Genetically determined resistance to listeriosis is associated with increased accumulation of inflammatory neutrophils and macrophages which have enhanced listericidal activity.

The C57BL/6 and A/J inbred strains of mice differ markedly in their resistance to the facultative intracellular bacterium Listeria monocytogenes. One possible explanation for this genetically determined resistance is that phagocytes from Listeria-resistant strains of mice can kill L. monocytogenes more effectively than phagocytes from Listeria-susceptible strains of mice. We report here that inflammatory neutrophils and macrophages from Listeria-resistant mice (C57BL/6) exhibit a slight but significantly enhanced ability to kill L. monocytogenes in vitro as compared to inflammatory phagocytes from Listeria-susceptible mice (A/J). More importantly, however, Listeria-resistant mice recruited more inflammatory neutrophils and macrophages to the peritoneal cavity in response to i.p. injection of heat-killed Listeria than did Listeria-susceptible mice. These data suggest that genetically determined resistance to listeriosis is dependent on the enhanced inflammatory responsiveness of Listeria-resistant mice. Further support for this hypothesis was provided by experiments in which the passive transfer to A/J mice (C5-deficient) of plasma from C57BL/6 mice (C5-sufficient) enhanced the ability of the recipient A/J mice both to recruit inflammatory neutrophils to the peritoneal cavity in response to i.p. injection of heat-killed Listeria, and to clear L. monocytogenes from the spleen after a sublethal challenge of viable Listeria.     

A/J mice are susceptible and C57BL/6 mice are resistant to Listeria monocytogenes infection by intragastric inoculation

Previous studies demonstrated that the innate resistance of mice to Listeria monocytogenes infection by intravenous or intraperitoneal inoculation is regulated principally by the Hc locus on mouse chromosome 2. The A/J and C57BL/6 mouse strains were identified as prototype L. monocytogenes-susceptible and -resistant strains, respectively. In the present study, we compared the relative susceptibilities of A/J and C57BL/6 mice to intragastric (i.g.) inoculation with L. monocytogenes. The results of our study indicate that A/J mice are significantly more susceptible than C57BL/6 mice to an i.g. challenge with L. monocytogenes. This was reflected in the estimated 50% lethal doses for the two strains (10(6) and 10(8) CFU for A/J and C57BL/6 mice, respectively) and a more rapid and severe dissemination of the infection to the spleen and liver in A/J mice than in C57BL/6 mice. Histopathological examination of tissues from the infected mice confirmed the greater severity of disease in A/J mice. Clearance of a primary infection enhanced the resistance of both A/J and C57BL/6 mice to reinfection with L. monocytogenes via the gastrointestinal tract. However, the relative difference in susceptibility between the two strains was evident even after immunization. The A/J mouse holds promise as a model for investigating the pathogenesis of gastrointestinal listeriosis because of its ability to develop systemic infection following challenge with numbers of organisms similar to those recovered from some L. monocytogenes-contaminated food products.     

Phenotypic expression of genetically controlled host resistance to Listeria monocytogenes.

Several inbred mouse strains, all of them derived from the C57BL background, have genetically determined increased resistance to infection with Listeria monocytogenes, whereas a variety of other strains are relatively sensitive to this infection. Comparison of the host response to L. monocytogenes in the sensitive A strain and the resistant C57BL/6 (B6) strain revealed that the B6 mice were superior to A mice both in the T-cell-independent and in the T-cell-dependent phase of the response. Although animals of both strains had equal ability to clear their circulation of intravenously administered Listeria and to take up comparable amounts of bacteria in their livers and spleens, already 24 to 48 h after infection the genetic advantage of B6 strain mice to suppress bacterial proliferation was apparent. Both the primary (early and late) and the secondary responses as well as the ability to inactivate the bacterial load after adoptive protection by syngeneic immune lymphocytes were more efficient in the B6 animals, suggesting that the common effector macrophage arm of the antilisterial resistance rather than the lymphocyte arm (mediating the T-cell-dependent phase of response) is genetically controlled.     

Mechanism of the bactericidal action of myeloperoxidase: increased permeability of the Escherichia coli cell envelope.

