Growth of Brucella abortus in macrophages from resistant and susceptible mouse strains

C57Bl/10 mice have a superior ability to control chronic infections with virulent strains of the intracellular bacteria Brucella abortus compared with BALB/c mice. While a number of differences in the cytokines produced by lymphocytes following infection of these two strains of mice have been shown, macrophages have not been evaluated for their role in conveying relative resistance. The importance of macrophages in control of brucella infections is demonstrated by the observations that intracellular survival of various strains of B. abortus directly correlates with their virulence in vivo, and the ability of macrophages to control brucellae in vitro has been shown to correlate with a brucella-resistant phenotype in ruminants. While both BALB/c and C57Bl are Nramp-susceptible mouse strains, additional differences in macrophage function outside of the Nramp1 gene effects could influence susceptibility to brucellosis. The studies conducted here comparing the ability of macrophages from C57Bl/10 and BALB/c mice indicate that the macrophages from resistant mice did not control intracellular growth of B. abortus strain 2308 more efficiently than those from the susceptible mice, either in the absence of, or following, interferon-gamma activation or iron supplementation. A number of different conditions for culturing macrophages were evaluated to rule out the influence of antibiotics on the conclusions drawn from the results.     

Quantitative and qualitative differences in bronchoalveolar inflammatory cells in Pseudomonas aeruginosa-resistant and -susceptible mice

The difference in severity of Pseudomonas aeruginosa-induced chronic lung infection may be determined by differences in host inflammatory responses. In the present study we investigate this possibility using BALB/c and C57Bl/6 mice, resistant and susceptible, respectively, to chronic lung infection with P. aeruginosa. Following intratracheal inoculation of P. aeruginosa-impregnated agar beads, C57Bl/6 mice mounted a stronger inflammatory response with significantly higher total cell numbers in the bronchoalveolar lavage fluid compared with BALB/c mice. While polymorphonuclear leucocytes were the predominant cell in C57Bl/6 mice, macrophages constituted the majority in BALB/c mice at day 7 post-infection. Alveolar macrophages from C57Bl/6 mice showed significantly higher spontaneous production of nitric oxide (NO) at day 7 post-infection compared with BALB/c mice. Following in vitro stimulation with heat-killed Pseudomonas antigen, these cells produced significantly higher NO compared with cells from BALB/c mice at day 21 post-infection. Production of tumour necrosis factor-alpha (TNF-α) by alveolar macrophages was significantly higher at day 7 in BALB/c mice compared with C57Bl/6 mice, which showed significantly higher levels at day 28 post-infection. Taken together, these results suggest that defects in the host inflammatory process contribute to the variable outcome of chronic lung infection with P. aeruginosa. An exaggerated inflammatory response dominated by polymorphonuclear cells correlates with susceptibility to infection, whilst a modest inflammatory response dominated by macrophages correlates with resistance. Moreover, the quantity and timing of production of NO and TNF-α by alveolar macrophages may modulate the course and outcome of infection.     

Trypanosoma rhodesiense: analysis of the genetic control of resistance among mice.

Inbred mouse strains differ in their resistance to infection with the human pathogen Trypanosoma rhodesiense. Of the strains tested, C57BL/6 (B6) mice were the most resistant, and BALB/c (C) mice were among the most susceptible. The genetic basis underlying the different susceptibility of these two strains was analyzed. (CXB6)F1 progeny of either sex were more resistant than the BALB/c parent. Also, the backcross of F1 mice to the susceptible male or female BALB/c parent resulted in 52.0% susceptible (i.e., death on or before day 24) progeny, as compared with only 0.64% susceptible F1 progeny. The data suggested that resistance was the dominant phenotype and that the resistant allele was carried by the B6 parent. The presence of another locus regulating resistance to death was suggested by the facts that only a small percentage of F2 mice were susceptible and that a number of F1 and F2 mice were more resistant than their B6 parent. The locus responsible for these phenomena was presumably hypostatic in nature and carried by BALB/c mice, and its effects were only evident in the presence of other resistance genes. In addition, the observation that many of the susceptible individuals among F2 and backcross mice were more resistant than the BALB/c mice suggested that other minor genes also modulated the response of mice to infection. A set of CXB recombinant inbred mice was tested as well, and the individual strains within this set could also be placed into four groups: susceptible, intermediate, resistant, or hyperresistant. These findings are compatible with the multigenic model suggested by the Mendelian analyses.     

