003 1-3998/85/1908-0846$02.00/0 PEDIATRIC RESE,ARCH Vol. 19, No. 8, 1985 Copyright 0 1985 International Pediatric Research Foundation, Inc. Primed in Cr.S.A.

Relation of Age, Race, and Allotype to Immunoglobulin Subclass Concentrations

PENELOPE G. SHACKELFORD, DAN M. GRANOFF, MOON H. NAIIM, MITCHELL G. SCOTT, BRIAN SUAREZ, JANARDAN P. PANDEY, AND SUSAN J. NELSON

Depur/mcnrs of Pediulric~[P.G.S.. D.M. G., S.J.N.], pa tho log,^ [M.H.N., M.G.S.], und Psychiulry [B.S.], Wu.sl?injironI;ni\w.c.ily Scl~oolof Mc~dicine;Divi.cion qf lr~fec~ioztsDismsc.~, St. Louis Children's Hospital, St. Lozris, M~ssollri63 1 78 and Depur?~nenrc?f Rusic & Clinicul Irnmzrnology and Microbiology [J.P. P.], hledicul Universi/.v qf'Souih Curolina, Cf7urlc~.~/on,Soulh Curolinu

ABSTRACT. Concentrations of IgG,, IgG,, and total IgG It is known that antipolysaccharide antibodies play an impor- were measured by a solid phase radioimmunoassay in sera tant role in protective immunity against polysaccharide encap- from 36 healthy adults and 114 healthy children. As ex- sulated bacterial pathogens (1 4- 16). Recent studics indicate that pected, IgC, and total IgG had a positive correlation with genetic factors, particularly genes associated with certain Gm age in children. In addition to age, several other factors and/or Km immunoglobulin allotypes, may be associated with were associated with significant differences in serum sub- altered responsiveness to vaccines containing bacterial polysac- class concentrations. Female children had higher concen- charides (17-19). In addition, black children lacking the Km(1) trations of IgG, than males, and black subjects had signif- allotype have an increased relative risk of developing meningitis icantly higher concentrations of IgG,, IgG2, and total IgG caused by Hib (1 7). Since human antipolysaccharide antibodies than whites. Although Km(1) and Gm(23) immunoglobulin have been reported to be relatively restricted to the IgG2 subclass, allotypes had no relation to subclass concentrations when it was of interest to determine if factors which appear to influence tested as single factors, the Km(1) allotype interacted the production of antipolysaccharide antibodies, i.e. race and significantly with race so that Km(1)-positive black chil- immunoglobulin allotype, might do so by virtue of their relation dren had higher IgG2 concentrations than other subjects. to the maturation and distribution of lgG subclasses in serum. Our findings may explain, in part, recent observations of In this study, we first established normal age-group values for an association of the Km(1) allotype with altered immune serum concentrations of IgC, IgG,, and IgG, in our laboratory. responses of blacks to certain vaccines containing bacterial We then examined the effects of sex, race, and Km(l) and polysaccharides. In addition, our data indicate the need to Gm(23) allotypes on these immunoglobulin concentrations in a control factors such as sex, race, and allotype in studies of group of children ages 7 to 38 months and in adults. We found subclass concentrations or immune responses. (Pediatr Res that black subjects, particularly those with certain immunoglob- 19: 846-849,1985) ulin allotypes, had higher serum concentrations of IgC, IgG,, and IgGl. Abbreviations

ANOVA, analysis of variance METHODS ANCOVA, analysis of covariance Hib, Haemophilus infruenzae type b Subjects. Normal subjects included 41 healthy adults and 1 14 healthy children, 7 months through 16 yr of age. These subjects were participants in various vaccine studies, or were recruited sequentially from among children presenting to a pediatric office for routine preventive care. Subjects in vaccine studies were Human IgG immunoglobulins have been divided antigenically into four subclasses, IgC,, IgG2, IgG3, and IgG4 (1-3). Of these, included based on availability of sera and race (to include equal numbers of blacks and whites). In