Serum levels of IgA1 and IgA2 in children and in patients with IgA deficiency

Moleeuiur Z~~t~un~i0g.y Vol. 20, No. 9, pp. 977-981. Printed in Great Britain. 0161-5890/83 $3.00 + 0.00 Q 1983 Pergamon Press Ltd. 1983 SERUM LEVELS OF IgA1 AND IgAz IN CHILDREN AND IN PATIENTS WITH IgA DEFICIENCY zyxwvutsrqponmlkjihgfed M ARY ELLEN CONLEY*, ALLAN ARBETER and STEVEN D. DOUGLAS The Divisions of Allergy-Immunology and Infectious Diseases of The Children’s Hospital of Philadelphia, The Joseph Stokes Jr, Research Institute, and The Department of Pediatrics, University of Pennsylvania School of Medicine; Philadelphia, PA 19104, U.S.A. (Received 14 ~ece~~ber 1982) Abstract-Serum levels of IgA, and IgAz were measured by solid phase radioimmunoassay in samples from 110 children between 3 months and 10 years of age. Both IgA, and IgAz were detectable in all samples, and both IgAi and IgAz increased with increasing age. The percent of total serum IgA that was IgA, did not change with age and was the same in samples from children (15.05 f 10.2%) as in samples from adults (15.86 + 7.98%). The proportion of serum IgA that was IgA, was much less variable within sibships than within the group at large (P < 0.005). In the 16 patients with IgA deficiency, the proportion of serumIgA that was IgA, or IgA, was highly variable. IgAt constituted more than 50% of the IgA in 5 patients and less than 7% of the IgA in an additional 5 patients. These findings suggest that regulation of serum concentrations of IgA, and IgA, is complex and influenced by genetic factors and probably other unidentified factors. INTRODUCTION Studies on the IgA subclasses, IgA, and IgA,, are providing a useful approach for investigation of the source, function and regulation of the IgA in serum. Only 6-35x of the IgA in serum is IgA, (Vaerman et al., 1968; Grey et al., 1968; Morel1 et al., 1973; Conley & Koopman, submitted) whereas up to 4&60x of the IgA in secretions is IgAz suggesting that the IgA in serum may be derived from a different source than IgA in secretions. We have recently demonstrated that in the serum of healthy adults, the concentration of IgA, does not correlate with the concentration of IgA, (Conley submitted). Furthermore, in & Koopman, patients with systemic lupus erythematosus the serum levels of IgA, are significantly elevated but the levels of IgAz are not different from controls (Conley & Koopman, submitted). These observations suggest that there are factors that independently regulate the serum levels of IgA, and IgA2. During ontogeny there are shifts in the proportions of IgA B cells expressing IgA, and IgAz in the peripheral circulation. In the new* Correspondence to be addressed to Mary Ellen Conley, M.D., Division of Allergy-Immunology, The Children’s Hospital of Philadelphia, 34th Street and Civic Center Boulevard, Philadelphia, PA 19104-4399, U.S.A. born there are equal numbers of IgA, B cells and IgAz B cells (Conley et al., 1980). These cells express small amounts of IgA but also express surface IgM and IgD and probably represent immature or virgin B cells. In 3- to 5-month-old infants there is a shift toward IgAl predominance that can be attributed to a population of large lymphoblastoid cells that are almost all positive for IgA 1. These are most likely cells that have recently encountered antigen. In the peripheral blood of an adult, 80% of the IgA B cells express IgA, and 20% express IgAz (Conley et al., 1980). These cells can be brightly stained for IgA but are negative for IgM and IgD and probably represent memory B cells. Studies on the IgG subclasses in humans and in mice have demonstrated differences between the subclasses in their dependence on T cell help; shifts in the ratio of one subclass to another that occur with increasing age; and clinical disorders in which one or more IgG subclass is deficient. In the mouse, synthesis of IgG, and IgG,, are more dependent on T cell help than IgGzB and IgG, (Bankhurst et al., 1975; Mongini et al., 1982; Isakson et al., 1982). Several investigators have shown that although the majority of IgG B cells in the peripheral circulation