Humoral immunity in steroid-dependent children with asthma and hypogammaglobulinemia

Humoral immunity in steroid-dependent children with asthma and hypogammaglobulinemia G i d e o n Lack, MD, Hans D, Ochs, MD, a n d Erwin W, G e l f a n d , MD From the Division of Basic Sciences, Department of Pediatrics, National Jewish Center for Immunology and Respiratory Medicine, Denver, Colorado, and the Department of Pediatrics, Universityof Washington, Seaffle

Objective: To determine primary and secondary antibody responses in children with hypogammaglobulinemia attributed to corticosteroid use. Results: In seven patients with steroid-dependent asthma and significant hypogammaglobulinemia (IgG concentration, 275 to 443 mg/dl), antibody responses to protein and polysaccharide antigens were shown to be normal, as were primary and secondary responses to a neoantigen, bacteriophage ~X174. Conclusions: Patients with asthma, andwith hypogammaglobulinemia resulting from steroid therapy, have normal humoral immunity, and immunoglobulin replacement therapy is not indicated. (J Pediatr 1996;129:898-903) Orally administered corticosteroids play an important role in the control of severe asthma. The inmmnosuppressive effects of steroids place these patients at risk of life-threatening primary viral infections, most notably varicella, presumably because of T-cell suppression. 1-3 However, pharmacologic doses Of corticosteroids are also associated with decreased immunoglobulin levels in some individuals. This has been described in patients with asthma during short-term therapy4, 5 and in those receiving long-term steroid treatment,6, 7 with the lowest levels occurring in the latter group. We identified a group of steroid-dependent asthma patients with serum IgG levels much lower than 2.5 SD below the mean, in a range denoting hypogammaglobulinemia. These levels are lower than the moderately decreased levels previously reported to be associated with corticosteroid therapy in similar patient populations. Although previous studies have assessed functional antibody production in pa-

Supported by grants AI-29704 (_Dr. Gelfand), HL-36577 (Dr. Gelfand), and HD17427 (Dr. Ochs) from the National Institutes of Health. Submitted for publication Dec. 23, 1995; accepted July 2, 1996. Reprint requests: Erwin W. Gelfand, MD, National Jewish Center for Immunology and Respiratory Medicine, 1400 Jackson St., Denver, CO 80206. Copyright © 1996 by Mosby-Year Book, Inc. 0022-3476/96/$5.00 + 0 9/21/76237

898

tients with asthma and low IgG levels in association with prolonged Steroid use, humoral immunity has not been assessed in pediatric patients with steroid-dependent asthma and a severe degree of immunoglobulin deficiency. Hamilos et al. 8 studied adults with asthma who were receiving long-term steroid therapy and who had severe hypogammaglobulinemia; a small proportion of these patients demonstrated impaired pneumococcal antibody responses. The majority Of investigators focused on recall antibody responses, except for Tuchinda et at., 9 who showed normal primary antibody responses to keyhole limpet hemocyanin in children with asthma who were receiving steroids and who had normal immunoglobulin levels. We therefore elected to study both primary antibody responses and immunoglobulin isotype switching during a secondary response in these children with severe hypogammaglobulineinia. This is important for several reasons: (1) the immune system in children is maturing, and children, unlike adults, are therefore exposed to more neoantigens; (2) primary antibody responses and isotype switching during secondary responses are T cell-dependent processes and could potentially be impaired because of the effect of steroids on T-cell immunity; (3) impaired immunity in such children could be associated with susceptibility to an increased number of respiratory infections, further worsening asthma; and (4) a rational decision concerning the need for immunoglobulin replacement therapy in these children should not be undertaken

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T a b l e . Prolonged steroid use in children with asthma, in association with various immunoglobulin isotype deficiencies

Patient No.

