Extracellular deposition of eosinophil granule major basic protein in pressure urticaria

Clinical a n d l a b o r a t o r y s t u d i e s I

I

Illl

Extracellular deposition of eosinophil granule major basic protein in pressure urticaria Margot S. Peters, M.D.,* Richard K. Winkelmann, M.D., Ph.D.,* Malcolm W. Greaves, M.D.,** Gail M. Kephart, B.S.,* and Gerald J. Gleich, M.D.* Rochester, MN, and London, England Utilizing affinity chromatography-purified antibody to the eosinophil granule major basic protein and formalin-fixed paraffin embedded tissue, we investigated the localization of major basic protein by immunofluorescence in twenty-four skin biopsy specimens from ten patients with pressure urticaria. Fourteen of twenty-four biopsy specimens were obtained from spontaneously occurring urticarial lesions of 4 to 48 hours' duration, and ten of the twentyfour specimens were from dermographometer-induced lesions that had been present from 40 minutes to 24 hours. Twenty-one of twenty-four biopsy specimens showed extracellular fluorescence of eosinophil granule major basic protein within the dermis. The extent and intensity of extracellular staining were not related to the presence or degree of tissue eosinophilia. Serial section controls from each block were stained with protein A purified rabbit IgG and were negative. Previous immunofluorescence studies have demonstrated deposition of major basic protein in lesions of chronic idiopathic urticaria, episodic angioedema, and facial edema. Major basic protein causes release of histamine from human basophils and induces wheal-and-flare reactions on intradermal injection. The present observations add further evidence to support a role for eosinophil mediators, particularly major basic protein, in the pathogenesis of cutaneous disease characterized by edema. (1 AM ACAD DERMATOL 1987;16:513-7.)

Eosinophils may be observed in skin biopsy specimens from patients with various types of urticarial lesions. However, the histologic picture, including the pattern of eosinophil infiltration, has been considered nonspecific. 1-3The eosinophil has numerous distinctive granules, each containing an electron-dense core and a relatively electron-lucent From the Departments of Dermatology, Immunology, Medicine, and the Allergic Diseases Research Laboratory, Mayo Clinic and Mayo Foundation, Rochester,* and St. John's Hospital for Diseases of the Skin, London.** Supported by grants from the National Institutes of Health, Nos. AI 15231 and CA 09127, and by the Mayo Foundation, the Eleanor Naylor Dana Charitable Trust, and the Kieckhefer Foundation. Accepted for publication Aug. 29, 1986. Reprint requests to: Dr. Margot S. Peters, Department of Dermatology, Mayo Clinic, Rochester, MN 55905.

matrix. 4 Major basic protein comprises the core of the eosinophil granule and 50% of the total granule protein. 4'5 A role for major basic protein in the production of tissue injury is supported by studies that demonstrate its cytotoxic properties against parasites and mammalian cells. 4,6: Previous studies have provided evidence that major basic protein may mediate cutaneous tissue damage and, in particular, play a role in chronic idiopathic urticaria and certain types of angioedema. 8t~ Utilizing an indirect immunofluorescence assay, we investigated the pattern of eosinophil degranulation in lesions of pressure urticaria from the patients included in the study of Winkelmann et al. 12 These data provide insight into the participation of eosinophils in the pathogenesis of cutaneous edema. 513

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Figs. 1-6. For legends, see opposite page.

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MATERIAL AND METHODS

ophils and were finally stained with fluorescein isothiocyanate-conjugated goat antirabbit IgG.* After the immunofluorescence findings were reviewed and photographed, sections stained with anti-major basic protein were counterstained with hematoxylin and eosin to allow more precise verification of the localization of major basic protein deposition and correlation with the cellular infiltrates.