A sensitive and highly reproducible assay was utilized to study in vitro interactions of Listeria monocytogenes with resident and activated macrophages. The technique is not compromised by extracellular events and can readily differentiate between the efficiency of ingestion and the postphagocytic fate of bacteria. Heat-labile factors in human or homologous serum markedly enhanced the phagocytosis of Listeria without noticeably affecting the intracellular fate of the microorganisms. The behavior of Listeria within macrophages cultivated from C57BL/6 and BALB/c mouse strains corresponded to previous reports of in vivo growth patterns in inbred mice. Thioglycolate- or caseinate-elicited macrophages, although highly phagocytic, were unable to prevent the proliferation of Listeria. A bactericidal macrophage population was derived from from C57BL/6 mice which had been immunized intraperitoneally with a sublethal dose of L. monocytogenes and subsequently boosted with heat-killed homologous organisms. Elicitation of immune animals produced an increase in the percentage of peroxidase-positive macrophages, but this activity could not be correlated with restriction of intracellular bacterial growth.     

Analysis of macrophage bactericidal function in genetically resistant and susceptible mice by using the temperature-sensitive mutant of Listeria monocytogenes.

Innate resistance to infection by Listeria monocytogenes is genetically controlled and is critically dependent on prompt macrophage recruitment to the sites of infection. Experiments reported here were designed to examine whether there was an additional, qualitative difference between the intrinsic bactericidal activity of the inflammatory macrophages of genetically resistant (C57BL/6J) and susceptible (A/J) hosts. To critically evaluate the bactericidal (rather than bacteriostatic) function of the macrophage, a temperature-sensitive (ts) mutant of L. monocytogenes was developed. Mutagenesis was induced with nitrosoguanidine, and the ts mutants were isolated following enrichment with penicillin-gentamicin combinations. The ts mutants were found to carry the cell surface and biochemical characteristics of the original wild-type strain of L. monocytogenes. Inflammatory peritoneal macrophages from resistant C57BL/6J mice were found to have enhanced listericidal activity when compared with inflammatory macrophages from susceptible A/J mice. However, further analysis of the macrophage populations revealed that this seemingly qualitative advantage was due to the relatively greater proportion of inflammatory macrophages present in the inflammatory exudates of resistant C57BL/6J mice. When homogeneous populations of pure inflammatory macrophages were compared, no interstrain differences in their listericidal activity in vitro were seen. These results suggest that the susceptibility of A/J strain mice to L. monocytogenes is not due to an intrinsic deficiency of the listericidal activity of the inflammatory macrophage. The slight increase in bactericidal activity of macrophages from resistant mice that was reported by others (C. J. Czuprynski, B. P. Canono, P. M. Henson, and P. A. Campbell, Immunology 55:511-518, 1985) is caused by the difference in the relative percentage of resident cells present in the peritoneal exudates from resistant and susceptible mice.     

Antibacterial resistance in mice infected with Mycobacterium lepraemurium.

The differences in susceptibility among C57Bl/6, DBA/2 mice and their F1 hybrids to infections with M. lepraemurium were shown to depend upon the route of infection and size of the inoculum. A method was developed to measure the ability of lymphocytes obtained from M. lepraemurium-infected donors to effect adoptive immunization of syngeneic naive mice against infection with M. tuberculosis. This required sublethal irradiation of recipient mice prior to cell transfer and bacterial challenge. Using this method, it was found that mice infected subcutaneously generated antituberculous immune mechanisms concordantly with the development of delayed-hypersensitivity to antigens of M. lepraemurium. In contrast, intravenously infected mice demonstrated only a transient from of delayed hypersensitivity and little or no antimycobacterial immunity in that progression of infection was associated with a rapid decay of both these functions. Moreover, during the terminal stage, M. lepraemurium-infected mice lost the ability to control the growth of a sublethal intravenous inoculum of the antigenically unrelated bacterium. Listeria monocytogenes.     

Innate anti-listerial resistance of mice differing in their susceptibility to listeriosis

The work was designated to compare the influence of an active immunization on the expression of anti-listerial resistance of relatively resistant to listeriosis C57B1/6 mice as compared with more susceptible to the infection DBA/2 mice. Although, specific immunization of DBA/2 mice enhanced their anti-listerial resistance but immunized DBA/2 mice still eliminated Listeria rods less effectively than immunized C57B1/6 mice. It means that innate difference in anti-listerial resistance between C57B1/6 and DBA/2 mice was maintained after immunizing them with the same number of alive bacteria. Greater anti-listerial resistance of C57B1/6 versus DBA/2 mice is associated with an increased accumulation of inflammatory Ms and PMNs in their peritonea and an increased capacity of their PMNs to restrict Listeria growth.     

