Central role of endogenous gamma interferon in protective immunity against blood-stage Plasmodium chabaudi AS infection

The role of endogenous gamma interferon (IFN-gamma) in protective immunity against blood-stage Plasmodium chabaudi AS malaria was studied using IFN-gamma gene knockout (GKO) and wild-type (WT) C57BL/6 mice. Following infection with 10(6) parasitized erythrocytes, GKO mice developed significantly higher parasitemia during acute infection than WT mice and had severe mortality. In infected GKO mice, production of interleukin 12 (IL-12) p70 and tumor necrosis factor alpha in vivo and IL-12 p70 in vitro by splenic macrophages was significantly reduced compared to that in WT mice and the enhanced nitric oxide (NO) production observed in infected WT mice was completely absent. WT and GKO mice had comparable numbers of total nucleated spleen cells and B220(+) and Mac-1(+) spleen cells both before and after infection. Infected WT mice, however, had significantly more F4/80(+), NK1.1(+), and F4/80(+)Ia(+) spleen cells than infected GKO mice; male WT had more CD3(+) cells than male GKO mice. In comparison with those from WT mice, splenocytes from infected GKO mice had significantly higher proliferation in vitro in response to parasite antigen or concanavalin A stimulation and produced significantly higher levels of IL-10 in response to parasite antigen. Infected WT mice produced more parasite-specific immunoglobulin M (IgM), IgG2a, and IgG3 and less IgG1 than GKO mice. Significant gender differences in both GKO and WT mice in peak parasitemia levels, mortality, phenotypes of spleen cells, and proliferation of and cytokine production by splenocytes in vitro were apparent during infection. These results thus provide unequivocal evidence for the central role of endogenous IFN-gamma in the development of protective immunity against blood-stage P. chabaudi AS.     

Interferon-gamma and tumor necrosis factor-alpha determine resistance to Paracoccidioides brasiliensis infection in mice

To investigate the role of interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) in the resistance to Paracoccidioides brasiliensis (Pb) infection, mice with homologous disruption of the IFN-gamma (GKO) or TNF-alpha receptor p55 (p55KO) were infected with the parasite. GKO and p55KO, but not wild-type (WT) mice, were unable to control the growth of yeast cells and the mice succumbed to infection by days 16 and 90 after infection, respectively. Typical inflammatory granulomas were found only in WT mice. In contrast, knockout mice presented an inflammatory infiltrate composed of a few neutrophils, mononuclear, epithelioid, and multinuclear giant cells forming incipient granulomas in GKO mice and without granuloma formation in p55KO mice. Besides, both groups of knockout mice exhibited elevated numbers of yeast forms in agreement with colony-forming unit counts in organs. Compared with WT, splenocytes from infected GKO mice cultured with the Pb F1 fraction produced lower TNF-alpha levels, whereas leukocytes from infected p55KO mice produced similar amounts of TNF-alpha but higher levels of IFN-gamma. Moreover, splenocytes from infected WT mice produced higher levels of nitric oxide (NO) resulting in a lower T-cell proliferative response to Con A than uninfected WT, or infected p55KO and GKO mice. On the contrary, the addition of IFN-gamma to splenocytes from infected GKO mice resulted in higher NO production and lower T cell proliferation. Taken together, these findings suggests that endogenous TNF-alpha, acting through the p55 receptor, and IFN-gamma mediate resistance to Pb infection and induce NO production that determines marked T cell unresponsiveness.     

