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.     

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.     

Strongyloides ratti: the role of interleukin-5 in protection against tissue migrating larvae and intestinal adult worms

To determine the role of interleukin-5 (IL-5) and eosinophils in protection against Strongyloides ratti, mice treated with anti-IL-5 monoclonal antibody (mAb) were infected with S. ratti larvae. Strongyloides ratti egg numbers in faeces (EPG) in mAb treated mice were higher than those in control mice on days 6 and 7 after inoculation. The numbers of migrating worms in mAb treated mice 36 h after inoculation were higher than those observed in control mice. Intestinal worm numbers in mAb treated mice 5 days after inoculation were higher than those in control mice. These results show that eosinophils effectively protected the host against S. ratti infection by mainly the larval stage in primary infections. The involvement of eosinophils in protection against secondary infection was also examined. Before secondary infection, mice were treated with anti-IL-5 mAb and infected with S. ratti. Patent infections were not observed in either mAb treated or control Ab treated mice. The numbers of migrating worms in the head and lungs of mAb treated mice increased to 60% of that in primary infected mice. Intestinal worms were not found in mAb treated mice or in control mice after oral implantation of adult worms. Eosinophils were therefore mainly involved in protection against tissue migrating worms in secondary infections.     

Ablation of eosinophil and IgE responses with anti-IL-5 or anti-IL-4 antibodies fails to affect immunity against Schistosoma mansoni in the mouse

To investigate the role of anaphylactic immune responses in protective immunity against schistosomiasis, mice vaccinated with irradiated cercariae of Schistosoma mansoni were treated with neutralizing mAb antibodies against either IL-5 or IL-4 before and during challenge infection. Anti-IL-5-treated vaccinated mice showed a complete ablation of circulating as well as tissue eosinophils present in inflammatory reactions to migrating schistosomula in the skin and lungs but nevertheless eliminated challenge infections as effectively as vaccinated animals treated with a control mAb. Similarly, treatment of vaccinated mice with an anti-IL-4 mAb markedly reduced serum IgE although failing to diminish immunity. The effect of anti-IL-5 mediated eosinophil depletion was also assessed in a second model in which resistance is induced by concomitant chronic infection. Again, normal, unaltered protection was observed in the absence of circulating and tissue eosinophils. In contrast to the above findings, treatment with anti-IFN-gamma was found to cause a partial depletion of immunity in vaccinated mice whereas, paradoxically, increasing the numbers of inflammatory reactions against invading schistosomula in the lungs. These observations argue against a requirement for either eosinophils or IgE in the anti-schistosome immunity induced by vaccination with irradiated cercariae or for eosinophils in the resistance resulting from previous infection in mice and support previous data suggesting a role for an IFN-gamma dependent cell-mediated effector mechanism in vaccine-induced resistance.     

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

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

Accumulation of gammadelta T cells in the lungs and their regulatory roles in Th1 response and host defense against pulmonary infection with Cryptococcus neoformans

The present study was designed to elucidate the role of gammadelta T cells in the host defense against pulmonary infection with Cryptococcus neoformans. The gammadelta T cells in lungs commenced to increase on day 1, reached a peak level on day 3 or 6, and then decreased on day 10 after intratracheal infection. The increase of these cells was similar in monocyte chemoattractant protein (MCP)-1-deficient mice, although that of NK and NKT cells was significantly reduced. The number of live microorganisms in lungs on days 14 and 21 was significantly reduced in mice depleted of gammadelta T cells by a specific mAb compared with mice treated with control IgG. Similarly, elimination of this fungal pathogen was promoted in gammadelta T cell-deficient (TCR-delta(-/-)) mice compared with control littermate mice. Finally, lung and serum levels of IFN-gamma on days 7 and 14 and on day 7 postinfection, respectively, were significantly higher in TCR-delta(-/-) mice than in littermate mice, whereas levels of TGF-beta showed the opposite results. IL-4 and IL-10 were not different between these mice. IFN-gamma production by draining lymph node cells upon restimulation with cryptococcal Ags was significantly higher in the infected TCR-delta(-/-) mice than in control mice. Our results demonstrated that gammadelta T cells accumulated in the lungs in a manner different from NK and NKT cells after cryptococcal infection and played a down-modulatory role in the development of Th1 response and host resistance against this fungal pathogen.     

