Development of eosinophilic conjunctival inflammation at late-phase reaction in mast cell–deficient mice

476 Letters to the Editor

J ALLERGY CLIN IMMUNOL AUGUST 2007

Supported by Programmi di Ricera di Interesse Nazionale (PRIN)-Ministero dell’Universita` e della Ricerca (MIUR) and Associazione Immunodeficienze Primitive. M.L.R. was supported by a Doctor in Research on Immunology and Applied Biotechnologies Grant, University of Rome Tor Vergata. Disclosure of potential conflict of interest: The authors have declared that they have no conflict of interest.

REFERENCES

FIG 2. IgM memory B cells and switched memory B cells in 6 children with definitive THI diagnosis and in 7 children with persistent hypogammaglobulinemia after 3 years of follow-up. Bar represents the median. Statistical analysis was performed by the Student t test, and P values are given.

In summary, our data suggest that, before 2 years of age, a child with a low level of serum immunoglobulins may be cautiously defined as having hypogammaglobulinemia during infancy. Only after 2 years of age, the evaluation of B cell memory subsets can be used to predict the evolution of the disease and discriminate, in the group of children with hypogammaglobulinemia during infancy, those who really were patients with THI from those who are likely to have permanent immunodeficiency later in life. We thank Prof Anne Durandy and Dr Maria Antonietta Avanzini for critical reading of the manuscript. Viviana Moschese, MD, PhDa Rita Carsetti, MDb Simona Graziani, MDa Loredana Chini, MDa Anna Rosa Soresina, MDc Maria La Rocca, MDa Grazia Bossi, MDd Silvia Di Cesare, BSca Alessandro Plebani, MDc For the Italian Primary Immunodeficiency Network

Letters to the Editor

From athe Department of Pediatrics, University of Rome Tor Vergata, Rome; b the Research Center, Ospedale Pediatrico Bambino Gesu` (Istituto di Ricovero e Cura a Carattere Scientifico), Rome; cthe Department of Pediatrics, University of Brescia; and dthe Department of Pediatrics, IRCCS Policlinico San Matteo, Pavia, Italy. Reprint requests: Viviana Moschese, MD, PhD, Department of Pediatrics, University of Rome Tor Vergata, Policlinico Tor Vergata, Viale Oxford, 81, Rome 00133, Italy. E-mail: [email protected].

1. International Union of Immunological Societies. Primary immunodeficiency diseases: report of an IUIS Scientific Committee. Clin Exp Immunol 1999;118(suppl 1):S1-28. 2. Stiehm ER, Ochs HD, Winkelstein JA. Immunodeficiency disorders: general considerations. In: Stiehm ER, Ochs HD, Winkelstein JA, editors. Immunologic disorders of infents and children. 5th ed. London: Elsevier Saunders; 2004. p. 289-355. 3. Bonilla FA, Bernstein L, Khan DA, Ballas ZK, Chinen J, Frank MM, et al. Practice parameter for the diagnosis and management of primary immunodeficiency. Ann Allergy Asthma Immunol 2005;94(suppl 1):S1-63. 4. Walker AM, Kemp AS, Hill DJ, Shelton MJ. Features of transient hypogammaglobulinemia in infants screened for immunological abnormalities. Arch Dis Child 1994;70:183-6. 5. Warnatz K, Denz A, Drager R, Braun M, Groth C, Wolff-Vorbeck G, et al. Severe deficiency of switched memory B cells (CD27(1)IgM(-)IgD(-)) in subgroups of patients with common variable immunodeficiency: a new approach to classify a heterogeneous disease. Blood 2002;99:1544-51. 6. Carsetti R, Rosado MM, Donnanno S, Guazzi V, Soresina AR, Meini A, et al. The loss of IgM memory B cells correlates with clinical disease in common variable immunodeficiency. J Allergy Clin Immunol 2005;115: 412-7. 7. Alachkar H, Taubenheim N, Haeney MR, Durandy A, Arkwright PD. Memory switched B cell percentage and not serum immunoglobulin concentration is associated with clinical complication in children and adults with specific antibody deficiency and common variable immunodeficiency. Clin Immunol 2006;120:310-8. 8. Kruetzmann S, Rosado MM, Weber H, Germing U, Tournilhac O, Peter HH, et al. Human immunoglobulin M memory B cells controlling Streptococcus pneumoniae infections are generated in the spleen. J Exp Med 2003;197:939-45. 9. McGeady SJ. Transient hypogammaglobulinemia of infancy: need to reconsider name and definition. J Pediatr 1987;110:47-50. 10. Tiller TL Jr, Buckley RH. Transient hypogammaglobulinemia of infancy: review of the literature, clinical and immunologic features of 11 new cases, and long-term follow-up. J Pediatr 1978;92:347-53. Available online May 26, 2007. doi:10.1016/j.jaci.2007.04.002

