A Novel Murine Model of Allergic Conjunctivitis

CLINICAL IMMUNOLOGY AND IMMUNOPATHOLOGY

Vol. 87, No. 1, April, pp. 75–84, 1998Article No. II974507

A Novel Murine Model of Allergic Conjunctivitis

M. Teresa Magone,1 Chi-Chao Chan, Luiz V. Rizzo, Alexander T. Kozhich, and Scott M. Whitcup

National Eye Institute, National Institutes of Health, Bethesda, Maryland 20892-1858

followed by cellular infiltration into the conjunctiva.Allergic conjunctivitis affects over 40 million pa- These cellular and humoral components of allergic con-

tients per year in the United States. Here we present junctivitis are crucial for the pathogenesis of the dis-the first murine model that incorporates the clinical, ease and for potential therapeutic targets.cellular, and humoral parameters of allergic conjunc- Current animal models for allergic conjunctivitis intivitis, including a ragweed-induced Th2-type cytokine the mouse (10, 11), rat (12, 13), guinea pig (14), orproduction by lymphocytes. SWR/J mice were immu- rabbit (15) do not incorporate all the cellular and hu-nized with short ragweed pollen in aluminum hydrox- moral parameters of the disease. In a number of theseide. Ten days after immunization, allergic conjunctivi-

models allergic disease is induced without sensitizationtis was induced by one topical application of ragweedof the animal, by topical application of a mast cell de-pollen onto the eye. Immediate response was charac-granulator (10, 12, 15). In others, pretreatment of theterized by chemosis, redness of the conjunctiva, andeye with a mucolytic agent is necessary before the in-lid edema. Histopathology and immunohistochemistryduction of an immediate response with the antigen (13).showed dense conjunctival infiltration with polymor-Existing murine models of allergic conjunctivitis usingphonuclear leukocytes, macrophages, and CD4/ Tragweed focus on topical immunization and inductionlymphocytes. In addition, ragweed-specific IgG1 andof clinical disease, but this model lacks systemic surro-IgE serum levels were significantly higher in immu-gate markers of disease (11). In addition, the multiplenized animals, and high levels of IL-4 and IL-5 were

detected in supernatants from ragweed-activated lym- pipetting of high amounts of ragweed pollen onto thephocytes. This reproducible model is a well-suited in- eye exposes not only the mouse but also the researcherstrument for testing the pathophysiology and future to this airborne, high allergenic substance.therapies of allergic conjunctivitis. We have developed a new murine model of allergic

Key Words: allergy; animal model; conjunctivitis; cy- conjunctivitis with clinical, cellular, and humoral pa-tokines; mouse. rameters for this disease. The development of a clini-

cally relevant murine model of allergic conjunctivitis isimportant since it allows investigators to study ocular

INTRODUCTIONallergy in transgenic and knockout mice. The presentmodel resembles human disease and is an accurate in-Seasonal allergic conjunctivitis is the most commonstrument for studying the pathogenesis of ocular al-allergic ocular disease, affecting yearly over 14% of thelergy and for testing therapies for allergic conjunctivi-population of the United States (1–3). Short ragweedtis.pollen is a clinically important seasonal airborne aller-

gen in North America; it is the major cause of late MATERIALS AND METHODSsummer rhinoconjunctivitis (3). Allergic conjunctivitis

Animalsrepresents a complex disorder of the immune systeminvolving macrophage processing and presentation of Female 6–8-week-old SWR/J mice were obtainedthe allergen (4, 5), activation of T cells (6–9), and IgE from Jackson Laboratories, Bar Harbor, Maine. Theproduction by B cells (8, 9). Allergen interaction with mice were kept under standard pathogen-free condi-mast cell-bound antigen-specific IgE then causes mast tions and given water and chow ad libitum. The presentcell degranulation in the conjunctiva and the release study was approved by the Institutional Animal Careof inflammatory mediators and cytokines. This acute and Use Committee of the National Eye Institute andphase of the disease is characterized clinically by itch- complies to the ARVO statement for the Use of Animalsing, erythema, and edema of the conjunctiva, and is in Ophthalmic and Vision Research.

Reagents1 To whom correspondence should be addressed at National Eye

Ragweed pollen (RW) was purchased from ICN Bio-Institute, NIH, Bldg 10, Rm 10B22, Bethesda, MD 20892-1858. Fax:301-496-7295. E-mail: [email protected]. medicals Inc. (Aurora, OH). Upon arrival the pollen

