Release of reactive nitrogen intermediates and reactive oxygen intermediates from mouse peritoneal macrophages. Comparison of activating cytokines and evidence for independent production

The capacity of 12 cytokines to induce NO2- or H2O2 release from murine peritoneal macrophages was tested by using resident macrophages, or macrophages elicited with periodate, casein, or thioglycollate broth. Elevated H2O2 release in response to PMA was observed in resident macrophages after a 48-h incubation with IFN-gamma, TNF-alpha, TNF-beta, or CSF-GM. Of these, only IFN-gamma induced substantial NO2- secretion during the culture period. The cytokines inactive in both assays under the conditions tested were IL-1 beta, IL-2, IL-3, IL-4, IFN-alpha, IFN-beta, CSF-M, and transforming growth factor-beta 1. Incubation of macrophages with IFN-gamma for 48 h in the presence of LPS inhibited H2O2 production but augmented NO2- release, whereas incubation in the presence of the arginine analog NG-monomethylarginine inhibited NO2- release but not H2O2 production. Although neither TNF-alpha nor TNF-beta induced NO2- synthesis on its own, addition of either cytokine together with IFN-gamma increased macrophage NO2- production up to six-fold over that in macrophages treated with IFN-gamma alone. Moreover, IFN-alpha or IFN-beta in combination with LPS could also induce NO2- production in macrophages, as was previously reported for IFN-gamma plus LPS. These data suggest that: 1) tested as a sole agent, IFN-gamma was the only one of the 12 cytokines capable of inducing both NO2- and H2O2 release; 2) the pathways leading to secretion of H2O2 and NO2- are independent; 3) either IFN-gamma and TNF-alpha/beta or IFN-alpha/beta/gamma and LPS can interact synergistically to induce NO2- release.     

IFN-gamma enhances bovine macrophage responsiveness to Mycobacterium bovis: Impact on bacterial replication, cytokine release and macrophage apoptosis

We sought to determine the impact of bovine IFN-gamma on the interaction between Mycobacterium bovis and bovine macrophages. Bovine macrophages released small amounts of nitric oxide (NO), TNF-alpha, IL-1beta and IL-12 upon infection with bacille Calmette-Guérin (BCG). Prior pulsing of cells with IFN-gamma significantly enhanced the release of NO and IL-12. Infection of bovine macrophages with virulent M. bovis led to the release of higher levels of pro-inflammatory mediators, compared to levels released upon BCG infection. IFN-gamma treatment of macrophages enhanced the release of pro-inflammatory mediators, but did not modify bacterial replication in M. bovis-infected macrophages. Treatment of macrophages with a combination of IFN-gamma and LPS led to a reduction in bacterial replication. Infected cells treated with IFN-gamma/LPS progressed mostly through an apoptotic pathway, whereas untreated infected cells eventually died by necrosis. Agents that prevented the acquisition of bacteriostatic activity by activated macrophages also prevented the induction of apoptosis in infected macrophages (IL-10 and neutralizing anti-TNF-alpha). We conclude that virulent M. bovis is a major determinant of release of pro-inflammatory cytokines by macrophages. IFN-gamma amplifies the macrophage cytokine release in response to M. bovis. Induction of apoptosis is closely linked to the emergence of macrophage resistance to M. bovis replication, which is dependent on endogenous TNF-alpha release.     

Quantitative aspects of lipopolysaccharide and cytokine requirements to generate nitric oxide in macrophages from LPS-hyporesponsive (<Emphasis Type="Italic">Lps</Emphasis><Superscript>d</Superscript>) C3H/HeJ mice

Due to a gene defect (Lps(d)), C3H/HeJ mice are known to be hyporesponsive to the immunobiological potential of lipopolysaccharide (LPS). We studied dose requirements for LPS, IFN-gamma, and cytokines TNF-alpha and IL-10 to produce nitric oxide (NO) in peritoneal macrophages (Mphi) from these animals. In contrast to the Lps(n) C3H/HeN mice, high concentrations of LPS (up to 5 microg/mL) or IFN-gamma (up to 5 ng/mL) by themselves were unable to activate NO production in C3H/HeJ Mphi. The failure to produce NO could not be overcome by addition of L-arginine or tetrahydropterin. The high-output NO biosynthesis was dose-dependently stimulated by combined administration of varying concentrations of IFN-gamma (50-5000 pg/mL) and LPS (approximately 1 ng/mL) or to a lesser extent by IFN-gamma plus TNF-alpha or TNF-alpha/IL-10. Formation of NO in C3H/HeJ MCO triggered by high concentration of LPS (approximately 1 microg/mL) given together with IFN-gamma (0.2-5 ng/mL) reached the values typical for Lps(n) C3H/HeN mice. While Mphi from C3H/HeN mice secreted TNF-alpha, IL-10, and IL-10 upon contact with a low dose of LPS (1 ng/mL), C3H/HeJ Mphi required high concentration of LPS (5 microg/mL) to enhance the secretion of the cytokines. Yet, this dose remained ineffective to stimulate IFN-gamma in Mphi from C3H/HeJ mice. It can be presumed that one of the important factors influencing their deficient ability to form NO is a failure of Mphi to produce IFN-gamma upon LPS contact.     

