Interferon-gamma production and host protective response against Mycobacterium tuberculosis in mice lacking both IL-12p40 and IL-18

Interferon (IFN)-gamma plays an essential role in host defense against infection with Mycobacterium tuberculosis, and its synthesis is critically regulated by interleukin (IL)-12, IL-18 and the recently identified IL-23. The present study was designed to determine the roles of these cytokines in IFN-gamma-mediated host defenses against M. tuberculosis. For this purpose, we compared host protective responses in IL-12p40 and IL-18 double-knockout (DKO) mice (which lacked both IL-12/IL-18 and also IL-23) and IFN-gamma gene-disrupted (GKO) mice. DKO mice were more resistant to the infection than GKO mice, as indicated by their extended survival and reduced live colony numbers in spleen, liver and lung. IFN-gamma was detected by ELISA in liver and lung homogenates, but not in spleen and serum, and in all organs by RT-PCR in DKO mice at comparable or reduced levels to those in wild-type mice. IFN-gamma production was reduced by depletion of CD4+ T cells, but not of natural killer (NK), NKT, gammadeltaT and dendritic cells. Neutralization of IFN-gamma or TNF-alpha by specific monoclonal antibodies (mAbs) significantly shortened the survival time of the infected DKO mice. Furthermore, anti-TNF-alpha mAb partially attenuated IFN-gamma synthesis in the liver of these mice. Finally, the expression level of inducible nitric oxide synthase (iNOS) mRNA in the spleen, liver and lung was considerable in DKO mice but only marginal or undetected in GKO mice. Our results indicate the presence of IL-12-, IL-18- and IL-23-independent host protective responses against mycobacterial infection mediated by IFN-gamma, which was secreted from helper T cells.     

Endogenous cytokines during a lethal infection with Listeria monocytogenes in mice

It has been demonstrated that endogenous cytokines including gamma interferon (IFN-gamma), tumour necrosis factor-alpha (TNF-alpha), and interleukin-6 (IL-6) play protective roles but that IL-4 and IL-10 play detrimental roles in nonlethal Listeria monocytogenes infection in mice. In this paper, we studied the roles of endogenous cytokines in a lethal infection with L. monocytogenes in mice. TNF-alpha and IL-6 titres in the bloodstreams, spleens and livers paralleled bacterial numbers in the organs, and both these cytokines and the bacterial numbers peaked just before the mice died. The high titres of TNF-alpha notably detected in the circulation in lethal infection were different from those in nonlethal infection. The maximum production of IFN-gamma was observed before the peaks of TNF-alpha and IL-6, and IFN-gamma almost disappeared from the bloodstreams and organs just before the mice died. No notable difference of IFN-gamma titres between lethal infection and nonlethal infection in the specimens obtained from mice was observed. IL-10 was also detected in the bloodstreams earlier than the peaks of TNF-alpha and IL-6 during lethal infection, while IL-4 was never detected in the sera. The administration of monoclonal antibodies (mAbs) against TNF-alpha, IFN-gamma, IL-6, IL-4 or IL-10 failed to rescue mice from lethal L. monocytogenes infection, whereas anti-TNF-alpha mAb and anti-IFN-gamma mAb prevented mice from lethality by high-dose endotoxin shock. These results suggest that lethality in L. monocytogenes infection might not be determined solely by these cytokines.     

Regulation of lipopolysaccharide-induced NO synthase expression in the major organs in a mouse model. The roles of endogenous interferon-gamma, tumor necrosis factor-alpha and interleukin-10

Elevated NO production mediated by activation of the enzyme iNOS is thought to play a central role in the development of tissue damage observed during septic shock. IFN-gamma, TNF-alpha and IL-10 have been shown to be involved in the regulation of LPS-induced serum levels of the NO-oxidation products nitrate and nitrite. Therefore, in the present study, we investigated the role of endogenous IFN-gamma, TNF-alpha and IL-10 in the regulation of LPS-induced tissue iNOS expression in the major organs. To this end, mice were pre-treated with anti-IFN-gamma, anti-TNF-alpha, anti-IL-10 monoclonal antibodies, or combinations of these, two hours before intraperitoneal LPS-challenge. Immunohistochemical staining for iNOS and determination of iNOS activity indicated that iNOS expression was mainly upregulated in the small intestine, lung and heart, and that IFN-gamma, TNF-alpha as well as IL-10 are involved in the regulation of iNOS expression and enzyme activity. Whereas blocking either IFN-gamma or TNF-alpha did not affect iNOS expression, iNOS enzymatic activity seems to be inhibited. In contrast, blocking both mediators nearly completely prevents iNOS expression after LPS challenge, suggesting that the presence of either IFN-gamma or TNF-alpha is essential for LPS-induced iNOS expression in these organs. Combined treatment of these monoclonal antibodies revealed that whereas on the one hand IL-10 inhibits LPS-induced iNOS expression, on the other hand IL-10 or an IL-10 inducible factor is also involved in the upregulation of iNOS expression after LPS challenge.     