Age (yr)

Prednisone (rng/kg/day)

IgG (mg/dl)

IgA (mg/dl)

IgM (mg/dl)

foE 0U/ml)

1 2 3 4 5 6 7 8 9

16 16 13.5 12 15.5 15 10 7 5.5

0.4 (D) 0.2 (A) 0.6 (D) 0.2 (A) 0.3(A) 0.25 (D) 1.1 (A) 0.3 (A) 0.5 (A)

418 384 275 377 396 443 309 39t 309

63 62 14" 22* 130 90 6t 89 60

147 84 33* 72 87 94 59 113 10I

171 15 8 7 165 76 6 400 7

Nine subjectshad serum immunoglobulinlevelsmeasured at the start of the study, demonstratinghypogammaglobulinemia(IgG>2.5 SD belowthe mean for age). Shown are the serum immunoglobulinisotypelevels and mean daily steroiddosages. A, Alternate-daydosing;D, daily dosing. *IgA and IgM levelsmore than 2.0 SD belowthe age-appropriatemean. without adequate assessment of functional antibody production. We identified nine patients with steroid-dependent asthma and significant hypogammaglobulinemia. Memory responses to protein and polysaccharide antigen, as well as their primary and secondary antibody responses to a neoantigen, bacteriophage oPX174, were evaluated in seven patients.

METHOD, S Patients. Nine patients, aged 4 to 18 years, with steroiddependent asthma and hypogammaglobulinemia were selected. Patients had reactive airways disease according to the criteria of the American Thoracic Society. I° Steroid dependence was defined as requiring the equivalent of at least 20 mg of every-other-day prednisone at the time of the study aud for a minimum of 6 months at the time of entry into the study. Individuals with serum IgG levels less than 450 rag/ dl, and more than 2.5 SD below the age-appropriate mean, were identified from the entire pool of individuals tested for quantitative immunoglobulins at National Jewish Center during the previous 4 years. They were tested again before enrollment into the study, and if the IgG level was still less than 450 m J d l , they were included. Criteria for exclusion were as follows: (i) individuals currently receiving or with a history of cytotoxic drug therapy, (2) individuals known or thought to have low immunoglobulin lewfls before steroid therapy, and (3) individuals who had received gammaglobulin therapy. Individuals who had previously received pneumococcal vaccine and were not revaccinated but were, instead, immunized with the conjugated Haemophilus influenzae vaccine. The patients' steroid dosage was calculated in milligrams of prednisone per kilogram per day, averaged ove r the preceding 6 months. The average: was determined by calculating the patient's cumulative ,dose of steroids, dividing by 180, and then

dividing by the patient's weight. A careful infectious disease history was obtained from the patient's parents, referring physician, and medial records for occurrences of otitis media, sinusitis, pneumonia, septicemia, meningitis, and osteomyelitis. The study was approved by the institutional review board of the center, and consent was obtained. Measurement of serum immunoglobulin and IgG subclasses. Serum IgG, IgA, and IgM concentrations were measured in the clinical laboratory at the National Jewish Center by rate nephelometry, with the use of goat anti-human immunoglobulin. Values were compared with those of age-matched healthy subjects. Serum from patients with low IgA levels was further analyzed by radial imanunodiffusion to confirm the low level of IgA. In this assay the agar plates were coated with rabbit anti-human IgA to avoid the detection of anti-bovida antibodies. Further analysis by immunoelectrophoresis was done to exclude the presence of anti-bovida antibodies. Serum IgE was measured by an enzyme immunoassay and was expressed in international units per milliliter. IgG subclasses were measured by ELISA and compared with those of age-matched healthy subjects. Immunization protocol Bacteriophage OPX174. This antigen was used as described by Smith et al.ll and Wedgewood et at.t2 It allows delineation of antigen clearance, primary antibody responses, secondary responses with amplification, and isotype switching from IgM to IgG. It was given intravenously at a dose of 2 x 109 plaque-forming units per kilogram for the primary response, and the same dose was administered again 6 weeks later to assess secondary responses. Serum was obtained before immunization and weekly for 4 weeks after primary and secondary immunization. Standard antigens. At the time of the first immunization with bacteriophage ~X174, subjects were vaccinated subcutaneously with 0.5 ml tetanus, diphtheria, and 0.5 ml pneumococcal polysaccharide antigens or conjugated H. in-

900

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The Journal of Pediatrics Deceember 1996

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Weeks After Injection with QX174 Fig. ]. Seven patients were immunized with bacteriophage dPX174and had serum determinations of anti-gPX174antibody at indicated time points. Shown are total serum anti-dPX174 antibody responses(comprising all immunoglobulin isotypes) for each patient (expressed as log 1000 x optical density [O.D.]). Table in lower right inset indicates the percentage contribution of IgG to total immunoglobulin levels during peak secondary antibody response for each patient and normal control values. Pri, Primary immunization; Sec, secondary immunization (booster); Pre, before immunization.

fluenzae type b. Serum samples were obtained 4 weeks later.

