Abstract
Abstract: :
Purpose: To analyze expression of TLR–4 signaling receptor for LPS, of various PKC isoenzymes with special attention to PKC–zeta and inducible nitric oxide synthase (NOS)–2 expression in inflammatory cells during LPS–induced ocular inflammation and its treatment with IL–13, a cytokine with potent anti–inflammatory properties. Methods: We studied TLR–4, PKCzeta (atypical PKC), PKC–epsilon and PKC–eta (novel PKCs), NOS–2 expression during EIU in rats treated with intracameral injection of saline, IL–13 or specific PKC–zeta inhibitor (PKC–zi) simultaneously, 2 hours before LPS injection or 6 hours after. Eyes from EIU rats were taken 24 hours after LPS injection and RT–PCR, immunohistochemistry on cryostat sections and western blotting of ocular tissues were performed using antibodies specific for PKC–zeta, PKC–epsilon, PKC–eta, TLR–4, NF–kappa–B, NOS–2, inflammatory cells (ED1, OX42). Results: As shown in immunoblot observation, LPS induced a degradation of PKC–zeta in ocular tissues. Intracameral injection of IL–13 or PKC–zi simultaneously or 6 hours after LPS injection allowed to preserve the constitutive expression of PKC–zeta and increased NFkappaB expression whereas when performed 2 hours before LPS injection, it altered the constitutive expression of PKC–zeta in the corneal endothelium resulting in corneal edema and in the retina inducing an apoptosis of some cells in the inner nuclear layer. In immunofluorescence studies, control rats injected with saline showed cytoplasmic coexpression of NOS–2 with PKC–zeta and PKC–epsilon but not with PKC–eta, and NFkappaB nuclear translocation mainly in macrophages. TLR–4 immunostaining revealed a cytoplasmic paranuclear distribution mainly in polymorphonuclear leukocytes (PMNs). Interestingly, PKC–zeta was expressed in vascular endothelial cells suggesting its role in the breakdown of vascular ocular barrier. The treatment with IL–13 or PKC–zi induced a nuclear translocation of PKC–zeta in macrophages that presented an apoptotic aspect and were NOS–2 negative. The cytoplasmic expression of TLR–4 was low in apoptotic PMNs. RT–PCR showed decreased TNF–a mRNA expression in treated rat eyes. Conclusions: Our results provide evidence for the role of TLR–4 and PKC–zeta expression in recruitment and apoptosis of ocular inflammatory cells during EIU and its inhibition by IL–13. Modulation of TLR–4/PKC–zeta signaling pathway at the level of macrophages and PMNs might represent a novel approach in the treatment of ocular inflammation.
Keywords: uveitis-clinical/animal model • inflammation • immunomodulation/immunoregulation