Twenty-four skin biopsy specimens obtained from lesions of ten patients with pressure urticaria were selected for study. There were six male and four female patients ranging in age from 27 to 51 years. Pressure urticaria had been present from l to 40 years (mean, 9) prior to evaluation at St. John's Hospital. A history of atopy was not elicited from any of the patients, and all had normal peripheral blood eosinophil counts. Fourteen of twenty-four skin biopsy specimens were obtained from spontaneously occurring lesions of 4 to 48 hours' duration, and ten specimens were taken from lesions that had been induced by a dermographometer, 40 minutes to 24 hours prior to biopsy. An indirect immunofluorescence assay, utilizing formalin-fixed, paraffin-embedded tissue, was performed as described in detail previously. 8'~3 Briefly, two 6-~m serial sections were obtained from each paraffin block and affixed to glass slides with LePage's Bond Fast resin glue (LePage's Ltd., Bramalea, Ontario, Canada). After deparaffinization in xylene, rehydration with alcohol and water, and trypsin digestion, sections were washed and then placed overnight in 10% normal goat serum (Pel-Freez Biologicals Inc., Rogers, AR). On day 2, one section from each block was incubated with affinity chromatography-purified rabbit antihuman major basic protein,* and the second (serial) section was incubated with protein A purified rabbit IgG as a control. On day 3, all sections were incubated in 1% chromotrope 2R to block nonspecific fluorescence of eosin-

Twenty-one of twenty-four biopsy sections stained with anti-major basic protein showed fluorescence outside the eosinophil. All fourteen biopsy specimens from spontaneously occurring lesions o f pressure urticaria and seven of ten biopsy specimens from dermographometer-induced lesions had extracellular deposition of major basic protein. The extent of extracellular fluorescence was unrelated to the presence or degree of intracellular fluorescence, that is, the extent of local tissue infiltration with eosinophils. Control serial sections stained with rabbit IgG were negative in all cases. There were three patterns of extracellular staining (Table I; Figs. 1 to 6). First, all twentyone specimens showed fluorescence of fine and/ or coarse connective tissue fibers of the dermis; fluorescence staining was seen in the superficial dennis alone in 7 biopsy specimens (4 of 7 were

*Mr. DavidLoegeringprepared the antisera.

*Ms. SharonCollyerprepared tissue sections.

RESULTS

Fig. 1. Photomicrograph showing immunofluorescence staining of eosinophil granule major basic protein on dermal connective tissue fibers. ( x 100.) Fig. 2. Counterstain of the section shown in Fig. 1 allows direct comparison of the fluorescence pattern with the histologic features. (Hemato×ylin-eosin stain; x 100.) Fig. 3. Photomicrograph illustrating staining with anti-major basic protein of granular material dispersed in the dermis. The arrow corresponds with arrow shown in Fig. 4 and facilitates direct comparison of intracellular staining of the eosinophil with fluorescence (this illustration) and with hematoxylin and eosin (Fig. 4). ( x 400.) Fig. 4. Counterstain of the section shown in Fig. 3 reveals that the granular material in this field is near or around vessels. The arrow corresponds with arrow shown in Fig. 3 and facilitates direct comparison of intracellular staining of the eosinophil with fluorescence (Fig. 3) and with hematoxylin and eosin (this illustration). ( x 400.) Fig. 5. Vessel wall is stained with immunoreactive major basic protein. ( x 400.) Fig. 6. Counterstain of the section shown in Fig. 5 verifies the location of the fluorescence and the absence of surrounding intact eosinophils. ( × 400.)

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Table L Locahzataon of major basic protein an pressure urhcana* Type of lesmn Site of fluorescence staining

Spontaneous (n = I4)

I Induced I Total (n = 10) I (n = 24,

Connectwe hssue 14 (100%) 7 (70%) 21 (88%) fibers Granular material 9 (64%) 2(20%) 11 (46%) Blood vessels 7 (50%) 1 (10%) 8 (33%) *Posltwe staamng m twenty-one of twenty-four (88%) biopsy specimens