Role of interleukin-18 (IL-18) in mycobacterial infection in IL-18-gene-disrupted mice

Immunity to mycobacterial infection is closely linked to the emergence of T cells that secrete cytokines, gamma interferon (IFN-gamma), interleukin-12 (IL-12), and tumor necrosis factor alpha (TNF-alpha), resulting in macrophage activation and recruitment of circulating monocytes to initiate chronic granuloma formation. The cytokine that mediates macrophage activation is IFN-gamma, and, like IL-12, IL-18 was shown to activate Th1 cells and induce IFN-gamma production by these cells. In order to investigate the role of IL-18 in mycobacterial infection, IL-18-deficient mice were infected with Mycobacterium tuberculosis and Mycobacterium bovis BCG Pasteur, and their capacities to control bacterial growth, granuloma formation, cytokine secretion, and NO production were examined. These mice developed marked granulomatous, but not necrotic, lesions in their lungs and spleens. Compared with the levels in wild-type mice, the splenic IFN-gamma levels were low but the IL-12 levels were normal in IL-18-deficient mice. The reduced IFN-gamma production was not secondary to reduced induction of IL-12 production. The levels of NO production by peritoneal macrophages of IL-18-deficient and wild-type mice did not differ significantly. Granulomatous lesion development by IL-18-deficient mice was inhibited significantly by treatment with exogenous recombinant IL-18. Therefore, IL-18 is important for the generation of protective immunity to mycobacteria, and its main function is the induction of IFN-gamma expression.     

Intrapulmonary response to Histoplasma capsulatum in gamma interferon knockout mice.

We examined the immunobiological responses to Histoplasma capsulatum in lungs of gamma interferon (IFN-gamma) knockout mice (GKO mice). Naive GKO mice succumbed by day 9 to intranasal challenge with 2.5 x 10(6) yeasts, whereas all wild-type (WT) mice survived for 45 days. Compared to lungs of WT mice, the lungs of acutely infected GKO mice exhibited dramatically elevated numbers of CFU in lungs and significantly higher levels of tumor necrosis factor alpha (TNF-alpha) and granulocyte-macrophage colony-stimulating factor (GM-CSF) but not interleukin-12 (IL-12) or IL-4. To determine if IFN-gamma is necessary in reexposure histoplasmosis, GKO and WT mice were inoculated with 10(4) yeasts intranasally and given amphotericin B for 3 weeks. Six weeks later, mice were rechallenged with 2.5 x 10(6) yeasts. All GKO mice died by day 6, whereas all WT mice survived for 45 days. Lungs of GKO mice contained substantially elevated numbers of CFU and higher TNF-alpha and GM-CSF levels but not IL-12 or IL-4. Thus, IFN-gamma is requisite for control of pulmonary histoplasmosis in naive and reexposed mice.     

Roles of interleukin-12 and gamma interferon in murine Chlamydia pneumoniae infection

BALB/c and strain 129 mice infected intranasally with Chlamydia pneumoniae displayed a moderate-to-severe inflammation in the lungs and produced interleukin-12 (IL-12), gamma interferon (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), and IL-10, with peak levels on days 1 to 3 postinfection (p.i.), returning to basal levels by day 16 p.i. Anti-IL-12 treatment resulted in less-severe pathological changes but higher bacterial titers on days 3 and 7 p.i. By day 16 p.i., the inflammatory responses of control antibody-treated mice subsided. The bacterial titers of both anti-IL-12- and control antibody-treated mice decreased within 3 weeks to marginally detectable levels. Anti-IL-12 treatment significantly reduced lung IFN-gamma production and in vitro spleen cell IFN-gamma production in response to either C. pneumoniae or concanavalin A. In gamma-irradiated infected mice, cytokine production was delayed, and this delay correlated with high bacterial titers in the lungs. Following C. pneumoniae infection, 129 mice lacking the IFN-gamma receptor alpha chain gene (G129 mice) produced similar IL-12 levels and exhibited similarly severe pathological changes but had higher bacterial titers than 129 mice. However, by day 45 p.i., bacterial titers became undetectable in both wild-type 129 and G129 mice. Thus, during C. pneumoniae lung infection, IL-12, more than IFN-gamma, plays a role in pulmonary-cell infiltration. IFN-gamma and IL-12, acting mostly through its induction of IFN-gamma and Th1 responses, play an important role in controlling acute C. pneumoniae infection in the lungs, but eventually all mice control the infection to undetectable levels by IL-12- and IFN-gamma-independent mechanisms.     

