PHA-induced Interleukin-2 Production Research Articles

Fatty Acid Ethyl Ester Effects on Interleukin‐2 Production, Cyclic AMP Synthesis, and Calcium Influx in Human Mononuclear Cells

Fatty acid ethyl esters (FAEE), nonoxidative ethanol metabolites, are produced by the esterification of fatty acids and ethanol. Plasma and serum FAEE correlate linearly with blood ethanol levels and are present in organs most commonly damaged by ethanol abuse. Our previous studies have shown that there is significant synthesis of FAEE by human mononuclear cells within seconds of exposure to physiologic doses of ethanol. We studied the effects of FAEE on selected early events of mononuclear cell activation that follow stimulation with phytohemagglutinin (PHA). Fatty acid ethyl esters induced changes in cytosolic free calcium ([Ca2+]) levels, the production and secretion of interleukin-2 (IL-2) by the cells, and intracellular cAMP concentrations. A mononuclear fraction of human white blood cells (WBC) was incubated with physiologic doses of FAEE. For experiments involving IL-2 production and calcium influx, PHA-stimulated cells were incubated with 10, 25, 50, or 100 microM ethyl oleate, a representative FAEE species, for 60 minutes. Interleukin-2 was measured by enzyme immunometeric assay and maximum levels of Ca2+ were monitored by spectrofluorimetry. In other experiments, mononuclear cells were incubated with 10, 25, and 50 microM ethyl oleate for 0.08 to 120 minutes, and then the concentration of cAMP was determined by a cAMP competitive enzyme immunoassay system. Fatty acid ethyl esters inhibited the PHA-induced IL-2 production and secretion in activated human mononuclear cells. Fatty acid ethyl esters also inhibited PHA-induced [Ca2+] influx into cells in a dose-dependent fashion. There was a rapid increase in the intracellular cAMP concentration of mononuclear cells induced by FAEE, with FAEE dose dependence. The cAMP concentration decreased as the incubation time with FAEE was increased. Fatty acid ethyl esters inhibited PHA-stimulated IL-2 production and Ca2+ influx into human mononuclear cells and elevated intracellular cAMP concentration. These changes in mononuclear cell signaling may be associated with the immunosuppression associated with alcohol abuse.

Ligation of RARγ inhibits proliferation of phytohaemagglutinin-stimulated T-cells via down-regulating JAK3 protein levels

The mechanisms whereby Vitamin A regulates the immune system are poorly understood. We have shown previously that retinoic acids, the Vitamin A derivatives, promote both apoptosis of neglected thymocytes and the activation-induced cell death of peripheral T-cells via ligating the nuclear retinoid receptor (RAR) gamma. In the present study, we found that human peripheral T-cells express RARα and γ, but not RARβ. Increasing concentrations of 9- cis RA inhibited phytohaemagglutinin (PHA)-induced proliferation of T-cells, an effect that could be mimicked only by addition of RARγ agonists and could be inhibited by an RARγ antagonist. Interleukin-2 (IL-2) produced is known to mediate PHA-induced proliferation of T lymphocytes. Ligation of RARγ did not affect the PHA-induced high affinity IL-2 receptor expression, slightly reduced the PHA-induced IL-2 production, but interfered with the IL-2-mediated signal transduction resulting in inhibition of PHA-induced phosphorylation of retinoblastoma protein and of up-regulation of Bcl-2. Janus kinases JAK1 and JAK3 play a determinant role in IL-2-dependent signal transduction. Ligation of RARγ did not affect the levels of JAK1, but prevented IL-2-induced expression of JAK3 resulting in inhibition of PHA-induced phosphorylation of Stat5 molecules. Our data suggest that the previously observed toxic effect of high concentrations of retinoids on the immune system might be mediated via formation of 9- cis RA, which via ligation of RARγ not only induces cell death in immature thymocytes, but inhibits proliferation of T-cells as well.

Stimulatory roles of muscarinic acetylcholine receptors on T cell antigen receptor/CD3 complex-mediated interleukin-2 production in human peripheral blood lymphocytes.

