Acute ventilator-induced vascular permeability and cytokine responses in isolated and in situ mouse lungs.

To determine the influence of experimental model and strain differences on the relationship of vascular permeability to inflammatory cytokine production after high peak inflation pressure (PIP) ventilation, we used isolated perfused mouse lung and intact mouse preparations of Balb/c and B6/129 mice ventilated at high and low PIP. Filtration coefficients in isolated lungs and bronchoalveolar lavage (BAL) albumin in intact mice increased within 20-30 min after initiation of high PIP in isolated Balb/c lungs and intact Balb/c, B6/129 wild-type, and p55 and p75 tumor necrosis factor (TNF) dual-receptor null mice. In contrast, the cytokine response was delayed and variable compared with the permeability response. In isolated Balb/c lungs ventilated with 25-27 cmH(2)O PIP, TNF-alpha, interleukin (IL)-1 beta, IL-1 alpha, macrophage inflammatory protein (MIP)-2, and IL-6 concentrations in perfusate were markedly increased in perfusate at 2 and 4 h, but only MIP-2 was detectable in intact Balb/c mice using the same PIP. In intact wild-type and TNF dual-receptor null mice with ventilation at 45 cmH(2)O PIP, the MIP-2 and IL-6 levels in BAL were significantly increased after 2 h in both groups, but there were no differences between groups in the BAL albumin and cytokine concentrations or in lung wet-to-dry weight ratios. TNF-alpha was not be detected in BAL fluids in any group of intact mice. These results suggest that the alveolar hyperpermeability induced by high PIP ventilation occurs very rapidly and is initially independent of TNF-alpha participation and unlikely to depend on MIP-2 or IL-6.

Synthesis of type II interleukin-1 receptors by human corneal epithelial cells but not by keratocytes.

PURPOSE: The purpose of this study was to determine whether human corneal epithelial cells and keratocytes synthesize both the soluble and membrane forms of the type II IL-1 receptor (IL-1RII). METHODS: Primary cell cultures of human corneal epithelial cells and keratocytes were established from human corneas. RT-PCR was used to analyze cell cultures for expression of IL-1RII mRNA. The capacity of corneal cells to synthesize membrane-bound IL-1RII was determined by immunofluorescence microscopy, whereas ELISA was used to quantitate synthesis of soluble IL-1RII after IL-1alpha and TNF-alpha stimulation. RESULTS: Corneal epithelial cells expressed IL-1RII mRNA. The cells also stained positive for membrane-bound IL-1RII, and media harvested from epithelial cell cultures contained up to 50 pg/ml of soluble IL-1RII. Both IL-1alpha and TNF-alpha significantly enhanced the amounts of soluble IL-1RII released from epithelial cell surfaces. In contrast to epithelial cells, corneal keratocytes did not express IL-1RII mRNA. Membrane-bound IL-1RII was not detected on keratocytes, nor was soluble IL-1RII detected in culture media harvested from these cells. CONCLUSIONS: Human corneal epithelial cells but not corneal keratocytes synthesize both membrane and soluble forms of IL-1RII. Because both forms of IL-1RII can function as IL-1alpha antagonists, the results suggest that human corneal epithelial cells but not corneal keratocytes have evolved the capacity to dampen IL-1alpha responses through the production of IL-1RII.

Role of macrophages in restricting herpes simplex virus type 1 growth after ocular infection.

PURPOSE: To investigate the role macrophages play in controlling herpes simplex virus (HSV)-1 replication after infection of the murine cornea. METHODS: Macrophage depletion in selected tissues before or after virus infection was achieved by repeated subconjunctival (SCJ) and/or intravenous (IV) injection of liposomes containing dichloromethylene diphosphonate (L-Cl2MDP). Controls received liposomes containing phosphate-buffered saline (L-PBS). The efficiency of depletion was evaluated by histologic examination. Virus content in infected tissues was determined by standard plaque assay. Delayed-type hypersensitivity (DTH) responsiveness was assessed using the ear-swelling assay. Antibody isotype responses to virus antigens and cytokine production were monitored by enzyme-linked immunosorbent assay. RESULTS: Balb/c mice given SCJ injection of L-Cl2MDP 4 and 2 days before HSV-1 corneal infection were found to have ocular virus titers as much as 10(5)-fold higher than that seen in the L-PBS-treated controls 8 days after infection. When L-Cl2MDP treatment was delayed until 2 and 4 days after infection, virus titers in the eye were analogous to those in the control animals. Subconjunctival and submandibular lymph node macrophages in mice given local (SCJ) L-Cl2MDP pretreatment were profoundly reduced, whereas the number of corneal Langerhans' cells and lymph node dendritic cells remained unchanged. Local L-Cl2MDP pretreatment was associated with significantly reduced DTH responsiveness to HSV-1 antigen, and an alteration in selected antibody isotype production. Depletion of macrophages in the subconjunctival tissue before corneal infection was not accompanied by enhanced virus growth at early times (2 or 4 days) after infection. CONCLUSIONS: Macrophages play an important role in restricting HSV-1 growth after corneal infection. These cells appear to be required for the development of an acquired immune response, presumably by functioning in antigen processing and presentation. The hypothesis that macrophages are major participants in innate immunity to HSV-1 corneal infection was not supported.