Development of eosinophilic conjunctival inflammation at late-phase reaction in mast cell–deficient mice To the Editor: Mast cells play a central role in immediate allergic reactions and in the early phase of allergic conjunctivitis.1 However, their role in the late-phase response is not clearly defined. The magnitude of eosinophil infiltration in the conjunctiva reflects the degree of its late-phase reaction. Using our model of allergic conjunctivitis and genetically mast cell–deficient (W/Wv) mice, we performed direct assessment of the role of mast cells in conjunctival eosinophil infiltration. Mast cells and the products they release are widely thought to contribute to the development of allergic conjunctivitis. It is triggered by IgE cross-linking on mast cells and produces early-phase reactions in the

conjunctiva. A panel of preformed or newly synthesized mediators, including histamine, with clinical effects on conjunctival redness, the degree of eye itching, and the amount of tearing from the conjunctiva, is released from mast cells1 in the acute phase of the allergic reaction. However, much less is known about the involvement of mast cells in the late phase. We used 6- to 12-week-old female mast cell–deficient ([WB-W/1 3 C57BL/6-Wv /1]F1; W/Wv) mice and congenic WBB6F1 normal mice (1/1; Japan SLC, Inc, Hamamatsu, Shizuoka, Japan). The experiments were conducted under a protocol approved by the Institutional Animal Care and Use Committee of the Kyoto Prefectural University of Medicine. Short ragweed pollen (RW) was purchased from Polysciences, Inc (Warrington, Pa), and aluminum hydroxide (alum) was purchased from Sigma (St Louis, Mo). The mice were immunized with an intracutaneous injection into the left hind footpad of RW adsorbed on alum (200 mg of RW and 2.6 mg of alum) on day 0, followed by intraperitoneal injections of RW adsorbed on alum on days 7 and 14. On days 21, 22, 23, and 25, their eyes were challenged with RW in PBS (500 mg, 5 mL per eye). At 24 hours after the last challenge, their eyes, including the conjunctiva, were harvested, fixed in 10% neutral buffered formalin, and embedded in paraffin blocks for histologic analysis. Vertical 6-mmthick sections were affixed to microscope slides, deparaffinized, and stained with Luna stain, which identifies erythrocytes and eosinophil granules. We counted infiltrating eosinophils in the lamina propria mucosae of the tarsal and bulbar conjunctiva in the entire section from the central portion of the eye, which included the pupil and optic nerve head. Cell counts were expressed as infiltrating eosinophil number per unit area (0.1 mm2 area) measured with Scion Image software (Scion Corp, Frederick, Md). For quantitative RT-PCR of eotaxin-specific mRNA in the eyelids, the upper and lower lids were collected 4 hours after the last RW challenge and homogenized in liquid nitrogen. Total RNA was extracted with the RNeasy mini kit (Qiagen, Tokyo, Japan). For reverse transcription, we used ReverTra Ace (TOYOBO, Otsu, Japan). The primers and probes for mouse eotaxin and glyceraldehyde3-phosphate dehydrogenase were from Applied Biosystems (Foster City, Calif). The results were analyzed with sequence-detection software (Applied Biosystems). Data were expressed as means 6 SEs, and statistical analyses were performed by using ANOVA or the Student t test, as appropriate. First, we compared eosinophil infiltration in 1/1 and W/Wv mice. Although no sensitization and sensitization without challenge did not affect the number of eosinophils, after sensitization with challenge, the number of eosinophils in the lamina propria mucosae of the conjunctiva was significantly increased in both the mast cell– deficient mice and their congenic littermates. There was no difference between mast cell–deficient and mast cell– sufficient mice (Fig 1, A and B). We next compared the expression of eotaxin-specific mRNA in the eyelids of 1/1 and W/Wv mice because

