Veterinary Immunology and Immunopathology 132 (2009) 53–61

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Veterinary Immunology and Immunopathology

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Sensitization of skin mast cells with IgE to Culicoides occurs frequently in clinically healthy horses

Bettina Wagner a,*, William H. Miller Jr.b, Hollis N. Erb a, D. Paul Lunn c, Douglas F. Antczak d a Department of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA b Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA c Department of Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO 80523, USA d Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA

ARTICLE INFO ABSTRACT

Keywords: IgE antibodies are mediators of mast cell degranulation during allergic diseases. The IgE binding of IgE to its high-affinity IgE receptor on mast cell surfaces is called ‘‘sensitization’’ IgG and precedes the development of clinical . Previously, intradermal injection of anti- Allergy IgE or the anti-IgG(T) CVS40 induced immediate skin reactions in horses. This Sensitization suggested that both IgE and IgG(T) sensitize equine skin mast cells. Here, we investigated Horse sensitization to and with IgE or IgG(T) in clinically healthy horses of different age groups. In addition, immediate skin reactions to Culicoides were determined by intradermal testing in non-allergic horses. A total of 14% of the young horses 1–3 years old and 38% of the adult animals showed skin reaction to Culicoides allergen extract. Sensitization with IgE and IgG(T) was evaluated in skin mast cells and peripheral blood basophils to determine whether sensitization with IgG(T) preceded that with IgE in young horses. Anti-IgE stimulated immediate skin reactions in 18 of 21 young horses, but only 7 of them reacted to the anti-IgG(T) antibody CVS40. The equine IgG(T) fraction is composed of IgG3 and IgG5. We used several newly developed monoclonal antibodies to IgG3 and IgG5 for intradermal testing to improve our understanding about the mast cell reaction induced by the anti-IgG(T) antibody CVS40. None of these antibodies induced a skin reaction in young or adult horses. To determine sensitization with IgE in neonates and foals at 6 and 12 weeks of age an in vitro release assay was performed using peripheral blood cells. The histamine concentration released by anti-IgE stimulation from foal basophils increased between birth and 12 weeks of age, while almost no histamine release was observed after anti-IgG(T) treatment of the cells. In summary, IgE was the major immunoglobulin involved in the sensitization of mast cells and basophils in horses at various ages. IgG(T) antibodies did not play a major role in the activation of mast cells or basophils in young horses and their role in the sensitization of adult horses remains unclear. Sensitization to Culicoides allergen in the absence of clinical disease was frequently found in horses of all age groups. Because many clinically healthy horses developed skin reactions to this allergen, sensitization results are useful to diagnose Culicoides-induced allergy only in horses with allergic conditions. ß 2009 Elsevier B.V. All rights reserved.

1. Introduction

IgE is a key molecule in type-I hypersensitivities. This * Corresponding author. Tel.: +1 607 253 3813; fax: +1 607 253 3440. form of allergic disease is characterized by an immediate E-mail address: [email protected] (B. Wagner). onset of clinical signs after exposure to allergen. Examples

