Early neutrophil recruitment and aggregation in the murine lung inhibit germination of Aspergillus fumigatus Conidia

Several types of polymorphonuclear neutrophil (PMN) deficiency are a predisposing condition for fatal Aspergillus fumigatus infection. In order to study the defensive role of PMNs in the lungs, with particular reference to PMN recruitment and antimicrobial oxidant activity, responses to pulmonary instillation of A. fumigatus conidia were examined. Responses in BALB/c and C57BL/6 mice were compared with those in CXCR2(-/-) and gp91(phox-/-) mice, which are known to have delayed recruitment of PMN to the lungs in response to inflammatory stimuli and inactive NADPH oxidase, respectively. In BALB/c mice, PMNs were recruited to the lungs and formed oxidase-active aggregates with conidia, which inhibited germination. In C57BL/6, gp91(phox-/-), and CXCR2(-/-) mice, PMN recruitment was slower and there was increased germination compared to that in BALB/c mice at 6 and 12 h. In gp91(phox-/-) mice, germination was extensive in PMN aggregates but negligible in alveolar macrophages (AM). Lung sections taken at 6 and 48 h from BALB/c mice showed PMN accumulation at peribronchiolar sites but no germinating conidia. Those from C57BL/6 and CXCR2(-/-) mice showed germinating conidia at 6 h but not at 48 h and few inflammatory cells. In contrast, those from gp91(phox-/-) mice showed germination at 6 h with more-extensive hyphal proliferation and tissue invasion at 48 h. These results indicate that when the lungs are exposed to large numbers of conidia, in addition to the phagocytic activity of AM, early PMN recruitment and formation of oxidative-active aggregates are essential in preventing germination of A. fumigatus conidia.     

Characterization of a Murine Model of Melioidosis: Comparison of Different Strains of Mice

Melioidosis is an infectious disease caused by the saprophytic gram-negative rod Burkholderia pseudomallei. The aim of this study was to establish and characterize a murine model of melioidosis to provide a basis for further investigations on the pathogenesis of the disease. After intravenous infection with B. pseudomallei, C57BL/6 mice were found to be significantly more resistant than BALB/c mice. There was a marked organotropism of B. pseudomallei for the spleen and liver in both strains of mice, with the highest bacterial load in the spleen. Electron microscopic investigations of the spleen clearly demonstrated intracellular replication within membrane-bound phagosomes. Electron micrographs of the liver provided evidence that B. pseudomallei-containing phagosomes in hepatocytes fuse with lysosomes, leading to degradation of bacteria. In both strains of mice, the course of infection was highly dependent on the infective dose and the bacterial strain used, ranging from death within a few days to death after several weeks. In comparison with BALB/c mice, the bacterial counts in C57BL/6 mice were decreased 12 h after infection, which is suggestive of an innate immune mechanism against B. pseudomallei in this early phase of infection contributing to the lower susceptibility of C57BL/6 mice. BALB/c mice developed a more pronounced lymphopenia, granulocytosis, and splenomegaly at a lower infective dose compared to C57BL/6 mice. Analysis of the antibody response against B. pseudomallei 11 days after infection revealed a significantly higher immunoglobulin G2A (IgG2a)/IgG1 ratio in C57BL/6 mice than in BALB/c mice, indicating that a T helper type 1 immune response is associated with resistance to infection with B. pseudomallei.     

The role of nicotinamide adenine dinucleotide phosphate oxidase-derived reactive oxygen species in the acquisition of metastatic ability of tumor cells