Correlation of persistent mouse hepatitis virus (MHV-3) infection with its effect on mouse macrophage cultures

Summary MHV3 has three distinct effects in different strains of mice: strain A mice are completely resistant, most strains (including C57BL, DBA/2, BALB/c and NZB strains) die of acute hepatitis whereas in certain strains (eg. C3H and A2G) the virus produces a persistent infection with neurological symptoms. In cultures of peritoneal macrophages from susceptible strains, MHV-3 replicated freely, with giant cell formation. No replication was observed in macrophages from strain A mice. In contrast to this full susceptibility or resistance, macrophage cultures from strains of mice in which persistent infections occur showed an intermediate susceptibility, as judged by the intensity of the cytopathic effect, the presence of viral antigens in the cytoplasm and levels of viral replication. Possible ways in which the intermediate susceptibility of macrophages and persistent infections might be related are discussed.With 3 Figures     

Response of scid mice to establishment of Leishmania major infection.

The initiation of Leishmania major infection in susceptible BALB/c mice is regulated by interferon-gamma (IFN-gamma). To examine further the mechanisms of IFN-gamma-dependent regulation of the establishment of L. major, we studied the characteristics of the infection in severe combined immunodeficient (scid) mice. In the first 2 weeks of infection, we observed a delay in the development of the lesions in the footpads and lower numbers of parasites in scid compared with BALB/c mice. By week 5 after infection, the size of the leishmanial lesion was similar in both strains of mice, but the number of parasites in scid mice was 100-fold higher than in BALB/c. Treatment with anti-IFN-gamma during the establishment of L. major did not alter the course of infection in scid mice, while it exacerbated lesion development in BALB/c mice. Macrophages from scid mice were unable to kill L. major when stimulated with IFN-gamma in vitro, and produced lower levels of nitric oxide compared with macrophages from susceptible BALB/c or the resistant C57Bl/6 mice. We examined whether delayed lesion development in scid mice was due to their inability to mount appropriate inflammatory responses. While significantly fewer nucleated cells were present in the footpads of scid mice compared with BALB/c, 2 and 3 weeks after infection, no difference in inflammatory response between scid and BALB/c mice was observed in response to L. major antigen in the footpads. In contrast, there was a dramatic increase in the number of cells in the popliteal lymph nodes of BALB/c mice. Decreased inflammatory responses of scid mice in the footpad (at the site of infection) may contribute to slower development of leishmanial lesions during the first 2 weeks of infection.     

Genetic control of murine listeriosis expressed in the macrophage response

Susceptibility to murine listeriosis is genetically regulated. For example, A/J, C3H, CBA, DBA/1, DBA/2 and 129/J mouse strains are classed as susceptible and demonstrate an early net bacterial growth rate which is significantly higher than that seen in strains classed as resistant, namely, C57BL-derived strains, NZB and SJL. These strain differences in susceptibility are expressed during the phase of natural resistance, as a property of the macrophage response. Genetic analysis in progeny derived from resistant C57BL-derived strains and susceptible A/J or BALB/c strain mice has indicated that a major gene is responsible for determining resistance/susceptibility to listeria. The genetic advantage of the resistant phenotype is attributed to a prompt influx of young (radiosensitive) inflammatory macrophages which control the early bacterial multiplication in infective sites. Such cells reportedly have superior listericidal activity in vitro, as compared to mature macrophages. Mononuclear phagocyte production, emigration and accumulation at infective foci are all increased in resistant C57BL, but not in susceptible A/J mice, shortly following infection. Thus, resistance to listeriosis is associated with an efficient macrophage inflammatory response and, conversely, susceptibility is attributed to a sluggish response. Genetic studies have demonstrated linkage between these two traits (listeria resistance/susceptibility and the macrophage inflammatory response). In all probability, different gene loci are responsible for susceptibility amongst the various mouse strains. In A/J mice, susceptibility is attributed to C5 deficiency (specified by Hc locus) while, for C5-sufficient strains, another genetic defect is presumably responsible.(ABSTRACT TRUNCATED AT 250 WORDS)     

