Hypereosinophilic syndrome with generalized myasthenia gravis

The Journal of Pediatrics Volume 128, Number 3

6. Cuttica RJ, Scheines EJ, Garay SM, Romanelli MC, Maldonado Cocco JA. Juvenile onset Reiter's syndrome: a retrospective study of 26 patients. Clin Exp Rheumatol 1992; 10: 285-8. 7. Lockie GN, Hunder GG. Reiter' s syndrome in children. A case report and review. Arthritis Rheum 1971;14:767-72. 8. Rosenberg AM, Petty RE. Reiter's disease in children. Am J Dis Child 1979;133:394-8. 9. Ingrain GJ, Scher RK. Reiter's syndrome with nail involvement: Is it psoriasis? Curls 1985;36:37-40. 10. Fink CW. Reactive arthritis. Pediatr Infect Dis J 1988;7:5865.

Ishida

369

I 1. Michet CJ, Machado EB, Ballard DJ, McKenna CH. Epidemiology of Reiter's syndrome in Rochester, Minnesota: 19501980. Arthritis Rheum 1988;31:428-31. 12. Brewerton DA, Caffrey M, Nicholls A, Walters D, Oates JK, James DC. Reiter's disease and HL-A 27. Lancet 1973;2: 996-8. 13. Hammer RE, Maika SD, Richardson JA, Tang JP, Taurog JD. Spontaneous inflammatory disease in transgenic rats expressing HLA-B27 mad human beta 2m: an animal model for HLA-B27-associated human disorders. Cell 1990;63:1099112.

Hypereosinophilic syndrome with generalized myasthenia gravis Yasushi Ishida, MD, Masatoshi Hayashi, MD, Aki Higaki, MD, Kenji Matsumoto, MD, Yoji likura, MD, Junichi Ishikawa, MD, a n d Kaichi Kida, MD From the Department of Pediatrics, Ehime University School of Medicine, and the Department of Allergy, National Children's Hospital, Tokyo, Japan

A patient with hypereosinophilic syndrome simultaneously had generalized myasthenia gravis. Ultrastructural and functional studies demonstrated an increase of the activated hypodense eosinophils. The eosJnophilic cationic protein level in plasma and whole blood paralleled the severity of congestive heart failure. A high level of interleukin-5 was detected in both serum and in conditioned medium of T cells by interleukin-2 stimulation before therapy. The findings indicate that both diseases may be associated with a common T-ceil abnormality. (J PEDIATR1996; 128:369-72)

Hypereosinophilic syndrome is a rare idiopathic disease characterized by persistent elevation of eosinophils with organ infiltration] 3 Cardiac disease is a major cause of morbidity" and death. 4 The neurologic complications may be divided into three typesS---cerebrovascular accidents, primary central nervous system dysfunction, and peripheral neuropathy. 5 Other organ dysfunctions include asthma-like symptoms, hepatosplenomegaly, visual disturbance, arthralgia and myalgia, diarrhea and malabsorption, and exanthema.1, 3

Submitted for publication April 17, 1995; accepted Oct. 20, 1995. Reprint requests: Yasushi Ishida, MD, Department of Pediatrics, Ehime UniversitySchool of Medicine, Shitsukawa, Shigenobu-cho, Onsen-gun, Ehime 791-02, Japan. Copyright © 1996 by Mosby-Year Book,. Inc. 0022-3476/96/$5.00 + 0 9/22/70012

To our knowledge there has been no case report of HES associated with myasthenia gravis. 6 CASE REPORT

A 9-year-old girl was referred to our hospital because of dysphagia and speech disturbance. Her mother had died of idiopathic collagen disease and an unknown hematologic abnormality 6 years ECP lIES IL MG

Eosinophilic cationic protein Hypereosinophilic syndrome Interleukin Myasthenia gravis

previously. Mild ptosis was recognized by the father 8 months before admission, and dysphagia and speech disturbance 6 months before admission. She also had had anorexia, general fatigue, and weight loss since 3 months before admission. Physical examination revealed a thin girl with severe ptosis who

370

Ishida

The Journal of Pediatrics March 1996

Table. Interleukin-5 production of peripheral T cells stimulated by interleukin-2 IL-2

Patient without symptoms (remission) Control Patient with symptoms (before therapy) Control

0 U/ml

0.2 U/ml

2 U/ml

0.30

0.66

0.77

0.33 1.14

0.31 17.1

0.24 13.1

0.74

1.77

1.20

The IL-5 productionof T lymphocyteswas determinedaccordingto the methodof Enokiharaet al,s fromheparinizedvenousbloodfromthe patient before therapyand duringclinicalremission.The controlvalueis that of healthy adultvolunteersexaminedat the same time.