Circulating soluble CD4 directly prevents host resistance and delayed-type hypersensitivity response to Cryptococcus neoformans in mice

In the present study, we examined the effect of soluble CD4 (sCD4) on host resistance and delayed-type hypersensitivity (DTH) response to Cryptococcus neoformans using a novel mutant mouse that exhibits a defect in the expression of membrane-bound CD4 but secretes high levels of sCD4 in the serum. In these mice, host resistance to this pathogen was impaired as indicated by an increased number of live pathogens in the lung. To elucidate the mechanism of immunodeficiency, three different sets of experiments were conducted. First, administration of anti-CD4 mAb restored the attenuated host defense. Second, in CD4 gene-disrupted (CD4KO) mice, host resistance was not attenuated compared to control mice. Third, implantation of sCD4 gene-transfected myeloma cells rendered the CD4KO mice susceptible to this infection, while similar treatment with mock-transfected cells did not show such an effect. These results indicated that immunodeficiency in the mutant mice was attributed to the circulating sCD4 rather than to the lack of CD4+ T cells. In addition, DTH response to C. neoformans evaluated by footpad swelling was reduced in the mutant mice compared to that in the control, and the reduced response was restored by the administration of anti-CD4 mAb. Finally, serum levels of IFN-gamma, IL-12 and IL-18 in the mutant mice were significantly reduced, while there was no difference in Th2 cytokines, such as IL-4 and IL-10. Considered collectively, our results demonstrated that sCD4 could directly prevent host resistance and DTH response to C. neoformans through interference with the production of Th1-type cytokines.     

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.     

Neutralization of endogenous granulocyte-macrophage colony-stimulating factor subverts the protective immune response to Histoplasma capsulatum.

We examined the influence of endogenous GM-CSF on the course of primary and secondary pulmonary histoplasmosis. A high proportion (>/=75%) of C57BL/6 mice given mAb to GM-CSF did not survive primary infection, whereas 88-94% of infected controls survived. Analysis of leukocytes revealed significantly fewer CD4+ and CD8+ cells in lungs, but not airways, of anti-GM-CSF-treated mice as compared with infected controls. However, the histopathology was similar between the two groups. Lungs of mice given mAb to GM-CSF manifested depressed levels of TNF-alpha, IFN-gamma, and reactive nitrogen intermediates and elevated levels of IL-4 and IL-10. Administration of mAb to IL-4, to IL-10, or both restored protective immunity in GM-CSF-neutralized mice. In secondary infection, administration of mAb to GM-CSF exacerbated infection but did not alter survival over 30 days. The character of the inflammatory response was similar, and no differences were detected in Th1 or Th2 cytokine production between the two groups. Thus, endogenous GM-CSF is essential for survival in primary but not secondary infection, and blockade perturbs protective immunity. These findings reveal a new mechanism whereby GM-CSF contributes to host protection and demonstrate differences in control of primary and secondary histoplasmosis.     

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

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

Protection from direct cerebral cryptococcus infection by interferon-gamma-dependent activation of microglial cells

The brain represents a significant barrier for protective immune responses in both infectious disease and cancer. We have recently demonstrated that immunotherapy with anti-CD40 and IL-2 can protect mice against disseminated Cryptococcus infection. We now applied this immunotherapy using a direct cerebral cryptococcosis model to study direct effects in the brain. Administration of anti-CD40 and IL-2 significantly prolonged the survival time of mice infected intracerebrally with Cryptococcus neoformans. The protection was correlated with activation of microglial cells indicated by the up-regulation of MHC II expression on brain CD45(low)CD11b(+) cells. CD4(+) T cells were not required for either the microglial cell activation or anticryptococcal efficacy induced by this immunotherapy. Experiments with IFN-gamma knockout mice and IFN-gammaR knockout mice demonstrated that IFN-gamma was critical for both microglial cell activation and the anticryptococcal efficacy induced by anti-CD40/IL-2. Interestingly, while peripheral IFN-gamma production and microglial cell activation were observed early after treatment, negligible IFN-gamma was detected locally in the brain. These studies indicate that immunotherapy using anti-CD40 and IL-2 can augment host immunity directly in the brain against C. neoformans infection and that IFN-gamma is essential for this effect.     