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 x 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 beta-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.

Human corneal cells and other fibroblasts can stimulate the appearance of herpes simplex virus from quiescently infected PC12 cells.

A two-cell system for the stimulation of herpes simplex virus type 1 (HSV-1) from an in vitro model of long-term (quiescent) infection is described. Rat pheochromocytoma (PC12) cells differentiated with nerve growth factor were infected with HSV-1 strain 17. Little, if any, cytotoxicity was observed, and a quiescent infection was established. The long-term infection was characterized by the absence of all detectable virus in the culture medium and little, if any, detectable early or late viral-gene expression as determined by reverse transcriptase PCR analysis. The presence of HSV-1 DNA was determined by PCR analysis. This showed that approximately 180 viral genomes were present in limiting dilutions where as few as 16 cells were examined. The viral DNA was infectious, since cocultivation with human corneal fibroblasts (HCF) or human corneal epithelial cells (HCE) resulted in recovery of virus from most, if not all, clusters of PC12 cells. Following cocultivation, viral antigens appeared first on PC12 cells and then on neighboring inducing cells, as determined by immunofluorescent staining, demonstrating that de novo viral protein synthesis first occurred in the long-term-infected PC12 cells. Interestingly, the ability to induce HSV varied among the cell lines tested. For example, monkey kidney CV-1 cells and human hepatoblastoma HepG2 cells, but not mouse neuroblastoma cells or undifferentiated PC12 cells, mediated stimulation. This work thus shows that (i) quiescent HSV infections can be maintained in PC12 cells in vitro, (ii) HSV can be induced from cells which do not accumulate significant levels of latency-associated transcripts, and (iii) the activation of HSV gene expression can be induced via neighboring cells. The ability of adjacent cells to stimulate HSV gene expression in neuron-like cells represents a novel area of study. The mechanism(s) whereby HCF, HCE, and HepG2 and CV-1 cells communicate with PC12 cells and stimulate viral replication, as well as how this system compares with other in vitro models of long-term infection, is discussed.

Role of MIP-2 in neutrophil migration and tissue injury in the herpes simplex virus-1-infected cornea.

PURPOSE: Neutrophils are the most prominent cell type to migrate initially into the herpes simplex virus type 1 (HSV-1)-infected murine cornea. The role the C-X-C chemokines macrophage inflammatory protein (MIP)-2 and KC play in promoting this response was investigated. METHODS: MIP-2 and KC were quantitated by enzyme-linked immunosorbent assay. Neutralization of endogenous MIP-2 and KC was achieved by subconjunctival inoculation of the appropriate antibody. Infected corneas were examined immunohistochemically for infiltrating leukocytes and assayed for myeloperoxidase activity using the dye o-dianisidine. Depletion of neutrophils and natural killer cells was accomplished by intraperitoneal administration of RB6-8C5 and asialo GM1 antibodies. RESULTS: Herpes simplex virus type 1, when introduced intracorneally, stimulated the production of MIP-2 and KC, with peak synthesis occurring 48 hours after infection. Dose-response studies showed that exogenous MIP-2 was three to four times more potent than KC in attracting neutrophils as assessed by myeloperoxidase assay and immunohistochemical staining. Subconjunctival administration of neutralizing antibody to MIP-2 resulted in a sharp decrease in neutrophil infiltration and significantly reduced corneal opacity scores. In contrast, in vivo treatment with neutralizing antibody to KC did not suppress ocular inflammation. Additional studies indicated that MIP-2 and KC could be made by corneal epithelial cells and that production was promoted by interleukin (IL)-1. In vivo depletion of neutrophils sharply reduced MIP-2 levels but did not affect KC levels. CONCLUSIONS: Collectively, the results suggest that MIP-2 is the major chemokine that attracts neutrophils into the HSV-1 infected cornea, where the cells directly or indirectly cause tissue injury. Resident corneal cells and inflammatory cells contribute to MIP-2 synthesis, whereas KC production seems to be confined largely to corneal cells.

