herpes simplex virus - type 1 ( hsv-1 ) infection of the human cornea can lead to a damaging inflammatory response known as herpes stromal keratitis ( hsk ) . according to the national eye institute , 50,000 new and recurring hsv-1 ocular infections are reported annually , and hsk is the leading cause of infectious blindness in the united states . during physical trauma or hsv-1 infection , cells resident in the cornea initiate an immune response through the production of proinflammatory mediators such as il-1 , il-6 , and the neutrophil chemoattractant cxcl8 ( homologous to mip-2 in the mouse ) [ 25 ] . mice also exhibit an inflammatory response to hsv-1 infection of the cornea which is marked by neutrophil infiltration at days 2 and 9 after infection ( p.i . ) cd8 t cells are required for viral clearance around day 8 after infection , while the cd4 t - cell subset has been described as having a role in the development of hsk and cd8 cell regulation after viral clearance has occurred [ 8 , 10 , 11 ] . the role of the neutrophil in the immune response to hsv-1 infection has not yet been fully explored . however , neutrophil depletion studies have demonstrated that in the absence of neutrophils cd8 , t - cell levels are reduced and viral clearance is limited leading to more severe disease development [ 12 , 13 ] . divito and hendricks   reported that neutrophil accumulation in addition to cd4 t - cell infiltration was necessary for hsk development . previous studies reported from our lab and others indicate a role for neutrophils in the secretion of t - cell - recruiting chemokines ip-10 ( interferon gamma - induced protein 10 or cxcl10 ) and mig ( monokine induced by gamma interferon or cxcl9 ) [ 15 , 16 ] . it is known that t lymphocytes express the receptor cxcr3 and may be recruited by the ligands ip-10 and mig and that antibody neutralization of ip-10 results in increased viral titers during hsv-1 corneal infection [ 17 , 18 ] . we previously used a model for delayed type hypersensitivity ( dth ) , a secondary immune response to hsv-1 antigen in the skin of mice , to demonstrate that the neutrophil acts as a source for both ip-10 and mig in the model . in the absence of neutrophil recruitment , thus , although the neutrophil has traditionally been described as merely a phagocytic cell , there is now increasing evidence to support the hypothesis that it may also function to bridge the response between the innate and adaptive immune system .   in the study presented here , we hypothesize that neutrophils play a role in t - cell recruitment into the hsv-1 infected cornea through the production of ip-10 . we describe the use of a model for the inflammatory response to a primary hsv-1 infection of the murine cornea and a novel ex vivo system for the study of primary hsv-1 infection in human corneas . data is presented from experiments designed to investigate the production kinetics of chemokine ip-10 and its receptors during infection . we also demonstrate the effect of cellular depletion of neutrophils , natural killer cells , and cd4 t cells on the level of ip-10 production and provide evidence for secretion of ip-10 by neutrophils , in vitro and in vivo , in both murine and human corneal tissue infected by hsv-1 . six - week - old female c57bl/6 mice were obtained from jackson laboratories ( bar harbour , me ) . all animals were cared for in accordance with federal , state , and local regulations . anti - mouse granulocyte mab rb6 - 8c5 was used for the depletion of neutrophils and was a gift from robert coffman ( dnax research institute , palo alto , ca ) . anti - mouse cd4 hybridoma clone gk1.5 was obtained from the american type culture collection ( atcc , manassas , va ) . antibodies were prepared from hybridoma lines as previously described . a combination of rabbit antiasialo gm1 ( wako pure chemical industries , ltd . , richmond , va ) and mab nk1.1 ( bd biosciences , san diego , ca ) were used for nk cell depletion . recombinant mouse il-1 and ifn- ) were purchased from r&d systems ( minneapolis , mn . ) .   for experiments investigating the protein kinetics of ip-10 , infections were achieved topically by applying 2  10  pfu/2  l hsv-1 strain re on the scarified cornea of the mice . for mrna investigation and cellular depletion studies , infection was performed using an intrastromal route to reduce intragroup variability . in brief , a pilot hole was formed through the epithelium of the cornea , with a 30-gauge disposable needle , through which a 32-gauge , 30  cm needle attached to a repeating dispenser ( hamilton , reno , nv ) was then inserted . 