It is known that there are some bidirectional interactions between the nervous and the immune systems via neurotransmitters and cytokines. To clarify whether any neurotransmitters modulate lymphocyte functions, we examined the effects of oxotremorine-M (Oxo-M) on interleukin-2 (IL-2) production in human peripheral blood lymphocytes by using enzyme-linked immunosorbent assays, Northern blot analyses, reverse transcriptase-polymerase chain reaction, and fluorescence-activated cell sorter. Pretreatment of cells with Oxo-M (10 nM to 10 microM) for 4-24 hr enhanced phytohemagglutinin (PHA)-induced IL-2 mRNA expression and markedly increased IL-2 production compared with those induced by PHA alone. Oxo-M alone did not affect IL-2 mRNA expression and IL-2 production. In CD3-positive T cells, pretreatment with Oxo-M for 24 hr enhanced PHA-induced IL-2 production. Furthermore, pretreatment with Oxo-M enhanced PHA-induced mRNA expression of the alpha and beta subunits of IL-2 receptors and DNA synthesis. Cytometric analysis showed Oxo-M treatment did not up-regulate expression of cell surface molecules such as CD3, CD2, CD4, CD8, and IL-2 receptors. These results suggest that activation of muscarinic receptors enhances T cell antigen receptor/CD3-induced IL-2 production.

Natural Human Interferon-α Augments Apoptosis in Activated T Cell Line

We have previously shown that interferon-α (IFN-α) augments interleukin-2 (IL-2) production in mitogen-activated but not unstimulated T cells (1). Here, we studied the effect of IFN-α on activation-driven cell death (apoptosis) using a human leukemia T cell line, MOLT-16, as a T cell activation model. IFN-α alone had no effect on either the IL-2 production or apoptosis of MOLT-16 cells, but significantly increased both the IL-2 production and apoptosis in the MOLT-16 cells after phytohemagglutinin (PHA) stimulation as determined by CTLL-2 assay and by flow cytometric analysis using propidium iodide (PI) staining. Since IL-2 has been shown to induce apoptosis in some systems, we next evaluated whether the apoptosis in MOLT-16 cells was due to endogenous IL-2 production upon PHA stimulation. However, the addition of exogenous IL-2 to unstimulated cultures of MOLT-16 did not induce DNA fragmentation, a characteristic feature of apoptosis, as determined by DNA electrophoresis and by flow cytometric analysis with PI-stained cells. Furthermore, anti-IL-2 antibody did not prevent PHA-induced DNA fragmentation in MOLT-16 cells. Thus, we conclude that both PHA-induced IL-2 production and apoptosis are outcomes of T cell activation and that IFN-α may exert immunoregulatory effects on T cell activation by augmenting both processes.

Effect of betamethasone on maternal, fetal and neonatal rat cellular immunity

Objective: We evaluated the effect of maternal administration of betamethasone (0.2 mg/kg per day) on mitogen-induced lymphocyte proliferation and interleukin-2 (IL-2) production by maternal, fetal, and neonatal rat splenic lymphocytes. Study Design: Betamethasone was injected intramuscularly on days 19 and 20 of gestation to timed-pregnant rats (Sprague-Dawley). Fetuses were delivered on day 21 of gestation, or allowed to deliver spontaneously at term (22 days), followed by sacrifice at various intervals after birth. Lymphocyte proliferation was determined by 3H-thymidine incorporation with and without phytohemagglutinin (PHA), and IL-2 by proliferation of IL-2 dependent CTLL-2 cells. Results: Maternal lymphocytes had higher spontaneous proliferation than lymphocytes from nonpregnant female rats. Betamethasone use resulted in a decrease in PHA-induced lymphocyte proliferation and IL-2 production by maternal lymphocytes. These effects were observed until 4 days after delivery. Significant decreases in these parameters were also seen in 21-day fetuses of betamethasone-treated mothers. These effects were still present 6 days after birth but not at 12 days of age. Conclusion: These findings suggest that, in the rat, exposure to betamethasone during late pregnancy results in marked, but transient decreases in PHA-induced lymphocyte proliferation and IL-2 production in both the mothers and their offspring.