0165-2427/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.vetimm.2009.09.015 54 B. Wagner et al. / Veterinary Immunology and Immunopathology 132 (2009) 53–61 of type-I hypersensitivities include hay fever, asthma and This study focused on the sensitization of skin mast atopic dermatitis in humans and skin hypersensitivity in cells and peripheral blood basophils to Culicoides allergen horses. The development of allergic diseases is initiated by and with IgE and IgG(T) in clinically healthy, young and the production of allergen-specific IgE by B-cells. IgE adult horses. The questions to be answered by the molecules are subsequently bound to the surfaces of mast experiments were: (1) What is the prevalence of sensitiza- cells and basophils via high-affinity IgE receptors (FceRI), a tion to Culicoides allergen in a population of horses without process called ‘‘sensitization’’. In the absence of the allergen, clinical signs of allergy kept in a defined environment sensitization itself does not induce clinical signs of allergy. with allergen exposure? (2) Do adult horses have higher Only crosslinking of cell-bound IgE with specific allergen prevalence of sensitization to Culicoides than younger triggers cellular degranulation and results in the immediate horses? (3) Are there differences in sensitization with IgE release of inflammatory mediators followed by a rapid onset or IgG(T) in young and adult horses? (4) Does sensitization of clinical signs (for review: Ravetch and Kinet, 1991). with IgG(T) precede sensitization with IgE in young Skin hypersensitivity is a common type-I allergy in horses? (5) Does stimulation with anti-IgG(T) activate horses. Culicoides allergens are the most important mast cells or basophils in foals and young horses? inducers of skin hypersensitivities, as shown using intradermal (i.d.) testing with Culicoides extracts (Braver- 2. Materials and methods man, 1988; Larsen et al., 1988; Greiner et al., 1990; Anderson et al., 1993; Littlewood, 1998; Steinman et al., 2.1. Horses used for intradermal testing 2003). More recently, direct evidence for the crucial role of Culicoides-specific IgE antibodies as mediators of the Intradermal testing was performed on clinically healthy disease was found by transferring IgE antibodies from horses without signs of skin disease or other allergic allergic donors to clinically healthy, non-sensitized reci- conditions before or during this study. The adult group was pient horses. After IgE transfer, challenge with Culicoides composed of 24 horses 4–18 years old (median 11 years). allergen extract induced immediate skin reactions in the The horses were of mixed breeds (11 Thoroughbreds, recipient horses (Wagner et al., 2006a). 8 Warmbloods, and 5 ponies) and sexes (21 mares and Intradermal injections of monoclonal anti-IgE and anti- 3 geldings). Intradermal testing was also performed on IgG(T) antibodies suggested that IgE and also IgG(T) can 21 young horses 1–3 years old. The group of young horses sensitize skin mast cells in horses (Wagner et al., 2006a). In consisted of 11 Thoroughbreds, 7 ponies, 2 Quarter Horses humans, IgG can be bound to the surface of mast cells and and 1 Appaloosa, 16 of which were mares and 5 were basophils via Fc-receptors for IgG (FcgRs). Depending on stallions. the type of the FcgR, receptor-bound IgG either increased All horses were kept in the herd of the Baker Institute mast cell activation or provided inhibitory signals after IgG for Animal Health at Cornell University (Ithaca, NY, USA) in crosslinking (Miyajima et al., 1997; Ujike et al., 1999; an environment with run-in sheds and daily access to Tkaczyk et al., 2004). pasture. The herd was naturally exposed to Culicoides Horses generally do not develop clinical signs of skin midges during the summer months as indicated by one hypersensitivity before the age of 3–4 years (Wilson et al., animal with Culicoides-induced skin hypersensitivity in the 2001). Skin are rarely observed in younger herd. This horse was kept in the experimental environment animals, although the horses are often kept in the same for many years and developed severe signs of clinical environment with clinically affected, allergic individuals allergy by the end of June during both years of the study. (Wagner et al., 2003). The recent observation that foals did The allergic animal was not included in this sensitization not produce IgE for at least 6–9 months after birth (Wagner study. Skin testing on adult, healthy horses was performed et al., 2006b; Marti et al., 2009) might provide some in May or early June, 2006. Skin tests on young horses were explanation for the late onset of allergic diseases in horses, performed in May and November of 2006 and 2007. All despite early exposure to allergen. However, IgE can be experimental procedures were approved by the Animal detected in the serum and on peripheral blood leukocytes Care Committee of Cornell University and were in of foals for the first 8–12 weeks after birth. These IgE accordance with the guidelines established by the NIH. antibodies are solely of maternal origin and are transferred with colostrum after birth (Wagner et al., 2006b; Marti 2.2. Intradermal testing et al., 2009). The highest concentrations of maternal IgE bound to the peripheral blood cells of foals were found on Skin testing was performed and evaluated as described days 2–5 after birth and declined constantly afterwards previously (Wagner et al., 2006a). Briefly, for each horse, a (Wagner et al., 2006b). In contrast to the late onset of negative (saline) and positive control (histamine, 27.5 mg/ endogenous IgE production in foals, IgG(T) production was ml), various allergen extracts and monoclonal antibodies detectable after 8–10 weeks of age (Sheoran et al., 2000; were injected i.d. in the lateral neck. The neck area was Holznagel et al., 2003). Because previous findings sug- clipped the day before skin testing was performed. The gested that IgG(T) could play a role in sensitization of adult volume per i.d. injection was 100 ml for adult and 50 ml for horses (Wagner et al., 2006a), we hypothesized that young horses. Skin reactions were read after 20–30 min. sensitization with IgG(T) might precede that with endo- The reactions were visible as wheals at the injection sites genous IgE in young horses and might influence the and were evaluated by inspection and palpation. A score development of allergen-specific immune responses in the (0–4) was recorded for the wheal sizes at each i.d. injection absence of IgE. site. The score of 0 was always given for the saline control B. Wagner et al. / Veterinary Immunology and Immunopathology 132 (2009) 53–61 55 and the score of 4 was applied to the wheal of the in the degranulation assay. Blood samples were obtained in histamine control. The remaining reactions were rated heparinized tubes and processed for the in vitro degranula- according to these controls for each horse. The evaluation tion assay as previously described (Wagner et al., 2008). of skin reactivity and the scoring were performed blindly by a board-certified dermatologist. 2.6. In vitro degranulation assay and histamine ELISA