Disassociation between the in vitro and in vivo effects of nitric oxide on a neurotropic murine coronavirus.

Intranasal inoculation of the neuroattenuated OBLV60 strain of mouse hepatitis virus results in infection of mitral neurons in the olfactory bulb, followed by spread along olfactory and limbic pathways to the brain. Immunocompetent BALB/c mice were able to clear virus by 11 days postinfection (p.i.). Gamma interferon (IFN-gamma) may play a role in clearance of OBLV60 from infected immunocompetent BALB/c mice through a nonlytic mechanism. Among the variety of immunomodulatory activities of IFN-gamma is the induction of expression of inducible nitric oxide synthase (iNOS), an enzyme responsible for the production of nitric oxide (NO). Studies were undertaken to investigate the role of IFN-gamma and NO in host defense and clearance of OBLV60 from the central nervous system (CNS). Exposure of OBLV60-infected OBL21a cells, a mouse neuronal cell line, to the NO-generating compound S-nitroso-L-acetyl penicillamine resulted in a significant decrease in viral replication, indicating that NO interfered with viral replication. Furthermore, infection of IFN-gamma knockout (GKO) mice and athymic nude mice with OBLV60 resulted in low-level expression of iNOS mRNA and protein in the brains compared to that of OBLV60-infected BALB/c mice. Nude mice were unable to clear virus and eventually died between days 11 and 14 p.i. (B. D. Pearce, M. V. Hobbs, T. S. McGraw, and M. J. Buchmeier, J. Virol. 68:5483-5495, 1994); however, GKO mice survived infection and cleared virus by day 18 p.i. These data suggest that IFN-gamma production in the olfactory bulb contributed to but may not be essential for clearance of OBLV60 from the brain. In addition, treatment of OBLV60-infected BALB/c mice with aminoguanidine, a selective inhibitor of iNOS activity, did not result in any increase in mortality, and the mice cleared the virus by 11 days p.i. These data suggest that although NO was able to block replication of virus in vitro, expression of iNOS with NO release in vivo did not appear to be the determinant factor in clearance of OBLV60 from CNS neurons.     

Expression of inflammatory markers in pig amnion after intraamniotic infection with nonpathogenic or enteropathogenic<Emphasis Type="Italic">Escherichia coli</Emphasis>

The pig amnion was in vivo intraamniotically infected with E. coli for 10 h at 80-85 d of gestation either with the nonpathogenic O86 strain or enteropathogenic O55 strain. TNF-alpha, IL-10, IL-1beta and IFN-gamma were determined in amniotic fluids by ELISA, the expression of cytokines and some other inflammatory markers was determined by immunohistochemistry. Intraamniotic infection induced high levels of TNF-alpha in amniotic fluids which correlated with bacterial virulence whereas IL-10 was induced only by O86. The IL-1beta level did not increase significantly and was expressed in all infected membranes. IFN-gamma was negligible or absent. TNF-alpha, IL-12p40, calprotectin, HSP65 and gp91phox were found by immunohistochemistry only in amnion membranes infected with the enteropathogenic strain 055.     

Pretreatment with granulocyte-colony stimulating factor decreases lipopolysaccharide-induced interferon-gamma production in mice in association with the production of interleukin-18