Strain-Dependent Differences in Murine Susceptibility to Coccidia

Differences in susceptibility of strains of mice to Eimeria ferrisi were observed by infecting eight strains of mice with six infectious dose levels and comparing the mortality rate among the strains for a period of 12 days. Mice of the C57BL/6 and HA/ICR strains were susceptible, and those of A/He, AKR, BALB/c, CBA, C3H/Anf, and DBA/2 strains were resistant to coccidial infection. Resistance was a dominant genetic expression, as indicated by the resistant response of F(1) hybrids of susceptible C57BL/6 and resistant CBA, C3H/Anf, or DBA/2 strains. An E. ferrisi infection in congenitally athymic nu/nu mice and phenotypically normal heterozygous nu/+ mice was used to determine how thymus-dependent immunoincompetence in cell-mediated immunity of the nu/nu mouse affected resistance to infection in a genetic background of the resistant BALB/c mouse. Results of primary and challenge infections in these two strains of mice suggested that resistance is thymus dependent. Furthermore, impairment of thymus-dependent cell-mediated immunity in resistant AKR mice by treatment with mouse antithymus serum led to partial susceptibility. However, susceptible C57BL/6 and HA/ICR strains are phenotypically normal mice, and previous evidence showed that C57BL/6 mice are not completely immunoincompetent in cell-mediated reactivity to coccidia. Collectively, our data show that cell-mediated immunity is necessary for resistance but may be subjected to modification by genetic expression of the host. The possible role of immune response genes in the control of coccidial immunity is discussed.     

Role of tumor necrosis factor alpha in innate resistance to mouse pulmonary infection with Pseudomonas aeruginosa.

In the present study, we have investigated the mechanisms underlying mouse resistance to endobronchial infection with Pseudomonas aeruginosa enmeshed in agar beads. This was done by monitoring macrophage activation-associated gene expression in lung and alveolar cells harvested from resistant (BALB/c) and susceptible (DBA/2, C57BL/6, and A/J) strains of mice over the course of infection with P. aeruginosa. Interleukin-1 alpha, interleukin-1 beta, macrophage inflammatory protein-1 alpha, JE, and tumor necrosis factor alpha (TNF-alpha) mRNA expression levels were up-regulated in all strains of mice during the early phase of the infection. The level of TNF-alpha mRNA expression was increased to a greater extent in resistant BALB/c mice than in susceptible DBA/2, C57BL/6, and A/J strains of mice. This observation paralleled a higher secretion of TNF-alpha into the alveolar space of BALB/c mice at 3 and 6 h postinfection. The concentration of TNF-alpha released in alveoli returned to basal levels within 24 h of infection in mice of all strains, even though the TNF-alpha mRNA expression remained high until 3 days after infection. In vivo treatments with either anti-murine TNF-alpha monoclonal antibodies or with aminoguanidine significantly increased the number of P. aeruginosa bacteria detected in the lungs of resistant mice at 3 days postinfection. Overall, these findings indicate that both TNF-alpha and nitric oxide exert a protective role in response to pulmonary infection with P. aeruginosa.     

Experimental African trypanosomiasis: differences in cytokine and nitric oxide production by macrophages from resistant and susceptible mice.

Immunosuppression in experimental infections with Trypanosoma congolense is mediated by the synergistic action of macrophages and a novel lymphocyte(s), which involves the activity of IFN-gamma as well as IL-10. BALB/c mice are highly susceptible while C57Bl/6 mice are relatively resistant to T. congolense infections. Plasma and/or supernatants of spleen cell cultures of infected susceptible BALB/c mice have more IL-10 but less IL-12 than those of infected relatively resistant C57Bl/6 mice. Cells of a BALB/c macrophage cell line, when pulsed with T. congolense, produce more IL-10 and IL-6, but have less TNF-alpha mRNA, than equally treated cells of a C57Bl/6 cell line. Peritoneal and/or bone marrow-derived macrophages obtained from BALB/c mice, pulsed with T. congolense in culture, produce less nitric oxide, TNF-alpha and IL-12, but more IL-6 and IL-10 than equally treated macrophages isolated from C57Bl/6 mice. We suggest that genetic resistance to African trypanosomiasis is expressed at the level of the macrophage.     

Low response of BALB/c macrophages to priming and activating signals.

Trehalose dimycolate (TDM), a mycobacterial glycolipid, is a powerful macrophage-priming agent. However, its efficiency seems limited in the case of BALB/c mice. Peritoneal macrophages harvested from TDM-treated BALB/c mice did not control BCG growth in vitro as efficiently as similar macrophages from two other mouse strains, (B6 x D2)F1 and C57BL/6, which are respectively Bcgr and Bcgs. BALB/c macrophages elicited by TDM also exhibited a low capacity to produce hydrogen peroxide and, after activation by lipopolysaccharide (LPS), weak cytostatic activity against P815 mastocytoma cells. Finally, alkaline phosphodiesterase, a marker of resident and inflammatory macrophages, was still expressed at a high level in macrophages of BALB/c mice treated with TDM. Low responsiveness of BALB/c macrophages to stimuli was not observed with TDM only; activation for tumor cytotoxicity of thioglycolate-elicited macrophages from BALB/c mice required also higher doses of interferon-gamma, and LPS. L-Arginine-dependent production of nitric oxide was inducible in macrophages from BALB/c mice, but the conditions required for its induction were more stringent. Thus, the reduced antiproliferative effects of BALB/c macrophages may be due to uncomplete induction of NO synthase after suboptimal stimulation.     