1  10 or 1  10  pfu hsv-1 re ( as indicated in the figure legend ) was injected intrastromally in a volume of 1  l . eyes were monitored for corneal opacity by visual observation under the dissecting microscope and graded as follows : 0 = clear cornea , 1 = slight corneal haze , 2 = moderate corneal opacity , 3 = severe corneal opacity but iris visible , 4 = severe corneal opacity with iris obscured , and 5 = necrotizing stromal keratitis . corneas were titrated for infectious virus on vero cell monolayers in a standard 48  h plaque assay .   in experiments where upregulation of mrna levels was investigated , corneas from 5 mice of the same experimental group were excised and pooled . the pooled corneas were homogenized for 30  s in 0.5  ml of rnawiz ( ambion inc . , austin , tx ) , while total rna purity and quantity were determined using the bio - rad smartspec 3000 spectrophotometer ( hercules , ca ) . ambion message sensor rt ( reverse transcriptase ) kit was used to convert 0.5  g total rna to cdna for real - time pcr analysis . bio - rad iq sybr green supermix in a 96-well plate format was used for analysis with a bio - rad icycler iq system . gapd mrna levels were used to normalize template loading variations and negative rt reactions were performed to ensure the absence of genomic contamination in the samples . mrna levels are reported as a fold increase over expression levels in untreated control corneas . sixteen corneas from 8 uninfected mice were excised , pooled , and incubated at 37c , 5% co2 in 5  mm edta / pbs for 20  min . after this time , epithelial sheets were removed with forceps under the dissecting microscope . the stromal layers of each cornea were minced and incubated in 1500  u / ml of collagenase type i ( sigma ) at 37c , 5% co2 , for 1  h. the digested stromal layers were washed repeatedly in dmem containing 20% fbs . resultant cells were cultured in a t25 tissue culture flask in minimal volume of dmem + 20% fbs medium . after 1 week of culture , cells were trypsinized and redistributed to the same flask to avoid clumping . at 90% confluency cells were passaged to a 12-well tissue culture plate at a density of 4  10  cells / well . cells were serum starved for 3 days prior to stimulation for 24  h with ifn- or il-1. each stimulation was performed in triplicate and chemokine production was quantitated by elisa . neutrophils were isolated from the bone marrow of mice as previously described . in brief , bone marrow cells extracted from the hind limbs of mice were gradient purified over histopaque 1119 and 1077 ( sigma , st . louis , mo ) . the enriched neutrophil band was washed in medium and treated with red blood cell lysis buffer ( sigma ) . neutrophil purity was established to be consistently > 99% , by hema3 ( biochemical sciences ) staining of cytospin slides . for in vitro stimulation assays , 1  10 neutrophils in 0.5  ml medium were placed in triplicate in 24-well tissue culture plates ( corning , new york , ny ) previously coated with newborn calf serum ( ncs ) . neutrophils were stimulated for 8  h at 37c , 5% co2 , with il-1 , ifn- , or hsv-1 . corneas were excised and cleaned of limbal tissue at the indicated times and processed by homogenization ( 30 seconds in a tissue tearer ; biospec products , bartlesville , ok ) and sonication ( 15  s ) in a total of 0.5  ml rpmi + 10% ncs unless stated otherwise . corneal samples were clarified by centrifugation at 150  g for 10  min and supernatants were analyzed by elisa . in cell studies , supernatants were collected and assayed for secreted chemokine levels by elisa . murine ip-10 and mip-2 elisa assay kit sensitivities were 2.2 and 1.2  pg / ml , respectively , and the human ip-10 assay kit had a mean minimal detectable dose of 1.67  pg / ml . all kits were obtained from r&d systems ( minneapolis , mn . ) .   to achieve in vivo depletion of cellular subsets , 0.5  mg of mab rb6 - 8c5 ( neutrophil depletion ) , 0.5  mg gk1.5 ( cd4 t - cell depletion ) , or 1  mg antiasialo gmi admixed with 0.1  mg nk1.1 ( nk cell depletion ) were administered by intraperitoneal injection to mice 3  h prior to hsv-1 challenge [ 13 , 2123 ] . mice were challenged by intrastromal injection of 1  10  pfu of hsv-1 and corneas excised at the times indicated . facs analysis of spleen cells was performed using gk1.5 and nk1.1 to quantify cd4 t - cell and nk - cell depletion levels , respectively . excised corneas were processed by homogenization and sonication to produce a lysate for chemokine analysis by quantitative elisa . four , 4  mm corneal buttons were punched from each cornea