order to increase the sample IgGl and IgG2 comprise the major portion of total IgG, 60 and size of sera from subjects with allotypes present in low numbers 30%~espectively.Although the biological importance of the [e.g. four IgCi subclasses is unknown, studies of myeloma proteins in the above sample Km(1) positive whites and Km(1) have revealed that these subclasses differ in their relative abilities negative blacks], we also included 10 serum samples that had to fix complement, to cross the placenta, and to bind to macro- previously been typed for Gm(23) and Km(1). After obtaining infbrmed consent, serum was collected by venipuncture and phages (4-6). In addition, antibodies to certain types of antigens, stored at -20" C. such as polysaccharides, are found to be relatively restricted to Laboratory methods. Serum IgG, and IgG, subclass concen- particular subclasses, i.c. IgG2 in humans (7, 8j and IgG, in mice (9, 10). Finally, striking differences in the ontogeny of subclasses trations were measured by a solid phase radioimunoassay (20) employing subclass specific reagents. In brief, a monoclonal have been reported, with IgG2 and IgG4 maturing much later antihuman IgG, reagent was prepared by immunization of a than IgGl and IgG3 in normal children (1 1-13). BALB/C mouse with "Cal," an IgG, myeloma protein (20). An Received October 12. 1984: acccpted March 27. 1985. antihuman IgG, reagent was prepared by immunization of a Address reprint requests to Penelope G. Shackelford. Department of Pediatrics. monkey (Mucacufasicuiaris) with two IgG, myeloma proteins, Washington University School of Medicine. Box 14871. St. Louis, MO 63 178. "Ziem" (IgGlk) and "Gar" (IgG,X) (20). The animal was primed Supported by Public Health Service Grants ROI-A1 19350, ROI-A1 17962, A1 18727, A1 19676, MH 3 i 302 and MH 14677 from the National Institutes of Health with Fc fragments and boosted with intact protein. Seruni was and by RR-36 from the General Clinical Research Center Branch. rendered IgG, specific by extensive absorption and affinity pu- SUBCLASSES IN NOKMAI. CM1LE)KEN 847 rification using lgGi and IgG2 conjugated sepharose 4B (Phar- Table 1, &GI, IgC,, and IgC concrntrations in sera from 114 macia, Piscataway, NJ) columns, respectively. Polyvinyl 96-well normal chi1drr.n and 41 healthy adults microtiter plates (Dynatech, Alexandria, VA) were coated with Geometric mean (pg/ml) subclass-specific antisera in phosphate-buffered saline. After coating with antiserum, plates were washed with 0.05% Tween 20 in normal saline and uncoated sites were blocked with 1% bovine serum albumin. Serum and myeloma standards were added to the plates and serial 4-lbld dilutions were performed in the plate. A proband myeloma, radiolabeled with "'I by the chloramine T method (21), was added to each well. The plates were incubated overnight at room temperature, washed, and individual wells were counted. The concentration of IgGi or IgG2 was calculated by comparison with the myeloma standard. The specificity of the IgGl and IgG, assays was confirmed using a panel of 39 purified human myeloma proteins (20). The World Health Organization reference serum 67/97 (IgG content 7700 pg/ml) was included in each assay as an internal control. The mean i SD value (pg/ml) determined for IgG, IgGi and IgG, in this reference serum, using our assays, compared well to those previously published (22) (in parentheses): IgG 8050 +- 332 (9000), IgGl 61 13 i 1273 (5 loo), and IgG2 2390 ? 