Distinct host-dependent pathogenic mechanisms leading to a similar clinical anemia after infection with lymphocytic choriomeningitis virus

The Docile strain of lymphocytic choriomeningitis virus (LCMV) induces anemia in a number of inbred strains of mice, including C3HeB/FeJ and CBA/Ht animals. A difference in the kinetics of anemia and in compensatory reticulocytosis suggested that impaired erythropoiesis was the major pathogenic mechanism involved in CBA/Ht mice, but not in C3HeB/FeJ mice. In both mouse strains an antierythrocyte autoantibody production that depended on the presence of functional CD4+ T lymphocytes was observed. Although depletion of T helper lymphocytes prevented anemia in C3HeB/FeJ mice, this treatment largely failed to inhibit the development of the disease in CBA/Ht animals. This observation indicated that the antierythrocyte autoimmune response induced by the infection was at least partly responsible for the anemia of C3HeB/FeJ mice, but not of CBA/Ht mice. Erythrophagocytosis was enhanced in both mouse strains after LCMV infection, but did not appear to be a major cause of anemia. These data clearly indicate that similar disease profiles induced by the same virus in two different host strains can be the result of distinctly different mechanisms.     

Cellular and metabolic requirements for induction of macrophage procoagulant activity by murine hepatitis virus strain 3 in vitro.

The cellular basis for the variation in induction of monocyte procoagulant activity (PCA) by murine hepatitis virus strain 3 (MHV-3) was examined using a set of recombinant inbred strains of mice derived from the resistant (A/J) and susceptible C57B1/6J (B) progenitors. Induction of PCA by MHV-3 required live virus and host protein and RNA synthesis. Absolute restriction for induction of PCA was observed at the level of the macrophage. Peritoneal macrophages from resistant parental A/J and RI strains (AXB5) could not be induced to express PCA when stimulated by MHV-3 alone or in the presence of lymphocytes from susceptible and H-2 compatible RI mice (AXB3) although they did respond to endotoxin (LPS). In contrast, macrophages from both susceptible (AXB3) and semisusceptible (AXB1) RI strains of mice expressed a similar increase in PCA after stimulation with MHV-3 in the absence of lymphocytes. The levels of PCA expressed by macrophages in the presence of Thy-1.2+ lymphocytes correlated with susceptibility to disease. Thy-1.2+ lymphocytes from susceptible RI AXB3 mice could induce levels of PCA in macrophages from semisusceptible RI AXB1 mice equivalent to that seen in cultures of macrophages and lymphocytes from susceptible mice. Further subfractionation of Thy-1.2+ cells demonstrated that L3T4+ cells instructed macrophages to produce PCA. Thy-1.2+ cells from MHV-3 immunized resistant AXB5 mice, but not from non-immunized mice, were able to suppress induction of PCA. This suppressor cell activity could be detected 4 days after immunization, reaching maximal activity at day 7 with significant suppression even at 28 days. The PCA was shown to have direct prothrombin cleaving activity (prothrombinase) by ELISA and immunofluorescence staining using the mAb 3D4.3. These results demonstrate that induction of a unique PCA (prothrombinase) is restricted at the level of the macrophage and define a regulatory role for T lymphocytes in its induction.     

Association of mouse fibrinogen-like protein with murine hepatitis virus-induced prothrombinase activity.

Previously, we demonstrated induction of a unique macrophage prothrombinase during infection of BALB/cJ mice by mouse hepatitis virus strain 3 (MHV-3). By immunologic screening, a clone representing this prothrombinase was isolated from a cDNA library and sequenced. The sequence identified this clone as representing part of a gene, musfiblp, that encodes a fibrinogen-like protein. Six additional clones were isolated, and one clone, p11-3-1, encompassed the entire coding region of musfiblp. Murine macrophages did not constitutively express musfiblp but, when infected with MHV-3, synthesized musfiblp-specific mRNA. musfiblp mRNA induction was earlier and significantly greater in BALB/cJ than A/J macrophages. Prothrombinase activity was demonstrated when musfiblp was expressed from p11-3-1 in RAW 264.7 cells. These data suggest that musfiblp encodes the MHV-induced prothrombinase.     

A deficiency in the B cell response of C57BL/6 mice correlates with loss of macrophage-mediated killing of Leishmania amazonensis

Infection of C3HeB/FeJ and C57BL/6 mice with Leishmania major stimulates a healing cell-mediated immune response, while Leishmania amazonensis infection leads to chronic disease. Here we show C3HeB/FeJ mice co-infected with both species of Leishmania heal, while co-infected C57BL/6 mice do not. Using an in vitro killing assay we determined B cells from infected C57BL/6 mice are ineffective in promoting parasite killing compared with B cells from infected C3HeB/FeJ mice. Furthermore, infected C57BL/6 mice produce less antigen-specific antibodies compared with infected C3HeB/FeJ mice. These findings suggest B cells play a required role in the cell-mediated immune response against L. amazonensis.     