using an arch punch ( c.s . corneal buttons were placed in 24-well tissue culture plates containing 0.5  ml serum free rpmi 1640 media for incubation at 37c , 5% co2 . corneal button surfaces were scarified using an 18-gauge needle to mimic the murine topical infection protocol . for hsv-1 infected samples 1  10 pfu of virus was added to the corneal button in the well . infected and uninfected corneas were then incubated in the presence or absence of purified human neutrophils , 1  10 neutrophils / well at 37c , 5% co2 , for 24  h. media , cells , and corneal buttons were removed and processed by sonication ( 30  s ) . this model permits the study of the interaction between resident corneal cells , neutrophils , and virus in the absence of other cell types which could be recruited to the site of inflammation in vivo . gradient purification was achieved following a protocol equivalent to that described above for the murine neutrophils . student 's t test was performed to determine significant differences between experimental and control groups which each contained a minimum of three mice or three human corneal donors . a representative experiment is shown in each figure with experiments having been performed multiple times . it has been shown previously in the murine model for hsv-1 corneal infection that the virus is cleared within 8 days due to the recruitment of cd8 t lymphocytes . with the focus of this study being the t - cell recruiting chemokine ip-10 , it was necessary to establish that the t lymphocytes recruited to the hsv-1 infected cornea were expressing the cxcr3 receptor for ip-10 . mice were infected by intrastromal injection of 1  10  pfu hsv-1 and corneas harvested 26 days post infection ( p.i . ) for mrna analysis . in figure 1 , cxcr2 was determined to be upregulated > 240-fold above levels found in the controls at day 2 p.i . upregulation of cxcr2 expression was significantly reduced by day 4 and 6 ( 84- and 21-fold , resp . ) . cxcr3 mrna was observed to be most significantly upregulated at day 6 p.i with an expression level > 28-fold higher than that of the uninfected control . this coincides with a > 39-fold upregulation in the expression of the cd8 marker on infiltrating cells ( data not shown ) . these results indicate that a marker for neutrophil recruitment ( cxcr2 ) peaks in the cornea at day 2 p.i . with hsv-1 and that cxcr3 is present in the infected cornea at a time point associated with the recruitment of cd8 t cells . having demonstrated the time points for expression of a neutrophil receptor and t - cell receptor in the model , experiments proceeded to investigate the potential to produce ip-10 in a time frame appropriate for neutrophils to be the source for this t - cell - recruiting chemokine . figure 2 shows that levels for upregulation of the mrna for mip-2 were found to be elevated > 724-fold at day 2 p.i . dropping to > 93-fold at day 4 p.i . expression levels for ip-10 mrna were highly upregulated ( 1179-fold over control ) at day 2 after infection but the level of upregulation was reduced significantly by day 4 after infection ( decreased to 24-fold over control ) . thus , ip-10 message was found at the same time point as the peak in cxcr2 and mip-2 mrna expression which marked the infiltration of neutrophils . in addition , ip-10 mrna expression was observed to be elevated prior to the time point for infiltration of t cells as suggested by the presence of cxcr3 mrna ( day 6 p.i . ) and cd8a and cd8b mrna ( data not shown ) .   although chemokine and receptor mrna expression profiles provided supporting evidence for the hypothesis that neutrophils may act as a cellular source for the t - cell - recruiting chemokine ip-10 , it was still necessary to demonstrate the presence of the ip-10 protein within the model . the kinetics of ip-10 production were determined over an 8 day period after topical infection of corneas with 2  10  pfu hsv-1 . at each time point indicated on figure 3 , mice were monitored for corneal opacity as an indicator of the level of inflammation ( figure 3(a ) ,  ) . mice were then sacrificed and corneal lysates produced to determine virus titers by plaque assay ( figure 3(a ) ,  ) . ip-10 protein levels were quantitated by elisa and are shown in figure 3(b ) . figure 3(a ) demonstrates that viral load (  ) was significantly reduced by day 8 p.i . compared to the day 0 inoculum and that corneal opacity due to cellular infiltration was significantly increased (  ) in the same time frame . the kinetics of ip-10 production are marked by a significant peak of 861.8  pg / ml  136.2 sem at day 2 after