2.3. Allergens for intradermal testing To provoke histamine release from blood cells, anti-IgE 134, anti-IgG(T) (CVS40), and anti-IgGa (CVS45) mAbs A reduced panel of 36 allergens was used for skin were used in a concentration of 5 mg/ml in Tyrodes/BSA testing in young horses. The allergen extracts were Alfalfa, buffer (135 mM NaCl, 5 mM KCl, 1.8 mM CaCl2,1mM Alternaria, Ash, Aspergillus, Beech, Birch, Brome, Cocklebur, MgCl2, 5.6 mM glucose, 20 mM hepes, pH 7.4, containing Culicoides, Dandelion, Eastern Oak, Elm, Fescue, Goldenrod, 1% (w/v) BSA). Negative controls were obtained by June (Kentucky blue), Kochia, Lamb’s Quarter, Maple Elder, incubation of the cells with Tyrodes/BSA alone, and Marsh Elder, Mugwort, Orchard, Pigweed, Plantain, Poplar, positive controls by boiling the cells in Tyrodes/BSA for Quack, Ragweed, Red Cedar, Red Top, Rye, Sorrel/Dock, 5 min. The supernatants from the boiled samples were Sweet Vernal, Sycamore, Timothy, Walnut, White Pine, and used to determine the maximal histamine release from the Wormwood (Greer Laboratories, Lenoir, NC). For skin cells. The histamine concentration was determined in the testing in adult horses, three selected allergen extracts (cat supernatants of all samples using the Histamine EIA kit dander, Culicoides and Quack grass) were used for the (Immuno Biological Laboratories, Inc., Minneapolis, MN). following reasons: (1) The allergic horse that was used as Allergen extracts have previously been reported to be an indicator for allergen exposure in the herd showed irritating and histamine has been found in Culicoides consistent positive reactions to Culicoides and Quack grass extract (Wagner et al., 2008). To determine if certain extracts by i.d. testing. Because the horse had lived in the allergen extracts used in intradermal testing contained herd for many years, frequent exposure to both substances detectable histamine concentrations that might have was expected for all animals in this environment; (2) induced positive reactions by itself and independent of Culicoides allergen was the most frequent inducer of equine the respective allergen, we also measured the histamine skin hypersensitivity (Braverman, 1988; Larsen et al., concentrations in all allergen extracts using the Histamine 1988; Greiner et al., 1990; Ungar-Waron et al., 1990; EIA kit. Anderson et al., 1993; Wilson et al., 2001; Wagner et al., 2006a, 2008); (3) IgE antibodies to cat dander extract were 2.7. Statistical analysis common in a previous serological study performed in allergic horses (Morgan et al., 2007). For comparing sensitization results between young and adult horses a Mann–Whitney test was performed. A 2.4. Monoclonal antibodies for intradermal testing Spearman’s rank correlation was used to calculate whether the age of adult horses and the skin testing result to The following mAbs were used for skin testing: anti-IgE Culicoides allergen extract were associated. The immediate 134 (Wagner et al., 2003, 2006a), anti-IgG(T) (CVS38 and skin testing results after injection of anti-IgE or anti-IgG CVS40), anti-IgGa (CVS45), anti-IgGb (CVS39) (Sheoran antibodies between different age groups were analyzed by et al., 1998), anti-IgG3 159-4, and anti-IgG5 clones 416-2, Mann–Whitney test. A Kruskal–Wallis test followed by a 429-2, 522 and 586 (Wagner et al., unpublished). The anti- Dunn’s test for multiple comparisons against the adult IgG3 and IgG5 reagents were developed in our group using group was used to compare the histamine release from previously described recombinant equine IgG3 or IgG5 foals (birth, 6 and 12 weeks) and adult horses for different proteins (Lewis et al., 2008) for immunization of mice. The stimuli. Most statistical calculations were performed using mAbs were tested for their specificity to IgG3 and/or IgG5 the GraphPad Prism program, Version 5.01. The 95% using the recombinant proteins and detected specifically confidence intervals (CI) on prevalences and incidences IgG5 (clones 416-2 and 522) or both IgG3 and IgG5 (clones were calculated in Statistix 9 (2008, Analytical Software, 429-2 and 586) (Wagner et al., unpublished). The latter Tallahassee, FL). We used 2-sided a = 0.05 for significance recognition pattern was similar to the IgG(T) antibodies and did not make additional adjustments for multiplicity. CVS38 and CVS40 which also recognized both isotypes (Wagner, 2006; Lewis et al., 2008). A mAb concentration of 3. Results 50 mg/ml was used for all intradermal injections. All mAbs were murine IgG1 isotypes. 3.1. Sensitization of skin mast cells to allergen extracts in young and adult, clinical healthy horses 2.5. Blood samples for an in vitro degranulation assay The prevalence of sensitization to allergen extracts Peripheral blood samples were obtained from foals at was analyzed by i.d. testing in clinically healthy horses 12–96 h after birth or at 6 or 12 weeks of age. All animals that lived in an environment with natural exposure to were clinically healthy throughout the sampling period. Culicoides midges. In young horses, the most frequent Foals were born in spring 2007 and kept in the same reaction was observed to Culicoides allergen extract (14%). environment at the Equine Park at Cornell University. The young horses showed a very low reactivity of overall Samples from 12 adult, unrelated horses were also included 2% to the remaining 35 allergens (Table 1). In all 21 horses, 56 B. Wagner et al. / Veterinary Immunology and Immunopathology 132 (2009) 53–61

