Effects of Differential Housing on the Primary and Secondary Antibody Responses of Male C57BL/6 and BALB/c Mice

The influence of the number of animals housed together in a cage on antibody production in response to keyhole limpet hemanocyanin (KLH) was evaluated among male C57B1/6 and BALB/c mice. In Experiment 1, male C57B1/6 mice were housed 1, 6, or 12 animals per cage and primary and secondary responses were evaluated. The secondary, but not the primary, antibody responses of male C57B1/6 mice were higher among mice housed alone compared to mice housed in groups; differences were observed for both IgM and IgG anti-KLH antibodies. The differential housing effects on secondary IgM antibody responses interacted with the priming dose of KLH. In Experiment 2, the primary and secondary responses to KLH of male C57B1/6 and BALB/c mice were compared among mice housed alone or 4 per cage. Among both strains, the secondary, not the primary, response was influenced by the number of mice housed in a cage; both secondary IgM and IgG titers were higher among the C57B1/6 mice housed alone but only secondary IgM titers were higher among the BALB/c mice housed alone. These experiments confirm previous observations that the primary antibody responses of C57B1/6 mice are not influenced by the number of animals housed in a cage and extend these findings by demonstrating that the secondary antibody responses of C57B1/6 and BALB/c are influenced by housing condition.     

3-aminobenzamide prevents restraint-evoked immunocompromise

Chronic stressors compromise immune function, which may affect disease state in rats and mice. Although the molecular mechanism(s) underlying the link between psychological stressors and physiological responses remain elusive, one putative mechanism is oxidative stress. DNA damage activates poly(ADP-ribose) polymerase (PARP), a nuclear enzyme that participates in DNA repair; if DNA damage is extensive, however, then PARP becomes cytotoxic. Because PARP-1-/- transgenic mice are resistant to chronic stress-induced immunocompromise, we tested the hypothesis that pre-restraint administration of 3-aminobenzamide (3-AB), a PARP inhibitor, would prevent restraint-evoked suppression of antibody production to the novel protein, keyhole limpet hemocyanin (KLH). Mice were physically restrained for 3 h daily for 14 consecutive days, then immunized with KLH. Daily restraint continued for an additional 21 days and anti-KLH IgG production was assessed. Mice exposed to repeated restraint reduced concentrations of anti-KLH IgG, whereas, mice treated with 3-AB (0.5, 5.0, or 20.0 mg/kg) prior to each bout of restraint displayed anti-KLH IgG concentrations similar to those of unrestrained mice. Treatment with 3-AB (0.5 and 5.0 mg/kg) during the restraint paradigm also facilitated habituation of the corticosterone response to restraint, and 3-AB (0.5 mg/kg) reduced the effect of repeated restraint on body mass. However, the immunoprotective effects of 3-AB and the endocrine and metabolic effects appear to be distinctly regulated because, unlike the endocrine and metabolic effects, the immunoprotective effects of 3-AB were independent of dose. These data suggest that PARP inhibitors may be useful to prevent compromised immune function in response to stressors.     

Attractiveness of illness-associated odorant cues in female rats is modulated by ovarian hormones, but not associated with pro-inflammatory cytokine levels

Odorant cues released by rodents play a key role in mate preference/selection. The goal of the following series of studies was to determine the impact of acute illness, and the potential role of the inflammatory response, on the release of illness-associated odor cues from female rats. Adult female Sprague-Dawley rats were injected with lipopolysaccharide (LPS, 100 μg/kg) and their soiled bedding was used as a stimulus to naïve male odor recipients. While odored bedding from sick males elicited a robust avoidance response evidenced by decreased sniffing, avoidance and burying behavior, odored bedding from sick females elicited only a reduction in sniffing, indicating a reduction in odor attractiveness. Odor cues from ovariectomized, but not sham-operated females decreased sniffing behavior and increased avoidance in male odor recipients. Acute estradiol benzoate (EB, 20 μg/kg) replacement into ovariectomized females restored the investigatory response of male recipients toward odor cues, while LPS administration into ovariectomized oil or EB treated females had little impact on odor attractiveness. Measurement of cytokines in both brain (the paraventricular nucleus of the hypothalamus) and blood from female odor donors indicated increased expression of TNF-α, IL-1β, and IL-6 following LPS, which was not affected by EB treatment. These findings illustrate a critical sexual dimorphism by demonstrating that acute illness reduces the attractiveness of female odor, whereas odor cues from sick males are highly aversive. Moreover, the attractiveness of female odor appears to be associated with circulating ovarian hormone levels, but not central or peripheral inflammatory cytokines.     

