Amphotericin B Selectively Stimulates Macrophages from High Responder Mouse Strains

Lymphoid cells from most inbred mouse strains respond to amphotericin B (AmB)-induced immunostimulation. However, C57BL/6 mice and related strains display low or absent lymphoid cell stimulation by AmB and enhanced susceptibility to AmB toxicity. Experiments reported here show that in vitro incubation with AmB can stimulate AKR (AmB-high responder strain) macrophage proliferation. Intraperitoneal injection of AKR mice with AmB also elicits a population of macrophages primed for enhanced oxidative burst activity after triggering by zymosan particles. Under the same experimental conditions, AmB elicits a population of very weakly responsive macrophages from C57BL/6 mice. the low responsiveness of C57BL/6 macrophages correlates with previous observations that AmB is a potent immunoadjuvant and B cell mitogen in most inbred strains, but it selectively lacks immunoadjuvant effects in C57BL/6 mice and it also fails to induce polyclonal B cell stimulation in their spleen cell suspensions. Similarly, in measurements of protein synthesis in vitro, high concentrations of AmB produce a greater inhibition of protein synthesis in C57BL/6 peritoneal macrophages than in parallel cultures of AKR macrophages. These findings support the hypothesis that the macrophage is an important target cell in the mediation of AmB-induced immunomodulation.     

Histocompatibility-linked genetic control of susceptibility to age-dependent polioencephalomyelitis in mice.

Susceptibility to induction of immune polioencephalomyelitis (IPE) was found to be controlled by a gene that is closely linked to the H-2 complex. Whereas mice of the AKR (H-2k) strain were susceptible to IPE induction, H-2-congenic mice, AKR.H-2b (H-2b from C57BL/6) and AKR.M (H-2m), were resistant. However, susceptibility to IPE may be under additional control by a gene(s) outside of the H-2 region, since both C57BL/6 (H-2b) mice and congenic B6.H-2k mice (H-2k from AKR) were resistant to IPE induction. F1 hybrid mice derived from AKR (susceptible) and DBA/2 (resistant) mice were susceptible to IPE induction, indicating that susceptibility is dominant in at least one gene, but susceptibility developed at a later age in the hybrid mice than in AKR mice. B6.PL-Ly-2a Ly-3a/Cy, C57BR, C57L, PL, and RF strain mice were resistant to ipe induction. Thus, of the 12 inbred strains tested so far, only two (C58 and AKR) are susceptible to IPE.     

Ethanol intake and motor sensitization: the role of brain catalase activity in mice with different genotypes

The C57BL/6J strain of inbred mice shows a characteristic pattern of ethanol-induced behaviors: very weak acute locomotor stimulation, a lack of locomotor-sensitizing effect of ethanol, and a high level of ethanol intake. This strain has relatively low levels of activity of the ethanol metabolizing enzyme catalase, and it has been proposed that brain catalase plays a role in the modulation of some behavioral effects of ethanol. In the first study of the present paper, we investigated the effects of pharmacological manipulations of brain catalase activity on C57BL/6J mice in acute ethanol-induced locomotion and ethanol intake. Results indicated that the reduction in motor activity produced by ethanol was reversed by pretreatment with catalase potentiators and it was enhanced by catalase inhibitors. In addition, ethanol intake was highly correlated with brain catalase activity in mice treated with a catalase potentiator. In the second study, F1 hybrid mice (SWXB6) from the outbred Swiss-Webster mice and the inbred C57BL/6J mice were used. Basal brain catalase activity levels of F1 mice were intermediate between to those of the two progenitor genotypes. That profile of catalase activity was parallel to the acute-ethanol-induced locomotion and to repeated-ethanol-induced motor sensitization effects observed across the three types of mice. These data suggest that brain catalase activity modifications in the C57BL/6J strain change the pattern of several ethanol-related behaviors in this inbred mouse.     

Tumorigenicity and tumour-graft rejection of polyoma virus-transformed fibroblast-T-lymphocyte hybrids.