Table 1 Immediate skin reactions (30 min) to monoclonal anti-IgE and anti-IgG antibodies and to allergen extracts in 21 clinically healthy horses 1–3 years old.

Number of reactions Positive reactions Score: median (min/max) 1st year 2nd year 3rd year Total % 95% CI

N (horses) 5 7 9 21 Saline 0/5 0/7 0/9 0/21 0 0, 19 0 (0/0) Histamine 5/5 7/7 9/9 21/21 100 81, 100 4 (4/4)

Anti-IgE 4/5 6/7 8/9 18/21 86 63, 96 3 (0/4) Anti-IgG(T) (CVS40) 1/5 3/7 3/9 7/21 33 15, 57 0 (0/4) Anti-IgG(T) (CVS38) 0/5 0/7 0/9 0/21 0 0, 19 0 (0/0) Anti-IgGa (CVS45) 0/5 1/7 0/9 1/21 5 0, 26 0 (0/2) Anti-IgGb (CVS39) 0/5 2/7 1/9 3/21 14 4, 37 0 (0/2)

Culicoides extract 1/5 1/7 1/9 3/21 14 4, 37 0 (0/4) Other allergen extracts (35 per horse) 4/175 6/245 4/315 14/735 2 1, 3 0 (0/2)

Fig. 1. Immediate skin reactions 30 min after intradermal injection in two horses. The reactions were scored according to the negative control (saline, score = 0) and the positive control (histamine, score = 4) for each horse. (A) Skin testing result of a horse not sensitized to the allergens tested. The i.d. injection of all solutions induced a small wheal. For this horse, the wheals of all three allergen extracts were not different from the saline control. (B) Skin testing result of a horse sensitized to Culicoides allergen. The size of the wheal at the injection site of Culicoides extract was similar or even exceeded the size of the histamine control. a single reaction was observed to Ash, Aspergillus, Brome, healthy, adult horses showed sensitization to Culicoides Eastern Oak, Marsh Elder, Quack, Ragweed, Walnut and extract with a score of 2 or higher (Table 2). Only 17% of Wormwood. Two reactions each were found to Alfalfa and the horses reacted to Quack grass extract, and none to cat Plantain. dander extract. The adult horses had a higher prevalence The adult horses were tested with three selected of sensitization to Culicoides than the young horses allergen extracts (cat dander, Culicoides and Quack grass) (p = 0.047; Fig. 2A), but sensitization to Quack grass extract and the controls (Fig. 1). A total of 38% of the clinically did not differ (p = 0.43; Fig. 2B). Sensitization to Culicoides

Table 2 Immediate skin reactions to selected allergen extracts (Culicoides, Quack grass and cat dander), and monoclonal antibodies to IgE and IgG(T) in adult, clinically healthy horses 30 min after i.d. injection.