We examined the role of phagocyte-derived oxygen radicals in tumor cell acquisition of metastatic phenotype by comparing gp91(phox-/-) mice and C57BL/6J wild-type (WT) mice. The gp91(phox-/-) mouse is deficient in the gp91(phox) gene, an essential subunit of the phagocyte nicotinamide adenine dinucleotide phosphate oxidase that generates superoxide anion. QR-32 fibrosarcoma cells are nonmetastatic but are converted into metastatic tumors once in contact with foreign body (gelatin sponge)-induced phagocytes in vivo. Compared to QR-32 cells co-implanted with the foreign body in WT mice, those in gp91(phox-/-) mice exhibited reduced metastasis. There was no difference in the incidence of primary tumors after injection of B16BL6 melanoma cells in WT and gp91(phox-/-) mice. However, after resection of the primary tumors, metastases were reduced in gp91(phox-/-) mice. Thymosin beta4 gene expression and cell motility/invasion were seen in the tumors from WT mice but not in those from gp91(phox-/-) mice. Adoptive transfer of phagocytes from WT mice, but not those from gp91(phox-/-) mice, restored the metastatic ability of tumors grown in gp91(phox-/-) mice. These findings show that tumor metastatic behavior can primarily be endowed by phagocyte-derived superoxide anion and its oxidative metabolites, which are generated through activation of nicotinamide adenine dinucleotide phosphate oxidase.     

Susceptibility to Yersinia pseudotuberculosis Infection is Linked to the Pattern of Macrophage Activation

T helper 1 cells play a crucial role in the clearance of Yersinia pseudotuberculosis infection. By producing cytokines and presenting antigens to T cells, activated macrophages can orientate the adaptive immune response. The pathway used by macrophages to metabolize arginine has been employed as an important parameter to discriminate their activation state. In this study, the pattern of macrophage activation in Y. pseudotuberculosis- infected BALB/ c ( Yersinia -susceptible) and C57BL/6 ( Yersinia -resistant) mice and their immunostimulatory capacity were analysed. In the early phase of infection, macrophages obtained from C57BL/6 mice produced higher levels of NO, lower arginase activity, and larger amounts of IL-12 and TNF-α than macrophages from BALB/ c mice. On the other hand, macrophages derived from BALB/ c mice produced higher levels of IL-10 and TGF-β than C57BL/6 mice. The Y. pseudotuberculosis infection leads to a fall in the macrophage immunostimulatory capacity of both strains of mice, with T-cell proliferation significantly reduced 12 h after infection. Moreover, we observed in the supernatant of co-culture of macrophages from infected mice with T lymphocytes from heat-killed Yersinia -immunized mice lower IFN-γ production by cells from BALB/ c mice than by C57BL/6 mice, and IL-4 was produced only by BALB/ c mice on the first- and third-day post-infection. These results suggest that the pattern of macrophage activation is associated with susceptibility and resistance to Y. pseudotuberculosis infection in BALB/ c and C57BL/6 mice.     

Activation of non-specific cytotoxic cells in Listeria-susceptible and -resistant mouse strains.

An increase in macrophage tumouricidal activity in the spleen was demonstrated following intravenous infection of genetically resistant C57BL/10 mice with Listeria monocytogenes, but not after infection of BALB/c mice. However, tumouricidal macrophages appeared in the peritoneal cavity of both strains after infection, while NK cell activity was generally higher in BALB/c mice. The activation of tumouricidal macrophages and NK cells in the C57BL/B10 mice was T independent and, at least in the peritoneal cavity, radioresistant (600 rads). The relevance of these results to the genetic control of resistance to Listeria is discussed.     

The role of CD4+ and CD8+ T-cells in host morbidity and innate resistance to Angiostrongylus cantonensis in the mouse

Strain-dependent differences in host morbidity and mortality due to Angiostrongyluscantonensis infection have been established between C57BL/6 and BALB/c mice; C57BL/6 mice show rapid worm killing with low morbidity, whereas BALB/c mice indicate slow worm killing with high morbidity and mortality. To determine the possible roles of CD4⁺ and CD8⁺ T-cells in host morbidity and innate resistance to A. cantonensis infection we treated C57BL/6 and BALB/c mice with anti-CD4 or anti-CD8 monoclonal antibody and examined the changes in host morbidity and worm-killing activity. Our study indicates that anti-CD4 antibody treatment interferes with worm killing and improves the morbidity of A. cantonensis-infected BALB/c mice, whereas anti-CD8 antibody treatment fails to improve the morbidity. Tumor necrosis factor-α (TNF-α, or cachectin) production in infected mice was not correlated with host morbidity. Anti-IL-5 monoclonal antibody treatment also failed to affect the morbidity of infected BALB/c mice, although their worm-killing activity was restrained as shown in anti-CD4-treated mice. These findings clearly indicate that the morbidity of infected BALB/c mice is regulated by some unknown CD4⁺ T-cell-dependent mechanism but not by an IL-5-, eosinophil-, or TNF-α-dependent mechanism. Received: 9 June 1997 / Accepted: 11 July 1997     

