The Role of and in Helminth-Induced

Eric Pearlman,1'2 Laurie R. Hall,1 Alan W. Higgins,1 David S. Bardenstein,2 Eugenia Diaconu,2 Fred E. Hazlett,1 Jamie Albright,1 James W. Kazura,1 and Jonathan H. Lass2

PURPOSE. Intrastromal injection of mice with antigens from the parasitic helminth that causes river blindness (Onchocerca volvulus) induces recruitment to the corneal stroma at the time of maximum corneal opacification and neovascularization. The present study was conducted to examine the role of eosinophils and neutrophils in onchocercal keratitis in control C57B1/6 mice and in interleukin-5 knockout (IL-5"7"") mice. /- METHODS. C57B1/6 and IL-5~ mice were immunized subcutaneously and injected intrastromally with soluble 0. volvulus antigens. Mice were killed at various times thereafter. Development of keratitis was assessed by slit lamp examination, and inflammatory cells in the were identified by immunohistochemistry. RESULTS. A biphasic recruitment of inflammatory cells was observed in C57B1/6 mice; neutrophils predominated during the first 72 hours after intrastromal injection and subsequently declined, whereas eosinophil recruitment increased as time elapsed and comprised the majority (90%) of cells in the cornea by day 7. In contrast, neutrophils were the predominant inflammatory cells in IL-5~/~ mice at early and late time points and were associated with extensive stromal damage and corneal opacification and neovascularization. Eosinophils were not detected in these mice at any time. CONCLUSIONS. In the absence of eosinophils, neutrophils can mediate keratitis induced by helminth antigens. Together with the early neutrophilic infiltrate in control animals, these observations indicate that neutrophils have an important role in onchocercal keratitis. (Invest Ophthalmol Vis Sci. 1998;39:1176-1182)

n estimated 18 million persons are infected with the developed.4 Corneal opacification and neovascularization can parasitic helminth Onchocerca volvulus, which is the be induced after intrastromal or subconjunctival injection of second leading cause of infectious blindness world- live parasites5"8 or soluble O. volvulus antigens.9"11 Using a A1 wide. Blindness develops in response to larval penetration murine model for O. volvulus-induced keratitis in which hel- from the skin to the cornea. The parasites elicit little or no minth antigens are injected intrastromally, we demonstrated in inflammatory response while alive; however, after larval death a previous study that development of keratitis is dependent on and degeneration, a localized is induced that prior immunization and the presence of sensitized T cells and results in corneal opacification and neovascularization.2'3 A is associated with a predominant T-helper type 2 (Th2) re- chronic inflammatory response to repeated larval invasion sponse (interleukin [IL]-4 and IL-5 > interferon-y [LFN-y]).12 causes sclerosing keratitis, which is the most common form of We also showed that eosinophils are the predominant inflam- river blindness.2'3 matory cells in the cornea after injection of parasite antigens Because of the paucity of from infected people, and that the severity of keratitis is associated with the number animal models for O. volvulus-induced keratitis have been of eosinophils in the cornea. For example, IL-4 - deficient mice develop less severe keratitis than do control animals, and sig- nificantly fewer eosinophils are present in corneas.1213 Con- From the Division of Geographic Medicine, the 'Departments of versely, systemic administration of recombinant IL-12 results in Medicine and 2Ophthalmology, Case Western Reserve University, and exacerbated keratitis, which is associated with increased eo- University Hospitals of Cleveland, Ohio. sinophils in the cornea.14 Furthermore, ultrastructural analysis Supported by grants EY10320 (EP), EY11373 (JHL), AI35938 (JWK) from the National Institutes of Health, Bethesda, Maryland; by of these corneas shows extensive infiltration of eosinophils, new investigator award 0720 (EP) from Burroughs Wellcome, Triangle some of which have the appearance of degranulated cells.15 Park, North Carolina; by a grant from the Research to Prevent Blind- In the present study, we examined the temporal recruit- ness, New York, New York; and by a grant from the Ohio Lions Eye Research Foundation, Columbus. ment of to the corneas of control, immunocom- -/ Submitted for publication October 16, 1997; revised February 3, petent C57B1/6 mice and IL-5 gene knockout (IL-5 ~) mice 1998; accepted February 11, 1998. that are deficient in their ability to produce eosinophils.16 We Proprietary interest category: N. found that neutrophils are prominent cells early in develop- Reprint requests: Eric Pearlman, Division of Geographic Medicine, Case Western Reserve University School of Medicine, W137, 2109 ment of onchocercal keratitis and can mediate corneal disease Adelbert Road, Cleveland, OH 44106-4983. in the absence of eosinophils.