N (horses) Number of reactions Positive reactions Score: medium (min/max)

% 95% CI

Saline 24 0/24 0 0, 17 0 (0/0) Histamine 24 24/24 100 83, 100 4 (4/4)

Anti-IgE 10 10/10 100 66, 100 3 (2/4) Anti-IgG(T) (CVS40) 10 9/10 90 54, 99 2 (0/3)

Cat dander extract 18 0/18 0 0, 22 0 (0/0) Quack grass extract 24 4/24 17 5, 38 0 (0/4) Culicoides extract 24 9/24 38 20, 59 0 (0/4) B. Wagner et al. / Veterinary Immunology and Immunopathology 132 (2009) 53–61 57

Fig. 3. Different concentrations of histamine were injected into the skin of four adult horses. The scores of the immediate skin reactions for each horse and histamine concentration are shown. The histamine concentration of 27,500 ng/ml corresponded to that of the positive control used for all skin testing experiments in this study.

was found throughout the group of adult horses and was not age-dependent (Fig. 2C).

3.2. Histamine concentrations in allergen extracts

Histamine was detected in 5 out of the 36 extracts used for skin testing in young horses and also in the cat dander extract (Table 3). The final histamine concentration injected with these extracts during skin testing was at least 1800-fold lower than the histamine concentration of the positive control. Skin testing using various concentra- tions of histamine was performed on four of the adult horses to identify the lowest histamine concentration that still induced a positive reaction and to assist in interpreting the influence of the histamine in some of the allergen extracts on the intradermal testing results. The histamine concentration used for the positive control in skin testing was 27,500 ng/ml. This concentration and also a histamine injection of 2750 ng/ml induced clearly positive reactions in all four horses (Fig. 3). At histamine concentrations of 27.5 ng/ml or lower immediate skin reactions were not observed. Because the histamine concentrations injected with the allergen extracts, including Culicoides extract, ranged between 3.8 and 15.1 ng/ml, we concluded that the Fig. 2. Immediate skin reactions to Culicoides (A) and Quack grass (B) histamine content of the allergen extracts alone was not allergen extracts in clinically healthy horses. Skin reactions were scored 30 min after i.d. injection and were compared by Mann–Whitney test sufficient to induce an immediate skin reaction in horses between 21 young (1–3 years old) and 24 adult horses (4yearsold) and that positive skin reactions resulted at least partially living in the same environment. The bars indicate the median score per from Culicoides allergen. Thus, the immediate skin reac- group. (C) Immediate skin reactions to Culicoides allergen extract of tions observed after intradermal injection of Culicoides individual adult horses between 4 and 18 years old (rsp = À0.26, extract in clinically healthy horses were likely to represent p = 0.21). true sensitization of skin mast cells to Culicoides allergen.

Table 3 Histamine concentrations (ng/ml) in various allergen extracts were detected in 6 out of 37 extracts by ELISA.

Allergen extract

Cat dander Culicoides Maple Elder Marsh Elder Quack grass Wormwood

Histamine concentration (undiluted extract) 302 165 136 76 172 184 Allergen dilution for i.d. testing 1:20 1:20 1:20 1:20 1:20 1:20 Final histamine concentration used for i.d. testing 15.1 8.25 6.8 3.8 8.6 9.2 58 B. Wagner et al. / Veterinary Immunology and Immunopathology 132 (2009) 53–61