Differences in expression of toll-like receptors and their reactivities in dendritic cells in BALB/c and C57BL/6 mice

We have previously reported that differences in early production of interleukin 12 (IL-12) by dendritic cells (DC) underlies the difference between the susceptibilities to Listeria monocytogenes of C57BL/6 and BALB/c mice. To elucidate mechanisms for the different abilities of DC to produce cytokine in C57BL/6 and BALB/c mice, we examined Toll-like receptor (TLR) expression by DC and their responses in vitro to known microbial ligands for TLRs. We found that DC isolated from the spleens of naive C57BL/6 mice preferentially expressed TLR9 mRNA, whereas DC from naive BALB/c mice strongly expressed TLR2, -4, -5, and -6 mRNAs. C57BL/6 DC produced a higher level of IL-12p40 in response to the ligands for TLR4 (lipopolysaccharide), TLR2 (lipoprotein), and TLR9 (CpG), whereas BALB/c DC responded to these ligands by producing a larger amount of monocyte chemoattractant protein 1. C57BL/6 DC expressed higher levels of CD40 and Stat4 than BALB/c DC did, suggesting that naive C57BL/6 mice contained more-mature subsets of DC than naive BALB/c mice. Differences in reactivities of DC to microbial molecules through TLRs may be associated with susceptibility and resistance to Listeria infection in BALB/c and C57BL/6 mice.     

Mouse genes influence antiviral action of interferon in vivo.

BALB/c mice are more sensitive to the antiviral effect of interferon than C57BL/6 mice, as demonstrated by experiments involving protection against lethal infection with encephalomyocarditis virus. This greater sensitivity of the BALB/c genotype to interferon action is in accord with previous observations that the bone marrow-derived erythroid precursors and macrophages of BALB/c mice are more sensitive to the anti-proliferative action of interferon than those of C57BL/6 mice. An analysis of the loci involved in the modulation of the activity of interferon against encephalomyocarditis virus infection was carried out in (BALB/c x C57BL/6)F1 progeny and in six recombinant inbred lines originally derived from a BALB/c x C57BL/6 cross. The antiviral effect of exogenous interferon in the F1 progeny was comparable to the effect in BALB/c mice, indicating dominance of the greater sensitivity to interferon action. The results obtained with the six recombinant inbred lines suggested a multifactorial influence. In vitro, interferon pretreatment of encephalomyocarditis virus-infected BALB/c and C57BL/6 fibroblast cultures did not reveal a difference in sensitivity between the two mouse genotypes. This finding demonstrates that it is not always possible to extrapolate from in vitro to in vivo when sensitivity to interferon action is studied.     

A novel regulatory role of gp49B on dendritic cells in T-cell priming

Dendritic cells (DC) play pivotal roles in the induction and regulation of both innate and acquired immunity. DC express several cell-surface immune inhibitory receptors. However, little is known about their potential immunoregulatory functions in the context of T-cell activation. Here we report that murine gp49B, a member of the immunoglobulin superfamily, harboring immunoreceptor tyrosine-based inhibitory motifs, is expressed on DC and downregulates cellular activity to prevent the excessive activation of T cells in vitro and in vivo. Bone marrow-derived DC (BMDC) from newly generated gp49B-deficient (gp49B(-/-)) mice induced enhanced proliferation and IL-2 release of antigen-specific CD4(+) and CD8(+) T cells compared with BMDC from wild-type mice, in a cell-cell contact manner. The enhanced proliferation by gp49B(-/-) BMDC was also observed in allogeneic CD4(+) and CD8(+) T cells. Moreover, the transfer of allogeneic BALB/c splenocytes into C57BL/6 gp49B(-/-) mice induced severe acute graft-versus-host disease with an augmented upregulation of CD86 on CD11c(+) splenic gp49B(-/-) DC, while transfer of C57BL/6 gp49B(-/-) splenocytes into BALB/c mice did not, suggesting the exacerbation of the disease was due, at least in part, to augmented activation of recipient gp49B(-/-) DC. These findings demonstrate a novel regulatory role of gp49B in the function of DC.     