Investigative Ophthalmology & Visual Science, June 1998, Vol. 39, No. 7 1176 Copyright © Association for Research in Vision and Ophthalmology

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MATERIALS AND METHODS reacts with a surface component of murine neutrophils but not with eosinophils.19 Animals Anti-major basic protein (1:1000) and 7/4 mAb (1:100) Interleukin-5 gene knockout mice were produced as described were diluted in 1% fetal calf serum in 0.05 M Tris-buffered by Kopf et al.16 and were back-crossed to a C57B1/6 back- saline (pH 7.6). Slides were incubated at room temperature in ground. Breeding pairs were kindly sent to us by Bruce Blazar a humid chamber for 2 hours. Biotinylated goat anti-rabbit Ig and Edward Pearce (Cornell University, Ithaca, NY) with per- (Dako, Carpenteria, CA) diluted 1:200, or prediluted biotinyl- mission from Manfred Kopf (Basel Institute of Immunology, ated rabbit anti-rat Igs (Rat Link; BioGenex, San Ramon, CA) Switzerland). Animals were bred in the specialized animal was added for 30 minutes, followed by a similar incubation module in the Animal Resource Center at Case Western Re- with prediluted conjugated streptavidin serve University, and females were immunized when aged 6 (BioGenex). Positive reactivity was detected using substrate weeks. Age and sex-matched C57B1/6 mice (Charles River (Vector Red; Sigma, St. Louis, MO) containing 12 mg levami- Laboratories, Wilmington, MA) were used as control animals. sole (Sigma), followed by counterstaining with modified Harris' All procedures conformed to the ARVO Statement for the Use hematoxylin (Richard-Allen, Kalamazoo, MI). At least two of Animals in Ophthalmic and Vision Research. 5-jLtm sections per cornea were counted directly for positive reactivity with anti-MBP. Parasite Antigens Subcutaneous nodules containing adult O. volvulus parasites Cytokine Gene Expression in Mouse Corneas were surgically removed from patients in Cameroon. The pro- The method for extracting corneal RNA and reverse transcrip- cedure reduces the number of circulating microfilarial larvae, tion-polymerase chain reaction conditions have been de- thereby reducing levels of disease in the person and transmis- scribed in detail previously.12"14 Briefly, corneas were dis- sion in the community.' Nodules were kindly provided by Sara sected from four mice with similar clinical responses, and RNA Lustigman at the New York Blood Center (New York, NY), and was extracted in 500 JLLI RNA STAT60 (Tel-Test; Friendswood, Thomas Unnasch at the University of Alabama, Birmingham. TX) using micro—tissue grinders (Kontes, Vineland, NJ). Com- Adult worms were isolated after digestion of the plementary DNA (cDNA) was synthesized using Superscript II nodular material as described.12 Parasites were homogenized reverse transcriptase (Gibco), and the polymerase chain reac- using a tissue grinder and were centrifuged to remove insolu- tions were performed with Taq DNA polymerase (Gibco) using ble material. Soluble O. volvulus antigens were sterile filtered, a thermal cycler (Omnigene; National Labnet, Woodbridge, and protein concentration was adjusted to 1 mg/ml. NJ). Reaction conditions were as follows: 1 minute at 95°C, 1 minute at 62°C, and 1.75 minutes at 72°C. For each set of