Impaired Eosinophil Recruitment to the Cornea in P-Selectin–Deficient Mice in Onchocerca volvulus Keratitis (River Blindness)

Abstract

purpose. A murine model of helminth-induced keratitis (river blindness) that is characterized by a biphasic recruitment of neutrophils (days 1–3) and eosinophils (days 3+) to the cornea has been developed. The purpose of this study was to determine the relative contribution of P- and E-selectin in recruitment of these inflammatory cells from limbal vessels to the corneal stroma.

methods. P- and E-selectin gene knockout (−/−) mice were immunized with antigens extracted from the parasitic helminth Onchocerca volvulus. One week after the last immunization, parasite antigens were injected directly into the corneal stroma. Mice were killed on days 1 and 3 postchallenge, and eyes were immunostained with either anti-eosinophil major basic protein (MBP) or with anti-neutrophil Ab. The number of cells in the cornea was determined by direct counting.

results. Recruitment of eosinophils to the cornea was significantly impaired in P-selectin^−/− mice (63.9% fewer eosinophils on day 1[ P = 0.0015], and 61% fewer on day 3[ P < 0.0001]) compared with control C57BL/6 mice. In contrast, P-selectin deficiency had no effect on neutrophil recruitment to the cornea. There was no inhibition of eosinophil and neutrophil migration to the corneas of E-selectin^−/− mice, indicating that there is no direct role for this adhesion molecule in helminth-induced keratitis.

conclusions. The present study demonstrates that P-selectin is an important mediator of eosinophil recruitment to the cornea. P-selectin interactions may therefore be potential targets for immunotherapy in eosinophil-mediated ocular inflammation.

Leukocyte migration into the tissue is a multistep process, beginning with selectin–mediated, low-affinity rolling of leukocytes along activated vascular endothelium. ¹ Continued stimulus leads to high-affinity adhesion mediated by integrins and members of the immunoglobulin superfamily. ¹ In the presence of chemotactic signals, subsets of leukocytes migrate across the endothelial cell layer into the tissue, and the nature of the leukocyte infiltrate depends on the specific vascular adhesion cell molecules and chemotactic cytokines that are expressed at the site. In the cornea, for example, migration of neutrophils in a murine model of herpes simplex keratitis is regulated by expression of platelet endothelial cell adhesion molecule (PECAM) 1. ²  

In contrast to herpes simplex and Pseudomonas keratitis, the murine model for Onchocerca volvulus keratitis (river blindness) is characterized by recruitment of eosinophils and neutrophils to the cornea in a biphasic manner, with neutrophils preceding and being replaced by eosinophils. ^{3 4 5} Our previous studies using this model demonstrated that eosinophil migration to the cornea is tightly regulated by chemotactic and regulatory cytokines, including IL-4, IL-12, and eotaxin ^{6 7 8 9} and that recruitment of neutrophils and eosinophils to the central cornea is dependent on the presence of specific antibody. ³  

In the present study, we used mice deficient in P- or E-selectin to examine the role of these adhesion molecules in recruitment of neutrophils and eosinophils from the limbal vessels to the cornea. We found that the absence of P-selectin significantly impaired recruitment of eosinophils but not neutrophils to the cornea, whereas E-selectin deficiency had no effect on migration of either cell type. These findings may have implications for other causes of ocular inflammation in which neutrophils and eosinophils are involved. 

Methods

Source of Mice

Mice deficient in E-selectin ¹⁰ and P-selectin ¹¹ on a C57BL/6 background were obtained from Taconic (Germantown, NY). Age- and sex-matched C57BL/6 mice were obtained from Taconic as controls. All mice were genotyped by PCR analysis using P-selectin specific primers (sense: TTG TAA ATC AGA AGG AAG TGG; antisense: AGA GTT ACT CTT GAT GTA GAT CTC C) and E-selectin primers (sense: GGA CTG TGT AGA GAT TTA CAT CC; antisense: GCA GGT GTA ACT ATT GAT GGT). All mice were treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 

Preparation of O. volvulus Antigens

O. volvulus parasites were isolated from s.c. nodules that had been surgically removed from infected patients in Cameroon (nodules were kindly provided by Janet Bradley at Salford University, United Kingdom). Worms recovered after digestion with collagenase (Sigma, St. Louis, MO) were homogenized in HBSS using a mortar and pestle, insoluble material was separated by centrifugation, and the protein concentration was determined using BioRad protein assay (BioRad, Richmond, CA). 

