Loss of TNFa in Macrophages Potentiates TGFbeta–Mediated Pathogenic Corneal Fibroblast Response in vitro and in vivo During Wound Healing in Mice

Abstract

Purpose: : To examine the role of tumor necrosis factor a (TNFa) in cornea wound healing following an alklai burn by using TNFa–null (KO) mice. Effect of loss of TNFa in ocular fibrobalsts or macrophages on fibrogenic reaction by the fibroblasts were also investigated in co–culture system.

Methods: : NaOH solution (1 N, 3micro L) was applied to one eye of adult KO (n=98) or wild–type (WT) mice (n=98) under general and topical anesthesia. Ofloxacin ointment was then topically administered. Healing corneas were histologically or immunohistochemically examined at up to 8 weeks. mRNA expression of cytokines and collagen Ia2 in healing corneas was assayed by real–time RT–PCR at each timepoints. The MMP activity was assayed with in situ zymography. To examine the roles of TNFa in bone marrow–derived inflammatory cells in the healing process by employing bone marrow transplantation (BMT) between WT and KO animals. Expression of phospho–Smad2 in tissue was examined by western blotting. The roles of TGFbeta/Smad signal in healing process was further examined by Smad7–adenoviral gene transfer to the cornea. Co–culture experiments with WT/KO ocular fibroblasts and macropahges, with or without TGFbeta– or TNFa–neutralizing anitbody, were performed to know the role of TNFa of each cell type in fibroblast fibrogenic gene expression.

Results: : Loss of TNFa resulted in corneal tissue destruction during healing post–alkali burn, but not in WT corneas. Up–regulation of growth factors, excess macrophage invasion and subsequent formation of a vascularized scar tissue were much more marked in KO mice than in WT mice. Phospho–Smad2 expression was more marked in KO tissue than in WT cornea, suggesting over–action of TGFbeta in the absence of TNFa. Such unfavorable outcome in KO mice was abolished by Smad7 gene introduction and BMT from WT mice, suggesting that TNFa in bone marrow–derived inflammatory cells suppresses TGFbeta over–action in the healing tissue. Co–culture experiments showed that loss of TNFa in macrophages, but not in fibroblasts, augments the fibrogenic gene expression in fibroblasts.

Conclusions: : TNFa in macrophages is required to suppress undesirable excessive inflammation and scarring, both promoted by TGFbeta and needed for restoration of tissue architecture in a healing alkali–burned cornea in mice.

Keywords: cornea: basic science • cytokines/chemokines • wound healing