appeared chronically ill. Ocular movements were normal, and she had no facial weakness. The heart was enlarged. She had a gallop rhythm and a late systolicmurmur. No rales were present. Her liver was palpable 3 cm below the right costal margin but the spleen was not palpable. There was no edema. Results of the neurologic examination was normal except for generalized muscle weakness. A myasthenic decrement was demonstrated in her facial muscles by electromyogram. Administration of edrophonium promptly improved the speech and ptosis. Laboratory values were as follows: hemoglobin 131 gin/L, leukocyte count 9.4 x 109/Lwith 21.6% eosinophils,and platelet count 397 x 109/L.Follow-up studies showed eosinophiliaup to 13 x 109/ L in peripheral blood. Liver and kidney function were normal. The IgE level was 395 IU/ml and increased to 2500 IU/ml before therapy. Antinuclear antibody was not present. Results of examination of stool for ova and parasites, serum antibody studies for toxocariasis, toxoplasmosis,and coccidioidomycosiswere all negative.The bone marrow showed hypercellularity with eosinophilic myelocytes, metamyelocytes45.4%, and mature eosinophils 21.8%. Cytogenetic analysis revealed no abnormalities (46,XX). An x-ray study of the chest and echocardiogramdisclosedenlargementof the heart with prominent pericardialeffusion and mild pleural effusion. Pericardiocentesis revealed eosinophilic infiltration. A chest x-ray study and computed tomographic scan of the thymus demonstrated no abnormality. The diagnoses of idiopathic HES and generalized MG6 were made. We started initial treatment according to the recommendation of Parrillo et al.7 because congestive heart failure was progressive; prednisolone therapy, 60 mg/m2 dally for 2 weeks, followed by alternate-day administration of 60 mg/m2 was begun. Relief of her symptoms occurred within 1 week. Diarrhea and eosinophilia recurred 1 month after alternate-day administration was begun, and required daily administration of prednisolone. Ptosis and speech disturbance occasionallyrecurred, depending on the dose of prednisolone. Her symptoms were subsequently controlled by increasing the dose of prednisolone to 40 mg per day and adding cyclophosphamide (2 mg/kg per day).

METHODS Electron microscopy was used to study the eosinophils in peripheral blood, bone marrow, and pericardial effusion from the patient, according to Peters et als. Superoxide anion was measured by the superoxide dismutase-inhibitable ferricytochrome C reduction method, 9 and flow cytometric study for hydrogen peroxide production was measured by oxidative product formation of 2'7'-dichlorofluorescein. 10 Buffy coat was obtained from heparinized peripheral blood by using 4% dextran solution (Pharmacia LKB, Uppsala, Sweden). After washing three times with phosphatebuffered saline solution (Sigma Chemical Co., St. Louis, Mo.), eosinophils were purified by negative selection with anti-CD 16 monoclonal antibody (Dako, Carpenteria, Calif.). Purified eosinophils were suspended in RPMI-1640 culture medium (IBL, Gunma, Japan) supplemented with 10% fetal calf serum (Grand Island Biological Co., GIBCO, Grand Island, N.Y.) at a cell density of 0.5 x 106. Autoproliferation was determined by counting the cell number after 3 or 7 days. In some experiments, 1 ng/ml human recombinant interleukin-5 (R&D Systems, Inc., Minneapolis, Minn.) was added to the culture medium. Eosinophil viability was determined with the trypan blue (Sigma) dye exclusion method. The eosinophilic cationic protein content in plasma and whole blood was measured by radioimmunoassay (ECP RIA, Pharmacia). Whole blood ECP was measured after freezing and thawing peripheral blood. We examined the serum IL-5 level and production of IL-5 by peripheral T lymphocytes according to Enokihara et al. 9 (Table). One xl06 T cells/ml were cultured with IL-2 (Takeda Chemical Industries, Ltd., Osaka, Japan) at 37 ° C in a humidified atmosphere of 5% CO2 in air. Cell-free conditioned medium was collected after 5 days of incubation. The measurement of IL-5 was performed by enzyme-linked immunosorbent assay. Ace@choline receptor antibody was measured by radioimmunoassay (Cosmic Kit, RSR Co. Ltd., Cardiff, Wales). it RESULTS Ultrastructural study revealed that eosinophils of the patient had small granule size ( 0 . 1 5 - 0.05 prn2, control 0.30 _ 0.10 pm2) and smaller total granule area (4.5 gm 2, control 8.5 -+ 4.0 Nn2). Superoxide anion (O2-) production of eosinophils was normal: 10-6 mol/L formyl methionine, 2.32 nmol/min x l 0 -6 cells (control, 6.0 to 10.7 nmol/min xl0-6); 1 mg/ml opsonized zymosan, 5.02 nmol/min x 10.6 cells (control, 1.0 to 5.0 nmol/min x 10-6); 500 ng/ml phorbol myristate acetate, 9.41 nmol/min x 10.6 cells (control, 5.0 to 7.5 nmol/min x 10-6). The percentage of hydrogen peroxide- producing cells was 16.2% at rest (control, 0 to

The Journal of Pediatrics Volume 128, Number 3

Ishida

1993 Oct.

I

NOV.