Effects of gonadotrophin on resistance to Listeria monocytogenes infection in mice.

It was demonstrated that exogenous GH suppressed the resistance to L. monocytogenes infection in Listeria resistant C57Bl/6 and susceptible A/J mice. However, different parameters of the immunological reaction to Listeria were affected by GH treatment in these mouse strains. In C57Bl/6 mice GH decreased accumulation of macrophages at the inflammatory site. On the contrary, a depression of anti-listerial activity of the phagocytes and a reduction of DTH reaction to Listeria antigen was demonstrated in GH treated A/J mice.     

Impaired resistance to Listeria monocytogenes in mice chronically exposed to cadmium.

It is shown in this work that resistance to Listeria monocytogenes is greatly impaired in C57BL/6 mice chronically exposed to cadmium (Cd) chloride. Animals received 0.5 mg/kg Cd by an intraperitoneal route three times a week during a 4-week period and were then infected with L. monocytogenes. Susceptibility to this pathogenic bacteria was not due to a defect of the specific immune response, since mice developed normal levels of anti-Listeria T cell-mediated immunity and did not show any impairment of macrophage activation. In fact, bacterial growth in organs was rapid in Cd-exposed mice during the early phase of infection, suggesting an impairment of non-specific defence mechanisms. Experimental data indicate that the susceptibility to L. monocytogenes might be due to a defect of macrophage recruitment in sites of infection during the early phase of the host response.     

Igh-6(-/-) (B-cell-deficient) mice fail to mount solid acquired resistance to oral challenge with virulent Salmonella enterica serovar typhimurium and show impaired Th1 T-cell responses to Salmonella antigens

In the present study we evaluated the role of B cells in acquired immunity to Salmonella infection by using gene-targeted B-cell-deficient innately susceptible mice on a C57BL/6 background (Igh-6(-/-)). Igh-6(-/-) mice immunized with a live, attenuated aroA Salmonella enterica serovar Typhimurium vaccine strain showed impaired long-term acquired resistance against the virulent serovar Typhimurium strain C5. Igh-6(-/-) mice were able to control a primary infection and to clear the inoculum from the reticuloendothelial system. However, Igh-6(-/-) mice, unlike Igh-6(+/+) C57BL/6 controls, did not survive an oral challenge with strain C5 at 4 months after vaccination. Transfer of immune serum did not restore resistance in Igh-6(-/-) mice. Total splenocytes and purified CD4(+) T cells obtained from Igh-6(-/-) mice 4 months after vaccination showed reduced ability to release Th1-type cytokines (interleukin 2 and gamma interferon) upon in vitro restimulation with serovar Typhimurium soluble cell extracts compared to cells obtained from Igh-6(+/+) C57BL/6 control mice. Therefore, the impaired resistance to oral challenge with virulent serovar Typhimurium observed in B-cell-deficient mice, which cannot be restored by passive transfer of Salmonella-immune serum, may be in part due to a reduced serovar Typhimurium-specific T-cell response following primary immunization.     

灭活自体T细胞增强小鼠免疫功能作用机制的研究

接种灭活自身反应性T细胞用于治疗自身免疫性疾病已获初步成效。本研究采用辐射方法灭活ConA活化自体T细胞,经皮下和腹腔免疫正常的C57BL/6J小鼠,结果发现,该免疫方案可明显上调T细胞功能,表现为:免疫小鼠体内活化T细胞增多且Th1型细胞因子分泌增加,淋巴细胞体外增殖能力增强且凋亡细胞明显减少。进一步研究结果提示,接种灭活自体T细胞可能通过上调Fas、GADD45β及下调FasL基因表达而促进T细胞的活化和存活,并增加其生物学功能。这些结果表明,自体T细胞免疫具有增强机体的细胞免疫功能,为其抗肿瘤免疫作用提供了一定的实验依据。     

T cell-mediated immunity to oncornavirus-induced tumors IV. Preliminary evidence for a specific suppression of anti-Moloney cell-mediated immune response by autoimmune T cells