Differential CD86 and CD40 co-stimulatory molecules and cytokine expression pattern induced by Trypanosoma cruzi in APCs from resistant or susceptible mice

Differential aspects of the host immune response generated by Trypanosoma cruzi infection were examined in two different mouse strains, BALB/c (haplotype H2-Kd) which does not overcome the acute phase of the infection and C57BL/6 (haplotype H2-Kb) which survives to the acute phase. After infection an increase in CD3+ T cells was observed in both mouse strains in the peritoneal cavity. However, while the CD3+ T cells from the BALB/c mice showed an increase in the IL-4 cytokine expression level, the same type of cells from the C57BL/6 mice showed an increase in IFN-gamma expression. In addition, only the macrophages from the C57BL/6 mice were activated secreting IL-12 and TNF-alpha and producing, moreover, high levels of nitrites. It was observed that also after parasite infection the expression of macrophage and dendritic cells CD40 and CD86 co-stimulation molecules from the spleen were diminished in BALB/c but not in C57BL/6 mice. In correlation with this observation the macrophages from the spleen of infected BALB/c mice secreted lower concentrations of nitrites than the C57BL/6 mouse cells. Also, the spleen dendritic cells from infected BALB/c mice had a small potential to present alloantigens in contrast to that observed in the infected C57BL/6 mouse cells.     

Macrophage-T cell interaction in experimental visceral leishmaniasis: failure to express costimulatory molecules on Leishmania-infected macrophages and its implication in the suppression of cell-mediated immunity

The most important immunopathological consequence of infection with Leishmania seen in murine and human hosts is the suppression of T cell-mediated immune responses to both mitogens and leishmanial antigens. It has been suggested that this suppression is mediated by macrophages, either by defective antigen processing and presentation or by the elaboration of suppressive mediators like prostaglandins. Optimum activation of T helper cells requires not only T cell receptor occupancy by the antigen-Ia complex, but also costimulatory signals provided by the antigen-presenting cells. We investigated the status of several costimulatory molecules on infected macrophages from both genetically susceptible BALB/c and resistant C57BL/6 mice. Our results demonstrate that upon parasitization, the macrophages become unable to deliver costimulatory signals to T helper cells, and that this effects is mediated by prostaglandins, as the inhibition of its synthesis by indomethacin recovered the defect. Upon infection with L. donovani, B7-1 expression was decreased, while ICAM-1 was marginally increased in BALB/c macrophages and there was no significant change in the expression of B7-1 and ICAM-1 in Leishmania-infected C57BL/6 macrophages. Expression of VCAM-1 did not change during infection. This selective alteration in the expression of costimulatory molecules on L. donovani-infected BALB/c macrophages was caused by the living parasite, as shown by the fact that killing of the parasites by stibogluconate led to no alteration in the levels of costimulatory molecules. We found that the change in B7-1 expression on the surface of infected macrophages resulted in the inhibition of delayed-type hypersensitivity-mediating functions of T helper cells from BALB/c mice. The results described in this study not only throw light on the possible mechanism of leishmanial pathogenesis, but also open up the possibility of immunotherapy of leishmaniasis by selective manipulation of costimulatory molecules.     

Strain differences in mouse cellular responses to Mycobacterium lepraemurium and BCG subcutaneous infections. I. Analysis of cell surface phenotype in local granulomas.

