Differences in initial rate of intracellular killing of Salmonella typhimurium by granulocytes of Salmonella-susceptible C57BL/10 mice and Salmonella-resistant CBA mice

The contribution of granulocytes to differences in the innate susceptibility of mouse strains to infection by Salmonella typhimurium was assessed on the basis of the size and composition of the inflammatory exudate after i.p. injection of bacteria and the intracellular killing of the bacteria by exudate peritoneal cells and blood granulocytes of resistant CBA and susceptible C57BL/10 mice. The increase in the numbers of both peritoneal granulocytes and macrophages 24 hr after i.p. injection of various numbers of live S. typhimurium was two to four times higher in C57BL/10 mice (p less than 0.05) than in CBA mice. However, despite the larger number of phagocytes in the inflammatory exudate, the numbers of viable S. typhimurium in the peritoneal cavity 24 hr after injection was higher (p less than 0.01) in C57BL/10 mice than in CBA mice. Because the proportion of noningested bacteria was similar in the two mouse strains (less than 30%), these findings indicate a difference in the rate of intracellular killing of the bacteria by exudate peritoneal cells (greater than 75% granulocytes) of the two mouse strains. Subsequent determination of the initial rate of intracellular killing (Kk) of S. typhimurium revealed that after phagocytosis of the bacteria in vivo, exudate peritoneal granulocytes (harvested 24 hr after i.p. injection of 10(3) live S. typhimurium) of CBA mice killed S. typhimurium twice as efficiently (Kk = 0.014 min-1; p less than 0.01) as exudate granulocytes of C57BL/10 mice (Kk = 0.008 min-1) did. Similarly, the initial rate of intracellular killing of the ingested S. typhimurium by blood granulocytes of CBA mice (Kk = 0.017 min-1) was two times higher (p less than 0.01) than that of C57BL/10 mice (Kk = 0.007 min-1). These findings may be specific for S. typhimurium, because L. monocytogenes were killed with equal efficiency by exudate granulocytes and blood granulocytes of these mouse strains (p greater than 0.20). The results of the present study are relevant with respect to the innate resistance of mice to S. typhimurium, particularly during the initial phase of infection when the inflammatory exudate contains predominantly granulocytes.     

Salmonella-typhimurium-specific difference in rate of intracellular killing by resident peritoneal macrophages from salmonella-resistant CBA and salmonella-susceptible C57BL/10 mice

The aim of the present study was to determine whether the difference between the rate of intracellular killing of Salmonella typhimurium by macrophages of salmonella-resistant CBA and salmonella-susceptible C57BL/10 mice also holds for other salmonellae and other bacteria species. After in vivo phagocytosis, the initial rate of in vitro intracellular killing of S. typhimurium phagetype 505, S. typhimurium phagetype 510, and S. typhimurium M206 by macrophages of CBA mice amounted always to approximately 1.7 times the value found for macrophages of C57BL/10 mice (p less than 0.001), indicating that the difference in killing efficiency between CBA and C57BL/10 macrophages holds for various strains of S. typhimurium. However, some other salmonella species, i.e., S. dublin and S. heidelberg, as well as E. coli 054 and 02K1+, Listeria monocytogenes EGD and L347, and Staphylococcus aureus were killed equally efficiently by macrophages of both mouse strains. These findings indicate that the difference between the rates of intracellular killing by macrophages of salmonella-resistant CBA and salmonella-susceptible C57BL/10 does not hold for several other bacteria species and thus might be specific for S. typhimurium. Subsequent experiments showed that the in vivo proliferation of S. typhimurium 510 in the first 2 days after i.v. injection was 2.0-fold to 3.0-fold higher in the spleens and livers of C57BL/10 mice than in those of CBA mice, whereas the in vivo proliferation of S. dublin and S. heidelberg was between 1.0-fold to 1.4-fold higher in the C57BL/10 mice. These findings suggest that the differences between the rate of in vitro intracellular killing of salmonella by CBA and C57BL/10 macrophages are reflected in differences in the rate of in vivo proliferation of these microorganisms in CBA and C57BL/10 mice. To gain insight into the involvement of the oxidative metabolism of CBA and C57BL/10 macrophages in the difference in the rate of intracellular killing of S. typhimurium, the O2 consumption and H2O2 release by resident peritoneal macrophages was determined. The amplitudes of the respiratory burst and the release of H2O2 was identical in macrophages of the two mouse strains after triggering by either preopsonized heat-killed S. typhimurium or phorbol myristic acetate. These findings indicate that the mouse species-associated difference in the intracellular killing of S. typhimurium is not caused by a difference in the oxidative metabolism of CBA and C57BL/10 macrophages.     