Membranes of herpes simplex virus type-1-infected human corneal epithelial cells are not permeabilized to macromolecules and therefore do not release IL-1alpha.

Nanogram amounts of the proinflammatory cytokine interleukin-1alpha (IL-1alpha) were detected in uninfected cultures of human corneal epithelial cells (HCEC). Although HSV-1 replicated >10(4)-fold in these cells and caused extensive cytopathic effects, virus infection was not accompanied by significant extracellular release of IL-1alpha. Additional studies showed that release of radiolabeled cytosolic proteins from virus-infected HCEC was no greater than that released by mock-infected cells. These findings indicate that HSV-1 infection of HCEC does not result in IL-1alpha release because newly formed virus progeny can escape infected cells without disrupting cell membranes.

Differential induction of GRO alpha gene expression in human corneal epithelial cells and keratocytes exposed to proinflammatory cytokines.

PURPOSE: GRO alpha is an inducible neutrophil chemotactic factor synthesized in inflamed corneal tissues. In this study, the regulation of GRO alpha synthesis after exposure of human corneal cells to proinflammatory cytokines was investigated. METHODS: Pure cultures of human corneal epithelial cells and keratocytes were exposed to increasing concentrations of either interleukin-1 alpha (IL-1 alpha) or tumor-necrosis factor-alpha (TNF-alpha). At selected times postexposure, the amounts of GRO alpha produced by the cultures were quantitated by enzyme-linked immunosorbent assay. The RNA was harvested from stimulated cultures to monitor GRO alpha mRNA and pre-mRNA levels by the reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: Both IL-1 alpha and TNF-alpha induced significant levels of GRO alpha synthesis in keratocytes. However, IL-1 alpha-stimulated cells produced > 10 times more GRO alpha protein and 12 times more GRO alpha mRNA than did TNF-alpha-stimulated keratocytes. In contrast to the log differences in mRNA levels and protein synthesis, there was a less than twofold difference between IL-1 alpha and TNF-alpha in their capacity to induce GRO alpha specific pre-mRNA synthesis. When actinomycin D was added to stimulated keratocytes to inhibit transcription, the half-life of GRO alpha mRNA in TNF-alpha-treated cells was < 1 hour, whereas the half-life of the GRO alpha mRNA synthesized in IL-1 alpha-stimulated cells was > or = 9 hours. In contrast to keratocytes, exposure of corneal epithelial cells to IL-1 alpha and TNF-alpha did not induce significant increases in steady-state levels of GRO alpha pre-mRNA, mRNA, or protein. CONCLUSIONS: The results suggest that keratocytes are the major producers of GRO alpha in the human cornea. The capacity of IL-1 alpha to stimulate the synthesis of significantly higher quantities of GRO alpha in keratocytes compared to TNF-alpha is caused by the fact that GRO alpha-specific transcripts are more than nine times more stable in cells exposed to IL-1 alpha than in cells exposed to TNF-alpha.

Proinflammatory cytokines induce RANTES and MCP-1 synthesis in human corneal keratocytes but not in corneal epithelial cells. Beta-chemokine synthesis in corneal cells.

PURPOSE: To determine whether human corneal epithelial cells and keratocytes produce chemokines regulated upon activation, normal T-cell expressed and secreted (RANTES) protein and monocyte chemotactic protein-1 (MCP-1) after exposure to proinflammatory cytokines interleukin-1 alpha (IL-1 alpha) and tumor necrosis factor-alpha (TNF-alpha). METHODS: Pure cultures of human corneal epithelial cells and keratocytes were exposed to increasing concentrations of either human recombinant IL-1 alpha or TNF-alpha. At selected time intervals after exposure, culture supernatants were removed and assayed for RANTES and MCP-1 by the enzyme-linked immunosorbent assay. Total RNA was extracted from the cell cultures, and steady state mRNA levels were measured by reverse transcriptase-polymerase chain reaction. RESULTS: Exposure to keratocytes to either IL-1 alpha or TNF-alpha resulted in > 100-fold increases in RANTES protein secretion and > 150-fold increases in MCP-1 protein secretion, as well as in rapid sustained increases in intracellular levels of their corresponding mRNAs. Exposure of corneal epithelial cells to IL-1 alpha and TNF-alpha did not stimulate MCP-1 secretion nor intracellular levels of MCP-1 mRNA. Epithelial cells also failed to secrete RANTES protein even through the two inducing cytokines did stimulate increased expression of RANTES mRNA. CONCLUSIONS: These results suggest that RANTES and MCP-1 gene expression in human keratocytes differs markedly from their expression in human corneal epithelial cells and that the stroma of the cornea may be more important than the epithelium in recruitment of mononuclear leukocytes responsible for cell-mediated immunity.