I Dec. I 1994 Jan. I '~';;;;,"~'a';;"' PrednisoloneJ eo~ IIIIIlll ~llllllllllllllllllll 30mg

Feb.

371

I [

....................

Ptosis ~

Discharge

yspnea~~~bl=

~r

200001 ~ I) 150004

~

WBC

~ ,

/ 1500~U~)

IgE(IU/ml) 395 11_-5(ng/ml) 14.5 Plasma ECP(/zg/ml) Blood ECP(/zg/ml)

AChR Ab

(pmol/ml)

4.56

1520

753

279

8.6 65.1

2.2

>200

128 1.29

208

511 364

133

163

5.8 2.8

76.1 0.70

120

106 0.2

0.52

Figure. Clinical course. Prednisolone therapy resulted in improvement in the patient's symptoms within 1 week. When daily use of prednisolone was changed to alternate-day therapy, eosinophilia recurred, whereas daily use of prednisolone controlled eosinophilia. Serum acetylcholine receptor antibody (AChRAb) titer (normal <0.16 pmol/ml) was also decreased after prednisolone therapy.

0.5%) and 86.8% after stimulation (control, 85.9% to 95.8%). Mean fluorescence intensity of eosinophils was 400.7 at rest and 427.8 after stimulation (control 120 to 150). We could not demonstrate autonomic proliferation of eosinophils by liquid culture. When cultured with 1 ng/ml IL-5, the survival percentage of eosinophils was 86% at 24 hours, 52% at 48 hours, and 2% at 72 hours. Both plasma and whole blood ECP values were high before therapy and decreased to the normal range after treatment (Figure). A high level of IL-5 was detected in the patient's serum, but the level decreased to normal during clinical remission (Figure). The conditioned medium of T cells by IL-2 stimulation before therapy contained a high level of IL-5 (Table) and decreased to normal during clinical remission. The serum acetylcholine receptor antibody titer was 4.56 pmol/ml (normal <0.16 pmol/ml) before therapy and decreased to 0.52 pmol/ml at discharge (Figure).

DISCUSSION The diagnostic criteria of HES, as outlined by Chusid et al.]0 are as follows: (1) persistent eosinophilia of 1.5 x 109/ L for more than 6 months or death before 6 months associated with signs and symptoms of HES, (2) lack of evidence for other causes of eosinophilia, and (3) evidence of organ system involvement. Our patient did not have persistent eosinophilia for more than 6 months, but she had had clinical signs and symptoms for 8 months, which were likely caused by both HES and MG. The presence of organ system involvement without any evidence of other causes for eosinophilia led to the diagnosis of idiopathic HES. Eosinophils circulating in the blood of patients with HES are in an "activated state." These changes include increased metabolic activity, diminished density ("hypodense"), enhanced antibody-mediated cytotoxicity, increased peroxidase activity, and morphologic alterations. ~ Ultrastructural study of eosinophils reveals that the hypodense eosinophils have smaller granules and smaller total granular area than