Macrophage activity in resistant and susceptible mouse strains infected with Mycobacterium lepraemurium.

The level of activation of peritoneal macrophages following subcutaneous inoculation of resistant (C57BL) and susceptible (BALB/c) mice was assessed by monitoring superoxide anion and hydrogen peroxide production and also tumour cell cytostasis. The level of systemic macrophage activation appeared to correlate with bacterial load, rather than resistance to infection. It was observed that the more susceptible (BALB/c) strain developed higher and more sustained levels of systemic macrophage activation, whereas the more resistant (C57BL) strain showed only low transient levels of macrophage activation. In contrast, in vivo challenge of subcutaneously infected C57BL mice, via the intra-peritoneal route, with heat-killed Mycobacterium lepraemurium and thioglycollate resulted in a high level of macrophage activation compared with similarly treated uninfected mice. Similar treatment of susceptible BALB/c mice, however, did not result in enhanced macrophage activation. It was also observed that high levels of macrophage activation occurred in T-cell deprived C57BL mice following infection with M. lepraemurium.     

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.     

Induction of tumor necrosis factor alpha by Leishmania infantum in murine macrophages from different inbred mice strains.

The present study was undertaken to determine whether the viscerotropic species, Leishmania infantum, endemic in Italy, could induce tumor necrosis factor alpha (TNF alpha) in murine macrophages. Genetically susceptible (Lshs) and resistant (Lshr) mice were used in the attempt to correlate TNF alpha production with the ability to control parasite growth and replication. Resident peritoneal macrophages of C3H/HeN, DBA/2, CBA (Lshr), C57BL/10 and BALB/c (Lshs) mice were infected in vitro with promastigotes at a parasite to cell ratio of 8:1. No significant differences in the percentages of infected peritoneal cells of Lshs versus Lshr mice were observed until 72 h of in vitro culture. On the contrary, Kupffer cells from Lshr mice inhibited Leishmania replication. Peritoneal macrophages of resistant mice produced significantly higher amounts of TNF alpha as compared to susceptible mice. TNF alpha production of both resistant and susceptible mice peaked at about 5 h after the challenge with the parasite. No TNF alpha was found in supernatants of infected Kupffer cells from all the strains tested. The ability of macrophages from susceptible or resistant mice strains to produce TNF alpha after challenge with Leishmania infantum does not seem related to their capacity to control parasite replication in vitro.     

Macrophages in resistance to rickettsial infection: strains of mice susceptible to the lethal effects of Rickettsia akari show defective macrophage Rickettsicidal activity in vitro.

Activation of macrophages was assessed in strains of mice inoculated intraperitoneally with 1,000 times the 50% lethal dose of Rickettsia akari. Macrophages from mice resistant to R. akari infection (C3H/HeN, C57BL/10J, and BALB/cN) were nonspecifically tumoricidal 2 to 4 days after rickettsial inoculation. Moreover, these macrophages were microbial for R. akari in vitro; cells were resistant to infection with the bacterium and were capable of killing intracellular rickettsiae. In contrast, macrophages from strains of mice susceptible to R. akari (C3H/HeJ, C57BL/10SnCR, and A/J) failed to develop nonspecific tumoricidal activity over the course of lethal disease and became infected with R. akari in vivo within 2 days of rickettsial inoculation. Macrophages from uninfected mice of strains susceptible to R. akari also could not be activated for rickettsicidal or tumoricidal activities by treatment with macrophage-activating agents (Mycobacterium bovis BCG) in vivo or by treatment with lymphokines in vitro.     

Influence of genetic background on host resistance to experimental murine tularemia.

