Cutting edge: murine dendritic cells require IL-15R alpha to prime NK cells

NK cells protect hosts against viral pathogens and transformed cells, and dendritic cells (DCs) play important roles in activating NK cells. We now find that murine IL-15Ralpha-deficient DCs fail to support NK cell cytolytic activity and elaboration of IFN-gamma, despite the fact that these DCs express normal levels of costimulatory molecules and IL-12. By contrast, IL-15Ralpha expression on NK cells is entirely dispensable for their activation by DCs. In addition, blockade with anti-IL-15Ralpha and anti-IL-2Rbeta but not anti-IL-2Ralpha-specific Abs prevents NK cell activation by wild-type DCs. Finally, presentation of IL-15 by purified IL-15Ralpha/Fc in trans synergizes with IL-12 to support NK cell priming. These findings suggest that murine DCs require IL-15Ralpha to present IL-15 in trans to NK cells during NK cell priming.     

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.     

IL-4 induces in vivo production of IFN-gamma by NK and NKT cells

Although IL-4 and IFN-gamma often have opposite effects and suppress each other's production by T cells, IL-4 can stimulate IFN-gamma production. To characterize this, we injected mice with IL-4 and quantified IFN-gamma production with the in vivo cytokine capture assay. IL-4 induced Stat6-dependent IFN-gamma production by NK and, to a lesser extent, NKT cells, but not conventional T cells, in 2-4 h. Increased IFN-gamma production persisted at a constant rate for >24 h, but eventually declined, even with continuing IL-4 stimulation. This eventual decline in IFN-gamma production was accompanied by a decrease in NK and T cell numbers. Consistent with a dominant role for NK cells in IL-4-stimulated IFN-gamma secretion, IL-4 induction of IFN-gamma was B and T cell-independent; suppressed by an anti-IL-2Rbeta mAb that eliminates most NK and NKT cells; reduced in Stat4-deficient mice, which have decreased numbers of NK cells; and absent in Rag2/gamma(c)-double-deficient mice, which lack T, B, and NK cells. IL-4-induced IFN-gamma production was not affected by neutralizing IL-12p40 and was increased by neutralizing IL-2. IL-13, which signals through the type 2 IL-4R and mimics many IL-4 effects, failed to stimulate IFN-gamma production and, in most experiments, suppressed basal IFN-gamma production. Thus, IL-4, acting through the type 1 IL-4R, induces Stat6-dependent IFN-gamma secretion by NK and NKT cells. This explains how IL-4 can contribute to Th1 cytokine-associated immune effector functions and suggests how IL-13 can have stronger proallergic effects than IL-4.     

Autocrine regulation of IL-12 receptor expression is independent of secondary IFN-gamma secretion and not restricted to T and NK cells.

The biological response to IL-12 is mediated through specific binding to a high affinity receptor complex composed of at least two subunits (designated IL-12Rbeta1 and IL-12Rbeta2) that are expressed on NK cells and activated T cells. The selective loss of IL-12Rbeta2 expression during Th2 T cell differentiation suggests that regulation of this receptor component may govern IL-12 responsiveness. In murine assays, down-regulation of IL-12Rbeta2 expression can be prevented by treatment with IFN-gamma, indicating that receptor expression and hence IL-12 responsiveness may be regulated, at least in part, by the local cytokine milieu. In this study, we report that cellular expression of both IL-12Rbeta1 and beta2 mRNA is increased in the lymph nodes of naive mice following systemic administration of murine rIL-12 (rmIL-12). Changes in IL-12R mRNA were associated with increased IFN-gamma secretion following ex vivo activation of lymph node cells with rmIL-12, indicating the presence of a functional receptor complex. Expression of IL-12R mRNA was not restricted to lymph node T cells, and its autocrine regulation was independent of secondary IFN-gamma secretion. Data from fractionated lymph node cells as well as rmIL-12-treated B cell-deficient mice suggest that IL-12-responsive B cells may represent an alternative cellular source for IFN-gamma production. However, the strength of the biological response to rmIL-12 is not governed solely by receptor expression, as rmIL-12-induced IFN-gamma secretion from cultured lymph node cells is accessory cell dependent and can be partially blocked by inhibition of B7 costimulation.     