Cryptosporidium infection in an adult mouse model. Independent roles for IFN-gamma and CD4+ T lymphocytes in protective immunity.

Cryptosporidium is a protozoan parasite that can cause chronic life-threatening diarrhea in immunocompromised persons. Host immune responses are poorly understood, an impediment to development of effective therapy. In mice, normal adult BALB/c animals resist infection whereas chronic symptomatic cryptosporidiosis develops in adult nude mice and in neonatally infected BALB/c mice treated with anti-CD4 mAb. To define further the immune defects that allow mice to be infected with Cryptosporidium, adult BALB/c mice were treated with cytolytic anti-CD4 or anti-CD8 or with neutralizing anti-IFN-gamma or anti-IL-2 mAb. Chronic infection, manifested by continuous shedding of sparse but statistically significant numbers of oocysts, occurred with anti-CD4 +/- anti-CD8 mAb treatment although anti-CD8 mAb treatment alone did not allow infection. Treatment with anti-IFN-gamma mAb greatly enhanced oocyst shedding but infection was self-limited. Treatment with a combination of anti-CD4 and anti-IFN-gamma mAb permitted both chronic infection and shedding of large numbers of oocysts. Furthermore mice treated initially with anti-CD4 mAb showed a substantial increase in oocyst shedding when later treated with anti-IFN-gamma mAb; and mice treated initially with both mAbs showed a decline in oocyst shedding when anti-IFN-gamma mAb was stopped. Anti-IFN-gamma mAb treatment of congenitally athymic adult BALB/c mice led to an approximately a 75-fold increase in oocyst shedding. Treatment of adult BALB/c mice with anti-IL-2 mAb did not permit Cryptosporidium infection. These results suggest that redundant immunologic mechanisms limit Cryptosporidium infection such that both CD4+ cells and IFN-gamma are required to prevent initiation of infection whereas either alone can limit the extent (IFN-gamma) or duration (CD4+ T cells) of infection. They also suggest that production of IFN-gamma by a non-T cell contributes to host immunity.     

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.     

IL-10 is required for prevention of necrosis in the small intestine and mortality in both genetically resistant BALB/c and susceptible C57BL/6 mice following peroral infection with Toxoplasma gondii

The role for IL-10 in the immunopathogenesis of acute toxoplasmosis following peroral infection was examined in both genetically susceptible C57BL/6 and resistant BALB/c mice. C57BL/6-background IL-10-targeted mutant (IL-10-/-) mice all died in 2 wk after infection with 20 cysts of the ME49 strain, whereas only 20% of control mice succumbed. Histological studies revealed necrosis in the small and large intestines and livers of infected IL-10-/- mice. The necrosis in the small intestine was the most severe pathologic response and was not observed in control mice. Treatment of infected IL-10-/- mice with either anti-CD4 or anti-IFN-gamma mAb prevented intestinal pathology and significantly prolonged time to death. Treatment of these animals with anti-IL-12 mAb also prevented the pathology. Significantly greater amounts of IFN-gamma mRNA were detected in the lamina propria lymphocytes obtained from the small intestine of infected IL-10-/- mice than those from infected control mice. In common with C57BL/6-background IL-10-/- mice, BALB/c-background IL-10-/- mice all died developing intestinal pathology after infection. Control BALB/c mice all survived even after infection with 100 cysts and did not develop the intestinal lesions. Treatment with anti-IFN-gamma mAb prevented the pathology and prolonged time to death of the infected IL-10-/- mice. These results strongly suggest that IL-10 plays a critical role in down-regulating IFN-gamma production in the small intestine following sublethal peroral infection with Toxoplasma gondii and that this down-regulatory effect of IL-10 is required for prevention of development of IFN-gamma-mediated intestinal pathology and mortality in both genetically resistant BALB/c and susceptible C57BL/6 mice.     