depended on the antibody clone used for injection. While CVS40 injection induced a skin reaction in one-third of the young horses, CVS38 did not result in any skin reaction (Table 1). In addition, we performed skin testing on seven of the young horses (1–2 years of age) using one mAb to equine IgG3, two mAbs to equine IgG5, and two new mAbs to IgG3 and IgG5. None of these antibodies induced an immediate skin reaction in any of the horses. Ten of the adult horses were tested with the anti-IgG(T) antibody CVS40. In response to CVS40 injection, 9 out of 10 adult horses developed an immediate skin reaction (Table 2). Although the reaction to CVS40 confirmed previously results obtained by i.d. injection of this anti- body, skin reactions were not induced by the injected CVS38 or any of the anti-IgG3 or anti-IgG5 antibodies into the skin of adult horses (data not shown). Fig. 4. The sensitization of skin mast cells with IgE was evaluated by i.d. injection of a monoclonal anti-IgE antibody. Immediate skin reactions 3.5. Antibody mediated in vitro degranulation from were scored in young (1–3 years old, open circles) and adult horses (black circles) 30 min after anti-IgE injection. A Kruskal–Wallis test indicated basophils of foals and adult horses that sensitization of skin mast cells with IgE did not differ between the age groups (p = 0.12). The bars show the median scores for each group. Intradermal testing is difficult to perform in neonates and young foals. Sensitization of peripheral blood baso- 3.3. Sensitization of skin mast cells with IgE in clinically phils can also be tested by in vitro assays. Basophils have a healthy young and adult horses frequency of 0.5–1% in peripheral white blood cells. They are equipped with high-affinity IgE receptors, are sensi- Anti-IgE induced an immediate skin reaction with a tized with IgE and release histamine in response to score of 2 in 86% of the i.d. injection sites in young horses stimulation similar to mast cells (Langner et al., 2008; (Table 1). The same anti-IgE reagent induced an immediate Wagner et al., 2008). A degranulation assay was used skin reaction in each of the ten adult horses tested to test for anti-IgE and anti-IgG(T) (CVS40) mediated (Table 2). Although the median score of the anti-IgE- histamine release from basophils of foals and adult horses. mediated immediate skin reactions increased from 2 The histamine release induced by stimulation of the (horses 1 and 2 old) to 3 (horses 3 years old and adults) cells with anti-IgE was significantly lower at birth, 6 weeks with age, the reactions between young and adult horses and 12 weeks of age compared to cells from adult horses were not significantly different (Fig. 4). (Fig. 5A). Basophils from foals at birth and 6 weeks of age also contained significantly less histamine than basophils 3.4. Skin reactivity to anti-IgG(T) antibodies in clinically from adult horses (Fig. 5B). After stimulation with anti- healthy young and adult horses IgG(T), low concentrations of histamine were detected in 20% of the samples from foals at birth and 6 weeks of We previously showed that the anti-IgG(T) antibody age, in 29% of the foals at 12 weeks of age, and in 67% of CVS40 induced immediate skin reactions in adult horses. the adult horses. Overall, the median histamine concen- Here, we expanded the testing by using various antibodies trations induced by anti-IgG(T) were lower than those and found that immediate skin reactions to anti-IgG(T) induced by anti-IgE in all age groups (Table 4). In adult

Fig. 5. Histamine released by basophils from neonates (birth), foals (6 and 12 weeks) or adult horses was detected by ELISA. (A) The anti-IgE induced histamine release and (B) the total histamine content of the cells are shown. The bars show the median histamine concentrations and the asterisks indicate significant differences between groups; **p < 0.01, ***p < 0.001. B. Wagner et al. / Veterinary Immunology and Immunopathology 132 (2009) 53–61 59

Table 4 Histamine release (ng/ml; expressed as median (min/max)) induced from peripheral blood basophils of neonates (12–96 h after birth), foals 6 or 12 weeks old and adult horses.