Interferon gamma priming is not critical for IL-12 production of murine spleen cells

Interferon gamma (IFN-gamma) priming is considered to be critical for interleukin 12 (IL-12) production of murine macrophages and human monocytes by lipopolysaccharide (LPS) stimulation. In our present experiments, freshly prepared spleen cells (f-spleen cells) were confirmed not to produce detectable level of IL-12 by LPS stimulation, although they produced significant amount of IL-12 by the stimulation with LPS plus IFN-gamma. However, the stimulation only with LPS induced IL-12 production of spleen cells preincubated in the absence of IFN-gamma. Findings on IL-12 p40 mRNA accumulation were consistent with their IL-12 production. Essentially the same results were obtained using spleen cells from IFN-gamma deficient mice. In the presence of anti-IL-10, f-spleen cells produced IL-12 upon LPS stimulation, indicating that the failure of f-spleen cells in IL-12 production is caused by IL-10 produced by themselves upon LPS stimulation. In addition, f-spleen cells produced IL-12 upon CD40 ligand stimulation, and the production was hardly affected by the presence of IFN-gamma or preincubation. These results indicate that IFN-gamma priming is not critical for IL-12 production of spleen cells stimulated with LPS or CD40 ligand, although IFN-gamma enhances the production, especially, in response to LPS stimulation.     

Interleukin-10 suppresses natural killer cell but not natural killer T cell activation during bacterial infection

Interleukin (IL)-10 is an anti-inflammatory cytokine known to modulate the outcome of sepsis by decreasing pro-inflammatory cytokine production, including IL-12, a main activator of natural killer (NK) cells. We hypothesized that neutralization of IL-10 would increase NK and natural killer T (NKT) cell activation through increased IL-12 in a mouse model of bacterial peritonitis. NK and NKT cell activations were measured by CD69 expression on NK1.1+/CD3- and NK1.1+/CD3+ cells after cecal ligation and puncture (CLP). NK cells were significantly more activated in mice treated with anti-IL-10 antibodies, whereas no such effect was observed in NKT cells. Similarly, intracellular interferon gamma (IFN-gamma) levels were increased in NK cells of anti-IL-10-treated mice, but not in NKT cells. IL-12 and IL-18 levels were increased in both CLP groups, but in anti-IL-10-treated mice, early IL-12 and late IL-18 levels were significantly higher than in controls. Survival at 18 h after CLP was lower in anti-IL-10 mice, which was associated with increased liver neutrophil accumulation. In summary, these data show an activating effect of IL-10 on NK, but not on NKT cells after CLP, which corresponded with decreased survival, higher IFN-gamma production, and increased remote organ neutrophil accumulation. These effects were not mediated by IL-12 and IL-18 alone, and reinforce a role for NK cells in remote organ dysfunction following peritonitis.     

Overexpression of IL-15 in vivo enhances protection against Mycobacterium bovis bacillus Calmette-Guérin infection via augmentation of NK and T cytotoxic 1 responses.

To investigate the immunomodulating effects of IL-15 in vivo on mycobacterial infection, we used IL-15-transgenic (Tg) mice, which were recently constructed with cDNA-encoding secretable isoform of IL-15 precursor protein under the control of a MHC class I promoter. The IL-15-Tg mice exhibited resistance against infection with Mycobacterium bovis bacillus Calmette-Guérin (BCG), as assessed by bacteria growth. IFN-gamma level in serum was significantly higher in IL-15-Tg mice than in non-Tg mice after BCG infection. NK cells were remarkably increased, and Ag-specific T cytotoxic 1 response mediated by CD8+ T cells producing IFN-gamma was significantly augmented in the IL-15-Tg mice following BCG infection. Neutralization of endogenous IFN-gamma by in vivo administration of anti-IFN-gamma mAb deteriorated the clearance of the bacteria. Depletion of of NK cells or CD8+ T cells by in vivo administration of anti-asialo-GM(1) Ab or anti-CD8 mAb hampered the exclusion of bacteria. Thus, overexpression of IL-15 in vivo enhanced protection against BCG infection via augmentation of NK and T cytotoxic 1 responses.     