of the newborn, as well as the adult, express surface IgG, (Froland & Natvig, 977 978 MARY ELLEN CONLEY, ALLAN ARBETER 1972u, h) the serum concentrations of IgG, reach adult levels later than IgG, or IgG, (Giessen et al., 1975; Oxelius, 1979; Schur et a/., 1979). Selective deficiencies of IgG, (Beck & Heiner, 1981) and IgG, plus IgG, (Oxelius, 1974) have been associated with an increased risk of respiratory infections in patients with normal concentrations of total serum IgG. In some patients with panhypogammaglobulinemia there is selective preservation of IgG, or less commonly IgG, (Young rt al., 1970; Schur et ul., 1970; Giessen et ui., 1976). To further investigate the regulation of the IgA subclasses, we examined the influence of age, genetics and immunodeficiency on the proportion of total IgA that is IgA, or IgA,. Serum levels of IgA, and IgAz were measured by solid phase radioimmunoassay in samples from 110 children, 46 of whom were members of 21 sibships, and in samples from 16 patients with IgA deficiency. MATERIALS AND METHODS Adult sera were obtained from 36 healthy laboratory personnel between 18 and 45 years of age. The samples from babies less than 18 months of age were from the sibs of children with immunodeficiency who were later found to be normal, from babies evaluated for immunodeficiency who were not found to have a defect, and from infants with medical problems not related to the immune system. The samples from children between 18 months and 10 years of age were from middle to upper middle class children of a private pediatric practice who were enrolled in the study of varicella vaccines. The patients with panhypogamma~lobulinemia were between 18 and 43 years of age and had multiple medical problems. Patient No. 6 was treated with plasma infusions but had not received any plasma in the 5 weeks before the serum sample was taken. The other patients were treated only with intramuscular gammaglobulin. These patients all had serum levels of IgG, IgM and IgA that were less than two standard deviations below the mean. The patients with selective IgA deficiency were between 5 and 18 years of age. Patient No. 8 presented with chronic active hepatitis; the other 9 patients presented with allergies or frequent upper respiratory tract infections. These patients all had serum levels and STEPHEN D. DOUGLAS of IgA that were less than two standard deviations below normal for age and serum levels of IgG and IgM that were within normal range. Patients with serum levels of IgA less than 0.05% of normal (1 pg/ml) were excluded because it was felt that the proportions of IgA, and IgA, could not be determined accurately in this group. On the average, the patients with selective IgA deficiency had lower levels of IgA than the patients with panhypogammaglobulinemia. All individuals studied were Caucasians. Radioimnzunoassay The production and characterization of the monoclonal anti-IgA subclass antibodies (Conley et al., 1980) and the heterologous goat antihuman IgA (Conley & Koopman, submitted) have been previously described. The radioiodination of the heterologous goat anti-human IgA and radioimmunoassay have also been described (Conley & Koopman, submitted). The wells of microtiter plates were coated with a I:500 dilution of hybridoma anti-IgA, or anti-IgA, ascites or a 4 ng/ml solution of purified monoclonal antibody. After remaining protein binding sites were blocked with bovine serum albumin (Sigma, St. Louis, MO) the serum samples were added to duplicate wells at several dilutions. After overnight incubation, the wells were washed and 10 ng of ’ 251-labelled heterologous goat anti-IgA was added. After 4 hr the wells were washed, then counted in a Beckman Gamma 4000 (Beckman, Irvine, CA). A serum standard with known concentrations of IgA, and IgA, was run in all assays to generate a standard curve. The amounts of IgA, and IgA, in the serum samples are expressed as a percent of the adult standard which was made up of a pool of equal amounts of serum from 30 healthy individuals and contains 210 mg/dl IgA, 179 mg/ml of IgA i and 31 mg/dl IgAz (Conley & Koopman, submitted). RESULTS IgA, and IgA, were measured in serum samples by radioimmunoassay from 110 children under 10 years of age. Although there was a great deal of variability in the levels of IgA, and IgA,, both subclasses increased with increasing age (R = 0.459 for IgA,, P < 10W6, and R = 0.552 for IgA,, P < IO-‘; Fig. 1). 