Foals Adults

Birth 6 weeks 12 weeks

N 10 15 14 12 Minimal release 0 (0/3) 0 (0/0) 0 (0/5) 0.2 (0/3) Maximal release 4.4 (0/21) 21.2 (13/25) 27.3 (15/32) 30.9 (21/32) Anti-IgE 1.0 (0/24) 5.8 (0/22) 13.9 (0/32) 29.5 (17/32) Anti-IgG(T) (CVS40) 0 (0/4) 0 (0/7) 0 (0/7) 4.6 (0/8) Anti-IgGa (CVS45) 0 (0/4) 0 (0/0) 0 (0/4) 0 (0/8) horses, anti-IgG(T) stimulated an increased histamine firmed that the amount of IgE bound to mast cell release compared to foals (p < 0.05 at all time points). Anti- surface in young horses was sufficient to mediate IgGa served as a negative control and a histamine release degranulation and inflammatory mediator release. induced by anti-IgGa was not detectable for most foals and Thus, it is unlikely that the suppression of endogenous adult horses. No significant differences between the IgE production during the first 6–9 months of life histamine release induced by the two anti-IgG antibodies directly influences the late onset of clinical signs of were found for the foals or for the adult horses. allergy in horses. (2) The development of an IgE response to Culicoides could 4. Discussion simply require time and/or repeated exposure. The response would then gradually increase with age and at Sensitization of mast cells and basophils with IgE some point cross the threshold to develop clinical precedes degranulation and the development of clinical allergy. This was not supported by our findings, because signs of allergy (Ravetch and Kinet, 1991). Intradermal skin sensitization to Culicoides did not increase between 1 testing and in vitro degranulation assays with allergens are and 3 years of age. Only one positive skin reaction to based on the principle of mast cell or basophil sensitization Culicoides was observed for the young horses of each of and are used in the diagnosis of allergies to identify allergens the ages 1, 2 or 3 years. Similarly, an obvious increase in responsible for the disease. Nevertheless, sensitization to a sensitization to Culicoides with age was not observed for particular allergen does not necessarily mean that the the adult horses. We concluded that the production of individual is allergic or will develop clinical signs of allergy. allergen-specific IgE and sensitization of mast cells with Previous reports in horses found that clinically healthy these antibodies alone is not sufficient to explain the animals were sometimes sensitized to allergens. Sensitiza- development of allergy in horses. tion in healthy control horses was identified by i.d. testing (Lorch et al., 2001a,b; Kolm-Stark and Wagner, 2002; Lebis It is not yet fully understood which immune reactions et al., 2002) or by performing an in vitro sensitization assay determine whether a horse produces IgE in response to (Wagner et al., 2008). Here, we investigated the sensitiza- Culicoides exposure and becomes sensitized, and whether tion of skin mast cells to Culicoides allergen in clinically or not a sensitized animal develops clinical signs of allergy. healthy horses kept in a single environment. More than one- Regulatory immune mechanisms might play an important third of the adult horses were sensitized to Culicoides role in the development of clinical allergy in humans. extract. We concluded that exposed horses frequently Successful desensitization in human allergy patients was become sensitized to Culicoides allergen without showing linked to the induction of interleukin 10 (IL-10) and/or clinical signs of allergy. tumor growth factor-b (TGF-b) secreting regulatory The prevalence of sensitization to Culicoides allergen T-cells (Hawrylowicz and O’Garra, 2005; Akdis and Akdis, was lower in young horses (14%) than in adult horses 2007; Pereira-Santos et al., 2008; Ryanna et al., 2009). (38%). This is in agreement with the observation that skin Regulatory T-cells directly suppressed the allergen-specific hypersensitivity usually does not develop before 3–4 years T-cell development (Meiler et al., 2008) and the activation of age (Wilson et al., 2001). If the exposure of all horses to of mast cells (Gri et al., 2008). In horses, IL-10 and TGF-b Culicoides allergen in the same environment is considered down-regulation of IL-4 suggested an suppressive effect on as equal, two explanations could be offered for the higher the development of skin hypersensitivity by this mechan- prevalence of sensitization in healthy adult horses. ism (Hamza et al., 2008). Another regulatory mechanism influencing the out- (1) The delay in the endogenous IgE production in foals come of mast cell and basophil degranulation is provided (Wagner et al., 2006b; Marti et al., 2009) could result in by FcgRs mediating both activation or inhibition of the less IgE bound to mast cell and basophil surfaces and cells (Ujike et al., 1999; Tkaczyk et al., 2004; Bruhns et al., consequently less severe inflammatory reactions after 2005). Simultaneous crosslinking of FceRI and the inhibi- allergen exposure. However, sensitization of skin mast tory FcgRIIb resulted in decreased activation and degra- cells with IgE was observed for almost all horses nulation from human basophils (Kepley et al., 2000; between 1 and 3 years old and sensitization with IgE in Wigginton et al., 2008). In contrast, aggregation of the FcgRI young and adult horses was not different. The skin on human mast cells (Woolhiser et al., 2001)orFcgRIII on reactions observed after anti-IgE injection also con- murine mast cells (Miyajima et al., 1997) promoted 60 B. Wagner et al. / Veterinary Immunology and Immunopathology 132 (2009) 53–61 mediator release comparable to crosslinking of FceRI. In the mediated histamine release increased in the samples from horse, FcgRs on mast cells or basophils have not yet been the foals, although the amount of maternal IgE on basophil identified. Nevertheless, equine IgG antibodies could play a surfaces decreased during the same time (Wagner et al., role in modulating the