infection ip-10 levels remain significantly high until day 7 and return to constitutive levels at day 8 p.i . ( 130.4  pg / ml  70.2 sem and 30.1  pg / ml  7.6 sem , resp . ) . time point for peak ip-10 protein production agrees with that observed for peak ip-10 mrna and peak cxcr2 mrna expression . murine corneas have been reported to be productive for mrna for various chemokines including mip-2 and ip-10 in a balb / c hsv-1 infection model [ 25 , 26 ] . as reported by lundberg et al . is produced at high levels within the c57 bl/6 hsv-1 infected cornea , experiments were initiated to determine the potential of resident corneal cells from this strain of mouse to act as a source of ip-10 . mouse corneas were excised from uninfected hosts and incubated ex vivo for 24  h. uninfected corneas were devoid of lymphocytes normally recruited during the inflammatory response to hsv-1 . constitutive levels of ip-10 protein were measured from samples which were excised and processed immediately . in figure 4 , it can be observed that constitutive levels of ip-10 were low ( 38.6  pg / ml  2.8 sem ) but that incubation for 24 hours led to a > 5-fold increase in ip-10 levels . this data demonstrates that resident cells of the cornea have the potential to secrete ip-10 protein in response to excision trauma and may , therefore , contribute to the production of the chemokine during virus infection in the c57 bl/6 model . tissue culture experiments were performed to further explore the potential for production of ip-10 by resident corneal cells as opposed to infiltrating leukocytes . corneas from uninfected mice were excised and processed , as described in the materials and methods , to establish a primary corneal fibroblast cell line . 4  10 fibroblast cells were plated under serum - starved conditions and stimulated with the proinflammatory cytokines il-1 or ifn- at 10  ng / ml for 24  h. the cell supernatants were assayed and levels of ip-10 production are shown in figure 5 . within the time frame of the stimulation , expansion of the cell population was not observed to occur ( data not shown ) . fibroblast cells produced high levels of ip-10 after stimulation with ifn- ( 3383  pg / ml  306 sem ) . although il-1 stimulation also lead to significant ip-10 production compared to the media control , the level was > 24-fold lower than that observed for ifn- stimulation . the ability of murine corneal fibroblast cells to produce ip-10 in response to stimulation with proinflammatory mediators was confirmed . neutrophils are rapidly recruited to the hsv-1 infected cornea and are present in high numbers at 2 day after infection . we observe this to also be the time point for peak ip-10 production in the model . we performed in vitro stimulation of bone marrow - derived neutrophils with ifn- , il-1 or hsv-1 in order to determine the neutrophils potential to secrete ip-10 . figure 6 illustrates that neutrophils produce high levels of ip-10 ( 4068  pg / ml  50 sem ) after incubation with 10  ng / ml ifn- for 8  h but did not produce significant levels of ip-10 after stimulation with the same concentrations of il-1. neutrophils also responded to the presence of hsv-1 at a multiplicity of infection ( m.o.i ) of 0.1 by producing 937  pg / ml  126.5 sem ip-10 . a 10-fold increase in number of virus particles led to a 3.9-fold increase in chemokine production . thus , neutrophils were demonstrated to have the ability to produce ip-10 in response to both proinflammatory cytokines and the presence of hsv-1 . these results indicate a potential for neutrophils to contribute to the production of ip-10 within the hsv-1 infected cornea . having established that neutrophils were a potential cellular source of ip-10 in vitro , we performed in vivo depletion of these cells to determine their contribution to chemokine production during a primary hsv-1 corneal infection . nk and cd4 t cells infiltrate the hsv-1-infected cornea   and have the potential to secrete cytokines including ifn- which could in turn stimulate neutrophils to secrete ip-10 . we therefore included depletion of nk and cd4 cells in this investigation . peripheral neutrophil depletion was assessed by differential staining of tail vein blood smears to be reduced by > 84% of the cell number observed in the igg - control - treated mice . cd4 t - cell and nk - cell depletions were monitored by facs analysis of spleen cells and cell numbers determined to be reduced by > 89% and > 67% , respectively , when compared to the igg control . two and three days post infection , corneas were harvested and analyzed to determine the effect