Inability of recombinant interferon-gamma to activate the antibacterial activity of mouse peritoneal macrophages against Listeria monocytogenes and Salmonella typhimurium

The effect of recombinant murine interferon-gamma (rIFN-gamma) as single stimulus for the activation of antibacterial activity of macrophages was investigated on the basis of the rate of intracellular killing of Listeria monocytogenes and Salmonella typhimurium by normal and rIFN gamma-activated peritoneal macrophages of CBA and C57BL/10 mice, which differ in natural resistance to infection by these bacteria. Eighteen hours after i.p. injection of 10 to 1 X 10(4) U rIFN-gamma, resident and exudate peritoneal macrophages which had phagocytosed L. monocytogenes or S. typhimurium in vivo, killed both species in vitro just as efficiently as did resident macrophages of normal mice. Similar results were obtained after 18 hr of in vitro incubation of resident or exudate peritoneal macrophages with 0.1 to 1 X 10(4) U/ml rIFN-gamma. Consistent with the in vitro findings, two i.v. injections of 5 X 10(4) U rIFN-gamma did not affect the rate of in vivo proliferation of L. monocytogenes or S. typhimurium in the spleens of mice during the first 2 days after i.v. injection of the bacteria. Compared with the effect on the controls, two i.p. injections of 5 X 10(2) to 5 X 10(4) U rIFN-gamma did not decrease the numbers of viable S. typhimurium in either the peritoneal cell suspension or the spleen 24 hr after i.p. injection of the bacteria. Checking the state of activation of rIFN-gamma-activated macrophages on the basis of two commonly used criteria for macrophage activation showed that rIFN-gamma-activated macrophages inhibited the intracellular replication of Toxoplasma gondii and displayed enhanced O2 consumption and H2O2 release after stimulation with phorbol myristate acetate compared with macrophages from normal CBA and C57BL/10 mice. The present findings show that as single activating stimulus, rIFN-gamma is not capable of activating the antibacterial effector functions of peritoneal macrophages against facultative intracellular pathogens such as L. monocytogenes and S. typhimurium.     

Fate of Legionella pneumophila in macrophages of C57BL/6 chronic granulomatous disease mice

We compared the intracellular survival and growth of Legionella pneumophila Philadelphia-1 in peritoneal macrophages obtained from A/J, C57BL/6, and X-linked chronic granulomatous disease (CGD) mice produced from C57BL/6 strain. The initial killing was observed in A/J and C57BL/6 macrophages at 2, 4 and 6 hr after in vitro phagocytosis, but not in the CGD macrophages. Thereafter, there was a 10-fold increase of CFU in A/J macrophages. The bacteria, however, did not proliferate in C57BL/6 and CGD macrophages at 24 or 48 hr after in vitro phagocytosis. These results suggest that effector molecules for the initial killing are a superoxide anion and its metabolites, and Lgn1 gene product inhibits the intracellular growth of L. pneumophila independently of NADPH oxidase.     

Different Sensitivity of Complement to Salmonella typhimurium Accounts for the Difference in Natural Resistance to Murine Typhoid between A/J and C57BL/6 Mice

