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.     

Alternative induction of alpha/beta interferons and gamma interferon by listeria monocytogenes in mouse spleen cell cultures

Production of interferon (IFN) by Listeria monocytogenes (LM) in nonimmunized mouse spleen cell cultures was studied. IFN-gamma defined by virtue of its acid stability and antigenicity was produced in spleen cell cultures obtained from ddY mice, C57BL/6 mice, and BALB/c mice in response to heat-killed (HK) LM within 24 hr. On the other hand, production of IFN-alpha/beta was demonstrated in spleen cell cultures obtained from one of four nude mice (BALB/c, nu/nu). Therefore, it is important to know the reason why the spleen cells of mice other than nude mice did produce only IFN-gamma, but did not produce IFN-alpha/beta in response to HK-LM. Spleen cells obtained from ddY mice were fractionated, and the cellular source for IFN production of either IFN-alpha/beta or IFN-gamma induced by HK-LM was investigated. IFN-gamma was produced only by a mixture of T lymphocytes (nylon wool-nonadherent, Thy-1-positive cells) and macrophages by HK-LM. Neither T lymphocytes nor macrophages alone produced IFN by HK-LM. Macrophage-depleted spleen cells produced neither IFN-gamma nor IFN-alpha/beta, but these cells acquired the ability to produce IFN-alpha/beta, not IFN-gamma, only when they had been treated with IFN-alpha/beta. A possible mechanism of both IFN-gamma and IFN-alpha/beta induction by Listeria in mouse spleen cell cultures is discussed.     

Involvement of endogenously synthesized interleukin (IL)-18 in the protective effects of IL-12 against pulmonary infection with Cryptococcus neoformans in mice

We previously demonstrated that interleukin (IL)-12 protected mice against fatal pulmonary infection with a highly virulent strain of Cryptococcus neoformans, which correlated well with the production of interferon (IFN)-gamma as well as IL-18 in the primary infected site. In the present study, we examined the role of endogenously synthesized IL-18 in IL-12-induced host resistance to this pathogen. There was little or no production of IFN-gamma and IL-18 both at mRNA and protein levels in lungs of mice infected with C. neoformans, while treatment with IL-12 induced a marked production of these cytokines. Caspase-1 mRNA was expressed in infected mice even without IL-12 treatment. Administration of neutralizing anti-IFN-gamma monoclonal antibody (mAb) clearly inhibited production of IFN-gamma and IL-18 induced by IL-12, while control IgG did not show such an effect. However, administration of IFN-gamma did not induce the production of both cytokines in infected mice, although tumor necrosis factor (TNF)-alpha and IFN-gamma-inducible protein (IP)-10 were synthesized by the same treatment. Finally, neutralizing anti-IL-18 antibody (Ab) significantly interfered with the production of IFN-gamma and elimination of the microorganism from the lung induced by IL-12 treatment. Furthermore, both IFN-gamma synthesis and host protection caused by IL-12 were profoundly diminished in IL-18 gene-disrupted mice. Considered collectively, our results indicated that host protection against C. neoformans induced by IL-12 involved endogenously synthesized IL-18 and that the production of IL-18 was mediated at least in part by endogenous IFN-gamma.     

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.     

The relative contribution of IL-4 receptor signaling and IL-10 to susceptibility to Leishmania major