of cellular depletion on ip-10 production . neutrophil depletion by antibody treatment with rb6 8c5 produced a significant 2-fold reduction in ip-10 protein levels when compared to the igg group on day 2 p.i . by day 3 p.i . ip-10 levels in the control corneas were greatly reduced ( 299  pg / ml  103 sem ) when compared to those observed at day 2 p.i . ( 931.4  pg / ml  111 sem ) , and the effect of neutrophil depletion was abrogated . nk depletion also led to a 2-fold reduction in ip-10 levels at day 2 p.i . the levels of ip-10 in the nk cell depletion group were not significantly different from that of the control . cd4 t - cell depletion was not conducted at day 2 p.i . and does not significantly affect ip-10 production at day 3 p.i . in the model . these results indicate a role for both neutrophils and natural killer cells in the production of ip-10 in the model .   in order to relate the findings of the murine model to infection of human corneal tissue with hsv-1 , a novel ex vivo model was designed using donated human corneas . as described in the material and methods , this model permits the interaction of resident corneal cells , hsv-1 , and neutrophils to be studied in the absence of other recruited inflammatory leukocytes which may be present in vivo . corneal tissue for each experiment was obtained from a minimum of three independent donors and sectioned into 4  mm buttons . corneal buttons from a single cornea were placed in one of the 4 test groups : control incubation with media ( media column ) , incubation with 1  10 neutrophils ( pmn column ) , infection with 1  10  pfu hsv-1 , a m.o.i . = 1 with respects to the pmn ( hsv-1 column ) or infection with hsv-1-plus incubation with neutrophils ( pmn + hsv-1 column ) . corneal buttons incubated in isolation produced 33.7  pg / ml ip-10  13.3  sem . infection of corneal buttons with hsv-1 failed to significantly increase ip-10 levels above this control . incubation of corneal buttons in the presence of neutrophils led to a 3-fold increase in the production of ip-10 ( 96.8  pg / ml  21.9  sem ) . hsv-1-infected corneas incubated in the presence of neutrophils also produced significant levels of ip-10 ( 100.4  pg / ml  25.7  sem ) . neutrophils incubated in isolation under conditions equivalent to those of the corneal tests produced relatively low levels of ip-10 which were significantly elevated on addition of hsv-1 . levels of cxcl8 production were also monitored for each corneal button and found to be significantly higher in hsv-1-infected corneas compared to the uninfected control ( data not shown ) . cxcl8 is a proinflammatory chemokine produced at high levels during hsv-1 infection of the murine and human cornea . it is responsible for the recruitment of the neutrophil to the site of infection and marks the development of an inflammatory response .   corneal infection with hsv-1 in the absence of neutrophils levels of ip-10 were observed to be elevated when traumatized or hsv-1-infected corneas were incubated with neutrophils . these results indicate that cellular interaction between corneal cells and neutrophils contribute to ip-10 production in a novel ex vivo human model and are supportive of the findings described above for the mouse model . it is known that both neutrophils and cd8 t cells are key players in the inflammatory response to primary hsv-1 infection of the cornea and that cd8 t cells are required for viral clearance [ 12 , 13 , 24 ] . although it is reported that neutrophils are also involved in the process of viral clearance , their specific mode of action is not yet fully understood . has previously demonstrated that human neutrophils can be stimulated to produce ip-10 and that neutrophil secretions are potent chemoattractants for nk and th1 cells . the requirement for ip-10 and cxcr3 during t - cell recruitment and activation in various inflammatory models has been established with the use of cxcr3 knockout mice . a model for bordetella respiratory infection reported ip-10 induction and cxcr3 expression early during the inflammatory response and reported reduced lymphocyte and nk recruitment when cxcr3 knockout mice were investigated . the requirement for cxcr3 signaling for mobilization and activation of nk and cd8 t cells and the requirement for ip-10 for viral clearance during hsv-1 and hsv-2 infections has also been described [ 3033 ] . in addition , neutrophils have been identified as a cellular source for ip-10 during ulcerative colitis disease and are required to sustain cd8 cell recruitment during acute myocarditis [ 34 , 35 ] . we have previously published data which indicates that neutrophils contribute to production of ip-10 and recruitment of cd4 t cells in an inflammatory model of delayed type hypersensitivity ( dth ) to hsv-1 in the mouse . we now present data which confirms ip-10 is produced in a model for corneal inflammation due to hsv-1 infection and that neutrophils are a source of this chemokine in vivo .    