The difference in natural resistance to Salmonella typhimurium between S. typhimurium-resistant A/J mice and S. typhimurium-susceptible C57BL/6 mice was analyzed. In both strains, the growth of S. typhimurium was controlled in the spleen until 48 hr of infection, while serum C3b levels were increased in A/J mice immediately after infection but not in C57BL/6 mice. Incubation of A/J mouse serum with S. typhimurium or its lipopolysaccharide (LPS) generated sufficient amounts of C3b, but that of C57BL/6 mouse serum with them did not. A/J macrophages had higher intracellular killing activity in vitro than did C57BL/6 cells against S. typhimurium pre-opsonized with each corresponding fresh serum. However, the cells from both mice exhibited a similar level of killing activity against S. typhimurium pre-opsonized with fresh A/J serum or rabbit complement. The resistance of C57BL/6 mice was significantly increased by opsonizing S. typhimurium with fresh A/J serum or rabbit complement before inoculation. The serum level of interferon-γ (IFN-γ) in A/J mice was 2.7 times as high as in C57BL/6 mice at 48 hr post-infection. Recombinant murine IFN-γ enhanced the intracellular killing activity of macrophages from both mice when S. typhimurium was pre-opsonized with fresh A/J serum but not with fresh C57BL/6 serum. These findings suggest that A/J macrophages exhibit maximal killing activity against A/J serum-opsonized S. typhimurium in vivo when the cells are activated with IFN-γ. Therefore, the rapid and sufficient activation of complement by Salmonella LPS may render A/J mice more resistant against murine typhoid.     

Cell motility in a system of mononuclear phagocytes from different strains of mice in the presence of bacteria and their components

The motility of organospecific macrophages of the abdominal cavity, spleen, lungs and liver, observed in the presence of serogroup A Neisseria meningitidis polysaccharide, formalin-killed Bordetella pertussis and acetone-treated Salmonella typhimurium, has been studied. In these experiments CBA, C57BL/6 and (CBA X C57BL/6)F1 mice have been used. The in vitro study of the motility of cells in the system of mononuclear phagocytes in the presence of bacterial antigens has revealed that these cells are functionally heterogeneous, which is manifested differently in mice of varying strains.     

Comparative study of the phagocytosis of sheep erythrocytes by the macrophages of inbred mice differing in their sensitivity to immunogenic stimulation by the given antigen

The phagocytosis of 14C-labeled sheep red blood cells (SRBC) by the macrophages of isogeneic CBA, BALB/c and C57BL/6 mice was studied. In the presence of bovine serum the macrophages of CBA mice were found to ingest SRBC significantly less actively than the macrophages of BALB/c and C57BL/6 mice. In the presence of isologous serum the macrophages of mice belonging to the strains under study showed quite comparable characteristics with respect to their capacity of ingesting SRBC. The duration of the intracellular digestion of SRBC by the macrophages of mice of these strains did not vary in different strains irrespective of the type of serum.     

Mouse strain differences in resident peritoneal cells: a flow cytometric analysis

A flow-cytometric study of resident peritoneal cells among 8 mouse strains showed a more than twofold variation in the ratio of macrophages to macrophages plus lymphocytes, ranging from 27% in A/J to 62% in C57B/L10, with significant strain differences in a number of other cellular parameters. There was a particular deficiency of lymphocytes in strain CBA/N, which carries the xid mutation. Studies of the phagocytosis of fluorescent beads also revealed large differences in the number of beads taken up, ranging from 0.99 per cell in MFI to 1.64 per cell in BALB/c mice in a 20-min period. The total number of peritoneal cells collected also varied between strains, ranging from 2.75 x 10(6) in CBA/Ca to 5.85 x 10(6) in MF1. The total yield of macrophages per mouse ranged from 0.93 x 10(6) in A/J to 3.16 x 10(6) in C57BL/10. These differences should be taken into account when designing experiments which use resident peritoneal cells.     

Genetic control of the innate resistance of mice to Salmonella typhimurium: Ity gene is expressed in vivo by 24 hours after infection

The early response of inbred mice to infection with S. typhimurium is controlled by the mouse Chromosome 1 locus, Ity. To better understand the expression of this gene, the initial interactions between the reticuloendothelial system (RES) and i.v. injected salmonellae were compared in resistant (Ityr) and susceptible (Itys) mice. In both mouse strains 99% of the bacteria was cleared from the blood within 2 hr, and uptake of S. typhimurium by splenic and hepatic macrophages was similar regardless of Ity genotype. In vivo phagocytosis of bacteria was followed by a 30 to 60% decline in viable bacteria, which was attributed to the bactericidal activity of RES macrophages. Experiments with radiolabeled S. typhimurium strains TML and TML/TS27 (a temperature-sensitive mutant) confirmed that the efficiency of this early phase killing was not under Ity control. Despite the equivalent uptake and initial bactericidal activity by resident macrophages, bacterial numbers in the RES organs of Itys mice were significantly greater than in Ityr mice by approximately 24 hr after infection. These data suggest that Ity regulates the level of surviving intracellular bacteria that accumulate within resident macrophages of the liver and spleen.     