Induction of O. volvulus Keratitis

Animals received three weekly s.c. immunizations with 10 μg O. volvulus antigens in a 1:1 ratio with adjuvant containing squalene (Aldrich Chemical, Milwaukee, WI), Tween 80 (Fisher, Fair Lawn, NJ), and pluronic acid (BASF, Parsippany, NJ). ⁶  

To inject parasite material into the corneal stroma, mice were anesthetized by intraperitoneal injection of 200 μl of a 1.2% solution of 2,2,2-tribromoethanol (Aldrich) containing 2.5% 2-methyl-2-butanol (tertiary amyl alcohol; Aldrich) dissolved in dH₂O. ¹² The epithelial layer was scarified using a 30-gauge needle, and 10 μg of O. volvulus antigens in 5 μl were injected into the corneal stroma using a 33-gauge needle attached to a Hamilton syringe (Hamilton, Reno, NV). Corneal opacification was monitored daily by slit lamp examination and evaluated as described previously. ⁶ Briefly, clinical scores were graded on the intensity and extent of corneal opacity, measured in 0.5-U increments, using the following guidelines: 0, no pathology, cornea is transparent; 1, slight opacity; 2, moderate opacity; and 3, severe opacity, underlying iris not visible. 

Immunohistochemical Analysis of the Cornea

Eyes were fixed overnight in 10% formaldehyde (Sigma), processed by standard methods, and embedded in paraffin. To detect eosinophils, 5-μm sections were immunostained with rabbit antisera to major basic protein (MBP) as described previously. ^{3 5} Biotinylated goat anti-rabbit Ig (Dako, Carpenteria, CA) was used as the secondary Ab. Neutrophils were detected using the rat mAb 7/4 (Serotec, Oxford, United Kingdom) diluted 1/100, followed by biotinylated rabbit anti-rat Ig (BioGenex, San Ramon, CA). After incubation with secondary Ab, sections were incubated with alkaline phosphatase–conjugated streptavidin (Dako). Positive reactivity was visualized using Vector Red Substrate containing Levamisole (Vector Laboratories, Burlingame, CA) and counterstained with modified Harris’ hematoxylin (Richard-Allen, Kalamazoo, MI). Cells were visualized by fluorescence microscopy and counted in a masked fashion. To assess migration of cells to the central cornea, peripheral, paracentral, and central regions of the cornea were defined as distance from the peripheral (limbal) vessels as described. ³ Briefly, neutrophil and eosinophil numbers for each section were determined in the peripheral regions (0 to ∼500 μm from each limbus), paracentral regions (∼500–1000 μm from each limbus), and the central region of each cornea (∼1000–1500 μm from each limbus). Because there are two peripheral and paracentral regions per section, values from each region were combined. 

Differential Blood Leukocyte Counts

Blood was collected retro-orbitally, and differential counts were determined after staining with modified Wright-Giemsa stain (Diff-Quik; Dade Diagnostics, Aguada, Puerto Rico). 

Measurement of Antigen-Specific Isotype Responses in the Sera

Sera were collected when animals were euthanatized and assayed for antibody by ELISA as described. ³ Immulon-4 ELISA plates (Dynatech, Chantilly, VA) were coated overnight with 50 μl of 1μ g/ml parasite antigens. After blocking with 1% fetal bovine serum, dilutions of mouse sera were incubated for 2 hours at room temperature, washed, and incubated with biotinylated goat anti-mouse isotype-specific antibodies (Southern Biotechnology, Birmingham, AL). Reactivity was determined after incubation with peroxidase-labeled anti-goat IgG (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and tetramethyl benzidine (TMB; Zymed, San Francisco, CA) was used as a substrate. Reaction was stopped after 10 minutes with 1 N HCl. Absorbance was measured at 450 nm on a kinetic microplate reader (Molecular Devices Corporation, Sunnyvale, CA). 