Protective antibody therapy is associated with reduced chemokine transcripts in herpes simplex virus type 1 corneal infection.

Herpes simplex virus type 1 (HSV-1) infection on the murine cornea induces an intense inflammatory response which can lead to blindness. This disease, known as herpes stromal keratitis, can be prevented by the timely passive transfer of monoclonal antibody specific for viral glycoprotein D (gD). Precisely how antibody treatment prevents excessive corneal inflammation is not known. In this study we investigated whether chemokine mRNA expression is inhibited by antibody treatment. Total cellular RNAs isolated from normal corneas and at various times after virus infection were analyzed via reverse transcription-PCR for mRNA coding for seven different chemokines. Constitutive levels of IP-10, KC, MIP-2, MCP-1, MIP-1 beta, and RANTES mRNA were detected in uninfected corneas of BALB/c mice. When the cornea was mechanically traumatized, message for all six chemokines was transiently elevated above constitutive levels. In contrast, HSV-1 infection resulted in prolonged enhanced chemokine message expression. The kinetics of mRNA accumulation was distinctive for each chemokine analyzed. MIP-1 alpha message, not detected constitutively, was not evident until day 7 postinfection. Administration of anti-HSV gD monoclonal antibody 1 day after infection was associated with reduced message for MIP-2, MCP-1, MIP-1 alpha, and MIP-1 beta. IP-10, KC, and RANTES messages were not altered. Collectively, our results suggest that anti-gD treatment may protect, at least in part, by inhibiting production of chemokines believed to promote inflammation.

Early cytokine synthesis in the excised mouse cornea.

Corneas excised from normal BALB/c mice and incubated in vitro were analyzed for the production of "early-warning" cytokines via reverse transcription-polymerase chain reaction and ELISA. It was found that the trauma of excision stimulated rapid IL-1 alpha synthesis, with peak protein accumulation occurring at 6 h, whereas IL-6 synthesis was maximal at 18 h. Neither IL-1 beta protein nor message was detected at any point, and TNF-alpha synthesis never increased above constituted levels. Antibody neutralization of endogenous IL-1 alpha blocked IL-6 synthesis. Addition of exogenous IL-1 alpha induced IL-1 alpha and IL-6 synthesis in vitro. Inoculation of IL-1 alpha into the cornea induced IL-6 synthesis in vivo. Addition of IL-1 alpha could stimulate IL-1R, IL-1 alpha, and IL-6 mRNA synthesis in the epithelial, stromal, and endothelial components of the cornea. However, protein production was readily detected only in the epithelial layer. We concluded that mechanical trauma to the mouse cornea triggers the enhanced synthesis of IL-1 alpha and IL-1R, which in turn results in the production of IL-6 and more IL-1 alpha. That corneal excision did not stimulate the synthesis of IL-1 beta or TNF-alpha indicates that there is a selective induction of early cytokine expression in this specialized tissue.

Differences in interleukin-6 gene expression between cultured human corneal epithelial cells and keratocytes.

PURPOSE: To determine whether interleukin-6 (IL-6) can be synthesized by human corneal keratocytes and epithelial cells. METHODS: Epithelial cells and keratocytes isolated from the same donor corneas were grown in vitro. After 2 to 3 passages, the cultures were exposed to varying concentrations of recombinant human interleukin-1 (IL-1 alpha) or tumor necrosis factor (TNF-alpha). Culture supernatants subsequently underwent enzyme-linked immunosorbent assays for cytokine content. The levels of cytokine mRNA in cell lysates were monitored by the reverse transcription-polymerase chain reaction. RESULTS: Cultured human keratocytes stimulated with 100 U/ml IL-1 alpha for 18 hours produced more than 160 ng IL-6 per 10(6) cells. Under the same conditions 500 U/ml TNF-alpha induced approximately 5 ng IL-6. IL-6 mRNA, evident by 3 hours after exposure to either cytokine, accumulated and persisted through 18 hours. Exposure of epithelial cells to IL-1 alpha or TNF-alpha induced minimal and transient expression of IL-6 mRNA and < 0.5 ng protein product per 10(6) cells. The poor production of IL-6 did not reflect an inability of epithelial cells to respond to IL-1 alpha and TNF-alpha because both cytokines induced these cells to make copious amounts of IL-8. CONCLUSIONS: These results demonstrate that both IL-1 alpha and TNF-alpha could induce keratocytes to produce nanogram levels of IL-6 but IL-1 alpha was a 30-fold more effective inducer. In contrast, neither cytokine could stimulate epithelial cells to make more than picogram quantities of IL-6. The abundant IL-6 synthesized by keratocytes may promote various activities including specific immune responses in surrounding lymphoid tissues.