979 zyxwvu IgA, and IgAZ Serum Levels 210 - 210 7 ‘g*z ‘@I 180 - 180 - no g 150 - P pzj 150 - z2 - _ z 5 120 - 8 90 - c; 0 2 4 6 AGE 8 10 ADULT 120 0 2 L AGE (YEARS1 6 8 10 ADULT (YEARS1 Fig, 1. Age is plotted against serum concentration of IgAi (left) or IgA, (right), expressed as a percent of an adult standard. Each (0) represents a single individual. In the 16 patients with IgA deficiency both Both IgA, and IgA, were detectable in all IgA, and TgAz were depressed in 15 patients. samples. The mean percent of the total serum The percent of serum IgA that was IgAz IgA that was IgA, was the same in the samples from children (15.05 f 10.20) compared to the ranged from 0.6% to greater than 98% with a samples from adults (15.86 + 7.98) but the mean of 31.5 4 29.4. As shown in Tables 1 and variability was greater as is demonstrated by 2, IgA, constituted more than 50% of the IgA the larger standard deviation (Fig. 2). When the in 5 patients and less than 7% of the IgA in an additional 5 patients. Patients with an insamples from the 43 children less than three creased or decreased proportion of IgAz could years of age were compared to the samples from the adult group, the mean percent of the be found in both the group with selective IgA IgA that was IgAz was slightly lower (13.37 rt 10.50) but not significantly different from the adult group. Table 1. IgA subclass levels in the serum of patients with Within the 110 samples from children, there panh~ogammaglobulinemia were 46 samples from 21 families; 17 families IgAz/Total with 2 children and 4 families with 3 children. Pt. No. IgA x 100 IgAl* IgA,* Within these sibships the proportion of IgA that was IgAz was significantly less variable 1 2.7 0.8 5 2 13 39 34 than within the group at large (I; = 3.06, zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPO 3 1 3 33 P < 0.005) (Sokal & Rohif, 1969). 4 25 150 51 5 6 0.21 I3 3.1 1.8 72 2 *Expressed as a percent of an adult standard. 60 r Table 2. IgA subclass levels in the serum of patients with selective IgA deficiency 50 . 8 L 60 i Pt. No. 0 2 4 AGE 6 8 10 ADULT (YEARS) IgAl* IgA,* I 6.6 A.2 8 9 10 11 12 13 14 0.08 0.5 1.2 7.4 0.01 0.5 1.3 15 16 Fig. 2. Age is plotted against percent of total IgA that is IgA,. 0.08 20 IgA,/total IgA x 100 5 3 2.5 3.7 5 1 0.075 1 *Expressed as a percent of an adult standard. 2 30 61 24 6 98 61 zyxwvuts 12 13 0.6 980 MARY ELLEN CONLEY, ALLAN ARBETER deficiency, and in the group with panhypogammaglobulinemia. The proportion of IgA that was IgA, was not correlated with age, concentration of total IgA or any particular clinical symptom. and STEPHEN D. DOUGLAS geneous in race, social class and antigenic exposure it seems more likely that the similarities are due to genetics. It has been shown that the total proportion of serum IgG that is IgGz or IgG, (Giessen et al., 1975; Yount et al., 1967; Litwin & Balaban, 1972) is related to the allotype of that IgG subclass. However, no allotypes for IgA, have DISCUSSION been reported and of the two IgA, allotypes The results presented in this paper demon(Am2 ’ and Am2-) over 90% of Caucasians strate that even in the youngest babies, the IgA have the allotype Am2+ (Vyas & Fudenberg, in serum is predominantly IgA,. This obser1969; Kunkel et al., 1969). The group studied vation complements our previous studies, in included only Caucasians, therefore it is unwhich we noted a population of large lymlikely that the variation in the ratio of IgA, phoblastoid IgA B cells in the peripheral circuto IgA, seen in the group at large can be lation of infants, the majority of which were attributed to allotypic differences. It may be positive for surface IgA, (Conley et al., 1980). that regulatory genes closely linked to the heavy chain constant region play an important The