Regulation of inducible nitric oxide synthase in proinflammatory cytokine-stimulated human primary astrocytes

The present study was undertaken to investigate the mechanism of expression of inducible nitric oxide synthase (iNOS) in human primary astrocytes. Among IL-1beta, TNF-alpha, and IFN-gamma, only IL-1beta alone was capable of inducing iNOS. Similarly, among different cytokine combinations, the combinations involving only IL-1beta as a partner were capable of inducing iNOS. The combination of IL-1beta and IFN-gamma (IL-IF) induced the expression of iNOS at the highest level. All three cytokines alone induced the activation of AP-1 while IL-1beta and TNF-alpha but not IFN-gamma induced the activation of NF-kappaB. However, among the three cytokines, only IL-1beta was capable of inducing the activation of CCAAT/enhancer-binding proteinbeta (C/EBPbeta), suggesting an essential role of C/EBPbeta in the expression of iNOS in astrocytes. Although IL-1beta and IFN-gamma alone induced the activation of AP-1, the combination of these two cytokines (IL-IF) markedly inhibited the activation of AP-1. Consistently, JNK-I, a specific inhibitor of JNK, inhibited IL-1beta-mediated activation of AP-1 and expression of iNOS. On the other hand, JNK-I had no effect on (IL-IF)-induced expression of iNOS, suggesting that the activation of AP-1 is involved only during the low level of iNOS induction by IL-1beta but not during the high level of induction by IL-IF. In contrast, the activation of gamma-activation site (GAS) was involved only during the high level of induction by IL-IF but not during the low level of induction by IL-1beta. However, the activation of NF-kappaB and C/EBPbeta was involved in the induction of iNOS by IL-1beta as well as by IL-IF.     

Saccharated colloidal iron enhances lipopolysaccharide-induced nitric oxide production in vivo

We investigated the effects of iron on the production of nitric oxide (NO), inducible NO synthase (iNOS), and plasma cytokines induced by lipopolysaccharide (LPS) in vivo. Male Wistar rats were preloaded with a single intravenous injection of saccharated colloidal iron (Fesin, 70 mg iron/kg body weight) or normal saline as a control, and then given an intraperitoneal injection of LPS (5.0 mg/kg body weight). Rats, preloaded with iron, had evidence of both iron deposition and strong iNOS induction in liver Kupffer cells upon injection of LPS; phagocytic cells in the spleen and lung had similar findings. LPS-induced NO production in iron-preloaded rats was significantly higher than control rats as accessed by NO-hemoglobin levels measured by ESR (electron spin resonance) and NOx (nitrate plus nitrite) levels. Western blot analysis showed that iron preloading significantly enhanced LPS-induced iNOS induction in the liver, but not in the spleen or lung. LPS-induced plasma levels of IL-6, IL-1beta, and TNF-alpha were also significantly higher in iron-preloaded rats as shown by ELISA, but IFN-gamma levels were unchanged. We conclude that colloidal-iron phagocytosed by liver Kupffer cells enhanced LPS-induced NO production in vivo, iNOS induction in the liver, and release of IL-6, IL-1beta, and TNF-alpha.     

Inflammatory cytokine production in interferon-gamma-primed mice, challenged with lipopolysaccharide. Inhibition by SK&F 86002 and interleukin-1 beta-converting enzyme inhibitor