Interrelationship between signals transduced by phytohemagglutinin and interleukin 1

In the murine cell line LBRM-331A5, phytohemagglutinin (PHA) induces secretion of the T cell growth factor interleukin 2 (IL2). IL1 augments PHA-induced IL2 production. In this cell line, PHA stimulates a number of biochemical changes including phospholipid hydrolysis, increases in cytosolic free calcium [( Ca2+]i), membrane hyperpolarization, cytosolic alkalinization, and tyrosine phosphorylation of specific substrates. Using LBRM cells, we have studied the interrelationship between these events and the secretion of IL2. Increases in [Ca2+]i triggered by PHA or following addition of ionomycin result in membrane hyperpolarization but are not required for PHA-induced cytosolic alkalinization or tyrosine phosphorylation. Addition of IL1 to PHA-stimulated cells did not affect any of the biochemical parameters, although it significantly augmented PHA-induced IL2 secretion. Increasing [Ca2+]i with ionomycin did not trigger IL2 secretion, increases in cytosolic pH, or tyrosine phosphorylation in the presence or absence of IL1. Preventing increases in cytosolic pH did not alter PHA-induced changes in [Ca2+]i or membrane potential. These data are compatible with PHA including activation of phospholipase C and production of inositol phosphates resulting in both release of Ca2+ from internal stores and transmembrane uptake of Ca2+ as well as activation of protein kinase C. However, unlike other growth factor or mitogen-stimulated systems, the changes stimulated by PHA and IL1 in LBRM cells including IL2 secretion are not regulated by a pertussis toxin-sensitive G protein.

Mitogen-stimulated lymphocyte proliferation and pituitary hormones in major depression

To assess cellular immune status and the hypothalamic-pituitary (HP) axis in patients with major depression, we examined peripheral blood mononuclear cells (PBMC) and measured the plasma levels of cortisol, adrenocorticotropin hormone (ACTH), growth hormone (GH), andprolactin (PRL). Twenty patients with major depression were compared with 20 control subjects matched for age, sex, and race. The dose-response curves for concunavalin-A (Con-A) and phytohemagglutinin (PHA) stimulation were not significantly different between the two groups. The patients had decreased Con-A—stimulated T-lymphocyte proliferation when compared to the control subjects, but only at the lowest suboptimal concentration of Con-A. None of the four concentrations of PHA-stimulated proliferation were different between the two groups, neither was PHA-induced interleukin-2 production. Within the patient group only, plasma prolactin (PRL) correlated significantly with stimulated lymphocyte proliferation using two optimal concentrations of PHA and one optimal concentration of Con-A, when the proliferation was expressed using the stimulation index.

Theophylline and the immune response: In vitro and in vivo effects

We analyzed the in vitro and in vivo effects of theophylline on various immunological parameters including proliferation of peripheral mononuclear cells (PMNC) in response to phytohemagglutinin (PHA), anti-T3 and anti-T11 monoclonal antibodies (MAb), PHA-induced interleukin-2 (IL-2) and interferon-γ (IFN-γ) production by PMNC, interleukin-1 (IL-1) production by accessory cells, PHA-induced IL-2 production by T-cell clones, and PHA- and anti-T3 MAb-induced DNA synthesis by T cell clones. Results showed that theophylline inhibited PHA- and anti-T3-induced proliferation of both PMNC and T-cell clones, whereas the PMNC proliferation induced by MAb anti-T11 was not affected. The inhibition appeared to be dose-dependent and strictly related to the presence of the drug in the culture. Moreover, PHA-induced IL-2 production by both PMNC and T-cell clones also appeared to be reduced by theophylline. IL-1 production by accessory cells was not affected. These data suggest that the immunological inhibition exerted by theophylline is confined to the T-cell compartment, mainly by acting on structure(s) related to the T3 Ti complex, the primary site for T-cell activation. The alternative pathway of T-cell activation (i.e., via T11 site) seems unaffected. In addition, these results suggest possible clinical relations between the inhibition of the immune response and the plasma levels of the drug reached after a “once daily” or “twice daily” oral ingestion of slow-release theophylline products.