Total Serum IgE

Total serum IgE was detected by two-site ELISA using rat anti-mouse IgE mAbs R35-72 and biotinylated R35-92 (PharMingen, San Diego, CA). Immulon-4 ELISA plates were coated overnight with R35-72. After blocking with 10% fetal bovine serum, mouse sera were incubated for 1 hour at 37°C. Purified mouse IgE, κ (α-TNP), (PharMingen) was used to generate a standard curve. The plates were then incubated with biotinylated R35-92 and streptavidin-peroxidase (Sigma). Positive reactivity was determined using tetramethyl benzidine. 

IL-5 Production by Spleen Cells

Spleens were removed 3 days after intrastromal injection and homogenized, and erythrocytes were lysed using cold 0.01 M Tris (pH 7.2) containing 0.85% ammonium chloride. Cells were washed three times and resuspended at 1 × 10⁷/ml RPMI-1640 containing 10% heat-inactivated fetal bovine serum, 1 mM sodium pyruvate, 2 mM l-glutamine, 20 mM Hepes, 100 U/ml penicillin, and 100 μg/ml streptomycin. Duplicate wells containing 1 × 10⁶ cells were incubated with 10μ g/ml parasite antigens for 72 hours at 37°C in 5% CO₂. IL-5 in cell culture supernatant was measured by two-site ELISA using mAbs TRFK-5 and TRFK-4, and recombinant IL-5 (PharMingen) was used as a standard. Positive reactivity was determined using tetramethyl benzidine. 

Statistics

Statistical significance between control C57BL/6 mice and P- or E-selectin–deficient mice was determined using an unpaired Student’s t-test (Prism Graph Pad Software, San Diego, CA). A value of P < 0.05 was considered to be significant. 

Results

Impaired Eosinophil Recruitment to the Cornea in P-Selectin–Deficient Mice

To determine whether expression of P- and E-selectin is important in eosinophil migration to the cornea, C57BL/6 and mice deficient in either E- or P-selectin were immunized s.c. and injected intrastromally with O. volvulus antigens. Mice were killed on day 1 or day 3, and eosinophils were detected using Ab to major basic protein. Our previous studies demonstrated that eosinophil recruitment to the cornea is gradual, with more eosinophils in the cornea on day 3 than on day 1 after intrastromal injection. ^{3 5} As shown in Figure 1 , the total number of eosinophils per 5-μm section was not significantly different between C57BL/6 and E-selectin–deficient mice at either time point, indicating that E-selectin is not essential for eosinophil recruitment to the cornea. In contrast, eosinophil migration was significantly impaired in the P-selectin–deficient mice both on day 1 and day 3 after intrastromal injection (Figs. 1 2) . On day 1, the number of eosinophils per corneal section was reduced by 63.9%, and on day 3, eosinophil numbers were reduced by 61%. Although fewer eosinophils were detected in P-selectin^−/− mice, the distribution of these cells in the peripheral, paracentral, and central regions of the cornea was similar (day 3, peripheral, paracentral, central: C57BL/6: 60.4%, 30.8%, 8.8%, respectively; P-selectin^−/−: 57.4%, 32%, 10.6%, respectively). 

Neutrophil Recruitment to the Cornea in P- and E-Selectin–Deficient Mice

In contrast to eosinophils, neutrophil migration to the cornea in O. volvulus keratitis peaks in the first 24 hours after intrastromal injection and then rapidly declines. ^{3 5} To determine the effect of P- and E-selectin deficiency on neutrophil recruitment to the cornea, C57BL/6, P- and E-selectin gene knockout mice were immunized and injected intrastromally as described above. Animals were killed on either day 1 or day 3, and 5-μm corneal sections were immunostained with mAb 7/4, which is specific for murine neutrophils. The total number of neutrophils in E- and P-selectin gene knockout mice was not significantly different from C57BL/6 mice at either time point after intrastromal injection (Fig. 3) . Furthermore, there were no differences in the distribution of neutrophils throughout the peripheral, paracentral, and central part of the cornea (data not shown). These data indicate that neither P- nor E-selectin is required for neutrophil migration from the limbal vessels to the cornea. 