C57BL/6, BALB/c and CBA mice were subcutaneously infected with either Mycobacterium lepraemurium (MLM) or BCG, and studied for bacillary growth, granuloma size of infected footpads and draining lymph nodes (DLN), and DLN cell surface phenotype. Whereas, BCG-infected mice controlled the infection and developed early and large granulomas, MLM-infected mice exhibited major strain variations in their resistance to the infection, as well as in the granuloma size and kinetics. C57BL/6 mice, highly resistant, displayed early and regressive granulomas; BALB/c mice showed lower resistance and early granulomas that grew continuously; CBA mice, highly susceptible, developed late, soft, phagocyte-rich granulomas. Important strain differences in lymph node lymphocyte subset distribution could be observed prior to any infection: C57BL/6 mice displayed higher B cell percentages than both of the other strains and BALB/c mice showed the highest CD4/CD8 ratios, followed by CBA and C57BL/6 mice. BCG and MLM infections both induced similar changes of these parameters in all three strains: that is a decrease of the B cell percentage and a decrease of the CD4/CD8 ratio, and the strain differences observed in uninfected mice persisted. On the other hand, DLN cells stimulated by the infecting bacillus and interleukin 2 also displayed an increase of the CD8 T cell percentage as compared with normal lymph node cells, but this phenomenon was much less pronounced in BALB/c mice, whether infected by MLM or BCG, and in MLM-infected CBA mice, than in BCG- or MLM-infected C57BL/6 (B6) mice. Thus the ability of C57BL/6 mice to generate an early and persistent CD8 T cell response to mycobacteria may contribute to their resistance to MLM.     

TH1 and TH2 T-cell subsets are differentially activated by macrophages and B cells in murine leishmaniasis.

The role of antigen-presenting cells in the differential expansion of TH1 and TH2 T cells in murine leishmaniasis was investigated. In general, macrophages preferentially induced gamma interferon and interleukin-2 secretion by syngeneic Leishmania-specific T cells, whereas B cells were more efficient in activating interleukin 4 production. B cells from susceptible BALB/c mice were better in inducing TH2 responses than B cells from resistant C57BL/6 mice, whereas macrophages from C57BL/6 mice were superior to BALB/c macrophages in inducing TH1 responses.     

Phenotype of mice and macrophages deficient in both phagocyte oxidase and inducible nitric oxide synthase

The two genetically established antimicrobial mechanisms of macrophages are production of reactive oxygen intermediates by phagocyte oxidase (phox) and reactive nitrogen intermediates by inducible nitric oxide synthase (NOS2). Mice doubly deficient in both enzymes (gp91(phox-/-)/NOS2(-/-)) formed massive abscesses containing commensal organisms, mostly enteric bacteria, even when reared under specific pathogen-free conditions with antibiotics. Neither parental strain showed such infections. Thus, phox and NOS2 appear to compensate for each other's deficiency in providing resistance to indigenous bacteria, and no other pathway does so fully. Macrophages from gp91(phox-/-)/NOS2(-/-) mice could not kill virulent Listeria. Their killing of S. typhimurium, E. coli, and attenuated Listeria was markedly diminished but demonstrable, establishing the existence of a mechanism of macrophage antibacterial activity independent of phox and NOS2.     

The role of macrophage activation and of Bcg-encoded macrophage function(s) in the control of Mycobacterium avium infection in mice.

Following the intraperitoneal inoculation of 2.5 x 10(8) colony-forming units of Mycobacterium avium strain ATCC 25291, there was bacillary growth in the liver, spleen and peritoneal cavity of C57BL/6, C57BL/10, DBA/1 and BALB/c mice whereas DBA/2, C3H/He, CBA/Ca and CD-1 mice controlled the infection showing constant or slightly decreasing numbers of viable bacteria in the liver and spleen and effective clearance of the bacilli from the peritoneal cavities. The acquisition of non-specific resistance (NSR) to Listeria monocytogenes during the infection by M. avium was high in C57BL/6, BALB/c and C3H/He mice and negligible in DBA/2 and CD-1 mice. The magnitude of the acquisition of NSR was reduced in T cell-deficient mice and was directly proportional to the dose of the inoculum of M. avium. The production of hydrogen peroxide by phorbol myristate acetate-stimulated peritoneal macrophages of M. avium-infected mice was higher in C57BL/6 and BALB/c mice than in CD-1, DBA/2 and C3H/He animals. BALB/c. Bcgr (C.D2) mice, unlike their congenic strain BALB/c, restricted bacterial growth following the intravenous inoculation of 2.5 x 10(8) CFU of M. avium as efficiently as DBA/2 mice. C.D2 and BALB/c peritoneal macrophages from infected mice produced similar amounts of H2O2 but BALB/c mice developed higher levels of NSR to listeria than C.D2 mice. The production of nitrite by peritoneal macrophages from infected mice was found to be enhanced in DBA/2 and C3H/He but not in BALB/c, C57BL/6, DC-1 and C.D2 mice. Resident peritoneal macrophages from C.D2 mice were more bacteriostatic in vitro for M. avium than macrophages from BALB/c mice. The same relative differences between the two macrophage populations were observed when the cells were activated with lymphokines. The results show that the populations were observed when the cells were activated with lymphokines. The results show that the resistance to M. avium infection in mice is under the control of the Bcg gene and that susceptibility may be due to some defect in macrophage antibacterial function not completely overcome by the activation of this phagocyte in the susceptible strains of mice.     