Intracellular growth of Trypanosoma cruzi in cardiac myocytes is inhibited by cytokine-induced nitric oxide release

The effect of interleukin-1beta (IL-1beta), tumor necrosis factor alpha (TNF-alpha), and gamma interferon (IFN-gamma) on Trypanosoma cruzi multiplication and nitric oxide (NO) production in cardiac myocytes was investigated. Cardiac myocyte cultures were obtained from neonatal Wistar rat hearts, infected with T. cruzi, and treated with IL-1beta, TNF-alpha, IFN-gamma, or N-monomethyl-L-arginine (L-NAME) for 72 h. Parasite growth was calculated from the number of infected cells in Giemsa-stained smears. Nitric oxide production was determined with the Griess reagent. Inducible nitric oxide synthase (iNOS) expression by cardiac myocytes was detected by Western blot. The results showed that the percentages of cardiac myocytes containing T. cruzi amastigotes in cytokine-treated cultures were significantly lower than in nontreated cultures. The addition of L-NAME reversed the inhibitory effect on parasite growth of IL-1beta and TNF-alpha but not of IFN-gamma. Nitrite levels released by T. cruzi-infected and noninfected cardiac myocyte cultures after 72 h of stimulation with IL-1beta were significantly higher than those produced upon treatment with TNF-alpha, IFN-gamma, or medium alone, regardless of the infection status. Nitrite levels in TNF-alpha-stimulated infected cultures were significantly higher than in untreated infected cultures and TNF-alpha-treated noninfected cultures. L-NAME inhibited IL-1beta- but not TNF-alpha-induced NO production, indicating the presence of iNOS-dependent and iNOS-independent mechanisms for NO formation in this experimental system. iNOS expression was detected in infected and noninfected cardiac myocytes stimulated with IL-1 beta and TNF-alpha but not with IFN-gamma. These results suggest an important role for cardiac myocytes and locally secreted cytokines in the control of parasite multiplication in T. cruzi-induced myocarditis.     

Impaired macrophage responses may contribute to exacerbation of blood-stage Plasmodium chabaudi chabaudi malaria in interleukin-12-deficient mice.

Aiming to clarify the role of endogenous interleukin-12 (IL-12) in protective immunity against blood stages of Plasmodium chabaudi chabaudi (AS), we evaluated the course of infection in IL-12p40 gene knockout (IL-12p40KO) and wild-type (WT) C57BL/6 mice, focusing (1) on the ability of T cells to develop adequate type 1 responses and (2) on the potentiality of macrophages to respond to parasites, interferon-gamma (IFN-gamma), or both. We observed that IL-12p40KO mice develop significantly higher parasitemias during the acute infection, although mice from both groups clear the parasites within a month and similarly eliminate a secondary challenge. Thus, fully protective immunity to P. c. chabaudi can be generated in the absence of IL-12. However, this cytokine may promote parasite control during the early phase of infection. The increased acute parasitemia of IL-12p40KO mice was associated with both impaired IFN-gamma and nitric oxide (NO) response by spleen cells. Because stimulation with recombinant IFN-gamma (rIFN-gamma) failed to improve the NO response in IL-12p40KO macrophages, we investigated whether these cells have an intrinsic defect. Analysis of peritoneal macrophages revealed that IL-12p40KO cells produce higher levels of transforming growth factor-beta1 (TGF-beta1) compared with WT cells and respond to infected erythrocytes or rIFN-gamma by releasing little NO. Moreover, IL-12p40KO macrophages had a severely impaired ability to internalize opsonized infected erythrocytes, suggesting that the low effector profile assumed by these cells may compromise antibody-mediated immunity. Taken together, our results support the idea that the absence of IL-12p40 not only affects IFN-gamma production but also has deep consequences in macrophage effector functions that may contribute to exacerbation of the early phase of P. c. chabaudi malaria.     

