SCIENTIFIC CORRESPONDENCE Detection of cytokine mRNA production in infiltrating cells in proliferative vitreoretinopathy using reverse transcription polymerase chain reaction

Aims—To determine whether the infiltrating cells in the vitreous and subretinal fluid of patients with proliferative vitreoretinopathy (PVR) express messenger RNA for various cytokines found in this condition. Methods—The presence of mRNA coding for HPRT, IL-6, IL-1â, IL-8, and TNFá was investigated in 20 vitreous and subretinal fluid (SRF) samples from patients with PVR by reverse transcriptase polymerase chain reaction (RT-PCR). 16 samples from patients with retinal detachment and macular holes were used as controls. Results—HPRT was detected in all samples of PVR and in 11 (69%) control cases. Patients with PVR demonstrated mRNA for the cytokines tested more often than controls. The diVerence was statistically significant. Conclusion—The presence of mRNA encoding for IL-6, IL-1â, IL-8, and TNFá is significantly detected by RT-PCR in vitreous and SRF samples of patients with PVR, indicating local production of these cytokines by vitreous and SRF cells. (Br J Ophthalmol 1999;83:1296–1299) Proliferative vitreoretinopathy (PVR) is a process of cellular proliferation and contraction that complicates rhegmatogenous retinal detachment. PVR is the most common cause of failure when the primary break has been appropriately treated. 2 The exact pathogenic mechanisms involved in the formation of PVR are not completely understood. However, five distinct stages appear to be important in its development including breakdown of the blood-retinal barrier (BRB), chemotaxis and cellular migration, cellular proliferation, membrane formation, and contraction. It is believed that diVerent cytokines, produced as a consequence of immune or inflammatory reactions, are involved in the pathogenesis of PVR. Cytokines such as interleukin 1 (IL-1), interleukin 6 (IL-6), tumour necrosis factor á (TNFá), interferon ã (IFNã), interleukin 8 (IL-8), and monocyte chemotactic protein 1 (MCP-1) have been detected in the vitreous samples as well as epiretinal membranes obtained from PVR patients. It is not known whether these cytokines are produced locally by the cells infiltrating the vitreous or diVuse into the vitreous cavity following a breakdown of the BRB. We therefore tested the infiltrating cells in vitreous obtained from patients with PVR, for mRNA encoding for HPRT (housekeeping gene), IL-1â, IL-6, TNFá, and IL-8. Materials and methods VITREOUS AND SRF SPECIMENS Twelve vitreous and eight subretinal fluid (SRF) samples were obtained by vitrectomy from 18 patients with PVR (both vitreous and SRF were obtained from two patients). Ten vitreous samples obtained from patients with macular hole and six vitreous samples from patients with retinal detachment (RD), not complicated by PVR, were examined as a control group. The presence of mRNA coding for HPRT, IL-6, IL-1â, TNFá, and IL-8 was investigated by reverse transcriptase polymerase chain reaction (RT-PCR). The severity of PVR was graded according to the criteria of the Retina Society Terminology Committee. Samples were obtained through conventional three port closed vitrectomy technique by manual suction before opening the infusion line. SRF samples were obtained during the drainage procedure with a Charles flute needle connected to a 1 ml syringe with a side port. Samples with vitreous haemorrhage were excluded from the study. The study complied with the declaration of Helsinki. mRNA EXTRACTION AND cDNA SYNTHESIS Cellular pellets were obtained from the samples by centrifugation and mRNA extracted using Qiagen RN easy (UK) method following the manufacturer’s procedure. Eluted RNA was made up to 50 μl with DEPC water and cDNA was prepared using Oligo-(dT) priming in ready to go cDNA synthesis tubes (PharmaBr J Ophthalmol 1999;83:1296–1299 1296 Department of Ophthalmology, University of Nottingham