to generate further evidence linking the involvement of neutrophils in t - cell recruitment to the hsv-1-infected cornea due to the technical difficulties associated with the isolation and quantitation of cells infiltrating the cornea , we chose to use a semiquantitative real - time pcr screening assay to monitor receptor expression within the murine model . we established that the assay was predictive for cellular infiltration by confirming that cd3 and cd8 mrna ( t cell markers ) were significantly upregulated in corneas at 6 days p.i . ( data not shown ) . continued screening demonstrated upregulation of mrna for both the chemokine ip-10 and its receptor , cxcr3 , at day 2 and 6 p.i . of these cell types , the neutrophil is known to be recruited to the site of hsv-1 infection in the cornea . these results may be interpreted to predict neutrophil recruitment at day 2 p.i . which coincides with ip-10 mrna upregulation and that cxcr3 expressing cells infiltrate to peak numbers by day 6 p.i . these findings are in accordance with those of araki - sasaki et al . and cook et al . who reported ip-10 and cxcr3 message upregulation in the corneas of mice infected with hsv-1 [ 36 , 37 ] . taken together , these results confirm that neutrophils are recruited within an appropriate time frame to be a cellular source for ip-10 . although previous research has begun to explore the role of the neutrophil in production of t - cell - recruiting chemokines at the site of inflammation due to viral infection , other cell types in the corneal model may contribute to ip-10 production . studies on human corneal epithelial ( hce ) and fibroblast ( hcf ) cells by our research group have described the ability of these cells to express ip-10 after stimulation with proinflammatory mediators such as il-1 and ifn- [ 38 , 39 ] . corneal production of ip-10 during the inflammatory response to other viral infections such as respiratory syncytial virus has been established . in the study presented here , we confirm that murine corneas behave in a similar manner and secrete ip-10 constitutively at low levels and significantly elevated levels in response to proinflammatory mediators such as il-1 produced by physical trauma ( e.g. , excision of cornea for ex vivo incubation ) . furthermore , we show that the murine corneal fibroblast cells in isolation produce high levels of ip-10 when stimulated with ifn- and to a lesser extent il-1. the cells resident in the cornea can thus be demonstrated to have the potential to contribute to ip-10 levels during infection of the cornea . we have previously demonstrated the ability of murine neutrophils to secrete ip-10 in vitro in response to stimulation with ifn- but not il-1 . here we expand the study to investigate the ability of purified neutrophils to secret ip-10 after in vitro stimulation with hsv-1 . neutrophils produce increasing levels of ip-10 in response to hsv-1 stimulation in a dose - dependent manner . the level of ip-10 observed in the assay after stimulation with hsv-1 at a m.o.i of 1 were equivalent to those observed for stimulation with 10  ng / ml ifn- , a known signal for ip-10 production in other models . these in vitro stimulation experiments provide evidence that within the hsv-1-infected murine cornea there is potential for either resident corneal cells or infiltrating neutrophils to be a source of ip-10 . to investigate the relative contribution to ip-10 production by resident corneal cells versus infiltrating neutrophils we conducted in vivo cellular depletion experiments . antibody - mediated cellular depletion of neutrophils , natural killer cells ( nk ) or cd4 t cells has been shown to be an effective method for the study of cellular function within inflammatory models [ 13 , 2123 ] . we have demonstrated previously that such depletion leads to a reduction in numbers of the cells recruited to the site of inflammation within a dth model . here we describe how ip-10 production was significantly reduced in response to reduction in numbers of either neutrophil or nk cells from the host . nk cells are not reported to be a source of ip-10 but do express the receptor for the chemokine ( cxcr3 ) . this suggests that in our study the reduction in ip-10 production observed