Contribution of interferon-γ in protecting mice during pulmonary and disseminated infection with Cryptococcus neoformans

Abstract In the present study, the role of interferon-γ (IFN-γ) in the host resistance against Cryptococcus neoformans was examined using a murine model of pulmonary and disseminated infection. In this model, mice were infected intratracheally with live yeast cells, and the histological changes in the lungs and the number of microorganisms in the lung and brain were compared in mice treated and untreated with anti-IFN-γ monoclonal antibody (mAb) to define the contribution of endogenously synthesized IFN-γ in the natural course of infection. Administration of this mAb reduced the accumulation of inflammatory cells in the alveolar septa, peribronchial and perivascular areas, and promoted the expansive growth of microorganisms in the alveoli and destruction of alveolar structure. The neutralization of endogenous IFN-γ by mAb increased the number of microorganisms in the lung and brain, and significantly shortened the survival time of infected mice. On the other hand, administration of IFN-γ decreased the number of microorganisms in these organs, and significantly extended their survival time. Considered together, our results suggest that endogenous IFN-γ protects mice from infection with C. neoformans by inducing a cellular inflammatory response, potentiating the clearance of microorganism from the lungs and preventing its dissemination into the central nervous system.     

IL-13 induces disease-promoting type 2 cytokines, alternatively activated macrophages and allergic inflammation during pulmonary infection of mice with Cryptococcus neoformans

In the murine model of Cryptococcus neoformans infection Th1 (IL-12/IFN-gamma) and Th17 (IL-23/IL-17) responses are associated with protection, whereas an IL-4-dependent Th2 response exacerbates disease. To investigate the role of the Th2 cytokine IL-13 during pulmonary infection with C. neoformans, IL-13-overexpressing transgenic (IL-13Tg(+)), IL-13-deficient (IL-13(-/-)), and wild-type (WT) mice were infected intranasally. Susceptibility to C. neoformans infection was found when IL-13 was induced in WT mice or overproduced in IL-13Tg(+) mice. Infected IL-13Tg(+) mice had a reduced survival time and higher pulmonary fungal load as compared with WT mice. In contrast, infected IL-13(-/-) mice were resistant and 89% of these mice survived the entire period of the experiment. Ag-specific production of IL-13 by susceptible WT and IL-13Tg(+) mice was associated with a significant type 2 cytokine shift but only minor changes in IFN-gamma production. Consistent with enhanced type 2 cytokine production, high levels of serum IgE and low ratios of serum IgG2a/IgG1 were detected in susceptible WT and IL-13Tg(+) mice. Interestingly, expression of IL-13 by susceptible WT and IL-13Tg(+) mice was associated with reduced IL-17 production. IL-13 was found to induce formation of alternatively activated macrophages expressing arginase-1, macrophage mannose receptor (CD206), and YM1. In addition, IL-13 production led to lung eosinophilia, goblet cell metaplasia and elevated mucus production, and enhanced airway hyperreactivity. This indicates that IL-13 contributes to fatal allergic inflammation during C. neoformans infection.     

Chemokine responses and accumulation of inflammatory cells in the lungs of mice infected with highly virulent Cryptococcus neoformans: effects of interleukin-12

