Nikolay M. Filipov

Differential effects of age on circulating and splenic leukocyte populations in C57BL/6 and BALB/c male mice

BackgroundDespite several reports on age-related phenotypic changes of the immune systems cells, studies that use a multipoint age comparison between the specific and innate immune cell populations of prototypical Th1- and Th2-type polarized mouse strains are still lacking.ResultsUsing a multipoint age comparison approach, cells from the two major immune system compartments, peripheral blood and spleen, and flow cytometry analysis, we found several principal differences in T cell and professional antigen presenting cell (APC) populations originating from a prototypical T helper (Th) 1 mouse strain, C57BL/6, and a prototypical Th2 strain, BALB/c. For example, regardless of age, there were strain differences in both peripheral blood mononuclear cells (PBMC) and spleens in the proportion of CD4+ (higher in the BALB/c strain), CD8+ T cells and CD11b+/CD11c+ APC (greater in C57BL/6 mice). Other differences were present only in PBMC (MHC class II + and CD19+ were greater in C57BL/6 mice) or differences were evident in the spleens but not in circulation (CD3+ T cells were greater in C57BL/6 mice). There were populations of cells that increased with age in PBMC and spleens of both strains (MHC class II+), decreased in the periphery and spleens of both strains (CD11b+) or did not change in the PBMC and spleens of both strains (CD8+). We also found strain and age differences in the distribution of naïve and memory/activated splenic T cells, e.g., BALB/c mice had more memory/activated and less naive CD8+ and CD4+ T cells and the C57BL/6 mice.ConclusionOur data provide important information on the principal differences, within the context of age, in T cell and professional APC populations between the prototypical Th1 mouse strain C57BL/6 and the prototypical Th2 strain BALB/c. Although the age-related changes that occur may be rather subtle, they may be very relevant in conditions of disease and stress. Importantly, our data indicate that age and strain should be considered in concert in the selection of appropriate mouse models for immunological research.

Alteration of dopamine uptake into rat striatal vesicles and synaptosomes caused by an in vitro exposure to atrazine and some of its metabolites

Studies have shown that both in vivo and in vitro exposure to the herbicide atrazine (ATR) results in dopaminergic neurotoxicity manifested by decreased striatal dopamine (DA) levels. However, the mechanism behind this reduction is largely unknown. A decrease in striatal DA could be due to ATR exposure affecting vesicular and/or synaptosomal uptake resulting in disrupted vesicular storage and/or cellular uptake of DA. Hence, we investigated the effects of in vitro ATR exposure on DA uptake into isolated rat striatal synaptosomes and synaptic vesicles. In addition to ATR, effects of its major mammalian metabolites, didealkyl atrazine (DACT), desethyl atrazine (DE) and desiopropyl atrazine (DIP) were investigated. ATR (1-250 microM) inhibited DA uptake into synaptic vesicles in a dose-dependent manner. Of the three ATR metabolites tested, DACT did not affect vesicular DA uptake. DE and DIP, on the other hand, significantly decreased vesicular DA uptake with the effect of 100 microM DE/DIP being similar to the effect of the same concentration of ATR. Kinetic analysis of vesicular DA uptake indicated that ATR significantly decreased the V(max) while the K(m) value was not affected. Contrary to the inhibitory effects on vesicular DA uptake, synaptosomal DA uptake was marginally (6-13%) increased by ATR and DE, but not by DACT and DIP, at concentrations of <or=100 microM. As a result, ATR, DIP and DE increased the synaptosomal/vesicular (DAT/VMAT-2) uptake ratio. Collectively, results from this study suggest that ATR and two of its metabolites, DIP and DE, but not its major mammalian metabolite, DACT, decrease striatal DA levels, at least in part, by increasing cytosolic DA, which is prone to oxidative breakdown.

Direct effects of manganese compounds on dopamine and its metabolite Dopac: an in vitro study.