Macrophage-stimulating protein, the ligand for the stem cell-derived tyrosine kinase/RON receptor tyrosine kinase, inhibits IL-12 production by primary peritoneal macrophages stimulated with IFN-gamma and lipopolysaccharide

IL-12, produced by APCs during the initial stages of an immune response, plays a pivotal role in the induction of IFN-gamma by NK and gammadeltaT cells and in driving the differentiation of Th1 cells, thus providing a critical link between innate and acquired immunity. Due to the unique position occupied by IL-12 in the regulation of immunity, many mechanisms have evolved to modulate IL-12 production. We have shown previously that macrophage-stimulating protein (MSP), the ligand for the stem cell-derived tyrosine kinase/recepteur d'origine nantais (RON) receptor, inhibits NO production by macrophages in response to IFN-gamma and enhances the expression of arginase. Mice lacking RON exhibit increased inflammation in a delayed-type hypersensitivity reaction and increased susceptibility to endotoxic shock. In this study we demonstrate that pretreatment of macrophages with MSP before IFN-gamma and LPS results in the complete inhibition of IL-12 production due to suppression of p40 expression. This response is mediated by the RON receptor, and splenocytes from RON(-/-) animals produce increased levels of IFN-gamma. MSP pretreatment of macrophages resulted in decreased tyrosine phosphorylation of Stat-1 and decreased expression of IFN consensus sequence binding protein in response to inflammatory cytokines. In addition to IL-12, the expression of IL-15 and IL-18, cytokines that are also dependent on IFN consensus sequence binding protein activation, is inhibited by pretreatment with MSP before IFN-gamma and LPS. We also show that the ability of MSP to inhibit IL-12 production is independent of IL-10. Taken together, these results suggest that MSP may actively suppress cell-mediated immune responses through its ability to down-regulate IL-12 production and thus inhibit classical activation of macrophages.     

Lung-restricted macrophage activation in the pearl mouse model of Hermansky-Pudlak syndrome

Pulmonary inflammation, abnormalities in alveolar type II cell and macrophage morphology, and pulmonary fibrosis are features of Hermansky-Pudlak Syndrome (HPS). We used the naturally occurring "pearl" HPS2 mouse model to investigate the mechanisms of lung inflammation observed in HPS. Although baseline bronchoalveolar lavage (BAL) cell counts and differentials were similar in pearl and strain-matched wild-type (WT) mice, elevated levels of proinflammatory (MIP1gamma) and counterregulatory (IL-12p40, soluble TNFr1/2) factors, but not TNF-alpha, were detected in BAL from pearl mice. After intranasal LPS challenge, BAL levels of TNF-alpha, MIP1alpha, KC, and MCP-1 were 2- to 3-fold greater in pearl than WT mice. At baseline, cultured pearl alveolar macrophages (AMs) had markedly increased production of inflammatory cytokines. Furthermore, pearl AMs had exaggerated TNF-alpha responses to TLR4, TLR2, and TLR3 ligands, as well as increased IFN-gamma/LPS-induced NO production. After 24 h in culture, pearl AM LPS responses reverted to WT levels, and pearl AMs were appropriately refractory to continuous LPS exposure. In contrast, cultured pearl peritoneal macrophages and peripheral blood monocytes did not produce TNF-alpha at baseline and had LPS responses which were no different from WT controls. Exposure of WT AMs to heat- and protease-labile components of pearl BAL, but not WT BAL, resulted in robust TNF-alpha secretion. Similar abnormalities were identified in AMs and BAL from another HPS model, pale ear HPS1 mice. We conclude that the lungs of HPS mice exhibit hyperresponsiveness to LPS and constitutive and organ-specific macrophage activation.     