The roles of IL-10 and IL-4 receptor signaling were evaluated in a murine model of Leishmania major infection. In previous studies the L. major substrain LV39 caused progressive, nonhealing lesions in BALB/c mice deficient for IL-4R alpha-chain (IL-4R alpha), while substrain IR173 was highly controlled. To explore whether IL-10 is responsible for inducing susceptibility to LV39, wild-type and IL-4R alpha(-/-) mice were treated with anti-IL-10R mAb, and in a genetic approach, the IL-4R alpha(-/-) mice were crossed with BALB/c IL-10(-/-) mice. In contrast to the lack of resistance conferred by IL-4R alpha gene deletion, partial resistance to LV39 was conferred by IL-10 gene deletion or treatment of BALB/c mice with anti-IL-10R mAb. Lesion sizes and LV39 parasite numbers were further and dramatically reduced in both anti-IL-10R-treated IL-4R alpha(-/-) mice and IL-4R alpha x IL-10 double knockouts. Anti-IL-10R mAb treatment further suppressed parasite growth in IL-4R alpha(-/-) mice infected with L. major IR173. Production of IFN-gamma was only increased relative to wild-type or littermate controls in IL-4R alpha(-/-) mice with complementary defects in IL-10. Comparisons of IFN-gamma-treated infected macrophages in vitro indicated that LV39 required 25- to 500-fold greater concentrations of IFN-gamma than IR173-infected macrophages to achieve a similar efficiency of parasite killing. These studies suggest that regardless of parasite substrain, IL-10 is as important as IL-4/IL-13 in promoting susceptibility to L. major and even more so for those substrains that are relatively resistant to IFN-gamma mediated killing.     

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.     

In vitro analysis of interferon gamma (IFN-gamma) and interleukin-12 (IL-12) production and their effects in ileal Crohn's disease

Crohn's disease is an inflammatory disease of the gut in which tumour necrosis factor (TNF) and T helper 1 (Th1) cytokines (interleukin (IL)-12, interferon (IFN)-gamma) are thought to play a major role. After the successes obtained with neutralisation of TNF, interest is now growing for therapy aiming at neutralisation of Th1-associated cytokines. Since cytokines are linked in a delicate network, in vitro cultures of ileal lamina propria mononuclear cells (LPMC) were set up for evaluation of a) IFN-gamma and IL-12 production, b) effects of rhIFN-gamma and rhIL-12 and c) effects of anti-IFN-gamma and anti-IL-12 on pro-inflammatory cytokines and IL-10 production. LPMC were isolated from surgical specimens of a total of 27 Crohn's disease and 17 caecum carcinoma (control) patients. Cells were stimulated with CD40L (which triggers myeloid CD40-expressing cells) or anti-CD3 +CD80 (which triggers T cells). LPMC from involved ileal, Crohn's disease produced, in both non-stimulated and stimulated conditions, more IFN-gamma and IL-12p70 than LPMC from non-involved tissue or from control patients. rhIFN-gamma significantly enhanced TNF production in both controls and in ileal Crohn's disease patients, while rhIL-12 enhanced IFN-gamma but not TNF production. LPMC from involved tissue were more sensitive to IL-12 than control LPMC. LP-T cell-dependent activation of monocytes was then studied by co-culture of anti-CD3/CD80-stimulated LPMC with fresh monocytes, which resulted in high IL-12, IFN-gamma, TNF and IL-10 production. The data show that neutralisation of either IL-12 or IFN-gamma with mAb in these cultures also affects secretion of the reciprocal cytokine and (in the case of anti-IL-12) also that of the anti-inflammatory cytokine IL-10. However, no effect of anti-IL-12 or anti-IFN-gamma on production of TNF, a cytokine with an important pathogenic role in Crohn's disease, could be found. Therapies aiming at neutralisation of IFN-gamma or IL-12 are therefore unlikely to replace anti-TNF, but they might provide an additive or synergistic effect.     

A monoclonal antibody directed against the murine macrophage surface molecule F4/80 modulates natural immune response to Listeria monocytogenes.

Whole spleen cell cultures from SCID mice release high levels of IFN-gamma when exposed to heat-killed Listeria monocytogenes (HKL). This microbe-induced and T cell-independent response depends on both macrophages (MPhi) and NK cells: HKL-stimulated MPhi release TNF-alpha and IL-12, which together activate NK cells for IFN-gamma release. We show here that this cytokine-mediated activation cascade can be modulated by a mAb against the MPhi surface glycoprotein F4/80. HKL-induced IL-12, TNF-alpha, and IFN-gamma in SCID whole spleen cell cultures was inhibited by coincubation with anti-F4/80 mAb whereas IL-1 and IL-10 were enhanced. Both effects were apparent at mRNA and protein release levels. Whereas inhibitory activities were F4/80 Ag specific, stimulatory effects were Fc dependent and nonspecific. Furthermore, cytokine inhibition by anti-F4/80 was only apparent when MPhi and NK cells were present simultaneously and in close vicinity, indicating that direct cell-to-cell contact is a prerequisite. These data suggest a novel pathway for microbe-induced MPhi/NK cell interaction involving direct cell-to-cell signaling and give the first evidence for a functional role of the MPhi surface glycoprotein F4/80.     