We studied the effects of pretreatment with granulocyte-colony stimulating factor (G-CSF) on the production of pro- and anti-inflammatory cytokines induced by lipopolysaccharide (LPS). Mice received G-CSF or control saline once a day for 7 days or once at 1 h before the injection of LPS. Cytokines were measured by enzyme-linked immunosorbent assay or antibody-based electrochemiluminescence assay and cytokine mRNA was measured by RNAse protection assay. Mice pretreated with G-CSF for 7 days before LPS had lower serum levels of LPS-induced interferon-gamma (IFN-gamma) and higher levels of interleukin (IL)-6 and IL-10 than controls. G-CSF-pretreated mice also had lower mRNA levels of IFN-gamma and higher mRNA levels of IL-6 and IL-10 in the spleen and/or liver than controls. G-CSF-pretreated mice had serum levels of tumor necrosis factor, IL-12 p70 and IL-12 p40 similar to controls. G-CSF-pretreated mice had lower levels of spleen IL-18 than controls-serum IL-18 being undetectable in mice after LPS-and lower levels of IL-18 mRNA in the spleen. Mice pretreated with G-CSF 1 h before LPS had lower levels of serum IFN-gamma and spleen IL-18 than controls. G-CSF pretreatment alters the expression of LPS-induced cytokines with a decrease in pro-inflammatory IFN-gamma and an increase in anti-inflammatory IL-6 and IL-10. G-CSF decrease of IL-18 production may be a major mechanism explaining the effects of G-CSF on the production of IFN-gamma.     

Il-10 is a central regulator of cyclooxygenase-2 expression and prostaglandin production

IL-10 is a potent anti-inflammatory and immune regulatory cytokine. IL-10(-/-) mice produce exaggerated amounts of inflammatory cytokines when stimulated with LPS, indicating that endogenous IL-10 is a central regulator of inflammatory cytokine production in vivo. PGs are lipid mediators that are also produced in large amounts during the inflammatory response. To study the role of IL-10 in the regulation of PG production during the acute inflammatory response, we evaluated LPS-induced cyclooxygenase (COX) expression and PG production in wild-type (wt) and IL-10(-/-) mice. LPS-induced PGE(2) production from IL-10(-/-) spleen cells was 5.6-fold greater than that from wt spleen cells. LPS stimulation resulted in the induction of COX-2 mRNA and protein in both wt and IL-10(-/-) spleen cells; however, the magnitude of increase in COX-2 mRNA was 5.5-fold greater in IL-10(-/-) mice as compared with wt mice. COX-1 protein levels were not affected by LPS stimulation in either wt or IL-10(-/-) mice. Neutralization of IFN-gamma, TNF-alpha, or IL-12 markedly decreased the induction of COX-2 in IL-10(-/-) spleen cells, suggesting that increased inflammatory cytokine production mediates much of the COX-2 induction in IL-10(-/-) mice. Treatment of IL-10(-/-) mice with low doses of LPS resulted in a marked induction of COX-2 mRNA in the spleen, whereas wt mice had minimal expression of COX-2 mRNA. These findings indicate that, in addition to IL-10's central role in the regulation of inflammatory cytokines, endogenous IL-10 is an important regulator of PG production in the response to LPS.     

Cytokine and chemokine mRNA expression in neutrophils from CBA/NSlc mice infected with Plasmodium berghei ANKA that induces experimental cerebral malaria.

To investigate the role of neutrophils in experimental cerebral malaria (ECM), in a previous study we found that early neutrophil depletion prevented the development of ECM and down regulated the expression of Th1 cytokines in the brain. To further clarify the mechanisms responsible for these findings, in the present study, using RT-PCR, we examined the expression of cytokine and chemokine mRNAs in neutrophils and macrophages after PbA infection. We found that, after infection, neutrophils not only expressed cytokines IL-2, IL-12p40, IL-18, IFN-gamma and TNF-alpha mRNAs, but also mRNAs for Th1 chemoattractive chemokines, monokine-induced by IFN-gamma (MIG), macrophage-inflammatory protein-1alpha (MIP-1alpha) and IFN-gamma inducible protein-10 (IP-10). Neutrophil depletion down regulated the expression of IL-18 and MIG mRNAs in macrophages, but did not affect the expression of IFN-gamma, TNF-alpha, MIP-1alpha and IP-10 mRNAs. Therefore, this study confirms our hypothesis that neutrophils may play a role in the pathogenesis of ECM via their expression of cytokines or chemokines.     