allergic responses in adult horses and 2006b). Our current findings suggested that neonatal could be even more important in young horses before basophils had an impaired ability to provoke inflammatory endogenous IgE is produced. reactions because they contained very low concentrations That anti-IgG(T) mAb CVS40 was previously found to of histamine, and that both the histamine content and the induce immediate skin reactions in horses suggested direct ability of basophils to release histamine by IgE-mediated sensitization of mast cells with IgG(T) (Wagner et al., mechanisms increased during the first weeks of life. At 2006a). Here, we investigated whether IgG(T) might have a 12 weeks of age, the total histamine content of foal cells did role in the sensitization of skin mast cells and basophils not differ from cells of adult horses. early in life. Although the previous skin testing results with Crosslinking of maternal IgE antibodies on neonatal the anti-IgG(T) antibody CVS40 were repeatable in this basophils could be induced by environmental antigens study in adult horses, evidence for an activating role of after birth and might provide an initial antigen-specific IgG(T) in the sensitization of skin mast cells or basophils trigger of the foal’s immune response early in life. Such a early in life could not be found. The responsiveness of mechanism might induce an adaptive immune response in young horses and foals to the CVS40 mAb in skin testing the foal that depends on the mare’s acquired immunity. and the in vitro degranulation assay was lower than that This could be beneficial for the foal if IgE antibodies to for anti-IgE. This confirmed the primary role of IgE in parasites are transferred or it could induce a first allergen- sensitization of equine mast cells and basophils and its specific response if the mare is allergic. Nevertheless, the potential to mediate the age-independent activation of reduced ability of the neonatal and young foal’s basophils these cells. The inability of various other anti-IgG3 and/or to release histamine might prevent strong inflammatory anti-IgG5 mAbs to induce immediate skin reactions could responses very early in life. be interpreted as evidence that skin mast cells are not Horses provide a valuable animal model for the better likely to be directly sensitized with these isotypes and that characterization and understanding of the late onset of the skin reactions induced by CVS40 might be mediated by allergic diseases despite early exposure to allergen and other, IgG-independent or indirect mechanisms. Poten- the development of functional IgE and competent mast tially, CVS40 could have an indirect effect on degranulation cell and basophil responses during the first year of life. by formation of IgG(T)/anti-IgG(T) complexes followed by Investigations of the immune response induced by binding to activating FcgRs in adult horses. In humans, maternal IgE and the mechanisms that stimulate endo- IgG-mediated mast cell degranulation depended on the genous IgE production in foals could lead to a novel FcgR and on the mast cell type. Biological responses of understanding of the immune regulation during the mast cells were a result of the balance between positive preclinical development of allergic diseases and the and negative signals that were generated by various identification of natural resistance mechanisms in geneti- activating and inhibitory receptors on the mast cell surface cally predisposed individuals. (Malbec and Dae¨ron, 2007). Mast cells and basophils of young horses might have a different FcgR expression 5. Conclusion profile resulting in the observed age-dependent variations in degranulation induced by the CVS40 mAb. Sensitization of skin mast cells or basophils in young Cross-reactions of the anti-IgG(T) mAb CVS40 with IgE and adult horses occurs primarily by IgE antibodies. We have been previously excluded for soluble IgE by could not confirm our hypothesis that IgG(T) antibodies serological assays (Wagner et al., 2003, 2006a). However, have a role in sensitization early in life. Sensitization with it cannot be completely ruled out that IgE binding to FceRI maternal IgE was detected during the first 3 months of life. induces conformational changes of the IgE molecule that Sensitization with endogenous IgE could be observed as supported the binding of CVS40 to the receptor-bound IgE early as 1 year of age. Culicoides extract was the most and thereby mediated degranulation. If the induction of prevalent allergen in young horses. Until 3 years of age, the degranulation by the CVS40 mAb was a result of cross- sensitization status to Culicoides allergen did not vary with reactivity with receptor-bound IgE, the decreased degra- age but it increased in adult horses. nulation in young horses could be explained by a lower density of FceRI-bound IgE on the cell surface of mast cells Conflict of interest and basophils in young horses that was not sufficient to induce degranulation by CVS40 effectively. None. Foals do not produce IgE for the first months of life. However, maternal IgE antibodies are transferred to the foal with the colostrum and can be detected in the Acknowledgements circulation for several weeks. In neonates and foals, basophils are thus solely sensitized with maternal IgE We thank the barn crews at the McConville Barn and (Wagner et al., 2006b). At birth, the total histamine content the Equine Park for their help with the animals during and the IgE-mediated histamine release from neonatal intradermal testing and blood sampling, and the Harry M. cells was low or not detectable. Between birth and 12 Zweig Memorial Fund for Equine Research at Cornell weeks of life, the total histamine content and the anti-IgE- University for supporting this project. B. Wagner et al. / Veterinary Immunology and Immunopathology 132 (2009) 53–61 61

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