Influence of plasmid pR50 controlling bacterial protease activity on Salmonella experimental infection

The comparative analysis of the infectious process and immunological parameters in (CBA x C57BL/6)F1 mice infected with S. typhimurium isogenic strains differing by the presence of plasmid pR50 determining protease activity, was carried out. A growth in the expression level of antilactoferrin, anticomplementary and anti-immunoglobulin activity in bacteria isolated from the spleen in the course of the infectious process was detected. In mice infected with S. typhimurium having plasmid pR50, in contrast to nonplasmid recipients, a higher level of contamination of organs, the suppression of spontaneous, stimulated production of interferon-gamma and the bactericidal properties of peritoneal macrophages were noted. The data obtained in this investigation suggested that the acquisition of R-plasmid of 50 MD, controlling protease activity and multiple medicinal resistance, contributed to the persistence of intracellular bacteria.     

The in vitro bactericidal activity of peritoneal and spleen cells from Listeria-resistant and -susceptible mouse strains

Two days after Listeria-resistant (LrR) C57BL/10 mice were infected intraperitoneally with Listeria, their peritoneal macrophages demonstrated enhanced bactericidal activity beyond that seen in susceptible (LrS) BALB/c or CBA mice. Intravenous infection had no effect on peritoneal cell activity. The induction, but not expression, of the enhanced activity was radiosensitive. There was no significant difference between the strains with respect to the number of cells or cellular composition of the exudates. No difference in the in vitro chemotactic response of cells from the two strains could be demonstrated. Therefore there seems to be recruitment to the infected peritoneal cavity of C57BL/10 mice of young, efficiently bactericidal monocytes/macrophages. On the other hand, spleen cell bactericidal activity was intrinsically superior in C57BL/10 mice compared with BALB/c mice, possibly because, as a haemopoietic organ, the C57BL/10 spleen already contains high numbers of these efficient monocytes.     

Susceptibility of inbred mice to Leishmania tropica infection: correlation of susceptibility with in vitro defective macrophage microbicidal activities

Eleven mouse strains were inoculated in footpads with amastigotes of Leishmania tropica and observed for 12 weeks. Liver and spleen impression smears from infected mice were examined for the presence of intracellular parasites. Four strains (BALB/cJ, C57L/J, NZW/N, and P/J) failed to heal the subcutaneous lesion and showed evidence of systemic infection; the remaining seven strains (A/J, C3H/HeJ, C3H/HeN, C3HeB/FeJ, C57BL/6J, C57BL/10J, and C57BL/10ScN) were each resistant to infection and resolved their lesions by Week 10. Macrophages from the four susceptible strains could not be activated to kill L. tropica amastigotes by treatment with soluble lymphocyte products in vitro. In contrast, macrophages from all seven resistant strains responded to lymphokine treatment and eliminated 80-90% of intracellular parasites. These results suggest that in vitro macrophage microbicidal activities predict the course of systemic leishmanial disease.     

Bacterial lipoprotein induces resistance to Gram-negative sepsis in TLR4-deficient mice via enhanced bacterial clearance

TLRs are highly conserved pathogen recognition receptors. As a result, TLR4-deficient C3H/HeJ mice are highly susceptible to Gram-negative sepsis. We have previously demonstrated that tolerance induced by bacterial lipoprotein (BLP) protects wild-type mice against polymicrobial sepsis-induced lethality. In this study, we assessed whether pretreatment of C3H/HeJ mice with BLP could induce resistance to a subsequent Gram-negative Salmonella typhimurium infection. Pretreatment with BLP resulted in a significant survival benefit in TLR4-deficient C3H/HeJ mice (p < 0.0002 vs control C3H/HeJ) after challenge with live S. typhimurium (0.25 x 10(6) CFU/mouse). This survival benefit was associated with enhanced bacterial clearance from the circulation and in the visceral organs (p < 0.05 vs control C3H/HeJ). Furthermore, pretreatment with BLP resulted in significant increases in complement receptor type 3 (CR3) and FcgammaIII/IIR expression on polymorphonuclear neutrophils (PMNs) and macrophages (p < 0.05 vs control C3H/HeJ). There was impaired bacterial recognition and phagocytosis in TLR4-deficient mice compared with wild-type mice. However, a significant augmented uptake, ingestion, and intracellular killing of S. typhimurium by PMNs and peritoneal macrophages was evident in BLP-pretreated C3H/HeJ mice (p < 0.05 vs control C3H/HeJ). An up-regulation of inducible NO synthase and increased production of intracellular NO were observed in peritoneal macrophages from BLP-pretreated C3H/HeJ mice (p < 0.05 vs control C3H/HeJ). Depletion of PMNs did not diminish the beneficial effects of BLP with regard to both animal survival and bacterial clearance. These results indicate that BLP, a TLR2 ligand, protects highly susceptible TLR4-deficient mice from Gram-negative sepsis via enhanced bacterial clearance.     