In anticipation of the use of functional T-lymphocyte hybrids in adoptive immunotherapy, the differentiation and tumorigenicity of hybrid clones generated by fusion of a T lymphocyte derived from F1 (DBA/J2 x AKR) mouse spleen, and a polyoma virus-transformed fibroblast initiated from C3H mouse cells, were studied. The hybrid cells grew in suspension and had an appearance (by transmission and scanning electron microscopy) very similar to that of the lymphocytic line. The hybrid and the different clones could induce tumour grafts. Malignancy was dominant in newborn mice where tumours were obtained in all mouse strains (allogeneic or semi-allogeneic) inoculated. In adult mice, the hybrid cells were tumorigenic in C3H and F1 (DBA/J2 x AKR), whereas there was complete tumour rejection in allogeneic (C57/BL6) or semi-allogeneic (DBA/J2 and AKR) mice. The role played by major histocompatibility antigens in the graft rejection is discussed. The histology of the tumour grafts was intermediate between fibrosarcoma and lymphosarcoma.     

Tumorigenicity and tumour-graft rejection of polyoma virus-transformed fibroblast-T-lymphocyte hybrids

In anticipation of the use of functional T-lymphocyte hybrids in adoptive immunotherapy, the differentiation and tumorigenicity of hybrid clones generated by fusion of a T lymphocyte derived from F1 (DBA/J2 x AKR) mouse spleen, and a polyoma virus-transformed fibroblast initiated from C3H mouse cells, were studied. The hybrid cells grew in suspension and had an appearance (by transmission and scanning electron microscopy) very similar to that of the lymphocytic line. The hybrid and the different clones could induce tumour grafts. Malignancy was dominant in newborn mice where tumours were obtained in all mouse strains (allogeneic or semi-allogeneic) inoculated. In adult mice, the hybrid cells were tumorigenic in C3H and F1 (DBA/J2 x AKR), whereas there was complete tumour rejection in allogeneic (C57/BL6) or semi-allogeneic (DBA/J2 and AKR) mice. The role played by major histocompatibility antigens in the graft rejection is discussed. The histology of the tumour grafts was intermediate between fibrosarcoma and lymphosarcoma.     

Experimental models for prevention of graft-versus-host reaction in bone marrow transfution. III. Reversible and irreversible differentiation of lymphocytes destined for cytotoxicity to effector cells for splenomegaly.

When lymphoid cells were obtained from AKR donors 12 h after a treatment with C57BL/L cells in complete Freund's adjuvant and transferred to (AKR X C57BL/6) F1 mice, splenomegaly in F1 recipients was augmented but cytotoxicity was suppressed. The suppression of cytotoxicity was antigen-specific. When cell transfer was carried out at stages as early as 3 or 6 h after the treatment of donors, cytotoxicity was enhanced but splenomegaly was suppressed. Irreversible deviation of immune response from the generation of cytotoxicity to the development of splenomegaly appears to occur within 12 h after such a treatment of donors.     

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.     

Murine genotype influences the in vitro production of gamma (immune) interferon

Spleen cells from mice of 6 different strains were stimulated with pokeweed mitogen and the amount of gamma interferon in th supernatants was measured. Three strains (BALB/c, A/J and C3H/He) were found to be low producers (less than 50 units) whereas three other strains (C57BL6, CBA and AKR) were high producers (50 to 500 units). F1 hybrids between a low producer strain (A/J) and a high producer strain (C57BL6) were found to produce low amounts of interferon. Nude mice did not produce detectable amounts of pokeweed mitogen-induce gamma interferon. The level of production of this T cell lymphokine is thus under genetic control.     

Genetic modification of hearing in tubby mice: evidence for the existence of a major gene (moth1) which protects tubby mice from hearing loss.