The relationship between PROP and ethanol preferences: an evaluation of 4 inbred mouse strains

Ethanol's taste attributes undoubtedly contribute to the development of drug preference. Ethanol's taste is both sweet and bitter. Taster status for bitter 6-n-propylthiouracil (PROP) has been proposed as a genetic marker for alcoholism; however, human results are conflicting. We collected preference scores for both tastants in 4 mouse strains selected on the basis of previously reported taste preference, with the generally accepted idea that inbred mice show minimal within-strain variation. Eighty-eight male mice (22 per strain) participated. The strains were as follows: C57BL/6J, ethanol preferring; BALB/cJ, ethanol avoiding; SWR/J, PROP avoiding; and C3HeB/FeJ, PROP neutral. Using a brief-access (1-min trials) 2-bottle preference test, we assessed the taste response of each strain to PROP and ethanol on separate days. Although PROP avoiding versus neutral mice could be segregated into significantly different populations, this was not the case for ethanol avoiding versus preferring mice, and all strains showed high variability. On average, only BALB/cJ, SWR/J, and C3HeB/FeJ mice conformed to their literature-reported preferences; nonetheless, there were a substantial number of discordant animals. C57BL/6J did not conform to previous results, indicating that they are ethanol preferring. Finally, we did not observe a significant relationship between PROP and ethanol preferences across strains. The high variability per strain and the number of animals in disagreement with their respective literature-reported preference raise concerns regarding their utility for investigations underlying mechanisms of taste-mediated ingestive responses. Absent postingestive consequences, the brief-access results suggest a possible degree of previously masked polymorphisms in taste preferences or a more recent drift in underlying genetic factors. The absence of a relationship between PROP and ethanol indicates that the bitter quality in ethanol may be more highly related to other bitter compounds that are mediated by different genetic influences.     

Factors influencing the in vitro hatching of mouse blastocysts

The influence of bovine serum albumin (BSA) concentration on embryo hatching and the number of embryos cultured per drop of culture medium was examined in F1 (C57BL/6J × DBA/2J), C3HeB/FeJ strain and Line E mice. Embryos collected from F1 and Line E mice exhibited uniform hatching rates at BSA concentrations between 1 and 10 mg/ml, and embryo numbers ranging from 1 to 10 per 3 μ1 of culture medium. The hatching of C3HeB/FeJ blastocysts was greater at the higher concentrations of BSA and higher embryo densities. When the C3HeB/FeJ embryos were grown at high densities until morula and then cultured singly in fresh media they hatched at a low rate. However, when allowed to develop until the blastocyst stage before replotting, the embryos hatched at a high rate. C3HeB/FeJ embryos cultured singly until morula and then placed in groups of 10 hatched at a high rate. Single C3HeB/FeJ embryos, cultured in medium conditioned by the prior presence of embryos at high densities, hatched at a slightly higher frequency than those cultured in fresh medium. There was no tendency of embryos developing from the two-cell to the eight-cell stages to hatch when cultured in the presence of high densities of hatching blastocysts.     

Evidence for separate genetic defects in C3H/HeJ and C3HeB/FeJ mice, that affect susceptibility to gram-negative infections

Past studies have suggested a linkage between susceptibility to Salmonella typhimurium infection and the Lpsd genotype in C3H mice. Recently, this linkage was questioned by the finding that C3HeB/FeJ mice (Lpsn,Lpsn) were highly susceptible to systemic S. typhimurium infection. The present study shows a marked difference between C3H/HeJ and C3HeB/FeJ in their susceptibility to Gram-negative urinary tract infection. The number of E. coli and S. typhimurium recovered from the kidneys 24 hr after infection was 70 to 100 times higher in C3H/HeJ than in C3HeB/FeJ or C3H/HeN mice. Subsequently, in C3HeB/FeJ mice S. typhimurium multiplied to the level of C3H/HeJ mice, resulting in a shorter mean survival time of C3H/HeJ and C3HeB/FeJ compared with C3H/HeN mice. In contrast, E. coli remained localized to the urinary tract of C3H/HeJ mice but were eliminated from C3HeB/FeJ and C3H/HeN mice. Thus, experimental E. coli urinary tract infection appears to provide a method to differentiate the genetic defects of C3H/HeJ and C3HeB/FeJ mice. The results support an influence of the Lpsd genotype on clearance of Gram-negative bacteria from the kidneys of C3H mice.     