The host response to experimental murine tularemia was examined in different inbred mouse strains. The kinetics of growth of Francisella tularensis live vaccine strain (LVS) in the livers and spleens of A and C57BL/6 mice were monitored, and it was observed that mice of the A strain were more susceptible to the proliferation of LVS than were C57BL/6 mice. The difference was most marked 5 days following infection, when the number of bacteria isolated from the spleens of A mice was found to exceed that of C57BL/6 mice by 100-fold. In addition, the C57BL/6 strain exhibited a more pronounced splenomegaly 8 days after infection than did the A strain. When the response of other inbred strains was evaluated by determining the splenic count of LVS on day 5 postinfection, several levels of antiularemic resistance were observed. Mice of the AKR, BALB/cBy, C57BL/10, and SJL strains were found to be most resistant, while SM mice were most susceptible to the proliferation of LVS. The DBA/2, CBA, 129, C3H/HeJ, and A strains expressed a resistance phenotype which was intermediate between the two extremes, with A and C3H/HeJ mice being somewhat more susceptible than DBA/2, CBA, or 129 mice. The trait of resistance or susceptibility was analyzed genetically in (C57BL/6 x A)F1 hybrid mice and in F2 generation and recombinant inbred (RI) mouse strains derived from C57BL/6 (resistant) and A (susceptible) strain progenitors. The F1 progeny exhibited a level of resistance to infection which was similar to that of the resistant parent. In both the F2 generation mice and the RI strains, a continuous spectrum of resistance levels was observed. The results of these experiments indicate that the genetic background of the host influences host resistance to experimental murine tularemia and that multiple genetic loci are involved in this response.     

Resistance to murine cytomegalovirus linked to the major histocompatibility complex of the mouse.

Studies of the resistance patterns to infection with a murine cytomegalovirus in inbred strains of mice revealed the existence of resistant and susceptible strains. Resistance was found to be associated with possession of the H-2k allele at the major histocompatibility locus of the mouse. The F1 hybrid between a resistant strain (C3H/HeJ) and a susceptible strain (BALB/c) was found to have a resistance intermediate between that of both parents, indicating that the gene(s) controlling resistance is partly dominant. Susceptible BALB/c mice could be made resistant to lethal infection by pre-treatment with thioglycollate broth but not by pre-treatment with endotoxin or BCG. Resistant C3H/HeJ mice could be made susceptible to lethal infection by pre-treatment with cyclophosphamide.     

Role of mononuclear phagocytes in expression of resistance and susceptibility to Mycobacterium avium infections in mice.

The growth of Mycobacterium avium 702 in the spleens and livers of four inbred strains of mice varied such that the mice could be separated into naturally susceptible (BALB/c and C57BL/6) and naturally resistant (A/Tru and DBA/2) strains. This phenomenon was independent of the size of the infecting inoculum of bacteria in that both low (10(4))- and high (10(7))-dose inocula of M. avium grew progressively in susceptible strains and were eliminated from the target organs of resistant strains. Resistance and susceptibility were also demonstrated in in vitro preparations of macrophages from these strains of mice. Over a 7-day period, replication of M. avium in susceptible mouse macrophages was far greater than that in resistant macrophages. Evidence was obtained to suggest that toxic oxygen metabolites were not responsible for this difference. Though no difference was found in the rate of clearance of M. avium from the blood of susceptible or resistant mice, resident macrophages from susceptible mice ingested more M. avium in vitro than did resident macrophages from resistant animals. Growth of M. avium in spleens of susceptible mice induced a large influx of phagocytes, whereas this was not observed in resistant mice. In contrast to this it was found that, after injection of a variety of inflammatory agents, influx of leukocytes into the peritoneal cavity could not be used to distinguish susceptible and resistant strains of mice.     

Induction and expression of protective T cells during Mycobacterium avium infections in mice.

Mycobacterium avium is an opportunistic pathogen that infects individuals suffering from chronic lung disease or immunocompromised patients such as AIDS patients. Here we show that a highly virulent isolate of M. avium proliferated as extensively in T cell deficient as in immunocompetent mice. T cell deficient mice allowed a progressive growth of a less virulent AIDS-derived isolate of M. avium while immunocompetent mice arrested the growth of this isolate. Adoptive transfer of T cell enriched spleen cells between congenic strains of mice differing at the Bcg/Ity/Lsh locus showed that only naturally resistant BALB/c.Bcgr (C.D2) mice infected with the highly virulent strain of M. avium or the naturally susceptible BALB/c mice infected with the lower virulence isolate developed protective T cells and that these cells only mediated protection when transferred to naturally susceptible, but not to naturally resistant, mice. Both strains of M. avium proliferated in bone marrow-derived macrophages cultured in vitro and they were both susceptible to the bacteriostatic effects induced in the macrophages by crude lymphokines produced by concanavalin A-stimulated spleen cells.