Synergistic anti-herpes effect of TNF-alpha and IFN-gamma 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.

Interleukin-10 treatment can suppress stromal keratitis induced by herpes simplex virus type 1.

Herpes simplex virus type 1 (HSV-1) infection of the murine cornea induces an intense inflammatory response that can lead to permanent blindness. We tested whether IL-10, a cytokine that has anti-inflammatory properties, could suppress the development of stromal disease. Murine rIL-10 was inoculated intracorneally 4 before and again on days +2 and +5 relative to the time of topical HSV-1 corneal infection. Additionally, the mice received IL-10 i.p. at the time of virus administration and again 3 days post-infection. Four weeks post-infection, the incidence of blinding disease was 95% in the saline-treated controls but only 36% in the IL-10-treated animals. Histologic studies showed extensive cellular infiltrates in control corneas but not in those of the IL-10-treated eyes. Examination of the proinflammatory cytokine levels in the cornea 10 days after infection revealed that the presence of IL-2 was 10-fold lower and IL-6 some 50-fold lower than that found in the controls. IL-1 alpha levels were not reduced. The IL-10 treatment protocol employed did not suppress the systemic cellular or humoral immune responses to viral Ag, nor was the rate of HSV-1 clearance from the eye different from that seen in the controls. In vitro studies revealed that spontaneous production of IL-6 by excised normal corneas was inhibited by > 95% with low dose IL-10. IL-1 alpha synthesis was not inhibited. Collectively, these results indicate that IL-10 treatment can 1) suppress the production of certain cytokines produced by corneal cells, and 2) minimize ocular inflammation without compromising clearance of the infecting virus from the eye.

Differential regulation of granulocyte-macrophage colony-stimulating factor gene expression in human corneal cells by pro-inflammatory cytokines.

Neutrophils and Langerhans cells participate in inflammatory reactions within the human cornea. Because granulocyte-macrophage (GM)-CSF is a chemotactic and activating factor for these two cell types, we investigated whether this cytokine is produced by human corneal epithelial cells and corneal fibroblasts. Cultures of each cell type were exposed to increasing concentrations of IL-1 alpha or TNF-alpha. Culture supernatants were assayed for GM-CSF by using ELISA and cytokine mRNA levels were monitored by using reverse transcriptase-PCR. IL-1 alpha treatment of both cell types resulted in the appearance of GM-CSF mRNA and the production of > 480 pg protein/10(6) cells. However, TNF-alpha treatment yielded divergent results. Stimulation of epithelial cells with TNF-alpha resulted in the appearance of > 560 GM-CSF mRNA molecules per cell and production of > 1300 pg GM-CSF/10(6) cells. In contrast, stimulation of corneal fibroblasts resulted in < 16 GM-CSF mRNA molecules/cell and < 60 pg GM-CSF/10(6) cells. Binding studies with 125I-labeled TNF-alpha revealed that corneal fibroblasts had as many receptor sites as did corneal epithelial cells. Furthermore, corneal fibroblasts could respond to TNF-alpha-receptor-mediated signal transduction because they produced nanogram amounts of IL-6 after being treated with this cytokine. The results suggest that both cell types synthesize GM-CSF in response to IL-1 alpha, but that only corneal epithelial cells produce significant amounts of GM-CSF after TNF-alpha exposure. Differences in the responses of the two cell types to TNF-alpha may reflect a means of limiting accumulation of neutrophils and Langerhans cells and, thus, minimize corneal damage.

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.

Induction of interleukin-8 gene expression is associated with herpes simplex virus infection of human corneal keratocytes but not human corneal epithelial cells.