We examined the mechanisms involved in the development of lung lesions after infection with Cryptococcus neoformans by comparing the histopathological findings and chemokine responses in the lungs of mice infected with C. neoformans and assessed the effect of interleukin (IL) 12 which protects mice from lethal infection. In mice infected intratracheally with a highly virulent strain of C. neoformans, the yeast cells multiplied quickly in the alveolar spaces but only a poor cellular inflammatory response was observed throughout the course of infection. Very little or no production of chemokines, including MCP-1, RANTES, MIP-1alpha, MIP-1beta and IP-10, was detected at the mRNA level using RT-PCR as well as at a protein level in MCP-1, RANTES and MIP-1alpha. In contrast, intraperitoneal administration of IL-12 induced the synthesis of these chemokines and a marked cellular inflammatory response involving histiocytes and lymphocytes in infected mice. Our findings were confirmed by flow cytometry of intraparenchymal leukocytes obtained from lung homogenates which showed IL-12-induced accumulation of inflammatory cells consisting mostly of macrophages and CD4+ alphabeta T cells. On the other hand, C-X-C chemokines including MIP-2 and KC, which attract neutrophils, were produced in infected and PBS-treated mice but treatment with IL-12 showed a marginal effect on their level, and neutrophil accumulation was similar in PBS- and IL-12-treated mice infected with C. neoforman. Our results demonstrate a close correlation between chemokine levels and development of lung lesions, and suggest that the induction of chemokine synthesis may be one of the mechanisms of IL-12-induced protection against cryptococcal infection.     

The role of IL-4 in Heligmosomoides polygyrus-induced alterations in murine intestinal epithelial cell function

IL-4 and IL-13 promote gastrointestinal worm expulsion, at least in part, through effects on nonlymphoid cells, such as intestinal epithelial cells. The role of IL-4/IL-13 in the regulation of intestinal epithelial function during Heligmosomoides polygyrus (Hp) infection was investigated in BALB/c mice infected with Hp or treated with a long-lasting formulation of recombinant mouse IL-4/alphaIL-4 complexes (IL-4C) for 7 days. Separate groups of BALB/c mice were drug-cured of initial infection and later reinfected and treated with anti-IL-4R mAb, an antagonist of IL-4 and IL-13 receptor binding, or with a control mAb. Segments of jejunum were mounted in Ussing chambers, and short circuit current responses to acetylcholine, histamine, serotonin, PGE2, and glucose were determined. Although only modest changes in epithelial cell function were observed during primary Hp infection, IL-4C or a secondary Hp infection each induced more dramatic changes, including increased mucosal permeability, reduced sodium-linked glucose absorption, and increased Cl- secretory response to PGE2. Some, but not all, effects of IL-4C and Hp infection were dependent on enteric nerves. Hp-induced changes in epithelial function were attenuated or prevented by anti-IL-4R mAb. Thus, IL-4/IL-13 mediate many of the effects of Hp infection on intestinal epithelial cell function and do so both through direct effects on epithelial cells and through indirect, enteric nerve-mediated prosecretory effects. These immune system-independent effector functions of IL-4/IL-13 may be important for host protection against gastrointestinal nematodes.     

IFN-alpha/beta signaling is required for polarization of cytokine responses toward a protective type 1 pattern during experimental cryptococcosis

The antiviral activities of type I IFNs have long been established. However, comparatively little is known of their role in defenses against nonviral pathogens. We examined here the effects of type I IFNs on host resistance against the model pathogenic yeast Cryptococcus neoformans. After intratracheal or i.v. challenge with this fungus, most mice lacking either the IFN-alpha/beta receptor (IFN-alpha/betaR) or IFN-beta died from unrestrained pneumonia and encephalitis, while all wild-type controls survived. The pulmonary immune response of IFN-alpha/betaR-/- mice was characterized by increased expression of IL-4, IL-13, and IL-10, decreased expression of TNF-alpha, IFN-gamma, inducible NO synthetase, and CXCL10, and similar levels of IL-12 mRNA, compared with wild-type controls. Histopathological analysis showed eosinophilic infiltrates in the lungs of IFN-alpha/betaR-/- mice, although this change was less extensive than that observed in similarly infected IFN-gammaR-deficient animals. Type I IFN responses could not be detected in the lung after intratracheal challenge. However, small, but statistically significant, elevations in IFN-beta levels were measured in the supernatants of bone marrow-derived macrophages or dendritic cells infected with C. neoformans. Our data demonstrate that type I IFN signaling is required for polarization of cytokine responses toward a protective type I pattern during cryptococcal infection.