75 0090-1229/98 $25.00

AID Clin 4507 / a51e$$$$41 04-01-98 14:18:23 clina AP: Clin

76 MAGONE ET AL.

TABLE 1

Clinical Evaluation of Allergic Conjunctivitis

None 0 Mild 1/ Moderate 2/ Severe 3/

1. Chemosis Absent Mild chemosis Obvious chemosis Severe chemosis (lid margin 3–4 1 thicker than baseline)0 1 2 3

2. Conjunctival redness Absent Minimal redness Moderate redness Severe redness0 1 2 3

3. Lid edema Absent Mild edema Moderate edema Severe edema (unable to open the eye)0 1 2 3

4. Tearing and discharge Absent Mild Moderate Severe0 1 2 3

Total score 0 4 8 12

was aliquoted and stored at 47C. Ragweed extract was vasodilation; a score of 8 was given to eyes with moder-ate chemosis and lid edema; and a score of 4 was givenprepared as described in (16). Briefly, RW was defatted

with acetone and extracted in 50 mM Tris buffer over- to eyes with mild conjunctival edema and erythema.night. The extract was then dialyzed (Slide-a-lyzer Histology10K, Pierce Rockford, IL) against PBS for 48 h, filtered, Eyes were enucleated with the attached lids and in-and stored at 47C until use. Protein concentration was tact conjunctiva. The eyes were immediately fixed indetermined with Coomassie plus protein assay (Pierce). 4% glutaraldehyde for 30 min and then transferred toRW extract was biotinylated with a Kit from Pierce (Nr 4% formaldehyde for at least 24 h before processing.2199). Adjuvant, Al(OH)3 gel (alum), was prepared as The tissue was embedded in methyl acrylate, seriallydescribed by Levine and Vaz (17). The gel was centrifu- sectioned (4-mm-thick) through the central verticalgated for 15 min at 1000 RPM and stored at 47C. Conca- plane, and stained with giemsa, hematoxylin and eosin,valin A was purchased from Sigma Chemical Co. (St. and periodic acid-Schiff. Six conjunctival tissue sec-Louis, MO). tions from each individual eye were obtained and exam-

ined. The numbers of infiltrating neutrophils, eosino-Immunization and Induction of Conjunctivitisphils, goblet cells, and mast cells in one section were

RW and alum were mixed at a concentration of 50 counted by a masked observer trained in ocular pathol-mg of RW in 5 mg of alum 40 min before immunization. ogy (C.C.C.). In some animals one eye with the intactOn Day 0 mice were anesthetized with Metofane inha- conjunctiva was embedded in optimum cutting-temper-lation (Methoxyflurane, Pitman-Moore Inc., Mundel- ature compound (OCT, Miles, Elkhart, IN), snap fro-ein, IL) during the injection of RW and alum into one zen, and stored at 0707C until sectioning.hind footpad. On Day 10, conjunctivitis was induced Immunohistochemical staining was performed usingby topical application of 1.5 mg RW suspended in 10 rat primary antibodies (Sera-Lab, Crawley Down, UK)ml phosphate buffered saline (PBS), pH 7.2, into each of L3T4 (CD4), Lyt2 (CD8), M1/70.15 (macrophage),eye. Two groups of control mice were either not immu- and YN1/1.7.4 (ICAM-1) (ATCC, Rockville, MD) usingnized or immunized with alum alone; both groups were an avidin-biotin-peroxidase complex technique (18).challenged with the same dosage of RW on Day 10. The Rat IgG (2 mg/ml) was used as the control primary anti-

body, and biotin-goat anti rat IgG as the secondaryanimals were sacrificed at 20 min and 3, 6, 12, 24, 48,antibody (Vector Laboratories, Burlingame, CA). Afterand 72 h after topical RW administration.incubation with the avidin-biotin-peroxidase complex

Clinical Evaluation (Vector Laboratories), the slides were developed in 3,3-diaminobenzidine (Mallinckrodt, Paris, KY) and dehy-Animals were examined clinically for signs of imme-drated and counterstained with 1% methyl green indiate hypersensitivity response 20 min after the topicalmethanol. Macrophages and lymphocytes in an 8-mm-application of RW. Chemosis, conjunctival redness, lidthick section of the conjunctiva were then assessed byedema, and tearing were each graded on a 0–3/ scalea masked observer (C.C.C.).(Table 1). The total score consisted of the sum of scoresLymphocyte Proliferation Assayin each of these four categories. The highest score of

12 was given to eyes which had severe edema in con- Draining popliteal lymph node cells were collectedon Day 11 and pooled within each group. Proliferativejunctival and lid tissue, tearing, and redness due to


77NOVEL MURINE MODEL OF ALLERGIC CONJUNCTIVITIS

responses to 0.1–100 mg/ml of ragweed extract weretested. The highest response was observed at 10 mg RWextract/ml. The cells (3 1 106/ml) were cultured in flat-bottomed microplates, in a final volume of 0.2 ml ofRPMI 1640 medium, supplemented with 25 mM Hepesbuffer, 2 mM L-glutamine, 5 1 1005 M 2-mercaptoetha-nol, and HL-1 (Hycor Biomedical Inc., Irvine, CA). Thecultures were incubated for a total of 90 h and pulsedwith [3H]thymidine (0.5 mCi/10 ml/well) (3H-TdR) dur-ing the last 16 h, harvested on a Tomtec harvester(Tomtec Inc., Orange, CT), and counted with a scintilla-tion counter (Wallac Inc., Turku, Finland). The resultsare presented as the mean cpm of [3H]thymidine incor-poration from three separate assays.