IL-12 receptor beta 1 and Toll-like receptor 4 increase IL-1 beta- and IL-18-associated myocarditis and coxsackievirus replication

Th1-type immune responses, mediated by IL-12-induced IFN-gamma, protect the host from most viral infections. To investigate the role of IL-12 and IFN-gamma on the development of Coxsackievirus B3 (CB3)-induced myocarditis, we examined the level of inflammation, viral replication, and cytokine production in IL-12Rbeta1- and IFN-gamma-deficient mice following CB3 infection. We report that IL-12Rbeta1 deficiency results in decreased viral replication and inflammation in the heart, while IFN-gamma deficiency exacerbates CB3 replication. Importantly, decreased IL-1beta and IL-18 levels in IL-12Rbeta1-deficient hearts correlated directly with decreased myocardial inflammation. Because IL-1beta and IL-18 were associated with myocardial inflammation, we examined the effect of TLR4 deficiency on CB3 infection and myocarditis. We found that TLR4-deficient mice also had significantly reduced levels of myocarditis, viral replication, and IL-1beta/IL-18, just as we had observed in IL-12Rbeta1-deficient mice. This is the first report that TLR4 influences CB3 replication. These results show that IL-12Rbeta1 and TLR4 exacerbate CB3 infection and myocarditis while IFN-gamma protects against viral replication. The remarkable similarities between the effects of IL-12Rbeta1 and TLR4 suggest that these receptors share common downstream pathways that directly influence IL-1beta and IL-18 production, and confirm that IL-1beta and IL-18 play a significant role in the pathogenesis of CB3-induced myocarditis. These findings have important implications not only for the pathogenesis of myocarditis, but for other autoimmune diseases triggered by viral infections.     

IL-18 contributes to host resistance against infection with Cryptococcus neoformans in mice with defective IL-12 synthesis through induction of IFN-gamma production by NK cells

The aim of this study was to examine the contribution of IL-18 in host defense against infection caused by Cryptococcus neoformans in mice with defective IL-12 production. Experiments were conducted in mice with a targeted disruption of the gene for IL-12p40 subunit (IL-12p40-/- mice). In these mice, host resistance was impaired, as shown by increased number of organisms in both lungs and brains, compared with control mice. Serum IFN-gamma was still detected in these mice at a considerable level (20-30% of that in control mice). The host resistance was moderately impaired in IL-12p40-/- mice compared with IFN-gamma-/- mice. Neutralizing anti-IFN-gamma mAb further increased the lung burdens of organisms. In addition, treatment with neutralizing anti-IL-18 Ab almost completely abrogated the production of IFN-gamma and also impaired the host resistance. Host resistance in IL-12p40-/- IL-18-/- mice was more profoundly impaired than in IL-12p40-/- mice. Administration of IL-12 as well as IL-18 increased the serum levels of IFN-gamma and significantly restored the reduced host resistance. Spleen cells obtained from infected IL-12p40-/- mice did not produce any IFN-gamma upon restimulation with the same organisms, while those from infected and IL-12-treated mice produced IFN-gamma. In contrast, IL-18 did not show such effect. Finally, depletion of NK cells by anti-asialo GM1 Ab mostly abrogated the residual production of IFN-gamma in IL-12p40-/- mice. Our results indicate that IL-18 contributes to host resistance to cryptococcal infection through the induction of IFN-gamma production by NK cells, but not through the development of Th1 cells, under the condition in which IL-12 synthesis is deficient.     

Differentiation of murine NK cells into distinct subsets based on variable expression of the IL-12R beta 2 subunit

The cytokine IL-12 manifests its biological activity via interaction with a heterodimeric receptor (IL-12R) present on activated T and NK cells. The cDNAs for two IL-12R subunits have been cloned from human and mouse and designated IL-12Rbeta1 and IL-12Rbeta2. The expression of IL-12Rbeta2 on T cells is influenced by cytokines, particularly IL-4, IL-12, and IFN-gamma; however, little is known regarding regulation of IL-12R expression on NK cells. In this study we show that murine NK cells differentiate into IL-12Rbeta2(low) and IL-12Rbeta2(high) subsets after in vitro stimulation with IL-2 in the absence of exogenous polarizing cytokines. Subset development occurs gradually as NK cells expand in vitro and is generally complete by 8-12 days of culture. Once established, IL-12Rbeta2(low) and IL-12Rbeta2(high) subsets are highly stable in vitro and can be maintained for at least 20 days after FACS sorting. Formation of these NK subsets appears to be strain independent. Flow cytometric analyses demonstrate that both subsets express a number of NK-associated markers, including NK1.1, DX-5, Ly-49A, and Ly-49C, but that the Ly-49G2 class I inhibitory receptor is expressed predominantly on the IL-12Rbeta2(high) population. Both IL-12Rbeta2(low) and IL-12Rbeta2(high) NK cells respond to exogenous IL-12 by rapid production of high levels of IFN-gamma and increased lytic activity against NK-sensitive YAC-1 target cells. Analyses of cytokine gene expression by RNase protection assay indicated that similar to the recently described human NK1 subset, both IL-12Rbeta2(high) and IL-12Rbeta2(low) murine NK subsets expressed high levels of IFN-gamma, whereas neither subset expressed mRNA for the NK2-associated cytokines IL-5 and IL-13.     