Letters to the Editor 477

FIG 1. Eosinophilic conjunctival inflammation in mast cell–deficient mice. A, Increased eosinophils in the eyelids of mast cell–deficient mice (W/Wv) and their congenic littermates (1/1; 3 eyes from separate animals per group). B, Infiltration of eosinophils into the conjunctiva of mast cell–deficient mice (W/Wv) and their congenic littermates (1/1; bar 5 50 mm). C, Increased expression of eotaxin mRNA in the eyelids of mast cell–deficient mice (W/Wv) and their congenic littermates (1/1; 5 eyes from separate animals per group). *P < .05.

chemokines such as eotaxin recruit eosinophils. Sensitization with challenge of RW significantly increased the expression of eotaxin-specific mRNA compared with sensitization alone in both mast cell–deficient and mast cell–sufficient mice (Fig 1, C). As in previous studies,2,3 after sensitization of RW, serum total IgE, anti-RW IgE, and anti-RW IgG1 levels were comparable in mast cell–deficient mice and their congenic littermates (data not shown). Sensitization with challenge of mast cell–deficient mice produced an increase in IgE and IgG1 antigen-specific antibody responses, conjunctival eosinophil counts, and eotaxin-specific mRNA in the eyelids. In this respect, these mice were indistinguishable from their congenic littermates. However, mast cells were identified histologically in the submucosa of 1/1 mice but not W/Wv mice (see Fig E1 in this article’s Online Repository at www.jacionline.org), suggesting that the development of eosinophilic conjunctival inflammation in the late phase of allergic conjunctivitis is not dependent

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J ALLERGY CLIN IMMUNOL VOLUME 120, NUMBER 2

478 Letters to the Editor

J ALLERGY CLIN IMMUNOL AUGUST 2007

on the presence of functional mast cells. These results are similar to those reported for other systems. For example, in ovalbumin (OVA)–sensitized, iteratively challenged mice, mast cells had no effect on eosinophil influx in bronchoalveolar lavage fluid.4 Like their congenic littermates, after sensitization and airway challenge with OVA, mast cell–deficient mice had allergic antibody and pulmonary eosinophilic responses.3 On the other hand, in sensitized mast cell–deficient mice, OVA challenge produced fewer eosinophils in the bronchoalveolar lavage fluid and lungs than in similarly sensitized and challenged congenic littermates.2 However, others3,5 contended that in the murine asthmatic pulmonary eosinophilic response, mast cells might be of greatest importance when relatively weak stimulants or inducers are used to induce the response. They pointed out that because Kung et al2 used attenuated sensitization and challenge protocols, the number of eosinophils might have been significantly lower in their study than in others. We also used attenuated sensitization and challenge protocols to examine eosinophilic conjunctival inflammation. Sensitization was with 1 intracutaneous injection of RW adsorbed on alum on day 0, followed by 1 intraperitoneal injection on day 7; challenge was with only 1 exposure to RW in PBS on day 18. Although these attenuated protocols did not induce detectable anti-RW IgE in serum, we found that mast cell–deficient mice were capable of having eosinophilic conjunctival inflammation similar to that seen in their congenic littermates. Human conjunctival epithelial cells expressed eotaxin in vivo,6,7 and conjunctival fibroblasts produced eotaxin in vitro,8 suggesting that conjunctival epithelial cells, fibroblasts, or both might be implicated in the eosinophilic conjunctival inflammation seen in allergic conjunctivitis. Investigations are underway in our laboratory to identify the cells implicated in the eosinophilic conjunctival inflammation of allergic conjunctivitis. In summary, we showed that mast cell–deficient mice exposed to sensitization and eyedrop challenge had eosinophilic conjunctival inflammation similar to that seen in their congenic littermates. Although this does not exclude mast cell contributions to other aspects of latephase allergic conjunctivitis, our finding indicates that these cells do not play an essential role in the development of eosinophilic conjunctival inflammation in mice sensitized and challenged as described in this report. Mayumi Ueta, MD, PhDa Takao Nakamura, MD, PhDb Satoshi Tanaka, PhDc Kentaro Kojima, MDa Shigeru Kinoshita, MD, PhDa