Following combustion of fuel containing the additive methylcyclopentadienyl-manganese-tricarbonyl (MMT), manganese phosphate (MnPO(4)) and manganese sulfate (MnSO(4)) are emitted in the atmosphere. Manganese chloride (MnCl(2)), another Mn(2+) species, is widely used experimentally. Using rat striatal slices, we found that MnPO(4) decreased tissue and media dopamine (DA) and media Dopac (a DA metabolite) levels substantially more than either MnCl(2) or MnSO(4); antioxidants were partially protective. Also, both MnCl(2) and MnPO(4) (more potently) oxidized DA and Dopac even in the absence of tissue in the media, suggesting a direct interaction between Mn and DA/Dopac. Because aminochrome is a major oxidation product of DA, we next determined whether MnPO(4) will be more potent in forming aminochrome than MnCl(2) or MnSO(4) which, indeed, was the case. Thus, a potential additional mechanism for the neurotoxic effects of environmentally-relevant forms of Mn, MnPO(4) in particular, is the generation of reactive DA intermediates.

Strain-specific sensitivity to MPTP of C57BL/6 and BALB/c mice is age dependent.

Administration of MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) to adult (2-month to 4-month-old) male C57BL/6 mice (MPTP-sensitive) is a valuable Parkinsons disease model. At comparable age, other strains, such as BALB/c, are minimally affected by MPTP (MPTP-resistant). However, the maintenance of resistance to MPTP throughout aging in MPTP-resistant strains has not been studied. Here, we show that, as previously reported, 1-month and 18-month-old C57BL/6 mice are least and most sensitive to MPTP, respectively. MPTP, as expected, did not affect the younger (1-month and 3-month-old) BALB/c mice, but it markedly decreased striatal dopamine in the older (10-month and 18-month-old) BALB/c mice. These data suggest that the sensitivity to MPTP is age dependent and that mice from an MPTP-resistant strain lose their resistance as they age.

#39 Peripheral LPS enhances dopaminergic toxicity of the fungicide maneb in a strain-dependent manner in mice

role in the development of IFN-a (cytokine)-induced behavioral change. To further explore the potential role of CRF in IFN-a-induced behavioral alterations, we have examined IFN-a-induced neuroendocrine, immune, and behavioral responses in rhesus monkeys. To first determine whether IFN-a activates relevant signaling pathways in rhesus monkeys, we evaluated the expression of phosphoSTAT1 (a major signaling pathway activated by IFN-a) in monkey peripheral blood mononuclear cells (PBMCs) treated with rHu-IFN-a (1000 IU/ml) in vitro using both flow cytometry and Western blot. IFN-a increased phospho-STAT-1 in monkey PBMCs at 15, 30, and 60 min, a pattern similar to that seen in humans. We then investigated the acute and chronic effects of IFN-a on neuroendocrine, immune, and behavioral responses in these animals. Consistent with the activation of upstream neuroendocrine secretagogues including CRF, acute administration of IFN-a (10 and 20 MIU/m2) to rhesus monkeys markedly increased ACTH and cortisol as well as IL-6 compared to saline in 8 young adult (6 male, 2 female) animals. We additionally treated 4 monkeys (2 male and 2 female), with IFN-a (20 MIU/m2) or saline for 4 weeks. Interestingly, the neuroendocrine and immune responses to chronic IFN-a exposure in monkeys appeared to depend on the social status of the animal. Whereas acute elevations in cortisol, ACTH, and IL-6 concentrations returned to saline levels by week 2 in dominant animals, the concentration of these hormones remained high throughout IFN-a treatment in subordinant animals. Behavioral changes included the induction of huddling (2 out 4 animals) and marked aggressive behavior in one of the dominant monkeys. Neither aggression nor huddling was present during saline treatment. Of note huddling has been previously described in monkeys treated with icv CRF. Taken together, these data provide evidence that rhesus monkeys exhibit similar immune, neuroendocrine, and behavioral responses to IFN-a as humans, and support the idea that activation of CRF pathways may be involved. Future studies using CRF antagonists will further elucidate the role of CRF in IFN-a induced neuroendocrine, immune, and behavioral changes, thus providing insight into novel approaches to the treatment of cytokine-induced behavioral syndromes.