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.     

Differences in interleukin-12 and -15 production by dendritic cells at the early stage of Listeria monocytogenes infection between BALB/c and C57 BL/6 mice

The mechanisms responsible for the resistance of C57BL/6 mice and for the susceptibility of BALB/c mice to infection with Listeria monocytogenes were studied by comparing early IL-12 and IL-15 production by dendritic cells (DC) after infection with L. monocytogenes. Splenic DC expressing CD11b(low) and CD11c(+) obtained from C57BL/6 mice at 3 and 6 h after L. monocytogenes infection expressed higher levels of IL-12 p40 mRNA and IL-12 p40 protein than did those from BALB/c mice. Concurrently, a larger amount of IFN-gamma was produced by the splenic T cells from C57BL/6 mice in response to immobilized anti-TCRalphabeta mAb than by those from BALB/c mice, while the splenic T cells from BALB/c mice produced a higher level of IL-4 upon TCR alphabeta stimulation than did those of C57BL/6 mice. IL-15 mRNA and intracellular IL-15 protein were detected more abundantly in the DC from C57BL/6 mice than in those from BALB/c mice on day 3 after infection. CD3(+) IL2Rbeta (+) cells in the spleen were increased in C57BL/6 mice but not in BALB/c mice at the early stage after infection. Furthermore, IL-12Rbeta2 gene expression was up-regulated in T cells from C57BL/6 mice but not in those from BALB/c mice at the early stage after listerial infection. These results suggest that the difference in early production of IL-12 and IL-15 by DC may at least partly underlie the difference in susceptibility to L. monocytogenes between C57BL/6 and BALB/c mice.     

Interleukin-4 weakens host resistance to pulmonary and disseminated cryptococcal infection caused by combined treatment with interferon-gamma-inducing cytokines.

We examined the role of interleukin (IL)-4 in host resistance against infection with Cryptococcus neoformans. First, we examined the effects of a neutralizing anti-IL-4 monoclonal antibody (mAb) on survival of mice infected intratracheally with this fungal pathogen. We also compared the number of live C. neoformans in lungs and brains of treated and untreated mice. Treatment with anti-IL-4 mAb significantly prolonged survival of infected mice and reduced the lung and brain burdens of C. neoformans, which was associated with increased production of IFN-gamma in lungs. In the next experiments, infected mice were treated with two IFN-gamma-inducing cytokines, IL-12 and IL-18, known to enhance protection against infection. We then evaluated the effect of such treatment on the number of live microorganisms and concentration of IL-4 in lungs. These two parameters showed a statistically significant relationship, suggesting a negative regulation of host protection by IL-4. Finally, we examined the effects of IL-4 treatment and administration of neutralizing anti-IL-4 mAbs on host protection against C. neoformans and local production of IFN-gamma in lungs induced by treatment with IL-12/IL-18. The former treatment suppressed host protection and reduced IFN-gamma production, while the latter produced the opposite effects. Our results indicated that IL-4 suppressed the host defense mechanisms against infection with C. neoformans potentiated by IFN-gamma-inducing cytokines probably through the suppression of local production of IFN-gamma.     

Production of IL-10 by CD4+ T lymphocytes correlates with down-regulation of Th1 cytokine synthesis in helminth infection.

