesis and reactive oxygen species generation catalyzed by COX-2, without influencing COX-1 activity in cloned sf-9 cells. In PMA plus LPS-stimulated U937 cells, cryptotanshinone had negligible effects on the expression of COX-1 and COX-2, at either a mRNA or protein level. These results demonstrate that the anti-inflammatory effect of cryptotanshinone is directed against enzymatic activity of COX-2, not against the transcription or translation of the enzyme.

Immunosuppressive effect of 2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine (PhIP) through the inhibition of T-lymphocyte proliferation and IL-2 production

2-Amino-1-methyl-6-phenylimidazo[4,5- b]pyridine (PhIP) is the predominant heterocyclic amine formed in cooked meat and fish and causes cancers in the colon, the mammary glands, and the lymphoid organs. In the present study, we investigated the immunological impact of PhIP using thymocytes isolated from Balb/c mice and a murine thymocyte-derived cell line, EL4. Treatment of the thymocytes with PhIP moderately inhibited T-cell mitogen-induced cell proliferation and interleukin (IL)-2 secretion. Reverse transcription-polymerase chain reaction (RT-PCR) analysis demonstrated that PhIP attenuated IL-2 mRNA expression in the thymocytes and EL4 cells stimulated with phytohemagglutinin (PHA) plus phorbol 12-myristate 13-acetate (PMA). In vitro transient transfection assay using a reporter gene construct containing IL-2 promoter showed that the decrease in the steady-state IL-2 mRNA level by PhIP is partially due to the attenuation of IL-2 mRNA synthesis at the transcriptional level. Furthermore, an electrophoretic mobility shift assay showed that PhIP inhibited DNA binding activity of nuclear factor for immunoglobulin κ chain in B cells (NF-κB), activator protein-1 (AP-1) and nuclear factor of activated T cells (NF-AT), which are known to be responsible for IL-2 transcriptional activation. Concomitantly, PhIP inhibited the PMA/PHA-induced generation of reactive oxygen species (ROS) involved in activation of the transcription factors. These results suggest that PhIP has potential immunosuppressive effects by inhibiting T-cell proliferation and IL-2 expression through down regulation of ROS generation and thereby inhibiting NF-κB, AP-1 and NF-AT activation.

Role of cytosolic phospholipase A(2) in prostaglandin E(2) production by lung fibroblasts.

Prostaglandin (PG)E2 acts in an autocrine fashion to suppress proliferation of lung fibroblasts and production of collagen, and may negatively regulate pulmonary fibrosis. The role of Group IVA cytosolic phospholipase A2 alpha (cPLA2 alpha) in PGE2 production was investigated by comparing lung fibroblasts from wild-type and cPLA2 alpha-deficient mice. Arachidonic acid release from wild-type mouse lung fibroblasts (MLF+/+) was stimulated by serum, A23187 plus phorbol-myristate acetate (PMA), and lysophosphatidic acid (LPA) plus platelet-derived growth factor, but was > or = 80% lower from cPLA2 alpha-deficient MLF (MLF-/-). Transforming growth factor-beta increased cyclooxygenase-2 (COX2) expression to similar levels in MLF+/+ and MLF-/-, but MLF+/+ produced an order of magnitude more PGE2 than MLF-/- in response to A23187/PMA or platelet-derived growth factor/LPA. MLF+/+ synthesized less collagen than MLF-/-, supporting a role for PGE2 in suppressing collagen production. An SV40 immortalized line developed from MLF+/+ released arachidonic acid and expressed COX2 to levels similar to those of primary fibroblasts but produced 30-fold lower amounts of PGE2. Unlike primary fibroblasts, immortalized cells were deficient in microsomal PGE synthase (mPGES) but expressed slightly higher levels of cytosolic PGES. The results demonstrate a primary role for cPLA2 alpha in providing arachidonic acid for PGE2 production in mouse lung fibroblasts and support a role for this pathway in regulating collagen production.