predominance of IgA, B cells, and IgA, role in the control of subclass synthesis. molecules provide further evidence that early The patients with IgA deficiency provide after primary antigen exposure, IgA B cells expressing surface IgA, are selectively stimuanother perspective to examine regulation of lated to proliferate and differentiate. These patients could be IgA I and IgA,. The proportion of total IgA in the serum divided into two groups, those who had relathat was IgAz was similar in babies and adults, tive preservation of IgA, levels and those who indicating that there is not a delay in the had relative preservation of IgA,. Only 3 of the appearance of one subclass compared to the 16 IgA-deficient patients had proportions of other. The stable ratio of IgA, to IgA, also IgA, that were within one standard deviation indicates that chronic antigen exposure, as of the mean adult proportion of IgA,. There occurs over time, does not result in isotype were no apparent clinical or laboratory switching from IgA, and IgA, or vice versa. features that distinguished the patients with a During a child’s development the levels of shift towards IgA, predominance in the serum. IgA in secretions reach adult concentrations Patients with increased and decreased proporbefore the levels of IgA in serum (Haworth & tions of IgA, could be found in both the group Dilling, 1966; Bursio zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA et al., 1980). If the IgA with panhypogammaglobulinemia and in the made at mucosal surfaces contributed signifigroup with selective IgA deficiency. cantly to the serum pool of IgA, one might One possible explanation for the disproporexpect that in the infant a higher proportion of tionately low concentration of IgA, in some the serum IgA would be derived from the sepatients might be that there was less of a concretory system. Because a higher proportion of tribution from the secretory surfaces in these the IgA in secretions is IgAz (Grey et al., 1968; patients. Evidence against this hypothesis, Delacroix et al., in press) one might further however, is that patient No. 16, who had the expect babies to have relatively higher levels of lowest proportion of IgAz, had a normal conIgA,. The fact that this does not occur suggests centration of IgA in unstimulated saliva that either there is not an enrichment for IgA, (3.2 mg/dl). in the secretions of babies or that the secretory Several immunoregulatory defects have been surfaces do not contribute significantly to the reported in IgA deficiency. IgA specific T supserum IgA and serum and secretory pools of pressor cells (Waldmann et al., 1976; Atwater IgA are derived from relatively independent & Tomasi, 1978; Levitt & Cooper, 1981) a lack sources. of T cell help (Atwater & Tomasi, 1978; King The proportion of total serum IgA that was et al., 1979) and immature or defective B cells IgAz was much less variable within sibships (Cassidy et al., 1979; Conley & Cooper, 1981) than within the group at large. This indicates have been described. In all cases it is difficult to that genetics or environment play an important determine if the defect noted was primary or role in the regulation of IgA, and IgA,. secondary. However, it is possible that the Because the population studied is very homopatients with a higher proportion of IgA, in IgA, and IgA, Serum Levels the serum have a different immunoregulatory defect than those with a lower proportion of IgA,. The results presented in this paper indicate that the regulation of IgAl and IgAz in the serum is complex. Prolonged antigenic exposure does not seem to influence subclass distribution in the serum. Nor are contributions from the secretory immune system reflected in the serum levels of IgA, and IgA,. Genetic factors do seem to play an important role but the mechanism is unclear. Acknowledgements-The authors wish to thank Matthew Bartelt and Patricia Paciorek for unflappable technical assistance, Dr. Paul Coates for patient statistical assistance and Mary Swayne for ever cheerful secretarial assistance. 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