Determination of antibody responses Bacteriophage cbX174. Serum IgM and IgG antibodies to bacteriophage qbX174 were measured by ELISA in seven patients, as previously described. 12, 13 Tetanus. Serum IgG levels against tetanus were measured by ELISA in seven patients and expressed as international units per milliliter. A normal antibody response to tetanus was defined as a greater than tenfold increase in serum levels after vaccination, with a postimmunization level of 0.1 IU/ml or greater. Diphtheria. Anti-diphtheria IgG levels were measured by enzyme immunoassay (Specialty Laboratories, Santa Monica, Calif.). A normal response was defined as a postvaecination/prevaccination ratio of 4 or higher, with a postvaccination level of 0.01 absorption unit per milliliter or greater. Pneumococcal vaccine. Anti-pneumococcal IgG response against serotype 3 was assessed in five patients with a hemagglutinin inhibition assay. A normal response was defined as a greater than fourfold increase in serum titers. Haemophilus influenzae type b. Serum anti-Hib IgG was assayed in three patients who had previously received the pneumococcal vaccine. IgG antibody to polyribosylribitol, the capsular polysaccharide of Hib was measured by radioirnmunoassay and expressed as micrograms per milliliter

(Smith, Kline, Beecham Laboratories). A serum level greater than 1 ~g/ml was considered to be protective. RESULTS

Patient characteristics. We identified (by contact) 13 patients who initially met the inclusion criteria--7% of the total number of patients with steroid-dependent asthma identified during the same period. Four patients were excluded because of prior treatment with intravenously administered immune globulin. Nine patients with hypogammaglobulinemia were assessed for immunoglobulin levels and IgG subclasses. Complete humoral responses to bacteriophage dPX174 and conventional antigens were evaluated in seven of these patients. The mean age of the nine subjects was 12.4 years (range, 5.1 to 16.3 years). Seven boys and 2 gifts participated in the study. All patients bad clinical steroid side effects to varying degrees, involving different systems. There was no relationship between the dose of prednisone per kilogram of body weight per day averaged for the preceding 6 months and the degree of hypogammaglobulinemia (Table). There was similarly no relationship between the pattern of clinical steroid side effects and the presence or degree of hypogammaglobulinemia. However, all nine patients with hypogammaglobulinemia had biochemical abnormalities caused by steroids. Seven of nine patients had

The Journal of Pediatrics Volume 129, Number 6

hypercholesterolemia, and eight of the nine had marked adrenal suppression as determined by morning serum cortisol levels (data not shown). In this group of children, the incidence of bacterial infection did not differ from that in agematched, steroid-dependent patients with asthma who had normal levels of IgG (data not shown). Immunoglobulin isotypes. All nine patients had a marked reduction in serum IgG levels (Table). Two patients had low levels of IgA, confirmed by radial immunodiffusion and immunoelectrophoresis to exclude the presence of anti-bovida antibodies. The serum IgM concentration was decreased in one patient. IgG subclasses. Low serum concentrations of IgG subclasses were defined in absolute terms as greater than 2.0 SD below the age-appropriate mean, and relatively, in terms of the normal percentage distribution of IgG subclasses. The main decrease in immunoglobulin occurred in the IgG1 subclass and accounted primarily for the observed hypogammaglobulinemia. All nine patients showed a considerable decrease in serum IgG1 levels. Patient 6 (see Table I) in p~ticulax had a serum IgG1 level of 155 mg/dl, which was only 33% of his total IgG concentration. Various other decreases in IgG subclasses were also apparent. Two patients had low serum IgG2 levels, three patients had low IgG3 levels, and one patient had a low serum IgG4 level. Although low in absolute terms, these IgG subclass values fell within the normal percentage distribution (IgG2 ->15%, IgG3 ->5%, IgG4 >-2%). No association was observed between the average steroid dosage during the preceding 6 months and the pattern or extent of IgG subclass deficiency. Primary and secondary responses to bacteriophage ~X174. No adverse reactions were noted in the seven patients immunized with bacteriophage qbX174. All seven patients cleared antigen normally and mounted both normal primary and normal secondary immune responses. The primary responses consisted largely of IgM, whereas the memory response was characterized by both amplification and a switch to IgG production (Fig. 1). The mean primary and secondary responses for the seven patients were statistically identical to the mean for published control valuesJ 3 Five patients had completely normal responses. Patient 9 showed a primary and a secondary response just outside the normal range, but also showed strong amplification and a normal proportion of IgG production during the secondary response. This patient's slightly weaker response may have been due to a low phage titer at 15 minutes after primary immunization, which suggests a low antigen dose. Patient 3, who had a normal primary response and an amplified secondary response just below the normal range, was nevertheless able to switch to IgG during the secondary response.