Systemic Responses to O. volvulus Antigens in P- and E-Selectin–Deficient Mice

We recently demonstrated an essential role for antibody-mediated recruitment of eosinophils and neutrophils to the cornea. ³ To assess whether altered Ab responses contribute to the impaired eosinophil migration observed in P-selectin^−/− mice, we measured isotype responses in selectin-deficient mice immunized with parasite antigens. As shown in Figure 4A , IgG1 was elevated compared with IgG2a in all three strains of mice, consistent with development of a Th2 response. However, there were no differences in titer of parasite-specific IgG1, IgG2a, IgG2b, or IgG3 in P- and E-selectin^−/− mice compared with C57BL/6 mice. Furthermore, total serum IgE levels were increased but not significantly different among the three groups (C57BL/6, 2.41 ± 0.36 μg/ml; E-selectin^−/−, 3.36 ± 0.51 μg/ml; and P-selectin^−/−, 2.93 ± 0.53 μg/ml). 

To determine whether impaired eosinophil recruitment in P-selectin^−/− mice might be attributed to a deficiency in systemic IL-5 or eosinophil production, we examined IL-5 production in parasite antigen–stimulated splenocytes and determined blood eosinophilia. There was no effect of P-selectin deficiency on IL-5 production (Fig. 4B) . IL-4 and IFN-γ were also unaffected (data not shown). Furthermore, there was no significant difference (P > 0.05) in blood eosinophils between C57BL/6 and P-selectin^−/− mice or between C57BL/6 and E-selectin^−/− mice (Fig. 4B) . 

Taken together, these data indicate that P-selectin–dependent eosinophil recruitment to the cornea is not due to altered development of parasite-specific T- or B-cell responses. 

Corneal Opacification in P- and E-Selectin–Deficient Mice

To determine whether P- or E-selectin expression is important in development of corneal opacification, mice were examined by slit lamp microscopy, and the degree of corneal opacification was scored as described in the Methods. As shown in Figure 5 , the severity of corneal opacification was not significantly different in any of the three strains, indicating that expression of P- and E-selectin is not essential for development of corneal disease. 

Discussion

The selectin family contains P-, E-, and L-selectin and has a common structure with an N-terminal lectin domain. ¹ L-selectin is present constitutively on most leukocytes and has been shown to participate in leukocyte adherence to vascular endothelium. ¹ In contrast, P-selectin is present only in platelets and vascular endothelial cells, where it is prestored in granules for rapid translocation to the cell surface. E-selectin expression requires de novo synthesis; however, both P- and E-selectin are important molecules in transmigration of neutrophils and eosinophils into tissues. These selectins mediate low-affinity rolling of leukocytes, and blockade of selectin-mediated interactions has been shown to inhibit inflammatory responses in animal models of allergic asthma, peritonitis, and allergic conjunctivitis. ^{11 13 14 15 16 17}  

The rationale for the present study was to determine whether there is a role for P- and E-selectin in recruitment of eosinophils and neutrophils to the cornea. Our findings demonstrate that P-selectin is essential for maximal recruitment of eosinophils to the cornea, but has no apparent role in neutrophil migration to this site. E-selectin has no direct effect on recruitment of either cell type to the cornea. Furthermore, our observations show that P-selectin deficiency has no effect on development of parasite-specific T- and B-cell responses or on blood eosinophilia, indicating that the effect of P-selectin deficiency in this model is only on eosinophil recruitment to the cornea. 