dermographometer-mduced lesions), throughout the dernus m 11 of 21 (7 were spontaneously occumng), and m deep dermal fibers alone m 3 specimens (all from spontaneously occurring lesions) Second, &spers~on of fine granular material within the derrms was observed in 11 biopsy specimens (9 of 11 were from spontaneously occurring lesions) Thxrd, fluorescence of dermal vessels was noted m 8 lesions (7 of 8 were spontaneous) A correlation could not be made between the extent or pattern of staining and the duration of the lesion DISCUSSION The finding of extracellular deposition of eosmophd granule major basic protean m lesions of pressure urticaria documents the presence of eosmophfl degranulatlon m this disease and suggests that eosmophll mediators, particularly major basic protern, may be ~mportant m the development of such lesions Chmcal studies support a correlation between eoslnophlls and cutaneous edema Patients with the hypereosmophdlc syndrome frequently have urticaria and angaoedema, and over 75% of patients with hypereosmophfllc syndrome exhibit dermatographlsm t4 ~5 Furthermore, patients with eplso&c angloedema have marked peripheral blood eoslnophlha (up to 88%) and suffer recurrent attacks of generalized angloedema that wax and wane m concert w~th the peripheral blood eosinophd count 9 Htstologlc stu&es of the cellular Infiltrates in pressure urticaria have demonstrated that eosmophlls are often present, although m varmble numbers ~,2~6~7 Lesaons of pressure urticaria may ex-

hlblt a broad spectrum of microscopic changes, from sparse penvascular inflammation to vascuhtls a Wmkelmann et a112 performed quantltauve analyses of the cellular infiltrates m both spontaneously occurnng and dermographometer-mduced lesions of pressure urticaria and found that tissue eoslnophd counts may vary in number and location within the dermis and may also vary depending on the duration of the lesxon prior to biopsy Fluorescence lmmunostamlng for eosinophfl granule major basic protein demonstrates that attempts to assess the lmmunopathogenesls of urticarial eruptions by cell counts may be misleading Previous lmmunofluorescence studies of major bastc protem deposition m chromc ~d~opathlc urticaria, atoplc dermatms, facial edema, and eplso&c angloedema have shown that the extent and pattern of extracellular staining with anti-major basic protern are unrelated to the presence or degree of eosmophll infiltration 810 J8 Indeed, some cases showed copious extracellular fluorescence m the absence of intact eoslnophlls Our present study of pressure urticaria also shows that extracellular lmmunolocallzatxon of major basic protein m the dermis IS unrelated to the number of eosmophfls in the txssue In ad&tlon, early lesions, In comparison with late ones, and spontaneous lesions, in comparison with induced lesions, could not be discriminated by extent or pattern of major basic protein deposition Both spontaneously occurring lesions and those Induced by a dermographometer showed intense fluorescence staining of dermal connective tissue fibers, but staining of granular matenal and vessels was predominantly observed m the spontaneous group Eosmophils are capable of metabohzmg arachidomc acid, and they preferentially produce leukotnene C4, ~9 a known medmtor of vascular permeability Major basic protein can Induce histamine release from mast cells and basophds, 2° zJ and major basic protein, eosanophll catlomc protein, and eosmophll-denved neurotoxln ehclt a doserelated wheal-and-flare reaction on mtradermal rejection into human slon 22Eosmophd cationic protelns, ECP and EPX, share similar cytotoxlc propertles with major basic protein and also appear to play a role m hypersensmvIty reactions 4 23 The present data showing extracellular localization of

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Eosinophil granule protein in pressure urticaria

eosinophil granule major basic protein in pressure urticaria, in addition to the observations of major basic protein deposition in chronic idiopathic urticaria, s facial edema,l° episodic angioedema,9 and the late phase of the immediate wheal-and-flare reaction, 24 provide further immunocytochemical evidence that eosinophil degranulation is associated with cutaneous edema. REFERENCES