Previous reports have demonstrated that adult C 57 BL/6 mice infected with murine sarcoma virus (MSV) develop a strong cell-mediated immune response against Friend, Moloney, Rauscher virus-induced type-specific (FMR) antigens and reject their tumors. To demonstrate a possible role for auto-anti-MSV T blasts, syngeneic C 57 BL/6 mice were immunized with highly enriched anti-FMR cytolytic T cells. One of 3 pools of these autoimmune T cells prepared from 12 surviving immunized mice (a) inhibited specifically the in vitro anti-MSV cytolysis generation and (b) enhanced drastically the MSV tumor growth in vivo. The possibility for such an immunization procedure to induce anti-idiotype T cells, the repeatability of this effect and the relationship of the suppressor cells with antigen-specific suppressor cells and other components of the anti-MSV immune response are discussed.     

In vitro acquisition of resistance against herpes simplex virus by permissive murine macrophages

Summary This study has documented that peritoneal macrophages (PM) from adult mice of strains DBA/2J, BALB/c and AKR can support productive replication of herpes simplex virus (HSV) strains Thea and Haase. Simian virus 40 transformed PM of C57 BL/6J origin yielded high titers of Thea and Haase, whereas normal PM from adult mice of the same genetic background were infected abortively. The evidence obtained suggested that multiplication of PM per se is insufficient to allow HSV replication. PM from DBA/2J mice, immune to HSV or Listeria monocytogenes lacked the capacity to support productive replication of HSV. PM from nonimmune, adult DBA/2J or suckling C57BL/6J mice acquired the potential to effectively restrict HSV replication when preincubated for 24 hours with 72 hours old MLC supernatant. These macrophages could also inhibit HSV replication if pretreated with supernatant from cultures of specific antigen stimulated, HSV or Listeria immune spleen lymphocytes (SL). Likewise, supernatant from concanavalin A stimulated SL also possessed the capacity to confer resistance against HSV replication in otherwise permissive PM. Sensitized T cells proved to be essential for the generation of active supernatants. Active supernatants had the potential to confer resistance against HSV replication within mouse but not chick or hamster embryo cells. The other characteristics of the soluble mediator included: instability at pH 2, inactivation by trypsin but not by DNase or RNase and persistence of activity when exposed to 56° C for 20 minutes.With 3 Figures     

Protective immunity against Trypanosoma cruzi provided by oral immunization with Phytomonas serpens: role of nitric oxide

We have previously demonstrated that Phytomonas serpens, a tomato parasite, shares antigens with Trypanosoma cruzi, the protozoa that causes Chagas' disease. These antigens are recognized by human sera and induce protective immunity in Balb/c mice. In the present study, inducible nitric oxide synthase (iNOS) knockout (KO) mice and C57BL/6 mice treated with the nitric oxide inhibitor, aminoguanidine (AG, 50 mg kg(-1)) infected with T. cruzi, were used to demonstrate the role of nitric oxide (NO) to host protection against T. cruzi infection achieved by oral immunization with live P. serpens. A reduction in parasitaemia and an increase in survival were observed in C57BL/6 infected mice and previously immunized with P. serpens, when compared to non-immunized mice. iNOS (KO) mice immunized and C57BL/6 immunized and treated with AG presented parasitaemia and mortality rates comparable to those of infected and non-immunized mice. By itself, immunization with P. serpens did not induce inflammation in the myocardium, but C57BL/6 mice so immunized showed fewer amastigotes nests in the heart following an acute T. cruzi infection than those in non-immunized mice. These results suggest that protective immunity against T. cruzi infection induced by immunization with P. serpens is dependent upon enhanced NO production during the acute phase of T. cruzi infection.     

Toxicity and induction of resistance to Listeria monocytogenes infection by amphotericin B in inbred strains of mice.

Amphotericin B (AmB) treatment before infection with the bacterium Listeria monocytogenes prolonged survival of AKR mice but shortened survival of C57BL/6 mice compared with survival of untreated infected controls. C57BL/6 mice were also more sensitive to the acute toxic effects of AmB than AKR mice, as were (C57BL/6 X AKR)F1 hybrid mice. Spleen cells and erythrocytes (RBCs) from the C57BL/6 and the F1 hybrid mice were both more sensitive to the lytic and lethal effects of AmB than corresponding cells from AKR mice. Biochemical analysis indicated that catalase levels in RBCs from C57BL/6 and F1 hybrid mice were about 60% of those found in RBCs from AKR mice. The lysis by AmB of RBCs from all these strains of mice was inhibited by catalase or incubation in a low-oxygen environment. These findings suggest that (i) the low catalase levels in C57BL/6 and F1 hybrid mice may limit the protection of cells from the oxidant damage involved in AmB action, and (ii) the toxicity which occurs at low concentrations of AmB in the mouse strains with low intracellular catalase levels may interfere with or ablate the AmB-induced increases in mouse resistance to L. monocytogenes infection.     