Cold stress-induced modulation of inflammatory responses and intracerebral cytokine mRNA expression in acute murine toxoplasmosis

The effects of a physical stressor, cold water stress (CWS), within the central nervous system were investigated in the acute phase of infection with Toxoplasma gondii. Female BALB/c mice were subjected to CWS for 5 min each day for 8 days prior to oral infection with 20 cysts of the low virulent ME 49 strain. Animals were killed at 10-day intervals to detect inflammation, gliosis, and expression of intracerebral cytokine mRNAs. Zones of inflammation were detected by Nissl staining and gliosis by immunoreactivity to glial fibrillary acidic protein. Larger zones of inflammation and reactive astrogliosis were consistently observed in mice subjected to CWS and infected (CWS +INF) compared to control infected (INF) mice. Expression of interleukin (IL)-1beta, IL-2, IL-12, interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, and inducible nitric oxide synthase (iNOS) were decreased in CWS+INF mice at 10 days postinoculation (PI), followed by a gradual increase after day 20 PI. This was in contrast to increased expression of these cytokines at 10 days PI in INF mice with a gradual decline thereafter. Inflammation and astrogliosis in CWS+INF mice were associated with an increased expression of IL-1beta, IL-6, IL-12, and TNF-alpha between 20 and 30 days PI. These findings correlated with the continuous gene expression of tachyzoite surface antigen (SAG)-1 mRNA in CWS+INF mice compared to its sharp decline in INF mice after 20 days PI. These results suggest that CWS delays regulation and control of intracerebral Toxoplasma gondii during acute infection in BALB/c mice by decreasing the early expression of IFN-gamma, IL-2, TNF-alpha, iNOS, IL-1beta, and IL-12, while increasing the expression of IL-6, a counterregulatory cytokine.     

The role of IL-18 in blood-stage immunity against murine malaria Plasmodium yoelii 265 and Plasmodium berghei ANKA

A possible protective role of IL-18 in host defense against blood-stage murine malarial infection was studied in BALB/c mice using a nonlethal strain, Plasmodium yoelii 265, and a lethal strain, Plasmodium berghei ANKA. Infection induced an increase in mRNA expression of IL-18, IL-12p40, IFN-gamma, and TNF-alpha in the case of P. yoelii 265 and an increase of IL-18, IL-12p40, and IFN-gamma in the case of P. berghei ANKA. The timing of mRNA expression of IL-18 in both cases was consistent with a role in the induction of IFN-gamma protein expression. Histological examination of spleen and liver tissues from infected controls treated with PBS showed poor cellular inflammatory reaction, massive necrosis, a large number of infected parasitized RBCs, and severe deposition of hemozoin pigment. In contrast, IL-18-treated infected mice showed massive infiltration of inflammatory cells consisting of mononuclear cells and Kupffer cells, decreased necrosis, and decreased deposition of the pigment hemozoin. Treatment with rIL-18 increased serum IFN-gamma levels in mice infected with both parasites, delayed onset of parasitemia, conferred a protective effect, and thus increased survival rate of infected mice. Administration of neutralizing anti-IL-18 Ab exacerbated infection, impaired host resistance and shortened the mean survival of mice infected with P. berghei ANKA. Furthermore, IL-18 knockout mice were more susceptible to P. berghei ANKA than were wild-type C57BL/6 mice. These data suggest that IL-18 plays a protective role in host defense by enhancing IFN-gamma production during blood-stage infection by murine malaria.     

Cytokine milieu of atopic dermatitis,as compared to psoriasis,skin prevents induction of innate immune response genes

Atopic dermatitis (AD) and psoriasis are the two most common chronic skin diseases. However patients with AD, but not psoriasis, suffer from frequent skin infections. To understand the molecular basis for this phenomenon, skin biopsies from AD and psoriasis patients were analyzed using GeneChip microarrays. The expression of innate immune response genes, human beta defensin (HBD)-2, IL-8, and inducible NO synthetase (iNOS) was found to be decreased in AD, as compared with psoriasis, skin (HBD-2, p = 0.00021; IL-8, p = 0.044; iNOS, p = 0.016). Decreased expression of the novel antimicrobial peptide, HBD-3, was demonstrated at the mRNA level by real-time PCR (p = 0.0002) and at the protein level by immunohistochemistry (p = 0.0005). By real-time PCR, our data confirmed that AD, as compared with psoriasis, is associated with elevated skin production of Th2 cytokines and low levels of proinflammatory cytokines such as TNF-alpha, IFN-gamma, and IL-1beta. Because HBD-2, IL-8, and iNOS are known to be inhibited by Th2 cytokines, we examined the effects of IL-4 and IL-13 on HBD-3 expression in keratinocyte culture in vitro. We found that IL-13 and IL-4 inhibited TNF-alpha- and IFN-gamma-induced HBD-3 production. These studies indicate that decreased expression of a constellation of antimicrobial genes occurs as the result of local up-regulation of Th2 cytokines and the lack of elevated amounts of TNF-alpha and IFN-gamma under inflammatory conditions in AD skin. These observations could explain the increased susceptibility of AD skin to microorganisms, and suggest a new fundamental rule that may explain the mechanism for frequent infection in other Th2 cytokine-mediated diseases.     

