Gender and estrogen manipulation do not affect traumatic brain injury in mice.

As epidemiological data have suggested that female patients may have improved clinical prognoses following traumatic brain injury (TBI) compared to males, we designed experiments to determine the role of gender and estrogen in TBI-induced brain injury and inflammation in rodents. To this end, male and female C57Bl/6 mice were separated into the following four groups: intact males, intact females with vehicle supplementation, ovariectomized females with vehicle supplementation, and ovariectomized females with estrogen supplementation. All mice were subjected to a controlled cortical impact model of TBI, and cortical injury, hippocampal degeneration, microglial activation, and brain cytokine expression were analyzed after injury. Additionally, the spleens were harvested and cytokine release from cultured splenic cells was measured in response to specific stimuli. Data indicate that TBI-induced cortical and hippocampal injury, as well as injury-related microglial activation were not significantly affected by gender or estrogen manipulation. Conversely, brain levels of MCP-1 and IL-6 were significantly increased in males and intact females following TBI, but not in female mice that had been ovariectomized and supplemented with either estrogen or vehicle. Evaluation of splenic responses showed that the spleen was only moderately affected by TBI, and furthermore that spleens isolated from mice that had been given estrogen supplementation showed significantly higher release of the anti-inflammatory cytokine IL-4, regardless of the presence of absence of TBI. Overall, these data indicate that while estrogen can modulate immune responses, and indeed can predispose splenic responses towards and anti-inflammatory phenotype, these effects do not translate to decreased brain injury or inflammation following TBI in mice.

Estrogen and brain inflammation: effects on microglial expression of MHC, costimulatory molecules and cytokines.

To model the effects of estrogen on adaptive immunity in the brain, we examined the effects of 17beta-estradiol on microglial parameters related to antigen presentation and T cell activation. Specifically, the effects of 17beta-estradiol on basal and LPS-induced surface staining of Class I and II MHC, as well as CD40, CD80, CD86, CD152, CD28, CD8, CD11b, Fas, FasL, and also ERalpha and ERbeta, were examined in N9 microglial cells. Additionally, the effects of 17beta-estradiol on basal and LPS-induced release of cytokines (TNF-alpha, IFN-gamma, IL-2, IL-4, and IL-10) were determined. Data indicate that estrogen increases IL-10 while decreasing TNFalpha and IFNgamma release from resting and LPS-stimulated N9 cells. Additionally, LPS-induced surface staining of MHC Class I, CD40, and CD86 was significantly attenuated by estrogen pretreatment. The basal percentage of cells positive for MHC Class I and II, CD40, and CD152, Fas, and FasL was significantly decreased by estrogen exposure. However, CD8, CD86, CD11b, and CD28 were unaffected by estrogen, and CD80 cell surface staining significantly increased following estrogen exposure. Taken together, these data indicate that estrogen can significantly decrease components of adaptive immunity in microglial cells, and highlight the multi-faceted regulatory effects of estrogen on microglial parameters related to antigen presentation and T cell interaction.

Estrogen increases proteasome activity in murine microglial cells.

During inflammation, microglial cells go through phenotypic and functional changes that include the production and release of large amounts of oxygen and nitrogen radicals. As such, activated microglia are subject to heightened oxidative stress. The multicatalytic proteasome clears oxidized and damaged proteins from cells, and has been shown to be an important aspect of the microglial compensatory response to activation. The female sex steroid estrogen is both cytoprotective and anti-inflammatory, and has been shown to affect microglial signaling in particular. To determine if estrogen might affect the proteasome in microglial cells, we examined the effects of 17 beta-estradiol treatment on proteasome activity in N9 microglial cells. Specifically, we measured ATP-dependent and ATP-independent chymotrypsin-like, trypsin-like, and peptidyl glutamyl peptide hydrolase (PGPH)-like activities in response to both 17 beta-estradiol and interferon gamma. Data indicate that estrogen, but not interferon gamma, significantly increases ATP-dependent chymotrypsin-like and PGPH-like activity. Furthermore, this effect was blocked by the p44/42 MAPK inhibitor PD98059. Hence, these data demonstrate that through the MAPK pathway, estrogen can upregulate proteasome activity, suggesting a possible mechanism for estrogen's cytoprotective effects.

