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9. Ishizaki ‘T. The essenceof allergy skin testings and their application J Jap Med Assoc 1969;62:761-72. 10. Nelson H. Diagnostic procedure in allergy. I. Allergy skin testing. Ann Allergy 1983;51:411-7. 11. Belin LG A, Norman PS. Diagnostic testsin the skin and serum of workers sensitizedto Bacillus subtilis enzymes.Clin Allergy 1977;7:55-68. 12. Bielory L, Wright R, Nienhuis AW, Young NS, Kaliner MA.

thrombin-induced

anaphylaxis

Antithymocyte globulin hypersensitivity in bone marrow failure patients. JAMA 1988;21:3164-7. 13. Hoffmann RG. Statistics in the practice of medicine. JAMA 1963;185:864-73. 14. Grubbs FE, Beck G. Extensions of sample sizes and the percentagepoints for significance tests of outlying observations. Technometrics1972;14:847-54.

Episodic eosinophilia-myalgialike syndrome in a patient without L-tryptophan use: Association with eosinophil activation and increased serum levels of granulocytemacrophage colony-stimutatitlg factor Bruce S. Bochner, MD, Baruch Friedman, MD, Guha Krishnaswami, MD, Robert P. Schleimer, PhD, Lawrence M. Lichtenstein, MD, PhD, and Claus Kroegel, MD Baltimore, Md. We have studied a patient with recurrent bouts of angioedema, myalgia, and eosinophilia that was not due to L-tryptophan ingestion. Peripheral blood eosinophils (EOSs) during exacerbations of his illness displayed characteristics of “activation, ” including hypodense phenotype and increased responsiveness to platelet-activating factor (PAF) in vitro with respect to expression of CD1 1b su$ace adhesion proteins. Elevated serum levels of granulocyte-macrophage colony-stimulating factor (GM-CSF) bioactiviry were also detected, whereas interleukin-3 and interleukin-5 levels were not increased. During treatment with glucocorticoids, all clinical symptoms resolved, EOSs decreased in number and became normodense, PAF responsiveness diminished, and GM-CSF levels returned to normal. During glucocorticoid tapering, a subsequent clinical relapse was again associated with EOS hypodensity, increased PAF responsiveness, and increased serum GM-CSF levels. Although this patient satisfies the diagnostic criteria for eosinophilia-myalgia syndrome, the episodic and profound nature of exacerbations and response to therapy in the absence of L-tryptophan usage suggests a possible overlap with the syndrome of episodic angioedema and eosinophilia. In vitro studies suggest that GM-CSF may play a role in the eosinophilia, EOS activation, and pathophysiology of disease in this patient and demonstrate resolution of these abnormalities during glucocorticoid therapy. The efjicacy of glucocorticoid therapy in some hypereosinophilic states may therefore be mediated, at least in part, via reduction of GM-CSF production andlor EOS activation. (J ALLERGYCLIN IMUUNOL 1991;88:629-36.) Key words: Eosinophil activation, myalgia, GM-CSF

From the Department of Medicine, Division of Clinical Immunology.,Johns Hopkins Asthma and Allergy Center, Baltimore, Md. Supported by National Institutes of Health Grants A127429, A108270, and A120136. Received for publication Jan. 30, 1991. Revised May 6, 1991. Accepted for publication May 22, 1991. Reprint requests:Bruce S. Bochner, MD, JohnsHopkins Asthma

and Allergy Center, Clinical Immunology Unit Office 3, 301 Bayview Blvd., Baltimore, MD 21224. Publication No. 0.58 from Johns Hopkins Asthma and Allergy Center. Dr. Bruce S. Bochner was supportedin part by a New Investigator Award from the American Lung Association. Dr. Lawrence M. Lichtenstein was a recipient of a Pfizer Biomedical Award. l/1/31238 629