372

Ishida

normal. 8 Eosinophils of our patient were also activated from the point of hydrogen peroxide production, and did not show autonomic proliferation as in eosinophilic leukemia. We concluded that the eosinophils in our patient were activated, hypodense eosinophils. The eosinophil's cationic proteins (major basic protein, eosinophil-derived neurotoxin, ECP, and peroxidase) can damage host cells.12 The levels of ECP paralleled the severity of the disease in our patient and prednisolone therapy suppressed the increase of ECP. Three cytokines (IL-5, IL-3, and granulocyte-macrophage colony-stimulating factor) are active in stimulating eosinophilopoiesis. Among them, overproduction of IL-5 would be a candidate mechanism for explaining u g s , 1, 13 and its dysregulated production by T-cell clones could represent an underlying mechanism for the hypereosinophilia.1, 8 Cogan et al.14 found that a type 2 helper T- cell clone could be established from a man with HES and that it produced high levels of 1L-4 and IL-5. They concluded that clonal expansion of type 2 helper T cells can cause HES. Thus IL-5-producing helper T cells may play a central role in the pathogenesis of HES. We also demonstrated that T cells obtained from our patient could produce IL-5 by stimulation of IL-2. There is extensive evidence that the production of acetylcholine receptor antibody requires the intervention of T cells.6 Helper T cells respond to acetycholine receptor antigen in combination with major histocompatibility complex class 11.6 Several T-cell epitopes were noted to stimulate proliferation of lymphocytes from patients and experimental animal models with MG. 15 Recently Yi et al. 16 reported that T cells stimulated by acetylcholine receptor antibody secreted IL-4, interferon-,/, IL-2, or a combination, and that this T-cell response is monocyte-macrophage dependent and restricted by major histocompatibility complex class 11. They concluded that both Thl/Th2 or Th0 subpopulations are involved in the autoimmune response. The mechanism of simultaneous onset remains to be answered, but these data indicate that T-cell abnormalities probably cause both HES and MG. Future studies should elucidate the pathophysiology of both diseases. We thank Masahiro Migita, MD (Department of Pediatrics, Kumamoto University, School of Medicine, Kumamoto, Japan) for measurement of IL-5 concentration, and Tadatoshi Kuratsuji, MD (Department of Inflammation, National Children's Hospital, Tokyo, Japan) for measurement of eosinophil function.

The Journal of Pediatrics March 1996

REFERENCES

1. Weller PF, Bubley GJ. The idiopathic hypereosinophilic syndrome. Blood 1994;83:2759-79. 2. Flaum MA, Schhley RT, Fanci AS, Gralnick HR. A clinical correlation of the idiopathic hypereosinophilic syndrome. I. Hematologic manifestations. Blood 1981;58:1012-20. 3. Schhley RT, Flaum MA, Gralnick HR, Fauei AS. A clinicopathologic correlation of the idiophatic hypereosinophilic syndrome. II. Clinical manifestations. Blood 1981;58: 1020-6. 4. Parrillo JE, Lawley YTJ, Frank MM, Kaplan AP, Fauci AS. The cardiovascular manifestations of the hypereosinophilic syndrome: prospective study of 26 patients, with review of the literature. Am J Med 1979;67:572-82. 5. Moire PM, Harley JB, Fauci AS. Neurologic dysfunction in the idiopathic hypereosinophilic syndrome. Ann Intern Med 1985; 102:109-14. 6. Drachman DB. Myasthenia gravis. N Engl J Med 1994;330: 1797-810. 7. Parrillo JE, Fauci AS, Wolff SM. Therapy of the hypereosinophilic syndrome. Ann Intern Med 1978;89:167-72. 8. Peters MS, Gleich GJ, Dunnette SL, Fnkuda T. Ultrastmctural study of eosinophils from patients with the hypereosinophilic syndrome: a moI]ghological basis of hypodense eosinophils. Blood 1988;71:780-5. 9. Enokihara H, Furusawa S, Nakakubo H, et al. T cells from eosinophilic patients produce interleukin-5 with interleukin-2 stimulation. Blood 1989;73:1809-13. 10. Chusid MJ, Dale DC, West BC, Wolff SM. The hypereosinophilic syndrome: analysis of fourteen cases with review of the literature. Medicine 1975;54:1-28. 11. Hayashi M, Takaoka T, Manabe K, et al. Comparison of antibody titer to human and rat acetylcholine receptor in myasthenia gravis. Brain Dev 1995;17:38-41. 12. Grech M, Adolphson CR, Leiferman KM. Eosinophils. In: Gallin JI, Goldstein IM, Syderman R, eds. Inflammation: basic principles and clinical correlates. 2nd ed. New York: Raven, 1992:663. 13. Enokihara H, Kajitani H, Nagashima S, et al. Interleukin-5 activity in sera from patients with eosinophilia. Br J Haematol 1990;75:458-62. 14. Cogan E, Schandene L, Crusiax A, Cochaux P, Vein T, Goldman M. Brief report: clonal proliferation of type 2 helper T cells in a man with the hypereosinophilic syndrome. N Engl J Med 1994;330:535-8. 15. Protti MP, Manfredi AA, Horton RM, Bellene M, ContiTronconi BM. Myasthenia gravis: recognition of a human autoantigen at the molecular level. Immunol Today 1993;14: 363-7. 16. Yi Q, Ahlberg R, Pirskanen R, Lefvert AK. Acetylcholine receptor-reactive T cells in myasthenia gravis: evidence for the involvement of different subpopulations o f T helper cells. J Neuroimmunol 1994;50:177-86.