HIV-1 Tat and opiate-induced changes in astrocytes promote chemotaxis of microglia through the expression of MCP-1 and alternative chemokines.

Opiates exacerbate human immunodeficiency virus type 1 (HIV-1) Tat(1-72)-induced release of key proinflammatory cytokines by astrocytes, which may accelerate HIV neuropathogenesis in opiate abusers. The release of monocyte chemoattractant protein-1 (MCP-1, also known as CCL2), in particular, is potentiated by opiate-HIV Tat interactions in vitro. Although MCP-1 draws monocytes/macrophages to sites of CNS infection, and activated monocytes/microglia release factors that can damage bystander neurons, the role of MCP-1 in neuro-acquired immunodeficiency syndrome (neuroAIDS) progression in opiate abusers, or nonabusers, is uncertain. Using a chemotaxis assay, N9 microglial cell migration was found to be significantly greater in conditioned medium from mouse striatal astrocytes exposed to morphine and/or Tat(1-72) than in vehicle-, mu-opioid receptor (MOR) antagonist-, or inactive, mutant Tat(delta31-61)-treated controls. Conditioned medium from astrocytes treated with morphine and Tat caused the greatest increase in motility. The response was attenuated using conditioned medium immunoneutralized with MCP-1 antibodies, or medium from MCP-1(-/-) astrocytes. In the presence of morphine (time-release, subcutaneous implant), intrastriatal Tat increased the proportion of neural cells that were astroglia and F4/80+ macrophages at 7 days post-injection. This was not seen after treatment with Tat alone, or with morphine plus inactive Tat(delta31-61) or naltrexone. Glia displayed increased MOR and MCP-1 immunoreactivity after morphine and/or Tat exposure. The findings indicate that MCP-1 underlies most of the response of microglia, suggesting that one way in which opiates exacerbate neuroAIDS is by increasing astroglial-derived proinflammatory chemokines at focal sites of CNS infection and promoting macrophage entry and local microglial activation. Importantly, increased glial expression of MOR can trigger an opiate-driven amplification/positive feedback of MCP-1 production and inflammation.

Immune modulation by estrogens: role in CNS HIV-1 infection.

Experimental and epidemiological data suggest that estrogen can be protective in both brain injury and infection. While estrogens can act directly on neurons to promote neuronal survival, estrogen also has antiinflammatory properties that may contribute to overall neuroprotection. Accordingly, estrogens may have particular relevance in chronic neuroimmune disorders such as HIV dementia. As AIDS is now a leading cause of death among women in their reproductive years, understanding the role that female sex hormones might play in the physiology of HIV-1 infection is especially critical. Indeed, there is accumulating evidence that many manifestations of HIV differ in women. For instance, it is now well established that women present with a lower viral titer at the time of seroconversion, have lower HIV viral loads compared to men at similar stages of disease, and may have altered disease progression during pregnancy. Conversely, while epidemiological studies suggest that women may be more vulnerable to certain late-stage AIDS-related illnesses including HIV dementia, there is accumulating data that strongly suggest an estrogen-deficient state is associated with long-term HIV infection in some women. Evaluated as a whole, existing evidence indicates that estrogen can directly protect neurons from damage, can modulate brain inflammation, and could act to maintain low titers of the HIV-1 virus. Accordingly, it can be hypothesized that maintenance of serum estradiol levels could decrease the incidence of HIV dementia and other AIDS-related neurological syndromes in HIV-1 positive women. In this article, we both summarize current understanding and present new data related to the potential mechanisms whereby estrogen could modulate the mechanics and the consequences of HIV-1 infection in the brain and thereby thwart the development of HIV dementia.