Both IL-12 and IL-18 contribute to small intestinal Th1-type immunopathology following oral infection with Toxoplasma gondii, but IL-12 is dominant over IL-18 in parasite control

Oral infection of C57BL/6 mice with Toxoplasma gondii results in small intestinal Th1-type immunopathology mediated by local production of IFN-gamma, TNF-alpha, and NO. To analyze whether the proinflammatory cytokines IL-12 and IL-18 play a role in the induction of immunopathology, IL-12p35/p40(-/-) and IL-18(-/-) mice were orally infected with T. gondii. Wild-type mice developed massive necrosis in their small intestines and died 7-10 days post infection. Even though IL-12p35/40(-/-) mice did not develop the necrosis they all died between day 9 and 11 after infection. In contrast, 50% of IL-18(-/-) mice died during the acute phase of infection. Compared to wild-type mice, IL-12p35/p40(-/-) but not IL-18(-/-) mice showed significantly higher parasite numbers in their small intestines and significantly higher numbers of parasite-associated inflammatory foci in their livers. IFN-gamma production was similar in infected wild-type and IL-18(-/-) mice but significantly decreased in IL-12p35/p40(-/-) mice. Treatment of mice with anti-IL-12- or anti-IL-18 antibodies after infection prevented the development of intestinal necrosis. These results reveal that both IL-12 and IL-18 play an important role in the development of intestinal immunopathology following oral infection with T. gondii. However, IL-12 is dominant over IL-18 in the host defense against parasite replication. Therefore, neutralization of IL-18 (rather than TNF-alpha, IL-12, and IFN-gamma) may be a safe strategy for the treatment of Th1-associated diseases.     

Protective role of interleukin-1 in mycobacterial infection in IL-1 alpha/beta double-knockout mice

To understand the role of the proinflammatory cytokine interleukin-1 (IL-1) in mycobacterial inflammation, IL-1 alpha/beta double-knockout (KO) mice were produced. These mice were infected with either Mycobacterium tuberculosis H37Rv by the airborne route using an airborne infection apparatus, and their capacities to control mycobacterial growth, granuloma formation, cytokine, and nitric oxide (NO) production were examined. The IL-1 alpha/beta mice developed significantly larger (p < 0.01) granulomatous, but not necrotic, lesions in their lungs than wild-type (WT) mice after infection with H37Rv. Inflammatory lesions, but not granulomas, were observed in spleen and liver tissues from both IL-1 alpha/beta KO and wild-type mice. Granulomatous lesion development in IL-1 alpha/beta KO mice was not significantly inhibited by treatment with exogenous recombinant IL-1 alpha/beta. Compared with wild-type mice, splenic IFN-gamma and IL-12 levels were within the normal range. NO production by cultured alveolar macrophages from IL-1 alpha/beta KO mice was lower than in wild-type mice but were increased by the addition of recombinant IL-1 alpha/beta. Our data clearly indicate that IL-1 is important for the generation of early-phase protective immunity against mycobacterial infection.     

Role of endogenous IFN-gamma in macrophage programming induced by IL-12 and IL-18.

