Immunologic Parameters in Down s Syndrome

Transcription

1 ANNALS OF CLINICAL AND LABORATORY SCIENCE, Vol. 8, No. 1 Copyright 1978, Institute for Clinical Science Immunologic Parameters in Down s Syndrome PATRICIA F. JACOBS, M.S., NICHOLAS M. BURDASH, Ph.D.,* JOHN P. MANOS, M.D., and ROBERT C. DUNCAN, Ph.D. Department o f Basic and Clinical Immunology and Microbiology, Department o f Laboratory Medicine and Department o f Biometry, Medical University o f South Carolina, Charleston, SC ABSTRACT Down s syndrome children are known to have increased susceptibility to respiratory infections. Quantitative or qualitative differences in the various com ponents of the im m une system could account for increased susceptibility to infection involving the upper respiratory tract. In an effort to establish certain normal values and to determ ine if humoral immune abnormalities are associated with the chromosomal anomalies of Down s syndrome, imm unoglobulin levels, certain com plem ent com ponent levels, viral antibodies, hepatitis B surface antigen and milk precipitins from a population of inpatients and outpatients were compared with those of age, sex and race m atched control populations. It does not appear that the upper respiratory infections are associated with abnormally low levels of immunoglobulins or complement, with the possible exception of IgM. Both the inpatient and outpatient D ow n s syndrome populations had decreased levels of IgM, indicating a possible relationship with the syndrome itself. In addition, the symptomatology does not seem to be due to IgE m ediated atopic sensitivity. Hepatitis B surface antigen was found only in institutionalized Down s syndrome patients, but it did not seem to be related to the other immune components studied. Introduction Children with Down s syndrome (DS) are known to have an increased incidence of u pper respiratory infections and a sig n ifican t m ortality rate ow ing to pneumonia. It has been postulated that these infections may be associated w ith * Reprint requests: Department of Laboratory Medicine, Medical University of South Carolina, Charleston, SC an immunodeficiency or immunoincompetency. To determine w hether or not a hum oral im m unodeficiency exists, DS serum immunoglobulins G, M and A have been quantitated by many investigators w ith som ew hat conflicting re s u lts 1,5, 8,9,10,17,20,23,25 suggesting that more precisely selected DS and control populations should provide data for more definitive information. Alternatively, few studies have b een carried out in w hich 17

2 18 JACOBS, BURDASH, MANOS AND DUNCAN im m unoglobulins D and E and com plem ent com ponents 3 and 4 have b een quantitated. Evaluation of the humoral response in terms of residual antibodies to viral agents commonly responsible for upper respiratory infections is necessary to ascertain if a particular agent or agents are responsible for increased infections. Antibody titers to these agents could also disclose any tre n d tow ard im m unoincom petency in this population. The present study compares immunoglobulin levels, com plem ent levels and antibodies to certain respiratory agents in DS patients with age, sex and race m atched control populations. It also compares the incidence of the hepatitis B surface antigen (HBsAg) and milk precipitins in the same populations since DS patients are reported to have a high incidence of both. Materials and Methods Sera were obtained from 26 karyotyped DS patients ranging in age from 8 to 35 years and 26 non-down s patients with no known chrom osomal abnorm alities from the South Carolina Coastal Center in Ladson, SC. Sera were also obtained from 20 karyotyped DS children from one to nine years of age, who w ere outpatients of the Vince Moseley Clinic in Charleston, SC and from 20 children without known chromosomal abnormalities, some of whom had known atopic disease. Each DS p atien t had an age, race and sex m atched control. Im m unoglobulins G, M and A and complement components 3 and 4 were quantitated by radial immunodiffusion u tiliz in g th e M ancini e n d p o in t technique*.12 Im m unoglobulin D was measured by the Fahey technique!.6 IgE determ inations w ere m ade by radioim munoassay. All of the im m unoglobulin * Behring Tri-Partigen and M-Partigen plates. I Hyland Immuno Plates. I Pharmacia Phadebas Radioimmunoassay Kit and com plem ent determ inations were run in duplicate against known standards, and the m ean value of the two determ inations recorded. Duplicate samples with differences exceeding 10 percent of their mean value were repeated. Milk precipitin reactions were carried out using the O uchterlony double diffusion in agar technique. Serum samples were reacted with commercially obtained cow s milk and observed daily for 7 days for precipitation lines. H B sag determ i nations were carried out using a modified gel diffusion technique. Serum samples were reacted with anti-hbs to detect the presence of the H B sag. Plates w ere observed daily for 7 days for precipitation lines. The plates for both determinations were stained with 7.5 percent acetic acid to insure visibility of all precipitation lines. Antibody titrations were performed on the follow ing antigensll com m only associated with respiratory infections in pediatric populations: adenovirus, influenza A and B, parainfluenza 1, 2 and 3, resp irato ry syncytial, reovirus and M ycoplasm a pneu m o n ia e and to cytomegalovirus. All antibody determ i nations were done using the Laboratory Branch C om plem ent Fixation M ethod (LBCF).15 Sera w ere diluted from 1:8 through 1:512 and retitrated using higher dilutions when necessary. A titer equal to or greater than 1:8 was considered seropositive. The Paired Student t test was used to d etect differences in m ean values b e tween patient and control groups for all quantitative m easurem ents since each patient was m atched w ith his own control. The Chi Square test was used to find differences in the frequency of response to the respiratory agents and to milk. It Abbott Austect Rheophoresis. Quantitation of HBsAg by RIA was not done owing to insufficient serum volume. I Microbiological Associates.

