Ir gene control of immune responses to insulins. II. Phenotypic differences in T cell activity among nonresponder strains of mice

Immune responses by mice to heterologous insulins are controlled by H-2 linked Ir genes. In studies to determine the mechanisms responsible for nonresponsiveness, we found that although pork insulin failed to stimulate antibody or proliferative responses in H-2b mice, it did prime T cells that can express helper activity in adoptive recipient mice. This helper activity was insulin-specific in both elicitation and expression. In studies presented in this paper, we have extended this analysis to the response patterns of helper T cells stimulated by sheep, horse, and rat insulins in mice bearing different H-2 haplotypes. The results demonstrate that nonresponder forms of insulin, including rat insulin, prime T cells in H-2b and H-2d, but not H-2k, mice. These results suggest that regulation of nonresponsiveness to insulin appears to be through different pathways in mice bearing different H-2 haplotypes.     

Antigen-specific lymphocyte proliferative responses in inbred mice after Trichinella spiralis infection.

The in vitro antigen-specific lymphoproliferative response of spleen, mesenteric lymph node (MLN), and coeliac lymph node (CLN) cells taken from various strains of inbred mice infected with Trichinella spiralis was assessed. In most experiments cell populations were stimulated with excretory/secretory antigens (ESA) derived from adult and larval worms. Lymphoid cells collected 5-7 days postinfection were usually the most responsive to ESA as measured by [3H]thymidine uptake. Spleen cells were more responsive than either MLN or CLN cells. There was a correlation between in vitro ESA stimulation and worm rejection in strong- and weak-responder strains of mice. Spleen and MLN cells of NFS mice showed higher antigen-specific responsiveness, whereas the same cells from B10.BR (H-2k) and B10.Q (H-2q) strains of mice were less responsive. Among intermediate responder strains 2 patterns were observed. Spleen and MLN cells of BuB and DBA/1 mice responded more strongly than those of C3H mice. Dose-response experiments demonstrated that increasing the infective dose of larvae to the host usually increased subsequent in vitro antigen-specific lymphoproliferation. Furthermore, non-MHC-linked genes appear to be the primary determinant of antigen-specific T-cell-proliferative responses in inbred mice infected with T. spiralis.     

Immune responses to the 18-kDa protein of Mycobacterium leprae. Similar B cell epitopes but different T cell epitopes seen by inbred strains of mice.

Antibody responses to the 18-kDa protein of Mycobacterium leprae have been analyzed in different strains of mice. High, intermediate, and low responder strains have been identified and these response patterns show clear linkage to genes encoded in the H-2 complex. Three peptides, residues 1-50, 51-100, and 101-148 have been synthesized, as well as a series of 20-mer peptides, which span the entire 18-kDa protein. Repeated immunization of different strains of mice with the 18-kDa protein resulted in IgG responses to epitopes found on all three synthetic peptides. Immunization of BALB/cJ and B10.BR mice, two high responder strains, with 18-kDa protein resulted in high levels of IgG antibody to epitopes found on peptides 1-20, 16-35, 31-50, 46-65, and 76-95. B10.BR mice also contained IgG that bound peptide 61-80 and BALB/cJ mice produced IgG that bound peptide 91-110. Although B10.BR mice produced IgG that bound the 50-mer peptide 101-148, this IgG was not detected by binding to peptides 91-110, 106-125, 121-140, and 131-148. Immunization of B10.BR mice with individual overlapping 20-mer peptides as Ag revealed that peptides 1-20, 16-35, 31-50, and 76-95 elicited high titers of IgG that bound both the immunizing peptide as well as 18-kDa protein. As these peptides induce antibody synthesis they must contain both B cell and T cell epitopes. By contrast, immunization of BALB/cJ mice with the same 20-mer peptides, all of which contain B cell epitopes for this strain, failed to elicit IgG responses with one exception. Peptide 91-110 induced IgG that bound peptide 91-110, but not the intact 18-kDa protein. We conclude that peptides 1-20, 16-35, 31-50, and 76-95 either lack T cell epitopes for BALB/cJ mice, or activate different T cell subpopulations in the two strains. We suggest that the induction of IgG responses to small peptide Ag is an in vivo assay of the activity of Th2 cell subpopulations.     

