Activated inflammatory infiltrate in HSV-1-infected corneas without herpes stromal keratitis.

Herpes simplex virus type 1 (HSV-1)–induced stromal keratitis (HSK) is a blinding immunopathologic disease of the cornea characterized by recurrent bouts of destructive inflammation and progressive scarring in the corneal stroma.1 In mice, HSV-1 infection induces a transient neutrophilic primary infiltrate that dissipates 3 to 4 days postinfection (dpi). This primary polymorphonuclear (PMN) infiltrate contributes to control of replicating virus and coincides with the presence of a clinically apparent epithelial lesion.2 This primary neutrophilic infiltrate appears to occur in response to cytokines and chemokines produced by corneal parenchymal cells or cells of the innate immune system, or both, and is independent of T lymphocytes.2–5 A more chronic secondary leukocytic infiltrate is initiated after viral clearance from the cornea in normal mice but does not occur in T cell–deficient mice.6–8 The secondary infiltrate consists of neutrophils, CD4+ T cells, few CD8+ T cells, and antigen-presenting cells (APCs) consisting of CD11c+ dendritic cells (DCs) and F4/80+ macrophages.3,6,9,10 In most murine models of HSK, the secondary leukocytic infiltration of the cornea is orchestrated by CD4+ T cells. Activation of naive CD4+ T cells in different microenvironments can result in differentiation along pathways leading to cells with distinct cytokine profiles. The involvement in HSK of CD4+ T cells expressing the Th1 cytokines IL-2 and IFN-γ has been established in mouse models.11,12 Extravasation of neutrophils into the infected cornea is facilitated by IFN-γ, apparently through the up-regulation of platelet endothelial cell adhesion molecule 1 on the local vascular endothelium,13 and is regulated indirectly by IL-2,13,14 presumably through the induction of chemotactic factors. CD4+ Th17 T cells have also been implicated in HSK because IL-17 induces corneal fibro-blasts to produce the neutrophil chemoattractant IL-8,15 and IL-6 regulates angiogenesis through the induction of vascular endothelial growth factor (VEGF) and neutrophilic infiltration through the induction of chemokines.15–19 In contrast, HSK severity appears to be ameliorated by CD4+ T cells expressing the Th2 cytokine IL-420 and by regulatory CD4+ T cells (Tregs), at least in part through the production of IL-10,21 which separately has been shown to mitigate HSK.4,22 Several chemokines have also been implicated in the secondary leukocytic infiltrate associated with HSK, including Mig (CXCL9), IP-10 (CXCL10), macrophage inflammatory protein (MIP)-1α (CCL3), MIP-1β (CCL4), MIP-2 (CXCL2), macrophage chemotactic protein (MCP)-1 (CCL2), MIP-3α (CCL20), and keratinocyte chemoattractant (KC).4,23–27 Many of these chemokines are produced by corneal parenchymal cells and by infiltrating inflammatory cells, and the relative contribution of these cellular sources to the chemokine milieu within the infected cornea likely changes as inflammation progresses. The combined effect of cytokine and chemokine production is an inflammatory infiltrate that, on achieving a critical mass, initiates neovascularization and destruction of the corneal architecture. However, in most humans who shed virus at the corneal surface and in some mice receiving a low-dose HSV-1 corneal infection, HSK fails to develop, suggesting that this cycle of leukocytic infiltration and activation is interrupted at some unknown point before the initiation of clinical disease. Our previous study demonstrated that susceptibility to HSK was not associated with the magnitude of the HSV-specific CD4+ T-cell response generated in the draining lymph nodes or with the level of the delayed-type hypersensitivity response after HSV-1 corneal infection.28 However, HSK development was associated with a massive DC infiltration into the infected cornea between 7 and 14 dpi, which was abrogated by CD4+ T-cell depletion. These findings suggest that susceptibility to HSK is determined by the capacity of HSV-specific CD4+ T cells to infiltrate the cornea and to induce DC and neutrophil infiltration. We hypothesized that corneas that fail to develop HSK after HSV-1 infection would lack a critical mass of activated CD4+ T cells. However, this study demonstrates that HSV-1 infected corneas without HSK contained as many activated CD4+ T cells as those with HSK at the time of near maximal HSK severity. Corneas without HSK exhibited significantly fewer neutrophils and DCs and lower levels of known neutrophil and DC chemoattractants, suggesting that interference in the inflammatory process in these corneas occurs after CD4+ T-cell activation.