Oleic acid‐induced PKC isozyme translocation in RAW 264.7 macrophages

Fatty acids are important second messengers that mediate various cellular functions, but their role in the formation of macrophage foam cells is not known. High plasma levels of oleic acid (OA) in obese patients are often associated with a high risk for atherosclerosis. In this study, we investigated the protein kinase C (PKC) isozymes involved in OA-induced lipid accumulation in RAW 264.7 macrophages. The results show that OA induces translocation of PKC alpha, beta1, and delta from the cytosol to the cell membrane 5 min after the treatment. After 16 h incubation with OA, PKC delta was found to be colocalized with adipose differentiation-related protein (ADRP) on the surface of lipid droplets, but immunoprecipitation experiments showed that PKC delta was not biochemically associated with ADRP. After 16 h incubation with OA plus phorbol 12-myristate 13-acetate (PMA), PKC delta staining on the lipid droplet surface was not seen, whereas the accumulation of lipid droplets was unaffected. Furthermore, downregulation of PKC delta was confirmed by immunoblotting. These results demonstrate possible involvement of specific PKC isozymes in the early phase of lipid accumulation, possibly during the uptake of OA.

Inhibition of HIV-1 replication in chronically infected cell lines and peripheral blood mononuclear cells by retrovirus-mediated antitat gene transfer

Among potential genetic targets for intervention in the HIV-1 life cycle, the tat gene product is a key target. We investigated the ability of an antitat gene to inhibit HIV-1 activation and replication in chronically infected promonocyte (U1) and T cell (ACH-2) lines in vitro. U1 and ACH-2 cells were transduced with an antitat gene expressing RNA with dual (polymeric Tat activation response element and antisense-tat) function that interferes with HIV-1 replication. Tumor necrosis factor-alpha (TNF-alpha) plus phorbol 12- myristate 13-acetate (PMA)-induced HIV-1 expression, as determined by reverse transcribed PCR and reverse transcriptase (RT) assays, was significantly inhibited in U1 and ACH-2 cells transduced with the antitat gene, compared with the cells transduced with control vector and untransduced cells. This resistance to TNF-alpha plus PMA-induced HIV-1 expression was demonstrated in antitat gene-transduced U1 and ACH-2 cells maintained in G418-free media for 5 months, suggesting that functional antitat gene may persist for many months in transduced cells and their progeny. Most importantly, we demonstrate that the antitat gene, when introduced into peripheral blood mononuclear cells (PBMC) isolated from patients with HIV-1 infection, inhibited TNF-alpha plus PMA-induced viral replication as determined by RT-PCR and RT activity. In addition, the antitat gene enhanced the survival of CD4+ T lymphocytes from such patients. These data suggest the feasibility of utilizing antitat gene therapy to block activation and replication of HIV-1 in latently infected monocytes and T- lymphocytes in vivo. Gene Therapy (2000) 7, 321-328.

Bryostatin-1 and 1alpha,25-dihydroxyvitamin D3 synergistically stimulate the differentiation of NB4 acute promyelocytic leukemia cells.

One of the objectives of treatment for patients with acute promyelocytic leukemia (APL) is to induce tumor cell differentiation and block cell proliferation. Acute promyelocytic leukemia cells (NB4) responded to the combination treatment of 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] plus phorbol 12-myristate 13-acetate (PMA) and differentiated into monocyte/macrophage-like cells, as well as expressed strong alkaline phosphatase (ALP) activities. Since PMA has limited clinical application due to its tumor-promoting effect, another protein kinase C activator, bryostatin-1, was currently tested for its interaction with 1alpha,25(OH)2D3 to induce NB4 cell differentiation and block cell proliferation. Bryostatin-1 alone, but not 1alpha,25(OH)2D3 alone, significantly inhibited cell proliferation and induced NB4 cell differentiation into monocyte/macrophages; however neither bryostatin-1 nor 1alpha,25(OH)2D3 alone induced ALP expression. Like PMA, bryostatin-1 synergistically interacted with 1alpha,25(OH)2D3 to stimulate ALP expression 30-fold over the control (P < 0.001) and further promote appearance of monocyte/macrophage-like cells. The ALP stimulation was both time- and dose-dependent. Thus, we demonstrate for the first time that the combination of bryostatin-1 and 1alpha,25(OH)2D3 strongly affect NB4 cell differentiation and proliferation. Therefore, this proposed combination treatment may be an alternatively potential therapeutic regimen for APL patients and assay of ALP may be a more sensitive and facile way to monitor the possible remission of APL patients.