Immunization and Intrastromal Injections primers, a positive sample (cDNA from anti-CD3-stimulated Mice were sensitized to parasite antigens by three weekly spleen cells) and a negative sample (distilled H2O) were run in subcutaneous immunizations with 10 jug O. volvulus antigens parallel. in Hanks' balanced salt solution (Gibco, Gaithersburg, MD) Primer sequences and cycle numbers were as follows: together with adjuvant containing squalene (Aldrich Chemical, hypoxanthine phosphoribosyl-transferase (HPRT): sense: GTT Milwaukee, WI), Tween 20 (Fisher, Fair Lawn, NJ), and plu- GAA TAC AGG CCA GAC TTT GTT G; antisense: GAT TCA ACT ronic acid (Pluronic L-121; BASF, Parsippany, NJ) at a 1:1 ratio. TGC GCT CAT CTT AGG C; internal: GTT GTT GGA TAT GCC For intrastromal injections, a 30-gauge needle was vised to CTT GAC (27 cycles); IL-4: sense: TAG TTG TCA TCC TGC TCT scarify the cornea, and 10 /xg O. volvulus antigens in 10 /xl was T; antisense: CTC AGT ACT ACG AGT AAT CCA; internal: AGG injected into the corneal stroma of the right eye using a 33- GCT TCC AAG GTG CTT CGC A (30 cycles); IFN-y: sense: TGT gauge needle attached to a gas-tight syringe (Hamilton, Reno, TAC TGT CAC GGC ACA GTC ATT; antisense: GTG GAC CAC NV). This procedure is highly reproducible with less than 3 ju-1 TCG GAT GAG CTC ATT: internal: ACC TCA GAC TCT TTG of fluid leakage from the wound.12 Clinical disease was quan- AAG TCT TGA (33 cycles); and IL-5: sense: GTG AAA GAG ACC tified by determining corneal opacification and neovasculariza- TTG ACA CAG CTG; antisense: CAC ACC AAG GAA CTC TTG tion using the scoring system described previously.1217 CAG GTA; internal: GGG GGT ACT GTG GAA ATG CTA T (32 cycles). Polymerase chain reaction products were separated by Detection of Eosinophils in Blood agarose gel electrophoresis and visualized after Southern hy- bridization by using fluorescein-deoxyuridine triphosphate- Blood was collected retro-orbitally, and differential staining labeled probes (Enhanced Chemiluminescence; Amersham, Ar- was performed using a modified Wright-Giemsa Stain (Diff- lington Heights, IL). Quik; Dade Diagnostics, Aguada, PR). Immunohistochemistry Cytokine Production by Splenocytes Mouse eyes were removed and fixed in 10% formalin for at Spleens were removed by standard aseptic technique, and red least 24 hours, followed by processing in a tissue processor cells were lysed with 0.01 M Tris (pH 7.2) containing 0.75% (Tissue-Tek VIP; Sakura Finetechnical, Tokyo, Japan). Prepara- ammonium chloride. Splenocytes were then suspended in tion of eyes for paraffin embedding and sectioning was carried Rosewell Park Memorial Institute (RPMI) medium containing 1 out according to standard methods. Five-micrometer sections mM sodium pyruvate, 2 mM L-glutamine, 20 mM HEPES, 100 were immunostained with rabbit antiserum to murine eosino- U/ml penicillin, 100 /xg/ml streptomycin, and 10% heat-inacti- phil major basic protein (MBP)18 kindly provided by Kirsten vated fetal calf serum. Duplicate wells containing 5 X 105 cells Larson and Gerald Gleich at the Mayo Clinic (Rochester, MN) were stimulated with 2 )Ltg/ml anti-CD3 (2C11; kindly provided or rat monoclonal antibody 7/4 (Serotec, Oxford, UK), which by Thomas Forsthuber, Department of Pathology, Case West-