Characterization of a congenitally LPS-resistant, athymic mouse strain.

C57BL/10ScN (nu/nu) mice have B cells and macrophages unresponsive to a phenol-water extracted preparation of Escherichia coli K 235 LPS. This unresponsiveness was demonstrated in vitro by the inability of spleen cells to incorporate 3H-thymidine after a 48 hr incubation with LPS (Ph) and by the inability of LPS (Ph) to inhibit macrophage phagocytosis of 51 Cr-labeled, opsonized sheep erythrocytes. Furthermore, macrophage cultures stimulated with LPS (Ph) produced low levels of LAF and PGE2 when compared with macrophages from the LPS-sensitive C3H/HeN and C3H/HeN (nu/nu) strains. Therefore, the C57BL/10ScN (nu/nu) strain is similar in its LPS unresponsiveness to the well-characterized C3H/HeJ and C57BL/10ScCR strains. The combination of endotoxin unresponsiveness and the athymic nature of this mouse strain may provide a powerful new tool for studying the cellular events mediating endotoxicity.     

Effector function of leucocytes from susceptible and resistant mice against distinct isolates of Candida albicans

Neutrophils and macrophages were generated in vitro from mice that display either high or low tissue susceptibilities to Candida albicans infection and their ability to phagocytose and kill three isolates of the yeast with different virulence characteristics was evaluated. In the absence of opsonization, phagocytosis by BALB/c and CBA/CaH neutrophils was comparable, but the killing was very poor. Opsonization with normal serum slightly decreased phagocytosis, but it had markedly different effects on killing, either enhancing or inhibiting candidacidal activity, depending on the combination of yeast isolate and mouse strain. In contrast, BALB/c macrophages showed high levels of phagocytosis and killing of both unopsonized yeasts and opsonized yeasts; whereas killing of unopsonized yeasts by CBA/CaH macrophages was poor, it was markedly enhanced by opsonization.     

Immune responses of different mouse strains after challenge with equivalent lethal doses of Toxoplasma gondii

Most immunological studies that utilize different strains of inbred mice following T. gondii infection fail to compensate for differences in host susceptibility to the size of the parasite innoculum. To address this concern, susceptible C57BL/6 and resistant CBA/J mice were orally infected with either an equivalent 50% lethal dose (LD50) of brain cysts of the 76K strain of T. gondii (15 cysts in C57BL/6, 400 cysts in CBA/J) or the same dose of parasites in each mouse strain. C57BL/6 mice receiving 400 cysts (LD50 of CBA/J mice) died post infection, whereas CBA/J mice that received 15 cysts (LD50 of C57BL/6 mice) survived. Parasite loads in the brains and serum Toxoplasma-specific IgG1 titers of LD50-infected C57BL/6 mice were significantly higher than those in LD50- or 15 cysts-infected CBA/J mice, whereas splenocyte proliferation to Toxoplasma antigen and the percentage of CD8 alpha+ T cells were reduced in LD50-infected C57BL/6 mice. In contrast, serum IgG2a and IgM titers, the percentage of gamma delta T cells and IFN-gamma expression of spleen of LD50-infected CBA/J mice were higher than those of either 15 cysts-infected CBA/J mice or LD50-infected C57BL/6 mice. These observations demonstrate that the immune response between LD50-infected C57BL/6 and CBA/J mice was more prominent when compared to C57BL/6 or CBA/J mice receiving the same parasite inoculum. These observations would suggest that caution must be excersized in the planning and interpretation of data when the size of the parasite inoculum has not been adjusted for mouse strain.     

