J Bone Miner Metab (2000) 18:36–40

© Springer-Verlag 2000Case report

Treatment with recombinant IL-2 for recurrent respiratory infection ina case of cartilage-hair hypoplasia with autoimmune hemolytic anemia

Sayuri Matsumoto1, Keiichi Ozono2, Takehisa Yamamoto1, Kanji Yamaoka3, Takayuki Okamura1,Junichi Hara1, Masaaki Shima1, and Shintaro Okada1

1Department of Pediatrics, D-5, Developmental Medicine, Osaka University Graduate School of Medical Science, Suita 565-0871, Japan2Department of Environmental Medicine, Osaka Medical Center and Research Institute for Maternal and Child Health, Izumi, Japan3Osaka Prefectural Hospital, Osaka, Japan

following an elevation of the soluble IL-2 receptorlevels has been reported [6]. Although AIHA has beenknown as a complication of CHH [5,7], the mechanismby which CHH patients develop AIHA remains to bedetermined. In addition, soluble IL-2 receptor levelshave not been investigated in CHH. We describe herea CHH patient with a remarkably short stature andsevere immunodeficiency who received recombinanthuman IL-2 (rIL-2) for recurrent infections. The serumlevels of IL-2 and soluble IL-2 receptor were deter-mined to enable us to better understand immunodefi-ciency in CHH patients.

Methods

Mononuclear cells isolated from the patient werecultured in RPMI-1640 (Nikken Biomedical, Kyoto,Japan) supplemented with 10% fetal calf serum and2µg/ml phytohemagglutinin (PHA) for 24h. The pro-duction of IL-2 was measured following stimulation byPHA according to the method reported by Nakamura etal. [8]. IL-2 activity in the culture supernatant was deter-mined as described [1]. The proliferative response ofthe lymphocytes to IL-2 after activation by 20µg/mlconcanavalin A (Con A) was evaluated by [3H-]thymi-dine incorporation. Briefly, mononuclear cells were cul-tured with 20µg/ml Con A at a density of 3–5 3 106 cells/ml in 96-well round-bottom plates. After 48h incuba-tion at 37°C, the cells were pulsed for 18h with 1µCi/ml[3H-]thymidine. Cells were then harvested using a multi-channel cell harvester. The amount of radioactivity in-corporated was measured with a liquid scintillationcounter.

The expression of IL-2 receptor-â was examined byflow cytometry using an anti-Mikâ1 monoclonal anti-body (Nichirei, Tokyo, Japan). Peripheral blood lym-phocytes from the patient and healthy controls werecultured in RPMI 1640 with or without 1027 M 12-O-

Key words: cartilage hair hypoplasia, immunodeficiency,autoimmune hemolytic anemia, recombinant human IL-2,soluble interleukin-2 receptor

Introduction

Cartilage hair hypoplasia (CHH), synonymous with theMcKusick type of metaphyseal chondrodysplasia, is arare bone disease transmitted in an autosomal-recessivefashion that was first described in the Amish populationin Philadelphia by McKusick et al. [1]. This disease ischaracterized by metaphyseal chondrodysplasia leadingto short-limb dwarfism and thin, sparse, slowly grow-ing hair and short stature [2]. Radiological studiesperformed before closure of the epiphyses revealedgeneralized abnormalities at the metaphyseal ends ofevery tubular bone in all patients. In addition to thebone dysplasia, cell-mediated immunodeficiency is rec-ognized as an important feature of the disease. Theimpaired production of IL-2 as well as the lack of prolif-erative response to exogenously supplied IL-2 in thelymphocytes have been postulated as one of the modesof pathogenesis of immunodeficiency in CHH patients[3]. However, the extent of cell-mediated immunodefi-ciency varies widely between patients, even within afamily [2]. In 108 Finnish patients with CHH, 56% hadbeen unusually prone to infection; half of these hadsevere CHH, and primary infections had been fatal in5.5% [4]. In children with CHH, the immunodeficiencycan be life-threatening; thus, bone marrow transplanta-tion has been considered as a therapeutic option [5].

In autoimmune hemolytic anemia (AIHA) associ-ated with malignancy, infections, and autoimmune dis-eases, hyperactivation of CD4-positive lymphocytes

Offprint requests to: M. ShimaReceived: Dec. 27, 1997 / Accepted: April 5, 1999

tetradecanoylphorbol 13-acetate (TPA) (Sigma, St.Louis, MO, USA) and 5 µg/ml rIL-2 for 96h. The ex-pression of IL-2 receptor-α was then examined by flowcytometry using anti-CD25 and anti-CD2 monoclonalantibodies (Beckton Dickinson ImmunocytometrySystems, San Jose, CA, USA). Serum IL-2 (OtsukaAssay Laboratory, Osaka, Japan) and soluble IL-2 re-ceptor (Yamanouchi Pharmaceutical, Tokyo, Japan)levels were measured using an enzyme immunoassaykit [9].