Aging and sympathetic modulation of immune function in Fischer 344 rats: effects of chemical sympathectomy on primary antibody response

In aged Fischer 344 (F334) rats, sympathetic innervation of the spleen is markedly diminished compared with young rats. To determine if this diminished noradrenergic (NA) innervation maintains a functional connection with the immune system, 3- and 17-month-old male F344 rats were treated with the NA-selective neurotoxin, 6-hydroxydopamine (6-OHDA), to ablate peripheral NA nerve fibers. In sympathectomized rats immunized with keyhole limpet hemocyanin (KLH), a T-dependent protein antigen, anti-KLH IgM, IgG, IgG1, IgG2b antibody titers were increased in young and old rats 14 days after immunization compared to vehicle controls. Furthermore, the number of IgM and IgG anti-KLH antibody-secreting spleen cells was elevated 7 and 14 days post-immunization. These effects were prevented by pretreatment with desipramine, a catecholamine uptake blocker that blocks 6-OHDA uptake and subsequent sympathectomy. Chemical sympathectomy also increased KLH-induced proliferation in vitro by spleen cells from old, but not young animals. Isoproterenol (ISO), a beta-adrenergic receptor agonist, elicited a rise in cAMP in spleen cells from NA-intact young and old rats, but the increase was attenuated in spleen cells from old rats. These results demonstrate that, although NA innervation in the F344 rat spleen is diminished with age, sympathetic signaling of the immune system remains intact. Thus, the SNS can inhibit antibody produced in response to a protein antigen in both young and old F344 rats.     

Influence of a natural stressor (predator odor) on locomotor activity in the meadow vole (Microtus pennsylvanicus): modulation by sex, reproductive condition and gonadal hormones

Sex differences in a variety of non-reproductive behaviors have been indicated to occur in seasonally breeding polygynous promiscuous rodents such as the meadow vole, Microtus pennsylvanicus. The present study was designed to assess the effects of reproductive and hormonal status on the locomotor responses of meadow voles following brief exposure to the odors of a natural predator, the Red fox (Vulpes vulpes). Adult male and female meadow voles, which are seasonal photoperiodically-induced breeders, were housed in either mixed sex pairs under a long, reproductively stimulatory photoperiod (simulating breeding: long light cycle, paired: LLC + P) or in same-sex pairs under a short, reproductively inhibitory photoperiod (simulated non-breeding: short light cycle, non-paired: SLC-NP). On 2 consecutive days following 1 day of baseline activity monitoring, voles were exposed individually for 3 min to fox odor and a novel pungent control odor (extract of almond). The levels of various measures of activity that were displayed by the voles were assessed by an automated Digiscan activity monitoring system. LLC + P (simulated breeding) voles displayed higher basal levels of activity relative to SLC + NP (simulated non-breeding) voles, with males displaying greater activity than females. LLC + P (simulated breeding) males displayed a significant reduction in activity levels following exposure to fox odor relative to control odor. The reductions in activity following fox odor exposure were related to plasma testosterone levels such that a larger behavioral response (i.e. greater reduction) was associated with higher levels of testosterone. Furthermore, dividing males into high and low testosterone groups based on the median levels of testosterone revealed that high but not low testosterone males displayed reductions in activity following exposure to fox odor relative to control odor. No changes in activity levels following exposure to fox odor were noted in SLC-NP males, and either SLC-NP or LLC + P females. These results show that this sexually dimorphic non-reproductive behavior is significantly influenced by reproductive condition and gonadal hormone levels.     

Behaviour, chemosignals and endocrine functions in male mice infected with tick-borne encephalitis virus

Odour attractiveness, social behaviour and endocrine status of male mice (outbred ICR strain) were examined 6-7 days after inoculation with subclinical dose of tick-borne encephalitis virus (TBE). According to RT-PCR control of efficiency of infection, males injected with TBE were divided on the two subgroups: TBE+ (males with viral RNA) and TBE- (males without viral RNA). Susceptible males (TBE+ subgroup) showed the higher level of plasma testosterone in comparison with both control and nonsusceptible (TBE- subgroup) males. TBE+ males had also more odour attraction for oestrus females and more aggressiveness in social conflict. Higher sexual attractiveness and aggressiveness of the infected host benefit the pathogen's distribution in the host population.     