Quantitative trait locus (QTL) analysis of genetic crosses has proven to be a useful tool for identifying loci associated with specific phenotypes and for dissecting genetic components of complex traits. Inclusion of a mutation that interacts epistatically with QTLs in genetic crosses is a unique and potentially powerful method of revealing the function of novel genes and pathways. Although we know that a mutation within the novel tub gene leads to obesity and cochlear and retinal degeneration, the biological function of the gene and the mechanism by which it induces its phenotypes are not known. In the current study, a QTL analysis for auditory brainstem response (ABR) thresholds, which indicates hearing ability, was performed in tubby mice from F(2)intercrosses between C57BL/6J- tub / tub and AKR/J-+/+ F(1)hybrids (AKR intercross) and between C57BL/6J- tub / tub and CAST/Ei.B6- tub / tub F(1)hybrids (CAST intercross). A major QTL, designated asmodifieroftubbyhearing1 ( moth1 ), was identified on chromosome 2 with a LOD score of 33.4 ( P < 10(-33)) in the AKR intercross (181 mice) and of 6.0 ( P < 10(-6)) in the CAST intercross (46 mice). This QTL is responsible for 57 and 43% of ABR threshold variance, respectively, in each strain combination. In addition, a C57BL/6J congenic line carrying a 129/Ola segment encompassing the described QTL region when made homozygous for tubby also exhibits normal hearing ability. We hypothesize that C57BL/6J carries a recessive mutation of the moth1 gene which interacts with the tub mutation to cause hearing loss in tub / tub mice. A moth1 allele from either AKR/J, CAST/Ei or 129/Ola is sufficient to protect C57BL/6J- tub / tub mice from hearing loss.     

Specific suppression of the local GVH reaction by F1 spleen cells

The local graft-versus-host (GvH) reaction in (C57BL/6 X BALB/c) F1 hybrid mice, assayed by popliteal lymph node enlargement, was specifically depressed by an injection of parental lymphocytes mixed with spleen cells from F1 mice pretreated with the same parental lymphocytes. Suppressor activity of CBF1 spleen cells was obtained 7 days after inoculation of parental lymphocytes, and peaked on day 10. The suppressive activity was induced by only spleen cells from CBF1 which was inoculated Balb/c lymphocytes, but not C57BL/6 lymphocytes. The lymphocyte subpopulation responsible for the suppressive activity was noticed in T cell population.     

Analysis of immunosuppression generated by the graft-versus-host reaction. II. Characterization of the suppression cell and its mechanism of action.

Spleen cells from (CBA X C57/BL) F1 mice undergoing graft-versus-host (GVH) reaction induced by injection of parental cells 7-14 days previously are capable of suppressing an immune response by normal or primed F1 spleen cells to chicken erythrocytes and levan in vivo and sheep erythrocytes in vitro. The cells in these GVH spleens which were responsible for the suppression were sensitive to treatment with anti-0 serum, resistant to 900 rad irradiation in vivo and not retained by anti-immunoglobulin columns. Suppressor activity in vitro was present only in the non-adherent fraction of these GVH cell suspensions. Furthermore, the T-cell fraction, purified by affinity chromatography, suppressed the in vitro response of macrophage-depleted normal F1 cells to DNP-levan. Collectively, these observations imply that suppressor T cells generated by GVH reaction can affect B-cell functions directly without intermediary macrophage participation. Spleen cells from (CBA X C57/BL) F1 mice undergoing GVH reaction induced by C57/BL cells were depleted of their F1 content by treatment with anti-CBA alloantiserum. The suppressive activity of the residual donor component was still expressed against other F1 cells (AKR X C57/BL) which were H-2 compatible with the original host, but not against H-2-incompatible cells (DBA/1 X C57/BL) F1. However, the latter were suppressed in the presence of (CBA X C57/BL) F1 cells. Thus, interaction of donor T cells with F1 target cells containing those H-2 antigens towards which they are sensitized is mandatory for the subsequent manifestation of immunosuppressive activity. GVH cells suppressed the response of primed F1 cells in double Marbrook chambers when the two populations were separated were by a cell-impermeable membrane, provided the GVH suspension contained F1 cells to which donor T cells were sensitized. This suggests that soluble factors are involved in the mechanism of GVH-induced immunosuppression.     

Comparison of T cell-mediated immune responsiveness of NZB, (NZB x &NZW)F1 hybrid and other murine strains.