After the onset of parasite egg deposition, mice infected with the helminth Schistosoma mansoni mount strong Th2 cytokine responses in the absence of significant Th1 cytokine synthesis. To examine the basis of this immunoregulatory state, spleen or lymph node cells from schistosome-infected mice were stimulated with parasite-specific Ag and the supernatants tested for their capacity to suppress IFN-gamma synthesis by a Th1 cell line. Strong inhibition was observed that was neutralized by a mAb against IL-10, a cytokine previously shown to down-regulate Th1 cytokine synthesis. By means of ELISA measurements the production of IL-10 in schistosome infection was confirmed and shown to depend on CD4+ T cells. IL-10 synthesis stimulated by either mitogen or Ag was observed only at those stages of infection when Th2 response induction and Th1 cytokine down-regulation also occurred and was not detected in mice vaccinated with attenuated parasites. Moreover, the addition of the neutralizing anti-IL-10 mAb to Ag-stimulated spleen cell cultures from infected mice caused a dramatic augmentation in IFN-gamma synthesis. These findings suggest that IL-10 is responsible for the down-regulation of Th1 responses observed in schistosome infections, a phenomenon that may enable the parasite to escape potentially harmful cell-mediated responses.     

Roles of endogenous cytokines in liver apoptosis of mice in lethal Listeria monocytogenes infection

Various bacterial pathogens have been identified as mediators of apoptosis. Apoptosis reportedly shows both detrimental and beneficial effects on biological functions. We studied the role of liver apoptosis in lethal Listeria monocytogenes infection and the regulation of apoptosis by endogenous cytokines during infection. Apoptosis was observed in the spleen but not in the liver of infected mice, whereas the induction of liver necrosis was evident by rising levels of serum aminotransferases in these animals. Apoptosis was detected in the liver of L. monocytogenes-infected mice which had been treated with monoclonal antibody (mAb) against tumor necrosis factor-alpha (TNF-alpha) or interleukin-6 (IL-6), or in TNF-alpha(-/-) mice, but not in gamma- interferon (IFN-gamma)(-/-) mice or mice which had been treated with mAb against IL-4 or IL-10. Augmentation of liver apoptosis in mice treated with mAb against TNF-alpha or IL-6 or in TNF-alpha(-/-) mice correlated with the increase in bacterial numbers in the organ, while no augmentation of apoptosis was observed in the liver of IFN-gamma(-/-) mice irrespective of the marked increase in bacterial numbers in the organs, indicating that augmentation of liver apoptosis may not be merely due to the increase in bacterial growth in the organs. These results suggest that TNF-alpha and IL-6 may play an important role in protecting the liver from apoptosis in lethal L. monocytogenes infection.     

IL-23 enhances host defense against vaccinia virus infection via a mechanism partly involving IL-17

To investigate roles of IL-23 in viral infection, we have engineered recombinant vaccinia virus (VV) expressing IL-12 (VV-IL-12) and expressing IL-23 (VV-IL-23). We found VV-IL-23 was less virulent in BALB/c mice than wild-type VV (VV-WT), indicating that IL-23 enhances resistance to VV. VV-specific CTL activity in VV-IL-23-infected mice was slightly higher than activity in VV-WT-inoculated mice, although antiviral Ab production and NK activity were not increased. IL-12/23p40-deficient mice survived the infection with VV-IL-23, indicating that IL-23 promotes VV resistance independently of IL-12. The mechanism of the IL-23-mediated resistance was distinct from that of the IL-12-regulated resistance because IFN-gamma-deficient mice did not eliminate VV-IL-12, but did eradicate VV-IL-23. These data indicate that IFN-gamma is essential for the IL-12-mediated resistance, but dispensable for the IL-23-regulated resistance. Because IL-17 is a key in the IL-23-regulated resistance to bacteria, we hypothesized an involvement of IL-17 in the resistance to VV. Treatment with an anti-IL-17 mAb resulted in a significant increase of viral titers in VV-IL-23-infected IFN-gamma-deficient mice. In addition, VV-IL-17 was less virulent than VV-WT in BALB/c mice, and IL-17-deficient mice were more sensitive to VV-WT than control mice. However, the effect of neutralization with an anti-IL-17 mAb was limited, and IL-17-deficient mice survived the infection with VV-IL-23. Taken together, these data suggest that the IL-23/IL-17 axis plays a certain but subdominant role in the IL-23-mediated resistance to VV. Unveiling of an alternative pathway in the IL-23-regulated resistance might provide a novel strategy against infectious pathogens without side effects of autoimmunity.     