The development of diploid parthenogenetic mouse embryos of the inbred C57BL and CBA strains]

We studied preimplantation development in vitro and postimplantation development in vivo of diploid parthenogenetic mouse embryos of C57BL/6 and CBA strains, as well as of (CBA x C57BL/6)F1 hybrids. Development to blastocyst stage of diploid eggs obtained from C57BL/6, CBA, and hybrid mice was observed in 90, 15, and 73% cases, respectively. After implantation, C57BL/6 embryos did not develop to somite stages, while CBA and hybrid embryos reached various stages of somite formation in 45 and 30% cases, respectively. Cultivation of embryos beginning from one-cell stage in the medium containing 2% newborn calf serum increased the yield of blastocysts from 15 to 59% in CBA embryos and from 73 to 90% in hybrids; However, such effect was not observed with C57BL/6 embryos. The latest stages of development observed in CBA and hybrid diploid parthenogenetic embryos were 33-35 somites and 25-30 somites, respectively. Imprinting patterns in chromosomes of CBA and C57BL/6 gametes are discussed.     

Mouse chromosome 1 Ity locus regulates microbicidal activity of isolated peritoneal macrophages against a diverse group of intracellular and extracellular bacteria

The genotype of a mouse influences whether or not it will survive infection with the agent of murine typhoid, Salmonella typhimurium. The best-characterized murine salmonella response gene is a Chromosome 1 locus designated Ity. Inbred strains of mice that express the Itys allele are unable to contain the net growth of Salmonella typhimurium within their spleens and livers, and usually die early in the infection. By contrast, mice homozygous or heterozygous for the Ityr allele are able to control the net multiplication of Salmonella typhimurium within these organs. The Ity gene also appears to regulate the extent of replication within murine reticuloendothelial cell tissues of the obligate intracellular parasite Leishmania donovani, as well as the facultative intracellular bacteria Mycobacterium bovis and Mycobacterium lepraemurium. Previous studies from our laboratory strongly suggested that Ityr mice are more resistant to S. typhimurium infection than are Itys mice, because resident Ityr macrophages kill salmonellae more efficiently than do Itys macrophages. In this study, we used an in vitro macrophage assay to assess the specificity of the enhanced killing capacity of Ityr macrophages. We found that Ityr macrophages were better able than Itys macrophages to kill both intracellular bacteria (Salmonella typhi) and extracellular bacteria (Escherichia coli, Staphylococcus aureus, Corynebacterium diphtheriae). Thus, the diversity of organisms affected by Ity expression suggests that the product of this gene may play a key regulatory role in the initial interaction of mice with a variety of microbial agents.     

The effect of aging on the induction of humoral and cellular immunity and tolerance in two long-lived mouse strains.

Aging is a complex process that adversely affects most if not all components of the immune system. In this report, two long-lived mouse strains have been compared in ability to generate both antigen-specific immunity and tolerance. Although CBA/CaJ mice produced high levels of antibody following injection of aqueous preparations of aggregated human gamma-globulin (AHGG), C57BL/6 mice made only meager antibody responses to such preparations. Age dramatically affects the humoral anti-HGG response to aqueous AHGG in both strains, but the meager response of young C57BL/6 mice was at insignificant levels in aged C57BL/6 mice. Conversely, both mouse strains generated good responses following injection of HGG in complete Freund's adjuvant at both the T and B cell level as evidenced by in vitro antigen-specific T cell proliferation and anti-HGG antibody production. Aged mice of both strains showed a marked decrease in the production of serum anti-HGG antibody in comparison to young mice. Although the antigen-specific T cell proliferative response was significantly decreased in aged CBA/CaJ mice, such proliferation was not affected in aged mice of the C57BL/6 strain. Removal of CD8+ cells from lymph node T cells of either young or aged C57BL/6 mice did not increase the antigen-specific proliferative response, suggesting that loss of CD8+ suppressors during the aging process is not responsible for the high level of antigen-specific T cell proliferation in aged C57BL/6 mice. Tolerance to HGG was readily induced in both young and aged C57BL/6 and CBA/CaJ mice although aged mice demonstrate a modest resistance to tolerance induction when compared to their young counterparts. This resistance was observed in both antibody production and antigen-specific T cell proliferation.