Mice challenged with lipopolysaccharide (LPS) produce variable serum levels of pro-inflammatory cytokines, and particularly low levels of interleukin-1 beta (IL-1 beta). Interferon-gamma (IFN-gamma) has been shown to be an important mediator of bacteria-induced hypersensitivity to LPS in mice. In the present study, we show that mice pretreated with IFN-gamma exhibit an enhanced capacity to produce serum IL-1 beta, IL-1 alpha, tumour necrosis factor (TNF-alpha) as well as IL-6 in response to LPS. Priming with intraperitoneal (i.p.) injection of 15 mg rat recombinant IFN-gamma, 18 hours prior to the i.p. LPS (300 mg) challenge resulted in a 4-fold increase in the LPS-stimulated release of IL-1 beta and a 2- to 7-fold increase in the release of IL-1 alpha, TNF-alpha, as well as IL-6 into the serum. LPS induced a concentration-dependent increase in the release of IL-1 beta in isolated peritoneal macrophages from IFN-gamma-primed mice whereas macrophages from unprimed mice released minute amounts of IL-1 beta. In addition, nigericin markedly enhanced the release of IL-1 beta in unprimed mice but not in macrophages from IFN-gamma primed mice. The cytokine synthesis inhibitor SK&F 86002, administered per os (100 mg/kg), 1 hour prior to LPS challenge, strongly inhibited the rise in serum levels of the four cytokines. Furthermore, treatment with the IL-1 beta converting enzyme (ICE) specific reversible inhibitor YVAD-CHO resulted in a sharp dose- and time-dependent inhibition of IL-1 beta secretion in the serum, whereas the other cytokines were not affected. In conclusion, IFN-gamma priming strongly potentiates the release of proinflammatory cytokines in the serum of mice as compared to LPS stimulation alone, and provides therefore a useful way to test the in vivo potency and selectivity of cytokine synthesis inhibitors.     

Virus-induced interferon alpha/beta (IFN-alpha/beta) production by T cells and by Th1 and Th2 helper T cell clones: a study of the immunoregulatory actions of IFN-gamma versus IFN-alpha/beta on functions of different T cell populations

Spleen cells, resting T cells, activated T cells, and T cell clones characterized as type 1 (Th1) and type 2 (Th2) were investigated for their ability to produce interferon (IFN) following in vitro culture with Newcastle disease virus (NDV). All of the above cell populations, including both Th1 and Th2 T cell clones, produced high levels of IFN following in vitro culture with NDV. This IFN was characterized as a mixture of IFN-alpha and IFN-beta with IFN-alpha being the predominate species of IFN contained in the mixture. IL-2 greatly enhanced the production of IFN-alpha/beta by all cell populations in response to NDV. These different T cell populations responded very differently to the immunoregulatory actions of IFN-gamma versus IFN-alpha/beta. IFN-alpha/beta was shown to be a potent inhibitor of Con A or IL-2-induced proliferation of different T cell populations. This inhibition was not associated with a reduction in lymphokine production since spleen cells or Th1 T cell clones cultured with Con A and IFN-alpha/beta had no decrease in IL-2 or IFN-gamma production when compared to Con A-stimulated control cultures. IFN-gamma had little to no inhibitory activity on Con A-induced proliferation of spleen cells. In fact, Con A-induced proliferation was usually enhanced by IFN-gamma when nylon wool-enriched T cells were assessed. Different results were observed when IFN-gamma and IFN-alpha/beta were investigated for their ability to inhibit IL-2-induced proliferation of different T helper cell clones. IFN-gamma and IFN-alpha/beta were both capable of inhibiting IL-2-induced proliferation of T cell clones characterized as type 2 (Th2). In contrast, IFN-gamma had no effect on IL-2-induced proliferation of Th1 clones. IFN-alpha/beta, however, inhibited IL-2-induced proliferative responses of both Th1 and Th2 T cell clones. These results document the facts that (1) IFN-gamma and IFN-alpha/beta differ in their immunoregulatory actions, (2) different T cell subpopulations vary in their susceptibility to IFN-gamma regulation, and (3) virus induction of IFN-alpha/beta appears to be a ubiquitous function associated with different T cell populations.     

