HERPES-SIMPLEX VIRUS TYPE-1 ALTERS TRANSCRIPT LEVELS OF TUMOR-NECROSIS-FACTOR-ALPHA AND INTERLEUKIN-6 IN RETINAL GLIAL-CELLS

Purpose. Studies were performed to determine whether retinal Muller ce lls transcribe genes for the proinflammatory cytokines interleukin-6 ( IL-6) and tumor necrosis factor-alpha (TNF alpha). Isolated murine ret inas were used to test whether these cytokines were upregulated in the retina in vivo after anterior chamber inoculation of herpes simplex v irus type 1 (HSV-1). The effects of exposure to HSV-1 or interferon-ga mma (IFN gamma) on transcript levels of these cytokines in cultured re tinal glia also were examined. Methods. In situ hybridization (ISH) us ing digoxigenin (DIG)-labeled RNA probes was used to localize mRNA for IL-6 and TNF alpha in cultured retinal glial cells. Changes in IL-6 a nd TNF alpha relative transcript levels were assessed in cultured reti nal glial cells using a semiquantitative approach comprised of reverse transcription-polymerase chain reaction (RT-PCR) assay at low amplifi cation cycle number followed by slot blotting and hybridization with D IG-labeled internal sequence probes. In the murine model of herpetic r etinitis, the same methods were used to compare temporal changes in re lative cytokine transcript levels in retinas isolated from eyes 1 to 7 days after anterior chamber injection of live HSV-1 (KOS strain; 2 X 10(4) pfu/eye) or buffer with levels in retinas isolated from normal, uninjected eyes. Densitometry was used to quantify relative signal cha nges obtained with serial diluted samples in slot blot assays. Cytokin e signal was normalized to hypoxanthine phosphoribosyl transferase sig nal obtained from the same cDNA samples. Results. Under baseline cultu re conditions, ISH and RT-PCR indicated that both IL-6 and TNF alpha w ere transcribed by cultured retinal glia. In vitro exposure to either viral (HSV-1) or inflammatory (IFN gamma) stimulants increased levels of these transcripts in a time-dependent manner. Peak TNF alpha mRNA l evels 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, respect ively). Differential increases in TNF alpha and IL-6 transcript levels were detected in retinas isolated from BALB/c mice that received ante rior chamber injections of either HSV-I or Hanks' balanced salt soluti on (HBSS). By day 3 after HSV-1 injection, increases of 4.5-fold in TN F alpha and 17-fold in IL-6 were detected, whereas substantially small er changes in TNF alpha and IL-6 (1.5-fold and 6.3-fold, respectively) were observed in HBSS-injected eyes. Virus-induced changes in TNF alp ha mRNA levels occurred slightly earlier than for IL-6 because maximal levels of TNF alpha were detected 2 to 3 days after infection, but IL -6 peaked at day 3. Conclusions. Cultured retinal glial cells exhibit upregulated TNF alpha and IL-6 transcript levels after exposure to vir us or inflammatory mediators. HSV-1 infection of the anterior segment of the mouse eye markedly upregulates TNF alpha and IL-6 mRNA levels c ompared to smaller responses to nonspecific inflammation. Taken togeth er, these results identify retinal Muller cells as an intraretinal sou rce of TNF alpha and IL-6 and support the potential of these resident cells to act as intraretinal modulators of immune and inflammatory res ponses.