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.     

A relationship between synaptosomal GABA uptake and blood pressure in five inbred strains of mice

Synaptosomal GABA uptake, sodium-dependent GABA binding to synaptic membranes and GABA receptor binding were measured in the cerebral cortex of five inbred strains of mice. These were the SWR/J, BALB/cJ, DBA/1J, C57BL/10J and C3HeB/FeJ strains. A highly significant negative correlation was found to exist between GABA uptake and blood pressure (r = ?0.92). A similarly good negative correlation occurred between sodium-dependent GABA binding and blood pressure. A poor correlation was observed between blood pressure and GABA receptor binding (r = ?0.46). Taken together with other evidence that exists in the literature, these results suggest that in mice blood pressure could be under the influence of the GABA system.     

Induction of activated macrophages in C3H/HeJ mice by avirulent Salmonella

A single injection of viable Salmonella typhimurium SL3235, an avirulent organism blocked in the aromatic pathway, induced the generation of activated peritoneal macrophages in three different C3H mouse strains, including macrophage-defective C3H/HeJ mice. Macrophages obtained from immunized mice were cytotoxic for B16 melanoma cells, P815 mastocytoma cells, and TU-5 fibrosarcoma cells and microbicidal in vitro for the obligate, intracellular, protozoan parasite Leishmania major. The capacity of live SL3235 to activate C3H/HeJ macrophages contrasts with the failure of live Bacillus Calmette-Guérin to induce activated macrophages in this mouse strain. Although viable SL3235 were capable of fully activating cells of both normal and defective mice, a dose-dependent difference was observed in the number of organisms necessary for induction of tumoricidal macrophages in C3HeB/FeJ (normal) and C3H/HeJ (defective) animals. As few as 80 viable SL3235 were capable of activating C3HeB/FeJ macrophages whereas 5 X 10(4) organisms were required to activate C3H/HeJ macrophages. Maximal macrophage activation occurred 7 to 10 days after SL3235 inoculation in C3H/HeJ and C3HeB/FeJ mice. Acetone-killed cells of SL3235 had some but not all of the activity of the living Salmonella. A single in vivo injection of the nonviable preparation resulted in the induction of tumoricidal macrophages in C3HeB/FeJ but not in C3H/HeJ mice, even when tested over a wide dosage range. Injection of acetone-killed cells of SL3235 did, however, result in a population of primed macrophages in C3H/HeJ mice, as explanted cells could be induced to express activated macrophage effector activities after additional treatment in vitro with either LPS or IFN-gamma. Thus, in vivo administration of viable SL3235 is, by itself, capable of eliciting the full series of steps required for activation of C3H/HeJ macrophages, whereas killed SL3235 only provides signals sufficient to prime these defective macrophages for further activation in vitro. AI 15613     

Subchronic endotoxin inhalation causes persistent airway disease

The endotoxin component of organic dusts causes acute reversible airflow obstruction and airway inflammation. To test the hypothesis that endotoxin alone causes airway remodeling, we have compared the response of two inbred mouse strains to subchronic endotoxin exposure. Physiological and biological parameters were evaluated after 1 day, 5 days, or 8 wk of exposure to endotoxin [lipopolysaccharide (LPS)] in endotoxin-sensitive (C3HeB/FeJ) and endotoxin-resistant (C3H/HeJ) mice. After 5 days or 8 wk of LPS exposure, only C3HeB/FeJ had elevated airway hyperreactivity to inhaled methacholine. Only the C3HeB/FeJ mice had significant inflammation of the lower respiratory tract after 1 day, 5 days, or 8 wk of LPS exposure. Stereological measurements of small, medium, and large airways indicated that an 8-wk exposure to LPS resulted in expansion of the submucosal area only in the C3HeB/FeJ mice. Cell proliferation as measured by bromodeoxyuridine incorporation contributed to the expansion of the submucosa and was only significantly elevated in C3HeB/FeJ mice actively exposed to LPS. C3HeB/FeJ mice had significantly elevated levels of interleukin-1beta protein in whole lung lavage after 1 day and 5 days of LPS exposure and significantly elevated protein levels of total and active transforming growth factor-beta1 in whole lung lavage fluid after 5 days of LPS exposure. Our findings demonstrate that subchronic inhalation of LPS results in the development of persistent airway disease in endotoxin-responsive mice.     

A cell surface antigen, TER, expressed by embryos and germ cells.