The H2(b) haplotype modifies the production of pro-inflammatory cytokines: implications for immunopathology

Congenic strains of mice which differ only in their H2 haplotype were used to examine the effects of MHC genes on production of pro-inflammatory cytokines, as we have shown previously that H2(b) mice produce low levels of T cell cytokines compared to congenic H2(k) and H2(d) mice. RNase protection assays were used to assess cytokine mRNA and cytokine protein was assessed by ELISA or bioassay. Concanavalin A or phorbol myristate acetate/calcium ionophore/anti-CD3 stimulation of spleen cells from H2(b) congenic mice induced less IL-1, IL-2, IFN-gamma and MIF mRNA and/or protein than the equivalent cells from H2(d) mice. However, following stimulation with lipopolysaccharide or phorbol myristate acetate/calcium ionophore, peritoneal cells from H2(b) mice synthesised significantly more IL-1 beta, TNF-alpha, TNFR and IFN-gamma protein and IFN-gamma mRNA than cells from congenic H2(k) or H2(d) mice. These differences were evident in congenic C57BL/10 and/or BALB/c strains. We suggest that the low IL-1 production in H2(b) spleen cultures is secondary to lower T cell activation. Evidence that the H2(b) haplotype carries an immunoregulatory allele which affects cytokine production warrants further investigation.     

Molecular mechanisms of high-dose antigen therapy in experimental autoimmune encephalomyelitis: rapid induction of Th1-type cytokines and inducible nitric oxide synthase

High-dose Ag administration induces apoptotic death of autoreactive T cells and is an effective therapy of experimental autoimmune diseases of the nervous system. To explore the role of cytokines in Ag-specific immunotherapy, we analyzed mRNA induction and protein expression for the proinflammatory cytokines TNF-alpha and IFN-gamma, the anti-inflammatory cytokine IL-10, and the cytokine-inducible NO synthase (iNOS) during high-dose Ag therapy of adoptive transfer experimental autoimmune encephalomyelitis (AT-EAE) in the Lewis rat. Using semiquantitative and competitive RT-PCR, we found 5- to 6-fold induction of TNF-alpha mRNA and 3-fold induction of IFN-gamma mRNA in the spinal cord that occurred within 1 h after i.v. injection of Ag and was accompanied by a 2-fold increase of iNOS mRNA. Both IFN-gamma and iNOS mRNA remained elevated for at least 6 h, whereas TNF-alpha mRNA was already down-regulated 6 h after Ag injection. A comparable time course was found for circulating serum levels of TNF-alpha and IFN-gamma. IL-10 mRNA levels did not change significantly following Ag injection. Neutralization of TNF-alpha by anti-TNF-alpha antiserum in vivo led to a significant decrease in the rate of T cell and oligodendrocyte apoptosis induced by high-dose Ag administration, but did not change the beneficial clinical effect of Ag therapy. Our data suggest profound activation of proinflammatory but not of anti-inflammatory cytokine gene expression by high-dose Ag injection. Functionally, TNF-alpha contributes to increased apoptosis of both autoaggressive T cells and oligodendrocytes in the target organ and may thereby play a dual role in this model of Ag-specific therapy of CNS autoimmune diseases.     

Interferon regulatory factor 1 in mycobacterial infection

In order to understand the role of IRF-1 in the development of murine tuberculosis in vivo, IRF-1 knockout mice were infected with Mycobacterium tuberculosis by placing them in the exposure chamber of an airborne infection apparatus. These knockout mice developed multifocal necrotic lesions in the lung, liver and spleen tissues and died of disseminated tuberculosis within 43 days of infection. Compared with the levels in wild-type mice, the pulmonary inducible NO synthase (iNOS) mRNA expression level was significantly lower, but IL-18 and IL-6 mRNA levels were higher. There was no statistically significant difference in the expression of IFN-gamma and TNF-alpha mRNA between the IRF-1 knockout and wild-type mice. IRF-1 is indirectly responsible for iNOS mRNA expression and plays an important role in the pathogenesis of murine tuberculosis.     

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.     

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.     

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.