285 (2500). The sensitivity of each assay was equal to the amount of standard protein which resulted in 50% inhibition of maximum binding. For IgG1 this ranged from 50- 129 ng/ml and for IgG2 from 70- 200 ng/mI. Determination of the Gm(23) and Km(1) allotypes was per- formed on coded serum samples, using a hemagglutination in- Adults 4 1 hibition assay with reagents described previously (23, 24). Sufi- cient sera for analysis was available from 36 adults and 64 children less than 38 months of age. * Indicates a value for the age group which is significantly different by The concentration of total IgG in serum was measured by the Scheffe' procedure (p 5 0.05) from the corresponding adult values. radial immunodiffusion using the kinetic (Fahey) method and ? The normal bounds in parentheses were determined by taking the commercial kits (ICL Scientific: Fountain Valley, CA). antilog of (mean logarithm t 2 SD of the logarithms). Statistical analysis. Statistical analysis was performed using SPSS programs (25) on a Harris 500 computer. Correlation Table 2. Immunoglobulin concerltrutions in norrr~alsubjects: analysis was used to assess the association between age and relation to sex immunoglobulin concentration. ANOVA was used lo test for differences in immunoglobulin concentrations among all sub- Mean Ig concentrations (@g/ jects when grouped by age and among adults grouped by sex, ml) race, or allotype. To allow for m~iltiplecomparisons, the Scheffi: n IgGi IgG2 IgG procedure was used to compare the age group means. For chil------dren, ANCOVA was used to remove the effect of age when Chlldren testing for mean differences based on sex, race, or allotype. Male 25 5338* 1127 8445 Normal values for immunoglobulin concentrations for the Female 39 7044* 1140 8835 various age groups are expressed as geometic means. The normal Adults range for each age group was determined by taking the antilog Male 18 7437 3025 12,364 of mean logarithm (+- 2 SD of the logarithms). Female 18 6512 2865 13.520 *p = 0.02 by 1-way ANCOVA with age as a covariate RESULTS The geometric mean IgGI, IgG,, and IgG concentrations in the significant linear correlation between age and both IgG and. groups of normal subjects of various ages are shown in Table 1. IgGz serum concentrations in these subjects. This correlatior~ As previously reported (1 1, 12), serum concentrations of IgGi permitted the use of ANCOVA to test for the effects of sex, race., increase rapidly during the 1st yr of life. Even in the youngest or immunoglobulin allotype on immunoglobulin concentration:; age group in this study (7-1 2 months) the concentrations were in young children while controlling for age. not significantly lower than those of adults. Female children were found to have significantly higher (p = The maturation of serum 1gG2 concentration differed consid- 0.02) serum concentrations of IgGI than males (Table 2). In erably from that of IgGI. In children 7-12 months of age, the adults, there were no significant differences in the serum concell-. mean concentration of IgG2 was 30% of the adult value and rose trations of IgGI, IgG2, or total IgG between males and females. to only 47% of the adult value by 3 1-36 months of age. Another We used 2-way ANOVA and ANCOVA to test for both main measure of the prominent age-effect on IgG2 was the positive and interactive effects of race and immunoglobulin allotypes on correlation with age in children less than 38 months of age (r = serum concer~trationsof IgC,, IgG,, and IgC. As summarized in 0.46, p < 0.00 1); in contrast, the correlation (r = 0.07) between Table 3, 2-way analysis of the relation of immunogiobulins to age and IgGi was not significantly different from zero. Total Km(1) and race showed no significant differences in IgGl con- serum IgG correlated with age in children less than 38 months centrations between subjects of different races although there of age (r = 0.37, p = 0.002). was a trend for IgC, to be higher in blacks than in whites (for The effects of sex, race, and the Gm(23) and Km(1) immu- adults, p = 0.07, and for children, p = 0.09). IgG2 concentrations noglobulin allotypes on serum IgGl, IgGz, and IgG concentra- were significantly higher in black adults than in white adults (I;' tions were tested. Data were analyzed separately for children and = 0.02). However, in children, this difference was apparent only for