Robert N. Lausch

Chemokine synthesis in the HSV-1-infected cornea and its suppression by interleukin-10.

Herpes simplex virus type 1 (HSV‐1) infection of the murine cornea results in a tissue‐destructive inflammatory response. In this study we show that virus infection induces the synthesis of macrophage inflammatory protein‐2 (MIP‐2), MIP‐1α, and monocyte chemoattractant protein‐1 (MCP‐1). However, only the production of MIP‐2 and MIP‐1α coincided with the influx of leukocytes into the cornea. IL‐10 treatment markedly suppressed chemokine message and protein synthesis in vivo. Local administration of IL‐10 also dramatically reduced the number of T cells and neutrophils migrating into the cornea and suppressed the severity of corneal disease. The inflammatory response could also be suppressed by the passive transfer of neutralizing antibody to MIP‐1α but not MCP‐1. We conclude that local IL‐10 administration can suppress chemokine synthesis, thereby ameliorating corneal disease. Furthermore, our results indicate that MIP‐1α plays a major role in herpes stromal keratitis development, whereas MCP‐1 does not. J. Leukoc. Biol. 63: 486–492; 1998.

IL-17 receptor signaling influences virus-induced corneal inflammation

IL‐17 has been associated with selected inflammatory and autoimmune diseases. We characterized the expression of this proinflammatory cytokine following HSV‐1 corneal infection and investigated whether IL‐17R signaling modulated the host response to the viral pathogen at early time‐points postinfection. IL‐17 was elevated in the murine cornea 24 h after high‐dose virus infection and subsequently persisted at low levels during the first week. Immunofluorescent studies showed that the IL‐17R was expressed by cultured mouse corneal fibroblasts. Exposure of corneal cells to IL‐17 led to production of IL‐6 and MIP‐2 in vitro and in vivo, indicating that the IL‐17R was functional. Mice lacking IL‐17R displayed significantly reduced neutrophil infiltration and corneal opacity. However, this effect was transient, as corneal pathology and neutrophil influx resembled that of wild‐type (WT) hosts 4 days postinfection. HSV‐1 growth and clearance in IL‐17R−/− hosts were similar to that of the WT controls. Infection of IFN‐γ gene knockout mice was associated with elevated IL‐17 levels and accelerated corneal opacity, suggesting that IFN‐γ negatively regulated IL‐17 expression. Collectively, our results establish that IL‐17 is rapidly produced in the cornea after HSV‐1 infection and is regulated at least in part by IFN‐γ. The absence of IL‐17 signaling results in a transient decrease in the expression of proinflammatory mediators, neutrophil migration, and corneal pathology, but control of virus growth in the cornea and trigeminal ganglia is not compromised. Thus, IL‐17 actively influences early virus‐induced corneal inflammation.

A novel role for neutrophils as a source of T cell-recruiting chemokines IP-10 and Mig during the DTH response to HSV-1 antigen

Analogous to CD4+ T cells, neutrophils are essential participants in delayed‐type hypersensitivity (DTH) to Herpes simplex virus type 1 antigen. However, what role they play in this cellular immune response is unclear. The recent recognition that neutrophils are potent producers of chemokines led us to hypothesize that they may help recruit CD4+ effector T cells. In the present study, we show that neutrophil depletion was accompanied by a marked decrease in the numbers of CD4+ and CXC receptor 3+ (CXCR3+)‐expressing cells migrating to the DTH site and a sharp drop in the levels of interferon‐inducible protein 10 (IP‐10) and monokine induced by IFN‐γ (Mig). Purified mouse neutrophils were stimulated directly by IFN‐γ to secrete these chemokines, and neutrophils at the DTH site expressed IP‐10. IFN‐γ knockout mice, which manifested depressed ear‐swelling following DTH challenge, made little IP‐10 and no Mig. Reconstitution of these mice with IFN‐γ induced CXCR3 ligand synthesis. Depletion of neutrophils or CD4+ T cells but not CD8+ T cells markedly reduced IFN‐γ levels, suggesting the former were direct (or indirect) cellular sources of this cytokine. Collectively, our results support the hypothesis that neutrophil production of T cell‐recruiting chemokines contributes to the regulation and amplification of the DTH response.