Sex differences in photoperiod control of antigen-specific primary and secondary humoral immunity in Siberian Hamsters

Photoperiod was hypothesized to mediate T cell-dependent B cell production of IgM and IgG. Antigens induced production of specific immunoglobulins; serum IgM but not IgG, was higher in males in long vs. short days (16 vs. 8 h light/day) and similarly among all groups of females. A second immunization with KLH robustly enhanced serum IgM, as well as IgG; increases were blunted in short- vs. long-day males but not in females. Thus, in male but not female hamsters, winter-like short days restrain aspects of primary and secondary humoral immune responses to xenoantigens. Actions on lymphocyte activities or clonal expansion are in considerations.     

The relationship between distress and the development of a primary immune response to a novel antigen

Forty-five medical students were recruited to examine the effects of distress on the development of an immune response to the novel antigen, keyhole limpet hemocyanin (KLH). The subjects' level of distress was manipulated by immunizing them either at the time of an important viva voce examination (n=22) or during examination-free term time (n=23). This manipulation increased variance amongst the subjects, but the emphasis in this research was on individual distress as a predictor of immune function. In the group as a whole, the likelihood of developing DTH skin responses to KLH was reduced in the more distressed subjects (r=-.45; p=.002), independently of a number of behavioral (e.g., sleep disturbance) and demographic (e.g., sex) variables. Proliferation of T cells against KLH in vitro and the development of anti-KLH IgG antibodies were not related to levels of distress. Thus, cellular, rather than humoral, immune responses in vivo appear susceptible to the influence of distress. This immunization model provides the opportunity to further dissect the basis of these stress-immune pathways.     

Cardiovascular exercise intervention improves the primary antibody response to keyhole limpet hemocyanin (KLH) in previously sedentary older adults

Based upon a prior cross-sectional study, we hypothesized that an aerobic exercise intervention in sedentary older adults would improve a primary T cell-dependent immune response. Participants were a subset of older subjects from a large, ongoing exercise intervention study who were randomly assigned to either an aerobic exercise (Cardio, n = 30, 68.9 + 0.8 years) or flexibility/balance (Flex, n = 20, 69.9 + 1.2 years) intervention. The intervention consisted of either three aerobic sessions for 30–60 min at 55–70% VO_2max or two 60 min flexibility/balance sessions weekly for 10 months. Eight months into the intervention, samples were collected before intramuscular administration of KLH (125 μg), followed by sampling at 2, 3, and 6 weeks post-KLH. Serum anti-KLH IgM, IgG1, and IgG2 was measured by ELISA. Physiological and psychosocial measures were also assessed pre- and post-intervention. While there was no difference in the anti-KLH IgG2 response between groups, Cardio displayed significantly (p < 0.05) higher anti-KLH IgG1 (at weeks 2, 3, and 6 post) and IgM responses when compared to Flex. Despite cardiovascular intervention-induced improvement in physical fitness (11% vs. 1% change in VO_2peak in Cardio vs. Flex, respectively), we found no relationship between improved fitness and enhanced anti-KLH antibody responses. Optimism, perceived stress, and affect were all associated with enhanced immune response. We have shown for the first time that cardiovascular training in previously sedentary elderly results in significantly higher primary IgG1 and IgM antibody responses, while having no effect on IgG2 production.     

Differential immune responses in mice with left- and right-turning preference.