adults. Children 7-38 months of age were chosen because of in subjects with the Km(1) allotype (p = 0.02, by ANCOVA 848 SHACKELFORD ET AL. Table 3. Immunoglobulirz concenlrations in normal subjects: effect between race and Gm(23) allotype was found in black reiation to race and Km(1) imrnuno,globulin allorv~e adults but not black children. Black adults who lacked the Mean Ig concentrations (pg/rnl) Gm(23) allotype had very high serum concentrations of IgGz (mean = 4325 pg/ml), approxi~uatelytwice the mean concentra- n 1gG1 JgG, IgG tions of 1gG2in the other three adult groups. Finally, as in the 2------Children way analysis with Km(l), serum concentrations of IgG were significantly higher in black children than in white children (p Km( 1)- White 27 5832 1066 7806 = 0.006) and tended to be higher in black adults than in white Black 12 6473 904 9334 adults (p = 0.07). Km(l)+ White 8 6407 1003 769 1 DISCUSSION Black 17 7163 1470 10,082 Employing solid phase inhibition assays, we obtained values Probabilities (2-way ANCOVA) for IgG, and IgG2 which are similar to those previously reported Main NS* NS 0.02 (race) (1 1-1 3). We developed these assays in order to test large numbers Interaction NS 0.02 NS of samples, including some at low concentrations of IgG sub- classes using small quantities of antisera (20). Our data confirm Adults earlier observations of age-related changes in immunoglobulin Km(1)- concentrations, and indicate further that IgG, and IgG2 concen- White 16 6111 2113 11,591 trations are influenced by sex and race of the subject, and by the Black 7 7858 3450 13,337 presence or absence of the Km(1) and Gm(23) allotypes. Km(l)+ Specifically, IgG2 concentrations were lower in children up to White 3 6530 2278 10,823 108 months (9 yr) of age than in adults. On the other hand, IgG, Black 10 8005 4043 15,017 concentrations of only the youngest children (7- 12 months) were Probabilities (2-way ANOVA) less than those of normal adults, and in older children concen- Main NS 0.02 (race) NS trations of IgG, were higher than in adults. Higher concentrations Interaction hTS NS NS -- - of IgG, in older children have been noted in previous studies * No significant difference. ( 13) and may reflect response to hyperimmunization resulting from routine childhood immunization and frequent infections. Although normal age group values for IgG, TgA, and IgM have Table 4. Irnrnunogllohulin concentratiotzs in normal subjects: been established separately for males and females, only one reia~iotzto race and Gm(23) allotype ------previous study of IgG subclass concentrations in children has Mean Ig concentrations (@gjml) addressed the possible influence of gender. Van Der Giessen et a!. (13) found no significant differences in subclass concentra- 11 IgG, IgG2 IgG - tions between boys and girls more than 48 months of age. In our Children analysis of the values in children 7-38 months of age, we found Gm(23)- significantly greater concentrations of IgG, in females than in White 15 5337 1013 6769 males. This difference was not noted in adults. Black 23 6571 1314 9318 In 1968, Buckley et a/. (26) reported significantly higher con- Gm(23)+ centrations of IgG in black subjects 6 through 14 yr of age White 20 6434 1081 8538 compared to those in whites of comparable age. No significant Black 6 8055 937 989 1 differences in IgG concentrations were found in black and white Probabilities (2-way ANCOVA) children less than 6 yr of age; however, no correction was made Main hTS NS 0.006 (race) for age so the effect of race on IgG in this group of children may Interaction NS N S NS have been masked by inclusion of a greater proportion of bla.cks Adults who were very young (<9 months). Using ANCOVA to correct Gm(23)- for age, we found that black children between 7 and 38 months White 5 5693 1775 10,314 of age had significantly higher serum concentrations of 1gG than Black 13 7568 4325 15,043 white children (Table 