KM(+), a lectin from Artocarpus integrifolia, induces IL-12 p40 production by macrophages and switches from type 2 to type 1 cell-mediated immunity against Leishmania major antigens, resulting in BALB/c mice resistance to infection.

The outcome and severity of some diseases correlate with the dominance of either the T helper 1 (Th1) or Th2 immune response, which is stimulated by IL-12 or IL-4, respectively. In the present study we demonstrate that gamma interferon (IFN-gamma) secretion by murine spleen cells stimulated with KM(+), a mannose-binding lectin from Artocarpus integrifolia, is due to IL-12 induction, because (1) macrophages from several sources (including cell lines) produced IL-12 p40 in response to KM(+), and (2) lectin-free supernatants from J774 cell line cultures stimulated with KM(+) induced the secretion of IFN-gamma by spleen cell cultures, an effect blocked by the supernatant pretreatment with anti-IL-12 antibody. The known pattern of susceptibility of BALB/c mice to infection with Leishmania major, attributed to high levels of IL-4 production leading to a Th2 nonprotective immune response, was modified by administration of KM(+). Draining lymph node cells from these immunized BALB/c mice (in contrast to cells from animals immunized only with soluble leishmanial antigen [SLA]) secreted high levels of IFN-gamma and low levels of IL-4, which characterized a Th1 rather than a Th2 response pattern. The footpad thickness of BALB/c mice immunized with SLA plus KM(+) and challenged with L. major was similar to that of uninfected mice. This beneficial effect against leishmanial infection was blocked by pretreatment of these mice with anti-IL-12 antibody. These observations indicate that KM(+) induces IL-12 p40 in vivo and has a protective effect against L. major infection.     

Cutting edge: IL-15 costimulates the generalized Shwartzman reaction and innate immune IFN-gamma production in vivo

Sequential administration of LPS to SCID mice results in the generalized Shwartzman reaction, manifesting as rapid mortality via cytokine-induced shock. Here we demonstrate that in vivo neutralization of IL-15 before LPS priming significantly reduced lethality in this reaction (p = 0.0172). We hypothesize that LPS priming induces IL-12 and IL-15 that costimulate NK cell-derived IFN-gamma. Such IFN-gamma may then in turn sensitize macrophages to elicit the Shwartzman reaction following a subsequent LPS challenge. Supporting this, IL-12 and IL-15 synergized to induce murine NK cell IFN-gamma production in vitro. LPS stimulation of SCID mouse splenocytes resulted in measurable IFN-gamma production, which was reduced when IL-15 was neutralized or IL-2/15Rbeta was blocked. Pretreatment with either anti-IL-2/15Rbeta or anti-IL-15 Abs reduced serum IFN-gamma protein following LPS administration to SCID mice. Collectively, these data provide the first in vivo evidence that IL-15 participates in LPS-induced innate immune IFN-gamma production and significantly contributes to the lethal Shwartzman reaction.     

Neutralization of interleukin-18 reduces severity in murine colitis and intestinal IFN-gamma and TNF-alpha production