Immune factors influencing the course of infection with Neospora caninum in the murine host

This paper investigates the role of specific immune factors on the course of infection in genetic knockout (gko) mice infected with 3 different strains of Neospora caninum. Survival time and parasite persistence were examined in interferon-gamma (IFN-gamma), tumor necrosis factor receptor-2 (TNFR2), interleukin 10 (IL-10), beta 2 microglobulin (beta2M), and inducible nitric oxide synthase (iNOS2) gko or wild-type (wt) mice following infection with either pathogenic (NC-1 or NC-2) or attenuated (NCts-8) N. caninum strains. Infection with NC-1 was 100% lethal in IFN-gamma gko mice, as evidenced by mean survival times of 10-13 days, depending on the challenge dose used. TNFR2 and beta2M gko mice infected with NC-1 or NC-2 strain demonstrated partial susceptibility to disease, as evidenced by histopathology and survival curves. TNFR2 or beta2M gko mice were not susceptible to infection with NCts-8, on the basis of absence of pathology and lack of mortality. Lack of mortality and minimal histopathology scores demonstrated that NC-1, NC-2, and NCts-8 infections were avirulent in IL-10 and iNOS2 gko mice. Adoptive transfer of immune cells from NCts-8 vaccinated normal syngeneic mice into IFN-gamma gko mice significantly (P < 0.05) prolonged mean survival times at all 3 challenge doses of NC-1 but failed to protect against mortality. Interestingly, there was a notable change in the tissue tropism of tachyzoites from the lung and brain in immunocompetent wt, TNFR2 gko, IL-10 gko, beta2M gko, and iNOS2 gko mice to the liver and spleen in IFN-gamma gko mice when challenged with N. caninum. On the basis of these results in gko mice, IFN-gamma is a critical cytokine in the host response against acute neosporosis.     

Human cytokines, tumor necrosis factor, and interferons: gene cloning, animal studies, and clinical trials

Presented is a comprehensive program designed to isolate human cytokine genes and investigate their relative induction, and to analyze cytokine activities in cell culture, animal tumor models, and human clinical trials. Human cytokine cDNAs have been isolated from a cDNA library made from normal human peripheral blood leukocytes (PBLs) treated with Sendai virus and the relative induction of tumor necrosis factor (TNF), alpha and gamma interferons (IFN-alpha, IFN-gamma), and interleukin-1 beta IL-1 beta) genes has been analyzed. In the Sendai virus-induced PBL system, IL-1 beta mRNA was shown to be approximately twofold higher than TNF or IFN-alpha mRNA whereas IFN-gamma mRNA was 50-100-fold lower than TNF or IFN-alpha mRNA. The cytotoxic activity of TNF was analyzed on several cell lines and IFN-alpha and IFN-gamma were shown to potentiate TNF cytotoxicity about 2-200-fold depending on cell lines. The LD50 for recombinant TNF in BALB/c mice was determined to be 6 X 10(7) U/kg and the therapeutic dose of recombinant TNF in sarcoma 180 bearing BALB/c mice was 3 X 10(5) U/kg, indicating a wide therapetic index. Phase I clinical trials of recombinant TNF given I.V. indicated a tolerated dose of 150,000 U/kg with biphasic half-life (T-1/2) of 2 and 31 min following TNF injection. Phase II trials of TNF and trials of TNF combined with IFN-alpha are in progress. These studies indicate that cytokines such as TNF and IFN-alpha are subject to similar induction systems, potentiate each other's activities, and can be tolerated at specific doses for potential therapeutic use.     

Cytokine-induced inhibition of insulin release from mouse pancreatic beta-cells deficient in inducible nitric oxide synthase

Cytokines may participate in islet destruction during the development of type 1 diabetes. Expression of inducible nitric oxide synthase (iNOS) and subsequent NO formation induced by IL-1 beta or (IL-1 beta + IFN-gamma) may impair islet function in rodent islets. Inhibition of iNOS or a deletion of the iNOS gene (iNOS -/- mice) protects against cytokine-induced beta-cell suppression, although cytokines might also induce NO-independent impairment. Presently, we exposed wild-type (wt, C57BL/6 x 129SvEv) and iNOS -/- islets to IL-1 beta (25 U/ml) and (IL-1 beta (25 U/ml) + IFN-gamma (1000 U/ml)) for 48 h. IL-1 beta and (IL-1 beta + IFN-gamma) induced a significant increase in NO formation in wt but not in iNOS -/- islets. Both IL-1 beta and (IL-1 beta + IFN-gamma) impaired glucose-stimulated insulin release and reduced the insulin content of wt islets, while (IL-1 beta + IFN-gamma) reduced glucose oxidation rates and cell viability. IL-1 beta exposure to iNOS -/- islets impaired glucose-stimulated insulin release, increased insulin accumulation and reduced the insulin content, without any increase in cell death. Exposure to (IL-1 beta + IFN-gamma) had no effect on iNOS -/- islets except reducing the insulin content. Our data suggest that IL-1 beta may inhibit glucose-stimulated insulin release by pathways that are not NO-dependent and not related to glucose metabolism or cell death.     