Besides the established role of interleukin-12 (IL-12) and IL-18 on interferon-gamma (IFN-gamma) production by natural killer (NK), T, and B cells, the effects of these cytokines on macrophages are largely unknown. Here, we investigated the role of IL-12/IL-18 on nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha) production by CD11b(+) adherent peritoneal cells, focusing on the involvement of endogenously produced IFN-gamma. C57BL/6 cells released substantial amounts of NO when stimulated with IFN-gamma or lipopolysaccharide (LPS), but failed to respond to IL-12 or IL-18 or both. However, IL-12/IL-18 pretreatment was able to program these cells to release 6-8-fold more NO and TNF-alpha in response to LPS or Trypanosoma cruzi stimulation, with NO levels directly correlating with macrophage resistance to intracellular parasite growth. Analysis of IL-12/IL-18-primed cells from mice deficient in IFN-gamma, IFNGR, and IFN regulatory factor-1 (IRF-1) revealed that these molecules were essential for LPS-induced NO release, but TNF-alpha production was IFN-gamma independent. Conversely, the myeloid differentiation factor 88 (MyD88)-dependent pathway was indispensable for IL-12/IL-18-programmed LPS-induced TNF-alpha production, but not for NO release. Contaminant T and NK cells largely modulated the IL-12/IL-18 programming of LPS-induced NO response through IFN-gamma secretion. Nevertheless, a small population of IFN-gamma(+) cells with a macrophage phenotype was also identified, particularly in the peritoneum of chronically T. cruzi-infected mice, reinforcing the notion that macrophages can be an alternative source of IFN-gamma. Taken together, our data contribute to elucidate the molecular basis of the IL-12/IL-18 autocrine pathway of macrophage activation, showing that endogenous IFN-gamma plays an important role in programming the NO response, whereas the TNF-alpha response occurs through an IFN-gamma-independent pathway.     

Respiratory tract infection with Mycoplasma pneumoniae in interleukin-12 knockout mice results in improved bacterial clearance and reduced pulmonary inflammation

Mycoplasma pneumoniae is a leading cause of pneumonia and is associated with asthma. Evidence links M. pneumoniae respiratory disease severity with interleukin-12 (IL-12) concentration in respiratory secretions. We evaluated the microbiologic, inflammatory, and pulmonary function indices of M. pneumoniae pneumonia in IL-12 (p35) knockout (KO) mice and wild-type (WT) mice to determine the role of IL-12 in M. pneumoniae respiratory disease. Eight-week-old wild-type BALB/c mice and 8-week-old IL-12 (p35) KO BALB/c mice were inoculated once intranasally with 10(7) CFU of M. pneumoniae. Mice were evaluated at days 2, 4, and 7 after inoculation. Outcome variables included quantitative bronchoalveolar lavage (BAL) M. pneumoniae culture, lung histopathologic scores (HPS), BAL cytokine concentrations determined by enzyme-linked immunosorbent assay (tumor necrosis factor alpha [TNF-alpha], gamma interferon [IFN-gamma], IL-1beta, IL-2, IL-4, IL-5, IL-6, IL-10, and granulocyte-macrophage colony-stimulating factor) and plethysmography, before and after methacholine, to assess airway obstruction (AO) and airway hyperreactivity (AHR). IL-12 (p35) KO mice infected with M. pneumoniae were found to have significantly lower BAL M. pneumoniae concentrations compared with M. pneumoniae-infected WT mice. Lung HPS and the parenchymal pneumonia subscores (neutrophilic alveolar infiltrate), as well as AO, were significantly lower in infected KO mice. No difference was found for AHR. Infected KO mice had significantly lower BAL concentrations of IFN-gamma than WT mice; a trend toward lower BAL concentrations was observed for IL-10 (P = 0.065) and TNF-alpha (P = 0.078). No differences were found for IL-1beta, IL-2, IL-4, IL-5, or IL-6. The lack of IL-12 in experimental M. pneumoniae pneumonia was associated with less severe pulmonary disease and more rapid microbiologic and histologic resolution.     

Production of gamma interferon by natural killer cells from Toxoplasma gondii-infected SCID mice: regulation by interleukin-10, interleukin-12, and tumor necrosis factor alpha.