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1200n O Neutrophils Eosinophils 0) 800- 75 T

"55 O

Days after intrastromal injection

FIGURE 1. Temporal recruitment of eosinophils and neutrophils to corneas of C57B1/6 mice in helminth-mediated keratitis. C57B1/6 mice were sensitized by repeated subcutaneous immunization with Onchocerca volvulus antigens, as described in the Materials and Methods section. Ten days after the last immunization, parasite antigens were injected directly into the corneal stroma to induce keratitis. Animals were killed at indicated times after intrastromal injection, eyes were immersed in formalin, and adjacent 5-jLim paraffin sections were immu- nostained with antiserum to eosinophil major basic protein or with antineutrophil monoclonal antibody (mAb) 7/4. Sites of mAb reactivity were visualized using Vector Red, and eosinophils and neutrophils per 5-/xm section were counted at high magnification. Data are mean ± SD of five mice per group at each time point. Similar results were obtained in a repeat experiment.

ern Reserve University) in a final volume of 200 /xl and incu- hours after intrastromal injection and increased to 1106 ± 90 bated at 37°C in 5% CO2 for 72 hours. by day 1 (90% of total cells in the cornea). By day 3, the number Interferon-y, IL-4, and IL-5 were measured by two-site of neutrophils declined to 708 ±159 and represented 50% of enzyme-linked immunosorbent assay, as described, using the the cell infiltrate. By day 7, the number of neutrophils had mAbs R46A2 and XMG1.2 for IFN-y; BVD-6 and BVD-4 for IL-4; further decreased to 17 ± 10 per section, and they were absent and TRFK-4 and TRFK-5 for IL-512 Recombinant murine IFN-y in many corneas. (Genzyme, Cambridge, MA), and IL-4 and IL-5 (PharMingen, San Diego, CA) were used to generate standard curves. Diminished Blood Eosinophils and Elevated Blood Neutrophils in Interleukin-5 Gene Statistics Knockout Mice Statistical analysis was performed using Student's £-test. P < As a first step in determining the role of neutrophils in the 0.05 was considered significant. absence of eosinophils, we examined the effect of IL-5 defi- ciency on circulating eosinophils and neutrophils after immu- nization with parasite antigens. C57B1/6 and IL-5-/~ mice were RESULTS immvinized and injected intrastromally with O. volvulus anti- Eosinophil Recruitment to the Cornea of Control gens, and the percentage of blood eosinophils was determined C57B1/6 Mice 7 days later. C57B1/6 mice had 311 ± 1.2% eosinophils com- pared with 0.17 ± 0.2% in IL-5-/~ mice (P < 0.01). Con- Previous studies demonstrated that immunized mice injected versely, neutrophils were significantly higher in IL-5~/- mice intrastromally with O. volvulus antigens exhibit maximum than in C57B1/6 mice (34.6 ± 9-3% versus 16.8 ± 3.7%; P < corneal opacification and neovascularization after 4 to 10 days 0.01). The remaining blood leukocytes were mononuclear. and that eosinophils are prominent in the corneal stroma at this time.12 To determine the kinetics of recruitment to the cornea, immunized C57B1/6 mice were killed at 12 hours Corneal Opacification and Neovascularization in and 1, 3, or 7 days after intrastromal injection of O. volvulus, Interleukin-5 Gene Knockout Mice and immunostained with Ab specific for eosinophils and neu- To determine whether helminth-mediated keratitis is in- trophils. Cells were counted at high magnification. duced in animals that are deficient in eosinophils, IL-5-/~ Few or no eosinophils were detected in the corneal mice were immunized and injected intrastromally with O. stroma 12 hours and 1 day after intrastromal injection (Fig. 1). volvulus antigens, as described in the Materials and Methods However, the number of eosinophils increased to 455 ± 80 section. Clinical manifestations of onchocercal keratitis cells per 5-ju-m section by day 3 and 635 ± 106 by day 7. In were determined in a blinded manner by slit lamp examina- contrast, 352 ± 36 neutrophils were detected as early as 12 tion. IL-5-/~ mice exhibited corneal opacification and neo-

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Opacification Neovascularization

9* o 3" 3- o

75 2- •• 2" "E O 1- 1-

C57BI/6 IL-5 -/- C57BI/6 IL-5 -/- / / FIGURE 2. Corneal opacification and neovascularization in interleukin (IL)-5 gene knockout (IL-5 ) mice. C57B1/6 and IL-5 mice were immunized and injected intrastromally with Onchocerca volvulus antigens. Corneas were examined by slit lamp examination and were scored. Corneal opacification scores were as follows: 0, no opacification; 1 + , slight opacity; 2, moderate opacity; 3, severe opacity, and 4, total opacity. Neovascularization scores were determined on the basis of number of vessels and growth from the limbus. Scores are from 6 days after intrastromal injection, and each data point represents an individual animal. No statistical difference in scores was detected between the groups for corneal opacification or neovascularization (P > 0.05). Similar results were seen in a repeat experiment.