3 IMM UNOLOGIC PARAM ETERS IN DOW N 'S SYNDROME 19 TABLE I Serum Immunoglobulin Levels of Down's Syndrome and Control Populations (Based on 26 Inpatients and 20 Outpatients in Each Population) Im m unoglobulins i n / O ut P t s. Down ' s C o n tr o l S ig n if ic a n c e Range Mean SE* Range Mean SE L e v e l IgA (mg/dl) IgM (mg/dl) IgG (mg/dl) IgD (mg/dl) IgE (IU/ml) In NSt Out NS In P < 0.01 Out P < In P < Out NS In P < Out NS In NS Out P < Standard error. tnot significant. was also used to find differences in the frequency of the presence of the HBsAg. Results In table I are shown the mean immunoglobulin levels for the DS patients and their respective controls. The DS inpatients were found to have IgM levels that w ere significantly lower than their controls; however, IgG and IgD levels were, significantly higher than their control group. No significant difference was found betw een these groups for levels of IgA or IgE. The outpatients with DS were also found to have IgM levels that w ere significantly lower than their control group. DS outpatient IgE levels w ere also significantly lower than their controls which included atopic individuals. Levels for IgA, IgG and IgD in DS outpatients did not differ significantly from their control group. The mean C3 and C4 levels for the DS patients and their matched controls can be seen in table II. DS inpatients were found to have C3 levels that were significantly lower than the control patients, while no difference was found betw een inpatient groups for levels of C4. No significant difference was found betw een the outpatient groups for levels of C3 and C4. TABLE I I Serum Complement Levels of Down s Syndrome and Control Populations (Based on 26 Inpatients and 20 Outpatients in Each Population) C om plem ent C om ponent I n / O ut P t s. Down 's C o n tr o l S i g n i f i c a n c e Range Mean SE* R ange Mean SE L e v e l C3 (mg/dl) In P < 0.05 Out NSt In NS Out NS *Standard error. tnot significant.