Humoral and cell-mediated immune responses of inbred BALB/c male mice were assayed for differential reactivities associated with behavioral sidedness, which was evaluated by spontaneous rotational behavior in a circular cage model system. Mice with left-turning preference had lower in vivo primary IgM and IgG anti-Keyhole Limpet Hemocyanin (KLH) antibody responses, delayed-type hypersensitivity (DTH) responses, and host-resistance against the intracellular bacteria, Listeria monocytogenes, than mice with right-turning preference. The only immune parameter not shown to be associated with turning preference was the secondary humoral immune response to KLH. The weak innate immune response of left-turners for clearance of Listeria showed close intercorrelation with elevated serum IL-6 levels. Serum corticosterone and splenic norepinephrine levels were differentially increased and decreased by infection, respectively. We suggest that the observed differential immune reactivities of individual animals with same age, gender, and genetic background are associated with functional asymmetries within the brain, that the hypothalamic-pituitary-adrenal (HPA) axis and sympathetic innervation are involved in the regulatory brain: immune interconnection after infection, and that the HPA axis and sympathetic nervous system are involved in the brain laterality effects on immune responses.     

Estrogen receptors alpha and beta mediate different aspects of the facilitatory effects of female cues on male risk taking

Male risk taking and decision making are affected by sex-related cues, with men making poorer and riskier decisions in the presence of females and, or their cues. In non-human species, female cues can also increase male risk taking, reducing their responses to predator threat. As estrogen receptors alpha and beta (ERalpha and ERbeta) are involved in the mediation of social and sexual responses, we investigated their roles in determining the effects of female-associated cues on male risk taking. We examined the effects of brief pre-exposure to the odors of either a novel or familiar estrous female on the avoidance of, and aversive responses to, predator threat (cat odor) in ERalpha and ERbeta wild type (alphaERWT, betaERWT) and gene-deleted (knockout, alphaERKO, betaERKO) male mice. Exposure of alphaERWT and betaERWT males to the odors of a novel, but not a familiar, estrous female mouse resulted in enhanced risk taking with the males displaying reduced avoidance of, and analgesic responses to, cat odor. In contrast, alphaERKO male mice failed to show any changes in risk taking, while betaERKO males, although displaying greater risk taking, did not distinguish between novel and familiar females, displaying similarly reduced avoidance responses to cat odor after exposure to either a novel or familiar female odor. These findings indicate that the gene for ERalpha is associated with the sexual mechanisms (response to estrous female) and the genes for ERbeta and ERalpha with the social (recognition of novel female) mechanisms underlying the effects of female cues on male risk taking.     

Scent marking behavior in male C57BL/6J mice: sexual and developmental determination

The present study investigated urinary scent marking behavior in male C57BL/6J (C57) mice as olfactory social signaling. In Experiment 1, when compared scent marking toward adult males, C57 males showed substantial scent marking toward CD-1 males and even toward the odor alone of CD-1 males, but not toward C57 males. Experiment 2 explored scent marking in C57 males of different ages to males and females, and juveniles and adults of the same strain. C57 males deposited more marks than control conditions only toward an adult C57 female when tested at 100 days of age, but not at 60 days of age. Development of urine marking behavior was investigated in C57 males at the ages of 30, 60, 90, and 120 days in Experiment 3. When tested alone (control) or confronted with a C57 male, C57 males showed diminished scent marks throughout development. Compared to controls, marking toward a CD-1 male increased after the age of 60 days, while marks toward an adult female showed significant increases after the age of 90 days. This difference in scent marking depending on the sex of the stimulus animal is likely to be associated with development of sexual behavior, in which males need to set up territories against other males prior to advertising to females. Although highly inbred strains have similar odor components, C57 males are able to detect and deposit urine marks after puberty as social communication depending on age, sex, and genetic differences in the opponents.     

Suppression of specific antibody production by inescapable shock: stability under varying conditions

The effect of exposure to uncontrollable shock on the production of antibodies to a novel antigen, keyhole limpet hemocyanin (KLH), was studied in adult male Sprague-Dawley rats. Groups of rats were tested under one of four experimental conditions which included testing during either the light or dark portions of their light cycles and following either one or three daily exposures to tail shock. Control subjects were immunized with KLH in the absence of shock exposure during either the light or dark phases of their light cycle. A tertiary (memory) response was evoked 60 days following the initial immunization sequence in all animals in the absence of a shock exposure. Blood samples were obtained from the tip of the tail at the time of each immunization and at 1-week intervals for 3 weeks following immunizations. Specific IgG antibodies to KLH were determined by an enzyme-linked immunosorbent assay (ELISA). All animals exposed to shock showed reduced levels of IgG antibodies to KLH regardless of the experimental conditions of shock exposure. Antibody levels were highest among animals immunized during the dark phase of their cycle for both control and shocked animals. Antibody production to a novel antigen appears to be a robust and sensitive measure for studies of modulation of immunity by behavioral factors.     