Previous studies of mice have implicated natural killer (NK) cells as mediators of protective activity against Toxoplasma gondii through their production of gamma interferon (IFN-gamma). In the present study, we have compared NK-cell activity in infected and uninfected SCID mice. Our data reveal that infection results in increased levels of IFN-gamma in serum and elevated NK-cell activity but that these NK cells were not cytotoxic for T. gondii-infected P815 cells. Treatment with anti-IFN-gamma antibody abrogated the increase in NK-cell activity and resulted in earlier mortality of infected mice. In vivo treatment with anti-asialo GM1 antiserum reduced NK cell activity and levels of IFN-gamma in serum but did not alter time to death. Spleen cells from infected mice produced higher levels of IFN-gamma than those from uninfected mice when stimulated in vitro with live T. gondii or parasite antigen preparations. Further analysis revealed that interleukin 10 (IL-10) inhibited, whereas tumor necrosis factor alpha (TNF-alpha) and IL-12 enhanced, IFN-gamma production by spleen cells from infected or uninfected mice. The combination of IL-12 and TNF-alpha induced higher levels of IFN-gamma from whole spleen cells of infected mice than from those of uninfected mice. Depletion of the adherent cell population from the spleen cells of infected mice led to a significant reduction in the levels of IFN-gamma produced after stimulation with IL-12 plus TNF-alpha. Similar results did not occur with cells from uninfected mice. These data indicate that other cytokines produced by the adherent cell population from infected mice may be involved in maximal production of IFN-gamma by NK cells stimulated with IL-12 and TNF-alpha. To assess the importance of endogenous IL-12, a polyclonal anti-IL-12 was administered to infected SCID mice. This treatment led to earlier mortality, indicating that endogenous IL-12 mediates resistance to T. gondii.     

Pulmonary inflammation induced by Pseudomonas aeruginosa lipopolysaccharide, phospholipase C, and exotoxin A: role of interferon regulatory factor 1

Chronic pulmonary infection with Pseudomonas aeruginosa is common in cystic fibrosis (CF) patients. P. aeruginosa lipopolysaccharide (LPS), phosholipase C (PLC), and exotoxin A (ETA) were evaluated for their ability to induce pulmonary inflammation in mice following intranasal inoculation. Both LPS and PLC induced high levels of tumor necrosis factor alpha (TNF-alpha), interleukin 1 beta (IL-1 beta-6, gamma interferon (IFN-gamma), MIP-1 alpha MIP-2 in the lungs but did not affect IL-18 levels. ETA did not induce TNF-alpha and was a weak inducer of IL-1 beta, IL-6, macrophage inflammatory protein 1 alpha (MIP-1 alpha), and MIP-2. Remarkably, ETA reduced constitutive lung IL-18 levels. LPS was the only factor inducing IFN-gamma. LPS, PLC, and ETA all induced cell infiltration in the lungs. The role of interferon regulatory factor-1 (IRF-1) in pulmonary inflammation induced by LPS, PLC, and ETA was evaluated. When inoculated with LPS, IRF-1 gene knockout (IRF-1 KO) mice produced lower levels of TNF-alpha, IL-1 beta, and IFN-gamma than did wild-type (WT) mice. Similarly, a milder effect of ETA on IL-1 beta and IL-18 was observed for IRF-1 KO than for WT mice. In contrast, the cytokine response to PLC did not differ between WT and IRF-1 KO mice. Accordingly, LPS and ETA, but not PLC, induced expression of IRF-1 mRNA. IRF-1 deficiency had no effect on MIP-1 alpha and MIP-2 levels and on cell infiltration induced by LPS, PLC, or ETA. Flow cytometric evaluation of lung mononuclear cells revealed strongly reduced percentages of CD8(+) and NK cells in IRF-1 KO mice compared to percentages observed for WT mice. These data indicate that different virulence factors from P. aeruginosa induce pulmonary inflammation in vivo and that IRF-1 is involved in some of the cytokine responses to LPS and ETA.     