Our results are consistent with earlier reports demonstrating a role for P-selectin, but not E-selectin, in recruitment of eosinophils to the peritoneal or the pleural cavities in mouse models of lipopolysaccharide-mediated peritonitis and allergic asthma. ^{13 14 15 18} These observations are also consistent with a role for P-, but not E-selectin, in eosinophil tethering to vascular endothelial cells under in vitro flow conditions. ¹⁹  

Using a model of ragweed-induced conjunctivitis, Strauss and coworkers ¹⁶ showed that systemic injection of a soluble form of P-selectin glycoprotein ligand (PSGL)–1 abrogated eosinophil migration to the conjunctiva and completely ablated clinical signs of allergic conjunctivitis. However, in contrast to our findings, they also noted that coadministration of mAbs to E- and P-selectin inhibits eosinophil migration, but anti–P-selectin alone had no significant effect. ¹⁶  

With regard to neutrophils, our studies showed that neither P- nor E-selectin deficiency had any effect on neutrophil recruitment to the cornea. This observation differs from other models in which neutrophil migration to the skin or the peritoneal cavity is diminished in P-selectin^−/− mice. ^{11 17} The discrepancy between these and our findings may result from differences in the activation state of the vascular endothelial cells, kinetics of expression of P- and E-selectin in the different models, or differences in expression of other cellular adhesion molecules on limbal vessels. 

Our previous studies demonstrated that eosinophil migration to the cornea is regulated by specific cytokines, including IL-4, IL-5, and IL-12. ^{6 7 8} IL-4– and IL-5–deficient mice have fewer eosinophils in the cornea than controls, whereas mice given recombinant IL-12 have increased eosinophils along with elevated chemokine expression. ^{6 7 8} Although we have yet to determine the underlying mechanisms, it is possible that P-selectin is involved in IL-4–dependent eosinophil migration, because IL-4 upregulates P-selectin expression on endothelial cells in vitro, and eosinophil tethering to IL-4–stimulated endothelial cells is P-selectin–dependent. ²⁰ Furthermore, eosinophils bind with greater avidity to P-selectin than neutrophils under in vitro flow conditions. ²¹  

In the present study, we found that the severity of corneal opacification was not reduced in P-selectin^−/− mice, despite the pronounced effect on eosinophil recruitment. There are at least two explanations for this observation. First, the number of eosinophils was not completely diminished; therefore, the eosinophils that do migrate into the cornea in P-selectin^−/− mice may degranulate and induce stromal disease. Second, it is possible that in the absence of eosinophils, neutrophils mediate the stromal damage that results in corneal opacification. This notion is supported by our earlier findings that IL-5 gene knockout mice, which do not produce eosinophils, develop keratitis that is mediated by neutrophils. ⁵ Most likely, both cell types contribute to keratitis, because under conditions where neutrophil and eosinophil migration to the cornea is impaired, as in antibody-deficient (μMT) mice, corneal opacification is completely inhibited. ³  

We have yet to determine the basis for the reduction in neutrophil numbers in the cornea after eosinophils are recruited. It is likely that the neutrophils undergo IL-2–dependent apoptosis as described for herpes simplex keratitis. ²² In addition, mature neutrophils may be irrevocably committed to autocrine death because of coexpression of cell-surface Fas and FasL. ²³ Any contribution of eosinophils to neutrophil apoptosis is likely to be indirect, probably by modulating the local cytokine environment. ^{24 25} Because there is no difference in neutrophil numbers in the cornea in P-selectin–deficient mice, despite the paucity of eosinophils (i.e., neutrophil numbers decrease in both strains by day 3), it is unlikely that P-selectin expression is important in this phenomenon. 

Although the present study demonstrates a role for specific adhesion molecules in murine O. volvulus keratitis, we cannot extrapolate these findings directly to human disease, that is, river blindness, because it is not feasible to obtain corneas from infected individuals. However, as in the murine model, neutrophils and eosinophils are prominent in the skin of O. volvulus–infected individuals with dermatitis. ^{26 27} Furthermore, mediators such as eotaxin are implicated in onchodermatitis and the murine model of keratitis. ^{9 27} These observations are consistent with the notion that similar inflammatory mediators are involved in human disease and in mouse models. 

In summary, our studies demonstrate that P-selectin expression is essential for eosinophil recruitment to the cornea. Blockade of P-selectin interactions may therefore be important in ocular onchocerciasis and in other ocular allergic disorders in which eosinophils are implicated. 