Deficient phytohemagglutinin-induced interleukin-2 activity in patients with inactive systemic lupus erythematosus is correctable by the addition of phorbol myristate acetate.

In systemic lupus erythematosus (SLE) patients, the production of interleukin-2 (IL-2) by blood T lymphocytes in response to stimulation with phytohemagglutinin (PHA) either alone or with phorbol myristate acetate (PMA) or ionomycin, a Ca2+ ionophore, was examined. Deficiency in PHA-stimulated IL-2 production by cells from SLE patients was repaired by the addition of PMA, but not ionomycin. PMA alone did not stimulate IL-2 production but, in concert with PHA, induced IL-2 synthesis. Moreover, PMA was effective in the repair of the deficiency of PHA-induced IL-2 production by both T4+ and T8+ subsets. Thus, for effective IL-2 production, SLE T cells required signals either distinct from or in addition to those supplied by PHA.

Interleukin-1 and interleukin-2 production by peripheral blood mononuclear cells in multiple sclerosis patients

The production of interleukin-1 (IL-1) and interleukin-2 (IL-2) by peripheral blood mononuclear cells (PBM) was assessed in multiple sclerosis (MS) patients in relapse, chronic progressive MS patients, patients with other neurological diseases (OND) and healthy subjects. Production was defined as the level of IL-1 and IL-2 in PBM supernatants. Neither spontaneous nor LPS-induced IL-1 production differed significantly in MS, OND patients or healthy individuals. On the other hand PHA-induced PBM IL-2 production was significantly less in MS patients in relapse (130 ± 10.0 U/ml) than in chronic progressive MS patients (172 ± 9.8 U/ml), OND patients (192 ± 11.5 U/ml) and healthy subjects (215 ± 13.8 U/ml) ( P < 0.02). Spontaneous IL-2 production was also diminished in MS patients in relapse (31 ± 7.2 U/ml) as compared to chronic progressive MS patients (46 ± 8.8 U/ml) and healthy subjects (49 ± 11.1 U/ml) ( P < 0.01). Anti-Tac monoclonal antibody was used to study IL-2 receptor expression on the same sample of PBM that was used for IL-2 study. MS patients in relapse had significantly higher levels of IL-2 receptor-positive unstimulated PBM (6.0 ± 2.2%) as compared to chronic progressive MS (2.0 ± 0.9%), OND (2.5 ± 1.1%) and healthy subjects (1.5 ± 0.7%) ( P < 0.002). We postulate that reduced apparent IL-2 production by PBM of MS patients in relapse may result from immediate IL-2 binding to receptor expressed on activated T lymphocytes and internalization of IL-2-receptor complex.

In vitro effects of thymosin and lithium on lymphoproliferative responses of normal donors and HIV seropositive male homosexuals with AIDS-related complex

The in vitro effects of thymosin fraction 5 (TF5) and lithium chloride (LiCl) on the ability of peripheral blood mononuclear cells (PBMC) obtained from 37 normal male donors and 33 male patients with AIDS-related complex (ARC) to respond to alloantigenic stimulation (mixed leukocyte reaction, MLR) and to produce interleukin 2 (IL-2) in response to mitogens were studied. TF5 significantly increased MLR responses in normal donors ( P < 0.01) and in a group of 33 ARC patients with depressed cellular immunity ( P < 0.05). Similar effects were observed when LiCl was added to the MLR assays in both the normal and the ARC patient groups. Furthermore, TF5 and LiCl exhibited additive immunoenhancing properties. In 10 normal donors TF5 enhanced phytohemaggutinin (PHA)-induced IL-2 production as well as IL-2 production in response to pokeweed mitogen (PWM) ( P < 0.02). TF5-mediated enhancement of IL-2 production by PBMC obtained from ARC patients was observed in response to both mitogens, i.e., PHA and PWM. Additionally, LiCl increased PHA-induced IL-2 production in both normal subjects and ARC patients. LiCl and TF5 together had an additive effect in the enhancement of IL-2 production in both groups of subjects. Our data extend previous observations regarding the immunoregulatory activities of TF5 and LiCl and provide evidence that PBMC obtained from ARC patients have the potential to respond in vitro to these agents. The significance of these findings is discussed.