Arginine metabolic pathways determine its therapeutic benefit in experimental heatstroke: Role of Th1/Th2 cytokine balance

We have demonstrated that therapeutic administration of L-arginine (L-arg) (120 mg/kg) at +2 h of whole body hyperthermia (WBH) could rescue the mice from heatstroke-induced death. Studies were undertaken to elucidate the role of L-arg in the immunomodulation of the heat-stressed mice. Administration of L-arginine (L-arg), (120 mg/kg, i.p.), at +2 h of WBH, rescued the mice from heat-induced death and reduced the hypothermia. At +4 and +24 h of WBH, levels of IL-1beta, IFN-gamma, nitrite, TNF-alpha, IL-4, TGF-beta1, inducible form of nitric oxide synthase (iNOS), and corticosterone significantly increased compared to the sham group. The elevated levels of Th(1) cytokines, namely TNF-alpha, IL-1beta, IFN-gamma, nitrite, and iNOS, decreased significantly both at +4 and +24 h of WBH, following L-arg administration. However, L-arg administration did not reduce the increased levels of Th(2) cytokines, namely IL-4 and TGF-beta1, in WBH mice at +4 h of WBH. L-arg administration significantly increased the levels of Th(2) cytokines at +24 h of WBH, compared to the saline-treated WBH mice. L-arg administration significantly increased both the splenic and hepatic arginase activity at +4 and +24 h of WBH compared to the saline-treated WBH mice. L-NAME treatment at +2 h of WBH and anti-TGF-beta antibody treatment at 0 h of WBH significantly increased the mortality compared to the saline-treated WBH mice. Altered liver histopathology was attenuated following the administration of L-arg at +2 h of WBH. These results suggest that therapeutic administration of L-arg at appropriate concentration and time attenuates the acute inflammatory response, leading to the rescue of mice from heatstroke.     

IL-18-binding protein protects against lipopolysaccharide- induced lethality and prevents the development of Fas/Fas ligand-mediated models of liver disease in mice.

IL-18-binding protein (IL-18BP) is a natural IL-18 inhibitor. Human IL-18BP isoform a was produced as fusion construct with human IgG1 Fc and assessed for binding and neutralizing IL-18. IL-18BP-Fc binds human, mouse, and rat IL-18 with high affinity (K(D) 0.3-5 nM) in a BIAcore-based assay. In vitro, IL-18BP-Fc blocks IL-18 (100 ng/ml)-induced IFN-gamma production by KG1 cells (EC(50) = 0.3 microg/ml). In mice challenged with an LD(90) of LPS (15 mg/kg), IL-18BP-Fc (5 mg/kg) administered 10 min before LPS blocks IFN-gamma production and protects against lethality. IL-18BP-Fc administered 10 min before LPS blocks IFN-gamma production induced by LPS (5 mg/kg) with ED(50) of 0.005 mg/kg. Furthermore, IL-18BP-Fc (5 mg/kg) abrogates LPS (5 mg/kg)-induced IFN-gamma production even when administered 6 days before LPS but shows no effect when administered 9 or 12 days before LPS. Given 10 min before LPS challenge to mice primed 12 days in advance with heat-killed Propionibacterium acnes, IL-18BP-Fc prevents LPS-induced liver damage and IFN-gamma and Fas ligand expression. Given at the moment of priming with P. acnes, IL-18BP-Fc decreases P. acnes-induced granuloma formation, macrophage-inflammatory protein-1alpha and macrophage-inflammatory protein-2 production and prevents sensitization to LPS. IL-18BP-Fc also prevents Con A-induced liver damage and IFN-gamma and Fas ligand expression as well as liver damage induced by Pseudomonas aeruginosa exotoxin A or by anti-Fas agonistic Ab. In conclusion, IL-18BP can be engineered and produced in recombinant form to generate an IL-18 inhibitor, IL-18BP-Fc, endowed with remarkable in vitro and in vivo properties of binding and neutralizing IL-18.     

