Abstract
Purpose. Studies were performed to determine whether retinal Muller cells transcribe genes for the proinflammatory cytokines interleukin-6 (IL- 6) and tumor necrosis factor-α (TNFα). Isolated murine retinas were used to test whether these cytokines were upregulated in the retina in vivo after anterior chamber inoculation of herpes simplex virus type 1 (HSV-1). The effects of exposure to HSV-1 or interferon-γ (IFNγ) on transcript levels of these cytokines in cultured retinal glia also were examined. Methods. In situ hybridization (ISH) using digoxigenin (DIG)-labeled RNA probes was used to localize mRNA for IL-6 and TNFα in cultured retinal glial cells. Changes in IL-6 and TNFα relative transcript levels were assessed in cultured retinal glial cells using a semiquantitative approach comprised of reverse transcription-polymerase chain reaction (RT-PCR) assay at low amplification cycle number followed by slot blotting and hybridization with DIG-labeled internal sequence probes. In the murine model of herpetic retinitis, the same methods were used to compare temporal changes in relative cytokine transcript levels in retinas isolated from eyes 1 to 7 days after anterior chamber injection of live HSV-1 (KOS strain; 2 x 104 pfu/eye) or buffer with levels in retinas isolated from normal, uninjected eyes. Densitometry was used to quantify relative signal changes obtained with serial diluted samples in slot blot assays. Cytokine signal was normalized to hypoxanthine phosphoribosyl transferase signal obtained from the same cDNA samples. Results. Under baseline culture conditions, ISH and RT-PCR indicated that both IL-6 and TNFα were transcribed by cultured retinal glia. In vitro exposure to either viral (HSV-1) or inflammatory (IFNγ) stimulants increased levels of these transcripts in a time-dependent manner. Peak TNFα mRNA levels were detected 4 hours after exposure to HSV, whereas IL-6 peaked 4 hours later (increases of 10.3 and 8.7 times over baseline, respectively). Differential increases in TNFα and IL-6 transcript levels were detected in retinas isolated from BALB/c mice that received anterior chamber injections of either HSV-1 or Hanks' balanced salt solution (HBSS). By day 3 after HSV-1 injection, increases of 4.5-fold in TNFα and 17-fold in IL-6 were detected, whereas substantially smaller changes in TNFα and IL-6 (1.5-fold and 6.3-fold, respectively) were observed in HBSS-injected eyes. Virus-induced changes in TNFα mRNA levels occurred slightly earlier than for IL-6 because maximal levels of TNFα were detected 2 to 3 days after infection, but IL-6 peaked at day 3. Conclusions. Cultured retinal glial cells exhibit upregulated TNFα and IL-6 transcript levels after exposure to virus or inflammatory mediators. HSV-1 infection of the anterior segment of the mouse eye markedly upregulates TNFα and IL-6 mRNA levels compared to smaller responses to nonspecific inflammation. Taken together, these results identify retinal Muller cells as an intraretinal source of TNFα and IL-6 and support the potential of these resident cells to act as intraretinal modulators of immune and inflammatory responses.