Lymphocyte Culture for Cytokine AnalysisFIG. 1. Clinical score over time of mice challenged with 1.5 mg

of ragweed pollen onto each eye. Mice were either immunized withLymphocytes from draining popliteal lymph nodesRW/ alum (circle), alum alone (triangle), or not immunized (square).were collected 10 days after immunization and culturedImmunized mice (RW / alum) had a significantly higher score thanin RPMI 1640 medium supplemented with 25 mMboth control groups (P Å 0.0001; P Å 0.0001).

Hepes buffer, 2 mM L-glutamine, 5 1 1005 M 2-ME, 50mg/ml gentamicin sulfate, and 0.5% fresh-frozen mouseserum. Supernatants were collected at 48 h and stored

weed extract (3 mg/ml), and the sera were absorbedat 0207C until cytokine analysis. Cytokine levels inthree to four times in anti IgM-, IgA-, and IgE-coatedsupernatants were measured using ELISA as describedplates, to remove RW specific IgA, IgE, and IgM. Afterin (19). Briefly, IFN-g, IL-2, and IL-4 were measuredovernight incubation the plates were washed andusing antibody pairs obtained from Pharmingen (LaHRPO-conjugated anti IgG1 was added. StandardsJolla, CA). Biological activity in units or concentrationwere used as described above. In the RW-specific IgEin pg/ml was calculated from a standard curve estab-assay, the plates were coated with anti-mouse IgE anti-lished with the respective recombinant cytokine. IL-5body, blocked with BSA, and incubated with sera andwas measured using a minikit from Endogen (Cam-IgE-standards (Pharmingen) overnight. Then biotinyl-bridge, MA).ated ragweed extract (1 mg/ml) was added. After over-night incubation, the plates were washed and HRPO-Assay for Antigen-Specific Antibody Productionconjugated streptavidin (Southern Biotech) was added

Sera from animals collected 11 days after immuniza- before development with o-phenyldiamine (Sigmation were analyzed by ELISA to measure the levels of Chemical Co.). All plates were scanned with a mi-total IgE, RW specific IgE, total serum IgG1, RW-spe- croplate reader at 490 nm.cific IgG1, and total serum IgG2a. Total IgE, IgG1, andIgG2a assays were measured as described in (20, 21). Statistical AnalysisBriefly, 96-well microtiter plates were coated with anti-

All results are presented as the mean{ SE. Compar-mouse antibody (1 mg/ml) (anti-IgE from Pharmingen,isons between immunized mice and the control groupLa Jolla, CA; anti-IgG1 or anti-IgG2a from Southernwere made with the two-sided students t test. Compari-Biotech, Birmingham, AL). After the plates weresons between naive, immunized (RW / alum), andblocked with PBS buffer containing 0.5% BSA, pH 7.4,alum-immunized mice were done using ANOVA, andand overnight incubation with serum samples andthe P values are given where appropriate.standards, the plates were developed using HRPO-con-

jugated goat anti IgG subclass-specific (Southern Bio-RESULTStech) or anti-IgE monoclonal antibodies (Pharmingen).

In these assays the concentration of IgG1, IgG2a, or Challenge with Ragweed Pollen Induces anIgE was estimated using standard curves constructed Immediate-Type Response in the Eyeby coating wells with anti-IgG1, IgG2a, or IgE antibodyand adding polyclonal standards of the pertinent iso- Ten days after immunization with RW in alum,

SWR/J mice (n Å 55) were challenged with 1.5 mg oftype (Southern Biotech) or IgE (Pharmingen). In theRW-specific assays the samples were compared rela- RW in 10 ml of PBS into each eye. The mice developed

lid edema, conjunctival redness, and chemosis, as welltively to murine IgE or IgG1 standards. In the RW-specific IgG1 assay, the plates were coated with rag- as tearing and intensive scratching of the eye lids.


78 MAGONE ET AL.

FIG. 2. Clinical photograph of the immediate clinical response 20 min after the topical application of ragweed in a mouse immunizedwith alum alone (left) and in an immunized animal (RW / alum) (right).

This reaction started approximately 5 min after appli- was significantly higher than in mice immunized withalum alone (0.5 { 0.2) or in naive mice (0.2 { 0.05)cation of the RW and peaked at 15 to 20 min. The

mean score of disease was 8.9 { 0.5 (mean { SE). It (P Å 0.0001; P Å 0.0001). Clinical disease began to

TABLE 2

Kinetics of Cellular Infiltration into the Conjunctiva in Immunized SWR/J Mice (Top) and in Naive Controls (Bottom)(Results Are Presented as Mean { SE)

SWR/J Mice Immunized with RW/ Alum

BeforeCells/section challenge 20 min 3 h 6 h 12 h 24 h 48 h 72 h(mean { SE) n Å 5 n Å 4 n Å 6 n Å 6 n Å 5 n Å 20 n Å 5 n Å 4