Preventive oral supplementation with glutamine and arginine has beneficial effects on the intestinal mucosa and inflammatory cytokines in endotoxemic rats

The objective of this study was to evaluate the effect of oral supplementation with a combination of arginine and glutamine on the intestinal mucosa and inflammatory cytokines of lipopolysaccharide (LPS)-induced adult rats. Fifty Sprague-Dawley rats (average weight of 185 ± 15 g) were randomly divided into five groups: control group A (CA) and control group B (CB), both orally supplemented with 0.9% saline; group Arg, supplemented with 300 mg/kg day(-1) arginine; group Gln, supplemented with 300 mg/kg day(-1) glutamine; group AG, supplemented with 150 mg/kg day(-1) arginine and 150 mg/kg day(-1) glutamine. The experiment lasted for 2 weeks. Food intake and body weight were measured during the experiment. At 10.00 h of day 15, animals were injected with 4 mg/kg LPS (group CB, Arg, Gln, and AG) or sterile saline (group CA) after supplementation. Then at 14.00 h, all animals were killed and blood and tissue collected. The results showed that compared with group CB, arginine concentration tended to be increased (P > 0.05) in group Arg and AG, while there was no significant difference in glutamine concentration among the groups challenged with LPS. Oral supplementation with arginine or/and glutamine mitigated morphology impairment (lower villus height, P < 0.05) in the jejunum and ileum induced by LPS challenge. LPS administration resulted in a significant increase in TNF-α, IL-1β, IL-6 and IL-10 mRNA abundance. Arginine only significantly decreased TNF-α mRNA abundance in the ileum, while glutamine significantly decreased both TNF-α and IL-10 mRNA in the ileum. A combination of arginine and glutamine significantly decreased TNF-α and IL-1β mRNA abundance in both the jejunum and ileum, while they also significantly decreased anti-inflammatory IL-10 in the ileum. These results revealed that an oral supply of combined arginine and glutamine had more favorable effects on the intestinal mucosa and inflammatory cytokines than a supply of arginine or glutamine alone.     

Inhibition of inducible NO synthase by TH2 cytokines and TGF beta in rheumatoid arthritic synoviocytes: effects on nitrosothiol production.

The aim of this study was to compare the effects on NO production of IL-4, IL-10, and IL-13 with those of TGF-beta. RA synovial cells were stimulated for 24 h with IL-1 beta (1 ng/ml), TNF-alpha (500 pg/ml), IFN-gamma (10(-4)IU/ml) alone or in combination. Nitrite was determined by the Griess reaction, S-nitrosothiols by fluorescence, and inducible NO synthase (iNOS) by immunofluorescence and fluorescence activated cell sorter analysis (FACS). In other experiments, IL-4, IL-10, IL-13, and TGF beta were used at various concentrations and were added in combination with proinflammatory cytokines. The addition of IL-1 beta, TNF-alpha, and IFN-gamma together increased nitrite production: 257.5 +/- 35.8 % and S-nitrosothiol production : 413 +/- 29%, P < 0.001. None of these cytokines added alone had any significant effect. iNOS synthesis increased with NO production. IL-4, IL-10, IL-13, and TGF beta strongly decreased the NO production caused by the combination of IL-1 beta, TNF-alpha, and IFN-gamma. These results demonstrate that stimulated RA synoviocytes produce S-nitrosothiols, bioactive NO* compounds, in similar quantities to nitrite. IL-4, IL-10, IL-13, and TGF-beta decrease NO production by RA synovial cells. The anti-inflammatory properties of these cytokines may thus be due at least in part to their effect on NO metabolism.     