Synergistic proliferation and activation of natural killer cells by interleukin 12 and interleukin 18.

We investigated the effects of IL-12 and IL-18 on unstimulated murine splenocytes and observed that the two cytokines strongly synergized for their proliferation, whereas IL-12 and IL-18 alone were essentially inactive in this respect. Phenotypical and functional analyses of cells proliferating in response to IL-12 and IL-18 revealed that large granular Ly-49C(+)DX5(+)CD3(-)NK blasts were expanded in these cultures and that they displayed cytotoxic activity against Yac-1 cells, a murine NK cell target. Further analyses indicated three major differences between NK cells appearing in response to IL-12 and IL-18 and those derived in the presence of other NK cell growth factors, such as IL-2 or IL-15. First, a population of T-NK cells, i.e. expressing T cell (TCRalphabeta, CD3) and NK cell (Ly-49) markers, was detected amongst cells growing in IL-2 or IL-15 but not in cultures supplemented with IL-12 and IL-18. Second, most NK cells derived with IL-2 or IL-15 expressed the NK1.1 antigen, while those derived with IL-12 and IL-18 did not. Finally, striking differences were observed regarding cytokine production. Cells stimulated with IL-12 and IL-18 in combination, but not with IL-2 or IL-15, produced IFN-gamma, IL-3, IL-6 and TNF. IFN-gamma was not involved in the response of NK cells to IL-12 and IL-18, as indicated by experiments demonstrating that the combination of the two cytokines displayed similar effects on spleen cells from IFN-gammaR-knock-out mice. Receptor (IL-12Rbeta1, IL-12Rbeta2 and IL-18R) gene expression studies did not indicate that the mechanism underlying the synergy between IL-12 and IL-18 involved reciprocal induction of their receptors. Taken together, our results demonstrate that IL-12 and IL-18 exert striking synergistic activities for NK cell proliferation and activation, distinct from those induced by IL-2 or IL-15.     

IFN-gamma production is specifically regulated by IL-10 in mice made tolerant with anti-CD40 ligand antibody and intact active bone

We have developed a strategy to induce tolerance to allografts, involving cotransplantation of allogeneic intact active bone and transient anti-CD40 ligand mAb therapy. Tolerance induced by this approach in C57BL/6 mice receiving BALB/c hearts is not mediated by deletional mechanisms, but by peripheral regulatory mechanisms. Tolerance is associated with diminished ex vivo IFN-gamma production that is donor specific, and a reduction in the frequency of IFN-gamma-producing cells. Splenocytes from mice tolerant to BALB/c grafts, but sensitized to third-party C3H skin grafts, demonstrated normally primed ex vivo IFN-gamma responses to C3H stimulators. Neutralizing anti-IL-10 and anti-IL-10R, but not anti-TGF-beta, anti-IL-4, or anti-CTLA-4, Abs restored the ex vivo IFN-gamma response to BALB/c stimulators. There was no significant difference in IL-2 or IL-4 production between tolerant and rejecting mice, and anti-IL-10 mAbs had no effect on IL-2 or IL-4 production. The Cincinnati cytokine capture assay was used to test whether suppression of IFN-gamma production in vivo was also a marker of tolerance. In naive mice, we observed a dramatic increase in serum IFN-gamma levels following challenge with allogeneic BALB/c splenocytes or hearts. Tolerant mice challenged with allogeneic BALB/c splenocytes or hearts made significantly less or undetectable amounts of IFN-gamma. No IL-4 or IL-10 production was detected in tolerant or rejecting mice. Collectively, our studies suggest that active suppression of IFN-gamma production by IL-10 is correlated with, and may contribute to, tolerance induced with intact active bone and anti-CD40 ligand mAbs.     