An antiserum prepared in rabbits against the C3HeB/FeJ mouse ovarian teratocarcinoma E6496 was absorbed in vivo in C3HeB/FeJ mice. This absored antiserum identified an antigen, denoted TER, that is present on sperm, ova, embryonic germ cells, and cells of the early mouse embryo. TER was absent from all adult somatic cells tested, but found on several murine tumors.     

Endogeneous interferon α/β produced by murine Kupffer cells augments liver-associated natural killing activity

Summary Nonparenchymal liver cells from untreated C3HeB/FeJ mice, when incubated in medium containing-10% fetal bovine serum or portal serum, produced significant amounts of interferon alpha/beta (IFN/). In contrast, other cell populations (spleen, mononuclear blood cells and peritoneal cells) from C3HeB/FeJ mice or nonparenchymal liver cells from other strains of mice (C3H/HeJ, germ-free C3H/HeN and C57Bl/6J) produced little or no detectable IFN in fetal bovine serum under the same culture conditions. The cells in the nonparenchymal liver cell population responsible for IFN/ production were adherent, phagocytic, silica-sensitive, carbonyl-iron-sensitive, and Thy1.2^–, presumably Kupffer cells or resident liver macrophages. IFN/ production by cultured Kupffer cells was not observed if medium containing fetal bovine serum or portal serum was treated with polymyxin B or if Kupffer cells were cultured in serum-free medium. This suggested that small amounts of endotoxin in fetal bovine or portal serum stimulated Kupffer cells to produce IFN/. Possibly, Kupffer cells are in a different state of activation/maturation than peritoneal and splenic macrophages since the sensitivity of resident Kupffer cells from C3HeB/FeJ mice to the stimulatory effects of endotoxin. The endogenous production of IFN/ by Kupffer cells from C3HeB/FeJ mice can augment liver-associated natural killer (NK) activity against YAC-1 cells (4 h) and induce liver-associated cytotoxic activity, not restricted by the major histocompatibility complex, against NK resistant P815 mastocytoma cells (18 h).This work was supported by National Institutes of Health grant CA28835, VA Merit Grant and by the Margaret Duffy and Robert Cameron Troup Fund Abbreviations used: NPC, nonparenchymal liver cells; FBS, fetal bovine serum; IFN, interferon; -AsGm-1, anti-asialo-GM1; -Thy1.2, anti-Thy1.2; Hepes, 4-(2-hydroxyethyl)1-piperazineethanesulfonic acid; HBSS, Hanks' balanced salt solution; GBSS, Gey's balanced salt solution; SRBC, sheep red blood cells; Ab, mouse anti-SRBC; NK, natural killer; MHC, major histocompatibility complex; polyI·polyC, polyinosinec·polycytidylic acid     

Genetic control of responses to bacterial lipopolysaccharides in mice. II. A gene that influences a membrane component involved in the activation of bone marrow-derived lymphocytes by lipipolysaccharides.

C3H/HeJ mice contain a defect in a single autosomal locus which is not linked to the H-2 histocompatibility or the heavy chain allotype loci that restrict immune, mitogenic, and polyclonal responses to bacterial lipopolysaccharides (LPS). Adult thymectomized C3H/HeJ mice that have been irradiated and reconstituted with C3HeB/FeJ bone marrow cells respond well to LPS. Cell-mixing experiments using C3H/HEJ-C3HeB/FeJ spleen cultures show that the failure of C3H/HeJ spleen cells to support responses to LPS is not due to nonspecific or LPS-induced suppressive events, or the lack of accessory cell types. C3H/HeJ and C3HeB/FeJ spleen cells bind LPS and respond to other B cell mitogens equally well. We suggest that the B lymphocytes of C3H/HeJ mice have a defect in a membrane component that is activated via interaction with LPS, and initiates the intracellular events that lead to cell proliferation.     

Optimal N-ethyl-N-nitrosourea (ENU) doses for inbred mouse strains

ENU is a powerful germline mutagen in the mouse, providing the opportunity to analyze the functions of large numbers of genes in the mammalian genome. In many mutagenesis experiments, it would be beneficial to exploit the advantages of inbred mouse strains. To perform an effective ENU mutagenesis screen using inbred mice, a dosage regimen is required to determine the optimal dose of ENU for that inbred strain, a time-consuming preliminary process. We have carried out dosage regimens for mutagenizing doses of ENU in ten inbred strains of mouse: 129X1/SvJ, 129S6/SvEv, A/J, BALB/cJ, BTBR/N, C3He/J, C3HeB/FeJ, C57BL/6J, C57BR/cdJ, and CBA/CaJ, and determined an optimal dose for each strain, defined by length of sterile period and number of males to survive treatment. Three strains: A/J, BALB/cJ and C57BL/6J, are able to tolerate high doses, up to 300 mg/kg body weight, and are highly recommended for mutagenesis studies.     