PMN 1.5 { 0.5 9.2 { 6.6 4.5 { 1.9 43.6 { 5.3 44.4 { 4.6 55.9 { 6.8 45.0 { 8.9 45.7 { 23.8Eosinophils 0 { 0 0.7 { 0.7 0.3 { 0.3 2.0 { 0.3 2.6 { 0.2 4.8 { 0.8 12.6 { 1.7 3.5 { 1.4Mast cells 6.2 { 0.6 7.2 { 2.3 6.6 { 0.6 7.8 { 1.9 8.3 { 2.7 11.0 { 8.8 9.0 { 0.5 8.0 { 1.3Goblet cells 73.0 { 5.6 38.2 { 10.2 40.5 { 10.7 36.4 { 6.6 31.0 { 5.0 47.9 { 3.4 66.2 { 4.2 98.2 { 32.1

Naive SWR/J Mice

BeforeCells/section challenge 20 min 3 h 6 h 12 h 24 h 48 h 72 h(mean { SE) n Å 4 n Å 4 n Å 4 n Å 4 n Å 4 n Å 4 n Å 4 n Å 4

PMN 0.25 { 0.25 0.25 { 0.25 0.5 { 0.3 0.5 { 0.5 1.0 { 0.4 1.4 { 0.4 0.5 { 0.3 0.5 { 0.5Eosinophils 0 { 0 0 { 0 0 { 0 0 { 0 0.25 { 0.25 0 { 0 0.25 { 0.25 0 { 0Mast cells 5.9 { 0.3 5.7 { 0.4 5.8 { 0.3 6.5 { 0.6 6.2 { 0.9 6.5 { 0.5 5.7 { 0.4 7.5 { 0.5Goblet cells 72 { 6.6 70.0 { 5.1 70.0 { 0.75 67.0 { 2.3 71.0 { 0.75 64.2 { 2.4 68.5 { 1.4 69.5 { 0.5



FIG. 3. Photomicrograph of an eye lid of immunized mouse 20 min after topical challenge with ragweed. Ragweed pollen grains mixwith tear fluid and goblet cell secretion and the allergenic components are trapped in the fornix conjunctivae. Inset: Higher magnificationof the ragweed pollen grains. (Giemsa, 1200; inset, 1400).

regress after 40 min and reached a mean score of 0.8 consisted predominantly of polymorphonuclear leuko-cytes (PMN), mainly, neutrophils, and eosinophils;{ 0.1 (P Å 0.0001) at 1 h after the challenge in immu-

nized animals. Histologically, conjunctival edema and some macrophages were also noted. Eosinophillic infil-tration peaked at 48 h after challenge with RW andearly mast cell degranulation could be observed at 20

min after challenge. Figure 1 shows the clinical score decreased after that time point. Mast cell numbers re-mained stable over time. Most degranulating cells wereafter challenge with RW pollen in the different groups

over time. Figure 2 shows the photograph of the imme- observed at 3 h after the challenge. However, partiallydegranulated mast cells could be detected until 24 hdiate response to topical RW pollen 20 min after the

application onto the eye in an immunized mouse and after topical challenge. After total degranulation, mastcontrol animal. cells are not stained. Table 2 shows the kinetics of cellu-

lar infiltration over time after topical challenge in naiveCellular Infiltration Peaks at 24 h after Topical and immunized animals.

Application of the Allergen ICAM-1 expression was upregulated in the conjunctivalvascular endothelium of immunized animals after the top-Mice immunized with alum alone (n Å 10) or naive,ical challenge with RW (Fig. 5A) compared to naive ani-nonimmunized mice (n Å 32) did not show any cellularmals or animals immunized with alum alone (Fig. 5B).infiltration into the conjunctiva after the topical chal-

Immunohistochemistry showed that immunized ani-lenge with RW at any time point. In contrast, immu-mals had elevated numbers of CD4/ T lymphocytesnized mice (RW / alum) (n Å 55) had a strong cellular(Fig. 5C) and CD8 T lymphocytes in their conjunctivainfiltration into the conjunctiva, which started at 3 hwhereas control animals (not immunized) had no CD4after the topical application of RW, peaked at 24 h, andcells (Fig. 5D) and no detectable CD8/ T lymphocytesdecreased after 48 h (Table 2).

As shown in Figs. 3 and 4, the cellular infiltration in their conjunctiva.


80 MAGONE ET AL.

FIG. 4. Photomicrograph of an eye lid at 24 h. Cellular infiltration is at its peak with many neutrophils, eosinophils (arrow), and somemononuclear cells in the conjunctiva. Insets: higher magnification of the infiltrating inflammatory cells (hematoxylin and eosin, 1200; upperinset, 1400; lower inset, 1640).