The effects of stress on the development of immunological memory following low-dose antigen priming in mice

Observable stress effects on immune responses may be a function of the quantitative and qualitative characteristics of the stressor, and the outcome measurement of immunity. Further, the effects of stress on humoral immunity, in particular, may be sensitive to the concentrations of antigen used to elicit a response. We have studied the effects of footshock stress during the time of priming with low concentrations of antigen on the secondary response to another low dose of antigen. The secondary humoral immune response of C3H/HeJ mice to the protein antigen keyhole limpet hemocyanin was examined following footshock, exposure to the apparatus without shock, or exposure to the home cage. Footshock reproducibly depressed the IgG anti-KLH response, and the effect on the IgM response was sporadic. Initially, footshock was administered for 7 days before and 7 days after priming with low amounts of antigen. Subsequent studies demonstrated that a single footshock session delivered 24 h after priming could suppress the IgG anti-KLH response.     

The effects of exposure to dietary nickel and zinc upon humoral and cellular immunity in SJL mice

We are interested in potential interactions between environmental trace metal exposures and immune function. In particular, we have wondered whether dietary exposure to nickel and zinc cations can influence T and B cell proliferation and function. To study this question, we fed SJL female mice supplemental nickel and zinc sulfate from 4-8 weeks of age, and immunized the animals intraperitoneally (i.p.) with keyhole limpet hemocyanin (KLH) at 8 weeks. Eight days later, we measured antibody responses to KLH. Both IgG and IgM antibody responses to KLH were significantly depressed in vivo in the nickel fed animals (p less than 0.005). In vitro antigenic responsiveness to KLH of splenocytes from nickel fed animals was also depressed compared with control and zinc supplemented animals (p less than 0.002). This altered antigenic responsiveness persisted even after cells had been cultured for 5 days in standard media. The zinc supplemented diets did not seem to affect antibody responsiveness and proliferation. The proliferative responses of B cells to the mitogen lipopolysaccharide (LPS) were significantly depressed in Ni fed mice, but were not affected in the zinc fed animals. T cell mitogenic responses to concanavalin A were not affected in the nickel fed animals, and were enhanced in zinc fed animals. We conclude that dietary exposure to certain trace metals may induce persisting alterations in immunity in this animal model.     

Sex and repeated restraint stress interact to affect cat odor-induced defensive behavior in adult rats

The overall objective of the present experiment was to assess sex differences in the effects of repeated restraint stress on fear-induced defensive behavior and general emotional behavior. Groups of male and female Long-Evans rats received either daily restraint stress (stressed) or daily brief handling (nonstressed) for 21 consecutive days. On days 22-25, a number of behavioral tests were administered concluding with a test of defensive behavior in response to a predatory odor. Stressed and nonstressed males and females were exposed to a piece of cat collar previously worn by a female domestic cat (cat odor) or a piece of collar never worn by a cat (control odor) in a familiar open field containing a hide barrier. Rats displayed pronounced defensive behavior (increased hiding and risk assessment) and decreased nondefensive behavior (grooming, rearing) in response to the cat odor. Nonstressed females exposed to cat odor displayed less risk assessment behavior relative to nonstressed males exposed to cat odor. Restraint stress had little effect on defensive behavior in male rats but significantly increased risk assessment behaviors in females. Behavior on the Porsolt forced swim test (a measure of depression-like behavior) and the open field test (a measure of anxiety-like behavior) was not affected by stress or sex. These findings indicate the utility of the predator odor paradigm in detecting subtle shifts in naturally occurring anxiety-like behaviors that may occur differentially in males and females.     