vascularization after intrastromal injection of parasite anti- Corneal Disease in Interleukin-5 Gene Knockout gens, and clinical scores at day 6 were not significantly Mice different from those in control C57B1/6 mice (Fig. 2; P > -/ 0.05). Examples of individual animals are shown in Figure 3 To characterize the cellular infiltrate in IL-5 ~ mice, animals (left panels). No differences in keratitis were noted at any were killed at day 1 or day 7 after intrastromal injection of O. other time point, and no clinical manifestations were de- volvulus antigens, and adjacent corneal sections were immu- tected in nonimmunized mice (data not shown). nostained with antisera to eosinophil MBP or with anti-neutro-

C57BI/6 Eosinophils Neutrophils

IL-5 ko

FIGURE 3- Clinical and histologic appearance of corneas of interleukin (IL)-5 gene knockout (IL-5 ' ) mice after intrastromal injection of helminth antigens. C57B1/6 and IL-5-/~ mice were immunized and injected intrastromally with Onchocerca volvulus antigens as described in the Materials and Methods section. Seven days later, corneal opacification and neovascularization had developed in both mouse strains Qeft panels). Eyes were fixedi n formalin, and adjacent sections were immunostained with antisera to eosinophil major basic protein (MBP: center panels) or anti- monoclonal antibody 7/4 (right panels'). Note the presence of eosinophils in the epithelial layer and extracellular MBP (upper center panel) and the presence of neutrophils in the anterior chamber (lower right panel). Original magnifications: left panels X25; all others X.400. Sections are representative of two experiments conducted in groups of 15 mice each.

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C57BI/6 /L-5 ko contrast to the distribution of eosinophils in C57B1/6 mice, neutrophils in corneas of IL-5~/~ mice were generally clus- d1 d8 d1 d8 tered below the epithelium and were often detected in the anterior chamber in close association with corneal endothe- lial cells.

Similar Levels of Interleukin-4 and Interferon-y in Immunocompetent C57B1/6 Mice and Interleukin-5 Gene Knockout Mice Although eosinophil deficiency is the primary defect in 1L-57" IL-5 mice,16 Th cytokines, especially IL-4, are important in devel- opment of onchocercal keratitis.1213 We therefore examined FIGURE 4. Expression of T helper cell-associated cytokine the effect of IL-5 deficiency on cytokine production after sen- mRNA in corneas of C57B1/6 and interleukin (IL>5 gene knock- / sitization to O. volvulus antigens. out QL-5~ ~') mice after intrastromal injection of Onchocerca C57B1/6 and IL-5-/~ mice were immunized systemically volvulus antigens. C57B1/6 and after IL-5-deficient mice were immunized subcutaneously and injected intrastromally with 0. and injected intrastromally with O. volvulus antigens. Animals volvulus antigens. Animals were killed on day 1 or day 8 after were killed 1 day or 8 days later, and corneas were removed injection, and corneas from four mice per group were removed and processed for reverse transcription-polymerase chain re- and pooled. Expression of the housekeeping gene HPRT and action and Southern blot analysis. One day after injection, cytokines interferon (IFN>7, IL-4, and IL-5 was determined by C57B1/6 mice expressed high levels of IFN-y transcripts, but reverse transcription-polymerase chain reaction as described not IL-4 or IL-5 (Fig. 4). However, after 8 days, IFN-7 expression in the Materials and Methods section. Polymerase chain reac- was diminished and IL-4 and IL-5 were increased, consistent tion products were visualized after agarose gel electrophoresis with development of the local Th2-type response described and Southern transfer. Data are representative of two experi- 12 13 / ments. previously. ' Corneas from YL-5~ ~ mice showed the same changes in temporal expression of IFN-y and IL-4 toward a Th2-type response, and transcripts for IL-5 were not detected. phil mAb 7/4. Neutrophils were the predominant cells in the To determine the effect of IL-5 deficiency on systemic corneas of IL-5-/~ mice at day 1, and cell numbers were not production of Th-associated cytokines, animals were killed 8 days after intrastromal injection, and splenocytes from C57B1/6 significantly different from C57B1/6 mice (907 ± 168 versus -/ 790 ± 110, respectively; P > 0.05). However, at day 7, keratitis and IL-5 ~ mice were stimulated in vitro by polyclonal acti- in C57B1/6 mice was associated with extensive eosinophil vation with anti-CD3 mAb. C57B1/6 mice produced IFN-7, IL-4, infiltration, whereas neutrophils were the predominant cell and IL-5. Consistent with cytokine expression in the cornea, -/ type in lL-5-/~ mice (Tig. 3). Eosinophils were not detected in IL-4 and IFN-y production by IL-5 ~ mice was not significantly the corneas of IL-5-/~ mice at any time. different from C57B1/6 mice (P > 0.05; Fig. 5). Similar results In corneas of C57B1/6 mice, eosinophils were present were obtained from animals killed on day 1 (data not shown). throughout the corneal stroma and were occasionally de- Taken together, these results indicate that IL-5 deficiency did tected in the epithelium (Fig. 3). In addition, extracellular not significantly affect production of O. volvulus-induced Th- MBP was present, indicative of eosinophil . In associated cytokines.

