Defensin expression by the cornea: Multiple signalling pathways mediate IL-1β stimulation of hBD-2 expression by human corneal epithelial cells

Defensins are cationic antimicrobial peptides characterized by the presence of six cysteine residues linked to form three disulfide bridges. Two forms of human defensin, α and β, are recognized, depending on the location and connectivity of the cysteines. α-Defensins have been localized to neutrophils and Paneth cells of the intestine, whereas β-defensins are expressed by many epithelia.1,2 To date, six human β-defensins (hBD-1 through -6), have been identified.3–8 hBD-1 is constitutively expressed, whereas hBD-2 and -3 are inducible by cytokines and bacterial products. hBD-4 appears to have a more limited distribution than hBD-1,- 2, or -3; In addition, its expression can be upregulated by bacterial infection but not by inflammatory factors that upregulate hBD-2 and -3.7 The most recently identified family members are hBD-5 and -6, which have been localized to the epididymis.8 Defensins have a broad spectrum of antimicrobial activity, being effective against many Gram-positive and -negative bacteria, some fungi, and enveloped viruses.1,2 The antimicrobial activity of defensins has been attributed to permeabilization of microbial membranes and subsequent release of cellular contents. Exactly how this is achieved has yet to be determined, but two models have been suggested: one in which defensin monomers assemble to form pores within the microbial membrane9 and a second in which defensins disrupt the membrane by electrostatic interactions with the polar head groups of the bilayer.10 In addition to their antimicrobial effects, defensins have been shown to modulate a variety of cellular activities including, chemotaxis of T cells, dendritic cells,11,12 and monocytes13; stimulation of epithelial cell and fibroblast proliferation14; stimulation of cytokine production15,16; and stimulation of histamine release from mast cells.17,18 These effects, which typically occur at defensin concentrations much lower than those required for antimicrobial activity, suggest that defensins not only participate in the innate immune response system by virtue of their ability to kill microbes, but also that they can act as regulatory factors. Previous studies have shown that human corneal epithelial cells (HCECs) express hBD-1 constitutively and that the expression of hBD-2 is variable19,20 and can be upregulated by bacterial products.21 Work in our laboratory has shown that whereas hBD-1 is constitutively expressed, expression of hBD-2 is upregulated during reepithelialization of wounded corneas in organ culture.22 We have speculated that the upregulation of hBD-2 after injury may give added protection to the ocular surface at a time when it is particularly vulnerable to infection and that hBD-2 may, through its nonmicrobicidal activities, contribute to the wound-healing process, per se. In the present study, we examined the expression of the recently identified hBD-3, by HCECs. Also we began to investigate the mechanism underlying upregulation of hBD-2 during wound healing. Studies by others have shown that proinflammatory cytokines, such as interleukin (IL)-1 and tumor necrosis factor (TNF)-α increase expression of hBD-2 in a variety of cell types.23–28 Proinflammatory cytokines are known to increase during wound healing (Ayliffe W, Espaillat A, Foster CS, Lee SJ, ARVO Abstract 3325, 1993)29–31 thus providing a stimulus by which hBD-2 may be upregulated after injury. Therefore, here we have investigated the effect of exposure to proinflammatory cytokines on expression hBD-2 by corneal epithelial cells.