1. Monroe EW, Schultz CI, Maize JC, Jordan RE. Vasculitis in chronic urticaria: an immunopathologic study. J Invest Dermatol 1981 ;76:103-7. 2. Czarnetzki BM, Meentken J, Kolde G, Br6cker EB. Morphology of the cellular infiltrate in delayed pressure urticaria. J AM ACAD DERMATOL 1985;12:253-9. 3. Natbony SF, Phillips ME, Elias JM, Godfrey HP, Kaplan AP. Histologic studies of chronic idiopathic urticaria. J Allergy Clin Immunol 1983;71:177-83. 4. Gleich GJ, Loegering DA. Immunobiology of eosinophils. Ann Rev lmmunol 1984;2:429-59. 5. Peters MS, Rodriguez M, Gleich GJ. Localization of human eosinophil granule major basic protein, eosinophil cationic protein and eosinophil-derived neurotoxin by immunoelectron microscopy. Lab Invest 1986;54:656-62. 6. Gleich GJ, Frigas E, Loegering DA, Wassom DL, Steinmuller D. Cytotoxic properties of the eosinophil major basic protein. J Immunol 1979;123:2925-9. 7. Frigas E, Loegering DA, Gleich GJ. Cytotoxic effects of the guinea pig eosinophil major basic protein on tracheal epithelium. Lab Invest 1980;42:35-43. 8. Peters MS, Schroeter AL, Kephart GM, Gleieh GJ. Localization of eosinophil granule major basic protein in chronic urticaria. J Invest Dermatol 1983;81:39-43. 9. Gleich GJ, Schroeter AL, Marcoux JP, Sachs MI, O'Connell EJ, Kohler PF. Episodic angioedema associated with eosinophilia. N Engl J Med 1984;310: 1621-6. 10. Songsiridej V, Peters MS, Dor RJ, Ackerman SJ, Gleich GJ, Busse WW. Facial edema and eosinophilia: evidence for eosinophil degranulation. Ann Intern Med 1985; 103:503-6. 11. Leiferman KM, Peters MS, Gleich GJ. The eosinophil and cutaneous edema. J AM ACAD DERMArOL 1986; 15:513-7.

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12. Winkelmann RK, Black AK, Dover J, Greaves MW. Pressure urticaria: histopathological study. Clin Exp Dermatol 1986; 11:139-47. 13. Peters MS, Schroeter AL, Gleieh GJ. Immunofluorescenee identification of eosinophil granule major basic protein in the flame figures of Wells' syndrome. Br J Dermatol 1983;109:141-8. 14. Kazmierowski JA, Chusid MJ, Parrillo JE, Fauci AS, Wolff SM. Dermatologic manifestations of the hypereosinophilic syndrome. Arch Dermatol 1978;114:531-5. 15. Parrillo JE, Lawley TJ, Frank NM, Kaplan AP, Fauci AS. Immunologic reactivity in the hypereosinophilic syndrome. J Allergy Clin Immunol 1979;64:113-21. 16. Ryan TJ, Shim-Young N, Turk JL. Delayed pressure urticaria. Br 1 Dermatol 1968;80:485-90. 17. Illig L, Engelhardt A, Thielemann K. Histologische untersuchungen bei "physikalischer urticaria" der hatu. 1. Die cellulare reaktion und ihr ablauf bei dur druckurticarla. Hautarzt 1970;21:355-69. 18. Leiferman KM, Ackerman SJ, Sampson HA, Haugen HS, Venencie PY, Gleich GJ. Dermal deposition of eosinophil granule major basic protein in atopic dermatitis: comparison with onchocerciasis. N Engl I Med 1985; 313:282-5. 19. Shaw RJ, Cromwell O, Kay AB. Preferential generation of leukotriene C4 by human eosinophils. Clin Exp Immunol 1984;56:716-22. 20. O'Donnell MC, Ackerrnan SJ, Gleich GJ, Thomas LL. Activation of basophil and mast cell histamine release by eosinophil granule major basic protein. J Exp Med 1983;157:1981-91. 21. Zeutlin LM, Ackerman S J, Gleich GJ, Thomas LL. Stimulation of basophi[ and rat mast cell histamine release by eosinophil granule-derived cationic proteins. J Immunol 1984;133:2180-5. 22. Leiferman KM, Loegering DA, Gleieh (3I. Production of wheal-and-flare skin reactions by eosinophil granule proteins [abstract]. J Invest Dermatol 1984;82:414, 23. Venge P, Dahl R, Fredens K, Hallgren R, Peterson C. Eosinophil cationic proteins (ECP and EPX) in health and disease. In: Yoshida T, Torisu M, eds. Immunobiology of the eosinophil. New York: Elsevier, 1983: 163-79. 24. Leiferman KM, Haugen HS, Gleich GJ. Evidence for eosinophil degranulation in the late phase of the immediate wheal and flare skin reaction [abstract]. J Invest Dermatol 1986;86:488.

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