Differences in time-dependent hypoxic phrenic responses among inbred rat strains.

Hypoxic ventilatory responses differ between rodent strains, suggesting a genetic contribution to interindividual variability. However, hypoxic ventilatory responses consist of multiple time-dependent mechanisms that can be observed in different respiratory motor outputs. We hypothesized that strain differences would exist in discrete time-dependent mechanisms of the hypoxic response and, furthermore, that there may be differences between hypoglossal and phrenic nerve responses to hypoxia. Hypoglossal and phrenic nerve responses were assessed during and after a 5-min hypoxic episode in anesthetized, vagotomized, and ventilated rats from four inbred strains: Brown Norway (BN), Fischer 344 (FS), Lewis (LW), and Piebald-viral-Glaxo (PVG). During baseline, burst frequency was higher in PVG than LW rats (P < 0.05), phrenic burst amplitude was higher in PVG vs. other strains (P < 0.05), and hypoglossal burst amplitude was higher in PVG and BN vs. FS and LW (P < 0.05). During hypoxia, burst frequency did not change in BN or LW rats, but it increased in PVG and FS rats. The phrenic amplitude response was smallest in PVG vs. other strains (P < 0.05), and the hypoglossal response was similar among strains. Short-term potentiation posthypoxia was slowest in FS and fastest in LW rats (P < 0.05). Posthypoxia frequency decline was absent in PVG, but it was observed in all other strains. Augmented breaths were observed during hypoxia in FS rats only. Thus genetic differences exist in the time domains of the hypoxic response, and these are differentially expressed in hypoglossal and phrenic nerves. Furthermore, genetic diversity observed in hypoxic ventilatory responses in unanesthetized rats may arise from multiple neural mechanisms.     

Strain-dependent differences in responses to exercise training in inbred and hybrid mice

The aim of this study was to characterize the response to exercise training in several mouse strains and estimate the genetic contribution to phenotypic variation in the responses to exercise training. Male mice from three inbred strains [C57Bl/6J (BL6), FVB/NJ (FVB), and Balb/cJ (Balb/c)] and three hybrid F(1) strains [CB6F1/J (CB6 = female Balb/c x male BL6), B6F F(1) (female BL6 x male FVB), and FB6 F(1) (female FVB x male BL6)] completed an exercise performance test before and after a 4-wk treadmill running program. Distance was used as the primary estimate of endurance exercise performance. FVB mice showed the greatest response to training, with five- to sevenfold greater increases in distance run compared with BL6 and Balb/c strains. Specifically, BL6, FVB, and Balb/c strains increased distance by 33, 172, and 23%, respectively. A similar pattern of changes across strains was observed for run time (17, 87, and 11%) and work (99, 287, and 57%). As a group, F(1) hybrid mice derived from BL6 and FVB strains showed an intermediate response to training (61%). However, further analysis indicated that training responses in FB6 F(1) mice (80%) were approximately 2.5-fold greater than responses in B6F F(1) mice (33%, P = 0.08). A similar pattern of changes between FB6 and B6F F(1) mice was observed for run time (44.5 and 17%) and work (141 and 59%). These data demonstrate that there are large strain-dependent differences in training responses among inbred mouse strains, suggesting that genetic background contributes significantly to adaptation to exercise. Furthermore, the contrasting responses in B6F and FB6 F(1) strains show that a maternal component strongly influences strain-dependent differences in training responses.     

Variations in the behaviors to novel objects among five inbred strains of mice

Novelty stimuli cause various behavioral responses, such as exploration and avoidance, and contextual variables may contribute to the behavioral outputs. Here, we tried to compare the behavioral responses to novel objects of five inbred strains of mice (C57BL/6J, 129/svJae, C3H/HeJ, BALB/cJ and DBA/2J) by using a modified novel object test where a small light-weight cube wrapped with paper was presented to mice in a home cage without beddings. In response to these objects, the C57BL/6J, 129/svJae and C3H/HeJ mice showed mild exploratory behaviors, such as approaching, sniffing or brief contact. In striking contrast, the BALB/cJ and DBA/2J mice, which have been known to show high avoidance/low exploration in other behavioral paradigms, exhibited play-like secondary reactions toward the objects after initial primary exploratory behaviors. Specifically, DBA/2J mice would move the object around in the cage, holding it with their mouths, and BALB/cJ mice would gnaw the object, eventually stripping off the wrapping paper. Such behaviors decreased when similar objects were presented repeatedly. The present results suggest that active manipulations of novel objects may be a relevant parameter for measuring novelty-induced behaviors in mice and appear to be strongly influenced by genetic factors.     