after nk cell depletion is due to an indirect effect . nk cells are a source of ifn- which we demonstrate here to be an effective stimulant for the production of ip-10 by neutrophils . thus the reduction in ip-10 production after nk cell depletion may be explained in terms of a reduction in ifn- stimulation of neutrophils . this conclusion is supported by observations in an ifn- gene knockout mouse which demonstrate reduced control of virus replication during corneal infections with hsv-1 . although cd4 t cells may also secrete ifn- , results from depletion of this cell type do not indicate that the cd4 t cell is required for ip-10 production in the in vivo model within the time frame studied . the depletion studies thus support the hypothesis that neutrophils act as a major cellular source for ip-10 during hsv-1 infection of the cornea and that loss of ip-10 production due to neutrophil depletion can not be compensated for by ip-10 production by resident corneal cells .   in the final set of data presented we investigated ip-10 production within an ex vivo model for human corneal infection by hsv-1 . previous work from our research group has focused on the chemokine and cytokine expression patterns from cultured hce and hcf cells . in this study , we chose to maintain the potential for communication between these cell types by using corneal buttons comprised of all cell layers normally present in the human cornea . therefore , we anticipated that the human model would reflect the findings from the murine in vivo model . furthermore , the novel ex vivo human model described was designed to permit the study of interactions between resident corneal cells and purified cell populations , such as the neutrophil , in isolation from other cell types normally recruited to the site of inflammation in vivo .   in the human ex vivo model levels of ip-10 produced by corneas increase significantly when the cornea was incubated in the presence of neutrophils . an equivalent result was observed when the corneas were infected with hsv-1 and incubated with neutrophils . this data suggests that proinflammatory mediators secreted by resident corneal cells in response to either physical trauma ( excision ) or viral infection lead to neutrophil production of ip-10 . corneal cells are reported to produce a variety of cytokines during response to viral infection including il-1 , il-6 , cxcl8 , and low levels of ifn- . although we found that murine neutrophils secreted high levels of ip-10 due to exposure to hsv-1 ( figure 6 ) this was not observed to occur to the same extent when human neutrophils were incubated with hsv-1 ( figure 8) . however , the results from the human ex vivo assay do indicate that ip-10 production by neutrophils in vivo is stimulated in part by communication from the cornea rather than as a direct response to neutrophil contact with the virus . it should be noted that in the ex vivo human model availability of virus for direct contact with the neutrophil is limited when the virus infects the corneal cells ( viral eclipse ) . in conclusion , we present here evidence that neutrophils are a key source of the t - cell - recruiting chemokine , ip-10 , during hsv-1 infection of the cornea . our results suggest that cells resident in the cornea produce proinflammatory mediators in response to hsv-1 infection which recruit and stimulate neutrophils to produce ip-10 . the neutrophils thereby contribute to the production of ip-10 during the early time point after infection ( day 2 ) . we hypothesize that this ip-10 leads in part to recruitment of nk cells which promote the inflammatory cascade by further stimulation of neutrophils with ifn- to increase ip-10 levels .
in this study we show that murine and human neutrophils are capable of secreting ip-10 in response to communication from the hsv-1 infected cornea and that they do so in a time frame associated with the recruitment of cd8 + t cells and cxcr3-expressing cells . 
 cellular markers were used to establish that neutrophil influx corresponded in time to peak ip-10 production , and cellular depletion confirmed neutrophils to be a significant source of ip-10 during hsv-1 corneal infection in mice . a novel ex vivo model for human corneal tissue infection with hsv-1 
 was used to confirm that cells resident in the cornea are also capable of stimulating neutrophils to secrete ip-10 . 
 our results support the hypothesis that neutrophils play a key role in t - cell recruitment and control of viral replication during hsv-1 corneal infection through the production of the t - cell recruiting chemokine ip-10 .