Regulation by endogenous interferon of virus-induced cytokine gene expression in mouse macrophages.

In macrophages from inbred mice the magnitude of the interferon (IFN) response to Newcastle disease virus (NDV) infection is under genetic control of the locus If-1, with C57BL/6 carrying the 'high-producer' allele If-1h whereas BALB/c have the 'low-producer' allele If-1l. The IFN produced consists of 90% IFN-beta and there are 10-fold differences between macrophages from If-1h and If-1l mice. Recently, we observed that interleukin-6 (IL-6) is coinduced by NDV in macrophages and seems to be under the same genetic control. Noninduced macrophages have been shown to secrete low amounts of antiviral activity endogenously when cultured in the presence of the macrophage-colony-stimulating factor (M-CSF). Here, we report that the amount of this endogenous IFN varies between macrophages from different mouse strains. Macrophages from BALB/c were found to secrete 5-10 times more endogenous IFN compared to C57BL/6. The antiviral activity could be identified as IFN-beta. Interestingly, we observed that endogenous IFN specifically down-regulates NDV-induced IFN and IL-6 production. Preculture of BALB/c macrophages in M-CSF plus anti-IFN-beta to neutralize the biological effects of the endogenous IFN provoked a 30- to 50-fold increase in NDV-induced cytokine production, resulting in a nearly complete abrogation of the genetically determined difference since the same treatment only caused a 6-fold increase in C57BL/6 macrophages following NDV infection. This increase in cytokine gene expression was specific for NDV and marked by a strong additional activation of IFN-alpha genes. Addition of mouse recombinant IFN-alpha 4 to anti-IFN-beta-treated macrophages for 18 h prior to NDV infection down-regulated again IFN gene expression and reestablished the genetic differences between macrophages from If-1h and If-1l mice.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differences in innate immune responses correlate with differences in murine susceptibility to Chlamydia muridarum pulmonary infection

We investigated the phenotypic basis for genetically determined differences in susceptibility and resistance to Chlamydia muridarum pulmonary infection using BALB/c and C57BL/6 mice. Following C. muridarum intranasal inoculation, the intensity of infection was very different between BALB/c and C57BL/6 beginning as early as 3 days post‐infection. Intrapulmonary cytokine patterns also differed at early time‐points (days 2 and 4) between these two strains of mice. The early recruitment of neutrophils to lung tissue was greater in BALB/c than in C57BL/6 mice and correlated with a higher number of inclusion forming units (IFU) of C. muridarum. At day 12 post‐infection, BALB/c mice continued to demonstrate a greater burden of infection, significantly higher lung cytokine levels for tumour necrosis factor‐α and interleukin‐17 (IL‐17) and a significantly lower level for interferon‐γ than did C57BL/6 mice. In vitro, bone‐marrow‐derived dendritic cells (BMDCs) from BALB/c mice underwent less functional maturation in response to C. muridarum infection than did BMDCs from C57BL/6 mice. The BMDCs of BALB/c mice expressed lower levels of activation markers (CD80, CD86, CD40 and major histocompatibility complex class II) and secreted less IL‐12 and more IL‐23 than BMDCs from C57BL/6 mice. Overall, the data demonstrate that the differences exhibited by BALB/c and C57BL/6 mice following C. muridarum pulmonary infection are associated with differences in early innate cytokine and cellular responses that are correlated with late differences in T helper type 17 versus type 1 adaptive immune responses.