Evidence for the involvement of interleukin-1α during development of experimental cytomegalovirus retinitis in immunosuppressed mice

Abstract

Interleukin-1α (IL-1α) is an alarmin involved in the recruitment of macrophages and neutrophils during tissue inflammation. IL-1α can undergo cleavage by proteases, such as calpain-1, that enhances IL-1α binding to its receptor, although proteolytic cleavage is not necessary for biological activity. Macrophages and neutrophils are involved in the retinal inflammation associated with development of AIDS-related human cytomegalovirus (HCMV) retinitis. We therefore performed studies to test the hypothesis that IL-1α gene expression is stimulated intraocularly during retinitis development using two mouse models of murine cytomegalovirus (MCMV) retinitis that differ in method of immunosuppression, one by retrovirus-induced immunosuppression (MAIDS) and the other by corticosteroid-induced immunosuppression. MCMV-infected eyes of groups of retinitis-susceptible mice with MAIDS of 10 weeks duration (MAIDS-10 mice) and retinitis-susceptible corticosteroid-treated mice showed significant stimulation of IL-1α mRNA. Western blot analysis confirmed IL-1α protein production within the MCMV-infected eyes of MAIDS-10 mice. Whereas significant intraocular calpain-1 mRNA and protein production were also observed within MCMV-infected eyes of MAIDS-10 mice, the MCMV-infected eyes of retinitis-susceptible corticosteroid-treated mice showed a pattern of mRNA synthesis equivalent to that found within the MCMV-infected eyes of healthy mice that fail to develop retinitis. Our findings suggest a role for the alarmin IL-1α in the pathogenesis of MCMV retinitis in immunosuppressed mice. These findings may extend to the pathogenesis of HCMV retinitis in patients with AIDS or other forms of immunosuppression.

Keywords: AIDS; Calpain-1; Corticosteroid; Cytomegalovirus; Interleukin-1α; Retinitis.

Figure 1.. Intraocular IL-1α gene expression in retrovirus-immunosuppressed (MAIDS) mice versus cyclosporin-immunosuppressed mice.

Groups of MAIDS-10 mice, MAIDS-4 mice, healthy mice, and corticosteroid-treated mice were intraocularly (subretinally) injected with 10⁴ PFU of MCMV (left eyes) or DMEM (right eyes) as described in Materials and Methods. Whole eyes were harvested from MAIDS-10 mice at indicated times following intraocular MCMV injection and assessed by real-time RT-PCR assay using the comparative 2^−ΔΔCt method for quantification of IL-1α mRNA expression (A) or assessed by western blot analysis for detection of IL-1α protein production (B). Quantification of protein band intensities was done using the ImageJ software and normalizing the intensity of the bands relative to GAPDH levels. Real-time RT-PCR assay was also used for quantification of IL-1α mRNA expression within MCMV-infected eyes of MAIDS-4 mice (C), IL-1α mRNA expression within MCMV-infected eyes of healthy mice (D), and IL-1α mRNA expression within MCMV-infected eyes of corticosteroid-immunosuppressied mice (E). Means ±SD of n = 5 – 8 mice per group are shown. * p<0.05 and ** p<0.01 for MCMV-infected eyes compared with media-injected controls.

Figure 2.. Intraocular calpain-1 gene expression in retrovirus-immunosuppressed (MAIDS) mice versus cyclosporin-immunosuppressed mice.

Groups of MAIDS-10 mice, MAIDS-4 mice, healthy mice, and corticosteroid-treated mice were intraocularly (subretinally) injected with 10⁴ PFU of MCMV (left eyes) or DMEM (right eyes) as described in Materials and Methods. Whole eyes were harvested from MAIDS-10 mice at indicated times following intraocular MCMV injection and assessed by real-time RT-PCR assay using the comparative 2^−ΔΔCt method for quantification of calpain-1 mRNA expression (A) or assessed by western blot analysis for detection of calpain-1 protein production (B). Quantification of protein band intensities was done using the ImageJ software and normalizing the intensity of the bands relative to GAPDH levels. Real-time RT-PCR assay was also used for quantification of calpain-1 mRNA expression within MCMV-infected eyes of MAIDS-4 mice (C), calpain-1 mRNA expression within MCMV-infected eyes of healthy mice (D), and calpain-1 mRNA expression within MCMV-infected eyes of corticosteroid-immunosuppressied mice (E). Means ±SD of n = 5 – 8 mice per group are shown. * p<0.05 and ** p<0.01 for MCMV-infected eyes compared with media-injected controls.