The Role of Eosinophils and Neutrophils in Helminth-Induced Keratitis 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 eosinophil 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 gene 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 cornea 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 inflammation 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 corneas 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 granulocytes 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 Downloaded from iovs.arvojournals.org on 09/25/2021 IOVS, June 1998, Vol. 39, No. 7 Granulocytes in Helminth-Induced Keratitis 1177 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 alkaline phosphatase 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 collagenase 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