Immunization with Ragweed and Alum Results in the In contrast, immunization with RW in alum did notincrease IgG2a production compared to that of animalsInduction of a Systemic IgG1 and IgE Responseimmunized with alum alone (P Å 0.3). SummarizedTotal IgE levels were significantly higher in immu- results of IgE and, IgG1, and IgG2a production in im-nized mice (RW / alum) than in naive mice (6944.0 { munized animals and controls are shown in Table 3.1744 ng/ml, 240.6 { 83.2 ng/ml; P Å 0.002). Alum hasOnly Lymphocytes from Immunized Animalsbeen described as an adjuvant which enhances IgE pro-

Proliferate in the Presence of Ragweed Antigenduction (22, 23). Therefore we chose to immunize micewith alum alone, as a baseline control for immunoglob- We examined the lymphoproliferative response ofulin production in this model. Total serum IgE levels lymphocytes from popliteal, draining lymph nodes ofin mice immunized with alum were only 687.6 { 112.1 immunized animals (RW / alum) and control animals.ng/ml. The 10-fold increase in IgE production in mice All groups responded well to Con A, but only immu-immunized with RW in alum was statistically signifi- nized animals showed a proliferative response whencant compared to that in control mice (P Å 0.02). Total cultured in the presence of ragweed extract (RW /serum IgG1 levels were also significantly elevated after alum: 14189.0 { 3894.3 cpm; alum: 1390.3 { 95.2 cpm;immunization compared to that in mice immunized naive: 1378.6 { 102.6 cpm) (P Å 0.02). Figure 6 showswith alum alone (P Å 0.01). Immunization with RW in the results of the proliferation assays in the differentalum also increased the allergen-specific IgE and IgG1 groups. These results suggest that immunization withproduction significantly (IgE: 4445.6 { 1245 ng/ml; ragweed primes lymphocytes to this allergen.IgG1: 14141 { 1639 ng/ml) compared to immunization

High Levels of IL-4 and IL-5 in Lymphocyte Culturewith alum alone (IgE: 457.2 { 100.7 ng/ml; IgG1: 577.2Supernatants from Immunized (RW / Alum) Mice{ 109.2 ng/ml) (PÅ 0.005 and PÅ 0.0001, respectively)

or compared to that in naive mice (IgE: P Å 0.008, and After measuring high proliferative responses of rag-weed-activated lymphocytes, we examined superna-IgG1: P Å 0.0001).



FIG. 5. Immunohistochemistry of the conjunctiva. ICAM-1 expression is upregulated in the vascular endothelium of immunized animals(RW / alum) after challenge with RW (A), compared to nonimmunized controls (B). Also, many CD4/ T cells are present in the conjunctivaof immunized animals (RW / alum) (C), but not in mice immunized with alum alone (D) (avidin-biotin-complex immunostaining, 1200).

tants from lymphocyte cultures for detectable levels of sensitized lymphocytes with ragweed extract is neces-sary in immunized animals for a Th2-type cytokinecytokine production (Fig. 7). We found high levels of

IL-4 (330.9 { 84.8 pg/ml), IL-5 (386.0 { 52.0 pg/ml), response in the present model.but low levels of IFN-g (496.6 { 385.1 ng/ml) and IL-2 (12.1 { 2.3 U/ml) in supernatants from cells cultured DISCUSSIONin the presence of ragweed extract. Interestingly, IFN-g levels were ú101 higher in cells cultured with Con The present study describes a new murine model for

allergic conjunctivitis with clinical, cellular, and hu-A than in cells stimulated with ragweed extract. Inaddition, cells which were stimulated unspecifically moral signs of allergic conjunctivitis, induced by a sin-

gle immunization and one antigenic challenge into thewith Con A did not produce detectable levels of IL-4 and IL-5. These results suggest that stimulation of eye. Although a number of animal models of allergic

TABLE 3

Immunization with RW/Alum Induces a Significant Increase in IgG1, Total IgE, and RW-Specific IgE Production(Results Are Presented as Mean { SE)

Type ofimmunization Naive Alum alone (5 mg) RW (50 mg)/Alum (5 mg)

Total serum IgE 240.6 { 83.2 ng/ml (n Å 8) 687.6 { 112.1 ng/ml (n Å 5) 6944.0 { 1744 ng/ml (n Å 11)RW-specific IgE 176.3 { 88.4 ng/ml (n Å 5) 457.2 { 100.7 ng/ml (n Å 5) 4445.6 { 1245 ng/ml (n Å 7)Total serum IgG1 — 198.9 { 41.0 mg/ml (n Å 5) 798.3 { 97.1 mg/ml (n Å 7)RW-specific IgG1 145 { 13.7 ng/ml (n Å 3) 577.2 { 109.2 ng/ml (n Å 5) 14141 { 1639 ng/ml (n Å 5)Total serum IgG2a — 262.8 { 122.6 mg/ml (n Å 5) 140.0 { 29.0 mg/ml (n Å 7)


82 MAGONE ET AL.

Clinical disease in the present model was character-ized by chemosis, hyperemia, lid swelling, and continu-ing scratching of the eye lids, beginning immediatelyafter topical allergen exposure. Histologically, this re-action was paralleled by mast cell degranulation, whichpeaked at 3 h after the challenge. This mimics humandisease and is a Type I hypersensitivity reaction to RW.