Case report

A 2-year and 7-month-old girl was referred to our hos-pital with failure to thrive. On admission, her height andweight were 64.5 cm (28.8 SD) and 5892g (25.0 SD),respectively. Her height was below the 10th percentilefor the CHH standard (,66cm) (Fig. 1) [10]. She haddisproportionately short limbs, with an upper- to lower-segment ratio of 1.4. Her scalp hair was thin and sparse(Fig. 2). Roentgenograms revealed a disproportionatewidth at the metaphyseal ends of the long bones, whichwas marked at the knee (Fig. 3). She had been admittedto hospital ten times for severe respiratory or gas-trointestinal infections since the age of 3 months. Onthe basis of these clinical findings, a diagnosis ofCHH was made. Parenteral nutrition was started forintestinal malabsorption due to chronic intestinalpseudoobstruction syndrome.

She developed recurrent bronchitis on the 132nd hos-pital day, and was placed in an oxygen tent for dyspnea.Because conventional treatments with intravenous anti-biotics and γ-globulin were not effective, treatment withrIL-2 was started (see Results). Macrocytic anemia wasobserved following recurrent respiratory infections onthe 350th hospital day. At that time, her hemoglobinlevel decreased rapidly from 10.7 to 5.8 g/dl with a highfever. The direct Coombs test was positive, whereas theindirect Coombs test was not. Hepatosplenomegaly wasnot apparent, while her indirect bilirubin levels wereslightly increased. Based on these data she was diag-nosed as having AIHA. Parvovirus B19 was not de-tected in the patient serum by PCR. For the treatment

Fig. 1. Growth curve of the patient (circles). Broken linesrepresent the 10th and 90th percentile for cartilage-hairhyperplasia (CHH) patients

Fig. 2A,B. Hair from normal control(A) and the patient (B). That of thepatient is apparently thinner with nohair cuticle

S. Matsumoto et al.: Treatment with rIL-2 for a patient with CHH 37

of the anemia, oral administration of 2.5mg/kg pred-nisolone was started. On the 5th day of the steroidtherapy, her hemoglobin level began to increase, recov-ering to normal levels on the 19th day. The treatmentwith prednisolone was gradually tapered to discontinu-ation over the following 5 months.

Results

Immunological examination of this patient revealed acell-mediated immunodeficiency characterized by re-duced numbers of lymphocytes, especially CD4-positivecells, and a deteriorated proliferative response to PHAwith the response to ConA preserved (Table 1). Skintests for PHA and purified protein derivative werenegative. Thymic size was normal on the computed to-mography of the chest. IL-2 production in the patientwas very low (Table 1), and her lymphoproliferativeresponse to IL-2 in the presence of Con A was de-creased. The expression of IL-2 receptor-â on the pe-ripheral blood lymphocytes including T cells, NK cells,and B cells was within the normal range. IL-2 receptor-α expression on CD4-positive lymphocytes (Table 1),but not NK cells, was decreased on stimulation withTPA and IL-2.

We administered human rIL-2 subcutaneously to thepatient on the 165th hospital day for the treatment ofrecurrent respiratory infections with informed consentobtained from her parents. She developed recurrentbronchitis with high fever and dyspnea, but recoveredgradually. The high fever declined 40 days after thetreatment, with the respiratory rate decreasing from50 to 40 times/min. Vomiting, induced by intractable

Fig. 3. Leg and hip films of the patient on admission. Note thewide, irregular, sclerotic metaphyses

Table 1. Immunological findings in the CHH patient before rIL-2 therapy

Finding Value Range

WBC (/µl) 9 960 (3 300–9400)Neu (%) 83.9 (40–73)Ly (%) 7.1 (18–52)T cell (%) 23 (61–73.8)B cell (%) 17 (9.7–17.3)NK cell (%) 60 (10–12)CD4/CD8 0.41 (0.88–1.84)Cytokines and receptors

IL-2 secretion (U/ml) ,0.8 (4–25)IL-2 response (cpm) 11 259 (22 000–38000)IL-2 receptor-α expression (%) 9 (90)IL-2 receptor-â expression (%) 64 (94)Serum-soluble IL-2 receptor (U/ml) 1 640 (120–384)

Lymphoproliferative responsePHA (cpm) 359 (26 000–53000)Con A (cpm) 2 858 (2 000–4800)

Normal ranges are indicated in parenthesesCHH, cartilage-hair hyperplasia; rIL-2, recombinant interleukin-2; WBC, white bood cell count;Neu, neutrophils; Ly, lymphocytes; NK cell, natural killer cell

38 S. Matsumoto et al.: Treatment with rIL-2 for a patient with CHH

cough, improved 30 days after the treatment, and itbecame possible to supply more nutrients parenterally.During the treatment, CD16-positive NK cells in-creased in number, but the lymphoproliferative re-sponse to PHA in vitro was not significantly improved(data not shown). Serum levels of the soluble IL-2receptor were already high before the IL-2 therapyand were elevated following rIL-2 treatment (Fig. 4).Soluble IL-2 receptor levels increased further, from1675 to 2397 U/ml, when she developed AIHA.