Interleukin (IL)-18, initially described as interferon (IFN)-gamma-inducing factor, is expressed in the inflamed mucosa of patients with Crohn's disease. To investigate the role of IL-18 in intestinal inflammation, the effect of neutralizing antimurine IL-18 antiserum in dextran sulfate sodium (DSS)-induced colitis in BALB/c and C57BL/6 mice was examined. During a dose response of DSS, levels of colonic IL-18 increased parallel with clinical worsening. With the use of confocal laser microscopy, the increased IL-18 was localized to the intestinal epithelial layer. Anti-IL-18 treatment resulted in a dose-dependent reduction of the severity of colitis in both BALB/c and C57BL/6 mice. Colon shortening following DSS-induced colitis was partially prevented in the treatment groups. In the colon tissue homogenates, IFN-gamma concentrations were lower in the anti-IL-18-treated DSS-fed mice compared with untreated DSS-fed mice. This suppressive effect of anti-IL-18 administered in vivo was also observed on spontaneous tumor necrosis factor-alpha, IL-18, and IFN-gamma production from ex vivo colon organ cultures. The stimulation of lamina propria mononuclear cells by IL-18 and IL-12 resulted in a synergistic increase in IFN-gamma synthesis. These findings suggest that IL-18 is a pivotal mediator in experimental colitis.     

A single intradermal administration of soluble leishmanial antigen and plasmid expressing interleukin-12 protects BALB/c mice from Leishmania major infection.

In murine leishmaniasis, the induction of the T-helper type 1 (Th1) response contributes to infection resistance, whereas the establishment of the Th2 response makes the mice susceptible to infection. Interleukin-12 (IL-12) plays a pivotal role in the diversification of immune responses to the Th1 type. In this study, we tested whether the co-administration of IL-12 expression plasmid which compose p35 and p40 subunits and soluble leishmanial antigen (SLA) will skew the susceptible BALB/c mice to Th1 response and protect from leishmaniasis. When the mice were intradermally injected with the combination of IL-12 plasmid and SLA 7 days prior to the challenge with 1x10(6) promastigotes of Leishmania major, the local lesions completely healed and the parasite burden in the local lymph nodes significantly decreased. The cured mice attained long-term immunity, and were resistant to any subsequent rechallenge of the lethal dose of the parasite. The protective effect was associated with the development of a Th1 response, as demonstrated by the enhanced level of antigen-specific interferon-gamma (IFN-gamma) and dominant production of IgG2a in the serum. In contrast, the administration of empty plasmid plus SLA or IL-12 plasmid alone failed to protect the disease and shape the Th1 response. Furthermore, the protective efficiency induced by the vaccination was clearly prevented by the injection of either neutralizing anti-IL-12 mAb or anti-IFN-gamma mAb. The IL-12 expression plasmid is thus an effective adjuvant for the elicitation of a protective Th1 response against leishmaniasis and is therefore, considered to be appropriate for vaccinations that require the induction of Th1 type immunity.     

Mutual modulation between interleukin-10 and interleukin-6 induced by Rhodococcus aurantiacus infection in mice

The interaction between interleukin-10 (IL-10) and interleukin-6 (IL-6) was investigated in the inflammatory response to Rhodococcus aurantiacus (R. aurantiacus) infection, in which both cytokines act as anti-inflammatory cytokines. Compared with wild-type (WT) counterparts, IL-6 gene-deficient (IL-6(-)/(-)) mice mounted a more robust production of IL-10 and tumor necrosis factor-alpha (TNF-alpha) during the initial phase of infection. Administration of anti-IL-10 antibody resulted in all the mice dying within 3 days post-infection as well as a further elevated TNF-alpha release. In vitro challenge of the macrophages from IL-6(-)/(-) and WT mice with heat-killed R. aurantiacus also showed similar results. Addition of exogenous IL-6 depressed IL-10 and TNF-alpha production by either IL-6(-)/(-) mice or IL-6(-)/(-) mouse macrophages. Likewise, WT mouse macrophages pretreated with anti-IL-10 or anti-IL-6 antibody exhibited increased production of TNF-alpha and IL-6 or IL-10 respectively. Moreover, neutralization of both IL-10 and IL-6 induced a further increase in TNF-alpha production by WT mouse cells. Overall, we conclude that IL-10 is a key element in protecting mice against mortality, and that IL-10 and IL-6 production are negatively regulated by each other although they are additive in suppressing TNF-alpha release in R. aurantiacus-infected mouse model.