Characterization of T cell proliferative responses induced by anti-Qa-2 monoclonal antibodies.

The MHC class I Qa-2 Ag are attached to the cell surface by a glycanphosphatidylinositol (GPI) anchor. Crosslinking of Qa-2 and several other cell surface Ag attached by the GPI linkage has been shown to lead to cell activation. We have developed 10 new anti-Qa-2 mAb and characterized their capacity to induce proliferation of spleen cells. In the absence of anti-Ig-mediated crosslinking, none of the mAbs alone could induce activation. However, mAb 23.1 which reacts with the alpha 3 domain of Qa-2, when combined with most of the other mAbs (alpha 1, alpha 2 domain reactive), activated cells in the absence of anti-Ig crosslinking. The mAb pair 23.1 plus 24.16 was the most proficient and induced proliferation in the absence of any exogenous second signals. Responses were greatly enhanced and equivalent to those seen with anti-CD3 by the addition of phorbol myristate acetate (PMA). Ionomycin, rIL-2, or rIL-4 also potentiated anti-Qa-2 responses but less efficiently than PMA. Significant strain variation in the magnitude Qa-2-mediated proliferative responses was observed correlating with the levels of Qa-2 expressed on the cell surface. Crosslinking of Qa-2 molecules by the mAb combinations was required because monovalent Fab fragments failed to activate cells. F(ab')2 fragments of mAb 23.1 plus 24.16 induced vigorous proliferation indicating that accessory cell presentation of the mAb via Fc receptors was not required. Immobilized (plate bound) anti-Qa-2 mAb induced proliferation suggesting that the Qa-2 pathway may be distinct from that of other GPI molecules such as Thy-1 and Ly-6. Populations enriched for T cells (approximately 95%) responded as well as whole spleen cells, whereas B lymphocytes failed to proliferate to anti-Qa-2. Both CD4+ and CD8+ cells were activated following crosslinking of Qa-2. Finally, T cell activation mediated by Qa-2 induced elevation of [Ca2+]i, IL-2R expression, and the release of IL-2. These data demonstrate that crosslinking of Qa-2 on T lymphocytes represents a potent pathway for inducing cell activation.     

Genetic control of interleukin-2 production in inbred mice

BALB/c mice (H-2d haplotype) produced IL 2 better than C57BL/6 mice (H-2d haplotype). However genetic analysis in F2 generation demonstrated independent segregation of the IL 2 production intensity and H-2 haplotype. Investigation of the IL 2 production intensity in BALB/c, C57BL/6 and their F1 and F2 generation revealed that this was controlled by only one gene.     

Cryptosporidium infections in inbred strains of mice.

Cryptosporidium, a protozoan parasite of man and animals, is an important etiological agent of diarrhea throughout the world, particularly in children and immunocompromised individuals such as AIDS patients. Unfortunately, because of the lack of both in vivo laboratory models and reliable in vitro parasite culture systems, virtually nothing is known about the immunological events occurring during disease. In order to identify reliable animal models for infection, we studied C. parvum infections in 19 different strains of mice representing 12 H-2 haplotypes: A/J, AKR/J, B10.D2/J, B10.M/J, C3H/HeJ, C57BL/65, C57BL/6J-bgJ, CBA/NJ, DBA/1J, DBA/2J, HRS/J, HTG/J, NZB/B1NJ, NZW/J, P/J, RIII/J, SJL/J, SWR/J, and WB/ReJ, and in one gerbil: Meriones unguiculatus. Fecal samples and histological sections of the intestine taken on day 7 post-Cryptosporidium inoculation indicated that only the beige mouse (C57BL/6J-bgJ) harbored significant numbers of parasites compared to the other strains. The numbers of parasites harbored in these NK cell-deficient beige mice were, however, considerably lower than those seen in neonatal mice. Adult inbred mouse strains susceptible to Cryptosporidium infections are discussed.     