Overlapping polypeptide induction in human fibroblasts in response to treatment with interferon-alpha, interferon-gamma, interleukin 1 alpha, interleukin 1 beta, and tumor necrosis factor

Cytokine-induced polypeptides were identified in whole cell lysates of human fibroblasts by computer-based analysis of two-dimensional gels with the use of the PDQuest System. Treatment with interferon-alpha (IFN-alpha) and interferon-gamma (IFN-gamma) enhanced the synthesis of 12 and 28 polypeptides, respectively. Exposure to interleukin 1 alpha (IL-1 alpha) or interleukin 1 beta (IL-1 beta) resulted in the increased synthesis of seven identical polypeptides. Treatment with tumor necrosis factor (TNF) at 100 U/ml led to enhanced expression of seven polypeptides, whereas exposure to TNF at 1000 U/ml increased the levels of these seven plus two additional polypeptides. The antiviral and antiproliferative effects of these cytokines in strain 153 fibroblasts were also assessed. Both IFN-alpha and IFN-gamma exhibited antiviral activity, whereas both IL-1 and TNF stimulated fibroblast growth. IFN-gamma was alone in inhibiting proliferation. Thus, although these cytokines exhibit low degrees of structural homology, they share some common functions, and a number of polypeptides were induced in common by two or more of these agents. The greatest similarities in polypeptide induction occur between IFN-alpha and IFN-gamma and between the IL-1s and TNF. However, polypeptides were also induced in common by IFN-alpha and TNF, IFN-gamma and IL-1, and IFN-gamma and TNF. These similarities in polypeptide induction may reflect the overlapping functions of these cytokines and may be indicative of common biochemical pathways in their mechanisms of action.     

Interleukin-1 is not essential for expression of inducible NOS in hepatocytes induced by lipopolysaccharide in vivo

Interleukin (IL)-1 and tumor necrotic factor alpha (TNFalpha) are pivotal in the pathogenesis of endotoxemia. In spite of the in vitro finding that IL-1beta, but not TNFalpha, can induce iNOS mRNA and NO production as a single stimulus in hepatocytes in primary culture, the involvement of IL-1 in iNOS induction in the liver has been less clear in vivo. To address this, we challenged IL-1alpha/beta double-knockout (IL-1alpha/beta(-/-)) and TNFalpha(-/-) mice with lipopolysaccharide (LPS). As compared with wild-type mice, the increases in the plasma NO level measured as nitrite and nitrate and hepatic iNOS were significantly reduced in IL-1alpha/beta(-/-) and TNFalpha(-/-) mice 8 and 12h after the LPS challenge. In the wild-type mice, iNOS protein was first detected in Kupffer cells around the portal vein 2h after LPS challenge; and then it spread to hepatocytes throughout the intralobular region of the liver by 8h. Although the expression of iNOS protein was detected in Kupffer cells of both IL-1alpha/beta(-/-) and TNFalpha(-/-) mice, its level was moderate in hepatocytes of IL-1alpha/beta(-/-) mice, but negligible in those of TNFalpha(-/-) mice, 8h after LPS challenge. Concomitant with the expression of iNOS protein in the liver, Toll-like receptor 4, the signaling receptor for LPS, was expressed in hepatocytes of wild-type and IL-1alpha/beta(-/-) mice, but not of TNFalpha(-/-) mice. These results demonstrate that the expression of Toll-like receptor 4 is well correlated with that of iNOS protein in hepatocytes in vivo after LPS challenge and that IL-1 is not essential for the induction of iNOS in hepatocytes in vivo.     