Distinct modulatory effects of LPS and CpG on IL-18-dependent IFN-gamma synthesis

Innate cellular production of IFN-gamma is suppressed after repeated exposure to LPS, whereas CpG-containing DNA potentiates IFN-gamma production. We compared the modulatory effects of LPS and CpG on specific cellular and cytokine responses necessary for NK-cell dependent IFN-gamma synthesis. C3H/HeN mice pretreated with LPS for 2 days generated 5-fold less circulating IL-12 p70 and IFN-gamma in response to subsequent LPS challenge than did challenged control mice. In contrast, CpG-pretreated mice produced 10-fold more circulating IFN-gamma without similar changes in IL-12 p70 levels, but with 10-fold increases in serum IL-18 relative to LPS-challenged control or endotoxin-tolerant mice. The role of IL-18 in CpG-induced immune potentiation was studied in splenocyte cultures from control, LPS-conditioned, or CpG-conditioned mice. These cultures produced similar amounts of IFN-gamma in response to rIL-12 and rIL-18. However, only CpG-conditioned cells produced IFN-gamma when cultured with LPS or CpG, and production was ablated in the presence of anti-IL-18R Ab. Anti-IL-18R Ab also reduced in vivo IFN-gamma production by >2-fold in CpG-pretreated mice. Finally, combined pretreatment of mice with LPS and CpG suppressed the production of circulating IFN-gamma, IL-12 p70, and IL-18 after subsequent LPS challenge. We conclude that CpG potentiates innate IFN-gamma production from NK cells by increasing IL-18 availability, but that the suppressive effects of LPS on innate cellular immunity dominate during combined LPS and CpG pretreatment. Multiple Toll-like receptor engagement in vivo during infection can result in functional polarization of innate immunity dominated by a specific Toll-like receptor response.     

Essential role of MAPK phosphatase-1 in the negative control of innate immune responses

TLR-induced innate immunity and inflammation are mediated by signaling cascades leading to activation of the MAPK family of Ser/Thr protein kinases, including p38 MAPK, which controls cytokine release during innate and adoptive immune responses. Failure to terminate such inflammatory reactions may lead to detrimental systemic effects, including septic shock and autoimmunity. In this study, we provide genetic evidence of a critical and nonredundant role of MAPK phosphatase (MKP)-1 in the negative control of MAPK-regulated inflammatory reactions in vivo. MKP-1-/- mice are hyperresponsive to low-dose LPS-induced toxicity and exhibit significantly increased serum TNF-alpha, IL-6, IL-12, MCP-1, IFN-gamma, and IL-10 levels after systemic administration of LPS. Furthermore, absence of MKP-1 increases systemic levels of proinflammatory cytokines and exacerbates disease development in a mouse model of rheumatoid arthritis. When activated through TLR2, TLR3, TLR4, TLR5, and TLR9, bone marrow-derived MKP-1-/- macrophages exhibit increased cytokine production and elevated expression of the differentiation markers B7.2 (CD86) and CD40. MKP-1-deficient macrophages also show enhanced constitutive and TLR-induced activation of p38 MAPK. Based on these findings, we propose that MKP-1 is an essential component of the intracellular homeostasis that controls the threshold and magnitude of p38 MAPK activation in macrophages, and inflammatory conditions accentuate the significance of this regulatory function.     

Enhanced protection against fatal mycobacterial infection in SCID beige mice by reshaping innate immunity with IFN-gamma transgene.

Humans with immune-compromised conditions such as SCID are unable to control infection caused by normally nonpathogenic intracellular pathogens such as Mycobacterium bovis bacillus Calmette-Guérin. We found that SCID beige mice lacking both lymphocytes and NK cells had functionally normal lung macrophages and yet a selectively impaired response of type 1 cytokines IFN-gamma and IL-12, but not TNF-alpha, during M. bovis bacillus Calmette-Guérin infection. These mice succumbed to such infection. A repeated lung gene transfer strategy was designed to reconstitute IFN-gamma in the lung, which allowed investigation of whether adequate activation of innate macrophages could enhance host defense in the complete absence of lymphocytes. IFN-gamma transgene-based treatment was initiated 10 days after the establishment of mycobacterial infection and led to increased levels of both IFN-gamma and IL-12, but not TNF-alpha, in the lung. Lung macrophages were activated to express increased MHC molecules, type 1 cytokines and NO, and increased phagocytic and mycobactericidal activities. Activation of innate immunity markedly inhibited otherwise uncontrollable growth of mycobacteria and prolonged the survival of infected SCID hosts. Thus, our study proposes a cytokine transgene-based therapeutic modality to enhance host defense in immune-compromised hosts against intracellular bacterial infection, and suggests a central effector activity played by IFN-gamma-activated macrophages in antimycobacterial cell-mediated immunity.     