T lymphocytes may play an important role in theprocess of sensitization and the regulation of ocularallergy (26, 28). We detected accumulation of CD4 andCD8 lymphocytes in immunized animals, but not incontrol mice. Th2 lymphocytes have been shown to ac-cumulate in the conjunctiva of patients with ocular al-lergy (34). We did not determine which subsets of lym-phocytes infiltrated the conjunctiva in our model, but

FIG. 6. Lymphocyte proliferative response to ragweed extract, based on the cytokine profile from lymphocyte superna-Con A, and RPMI medium. Only lymphocytes from immunized mice tants, it is likely that the majority are Th2 lympho-(RW / alum) proliferated in the presence of ragweed extract (P Å

cytes.0.02). The maximum proliferation was present at a concentration ofThe cellular infiltration into the conjunctiva in our10 mg ragweed extract/ml. Lymphocytes from controls (naive mice,

mice immunized with alum alone) did not show a proliferative re- model consisted of neutrophils, macrophages, and eo-sponse. Error bars indicate SE. sinophils. Eosinophillic infiltration peaked at 48 h after

the topical challenge with RW. The presence of eosino-phils and neutrophils, but also lymphocytes is typicallyfound in the conjunctiva of patients with allergic con-conjunctivitis have been described in the literature,

none of them is perfectly suited for studying the immu- junctivitis after challenge with the allergen (35). Mastcell degranulation was observed in our model and itsnologic alterations in the course of this disease (10–

13). None of them has a significant humoral response presence paralleled ICAM-1 upregulation. It has beenshown that mast cells are potent regulators of ICAM-to the allergen, and cytokine production by activated

lymphocytes. Topical sensitization and topical chal- 1 and VCAM-1 expression (36). ICAM-1 expression onthe vascular endothelium was upregulated after chal-lenge with the same allergen may reflect the route of

sensitization in allergic conjunctivitis (11, 14). A limita- lenge with ragweed only in immunized animals (RW/ alum). These results correspond to human ICAM-1tion of this approach may be that high dose exposure

of the conjunctiva, the nasal mucosa, and the gut expression in allergic subjects after allergen challenge(37, 38).(through ingestion) to an allergen over many days may

induce tolerance to allergen-specific Th2 cells (24, 25). In our model lymphocytes from draining lymphnodes of immunized mice produced Th2-type cytokinesThe Th2 subset of lymphocytes may play an important

role in ocular inflammation by releasing selective cyto- (IL-4, IL-5) only when cultured in the presence of rag-weed extract. Allergen-specific T cell clones from atopickines which promote mast cell and B cell growth and

isotype switching from IgG1 to IgE production (26– patients have been shown to produce high amounts ofIL-4, but almost no IFN-g in the presence of the aller-29). However, anergy of Th2 cells may result in lack

of cytokine production and B cell help for IgE synthesis. gen (26, 27). In our study, lymphocytes from immu-nized mice that were stimulated unspecifically withSince the immune system of the mouse is well studied,

our new murine model will provide a useful and im- Con A produced high amounts of IFN-g, but had unde-tectable levels of IL-4 or IL-5 in the absence of ragweed.portant means for studying the topical and systemic fac-

tors involved in ocular allergy. This model mimics hu- These results suggest that the activation of memoryT cells and cytokine response to an allergen can beman allergic conjunctivitis and is accompanied by an

increase of both total serum and ragweed-specific serum modulated. IL-2 levels were low in supernatants fromcultures stimulated with Con A, probably due to self-IgE and IgG1 levels, which parallels findings in humans.

Because we compared our samples to a relative IgE and consumption of IL-2 by the proliferating cells in anautocrine and paracrine way, and by decreased IL-2IgG1 standard, our allergen-specific results are not ab-

solute. But the qualitative increase in immunoglobulin production at 48 h (39, 40).We also applied the same route of immunization andproduction, which mounts to 1–2 orders of magnitude,

clearly demonstrates a specific effect of the immuniza- challenge in different mouse strains (BALB/c mice andC57/BL). Although the responses were not as strong astion. It has been shown that serum IgE levels are ele-

vated in patients with allergic rhinoconjunctivitis, spe- in SWR/J mice, we were able to induce allergic conjunc-tivitis (unpublished observations). This may enable re-cially after the ragweed season (30–33).



FIG. 7. Supernatants from lymphocyte cultures of immunized animals (48 h) had high levels of IL-4 and IL-5 when cultured in thepresence of 25 mg/ml of ragweed extract (bottom graphs). However, when the cells were cultured with Con A, IFN-g was detected, but noIL-4 nor IL-5.

2. Friedlaender, M. H., Current concepts in ocular allergy. Ann.search of ocular allergy in transgenic mice and knock-Allergy 67, 5–14, 1991.out mice with different strain backgrounds.