Letters to the Editor

From athe Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan; bthe Department of Ophthalmology, Otemae Hospital, Osaka, Japan; and cthe Department of Immunobiology, School of Pharmaceutical Sciences, Mukogawa Women’s University, Hyogo, Japan. E-mail: [email protected]. Supported in part by grants-in-aid for scientific research from the Japanese Ministry of Health, Labour, and Welfare; the Japanese Ministry of Education, Culture, Sports, Science, and Technology; CREST from JST; a research grant from the Kyoto Foundation for the Promotion of Medical Science;

and the Intramural Research Fund of Kyoto Prefectural University of Medicine. Disclosure of potential conflict of interest: The authors have declared that they have no conflict of interest. REFERENCES 1. Graziano FM, Stahl JL, Cook EB, Barney NP. Conjunctival mast cells in ocular allergic disease. Allergy Asthma Proc 2001;22:121-6. 2. Kung TT, Stelts D, Zurcher JA, Jones H, Umland SP, Kreutner W, et al. Mast cells modulate allergic pulmonary eosinophilia in mice. Am J Respir Cell Mol Biol 1995;12:404-9. 3. Takeda K, Hamelmann E, Joetham A, Shultz LD, Larsen GL, Irvin CG, et al. Development of eosinophilic airway inflammation and airway hyperresponsiveness in mast cell-deficient mice. J Exp Med 1997;186:449-54. 4. Brusselle GG, Kips JC, Tavernier JH, van der Heyden JG, Cuvelier CA, Pauwels RA, et al. Attenuation of allergic airway inflammation in IL-4 deficient mice. Clin Exp Allergy 1994;24:73-80. 5. Tsai M, Grimbaldeston MA, Yu M, Tam SY, Galli SJ. Using mast cell knock-in mice to analyze the roles of mast cells in allergic responses in vivo. Chem Immunol Allergy 2005;87:179-97. 6. Abu El-Asrar AM, Struyf S, Al-Kharashi SA, Missotten L, Van Damme J, Geboes K. Chemokines in the limbal form of vernal keratoconjunctivitis. Br J Ophthalmol 2000;84:1360-6. 7. Martin AP, Urrets-Zavalia J, Berra A, Mariani AL, Gallino N, Gomez Demel E, et al. The effect of ketotifen on inflammatory markers in allergic conjunctivitis: an open, uncontrolled study. BMC Ophthalmol 2003;3:2-9. 8. Leonardi A, Curnow SJ, Zhan H, Calder VL. Multiple cytokines in human tear specimens in seasonal and chronic allergic eye disease and in conjunctival fibroblast cultures. Clin Exp Allergy 2006;36:777-84. Available online May 26, 2007. doi:10.1016/j.jaci.2007.04.024

Irritant skin test reactions to common vaccines To the Editor: Concerns about possible allergic reactions to immunizations are frequently raised by both patients/parents and primary caretakers. Estimates of true allergic reactions to routine vaccines range from 1 per 50,000 doses for diphtheria-tetanus-pertussis vaccine to about 1 per 500,000 to 1,000,000 doses for most other vaccines.1 Although these per-dose estimates suggest that reactions are extraordinarily rare, the large number of doses administered makes this a relatively common clinical problem. Skin testing for immediate-type hypersensitivity is indicated for some patients with suspected allergic reactions to immunizations.2,3 Standard skin testing to vaccines involves the use of both prick (puncture) and intradermal (intracutaneous) skin tests at varying concentrations. However, the interpretation of these skin test results is complicated in many instances by irritant reactions. Although it is clear from clinical experience that irritant reactions do occur to some vaccines, the frequency with which they occur has never been formally evaluated. Twenty healthy adult volunteers with no history of food or drug allergy or adverse vaccine reactions were tested to 10 common vaccines (Table I). Each vaccine was tested at a full-strength skin prick dose with a bifurcated needle and at 1:100, 1:10, and full-strength intradermal doses. All test results were read at 15 minutes and compared with results with positive (histamine) and negative (diluent) controls.