Abnormal angiogenesis plays a key role in diseases of aging such as heart disease, certain cancers, and eye diseases including age-related macular degeneration. Macrophages have been shown previously to be both anti-and proangiogenic, and their role in regulating angiogenesis at sites of tissue injury is critical and complex. In this study, we analyzed cytokine gene expression patterns of mouse macrophages by realtime quantitative PCR and tested the functional effects of senescence on gene expression and macrophage polarization. Following laser injury to the retina, IL-10 was upregulated and Fas ligand (FasL), IL-12, and TNF-α were downregulated in ocular macrophages of old mice (>18 months of age). Downregulation of FasL on macrophages led to a loss of the antiangiogenic phenotype, as evidenced by the inability of these macrophages to inhibit vascular endothelial cells. Our results demonstrate that senescence, FasL, and IL-10 are key determinants of macrophage function that alter the growth of abnormal postdevelopmental blood vessels in disease processes including blinding eye disease.

Autophagy is an important component of host innate and adaptive immunity to viruses. It is critical for the degradation of intracellular pathogens and for promoting antigen presentation. Herpes simplex virus type 1 (HSV-1) infection induces an autophagy response, but this response is antagonized by the HSV-1 neurovirulence gene product, ICP34.5. This is due, in part, to its interaction with the essential autophagy protein Beclin 1 (Atg6) via the Beclin-binding domain (BBD) of ICP34.5. Using a recombinant virus lacking the BBD, we examined pathogenesis and immune responses using mouse models of infection. The BBD-deficient virus (⌬68H) replicated equivalently to its marker-rescued counterpart (⌬68HR) at early times but was cleared more rapidly than ⌬68HR from all tissues at late times following corneal infection. In addition, the infection of the cornea with ⌬68H induced less ocular disease than ⌬68HR. These results suggested that ⌬68H was attenuated due to its failure to control adaptive rather than innate immunity. In support of this idea, ⌬68H stimulated a significantly stronger CD4 ؉ T-cell-mediated delayed-type hypersensitivity response and resulted in significantly more production of gamma interferon and interleukin-2 from HSV-specific CD4 ؉ T cells than ⌬68HR. Taken together, these data suggest a role for the BBD of ICP34.5 in precluding autophagy-mediated class II antigen presentation, thereby enhancing the virulence and pathogenesis of HSV-1.Autophagy is a conserved cellular pathway that eliminates defective proteins and organelles, prevents abnormal protein aggregate accumulation, and removes intracellular pathogens (11,22,32,56). This process begins with the formation and elongation of a double membrane that fuses to form an autophagosome. The cytoplasmic contents are nonspecifically sequestered inside the autophagosome and then are degraded once the autophagosome fuses with the lysosome. Autophagy is upregulated during starvation, growth factor withdrawal, hypoxia, and infection (10). Following metabolic stress, autophagy can generate metabolic precursors that can be recycled for the de novo synthesis of proteins. The autophagic pathway has important roles in development, immune defense, apoptosis, tumor suppression, and the prevention of neuronal degeneration (reviewed in references 21 and 31).Autophagy is not limited to the degradation of self proteins; it also can engulf and break down invading microorganisms in a process termed xenophagy (26). Xenophagy can limit the replication of pathogens (3, 4, 29, 34, 42), but some infectious agents can exploit autophagy to enhance their replication (2, 18, 38). There are pathogens that actively inhibit autophagy through interaction with the essential autophagy-promoting protein, Beclin 1 (24, 35). Beclin 1 is the mammalian homolog of yeast Atg6 and is required for the formation of the autophagosome membrane through its interaction with VPS34, a class III phosphatidylinositol 3-kinase (19,27). Autophagy/ xenophagy is also an important process for the adaptive...

IFN-γ plays a critical role during the immune response to infection with Listeria monocytogenes. Early in the innate response NK cells are thought to be a primary source of IFN-γ; however, protection can be mediated by the presence of significant numbers of primed IFN-γ-secreting CD8+ T cells. In this report, we examined the early response to Listeria and found that 18 h after infection spleens contain CD11b+, Gr-1high, or Ly6G+ cells that produce significant IFN-γ. Morphological analysis of sorted Gr-1highIFN-γ+ and Gr-1lowIFN-γ+ or Ly6G+IFN-γ+ cells confirmed that these cells were neutrophils. The importance of IFN-γ production by these cells was further tested using adoptive transfer studies. Transfer of purified neutrophils from Ifng+/+ mice led to increased bacterial clearance in Ifng−/− mice. Transfer of Ifng−/− neutrophils provided no such protection. We conclude that neutrophils are an early source of IFN-γ during Listeria infection and are important in providing immune protection.

CD154 (CD40 ligand) expression on CD4 T cells is normally tightly controlled, but abnormal or dysregulated expression of CD154 has been well documented in autoimmune diseases, such as systemic lupus erythematosus. Beyond regulation by NFAT proteins, little is known about the transcriptional activation of the CD154 promoter. We identified a species-conserved purine-rich sequence located adjacent to the CD154 transcriptional promoter proximal NFAT site, which binds early growth response (Egr) transcription factors. Gel shift assays and chromatin immunoprecipitation assays reveal that Egr-1, Egr-3, and NFAT1 present in primary human CD4 T cells are capable of binding this combinatorial site in vitro and in vivo, respectively. Multimerization of this NFAT/Egr sequence in the context of a reporter gene demonstrates this sequence is transcriptionally active upon T cell activation in primary human CD4 T cells. Overexpression of Egr-1, but not Egr-3, is capable of augmenting transcription of this reporter gene as well as that of an intact CD154 promoter. Conversely, overexpression of small interfering RNA specific for Egr-1 in primary human CD4 T cells inhibits CD154 expression. Similarly, upon activation, CD154 message is notably decreased in splenic CD4 T cells from Egr-1-deficient mice compared with wild-type controls. Our data demonstrate that Egr-1 is required for CD154 transcription in primary CD4 T cells. This has implications for selective targeting of Egr family members to control abnormal expression of CD154 in autoimmune diseases such as systemic lupus erythematosus.

PURPOSE. Current studies suggest that the immune system plays a critical role in blinding eye disorders. The eye is an immune-privileged site, and FasL expression is a major part of that mechanism because Fas/FasL interactions regulate inflammation and neovascularization, preventing damage to delicate ocular structures. These studies were undertaken to test the idea that modulating immune privilege might be an effective therapeutic approach to pathogenic angiogenesis in the eye. METHODS. C57BL/6 mice or FasL-defective B6-gld mice were laser treated to induce choroidal neovascularization (CNV). Mice were injected with cytotoxic FasL in the vitreous cavity or were treated with oral doxycycline in the drinking water. They were evaluated for CNV 7 days later. In some experiments eye tissue was harvested and evaluated for FasL expression, macrophage influx by immunohistochemistry, and release of sFasL. RESULTS. Injection of cytotoxic FasL successfully prevented neovascularization in a mouse model of CNV. Oral doxycycline increased functional FasL in the eye and substantially inhibited neovascularization. Doxycycline treatment increased FasL expression on the RPE cells and reduced circulating and tissue-associated sFasL. Treatment was ineffective in B6-gld mice, demonstrating that CNV inhibition was mediated by FasL. CONCLUSIONS. Targeting immune privilege using cytotoxic molecules or by increasing expression of the proapoptotic protein FasL may be a viable approach to treating neovascular eye disease.