Discussion

Although the phenotypes of CHH are variable, themost consistent finding is profound short stature [2].Examination of the long bones revealed a paucity ofchondrocytes at the growth plates with disorderlycolumnation in CHH patients [2]. A generalized defectin enchondral ossification is present in all long bonesexamined [11]. Patients with remarkable growth failurewere shown to develop recurrent infections as wellas lymphopenia in CHH [4]. Height was below the

Fig. 4. The clinical course for this patient. Recombinantinterleukin-2 (rIL-2) therapy was started on the 126th dayafter hospitalization. rIL-2 was given subcutaneously at a doseof 25000 JRU(WHO)/day. After 3 days, this dosage was in-creased to 50000 JRU/day for 26 days, then to 66 000 JRU/dayfor another 34 days. There were no remarkable side effectsfrom the administration of rIL-2. The treatment amelioratedher recurrent bronchitis, decreasing the respiratory rate,which enabled us to start enteral feeding. The lympho-

proliferative response by phytohemagglutinin (PHA) wasalso improved from 0 3 0/15 3 16mm to 9 3 10/21 3 16mmon skin reaction, and from 671 cpm to 2243 cpm in vitro. Thenumber of peripheral lymphocytes increased during rIL-2therapy. The number of Leu-11-positive natural killer (NK)cells increased from 143/µl to 545/µl. Soluble IL-2 receptorlevel was elevated before rIL-2 therapy and increased follow-ing the treatment. The normal range of serum level of solubleIL-2 receptor is 120–384 U/ml

10th percentile for the CHH standard, and recurrentinfections were observed in our patient. Ours can beconsidered one of the most severe cases becausethe patient had remarkably short stature and severeimmunodeficiency.

Lymphocytes from CHH subjects showed a markedimpairment in IL-2 production and inability to prolifer-ate in response to various mitogens [3]. IL-2 binds toIL-2 receptors on the cellular surface, and the IL-2receptors are linked to three distinct signaling path-ways: induction of c-fos/c-jun, c-myc, and bcl-2 [12]. Thehigh-affinity form of IL-2 receptor consists of α-, â-, andγ-subunits [13]. In the hematopoietic cells, the stimula-tion of these pathways promotes the transition of thecell cycle, resulting in the cell proliferation [14]. In ourcase, decreased IL-2 production and reduced IL-2response in the proliferation of mononuclear cellswere observed (see Table 1). However, the lymphopro-liferative response to exogenous IL-2 was relativelypreserved in vitro. We administered rIL-2 to the patientfor the treatment of recurrent respiratory infectionsbecause conventional therapy was not effective anda response to exogenous IL-2 by lymphocytes was

S. Matsumoto et al.: Treatment with rIL-2 for a patient with CHH 39

expected. Exogenous IL-2 administration has recentlybeen used in an attempt to improve defective T-cellproliferation in patients with severe combined immuno-deficiency whose lymphocytes were remarkably defi-cient in messenger RNA for IL-2 and exhibiteddiminished production of endogenous IL-2 [15]. Thistreatment has been reported to improve lymphopro-liferative responses and increase the percentage of Tcells. In our patient, the administration of rIL-2,although of limited effect, improved her general con-dition and ameliorated the intractable respiratoryinfections. To our knowledge, this is the first report ofthe administration of rIL-2 for immunodeficiency in apatient with CHH.

Soluble IL-2 receptor is known to inhibit immuneresponse in infections and be elevated in patients withautoimmune deseases [12]. In our case, serum solubleIL-2 receptor levels were increased, although lym-phopenia was observed. The reason for this discrepancyis not clear, but we speculate recurrent infectionscaused activated B cells to produce soluble IL-2 recep-tor, resulting in the patient’s increased serum levels [16].

AIHA has been often associated with primary immu-nodeficiencies in childhood [17]. AIHA has been re-ported to be one of the complications of CHH, althoughthe mechanism of AIHA in CHH remains unclear [5,7].Both the serum level of soluble IL-2 receptor and num-bers of IL-2 receptor on B cells were increased duringthe period of AIHA in our patient. In patients withAIHA, it has been reported that CD4-positive lympho-cytes are extremely activated and autoantibody produc-tion by B lymphocytes is increased [6]. Because solubleIL-2 receptor is released from activated human lympho-cytes [16], including CD-4-positive lymphocytes, it isunderstandable that the increased soluble IL-2 receptorlevels in our case were related to the elevated autoanti-body production resulting from AIHA. It is not clearwhether IL-2 administration induced AIHA in the pa-tient. The administration of rIL-2 is a likely treatmentfor T-cell type immunodeficiency in CHH, but the opti-mal dose and duration of IL-2 treatment remain to bedetermined.

Acknowledgments. We express our thanks to Drs. M.Nakayama and A. Miyano for technical assistance inmeasuring soluble IL-2 receptor and Dr. S. Nakajimafor critical reading of the manuscript. This work wassupported by grants from the Welfare Ministry ofJapan.

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40 S. Matsumoto et al.: Treatment with rIL-2 for a patient with CHH