The age-dependent capacity of NZB and (NZB x NZW)F1 hybrid, BALB/c, DBA/2, C57BL/6 and C3H mice to generate T cell-mediated immune responses was assessed qualitatively and quantitatively by measuring the following effector functions: (a) the time course of alloreactive cytotoxic T-cell activity triggered in vitro was comparable for NZ and other mouse strains; cell reactivity generated in vivo against EL4 tumour cells was low in young (NZB x NZW)F1 mice and in DBA/2 mice but was comparable for older (NZB x NZW)F1, NZB and other mouse strains; (b) the time-dependent, vaccinia virus-specific, cytotoxic T-cell activity after systemic infection was similar for all mouse strains; (c) the T cell-dependent primary footpad swelling after local injection with lymphocytic choriomeningitis virus was within the same range for all mouse strains tested with respect to size and kinetics of the reaction; (d) the cell-mediated immune protection against Listeria monocytogenes after systemic infection revealed that NZ mice are, independent of age, more susceptible than C3H or C57BL/6 mice and comparable to A strain mice. Therefore, these responses in young, or clinically relatively normal older, NZB or (NZB x NZW)F1 strains that are affected by a lupus-like autoimmune disease did not differ markedly from the range of responses of other mouse strains of 2-14 months of age, which are not known to be similarly diseased. Thus, overall cell-mediated immunity of NZ mice as assessed quantitatively and kinetically in these functional models is within normal ranges. Possible T-cell defects may therefore be selective and either do not occur or were not detected in these models.     

Mice bearing the ob/ob mutation have impaired immunity.

The obese mutant mouse C57BL/6J ob/ob showed impaired ability to reject skin grafts or react to a contact-sensitising agent in comparison with littermate controls (either +/ob or +/+). Ability of spleen cells from mice bearing the ob/ob mutation to produce a graft-versus-host reaction in C57BL/6J X DBA/2J F1 hybrid mice was not impaired.     

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.     

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.     

Effects of H-2 haplotype and gender on the lifespan of A and C57BL/6 mice and their F1, F2, and backcross offspring

The effects of H-2 type and gender on the lifespan of A and C57BL/6 mice and their F1, F2, and backcross offspring were studied. The proportion of mice remaining alive, percent survivors, was calculated at monthly intervals for each mating group. Statistical analyses of these survival data showed that, in agreement with studies from other laboratories, C57BL/6 (H-2b) mice lived significantly longer than A (H-2a) mice. When the survival curves for A and C57BL/6 backcross and F2 offspring were analyzed to test for the presence of an H-2 effect on mouse lifespan, no statistically significant association was detected, although a trend toward increased 10th decile survivorship among female H-2a mice was noted. The mice used in this study were also evaluated for the presence of an effect of gender on lifespan. Female mice of the A strain, and F1, F2, and backcross groups, but not the C57BL/6 mice, exhibited significantly longer lifespans when compared with their male counterparts. Thus these data show a significant gender effect, but only a trend towards an H-2 effect, on the lifespan of A and C57BL/6 mice and their F1, F2, and backcross offspring.     

Induction of persistent autoimmune haemolytic anaemia in mice by combined use of rat erythrocyte preimmunization and chronic GVHR

Female F1 mice (BDF1: C57BL/6 x DBA/2, BCF1: C57BL/6 x C3H/HeN, BBF1: C57BL/6 x BALB/c) were preimmunized with rat erythrocytes and then received splenic T cells from parental female C57BL/6 mice injected with hydrocortisone acetate before killing. In BDF1 and BCF1 mice, direct Coombs' test (DCT) positivity persisted for more than 4 months in almost all mice. They also showed haematological sign of anaemia, and the survival of infused 51Cr-labelled parental C57BL/6 erythrocytes was reduced. In BBF1 mice, DCT positivity returned to negative within 2 months. Autoantibodies were eluted from DCT-positive erythrocytes and their specificities were examined. They were capable of binding equally well to erythrocytes from all strains of mice tested, including the donor mice. They also cross-reacted with rat erythrocytes but not with sheep, rabbit or human erythrocytes. Isotypes of the autoantibodies eluted were also examined. Interestingly, isotypes of the autoantibodies obtained from individual mice were confined to one or other subclass of IgG isotype: IgG1, IgG2a or IgG2b.     