Association between nitric oxide synthesis and vaccination-acquired resistance to murine hepatitis virus by spf mice

Murine hepatitis virus strain 3 (MHV-3) produces a strain-dependent pattern of disease, with A/J and BALB/c mice being considered models of resistance and susceptibility, respectively. A role for nitric oxide in controlling infection remains debatable; thus, we monitored nitric oxide levels in blood and liver of immunized and nonimmunized spf mice during infection by electron paramagnetic resonance. In parallel, liver histology, virus titers, and plasma alanine aminotransferase (ALT) activity were monitored. Nitric oxide synthesis was barely detectable in BALB/c mice, which showed a progressive increase in virus titers and ALT activity. These animals died with a shorter survival time than A/J mice. The latter displayed a less severe infection and presented detectable levels of nitric oxide as nitrosyl complexes in blood and liver at 72 hpi. Immunized mice from both strains became resistant to MHV-3 and showed comparable levels of nitrosyl complexes in blood and liver at an early time (24 hpi). Thereafter, nitric oxide levels decreased but remained detectable in blood up to 96 hpi. Immunized mice were capable of clearing the virus and clearance was inhibited by administration of a nitric oxide synthase inhibitor. Overall, the results support a role for nitric oxide in controlling MHV-3 infection.     

Involvement of lipopolysaccharide in the pathogenicity of Treponema hyodysenteriae

Treponema hyodysenteriae, the etiologic agent of swine dysentery, caused gross and microscopic lesions in the large intestines of C3HeB/FeJ mice. No gross lesions were observed in the intestines of the closely related, but lipopolysaccharide-resistant, C3H/HeJ strain of mice, and microscopic lesions were mild, if present at all. In the presence of actinomycin D, 1 mg of T. hyodysenteriae lipopolysaccharide (LPS) was lethal for C3HeB/FeJ but not for C3H/HeJ mice. Also, the treponemal LPS was chemotactic for macrophages from C3H/HeJ mice but not for macrophages from C3HeB/FeJ mice. The difference between the two mouse strains in lesion development may be due to the nondestructive nature of LPS in C3H/HeJ mice, which suggests that the treponemal LPS is involved in the pathogenicity of T. hyodysenteriae. T. hyodysenteriae may prove to be a useful bacterium in the study of LPS-resistant C3H/HeJ mice, because resistance to the treponemal LPS and to the treponeme itself appear to correlate.     

Trichinella spiralis: role of non-H-2 genes in resistance to primary infection in mice

Two strains of mice which share identical H-2 genes but differ in their genetic backgrounds were compared for their ability to resist infection with Trichinella spiralis. The two strains of mice, C3HeB/FeJ and AKR/J, share the H-2k haplotype which is associated with susceptibility to primary infection with T. spiralis in H-2 congenic strains of mice. AKR/J mice, infected with 150 infective muscle larvae, harbored significantly fewer muscle larvae 30 days postinfection than did mice of the strain C3HeB/FeJ. Approximately equal numbers of worms establish in the small intestine of AKR and C3H mice, but the AKR mice expelled adult worms from the gut more rapidly than did mice of the C3H strain. By Day 9 postinfection, 50% of the worms had been expelled by the AKR mice whereas expulsion of worms from C3H mice was delayed beyond Day 9 and occurred primarily between Days 10 and 12. Over this same experimental period (Days 6-12), fecundity of female worms from AKR mice, measured as the mean newborn larvae/female/hour, was approximately one-half that of worms taken from C3H mice. These results support the conclusion that genes outside of the mouse H-2 complex regulate expulsion of adult worms from the gut. These background genes also markedly influence the fecundity of female worms.     

Immunopathology of chronic progressive hepatitis in nude mice infected with low-virulent mouse hepatitis virus

Progressive hepatitis in athymic nude (nu/nu) mice due to a low-virulent mouse hepatitis virus, MHV-2 cc, was examined for involvement of immunocytes and serum antibodies. At 3 to 6 weeks postinoculation (p.i.) a considerable number of Mac 1- and asialo GM1-positive cells were accumulated in the affected liver and spleen. There were also some Thy-1-positive cells. Later than 2 weeks p.i., serum IgG and IgM antibodies were detected in parallel with virus-neutralizing activity, while the IgG levels were lower than those of infected euthymic (nu/+) littermates. By transfer of the infected nu/nu mouse serum, the recipient euthymic mice acquired resistance to lethal challenge infection with a virulent virus, MHV-2.