The role of lipoprotein-associated phospholipase A2 in a murine model of experimental autoimmune uveoretinitis.

Macrophage activation is, in part, regulated via hydrolysis of oxidised low density lipoproteins by Lipoprotein-Associated phospholipase A2 (Lp-PLA2), resulting in increased macrophage migration, pro-inflammatory cytokine release and chemokine expression. In uveitis, tissue damage is mediated as a result of macrophage activation; hence inhibition of Lp-PLA2 may limit macrophage activation and protect the tissue. Utilising Lp-PLA2 gene-deficient (KO) mice and a pharmacological inhibitor of Lp-PLA2 (SB-435495) we aimed to determine the effect of Lp-PLA2 suppression in mediating retinal protection in a model of autoimmune retinal inflammation, experimental autoimmune uveoretinitis (EAU). Following immunisation with RBP-3 (IRBP) 1-20 or 161-180 peptides, clinical disease was monitored and severity assessed, infiltrating leukocytes were enumerated by flow cytometry and tissue destruction quantified by histology. Despite ablation of Lp-PLA2 enzyme activity in Lp-PLA2 KO mice or wild-type mice treated with SB-435495, the number of infiltrating CD45+ cells in the retina was equivalent to control EAU animals, and there was no reduction in disease severity. Thus, despite the reported beneficial effects of therapeutic Lp-PLA2 depletion in a variety of vascular inflammatory conditions, we were unable to attenuate disease, show delayed disease onset or prevent progression of EAU in Lp-PLA2 KO mice. Although EAU exhibits inflammatory vasculopathy there is no overt defect in lipid metabolism and given the lack of effect following Lp-PLA2 suppression, these data support the hypothesis that sub-acute autoimmune inflammatory disease progresses independently of Lp-PLA2 activity.

Tumour necrosis factor-mediated macrophage activation in the target organ is critical for clinical manifestation of uveitis.

Clinically available anti-tumour necrosis factor (TNF) biologics, which inhibit both soluble (sTNF) and transmembrane forms (tmTNF) of TNF, eliminating all TNF signalling, have successfully treated autoimmune diseases including uveitis. These have potentially serious side effects such as reactivation of latent Mycobacterium tuberculosis and, therefore, more specific inhibition of TNF signalling pathways may maintain clinical efficacy while reducing adverse effects. To determine the effects of specific pharmacological inhibition of sTNF on macrophage activation and migration, we used a mouse model of uveitis (experimental autoimmune uveoretinitis; EAU). We show that selective inhibition of sTNF is sufficient to suppress EAU by limiting inflammatory CD11b(+) macrophages and CD4(+) T cell migration into the eye. However, inhibition of both sTNF and tmTNF is required to inhibit interferon-γ-induced chemokine receptor 2, CD40, major histocompatibility complex class II and nitric oxide (NO) up-regulation, and signalling via tmTNF is sufficient to mediate tissue damage. In confirmation, intravitreal inhibition of sTNF alone did not suppress disease, and inflammatory cells that migrated into the eye were activated, generating NO, thus causing structural damage to the retina. In contrast, intravitreal inhibition of both sTNF and tmTNF suppressed macrophage activation and therefore disease. We conclude that sTNF is required for inflammatory cell infiltration into target tissue, but at the tissue site inhibition of both sTNF and tmTNF is required to inhibit macrophage activation and to protect from tissue damage.

Xenoestrogen action in breast cancer: impact on ER-dependent transcription and mitogenesis.

Several estrogen mimics (xenoestrogens) inappropriately activate the estrogen receptor (ER) in the absence of endogenous ligand. Given the importance of the ER in breast cancer growth and regulation, delineating the impact of these agents under conditions related to tumor treatment is of significant importance. We examined the effect of two prevalent xenoestrogens (bisphenol A and coumestrol) on ER activation and ER-dependent mitogenesis in breast cancer cells. We show that the ability of these agents to induce mitogenesis was restricted to conditions of estrogen depletion, and that these agents failed to cooperate with estradiol to induce MCF-7 breast cancer cell growth. These observations are consistent with the impact of each agent specifically on exogenous ER activation as monitored in HeLa cells, wherein the xenoestrogens activated the receptor in the absence of estradiol but failed to cooperate with estrogen. Tamoxifen blocked bisphenol A and coumestrol-mediated ER activation, indicating that exposure to these agents is unlikely to disrupt such therapeutic intervention. The response of tumor-derived ER alleles to these xenoestrogens was also examined. Although the xenoestrogens failed to alter ER-Y537S function, the ER-D351Y mutant demonstrated an enhanced response to bisphenol A. Moreover, tamoxifen enhanced the agonistic effects of xenoestrogens on ER-D351Y. Lastly, we examined the impact of ER co-activator overexpression on xenoestrogen response. Bisphenol A and coumestrol exhibited differential responses to co-activators with regard to ER activation. However, when using mitogenesis as an endpoint, these co-activators were insufficient to provide a significant growth advantage. Combined, these data demonstrate that bisphenol A and coumestrol can impact ER activity and ER-dependent proliferation in breast cancer cells, but the influence of these agents is restricted to conditions of estrogen depletion, selective mutation of the ER, and expression of specific co-activators.