Role of TNF-alpha in the induction of fungicidal activity of mouse peritoneal exudate cells against Cryptococcus neoformans by IL-12 and IL-18

We have recently demonstrated that two IFN-gamma-inducing cytokines, interleukin (IL)-12 and IL-18, synergistically induced the fungicidal activity of mouse peritoneal exudate cells (PEC) against Cryptococcus neoformans through NK cell production of interferon (IFN)-gamma and nitric oxide (NO) synthesis. In the present study, we further dissected these effects by examining the involvement of tumor necrosis factor (TNF)-alpha in the induction of IL-12/IL-18-stimulated PEC fungicidal activity. The addition of neutralizing anti-TNF-alpha mAb significantly suppressed IL-12/IL-18-stimulated PEC anticryptococcal activity. This effect was ascribed to the inhibition of macrophage NO synthesis, but not of IFN-gamma production by NK cells, because the same treatment inhibited the former response, but not the latter one. On the other hand, combined treatment with IL-12 and IL-18 synergistically induced the production of TNF-alpha by PEC and this effect was almost completely abrogated by neutralizing anti-IFN-gamma mAb. The cell type producing TNF-alpha among PEC was mostly macrophage. TNF-alpha significantly promoted macrophage NO production and anticryptococcal activity induced by IFN-gamma, and furthermore anti-TNF-alpha mAb partially inhibited these responses. Considered together, our results indicated that TNF-alpha contributed to the potentiation of IL-12/IL-18-induced PEC fungicidal activity against C. neoformans through enhancement of IFN-gamma-induced production of NO by macrophages, but not through increased production of IFN-gamma by NK cells.     

IRF-8/interferon (IFN) consensus sequence-binding protein is involved in Toll-like receptor (TLR) signaling and contributes to the cross-talk between TLR and IFN-gamma signaling pathways

Toll-like receptor (TLR) and interferon-gamma (IFN-gamma) signaling pathways are important for both innate and adaptive immune responses. However, the cross-talk between these two signaling pathways is incompletely understood. Here we show that IFN-gamma and LPS synergistically induce the expression of proinflammatory factors, including interleukin-1 (IL-1), IL-6, IL-12, NO, and tumor necrosis factor-alpha (TNF-alpha). Comparable synergism was observed between IFN-gamma and peptidoglycan (PGN; a TLR2 ligand) and poly(I:C) (a TLR3 ligand) in the induction of IL-12 promoter activity. IFN-gamma enhanced lipopolysaccharide (LPS)-induced ERK and JNK phosphorylation but had no effect on LPS-induced NF-kappaB activation. Interestingly, we found that IRF-8-/- macrophages were impaired in the activation of LPS-induced ERK and JNK and the production of proinflammatory cytokines induced by LPS or IFN-gamma plus LPS. Retroviral transduction of IRF-8 into IRF-8-/- macrophages rescued ERK and JNK activation. Furthermore, co-immunoprecipitation experiments show that IRF-8 physically interacts with TRAF6 at a binding site between amino acid residues 356 and 305 of IRF-8. Transfection of IRF-8 enhanced TRAF6 ubiquitination, which is consistent with a physical interaction of IRF-8 with TRAF6. Taken together, the results suggest that the interaction of IRF-8 with TRAF6 modulates TLR signaling and may contribute to the cross-talk between IFN-gamma and TLR signal pathways.     

Antifungal type 1 responses are upregulated in IL-10-deficient mice

C57BL/6 mice are highly resistant to infections caused by Candida albicans and Aspergillus fumigatus. To elucidate the role of IL-10 produced by C57BL/6 mice during these infections, parameters of infection and immunity to it were evaluated in IL-10-deficient and wild-type mice with disseminated or gastrointestinal candidiasis or invasive pulmonary aspergillosis. Unlike parasitic protozoan infection, C. albicans or A. fumigatus infection did not induce significant acute toxicity in IL-10-deficient mice, who, instead, showed reduced fungal burden and fungal-associated inflammatory responses. The increased resistance to infections as compared to wild-type mice was associated with upregulation of innate and acquired antifungal Th1 responses, such as a dramatically higher production of IL-12, nitric oxide (NO) and TNF-alpha as well as IFN-gamma by CD4+ T cells. Pharmacological inhibition of NO production greatly reduced resistance to gastrointestinal candidiasis, thus pointing to the importance of IL-10-dependent NO regulation at mucosal sites in fungal infections. These results are reminiscent of those obtained in genetically susceptible mice, in which IL-10 administration increased, and IL-10 neutralization decreased, susceptibility to C. albicans and A. fumigatus infections. Collectively, these observations indicate that the absence of IL-10 augments innate and acquired antifungal immunity by upregulating type 1 cytokine responses. The resulting protective Th1 responses lead to a prompt reduction of fungal growth, thus preventing tissue destruction and lethal levels of proinflammatory cytokines.     