Modulatory effect of zinc on the proliferative response of murine spleen cells to polyclonal T cell mitogens

To study the effect of zinc on the proliferative response to polyclonal T cell mitogens, spleen cells from C57BL/6 mice were cultured with or without ZnCl2 and stimulated with graded doses of concanavalin A or phytohemagglutinin. Addition of 10(-4) M ZnCl2 inhibited proliferation whereas 10(-5) to 10(-6) M ZnCl2 did not modify the response to suboptimal doses of mitogen but increased DNA synthesis in cultures stimulated with high doses of mitogen (10 or 20 micrograms/ml of concanavalin A and 10 or 25 microliters/ml of phytohemagglutinin) which are supraoptimal for C57BL/6 mice, and inhibited proliferation in cultures of spleen cells from animals of this strain, low responder to T cell mitogens. In contrast, supplementation with ZnCl2 did not enhance the response to mitogen of spleen cells from high responder BALB/c mice. The enhancing effects of ZnCl2 on the proliferative response of C57BL/6 cells were not observed following depletion of adherent cells or in cultures supplemented with 5 X 10(-5) M 2-mercaptoethanol, both conditions capable of abrogating the inhibitory effect of high mitogen doses on the response of C57BL/6 cells.     

Genetic and behavioral differences among five inbred mouse strains commonly used in the production of transgenic and knockout mice

Five strains of mice commonly used in transgenic and knockout production were compared with regard to genetic background and behavior. These strains were: C57BL/6J, C57BL/6NTac, 129P3/J (formerly 129/J), 129S6/SvEvTac (formerly 129/SvEvTac) and FVB/NTac. Genotypes for 342 microsatellite markers and performance in three behavioral tests (rotorod, open field activity and habituation, and contextual and cued fear conditioning) were determined. C57BL/6J and C57BL/6NTac were found to be true substrains; there were only 12 microsatellite differences between them. Given the data on the genetic background, one might predict that the two C57BL/6 substrains should be very similar behaviorally. Indeed, there were no significant behavioral differences between C57BL/6J and C57BL/6NTac. Contrary to literature reports on other 129 strains, 129S6/SvEvTac often performed similarly to C57BL/6 strains, except that it was less active. FVB/NTac showed impaired rotorod learning and cued fear conditioning. Therefore, both 129S6/SvEvTac and C57BL/6 are recommended as background strains for targeted mutations when researchers want to evaluate their mice in any of these three behavior tests. However, any transgene on the FVB/NTac background should be transferred to B6. Habituation to the open field was analyzed using the parameters: total distance, center distance, velocity and vertical activity. Contrary to earlier studies, we found that all strains habituated to the open field in at least two of these parameters (center distance and velocity).     

Comparative teratogenicity of phenytoin among several inbred strains of mice

Inbred strains of mice differ in their response to the embryopathic effects of phenytoin (PHT). A/J animals, the most susceptible strain, were mated to C57BL/6J mice, the most resistant strain. The susceptibility of the F1 hybrid offspring was determined by the susceptibility of the mother. B6AF1 animals were as resistant as C57BL/6J parental mice, and AB6F1 hybrids were as susceptible as A/J mice. This was especially evident when orofacial anomalies were tallied. (B6A)F2 hybrid offspring were as resistant as their C57BL/6J grandparents.     