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Significant elevationsin the number and percentage of peripheral blood EOSs is observed in a relatively limited number of diseases.For example, the recently described eosiuophilia-myalgia syndrome is defined by the presence of peripheral blood eosinophilia 2 1000 cells per cubic millimeter and disabling myalgias in the absenceof other known causes.‘7* This syndrome is distinct from episodic angioedemaand eosinophilia, a rare, recurrent condition accompanied by urticaria, fever, and weight gain,3 cyclic or relapsing eosinophilic myositis,4 and the so-called hypereosinophilic syndrome.5Although the toxic potential of EOS-derived products has been demonstrated in diseasesof the airways associated with eosinophilia,6T7 the mechanismsresponsible for the cyclic nature of eosinophilia and disease activity remain poorly understood. It is now clear that severalcytokines are capableof altering EOS differentiation and function. Interleukin3 (IL-3), interleukin-5 (IL-S), and GM-CSF possess EOS hematopoietic activity, enhance EOS degranulation, and convert these cells during culture to the so-called “hypodense” phenotype associatedwith enhancedsurvival and cell function, including cytotoxicity and leukotriene production.8,9Administration of GM-CSF in vivo results in increasednumbers of circulating EOSS,‘~~ I’ and GM-CSF production in vitro from mononuclearcells in patients with eosinophiliamyalgia syndrome reportedly releaseGM-CSF when they are incubated with L-tryptophan.” It has also recently been reported that in some patients with the idiopathic hypereosinophilic syndrome, episodic angioedema and eosinophilia, or eosinophilia-myalgia syndrome, elevations in serum interleukin-5 (IL-5) levels appearto parallel increasesin eosinophilia and diseaseactivity. 13-15 Werecently had the opportunity to evaluatea patient with recurrentepisodesof angioedemaassociatedwith disabling myalgias, fever, profound weight gain, and eosinophilia that was extremely responsive to glucocorticoid therapy. Despite some similarities between our patient’s clinical course and that of the eosinophilia-myalgia syndrome,this patient had never ingested L-tryptophan, and no clear explanation for his recurrent illness could be identified. We report in this article that during recurrenceof disease,in vitro measurements,including serum levels of GM-CSF bioactivity and measurementsof EOS activation, paralleled diseaseactivity; with glucocorticoid treatment, normalization of both clinical and in vitro parameters was observed.Thesedatademonstratethat the clinical presentation may overlap between eosinophiliamyalgia syndrome and episodic angioedemaand eosinophilia. In addition, in vitro studiesimplicate GM-

J. ALLERGY

CLIN. IMMUNOL. OCTOBER 1991

Abbreviations used

EOS: Eosinophil GM-CSF: Granulocyte-macmphage colonystimulatingfactor PAF: Platelet-activating factor PE: R-Phycoerythrin PIPES: Piperazine-iV-N’-bis (Zethanesulfonic acid)

PAG: PIPESbuffer(25mmol/L of PIPES,110 mmol/L of NaCl, 5 nuuol/L of KCl), of pH 7.4, containing 0.003% human serum albumin and 0.1% D-glucose IL: Interleukin Ab: Antibody

CSF in the pathophysiology of this patient’s disease and suggest that glucocorticoids may mediate their beneficial effects in somehypereosinophilic states,at least in part, via reduction of levels of GM-CSF. CASE REPORT A 55year-old white man was referred for evaluation of an 8-month history of recurrent episodesof fever, myalgias, swelling of the face, 8 to 10 kg weight gain, oliguria, and eosinophilia. He had been in good health until 8 months before admission (May 1989) when he abruptly developed the above symptomsthat resolved during 7 to 10 days without medical intervention. Furtherevaluationduring an exacerbation 3 months before admission (December 1989) revealedmarked facial edemawithout rash, a white blood cell count of 19,500 cells per cubic millimeter with 47% EOS, and a Westergrensedimentationrate of 2 mmlhr. Two months before admission (January 1990), he had another symptomatic relapse, and laboratory evaluation revealed a lactatedehydrogenaselevel of 878 U/L (normal, 297 to 537 U/ml), and a mild polyclonal elevation of IgM (332 rig/ml; normal, 45 to 250). The white cell count was 23,600 cells per cubic millimeter with 51% EOSs, 33% neutrophils, 8% monocytes,7% lymphocytes, and 1% band forms. Tests for rheumatoid and antinuclear factors were negative. One month before admission(February 1990), symptoms recurred, and he was startedon a regimen of hydroxyzine (10 to 40 mg/day) and prednisone (20 mg twice daily); within 48 hours there was dramatic improvement. Prednisonetherapy was gradually taperedduring the next 2 weeks, but 3 days after prednisonetherapy was stopped,the patient experienced shaking chills, fever, diffuse myalgias, facial swelling, and weight gain. He was referred to us and admitted for further evaluation. There was no personal or family history of atopy, and he had no known medication allergies. He denied the use of any other drugs, including L-tryptophan, and there was no recenttravel history or history of ingesting poorly cooked pork or meat. Physical examination on admission was remarkable for

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q aaaoanaaaaa aaaaaaaaonaaaaaaa

IL-

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FIG. 1. Clinical and hematologic features during and between episodes of angioedema; WBC, white blood cells; day 0, date of initial hospitalization (March 1990). Prednisone therapy was started on day 5.