3). In addition, black adults had higher Cim(23)' IgG, and IgG2 concentrations than white adults. Although IgG, White 14 6350 2269 11,883 tended to be higher in black children, serum IgG, and IgG2 Black 4 9167 2088 1 !,993 concentrations did not differ significantly in black and white Probabilities (2-way ANOVA) children when only the single effect of race was tested. Main 0.02 (race) 0.04 (race) NS Previous studies suggested an effect of immunoglobulin allo- Interaction NS 0.03 NS type on serum concentrations of IgG subclasses (13, 22, 27) but ------the results were conflicting. In one study, both IgG2 and rgG4 concentrations were higher in persons with the Gm(23) pheno- with age as the covariate). Black children had significantly higher type (22). In a subsequent study (27) IgG4 but not IgG2 concen- concentrations of total IgG (mean 9973 ,ug/ml) than white chil- trations were increased in subjects with Gm(23) compared with dren (7780 ,ug/ml) (p= 0.02). Although IgG concentrations also the corresponding values of subjects lacking Gm(23). In a third tended to be higher in black adults, this difference was not study (1 3), mean IgG2 concentrations were higher in sera positive statistically significant (p = 0.07). for Gm(23) than in sera lacking this allotype. In the present The effects of race and Gm(2.3) allotype on immunoglobulin study, with one exception, there were no significant differences concentrations are shown in Table 4. In this analysis, with in subclass concentrations related to this allotype. The exception Cim(23) as the second factor in the 2-way ANOVA, the higher was higher IgG2 concentrations in black adults lacking Gm(23) mean concentration of IgG, in black adults was statistically (Table 3). However, the Gm(23) allotype, frequent in whites, is significant (p = 0.02). IgG, concentrations also tended to be rarely found in African blacks (28). Thus, we believe that the higher in black children than in white children (p= 0.09). IgG2 relation of serum IgG2 concentrations with Gm(23) allotype in concentrations were again found to be significantly higher in our North American blacks reflects the influence of racial ad- black adults (p = 0.04). In addition, a significant interaction mixture rather than an allotype effect, since black race itself was ORMAP. CHILDREN 8441

associated with higher IgCz concentrations than white race, and 8. Riesen WF, Skvaril F, Braun DG 1976 Natural infection of man with group A streptococci. Scand J lrnmunol 5:383-390 Gm(23)was not associated with altered IgCz concentrations in 9. Perlmutter R, Hansburg D, Briles D, Nicolotti R, Davie JM 1978 Subclass whites. However, another posibility is that genes associated with restriction of murine anticarbohydrate antibodies. J Immunol 121 :566-5711 the Gm(23)locus are interacting with other gene products present 80. Slack J, DerBalian 6,Nahm M, Davie JM 1980 Subclass restriction of murine in blacks, but not whites, and affecting IgG2 levels. For example, antibodies. 11. The IgG plaque forming cell response to thymus independent type 1 ant1 type 2 antigens in normal mice and mlce expressing an x-linked there are reports of interactive eKecrs of allotype and major immunodeficiency. J Exp Med 151~853-862 histocompatibility loci on immune responses (29-311, and in I I. Morell A, SltvaPil F, Mitzig WH, Barandum S 1972 9gG subclasses: Develop- humans, the frequency of HEA specificities differsamong races. ment ofthe serum concentrations in "normal" infants and children. J Pedialr In this study, we found that black children who were Km(I) 80:960-964 12. Schur PH, Rosen F, Norman ME 1979 immunologic subclasses in normzil positive had significantly higher concentrations of IgGz than children. Pediatr Rcs 3 3:181-183 black children who lacked this allotype (Table 3). Antibody 13. Van Der Giessen M, Rossouw E, Algra-Van Vcen T, Loghem E Van, Zegcrs against the polysaccharide capsule of Wib is protective