Variability in empathic fear response among 11 inbred strains of mice

Empathy is an important emotional process that involves the ability to recognize and share emotions with others. We have previously developed an observational fear learning (OFL) behavioral assay to measure empathic fear in mice. In the OFL task, a mouse is conditioned for context‐dependent fear when it observes a conspecific demonstrator receiving aversive stimuli. In the present study, by comparing 11 different inbred mouse strains that are commonly used in the laboratory, we found that empathic fear response was highly variable between different strains. Five strains – C57BL/6J, C57BL/6NTac, 129S1/SvImJ, 129S4/SvJae and BTBR T⁺ Itpr3^tf/J – showed observational fear (OF) responses, whereas AKR/J, BALB/cByJ, C3H/HeJ, DBA/2J, FVB/NJ and NOD/ShiLtJ mice exhibited low empathic fear response. Importantly, day 2 OF memory was significantly correlated with contextual memory in the classical fear conditioning among the 11 strains. Innate differences in anxiety, locomotor activity, sociability and preference for social novelty were not significantly correlated with OFL. Interestingly, early adolescent C57BL/6J mice exhibited an increase in acquisition of OF. The level of OFL in C57BL/6J strain was not affected by sex or strains of the demonstrator. Taken together, these data strongly suggest that there are naturally occurring OFL‐specific genetic variations modulating empathic fear behaviors in mice. The identification of causal genes may uncover novel genetic pathways and underlying neural mechanisms that modulate empathic fear and, ultimately, provide new targets for therapeutic intervention in human mental disorders associated with impaired empathy.     

Paclitaxel-induced neuropathic hypersensitivity in mice: responses in 10 inbred mouse strains

Mechanical allodynia, or hypersensitivity to tactile stimuli, is a frequent clinical symptom of neuropathy. Large interindividual differences have been observed in neuropathic pain, both in susceptibility to its development and in its severity. Identification of genetic factors relevant to this variability would be of obvious utility. Although many animal models of neuropathic pain following peripheral nerve injury have been developed, most involve intricate surgeries and are thus poorly suited for large-scale linkage mapping investigations in the mouse. Recently, a schedule of intraperitoneal injections of the chemotherapeutic agent, paclitaxel (Taxol(R)), has been shown to produce a long-lasting, bilateral neuropathy in the rat, featuring hypersensitivity to mechanical, thermal and cold stimuli. We present here a survey of the responses of 10 inbred mouse strains to paclitaxel injections. Virtually all strains developed statistically significant mechanical allodynia, with one strain, DBA/2J, exhibiting especially robust changes. Strain sensitivities to paclitaxel-induced mechanical allodynia were similar to those obtained previously using a surgical model of neuropathic pain, supporting our contention that genetic sensitivity to mechanical allodynia is independent of the precise mode of induction. Using sensitive DBA/2 mice and a resistant strain, C57BL/6J, for comparison, we further characterized the paclitaxel model in mice by examining cold allodynia and thermal hyperalgesia. Both strains displayed equivalent cold allodynia but neither strain developed thermal hyperalgesia. The present data confirm a genetic component in mechanical allodynia using this model, while dissociating mechanical hypersensitivity from other pain modalities.     

Synthetic Plasmodium falciparum circumsporozoite peptides elicit heterogenous L3T4+ T cell proliferative responses in H-2b mice

The ability of synthetic P. falciparum (NANP)n circumsporozoite peptides to elicit murine T cell proliferative responses was studied. When C57BL/6, C3H, and DBA/2 mice were injected with (NANP)40, only C57BL/6 (H-2b)-immune lymph node cells proliferated on restimulation in vitro with the same peptide. By using anti-I-A monoclonal antibodies or spleen cells from congenic H-2b mice as a source of antigen-presenting cells, the T cell proliferative response was shown to be restricted to the I-Ab region of the C57BL/6 haplotype. These results are in agreement with previous experiments which demonstrated that the anti-(NANP)40 antibody response was uniquely restricted to C57BL/6 (H-2b) mice. Several C57BL/6 long-term (NANP)n-specific T cell lines and clones were derived. All of the clones exhibited the L3T4 helper T cell phenotype. A considerable heterogeneity of T cell responses was observed when the lines and clones were stimulated with different concentrations of the various peptides studied. The results, together with the observed genetic restriction for both antibody and T cell responses, suggest that perhaps not all individuals who receive a similar repetitive tetrapeptide sporozoite malaria vaccine will develop T cell and or antibody responses.     

Gene markers for alcohol-metabolizing enzymes among recombinant inbred strains of mice with differential behavioural responses towards alcohol

The genetic variability of alcohol dehydrogenase (C2 isozyme), aldehyde dehydrogenase (A2 isozyme) and aldehyde oxidase (A2 isozyme) has been examined among recombinant inbred strains of mice which have been previously studied concerning their differential behavioural responses towards alcohol. The results showed no correlation between biochemical phenotype for these loci and behavioural response.