Antibody response and allogeneic mixed lymphocyte reaction in mu-, delta-, and kappa-opioid receptor knockout mice

The implication of opioid receptors in immune response has been studied using mu-, delta- and kappa-opioid receptor knockout mice. The mutant animals were compared to their wild-type (WT) counterparts for antibody (Ab) response to the prototype Ag keyhole limpet hemocyanin (KLH). Kappa-receptor deficient mice displayed higher Ab titers for either total Ig, IgM, IgG1 or IgG2a isotypes, whereas mu and delta animals behaved as wild-type mice. Therefore, endogenous kappa-receptor activation would tonically inhibit Ab response. Opioid receptor deficient mice were also used to investigate the immunosuppressive action of naltrindole, a delta-opioid receptor antagonist, shown earlier to inhibit graft rejection and the allogeneic mixed lymphocyte reaction (MLR) in vitro. Naltrindole and two related compounds inhibited MLR performed with lymphocytes from wild-type and delta-opioid receptor knockout mice. These compounds also suppressed MLR assayed with cells from triple mu/delta/kappa-opioid receptor mutants. We therefore demonstrate that naltrindole immunosuppressive activity is not mediated by any of the three mu-, delta- or kappa-opioid receptors, but by a target which remains to be discovered.     

Alterations in male sexual behaviour, attractiveness and testosterone levels induced by an adult-onset calorie restriction regimen

Despite an abundance of research on calorie restriction (CR) altering gonadal and appetite regulating hormones, the sexual behavioural consequences of CR remain to be examined systematically. This study compared the sexual behaviour, partner preference, serum testosterone and leptin levels of male adult Hooded Wistar rats administered a CR (continuous 25%, 50% CR or a temporary restriction) with ad libitum fed controls. The temporary restriction (Previous CR) failed to alter sexual behaviour, partner preference and levels of testosterone and leptin. The moderately 25% CR males did not demonstrate an impairment in sexual behaviour but did demonstrate a reduced level of attractiveness to females in one measure of partner preference. Sexual performance was affected by a substantial CR, as the CR 50% group exhibited a longer latency to the first intromission, indicating alteration in sexual arousal. Females also consistently demonstrated a clear preference for the control group compared to the CR 50% group. These findings indicate a possible reduction in the overall reproductive potential of the substantially CR animals. Testosterone levels were equally suppressed by both the 25% and 50% CR, while leptin levels were only reduced in the CR 50% group. Leptin, rather than testosterone, may have influenced the impairment in sexual behaviour only demonstrated by the substantially CR animals. Testosterone, may, however, play a role in modulating the preference of control over CR males, as attractiveness was totally reduced by a substantial CR, and partially reduced by a moderate restricted regimen.     

Corticosterone rapidly reduces male odor preferences in female mice

There is accumulating evidence for rapid, non-genomic behavioral effects of various steroids including that of the glucocorticoid, corticosterone. Using an odor preference test, the responses of which are indicative of mate preferences and sexual interest, we examined the effects of acute corticosterone on the responses of oestrous female mice to male odors. Control female mice displayed an overwhelming preference for the odors of male mice. Peripheral administration of corticosterone elicited a significant dose-related (1.0-5.0 mg/kg) decrease in female preference for male odors at 10 min, but not at 60 min, after administration. These inhibitory effects of corticosterone on odor preferences were significantly reduced by the competitive NMDA antagonist, NPC 12626, and enhanced by the GABA antagonist bicuculline. This indicates that corticosterone has rapid inhibitory effects on olfactory mediated female mate preferences and responses to male odor that in part involve interactions with NMDA and GABA receptor mechanisms.     

Enhanced humoral response in kappa-opioid receptor knockout mice

Opiates are major analgesics and addictive drugs described also as immunomodulators. Here, we investigated the contribution of kappa-opioid receptor (KOR) activity in immunity in vivo by studying immune responses in KOR knockout mice. These animals displayed a modest reduction in thymus cellularity and CD4(+) cell ratio, parallel to a slight increase in immature CD4(+)CD8(+) lymphocytes. In spleen, KOR null animals showed augmented cell number with no change in cell distribution. T and B lymphocyte proliferative capabilities in vitro, Natural Killer activity and steady-state Ig levels were unchanged in KOR-/- mice. We immunized the mice with the antigen keyhole limpet hemocyanin (KLH). Compared to wild-type (WT) mice, KOR-/- animals produced significant higher levels of antigen-specific total Ig, IgM, IgG1 and IgG2a antibodies. This enhancement of humoral activity was not observed in mu-opioid receptor and delta-opioid receptor knockout animals. These results show that endogenous activation of kappa-opioid receptors may exert a tonic inhibition of antibody (Ab) response.