Cytokine Profiles in Infants with Congenital Cytomegalovirus (CMV) Infection† 814

CD4+ T lymphocytes comprise functionally heterogeneous populations with specific profiles of cytokine production. Th1 cells produce IL-2, IFN-γ and TNF-β, whereas Th2 cells produce IL-4, IL-5, and IL-10. To characterize the cytokine responses in intrauterine CMV infection, we investigated the cytokine production by PBL in response to polyclonal and antigenic responses. Production of IFN-γ, IL-2, IL-4 and IL-10 was measured in cultures of PBL from 15 children with congenital CMV infection (7 with symptoamtic and 8 with asymptoamtic infection) and 8 healthy neonates(controls) within the first 2 months of life using cytoscreen ELISA kits(Biosource International, Camarillo, CA). The results cytokine levels(mean±1 SD) are shown in the table below. Unstimulated cultures and those stimulated with the major envelope glycoprotein B (gB) of CMV produced similar levels of cytokines in both groups. Upon stimulation with phytohemagglutinin (PHA) plus phorbol-12-myristate-13-acetate (PMA), infants with congenital CMV infection produced less IFN-γ than control children (p<.01). Although PBL from infants with symptomatic congenital CMV infection produced lower amounts of IFN-γ than those with asymptomatic infection, the difference was not statistically significant (p=.08). Furthermore, the group of infants with symptomatic infection produced increased amounts of IL-10 in cultures than asymptomatic infants and the control group and the difference approached statistical significance (p=.055). The levels of IL-2 and IL-4 were not different between the congenitally infected infants and the control subjects. The results suggest that infants with congenital CMV infection exhibit decreased production of mitogen stimulated Th1 type cytokines and increased TH2 cytokines (IL-10). Although the significance of these findings is not entirely clear, the differences in cytokine response may explain the decreased or absent cell mediated immune responses to CMV antigens observed in most infants with congenital CMV infection. The patterns of cytokine expression is being investigated in the study children.

Use of the methylxanthine derivative A802715 in transplantation immunology: I. Strong in vitro inhibitory effects on CD28-costimulated T cell activities.

Recently, methylxanthines such as pentoxifylline (PTX) were shown to be immunosuppressive in vitro. Unfortunately, when used in transplant patients, PTX was poorly active as an immunosuppressant. Here we report that the new methylxanthine derivative A802715 not only is more active than PTX, it also suppresses the cyclosporine (CsA)-resistant "signal two"-dependent pathway of T cell proliferation, making it an interesting drug to associate with CsA. "Signal one"- and "signal two"-dependent T cell activation was investigated with purified human T cells stimulated with immobilized anti-CD3 or anti-CD28 monoclonal antibody (mAb) plus phorbol myristate acetate (PMA) or with a 3T6 mouse fibroblast cell line presenting anti-CD3 mAb on transfected human Fcgamma receptors II (FcgammaRII) in the presence or absence of transfected B7-1 (CD80) molecules. A802715 was more immunosuppressive in the mixed lymphocyte reaction (MLR) than PTX. A802715 dose-dependently suppressed polyclonal signal one-dependent T cell activation induced by anti-CD3 mAb/PMA. In addition, A802715 also suppressed signal two-dependent T cell proliferation induced by anti-CD28 mAb/PMA. The expression of the interleukin-2 receptor on T cells stimulated by anti-CD3 mAb presented on 3T6/FcgammaRII cells was equally well suppressed by A802715 and PTX. In contrast, interleukin-2 receptor or CD40L (gp39) expression by T cells after stimulation with the same anti-CD3 mAb- 3T6/FcgammaRII cells, but coexpressing transfected B7-1, was only suppressed by A802715. The anticipated synergism between A802715 and CsA was confirmed in MLR assays. Moreover, generation of cytotoxic T lymphocytes during MLR with Epstein-Barr virus-transformed B cells, which strongly express B7-1 and B7-2, was also inhibited by A802715. These in vitro data indicate that the A802715 (1) is a stronger immunosuppressant for T cells than PTX, (2) suppresses T cell activation pathways that are resistant to PTX or CsA, and (3) acts synergistically with CsA.

Inhibition of interleukin-2 receptor (CD25) expression induced on T cells from children with acute lymphoblastic leukemia.