Regulation of the RON receptor tyrosine kinase expression in macrophages: blocking the RON gene transcription by endotoxin-induced nitric oxide

Previous studies have shown that activation of the RON receptor tyrosine kinase inhibits inducible NO production in murine peritoneal macrophages. The purpose of this study is to determine whether inflammatory mediators such as LPS, IFN-gamma, and TNF-alpha regulate RON expression. Western blot analysis showed that RON expression is reduced in peritoneal macrophages collected from mice injected with a low dose of LPS. The inhibition was seen as early as 8 h after LPS challenge. Experiments in vitro also demonstrated that the levels of the RON mRNA and protein are diminished in cultured peritoneal macrophages following LPS stimulation. TNF-alpha plus IFN-gamma abrogated macrophage RON expression, although individual cytokines had no significant effect. Because LPS and TNF-alpha plus IFN-gamma induce NO production, we reasoned that NO might be involved in the RON inhibition. Two NO donors, S-nitroglutathione (GSNO) and (+/-)-S-nitroso-N-acetylpenicillamine (SNAP), directly inhibited macrophage RON expression when added to the cell cultures. Blocking NO production by NO inhibitors like TGF-beta prevented the LPS-mediated inhibitory effect. In Raw264.7 cells transiently transfected with a report vector, GSNO or SNAP inhibited the luciferase activities driven by the RON gene promoter. Moreover, GSNO or SNAP inhibited the macrophage-stimulating protein-induced RON phosphorylation and macrophage migration. We concluded from these data that RON expression in macrophages is regulated during inflammation. LPS and TNF-alpha plus IFN-gamma are capable of down-regulating RON expression through induction of NO production. The inhibitory effect of NO is mediated by suppression of the RON gene promoter activities.     

Cholera toxin B pretreatment of macrophages and monocytes diminishes their proinflammatory responsiveness to lipopolysaccharide

The cholera toxin B chain (CTB) has been reported to suppress T cell-dependent autoimmune diseases and to potentiate tolerance of the adaptive immune system. We have analyzed the effects of CTB on macrophages in vitro and have found that preincubation with CTB (10 microg/ml) suppresses the proinflammatory reaction to LPS challenge, as demonstrated by suppressed production of TNF-alpha, IL-6, IL-12(p70), and NO (p < 0.01) in cells of macrophage lines. Pre-exposure to CTB also suppresses LPS-induced TNF-alpha and IL-12(p70) formation in human PBMC. Both native and recombinant CTB exhibited suppressive activity, which was shared by intact cholera toxin. In cells of the human monocyte line Mono Mac 6, exposure to CTB failed to suppress the production of IL-10 in response to LPS. Control experiments excluded a role of possible contamination of CTB by endotoxin or intact cholera toxin. The suppression of TNF-alpha production occurred at the level of mRNA formation. Tolerance induction by CTB was dose and time dependent. The suppression of TNF-alpha and IL-6 production could be counteracted by the addition of Abs to IL-10 and TGF-beta. IFN-gamma also antagonized the actions of CTB on macrophages. In contrast to desensitization by low doses of LPS, tolerance induction by CTB occurred silently, i.e., in the absence of a measurable proinflammatory response. These findings identify immune-deviating properties of CTB at the level of innate immune cells and may be relevant to the use of CTB in modulating immune-mediated diseases.     

Tumor necrosis factor-alpha-dependent production of reactive nitrogen intermediates mediates IFN-gamma plus IL-2-induced murine macrophage tumoricidal activity.