against BJM, Sander PC 1975 Quantification of IgG subclasses in sera of norrn;il invasive disease (14- 16). Further, antibodies to many other adults and healthy children between 4 and 12 years of age. Clin Exp Imrnunol 21:501-509 polysaccharide antigens are reported to be relatively restricted to 14. Anderson P, Johnston RB Jr, Smith DH 1972 Human serum activities against the IgG2 subclass (7, 8). Recent studies suggest that genes asso- Naemophrliis injluenzae type b. J Clin Invest 51:31-44 ciated with the Km(l)immunoglobulin allotype may influence 15. Schneerson R, Rodrigues LP, Parke JC Jr, Robbins JB 1971 Immunity to the antibody response to vaccines containing bacterial palysac- disease caused by Maemophiius inj7uenzae type b. 11. Specificity and some chabides ( 17- 19). For example, black children with the Km( 1)- biologic characteristics of "natural," infection-acquired, and irnmunizationr- induced antibodies to the capsular polysaccharide ofNaemophi1ir.s influc,nn;ae positive allotype have increased antibody responses to Hib pol- tys b. J Immunol l07:1081-1089 ysaccharide-pertussis vaccine compared with those ofblacks who 16. Peltola PI, Icoylrty H, Sironen A, MBkclP PH 1977 Ifaemophiius influenz~re lack this allotype (17). More recently, blacks with Km(l) also type b capsular polysaccharide vaccine in children: A double-blind field urere found to have higher antibody respo~lsesto the purified study of 100,000 vaccinees three months to five years of age in Finland. Pediatrics 60:730-737 type b polysaccharide (32). In addition, Km(l)-positive black 17. Granoff DM, Pandey JP, Boies E, Squires J, Munson RS Jr, Suarez B 19E,4 children had a lower relative risk of Waemophilus meningitis Response to immunization with Haemophilli.~tnj7ilenzue type b polysaccha- than blacks who lack this allotype (14). Thus, in blacks, high nde pertussis vaccine and risk of Huemophiiirs meningitis in children wil h responsiveness to Hib vaccines and low susceptibility to Hib the Km( I) immunoglobulin allotype. J Clin Invest 74: 1708-1 7 14 18. Pandey JP, Fundenberg HH, Virella G, Kyong CU, Loadholt CB, Galbrai1.h disease appear to be related to genes associated with the Mm(1) RM, Gobschliclm EC, Parke JC Jr 1979 Association between immunoglobulin locus. The present data suggest that these genes in turn may be allotypes and immune responxs to I~aemopliilusinfluenzae and meningo- associated with high concentrations ofthe IgGz immunoglobulin coccus polysaccharides. Lancct 1: 190-1 92 subclass. 19. Ambrosino DM, Siber GR, Loghe E van, Schiffman G 198 1 Increased antibody In conclusion, IgC subclass concentrations in children are response to immunization with Harmophiizrs influcwzae type b and pneu- mococcal capsular polysaccharide vaccines in adults with G2M(n) allotype. influenced by many factors, the most important of which is age. Pediatr Res 16:234A(abstr) It appears that race and the KmjS) in~munogiobuPinallotype 20. Scott MG, IVahm MH 1984 Mitogen induced human IgG subclass expression. also may affect serum concentrations of the late maturing IgG, J lmmunol 133:2454-2460, 1984, subclass. These data may explain, in part, recent observations of 2 1. Hunter WM, Greenwood FC 1962 Preparation of iodine- 13 1 labeled human growth hormone of high specific gravity. Nature 194:495-496 the asssociation of Km(1) immunoglobulin allotypes with differ- 22. Morrell A, Skvaril F, Steinberg AG, Loghem E van, Terry WD 1972 Correla- ent immune responses to vaccines containing poiysaccharides. tions between the concentration of the four subclasses of IgG and Gm Our findings emphasize the need to control for factors such as allotypes in normal human sera. J lmmunol 108:195-206 race, sex, and immunoglobulin allotype in future studies assess- 23. Vyas GN, Fudenberg HH, Pretty HM, Gold ER 1968 A new rapid method fi~r genetic typing of human imunoglobulins. 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