Lack, Ochs, and Gelfand

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Fig. 2. Seven patients were immunized with tetanus toxoid and five with pneumococcal polysaccharide. Serum was obtained before and 4 weeks after immunization, and frozen at -70 ° C. A, Anti-tetanus IgG was measured by ELISA and is expressed as international units per milliliter. Dotted line reflects a protective level. B, Antipneumococcal IgG levels against serotype 3 are expressed along yaxis as reciprocal dilution of serum at which hemagglutination inhibition occurred. Symbols: &, patient 1; O, patient 3; &, patient 4; [-], patient 5; G, patient 6; [], patient 7; O, patient 9.

Responses to recall protein antigens. All seven patients immunized with tetanus were able to mount a protective antibody response to tetanus (Fig. 2, A). Patients 5 and 7 had decreased antibody responses (i.e., less than a tenfold increase in anti-tetanus IgG). However, this is probably because they had both been immunized during the 6 months preceding the most recent immunization and had relatively high levels of neutralizing antibodies before immunization; indeed, both mounted normal responses to diphtheria antigen (data not shown). Of note, patients 6 and 3, both of whom

902

Lack, Ochs, and Gelfand

had low IgG1 and IgG3 levels, mounted normal antibody responses to immunization with protein antigens. Responses to polysaeeharide antigens. All five of the patients who received pneumococcal vaccination mounted normal IgG antibody responses to pneumococcus serotype 3 (Fig. 2, B). Patients 7 and 9, both of whom had previously been immunized with the vaccine, were able to mount norreal responses to Hib immunization. Of note, patients 3 and 7, both with abnormally low serum concentrations of IgG2 subclasses, were able to mount normal antibody responses to polysaccharide antigens. DISCUSSION The immunomodulatory effects of steroids are complex. We identified a group of steroid-dependent children with asthma and with significant hypoganunaglobulinemia, presumably as a result of prolonged steroid use. We established that these patients have normal antibody responses to both protein and polysaccharide antigens. We further demonstrated that these patients had normal primary antibody responses to a neoantigen and amplified memory responses characterized by immunoglobulin isotype switching to IgG. Several mechanisms may contribute to hypogammaglobulinemia in these patients. Studies show both increased immunoglobulin catabolism 14, t5 and decreased immunoglobulin synthesis 16, 17 in response to steroid treatment. Moreover, these effects vary in different compartments of the immune system. 18 Immunization with bacteriophage dpX174 provides more information than does assessment with conventional antigens. We were able to show that the primary response and the secondary (memory) responses, with amplification and immunoglobulin-class switching to IgG, were all intact. Although two patients had mildly decreased responses, their ability to amplify and to undergo isotype switch clearly distinguishes them from patients with humoral immunodeficiencies. The ability to respond to different antigens was confirmed by responses to immunization with protein and polysaccharide recall antigens. Furthermore, despite low levels of IgM, IgA, and IgE, these lower levels were not associated with any degree of humoral immunodeficiency. Patient 3, who had an IgM level of only 33 mg/dl, was able to mount a normal primary IgM response to bacteriophage dPX174 and also had normal titers of isohemagglutinins in his serum (data not shown). Immediate hypersensitivity skin testing to numerous aeroallergens was preserved despite low or low-normal total serum IgE levels in four patients assessed for atopy (data not shown). Thus the ability to make functional antibody in these patients was not confined to the IgG isotype but extended as well to the IgM and IgE isotypes. We also noted combinations of low immunoglobulin subclass levels in these patients, in comparison with normal