a temperatureof 39.4” C and pitting edemainvolving the face, which was especially prominent over the temporal areaswhere his spectacleshad left an impression. The conjunctivae were injected bilaterally, and there was diffuse proximal muscle tenderness.There was no lymphadenopathy or other skin lesions, and examination of the heart, lung, abdomen, and neurologic system was normal. Laboratclry evaluation (3 days after prednisone therapy was stopped)(Fig. 1, day 1) revealeda white cell count of 19,5OO/mm’with 4% EOSs, 77% neutrophils, 17% lymphocytes, and 2% basophils. The hemoglobin was 18.2 gmldl, and the platelet count was 443,000/mm3. Serum electrolytes were normal as were the alanine and aspartate aminotransferaselevels. Serumcreatine phosphokinaseand aldolaselevels were normal. The lactatedehydrogenasewas 211 IUlL (normal for this laboratory, 0 to 200 IU/L) with a nonspecific isoenzyme distribution. Tests were negative for antinuc:lear Abs, rheumatoid factor, C3, C4, and Trichinella antigen by latex agglutination. The erythrocyte sedimentatitonrate was 0 mm&. Results of chest x-ray

film, electrocardiogram, and echocardiogramof the heart were unremarkable. During the 4-day hospitalization, his weight and white blood cell andeosinophil counts continued to increase (Fig. 1). A biopsy specimen of muscle and surrounding fascia of the right deltoid obtained on the third hospital day revealed no evidence of trichinosis, myositis, or vasculitis, but did demonstratediffuse thickening of the surrounding connective tissues along with an intense interstitial inflammatory cell infiltrate consisting of macrophages,mononuclearcells, and EOSs. The patient was discharged, and treatment with prednisone was initiated. Within 48 hours, the patient experienced a dramatic msolution of symptoms, associatedwith diuresis and weight loss; his subsequent clinical course is summarized in Fig. 1.

METHODS Reagents The following chemicalsand reagentswere obtainedfrom the sourcesindicated: PIPES, PAF, bovine serum albumin,

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human serum albumin, and ethylenediaminetetraacetic acid (Sigma Chemical Co., St. Louis, MO.); Percoll (Pharmacia, Uppsala, Sweden); fetal bovine serum (Hyclone Laboratories, Inc., Logan Utah); Dulbecco’s modified Eagle medium with 2 mmol/L of L-glutamine (Gibco Laboratories, Grand Island, N.Y.); penicillin and streptomycin (Whitaker Bioproducts, Inc., Walkersville, Md.); fluorescein isothiocyanate-conjugatedCD16 (Leu1la), PE-conjugatedCD1 lb (Leu-15) (Becton Dickinson, Mountainview, Calif.), and PE-conjugatedCD45 (KC56) (Coulter Electronics, Hialeah, Fla.); appropriateconjugated irrelevant isotypic controls (Coulter Electronics and Becton Dickinson) were also used. Recombinant human cytokines IL-3 and GM-CSF, and specific polyclonal rabbit and sheepantisera for these cytokines were generouslyprovided by Dr. StevenClark (Genetics Institute, Boston, Mass.). Buffers usedincluded Dulbecco’s phosphate-bufferedsaline containing 0.2% bovine serum albumin; PAG (PIPES buffer (25 mm01PIPES, 110 mM NaCl, 5 mM KCl) containing 0.003% human serumalbumin, 0.1% D-glucose,pH 7.4; and PAG containing 1 mmol/L of CaCl, and 1 mmol/L of MgCl,.

Purification

of EOSs

EOSswere repeatedlyobtainedfrom the peripheral blood of the patient or from eight normal adult subjects, and purified with a 7-step discontinuous Percoll density gradient as described previously.16EOSs at each density interface were counted in a Neubauerhemacytometer(American Optical Corp., Safety ProductsDivision, Southbridge, Mass.) with Kimura’s stain.” The viability of EOSs consistently exceeded97%, as determinedby trypan blue exclusion.

Flow cytometric analysis of EOS surface antigens With a protocol adaptedfrom previously describedtechniques,” aliquots of cells (zO.3 to 1 X lo6 per tube) were labeled (for 30 minutes at 4” C) with saturating concentrations of monoclonal Abs recognizing various cell-surface molecules. Negative controls consistedof cells labeled with irrelevant isotype-matchedcontrol monoclonalsor with secondary Ab alone; positive controls consisted of the use of Ab to CD45 antigens.Cells in preparationscontaining EOSs at purities