In vivo clearance of an intracellular bacterium, Francisella tularensis LVS, is dependent on the p40 subunit of interleukin-12 (IL-12) but not on IL-12 p70

To determine the role of interleukin-12 (IL-12) in primary and secondary immunity to a model intracellular bacterium, we have comprehensively evaluated infection with Francisella tularensis LVS in three murine models of IL-12 deficiency. Mice lacking the p40 protein of IL-12 (p40 knockout [KO] mice) and mice treated in vivo with neutralizing anti-IL-12 antibodies survived large doses of primary and secondary LVS infection but never cleared bacteria and exhibited a chronic infection. In dramatic contrast, mice lacking the p35 protein (p35 KO mice) of heterodimeric IL-12 readily survived large doses of primary sublethal LVS infection as well as maximal secondary lethal challenge, with only a slight delay in clearance of bacteria. LVS-immune wild-type (WT) lymphocytes produced large amounts of gamma interferon (IFN-gamma), but p35 KO and p40 KO lymphocytes produced much less; nonetheless, similar amounts of NO were found in all cultures containing immune lymphocytes, and all immune lymphocytes were equally capable of controlling intracellular growth of LVS in vitro. Purified CD4(+) and CD8(+) T cells from both WT and p40 KO mice controlled intracellular growth, even though T cells from WT mice produced much more IFN-gamma than those from p40 KO mice, and p40 KO T cells did not adopt a Th2 phenotype. Thus, while IL-12 p70 stimulation of IFN-gamma production may be important for bacteriostasis, IL-12 p70 is not necessary for appropriate development of LVS-immune T cells that are capable of controlling intracellular bacterial growth and for clearance of primary or secondary LVS infection. Instead, an additional mechanism dependent on the IL-12 p40 protein, either alone or in another complex such as the newly discovered heterodimer IL-23, appears to be responsible for actual clearance of this intracellular bacterium.     

Early increase in mRNA levels of pro-inflammatory cytokines and their interactions in the mouse hippocampus after transient global ischemia

There is convincing evidence that cytokines are involved in the inflammatory response following cerebral ischemia, but the interactions among the pro-inflammatory cytokines tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta and IL-6 in the early stage of ischemic reperfusion are not yet completely understood. In this study, we examined the early mRNA expressions of pro-inflammatory cytokines in the ischemic hippocampus after 30 min of bilateral common carotid artery occlusion in C57BL/6J wild-type (WT) and TNF-alpha, IL-1alpha/beta or IL-6 gene knockout (KO) mice utilizing real-time polymerase chain reaction. The mRNA expressions of the pro-inflammatory cytokines TNF-alpha, IL-6 and IL-1beta were significantly induced in ischemic WT mice compared with in the sham-operated mice. These increases peaked at 3 to 24 h for TNF-alpha, at 12 h for IL-1beta, and at 6 to 24 h for IL-6 after ischemia. The pattern of temporal expression of the cytokine mRNAs in ischemic gene KO mice, however, differed from that in WT mice. The TNF-alpha mRNA expression showed a similar temporal expression pattern in IL-6 KO mice compared to in WT mice following ischemic reperfusion, and the levels at all time points were lower than in WT mice. The IL-1beta mRNA level was very low in ischemic TNF-alpha KO mice and IL-6 KO mice in spite of a small peak observed in both at 24 h. The IL-6 mRNA level was significantly upregulated at all time points in both ischemic WT and TNF-alpha KO mice; however, the peak was delayed by 12-h in IL-1alpha/beta KO mice. In conclusion, the present study indicates that the rapid increases in cytokine levels are interdependent, interactive, and possibly modulate each other in the mouse hippocampus after transient global ischemia.     

Intrathecal production of interleukin-12 and gamma interferon in patients with bacterial meningitis.