We have previously established that IFN-gamma plus IL-2 induces murine macrophage tumoricidal activity. The purpose of this study was to identify the effector molecules that account for the IFN-gamma plus IL-2-induced macrophage cytotoxicity against P815 mastocytoma cells. ANA-1 macrophages and normal thioglycollate-elicited mouse peritoneal macrophages produced little or no detectable nitrite (NO2-) after incubation with IFN-gamma alone or IL-2 alone; however, IL-2 synergized with IFN-gamma for the production of NO2-. IFN-gamma plus IL-2 did not induce NO2- production or tumoricidal activity in ANA-1 macrophages that were cultured in medium devoid of L-arginine or in ANA-1 macrophages that were incubated with NG-monomethyl-L-arginine. As observed previously with ANA-1 macrophage tumoricidal activity, IL-4 inhibited IFN-gamma plus IL-2-induced, but not IFN-gamma plus LPS-induced, NO2- production. IL-4 also selectively decreased the ability of IFN-gamma and/or IL-2 to augment TNF-alpha mRNA expression in ANA-1 macrophages. Lastly, incubation of ANA-1 macrophages with anti-TNF mAb selectively inhibited the ability of IFN-gamma plus IL-2 to induce NO2- production and tumoricidal activity. These results indicate that IFN-gamma plus IL-2-induced tumoricidal activity is dependent upon the metabolism of L-arginine to reactive nitrogen intermediates, and they establish a role for TNF-alpha as a required intermediate for IL-2-dependent NO2- production and tumoricidal activity.     

Inherited IL-12 unresponsiveness contributes to the high LPS resistance of the Lps(d) C57BL/10ScCr mouse

LPS(d) mouse strains are characterized by the presence of a defective LPS/tlr4 gene that make them refractory to the biological activity of LPS. One of the mouse strains commonly used to study LPS defects is the C57BL/10ScCr (Cr) strain. However, unlike other LPS(d) strains, the Cr strain also has a heavily impaired IFN-gamma response to micro-organisms. As a consequence, unlike other LPS(d) mouse strains, they do not acquire a partial LPS susceptibility when treated with sensitizing bacteria. Because IL-12 is important for the microbial induction of IFN-gamma, we investigated whether the production or function of IL-12 might be defective in Cr mice. IL-12 mRNA (p35 and p40) was present in the spleen of untreated Cr mice, IL-12p40 mRNA was inducible in mice injected with live or killed Salmonella typhimurium, and IL-12 (p70) was inducible in macrophages by bacteria. Thus, Cr mice exhibit normal IL-12 responses. In functional tests, splenocytes of untreated or of S. typhimurium-infected mice failed to produce IFN-gamma when stimulated with murine rIL-12 or with a combination of IL-12 and murine rIL-18 or Con A. Furthermore, Cr mice were identical with IL-12p35/p40 and IL-12 receptor beta(1) knockout mice in their impaired in vivo and in vitro IFN-gamma responses to bacteria. Thus, Cr mice carry a second genetic defect unrelated to the Lps/tlr4 mutation that underlies the IL-12 unresponsiveness and contributes to the LPS resistance and impaired innate immune response in this strain.     

IL-10 inhibits cytokine production by activated macrophages

IL-10 inhibits the ability of macrophage but not B cell APC to stimulate cytokine synthesis by Th1 T cell clones. In this study we have examined the direct effects of IL-10 on both macrophage cell lines and normal peritoneal macrophages. LPS (or LPS and IFN-gamma)-induced production of IL-1, IL-6, and TNF-alpha proteins was significantly inhibited by IL-10 in two macrophage cell lines. Furthermore, IL-10 appears to be a more potent inhibitor of monokine synthesis than IL-4 when added at similar concentrations. LPS or LPS- and IFN-gamma-induced expression of IL-1 alpha, IL-6, or TNF-alpha mRNA was also inhibited by IL-10 as shown by semiquantitative polymerase chain reaction or Northern blot analysis. Inhibition of LPS-induced IL-6 secretion by IL-10 was less marked in FACS-purified peritoneal macrophages than in the macrophage cell lines. However, IL-6 production by peritoneal macrophages was enhanced by addition of anti-IL-10 antibodies, implying the presence in these cultures of endogenous IL-10, which results in an intrinsic reduction of monokine synthesis after LPS activation. Consistent with this proposal, LPS-stimulated peritoneal macrophages were shown to directly produce IL-10 detectable by ELISA. Furthermore, IFN-gamma was found to enhance IL-6 production by LPS-stimulated peritoneal macrophages, and this could be explained by its suppression of IL-10 production by this same population of cells. In addition to its effects on monokine synthesis, IL-10 also induces a significant change in morphology in IFN-gamma-stimulated peritoneal macrophages. The potent action of IL-10 on the macrophage, particularly at the level of monokine production, supports an important role for this cytokine not only in the regulation of T cell responses but also in acute inflammatory responses.