NKT cells are critical to initiate an inflammatory response after Pseudomonas aeruginosa ocular infection in susceptible mice

CD4(+) T cells produce IFN-gamma contributing to corneal perforation in C57BL/6 (B6) mice after Pseudomonas aeruginosa infection. To determine the role of NK and NKT cells, infected corneas of B6 mice were dual immunolabeled. Initially, more NKT than NK cells were detected, but as disease progressed, NK cells increased, while NKT cells decreased. Therefore, B6 mice were depleted of NK/NKT cells with anti-asialo GM1 or anti-NK1.1 Ab. Either treatment accelerated time to perforation, increased bacterial load and polymorphonuclear neutrophils, but decreased IFN-gamma and IL-12p40 mRNA expression vs controls. Next, RAG-1 knockout (-/-; no T/NKT cells), B6.TCR Jalpha281(-/-) (NKT cell deficient), alpha-galactosylceramide (alphaGalCer) (anergized NKT cells) injected and IL-12p40(-/-) vs B6 controls were tested. IFN-gamma mRNA was undetectable in RAG-1(-/-)- and alphaGalCer-treated mice at 5 h and was significantly reduced vs controls at 1 day postinfection. It also was reduced significantly in B6.TCR Jalpha281(-/-), alphaGalCer-treated, and IL-12p40(-/-) (activated CD4(+) T cells also reduced) vs control mice at 5 days postinfection. In vitro studies tested whether endotoxin (LPS) stimulated Langerhans cells and macrophages (Mphi; from B6 mice) provided signals to activate NKT cells. LPS up-regulated mRNA expression for IL-12p40, costimulatory molecules CD80 and CD86, NF-kappaB, and CD1d, and addition of rIFN-gamma potentiated Mphi CD1d levels. Together, these data suggest that Langerhans cell/Mphi recognition of microbial LPS regulates IL-12p40 (and CD1d) driven IFN-gamma production by NKT cells, that IFN-gamma is required to optimally activate NK cells to produce IFN-gamma, and that depletion of both NKT/NK cells results in earlier corneal perforation.     

Protective effect of NK1.1(+) T cells as well as NK cells against intraperitoneal tumors in mice

Peritoneal resident cells of mice normally contain small populations of NK cells and NK1.1(+) alphabetaT cells. These populations increased after either 3LL or EL4 tumor inoculations into the peritoneal cavity. In vivo depletion of NK cell alone by anti-asialo GM1 (ASGM1) Ab significantly decreased survival time of tumor-injected mice, while depletion of both NK cells and NK1.1(+) T cells by anti-NK 1.1 Ab greatly shortened mouse survival time. NK1. 1(+) T cells in peritoneal cavity consist of a larger proportion of double-negative T cells and smaller populations of CD4(+) T cells and Vbeta8(+) T cells compared with liver NK1.1(+) T cells and normally lack Vbeta2(+) T cells. Tumor inoculation induced rapid IL-12 and IFN-gamma mRNA in tumor-infiltrating mononuclear cells (TIM). Although anti-NK1 Ab pretreatment in vivo abrogated IFN-gamma mRNA expression and IFN-gamma production of TIM, NK cell depletion alone by anti-ASGM1 Ab pretreatment retained IFN-gamma mRNA expression and partly inhibited IFN-gamma production of TIM. Peritoneal NK cells as well as NK1.1(+) T cells but not NK1.1(-) T cells of 3LL cell- or EL4 cell-injected mice showed cytotoxicities against the same tumor cells. Further, either anti-IL-12 Ab or anti-IFN-gamma Ab ip injection significantly shortened EL4 cell-inoculated mouse survival time. Our findings suggest that peritoneal macrophages activated by tumors produce IL-12 which activates NK cells and NK1.1(+) T cells to produce IFN-gamma and both NK cells and NK1.1(+) T cells are important in suppressing the growth of the intraperitoneal tumors.     

Treatment with anti-IL-2 antibodies reduces hepatic pathology and eosinophilia in Schistosoma mansoni-infected mice while selectively inhibiting T cell IL-5 production.