3. King, T. P., Chemical and biological properties of some atopicIn summary, we believe that this new model hasallergens. Adv. Immunol. 23, 77–105, 1976.the immunological parameters of allergic conjunctivitis

4. Rankin, J. A., Hitchkock, M., Merrill, W. W., Bach, M. K.,and it provides a useful means for studying the patho- Brashler, J. R., and Askenase, P. W., IgE dependent release ofgenesis and future treatments of ocular allergy. leukotriene C4 from alveolar macrophages. Nature 297, 329–

331, 1982.5. Rouzer, C. A., Scott, W. A., Hamill, A. L., Liu, F. T., Katz, D. H.,ACKNOWLEDGMENT

and Cohn, Z. A., IgE immune complexes stimulate arachnidonicacid release by mouse peritoneal macrophages. Proc. Natl. Acad.We are grateful to Dr. Qian Li for many helpful discussions. Sci. 79, 5656–5660, 1982.

6. Sustiel, A., Rocklin, R. T cell responses in allergic rhinitis. Clin.REFERENCES Exp. Allergy 19, 11–18, 1989.

7. Gatien, J. G., Merler, E., and Colten, H. R. Allergy to ragweedantigen E: Effect of specific immunotherapy on the reactivity of1. Meltzer, E. O., Evaluating rhinitis: Clinical rhinomanometric,

and cytological assessment. J. Allergy Clin. Immunol. 82, 900– human lymphocytes in vitro. Clin. Immunol. Immunopathol. 4,32–37, 1975.908, 1988.


84 MAGONE ET AL.

8. Katz, D. H. Recent studies on the regulation of IgE antibody 24. Fasler, S., Aversa, G., Terr, A., Thestrup-Pedersen, K., de Vries,J. E., and Yssel, H., Peptide-induced anergy in allergen-specificsynthesis in experimental animals and man. Immunology 41, 1–

24, 1980. human Th2 cells results in lack of cytokine production and Bcell help for IgE synthesis. J. Immunol. 155, 4199–4206, 1995.9. Gauchat, J. F., Lebman, D. A., Coffman, R. L., Gascan, H., and

25. Joost van Neerven, R. J., Ebner, C., Yssel, H., Kapsenberg, M. L.,de Vries, J. E., Structure and expression of germline epsilonand Lamb, J., T-cell responses to allergens: Epitope-specificitytranscripts in human B cells induced by interleukin-4 to switchand clinical relevance. Immunol. Today 17, 526–532, 1996.to IgE production. J. Exp. Med. 172, 463–473, 1990.

26. Wierenga, E. A., Snoek, M., de Groot, C., et al., Evidence for10. Li, Q., Luyo, D., Hikita, N., Whitcup, S. M., and Chan, C. C.,compartmentilisation of functional subsets of CD4 T lympho-Compound 48/80-induced conjunctivitis in the mouse: kinetics,cytes in atopic patients. J. Immunol. 144, 4651–4656, 1990.susceptibility, and mechanism. Int. Arch. Allergy Immunol. 109,

277–285, 1996. 27. Parronchi, P., Macchia, D., Piccinni, M. P., et al., Allergen andbacterial component T cell clones established from atopic donors11. Merayo-Lloves, J., and Foster, C. S., Murine model of seasonalshow a different profile of cytokine production. Proc. Natl. Acad.allergic conjunctivitis. In ‘‘Advances in Ocular Immunology’’Sci. USA 88, 4538–4542, 1991.(R. B. Nussenblatt, S. M. Whitcup, R. Caspi, and I. Gery, Eds.),

28. Peltz, G., A role for CD4/ T cell subsets producing a selectivepp. 107–110, Elsevier, Amsterdam, 1994.pattern of lymphokines in the pathogenesis of human chronic12. Allansmith, M. R., Baird, R. S., and Ross, R. N., Ocular anaphy-inflammatory and allergic diseases. Immunol. Rev. 123, 23–35,laxis induced in the rat by topical application of compound 48/1991.80. Dose response and time course study. Acta Ophthalmol.

29. Umetsu, D. T., and De Kruyff, R. H. Th1 and Th2 CD4/ cells inSuppl. 192, 145–153, 1989.the pathogenesis of allergic diseases. Proc. Soc. Exp. Biol. Med.13. Trocme, S. D., Trocme, M. C., Bloch, K. J., and Allansmith, M. R.,215, 11–19, 1997.Topically induced ocular anaphylaxis in rats immunized with

30. Sherman, W. B., Stull, A., and Cooke, R. A., Serologic changesegg albumin. Ophthalmic Res. 18, 68–74, 1986.in hay fever cases treated over a period of one year. J. Allergy14. Merayo-Lloves, J., Calonge, M., and Foster, C. S., Experimental11, 225–244, 1940.model of allergic conjunctivitis to ragweed in guinea pig. Curr.