Peripheral blood lymphocytes obtained from children with acute lymphoblastic leukemia (ALL) at onset were studied for the expression of interleukin-2 (IL-2) receptor alpha-chain (CD25) by two-color flow-cytometric analysis. Stimulated with anti-CD3 monoclonal antibody (mAb) alone. CD25 expression was significantly suppressed in CD4+ T cells from 27 of 48 (56.3%) cases and in CD8+ T cells from 29 of 48 (60.4%) cases. When stimulated with anti-CD3 mAb plus phorbol 12-myristate 13-acetate (PMA), CD25 expression was clearly restored in certain cases of ALL. When PMA plus ionomycin were used for stimulation of T cells. CD25 was inducible in a majority of cases. Interestingly CD25 expression upon anti-CD3 mAb stimulation was recovered after complete remission had been achieved. These observations suggest the presence in ALL children at onset of an in vitro defect in the signal transduction pathway of the T-cell-receptor/CD3 complex, resulting in inefficient CD25 expression. However, immune-staining analysis indicated that protein kinase C was normally translocated from the cytosol fraction to the cell membrane fraction. The mobilization of cytoplasmic free calcium is also normal.

Type 1 and type 2 cytokine profiles in children exposed to or infected with vertically transmitted human immunodeficiency virus.

In human immunodeficiency virus (HIV)-infected adults, cytokine production profiles switch from predominantly type 1 (interleukin-2 [IL-2] and gamma interferon [IFN-gamma]) to type 2 (IL-4 and IL-10) cytokines with disease progression. To test this hypothesis in vertically HIV-infected children, we measured cytokine transcription and production in rapid progressors (RPs), seroreverters (SRs), and those children exposed to HIV in utero (P0s). Production of type 1 and type 2 cytokines was measured in peripheral blood mononuclear cell cultures of 8 SR, 25 P0, and 11 RP children. Unstimulated cultures, irrespective of infection and stage of disease, produced similar levels of IL-2, IFN-gamma, IL-4, and IL-10. Upon stimulation with phytohemagglutinin (PHA) plus phorbol-12-myristate-13-acetate (PMA), RP children produced less IL-2 (P < 0.01) and IFN-gamma (P < 0.02) than SR children and also expressed significantly less IFN-gamma mRNA (P < 0.01) than SR children. RP children expressed significantly higher levels of IL-4 mRNA than P0 children (P < 0.03). There were no differences in the production of IL-10 by PHA-PMA-stimulated peripheral blood mononuclear cell cultures among the three groups of children. Our data with these pediatric patients suggest that a deficiency in mitogen-stimulated type 1 cytokine production and excess type 2 cytokine (IL-4) transcription correlate with disease progression. Additional studies with larger sample sizes are needed to test further the hypothesis of the type 1-to-type 2 cytokine switch in children infected with HIV.

Persistent Activation of c-Jun N-terminal Kinase 1 (JNK1) in γ Radiation-induced Apoptosis

The c-Jun N-terminal kinases (JNK) are activated by various stimuli, including UV light, interleukin-1, tumor necrosis factor-alpha (TNF-alpha), and CD28 costimulation. Induction of JNK by TNF-alpha, a strong apoptosis inducer, implies a possible role of JNK in the regulation of programmed cell death. Present studies show that lethal doses of gamma radiation (GR) induced JNK activities at the early phase of apoptosis in Jurkat T-cells. We demonstrate that JNK1 was activated by either the T-cell activation signals, anti-CD28 monoclonal antibody plus phorbol 12-myristate 13-acetate (PMA), or the apoptosis-inducing treatment, GR; however, the induction patterns were different. In contrast to the rapid and transient JNK1 activation caused by CD28 signaling plus PMA, GR induced a delayed and persistent JNK1 activation. This implies a distinct regulatory mechanism and specific function of JNK1 in irradiated cells. The nuclear and cytosolic JNK1 activities were simultaneously increased in the irradiated cells without an evident change in the protein levels. The abilities of GR to induce JNK1 activation and DNA fragmentation were correlated. Peripheral blood lymphocytes were more sensitive to GR than Jurkat cells in JNK1 induction. The responsiveness of JNK1 to GR suggests the involvement of JNK1 in the initiation of the apoptosis process.