The Journal of Pediatrics Deeeember 1996

age-matched values. Although low in absolute values, the percentage distribution of these subclasses was normal with respect to percentage composition of total IgG. Such tow levels of IgG subclasses had no apparent clinical relevance in these patients, and there was no correlation between the pattern of subclass deficiency and the ability to mount a response to protein or polysaccharide antigens. Evaluation of the clinical histories did not indicate an increased frequency or severity of infections in these patients with hypogammaglobulinemia. The presence and severity of chronic and acute sinusitis in this group of patients were no different from those encountered in patients with severe asthma who had normal serum levels of immunoglobulins. In conclusion, prolonged steroid therapy in children with asthma may be associated with significant hypogammaglobulinemia, with low levels of lgG, IgA, and IgM as well as various combinations of abnormally low IgG subclass levels. These children do not appear unusually susceptible to infections, and humoral antibody responses to conventional antigens are normal. We further demonstrated that the children were able to respond normally to neoantigens and to switch to IgG production during secondary responses. In the face of normal humoral immunity, such children do not need immunoglobulin replacement therapy.

REFERENCES l. Sloman JC, Bell PA. Cell cycle-specific effects of glucocorticoids on phytohemagghitinin-stimulated lymphocytes. Clin Exp Immunol 1980;39:503-9. 2. Hahn BH, MacDermott RP, Jacobs SB, et al. Immunosuppressive effects of low doses of glucocorticoids: effects on autologous and allogeneic mixed leukocyte reactions. J Immunol 1980;125:2812-7. 3. Watson JD, Aarden LA, Lefkovits I. The purification a n d quantitation of helper T-cell replacing factors secreted by mufine spleen cells activated by concanavalin A. J Immunol 1979;122:207-15. 4. Butler WT, Rossen RD. Effects of corticosteroids on immunity in man. J Clin Invest 1973;52:2629-40. 5. Posey WC, Nelson HS, Branch B, Perlman DS. The effects of acute corticosteroid therapy for asthma on serum immunoglobulin levels. J Allergy Clin Immunol 1978;62:340-8. 6. Katz Y, Harbeck RJ, DeMichelle D, Mitchell B, Strunk RC. Steroid-treated asthmatic patients with low levels of IgG have a normal capacity to produce specific antibodies. Pediatr Asthma Allergy Inununol 1988;2:309-16. 7. Berger W, Pollock J, Kiechel F, Danning M, Pearlman DS. Immunoglobulin levels in children with chronic severe asthma. Ann Allergy 1978;41:67-74. 8. Hamilos DL, Young RM, Peter JB, Agopian MS, Ikle DN, Barka N. Hypogammaglobulinen~'a in asthmatic patients. Ann Allergy 68:472-81. 9. Tuchinda M, Newcomb RW, DeVald BL. Effect ofprednisone treatment on the human immune response to keyhole limpet hemocyanin. Int Arch Allergy 1972;42:533-44. 10. Spector SL. Definition of asthma. ATS News 1982;89:5-6. 11. Smith TF, Morris EC, Bain RP. IgG subclass in nonallergic

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children with chronic chest symptoms. J Pediatr 1984;105:896900. 12. Wedgewood RJ, Ochs HD, Starkey DD. The recognition and classification of immunodeficiency diseases with bacteriophage dPX174. Birth Defects Original Article Series 1975;11: 331-8. I3. Pyun KH, Ochs HD, Wedgewood RJ, Xiqiang Y, Heller SR, Reimer CB. Human antibody responses to bacteriophage qsX174: sequential induction of IgM and IgG subclass antibody. Clin Immunol Immunopathol 1989;51:252-63. 14. Levy AL, Waldman TA. The effect of hydrocortisone on immunoglobulin metabolism. J Clin Invest 1970;49:1679-84.

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15. Cupps TR, Fauci AS. Corticosteroid-mediated immunoregulation in man. Immunol Rev 1982;65:133-55. 16. Griggs RC, Condemi JJ, Vaugh JH. Effect of therapeutic dosages of prednisone on human immunoglobulin G metabolism. J Allergy Clin Immunol 1972;49:267-73. 17. Cupps TR, Edgar LC, Thomas CA, Fauci AS. Multiple mechanisms of B cell immunoregulation in man after administration of in vivo corticosteroids. J Immunol 1984;132:170-5. 18. McMillan K, Longmire R, Yelenosk S. The effect of corticosteroids on human IgG synthesis. J Immunol 1976;116:1592-5.

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