Adoptive transfer of immunity to oral challenge with virulent salmonellae in innately susceptible BALB/c mice requires both immune serum and T cells.

The mechanisms of immunity to salmonellae conferred by immunization with live vaccines were studied by adoptive transfer using the mouse-virulent strain Salmonella typhimurium C5 and innately susceptible BALB/c (ltys) mice. This organism cannot establish a sublethal infection in naive BALB/c mice. Animals immunized 2 to 3 months earlier with the S. typhimurium SL3261 aroA live vaccine were used as donors of serum, spleen cells, and mesenteric lymph node cells for naive recipients which were challenged orally with the virulent C5 strain. Simultaneous transfer of both immune serum and immune cells was necessary for protection. Simultaneously depleting the donors of CD4+ and CD8+ T cells by administration of antisera in vivo prior to cell harvesting showed that T cells were necessary for protection. The results demonstrate that both antibody and T cells are required for recall of immunity to oral challenge with virulent salmonellae in innately susceptible mice and suggest that the ability to elicit opsonizing antibody in addition to cell-mediated immunity is important for optimal protection induced by salmonella vaccines.     

Modulation of proteinase-K resistant prion protein by prion peptide immunization

Prion diseases are caused by conformational alterations in the prion protein (PrP). The immune system has been assumed to be non-responsive to the self-prion protein, therefore, PrP autoimmunity has not been investigated. Here, we immunized various strains of mice with PrP peptides, some selected to fit the MHC class II-peptide binding motif. We found that specific PrP peptides elicited strong immune responses in NOD, C57BL/6 and A/J mice. To test the functional effect of this immunization, we examined the expression of proteinase-K-resistant PrP by a scrapie-infected tumor transplanted to immunized syngeneic A/J mice. PrP peptide vaccination did not affect the growth of the infected tumor transplant, but significantly reduced the level of protease-resistant PrP. Our results demonstrate that self-PrP peptides are immunogenic in mice and suggest that this immune response might affect PrP-scrapie levels in certain conditions.     

Requirement of the initial production of gamma interferon in the generation of protective immunity of mice against Listeria monocytogenes.

Protective immunity of mice against Listeria monocytogenes, which is mediated mainly by gamma interferon (IFN-gamma)-producing T cells, was induced by immunization with viable bacteria but not with killed bacteria. By comparing mice immunized with either viable or killed L. monocytogenes, it was found that IFN-gamma was produced at the initial stage only after immunization with viable bacteria. This finding prompted us to investigate the effect of neutralizing the IFN-gamma on the final generation of protective T cells against L. monocytogenes. When endogenous IFN-gamma was neutralized by administration of anti-IFN-gamma monoclonal antibody for the initial 2 days in mice immunized with viable bacteria, the generation of protective T cells on day 6 was completely blocked, as revealed by T-cell adoptive transfer. The generation of listeria-specific IFN-gamma-producing T cells was also abolished. These results clearly demonstrated that endogenous IFN-gamma, which is produced at the initial stage of immunization, actually plays a critical role in the generation of protective T cells against L. monocytogenes in vivo. Moreover, this study suggested that the lack of IFN-gamma-inducing ability is responsible for the inability of killed L. monocytogenes to induce protective T cells in mice.     

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.     

Immune response to syngeneic or autologous testicular cells in mice. I. Augmented delayed footpad reaction in cyclophosphamide-treated mice.

A delayed footpad reaction against syngeneic or autologous testicular cells was detected in mice of inbred C57BL/6, AKR and C3H/He strains. The reaction was only provoked to a measurable level if the immunization was preceded by treatment with cyclophosphamide (CY). Footpad reaction was strongest on day 6 after immunization and was detected in both male and female mice. It was found that the reaction was elicited not only with the immunizing antigen, but also with allogeneic or xenogeneic testicular antigen in mice immunized with syngeneic testicular cells.