81 0.4 n 0.4 n m C57BI/6

V7 * 1

SSS IL-5 ko

£ 4- ssss> 0.2- 0.2- SSSSSS SSSSSS sssssss* I SSSSSS

ssss> SSSSSS .sss O SSSSSS

IFN IL-4 IL-5

/ / FIGURE 5- Cytokine production by splenocytes from interleukin (IL>5 gene knockout (IL-5 ) mice. C57B1/6 and IL-5 mice were immunized and injected intrastromally with Onchocerca volvulus antigens. Spleen cells (5 X 106/ml) were stimulated with anti-CD3 monoclonal antibodies 2C11, and cytokines were measured by two-site enzyme-linked immunosorbent assay, as described in the Materials and Methods section. Results are presented as mean ± SD from groups of 10 mice each. Similar results were obtained in a repeat experiment. IFN, interferon.

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4 DISCUSSION creased in IL-12-treated mice that have exacerbated keratitis,' and targeted disruption of the eotaxin gene results in signifi- The present study demonstrates that neutrophils comprise an cant reduction in eosinophil recruitment to the cornea.34 In early cellular infiltrate into the cornea and can mediate on- future studies, the role of chemokines and adhesion molecules chocercal keratitis in the absence of eosinophils. Neutrophils in recruitment of neutrophils and eosinophils to the cornea in have been implicated in the pathologic course of keratitis helminth-mediated keratitis will be examined. caused by Pseudomonas aeruginosa20 and herpes simplex The cytotoxic activity of eosinophils is caused primarily virus.2'>22 In herpes simplex keratitis, an early phase of neu- by production of highly cationic proteins, notably trophil recruitment occurs after the first 2 to 3 days and is MBP, which comprises the eosinophil granule core35 and has followed by a second, more intense neutrophil infiltrate that is been reported in biopsy specimens from patients with allergic 21 22 associated with corneal disease. ' A biphasic infiltrate of conjunctivitis.36'37 Major basic protein also inhibits corneal granulocyte recruitment was also apparent in murine helminth- wound healing38 and has a direct cytotoxic effect on cultured mediated keratitis, with an early phase of neutrophil recruit- human corneal epithelial cells.39 In addition, MBP and other -/ ment in C57B1/6 and IL-5 ~ mice. However, in immunocom- eosinophil granule proteins have been detected on O. volvulus petent C57B1/6 mice, the neutrophil infiltrate was replaced by larvae and surrounding tissues in skin lesions of infected -/ eosinophils, whereas neutrophils were present in IL-5 ~ mice persons after topical or systemic administration of anthelmin- at later time points. thics.40"44 The presence of each cell type in the cornea probably The latter study also demonstrated the presence of neu- reflects a balance between cell recruitment and survival within trophils and release of neutrophilic enzymes, including elas- the tissue. In further studies, the molecular events that regulate tase, , and , in O. volvulus skin le- infiltration of neutrophils and eosinophils in the cornea will be sions.44 These data indicate that worm and host tissue damage examined and the role of programmed cell death in develop- under these conditions is mediated by cytotoxic products from ment of onchocercal keratitis will be determined. Immune neutrophils and eosinophils. Taken together with our current privilege in the cornea is correlated with Fas ligand (CD95) observations in the murine model for onchocercal keratitis, it surface expression, especially in the corneal epithelium and appears likely that neutrophils and eosinophils contribute to endothelium, and contributes to allograft acceptance by induc- corneal disease in infected people. ing apoptosis in inflammatory cells that infiltrate the cor- In summary, our previous studies demonstrate that T cells 2324 nea. This mechanism may also contribute to neutrophil and eosinophils are important components in development of 25 apoptosis observed in