Antibody-mediated protection against Cryptococcus neoformans pulmonary infection is dependent on B cells

The pathogenesis of pulmonary Cryptococcus neoformans infection and the efficacy of passive immunoglobulin G1 (IgG1) administration were investigated in B-cell-deficient and C57BL/6J mice. C57BL/6J mice lived longer than B-cell-deficient mice after both intratracheal and intravenous infections. Administration of IgG1 prior to infection prolonged the survival of C57BL/6J mice but had no effect on the survival or numbers of CFU in the lungs of B-cell-deficient mice. C. neoformans infection in B-cell-deficient mice resulted in significantly higher levels of gamma interferon (IFN-gamma), monocyte chemoattractant protein-1 (MCP-1), and macrophage inflammatory protein-1alpha (MIP-1alpha) than in C57BL/6J mice. IgG1 administration reduced IFN-gamma and MCP-1 levels in C57BL/6J mice but not in B-cell-deficient mice. In addition, compared to its effect in C57BL/6J mice, C. neoformans infection in FcRgammaIII-deficient, athymic, and SCID mice significantly increased IFN-gamma and MCP-1 levels. IgG1 administration was associated with reduced IFN-gamma levels in C57BL/6J mice but not in FcRgammaIII-deficient, athymic, and SCID mice. These observations suggest that IgG1-mediated protection in this system is a consequence of alterations in the inflammatory response that translate into less damage to the host without directly reducing the fungal burden. For hosts with impaired immunities, the ineffectiveness of passive antibody (Ab) may reflect an inability to down-regulate inflammation and avoid self-damage. The results indicate an important role for B cells in host defense against C. neoformans infection and demonstrate a surprising dependence of Ab-mediated protection on B cells in this system.     

T cell independence of bleomycin-induced pulmonary fibrosis

The role of T cells and cytokines in bleomycin (BLM)-induced fibrosis was evaluated in susceptible and resistant strains of normal and SCID mice. Histology and hydroxyproline analysis showed that BLM induced pulmonary fibrosis in C57BL/6 and (C57BL/6 x BALB/c)F1 mice, whereas BALB/c mice were resistant to the disease. To test whether lymphocytes were required for the induction of BLM-induced pulmonary fibrosis, SCID mice were injected intratracheally with BLM and evaluated for the development of pulmonary inflammation and fibrosis. Similar morphological changes and increases in hydroxyproline were observed in both C57BL/6 SCID and (C57BL/6 x CB.17)F1 SCID animals compared to those seen in wild-type C57BL/6 and (C57BL/6 x BALB/c)F1 mice. In contrast, CB.17 SCID mice, which are genetically similar to BALB/c mice, were resistant to disease induction. Analysis of the cellular infiltrate in BLM-treated C57Bl/6 SCID mice confirmed a lack of T cells in the lungs of SCID mice and demonstrated a pronounced accumulation of eosinophils in areas of developing pulmonary fibrosis. NK cells were significantly elevated in untreated SCID mice and did not increase further after BLM treatment. Analysis of selected cytokines 1 day after initiation of BLM-induced pulmonary fibrosis indicated that the levels of TNF-alpha and IFN-gamma appeared to segregate with fibrosis in both the SCID and wild-type mice. The data demonstrate that T cells are not required for the induction of fibrosis by BLM and suggest that responses by non-lymphoid cells may be sufficient for the induction of fibrosis.     

The absence of one class of virus-induced protein in arginine-deprived cells infected with pig herpesvirus.

C57BL/6 mice responded to immunization with purified gp71 of Friend murine leukemia virus by mounting both humoral and cell-mediated responses. As measured by a number of tests, the responses were generally stronger than those obtained in BALB/c mice. However, in contrast to the BALB/c mice, immunization of C57BL/6 mice with gp71 did not result in the development of cytotoxic lymphocytes, although spleen lymphocytes were capable of undergoing blastogenesis when incubated with purified gp71. As in the BALB/c mice, the humoral response was type-specific.A unique feature of the response in gp71-immunized C57BL/6 mice was the accelerated activation of the endogenous virus as measured by the development of an immune response to its distinct envelope antigens. This resulted in the production of three distinguishable antibody populations: (1) type-specific antibodies to FLV gp71; (2) type-specific antibodies reacting with AKR gp71 (AKR virus being related to the endogenous virus of C57BL/6 mice); and (3) antibodies directed against p15 (E) which reacted both with AKR and Friend-Moloney-Rauscher viruses and are therefore considered group-specific in the murine system. The possible significance of the activation of the endogenous virus subsequent to gp71 immunization of C57BL/6 mice is discussed.