31. Wide, L., Bennich, H., and Johansson, S. G. O., Diagnosis of al-Eye Res. 14, 487–494, 1995.lergy by an in vitro test for allergen antibodies. Lancet 2, 1105,

15. Stenback, H., Krootila, K., Palkama, A., and Uusitalo, H., Mast 1967.cells in the anterior uvea of the rabbit: Intraocular effects of

32. Gleich, G. J., Zimmermann, E. M., Henderson, L. L., and Yun-compound 48/80 in the rabbit. Exp. Eye Res. 54, 247–252, 1992.ginger, J. W., Effect of immunotherapy on immunoglobulin E

16. Rafnar, T., Griffith, I. J., Kuo, M., Bond, J. F., Rogers, B. L., and and immunoglobulin antibodies to ragweed antigens: A six-yearKlapper, D. G., Cloning of Amb a I (antigen E) the major allergen prospective study. J. Allergy Clin. Immunol. 70, 261–271, 1982.family of short ragweed pollen. J. Biol. Chem. 266, 1229–1236,

33. Li, J. T. C., Yunginger, J. W., Reed, C. E., Jaffe, H. S., Nelson,1991.D. R., and Gleich, G. J., Lack of suppression of IgE production

17. Levine, B. B., and Vaz, N. M., Effect of combinations of inbred by recombinant interferon gamma: A controlled trial in patientsstrain, antigen and antigen dose on immune responsiveness and with allergic rhinitis. J. Allergy Clin. Immunol. 85, 934–940,reagin production in the mouse. Int. Arch. Allergy Appl. Immu- 1990.nol. 39, 156–171, 1970. 34. Maggi, E., Biswas, P., Del Prete, G., et al. Accumulation of TH2-

18. Hsu, S. M., Raine, L., and Fanger, H. The use of avidin-biotin- like helper T cells in the conjunctiva of patients with vernalperoxidase complex (ABC) in immunoperoxidase technique: a conjunctivitis. J. Immunol. 146, 1169–1174, 1991.comparison between ABC and unlabeled antibody (PAP) proce- 35. Bonini, S., Bonini, S., Vecchione, A., Naim, D. M., Allansmith,dure. J. Histochem. Cytochem. 29, 577–580, 1981. M. R., and Balsano, F., Inflammatory changes in conjunctival

19. Rizzo, L. V., Miller-Rivero, N. E., Chan, C. C., Wiggert, B., Nus- scrapings after allergen provocation in humans. J. Allergy Clin.senblatt, R. B., and Caspi, R. R., Interleukin-2 treatment po- Immunol. 82, 462–469, 1988.tentiates induction of oral tolerance in a murine model of autoim- 36. Meng, H., Tonnesen, M. G., Marchese, M. J., Clark, R. A. F., Ba-munity. J. Clin. Invest. 94, 1668–1672, 1994. hou, W. F., and Gruber, B. L., Mast cells are potent regulators

20. Rizzo, L. V., De Kruyff, R. H., Umetsu, D. T., and Caspi, R. R., of endothelial cell adhesion molecule ICAM-1 and VCAM-1 ex-Regulation of the interaction between Th1 and Th2 T cell clones pression. J. Cell. Physiol. 165, 40–53, 1995.to provide help for antibody production in vivo. Eur. J. Immunol. 37. Ciprandi, G., Buscaglia, S., Pesce, G. P., Villaggio, B., Bagnasco,25, 708–716, 1995. M., and Canonica, G. W., Allergic subjects express intercellular

adhesion molecule-1 (ICAM-1 or CD54) on epithelial cells of con-21. de Kruyff, R. H., Ju, S., Hunt, A. J., Mosmann, T. R., andjunctiva after allergen challenge. J. Allergy Clin. Immunol. 91,Umetsu, D. T., Induction of antigen specific responses in primed783–792, 1993.and unprimed B cells. Functional heterogeneity among Th1 and

Th2 T cell clones. J. Immunol. 142, 2575–2582, 1989. 38. Canonica, G. W., Cipriandi, G., Pesce, G. P., Buscaglia, S., Pao-lieri, F., and Bagnasco, M., ICAM-1 on epithelial cells of allergic22. Hamaoka, T., Katz, D. H., and Benacerraf, B., Hapten-specificsubjects: a hallmark of allergic inflammation. Int. Arch. AllergyIgE antibody responses in mice. II. Cooperative interactions be-Immunol. 107, 99–102, 1995.tween adoptively transferred T and B lymphocytes in the devel-

opment of IgE response. J. Exp. Med. 138, 538–556, 1973. 39. Smith, K. A., Interleukin-2: Inception, impact, and implications.Science 240, 1169–1176, 1988.23. Bomford, R., Aluminum salts: Perspectives in their use as adju-

vants. In ‘‘Immunological Adjuvants and Vaccines’’ (G. Gregori- 40. Paliard, X., De Waal Malefijt, R., Yssel, H., et al., Simultaneousproduction of IL-2, IL-4, and IFN-g by activated human CD4/adis, A. C. Allison, G. Poste, Eds.), Vol. 179, Plenum, New York,

1989. and CD8/ T cell clones. J. Immunol. 141, 849–855, 1988.

Received September 30, 1997; accepted with revision December 9,1997


A Novel Murine Model of Allergic Conjunctivitis

Documents

Transcript of A Novel Murine Model of Allergic Conjunctivitis