Granulomas around Schistosoma mansoni eggs are a principal cause of morbidity in mice infected with this helminth. In vivo treatment of infected mice with anti-IL-2 antibodies, with or without anti-IL-2 receptor antibodies, significantly diminished the size of circumoval granulomas in the liver and decreased hepatic fibrosis to half that in untreated mice. Antibody-treated animals also displayed a marked reduction in both peripheral blood and tissue eosinophilia while IgE levels were unchanged or increased. Spleen cell cytokine production in response to Ag or mitogen stimulation was selectively altered by in vivo anti-IL-2 administration. IL-5 responses were dramatically reduced, whereas IL-4, IL-2, and IFN-gamma responses were not consistently changed. These findings confirm previous observations, suggesting a role for IL-2 in egg-induced pathology but indicate that the primary function of this cytokine in schistosome-infected mice may be in the generation of Th2- rather than Th1-associated responses.     

Administration of anti-IL-4 monoclonal antibody 11B11 increases the resistance of mice to Listeria monocytogenes infection.

The role of endogenous IL-4 in resistance to Listeria monocytogenes infection was investigated by in vivo administration of an anti-IL-4 mAb (11B11). Mice treated with 0.01 to 0.4 mg of anti-IL-4 mAb before L. monocytogenes challenge demonstrated a significantly reduced peak bacterial burden in their livers and spleens and accelerated bacterial clearance from these organs. In addition, histopathologic damage to the liver was reduced. Maximal protection was achieved by i.p. injection of 0.1 mg of anti-IL-4 mAb 2 or 24 h before L. monocytogenes challenge; treatment with anti-IL-4 mAb after injection of L. monocytogenes had no effect on antilisterial resistance. Anti-IL-4 mAb-treated and control Listeria-infected mice exhibited similar patterns of IFN-gamma, IL-2, and IL-4 mRNA, as determined by polymerase chain reaction amplification of RNA extracted from spleen cells. In both anti-IL-4 mAb-treated and control mice, IFN-gamma, IL-2, and IL-4 mRNA were produced within 4 h after challenge. Cytokine mRNA levels were similar for anti-IL-4 mAb-treated and control mice, except for the greater amount of IFN-gamma mRNA in the anti-IL-4 mAb-treated mice at 4 h after L. monocytogenes challenge. IFN-gamma and IL-2 mRNA levels were sustained for at least 5 days, whereas IL-4 mRNA was undetectable by 3 days after challenge. There were no significant differences in the amounts of IL-4 and IFN-gamma detected in culture supernatants of spleen cells from anti-IL-4 mAb-treated and control Listeria-infected mice. These results suggest that endogenous IL-4, a cytokine thought to be produced principally by Th2 cells, has a deleterious effect on host defense against the facultative intracellular pathogen L. monocytogenes. Administration of an anti-IL-4 mAb increases antilisterial resistance without causing a detectable shift to a Th1 type of cytokine response.     

Endotoxin fails to induce IFN-gamma in endotoxin-tolerant mice: deficiencies in both IL-12 heterodimer production and IL-12 responsiveness

Mice exposed to sublethal endotoxemia develop short-term endotoxin tolerance, a state characterized by decreased monokine production and enhanced protection against endotoxic lethality. We confirmed that TNF-alpha production is markedly impaired in endotoxin-tolerant mice and additionally found 2- to 6-fold decreases in serum IFN-gamma in these animals following endotoxin challenge. The IFN-gamma deficiency of endotoxin tolerance correlated with 8-fold decreases in the bioactive p40/p35 heterodimeric form of IL-12. In contrast, total circulating IL-12 p40 was reduced by only 30-50%. Endotoxin-tolerant mice were less responsive to IL-12 than control mice, as evidenced by 3-fold lower levels of IFN-gamma inducible in vivo when rIL-12 was administered at the time of endotoxin challenge. Similarly, spleen cell cultures of endotoxin-tolerant mice produced 3-fold less IFN-gamma in the presence of optimal concentrations of both IL-12 and IL-18. Finally, levels of IL-12R beta 2 subunit mRNA and the percent composition of NK lymphocytes in the spleen were both decreased in endotoxin-tolerant mice relative to controls. We conclude that endotoxin-tolerant mice are profoundly impaired in their ability to produce IFN-gamma in response to endotoxin and that this is associated with acquired defects in both the production of circulating IL-12 heterodimer response and the response to IL-12 by NK cells.