Calcitonin Gene-Related Peptide Induces IL-8 Synthesis in Human Corneal Epithelial Cells

Calcitonin gene-related peptide (CGRP), a neuropeptide with proinflammatory activities, is released from termini of corneal sensory neurons in response to pain stimuli. Because neutrophil infiltration of the clear corneal surface is a hallmark of corneal inflammation in the human eye, we determined whether CGRP can bind to human corneal epithelial cells (HCEC) and induce expression of the neutrophil chemotactic protein IL-8. It was found that HCEC specifically bound CGRP in a saturable manner with a Kd of 2.0 × 10−9 M. Exposure of HCEC to CGRP induced a significant increase in intracellular cAMP levels and enhanced IL-8 synthesis nearly 4-fold. The capacity of CGRP to stimulate cAMP and IL-8 synthesis was abrogated in the presence of the CGRP receptor antagonist CGRP8–37. CGRP stimulation had no effect on the half-life of IL-8 mRNA while increasing IL-8 pre-mRNA synthesis >2-fold. In contrast to IL-8, CGRP did not induce monocyte chemotactic protein-1 or RANTES synthesis, nor did the neuropeptide enhance detectable increases in steady state levels of mRNA specific for these two β-chemokines. The results suggest that HCEC possess CGRP receptors capable of initiating a signal transduction cascade that differentially activates expression of the IL-8 gene but not the genes for monocyte chemotactic protein-1 or RANTES. The capacity of CGRP to stimulate IL-8 synthesis in HCEC suggests that sensory neurons are involved in induction of acute inflammation at the eye surface.

Role for Macrophage Inflammatory Protein 2 (MIP-2), MIP-1α, and Interleukin-1α in the Delayed-Type Hypersensitivity Response to Viral Antigen

ABSTRACT BALB/c mice sensitized to herpes simplex virus type 1 (HSV-1) develop a vigorous delayed-type hypersensitivity (DTH) response upon intradermal virus antigen challenge. Although CD4+ T cells are a key mediator of this response, neutrophils are the most abundant cells at the antigen challenge site both initially and at the peak of the reaction. We investigated what role, if any, neutrophils play in the DTH to a viral antigen. We show here that antibody-mediated depletion of neutrophils 1 day before antigen challenge significantly suppressed ear swelling and markedly reduced cellular influx. Additionally, neutrophil depletion was associated with decreased expression of macrophage inflammatory protein 2 (MIP-2) and MIP-1α, as well as with a >60-fold increase in HSV-1 replication. Neutralizing antibodies to neutrophil chemoattractants MIP-2 or MIP-1α but not KC significantly suppressed DTH and sharply reduced neutrophil accumulation in the ear pinna. Purified bone marrow-derived neutrophils exposed to interleukin-1α (IL-1α) produced chemokines in an 8-h assay. Administration of neutralizing antibody to IL-1α significantly reduced ear swelling and suppressed the levels of MIP-2, MIP-1α, MIP-1β, and RANTES. We conclude that neutrophils are a critical component of the DTH response to viral antigen. They are recruited to the DTH test site by MIP-2 and MIP-1α, where they can be activated by IL-1α. The infiltrating cells also help suppress virus replication in immunized mice.

Activation of cytokines and NF-kappa B in corneal epithelial cells infected by respiratory syncytial virus: potential relevance in ocular inflammation and respiratory infection

BackgroundRespiratory syncytial virus (RSV) is a major cause of lower respiratory tract infection, claiming millions of lives annually. The virus infects various cells of the respiratory tract as well as resident inflammatory cells such as macrophages. Infection activates a variety of cellular factors such as cytokines and the pro-inflammatory transcription factor, NF-kappa B, all of which are important players in the respiratory disease. However, the exact natural route of RSV infection and its etiology remain relatively unknown. In this paper, we test the hypothesis that human corneal epithelial cells, which constitute the outermost layer of the cornea, can be infected with RSV, and that the infection leads to the activation of proinflammatory macromolecules.ResultsCorneal swabs obtained from pediatric patients with acute respiratory disease were found to contain RSV at a high frequency (43 positive out of 72 samples, i.e., 60%). Primary corneal epithelial cells in tissue culture supported robust infection and productive growth of RSV. Infection resulted in the activation of TNF-α, IL-6 and sixteen chemokines as well as NF-κB. Three proinflammatory CXC chemokines (MIG, I-TAC, IP-10) underwent the greatest activation.ConclusionsThe ocular epithelium is readily infected by RSV. The pro-inflammatory cytokines are likely to play critical roles in the etiology of inflammation and conjunctivitis commonly seen in pediatric patients with respiratory infections. RSV-eye interactions have important implications in RSV transmission, immunopathology of RSV disease, and in the management of conjunctivitis.