herpes simplex keratitis. In helminth- helminth-mediated keratitis.12"15 In the present study, we mediated keratitis, CD95-mediated apoptosis of neutrophils found that neutrophils were also involved in corneal disease may contribute to the temporal shift in C57B1/6 mice from a They were prominent early in the inflammatory response and predominantly neutrophilic to an eosinophilic infiltrate. Con- mediated keratitis in the absence of eosinophils. An under- versely, inhibition of apoptosis may underlie the continued 7 standing of the molecular interactions underlying these obser- presence of neutrophils in IL-5~ ~ mice and may relate to vations may suggest approaches to immunologic intervention differential expression of cytokines that prolong neutrophil relevant to helminthic and atopic disease. survival, including IL-lj3, TNF-a, and IL-2.25'26 Temporal recruitment of neutrophils and eosinophils to Acknowledgments the corneas of these mice may be related to selective expres- The authors thank Bruce Blazar, Edward Pearce, and Manfred Kopf for sion of vascular adhesion molecules. For example, vascular cell interleukin-5 gene knockout mice; Sara Lustigman and Thomas Unn- adhesion molecule-1 (VCAM-1) is important in eosinophil trans- asch for Onchocerca volvulus adult worms; and Kirsten Larson and migration in vitro,27"29 whereas platelet endothelial cell adhe- Gerald Gleich for rabbit anti—major basic protein. The authors thank sion molecule-1 (PECAM-1) mediates neutrophil adhesion.3031 Robert Fairchild, Frederick Heinzel, and Christopher King for critical Cytokine regulation of expression of these adhesion molecules reading of the manuscript and Beth Ann Benetz for assistance with graphics. may contribute to selective recruitment of granulocytes to the 32 cornea. Tang and Hendricks showed that PECAM-1 expres- References sion on vascular endothelial cells was diminished after in vivo neutralization of IFN-y, which resulted in selective blockade of 1. World Health Organization Expert Committee on . neutrophil recruitment to the cornea and reduced severity of Onchocerciasis and its control. World Health Organ Tech Rept. 1995;852:1O3. herpes simplex keratitis. Conversely, IL-4 selectively upregu- 2. World Health Organization Expert Committee on Onchocerciasis. lates VCAM-1 expression and eosinophil transmigration across Third Report. World Health Organ Tech Rept. 1987;752:l67. 27 29 vascular endothelial cells in vitro. " In helminth-mediated 3. Ottesen E. Immune responsiveness and the pathogenesis of human keratitis, the observed early expression of IFN-y may contrib- onchocerciasis. / Infect Dis. 1995; 171:659 - 671. ute to neutrophil recruitment through PECAM-1, whereas later 4. Pearlman E. Experimental onchocercal keratitis. Parasitol Today. expression of IL-4 could upregulate VCAM-1 and enhance eo- 1996;12:26l-267. sinophil recruitment. 5. Duke BO, Anderson J. A comparison of the lesions produced in the cornea of the rabbit eye by micronlariae of the forest and Sudan- In addition to vascular cell adhesion molecules, chemo- savanna strains of Onchocerca volvulus from Cameroon. I. The tactic cytokines are important in cell recruitment to sites of clinical picture. Z Tropenmed Parasitol. 1972;23:354-368. inflammation.33 We demonstrated that expression of chemo- 6. Duke BO, Garner A. Reactions to subconjunctival inoculation of kines is important in eosinophil recruitment to the cornea in Onchocerca volvulus micronlariae in pre-immunized rabbits. Z Tropenmed Parasitol. 1975;26:435-448. helminth-mediated keratitis, because regulated upon activa- 7. Donnelly JJ, Rockey JH, Bianco AE, Soulsby EJL. Ocular immuno- tion, normal T-cell expressed and secreted (RANTES), macro- pathologic findings of experimental onchocerciasis. Arch Ophthal- phage inflammatory protein (MIP)-la, and eotaxin are in- mol. 1984; 102:628-634.

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