Interleukin-8 (IL-8) is a proinflammatory cytokine released at sites of tissue damage by various cell types. One important function of IL-8 is to recruit neutrophils into sites of inflammation and to activate their biological activity. Stromal keratitis induced by herpes simplex virus type 1 (HSV-1) is characterized by an initial infiltration of neutrophils. This study was carried out to determine whether cells resident in the cornea synthesize IL-8 after virus infection. Pure cultures of epithelial cells and keratocytes established from human corneas were infected with HSV-1, and the medium overlying the cells was subsequently assayed for IL-8 by an enzyme-linked immunosorbent assay. Cytokine mRNA levels in cell lysates were monitored by Northern (RNA) blot analysis. It was found that virus infection of keratocyte cultures led to the synthesis of IL-8-specific mRNA with more than 30 ng of IL-8 made per 10(6) cells. Neither UV-inactivated virus nor virus-free filtrates collected from HSV-1-infected keratocytes could induce IL-8 protein or mRNA, suggesting that viral gene expression was needed for induction of IL-8 gene expression. Unlike keratocytes, HSV-1-infected epithelial cells failed to synthesize IL-8 protein or mRNA. However, these cells readily produced both molecules following tumor necrosis factor alpha stimulation. HSV-1 had similar titers in both cell types. Thus, the failure to induce IL-8 synthesis was not due to an inability of the virus to replicate in epithelial cells. The capacity of HSV-1-infected corneal keratocytes to synthesize IL-8 suggests that these cells can contribute to the induction of the acute inflammatory response seen in herpes stromal keratitis.

Passive transfer of anti-herpes simplex virus type 2 monoclonal and polyclonal antibodies protect against herpes simplex virus type 1-induced but not herpes simplex virus type 2-induced stromal keratitis.

PURPOSE: To investigate whether passive transfer of antibodies to viral glycoproteins would protect against herpes simplex virus type 2-induced stromal keratitis. METHODS: Balb/c mice were infected on the scarified cornea with herpes simplex virus types 1 or 2 (HSV-1 and HSV-2, respectively), and monoclonal or polyclonal antibodies were administered intraperitoneally 24 hr later. Eyes were monitored for corneal opacity. Flow cytometry was used to examine the expression of glycoproteins on the surface of HSV-infected cells. RESULTS: Passive transfer of monoclonal antibodies to viral glycoproteins gB, gD, or gE or anti-HSV-2 hyperimmune serum were all highly effective (P < 0.005) at preventing blinding disease induced by HSV-1. In contrast, none of the antibody preparations could prevent stromal keratitis when the animals were challenged with various HSV-2 strains. However, antibody treatment could prevent the development of fatal encephalitis in the majority of HSV-2 infected hosts. Flow cytometry analysis revealed that gD and gB expression on the membranes of HSV-2 infected corneal epithelial cells isolated from excised corneas was substantially less (P < 0.005) than that detected on HSV-1 infected cells at both 24 and 48 hours postinfection. This antigenic difference was not due to the failure of HSV-2 to replicate in corneal epithelial cells in vivo. CONCLUSIONS: Decreased levels of membrane glycoprotein antigen expression may be one factor contributing to the refractiveness of HSV-2-induced ocular disease to humoral immunotherapy.

Cytokine expression in vivo during murine herpetic stromal keratitis. Effect of protective antibody therapy.

HSV-1 topical infection on the murine cornea can induce herpetic stromal keratitis (HSK), a T cell-mediated inflammatory response that results in blindness. To begin to decipher the molecular interactions involved in this infection, extracts of infected corneas were assayed for the presence of seven different cytokines by ELISA. The most prominent cytokines detected were IL-1 alpha and IL-6. Both were elevated by day 2 after infection, reached peak levels of 82 and 538 pg/cornea, respectively, at day 10, and then diminished over the next 10 days. In contrast, TNF-alpha concentrations were not elevated over that seen in uninfected corneas at any time during the 20-day observation period. IFN-gamma and granulocyte-macrophage CSF corneal concentrations were below the sensitivity of the assay. We investigated whether passive transfer of antibody to viral glycoprotein D, which prevents HSK, influenced the production of IL-1 alpha and IL-6. It was found that corneal concentrations of IL-1 alpha were reduced threefold and IL-6 was undetectable at day 10 in the antibody-treated hosts. The levels of IL-10 and IL-4 in uninfected control and antibody-treated hosts were also monitored. Neither of these two regulatory cytokines was associated with HSK development or effective antibody therapy. Naive corneas cultured in vitro spontaneously produced IL-1 and IL-6, indicating that cells resident in the cornea had the ability to synthesize these proinflammatory cytokines. Collectively, our results imply that IL-1 alpha and IL-6 may be important contributors to the development of HSK pathogenesis.