Synergistic anti-HSV effect of tumor necrosis factor alpha and interferon gamma in human corneal fibroblasts is associated with interferon beta induction

HSV-1 (17) replicated to high titer in human corneal fibroblasts (> 10(8) PFU/10(5) cells) following infection at one PFU per 100 cells. Pretreatment of the cells for 24 h with 50 U/ml recombinant human tumor necrosis factor alpha (TNF-alpha) or 5 IU/ml of human interferon gamma (IFN-gamma) resulted in only modest reduction (2- to 19-fold) in virus yield. However, when the two cytokines were combined the antiviral effect was dramatically increased. There was > 1000-fold reduction in virus titer in 8 of 8 trials. In contrast, the combinations of 50 U/ml TNF-alpha with 5 IU/ml IFN-alpha or IFN-beta did not produce a synergistic effect. The pronounced synergistic antiviral activity of TNF-alpha+IFN-gamma could be demonstrated in fibroblast cultures from different donors, and HSV-2 as well as HSV-1 strains were inhibited. There was no evidence that dual cytokine treatment was toxic for uninfected or HSV-infected cells. Insight into the mechanism responsible for the synergistic effect was provided by the observation that TNF-alpha+IFN-gamma induced IFN-beta. In addition, anti-IFN-beta but not anti-IFN-alpha antibodies could reverse the antiviral effect, and reconstitution with IFN-beta could duplicate the phenomenon. We conclude that the combination of TNF-alpha and IFN-gamma at low concentrations can exert a powerful anti-herpes effect in human corneal fibroblasts which can be chiefly attributed to the induction of IFN-beta.

Herpes simplex virus type 1 DNA sequences which direct spread of virus from comes to central nervous system

Abstract The virulence of a herpes simplex virus (HSV) intertypic recombinant possessing HSV-1 DNA sequences from map units 0.31 to 0.44 and HSV-2 sequences from map units 0 to 0.30 and 0.45 to 1.0 were compared with the virulence of the two parental strains. Following ocular inoculation, both the intertypic recombinant and the HSV-1 parent replicated at the infection site and spread to the peripheral and central nervous system (CNS) to produce fatal encephalitis. The HSV-2 parent also replicated at the infection site but failed to progress to the CNS. However, when inoculated intracerebrally, the HSV-2 strain was as lethal as the HSV-1 parent. Furthermore, the HSV-2 strain could produce thymidine kinase at 37 and 39° in levels comparable to the HSV-1 strain. The results indicate that transfer of the HSV-1 DNA sequences imparted to the recombinant virus the necessary genetic information to spread from the cornea into the central nervous system.

Quantitation of purified monoclonal antibody needed to prevent HSV-1 induced stromal keratitis in mice

A purified IgG2a monoclonal antibody with a neutralizing titer of 10(4) and specificity for gD was evaluated for its therapeutic potential in a murine ocular infection model. BALB/c mice, infected on the scarified cornea with 10 times the HSV-1 strain RE concentration needed to produce severe and persistent stromal opacity, were given a single inoculation of antibody intraperitoneally 24 hours later. The animals were then followed for corneal disease development. Antibody, at concentrations as low as 10 micrograms per mouse, was strikingly effective at preventing corneal opacity. Furthermore, the corneas, once clear, remained clear whereas the controls developed +3 to +5 stromal disease which was still present 60 days post-infection. Animals that had been treated and recovered from infection were resistant to subsequent HSV-1 challenge on the opposite cornea. These results demonstrate the therapeutic potential of systemically administered microgram quantities of anti-gD antibody.

Synergistic anti-herpes effect of TNF-α and IFN-γ in human corneal epithelial cells compared with that in corneal fibroblasts

In this study we compared how effectively the proinflammatory cytokines TNF-alpha and IFN-gamma could inhibit HSV-1 replication in human corneal tissue fragments and monolayers of epithelial cells and fibroblasts derived from intact corneas, and investigated the mechanism responsible for the inhibition. Pretreatment of corneal tissue or cells derived therefrom with TNF-alpha (50 U/ml) and IFN-gamma (5 IU/ml) consistently induced a synergistic antiviral effect. Inhibition of HSV-1 growth was most evident in fibroblasts (> 1000-fold reduction) and less apparent (7-25-fold reduction) when epithelial cells were the target. Virus suppression was correlated with the induction of IFN-beta because antibody to this cytokine but not IFN-alpha abrogated synergism. The more modest synergistic effect in epithelial cells was associated with a > or = 4-fold reduction in the synthesis of IFN-beta protein and mRNA, and decreased responsiveness of these cells to the antiviral effect of IFN-beta. We conclude that the combination of TNF-alpha and IFN-gamma induces a synergistic antiviral effect in corneal cells. The degree of synergism observed varies with the corneal cell type, and is correlated with the amount of IFN-beta induced and the target cell responsiveness to the antiviral action of this cytokine.