 Supported by National Institutes of Health Grants EY10320 (EP), EY06913 (LRH), and EY11373 (JHL); Burroughs Wellcome New Investigator Award 0720 (EP); Ohio Lions Research Foundation; and the Research to Prevent Blindness Foundation. JTK is a recipient of a scholarship award from the German National Merit Foundation, Studienstiftung des deutschen Volkes.

 Submitted for publication March 31, 2000; revised June 16, 2000; accepted July 21, 2000.

 Commercial relationships policy: N.

 

Figure 1.

 

The effect of E- and P-selectin deficiency on eosinophil migration to the cornea. C57BL/6, E-selectin^−/−, and P-selectin^−/− mice were immunized s.c. and injected intrastromally with antigens from the parasitic helminth Onchocerca volvulus as described in Methods. Mice were killed on day 1 and day 3, and eyes were fixed in formalin. Sections (5μ m) were immunostained with rabbit antisera to eosinophil major basic protein and visualized using Vector Red. Total eosinophil numbers per section were determined by direct counting and are presented as the mean ± SEM of five mice per group. There was no significant difference in eosinophils in C57BL/6 and E-selectin^−/− mice on day 1 or 3. However, the difference between C57BL/6 and P-selectin^−/− mice was highly significant at both time points (day 1: P = 0.0015; day 3: P < 0.0001). These experiments were repeated twice (total of three experiments) with similar results.

Figure 2.

 

Impaired eosinophil recruitment to the corneal stroma in P-selectin^−/− mice. C57BL/6 and P-selectin^−/− were immunized s.c. and injected intrastromally with helminth antigens, and eosinophils were detected as described in the legend to Figure 1 . Corneas from animals killed on day 3 were immunostained with antisera to eosinophil MBP and visualized by bright field (A, C) and fluorescence (B, D) microscopy. Photomicrographs show the limbal region of the corneal stroma of a C57BL/6 (A, B) and a P-selectin^−/− mouse (C, D). Note impaired migration of eosinophils to the cornea of P-selectin^−/− mouse compared with C57BL/6 mouse. V, limbal vessel. Original magnification, ×200.

Figure 3.

 

Neutrophil migration to the cornea in E- and P-selectin–deficient mice. C57BL/6, E-selectin^−/−, and P-selectin^−/− mice were immunized and injected intrastromally with parasite antigens (see Fig. 2 ). Mice were killed on day 1 (left) or day 3 (right). Eyes were fixed in formalin, and neutrophils were detected in 5-μm sections after immunostaining with mAb 7/4 and Vector Red. No significant differences were noted among any of the strains of mice at either time point (P > 0.05). Similar results were obtained in two repeat experiments.

Figure 4.

 

O. volvulus–stimulated antibody, IL-5 and eosinophil production in P- and E-selectin^−/− mice. C57BL/6, E-selectin^−/−, and P-selectin^−/− mice were immunized as described in Figure 1 . (A) Serial dilutions of sera from each group of mice were pooled and assayed for parasite-specific IgG isotypes by ELISA. (B) IL-5 production by antigen-stimulated splenocytes was assayed by two-site ELISA, and blood eosinophilia was determined by direct counting. Data are means ± SEM from five mice per group. There was no significant difference (P > 0.05) in isotypes, IL-5 production, or blood eosinophils between C57BL/6 and P-selectin^−/− mice or between C57BL/6 and E-selectin^−/− mice. These observations were reproduced in two additional experiments.

Figure 5.

 

O. volvulus–induced corneal opacification in E-selectin^−/− and P-selectin^−/− mice. C57BL/6, E-selectin^−/−, and P-selectin^−/− mice were immunized s.c. and injected into the corneal stroma with soluble O. volvulus antigens. Corneal opacification was monitored daily by slit lamp examination, and clinical scores were assessed based on the intensity and extent of opacity as described in Methods. Data are means ± SEM of five mice per group. These data are representative of three repeat experiments.

The authors thank Carol Luckey for performing the genotyping assays and Fred Heinzel and Richard Silver for critical review of the manuscript.