Innate immune response of the human host to exposure with herpes simplex virus type 1: in vitro control of the virus infection by enhanced natural killer activity via interleukin-15 induction

Infections with herpes simplex virus type 1 (HSV-1) in humans and in animal models are accompanied by enhanced natural killer (NK) activity. In vitro, HSV-1 also enhances the NK activity of human peripheral blood mononuclear cells (PBMC). The molecular basis of this enhanced NK activity, however, is not well characterized. We investigated the role of human interleukin-15 (IL-15) in this phenomenon and report here that HSV-1-mediated enhanced NK activity was abrogated by neutralizing antibodies for IL-15 but not for other cytokines (i.e., IL-2, IL-12, gamma interferon [IFN-gamma], tumor necrosis factor alpha, or IFN-alpha). Anti-CD122 antibodies which block signaling through IL-2 receptor beta chain, and therefore neutralize the effects of IL-15 (and IL-2), also abrogated this enhancement. Furthermore, HSV-1 increased the levels of IL-15 mRNA and the production of IL-15 in HSV-1-infected PBMC cultures. The neutralization of IL-15 in cocultures of PBMC with HSV-1-infected cells significantly increased HSV-1 production. These results strongly suggest a role for IL-15 in the HSV-1-mediated in vitro enhancement of NK activity and in the PBMC-mediated suppression of HSV-1 replication.     

Characterization of interleukin 2-dependent cytotoxic T-cell clones. IV. Production of alpha, beta and gamma interferons and interleukin 2 by Lyt-2+ T cells

The production of alpha, beta and gamma interferons (IFN) and interleukin 2 (IL-2) by Lyt-2+-dependent cytotoxic T-cell lines/clones was investigated. Cloned and uncloned T-cell lines specific for H-2Dd or the unique RL male 1 leukemia antigen were studied. After infection with Sendai virus (SV) or Newcastle disease virus (NDV) all cell lines produced IFN-alpha and -beta. Induction of IFN-gamma was attempted with the mitogens Con A, PHA, PWM, SEA, and SEB, with poly(I:C), with antibodies Lyt-1.2, -2.2, and Thy-1.2, or with the target cells Meth A (H-2Dd+) and RL male 1. All mitogens were effective inducers. However, the antibodies and poly(I:C) were not. One uncloned RL male 1-specific cell line CTLL-RP, produced IFN-gamma after induction with RL male 1. Production of IFN-alpha, beta depended on IL-2, whereas production of IFN-gamma did not, although addition of highly purified IL-2 increased IFN-gamma production even in the absence of other inducers. Crude IL-2 inhibited the production of IFN-gamma but not IFN-alpha, beta. In response to mitogens, some T-cell clones also produced IL-2. The results demonstrate that Lyt-2+ cells can produce a broad spectrum of lymphokine activities after appropriate stimulation. Their availability now affords us the opportunity to study the regulation of lymphokine production at the clonal level.     

Activation of macrophages for ADCC in vitro: effects of IL-4, TNF, interferons-alpha/beta, interferon-gamma, and GM-CSF.

Macrophages in varying states of activation differ in their ability to perform antibody-dependent cellular cytotoxicity (ADCC) and antibody-independent macrophage-mediated tumor cytotoxicity (MTC). To define further the activation requirements for macrophages to perform various cytolytic functions, we stimulated peptone-elicited peritoneal macrophages, which are only poorly cytolytic, with one of a panel of cytokines and then quantified three distinct cytolytic capacities. The peptone-elicited macrophages, after stimulation with IFN-alpha/beta, IL-4, or TNF, had increased ability to perform both the rapid and slow variants of ADCC but not to perform MTC. Stimulation with high doses of IFN-gamma, however, increased the macrophages' ability to perform all three cytolytic functions. GM-CSF had no effects on any cytolytic capacity. The effects of IL-4, TNF, IFN-gamma, and IFN-alpha/beta on the macrophages' capacity for both forms of ADCC were dose-dependent. IFN-gamma and IFN-alpha/beta increased the macrophages' capacity for both variants of ADCC within 4 hr of treatment, whereas IL-4 and TNF did so only after prolonged treatment. These results suggest that three forms of macrophage cytolytic capacity can be enhanced by cytokine treatment but that the requirements for enhancing each of the three forms of macrophage cytolytic capacity differ.