Inhibition of bovine lymphocyte response to phytohemagglutinin by a specific 5-lipoxygenase inhibitor

AA861, a specific 5-lipoxygenase inhibitor, inhibited bovine lymphocyte response to phytohemagglutinin (PHA). Mitogen-stimulated cultures of mononuclear cells produced leukotriene B 4 (LTB 4) in 24 hours. The production of LTB 4 was completely inhibited by concentrations of AA861 that inhibited mitogen-induced 3H-thymidine incorporation. The inhibition of lymphocyte proliferation was totally reversed by LTB 4 but not by leukotriene C 4 or leukotriene D 4. The inhibition of interleukin-2 (IL-2) production by AA861 was also completely reversed by addition of exogenous LTB 4 to lymphocyte cultures. Thus, endogenous LTB 4 production appeared to be necessary for PHA-induced IL-2 production and lymphocyte proliferation.

Bacampicillin and the immune response.

To evaluate the possible influence of a bacampicillin on the immune response, 16 subjects, out of a group of 60 patients with bacterial respiratory tract infections, had various tests of immune function determined, before and after treatment. The peripheral mononuclear blastogenic index (ratio between PHA-induced and spontaneous proliferation), PHA-induced interferon-gamma production, percentages of T and B lymphocytes, serum immunoglobulins (IgG, IgM, IgA) levels, failed to show any significative differences before and after the treatment with bacampicillin. The PHA-induced interleukin-2 production increased after treatment but just failed to reach statistical significance (0.1 less than P less than 0.05; t = 1.9). The clinical condition of 56 of the sixty (93.3%) treated patients improved and neither side-effects nor alterations of liver or kidney function were observed. This study has shown no inhibitory effect of bacampicillin on the immune response while confirming its clinical efficacy.

Effects of isoprinosine and NPT 15392 on interleukin-2 (IL-2) production

Two purine immunoenhancing drugs, isoprinosine and NPT 15392, were evaluated as inducers of IL-2 or enhancers of IL-2 induction by phytohemagglutinin (PHA). Both drugs were found to have significant enhancing activity on PHA induction of IL-2 by human lymphocytes over a wide range of concentrations. Alone they were unable to consistently induce IL-2 activity. NPT 15392 was able to enhance PHA-induced IL-2 production at 10–100 times lower concentrations than isoprinosine. The actions of these compounds to enhance IL-2 production may explain their effects on PHA-induced lymphocyte proliferation.

Impaired IL2 production by lymphocytes of patients with systemic lupus erythematosus

The aim of this study was to define as possible abnormality of interleukin 2 (IL2) production by PHA-stimulated peripheral blood lymphocytes (PBL) in systemic lupus erythematosus (SLE). Thirty-four SLE PBL samples were stimulated to produce IL2 and compared with PBL from healthy (age- and sex-matched) controls. A significant defect in IL2 production was observed in SLE patients. This defect was not restricted to corticosteroid-treated patients and was not correlated with the presence of lymphotoxic antibodies or with clinical disease activity. Although 5-day responsiveness to phytohaemagglutinin (PHA) decreased in SLE, the SLE PHA-blasts responded as well as control blasts to semipurified IL2, suggesting that the receptor for IL2 was normally expressed on SLE blasts. In 9 cases, the effect of addition of indomethacin (2 micrograms/ml) or of an optimal amount of IL1 on PHA-induced IL2 production was studied. Indomethacin increased (22%) the IL2 yield of healthy individual PBL. In SLE, indomethacin (but not IL1) was able to completely restore (41% increase) the IL2 production of lectin-stimulated PBL (P less than 0.01). These data suggest that, in SLE, the inhibition of IL2 production is mediated by prostaglandin, possibly produced by monocytes.