To assess the role of interleukin-12 (IL-12) and gamma interferon (IFN-gamma) in children with bacterial meningitis, bioactive IL-12 (p70) and the inactive subunit p40 and IFN-gamma were measured in serum and cerebrospinal fluid (CSF) from 35 children with bacterial meningitis and 10 control subjects. The production of IFN-gamma is induced by IL-12 with tumor necrosis factor alpha (TNF-alpha) as a costimulator and inhibited by IL-10. CSF concentrations of IL-12 p40 as well as those of IFN-gamma were markedly elevated, whereas IL-12 p70 was hardly detectable. Detectable CSF levels of IFN-gamma correlated positively with IL-12 p40 (r = 0.40, P = 0.02) and TNF-alpha (r = 0.46, P = 0.04) but not with IL-6, IL-8, or IL-10. In contrast to CSF levels of TNF-alpha, IL-12, and IL-10, those of IFN-gamma were significantly higher in patients with pneumococcal meningitis than in children with meningitis caused by Haemophilus influenzae and Neisseria meningitidis, presumably because of a high CSF TNF-alpha/IL-10 ratio in the former. We suggest that IL-12- and TNF-alpha-induced IFN-gamma production may contribute to the natural immunity against microorganisms in the CSF compartment during the acute phase of bacterial meningitis.     

Elevated interferon gamma expression in the central nervous system of tumour necrosis factor receptor 1-deficient mice with experimental autoimmune encephalomyelitis

Inflammation in the central nervous system (CNS) can be studied in experimental autoimmune encephalomyelitis (EAE). The proinflammatory cytokines interferon-gamma (IFN-gamma) and tumour necrosis factor (TNF) are implicated in EAE pathogenesis. Signals through the type 1 TNF receptor (TNFR1) are required for severe EAE to develop, whereas deficiency in IFN-gamma or its receptor result in more severe EAE. We investigated IFN-gamma expression in TNFR1-deficient (TNFR1-/-) mice. We describe here that there were more IFN-gamma-secreting T cells present in the CNS of TNFR1-/- mice during EAE compared to wild-type (WT) mice, despite that clinical symptoms were mild, with delayed onset. There was greater expression of IL-12/23p40 by antigen-presenting cells in these mice, and in vitro, TNFR1-/- antigen-presenting cells induced greater secretion of IFN-gamma but not interleukin (IL)-17 when cultured with primed T cells than did WT antigen presenting cells. TNFR1-/- mice with EAE had significantly higher expression of CXCL10 mRNA (but not CCL5 mRNA) in the CNS compared to WT mice with EAE. These data demonstrate that IFN-gamma expression is enhanced in the CNS of TNFR1-/- mice with EAE and suggest that IFN-gamma levels do not necessarily correlate with EAE severity.     

Enhanced gamma interferon production through activation of Valpha14(+) natural killer T cells by alpha-galactosylceramide in interleukin-18-deficient mice with systemic cryptococcosis

We showed recently that activation of Valpha14(+) natural killer T cells (NKT cells) by alpha-galactosylceramide (alpha-GalCer) resulted in increased gamma interferon (IFN-gamma) production and host resistance to intravenous infection with Cryptococcus neoformans. In other studies, interleukin-18 (IL-18) activated NKT cells in collaboration with IL-12, suggesting the possible contribution of this cytokine to alpha-GalCer-induced IFN-gamma synthesis. Here we examined the role of IL-18 in alpha-GalCer-induced Th1 response by using IL-18KO mice with this infection. In these mice, levels of IFN-gamma in serum and its synthesis in vitro by spleen cells stimulated with live organisms were not reduced, but rather enhanced, compared to those in wild-type (WT) mice, while such production was completely absent in IL-12KO mice. The enhanced production of IFN-gamma correlated with increased IL-12 synthesis but not with reduced production of IL-4, which was rather increased. IFN-gamma synthesis in IL-18KO mice was abolished by neutralizing anti-IL-12 antibody and significantly inhibited by neutralization of endogenous IL-4 with a specific monoclonal antibody. In addition, administration of recombinant IL-4 significantly enhanced the production of IFN-gamma in WT mice. Finally, the enhanced production of IFN-gamma in IL-18KO mice correlated with increased host defense against cryptococcal infection, as indicated by enhancement in alpha-GalCer-related clearance of microorganisms. Our results indicated that in IL-18KO mice, IFN-gamma synthesis was enhanced through overproduction of IL-12 and IL-4 after intravenous infection with C. neoformans and a ligand-specific activation of Valpha14(+) NKT cells.