4 2 0 JACOBS, BURDASH, MANOS AND DUNCAN The HBsAg was detected in 15.4 percent of the DS inpatient population, while none was found among the matched controls. The HBsAg was not found in any of the DS outpatients or their control group. The incidence of milk precipitins found in the DS inpatients and outpatients was 11.5 percent and 5.0 percent, respectively, w hile both control populations were negative. W hen the seropositivity o f the DS patients and the control patients to common respiratory agents and cytomegalovirus was compared, the total control group was seropositive to the parainfluenza 3 virus more often (p < 0.05)than the DS group (39 of 46 compared with 28 of 44, res pecti vely). Respiratory syncytial virus antibodies in the DS inpatients were found significantly more often (p < 0.05) than in the control inpatients (12 of 25 compared with 4 of 26, respectively). No significant differences were found betw een the groups for any of the other infectious agents. Also, within the four groups, levels of antibody titers to the various agents were not significantly different. Discussion It does not appear that the upper respiratory symptoms so common in DS patients are due to extremely low levels of immunoglobulins, C3 or C4, the inability to respond to certain viral infections or immediate hypersensitivity. However, IgM was decreased for both the inpatient and outpatient DS populations indicating a possible relationship with the syndrome itself. This deficiency could be due to several possibilities including decreased production or half life of the /x chains necessary for IgM synthesis. It is not thought that the IgM produced is defective since these individuals are know n to exhibit normal isoagglutinins16 and to produce a primary hum oral response to antigenic stimulation,7,19 both of which are primarily of the IgM class. The increased levels of IgG found in the institutionalized DS patients support the findings of Sutnick etal,23 who suggestedthatthis increase is a response to persistent infection common to DS institutionalized populations. This increase may also be a compensation for the low levels of IgM as proposed by Stiehm and Fudenberg.20 Serum levels of IgA were found to be norm al in b oth DS populations. H ow ever, since these children have chronic resp irato ry infections, secretory IgA levels could be of more significance. This form of the antibody is one of the primary means of defense in the respiratory and gastrointestinal tracts. Increased levels of IgD w ere found in the in p atien t DS group which is in agreem ent with the work of Rundle et al.18 Since little is known about IgD, and its role in the body s defense has not been firmly established, the significance of this increase remains unknown at present. It does not seem probable that IgE synth esis is d ire c tly re la te d to the chrom osom al ab n o rm ality since IgE levels were the same or lower than the control populations suggesting that the upper respiratory symptoms were not the result of immediate hypersensitivity. The mean IgE level for the inpatients was not significantly low er than the controls; however, only one DS inpatient had an IgE level greater than 800 IU per ml while the control group had six individuals exceeding this level. If this one DS patient is excluded,14 the mean value is reduced to IU per ml which is significantly lower than the control group (p > 0.05). T hese findings are in agreem ent with those of Lopez11 in his study on an institutionalized DS population. All of the im m unoglobulins measured, except IgD in the control group, were h ig h er in the in p a tie n t p o p u latio n s further supporting the theory of Sutnick et al,23 of increased immune responses in in stitu tio n a liz e d DS p o p u latio n s owing to persistent infections.

5 IMM UNOLOGIC PARAM ETERS IN DOW N S SYNDROME 21 Com plem ent should be assessed on patients w ith recurrent infections, especially if antibodies appear to be normal. C3, which is the com plem ent component present in largest amounts in the serum and generally reflects the total com plem ent activity, is often decreased in imm une com plex and autoim m une diseases. C4 is p re se n t in low er concentrations than C3 and may be used as a more sensitive indicator of complement consumption. C4 is also involved in the classical antigen-antibody activation of the com plem ent pathw ay b u t not the p ro p erd in or a lte rn a te co m plem ent pathway. In certain disease states, depression of C4 concentration may precede and is often more m arked than depression C3. Agarwal et al,2 did not find levels of C3 and C4 in DS inpatients to be significantly different from healthy controls. In the present study C4 levels did not differ significantly from control populations. Although decreased C3 levels were found in the DS inpatients which were significantly different from their controls, these data do not seem to be of critical clinical significance since these levels of C3 approximate the lower limits of the normal range. In this investigation the mean C3 level of the DS inpatients was 79.6 mg per dl while that of Agarwal s was 74.3 mg per dl. It does not seem, therefore, that these two major components of the com plem ent system are quantitatively involved in the increased susceptibility of DS individuals to upper respiratory infections. The question as to functional capability of com plem ent remains unanswered owing to our inability to freeze the sera before the complement was inactivated. Precipitating serum antibodies to milk are found in one to two percent of the general population.4 Children with these antibodies generally exhibit several clinical features including chronic respiratory disease. McCrea et al13 found these antibodies in 34 percent of DS individuals. In the present study, milk precipitins were found in 8.7 percent of the DS patients which is somewhat lower than that reported by M ccrea et al;13 how ever, these data do support the findings that there is an increased incidence of this antibody in the DS population. Buckley and Dees4 reported a correlation between serum IgA deficiency and an increased incidence of milk precipitins. Serum IgA levels in DS individuals displaying these antibodies, how ever, w ere all in the upper range of the accepted normal limits for the appropriate age of the patients indicating no correlation in the DS patients betw een IgA deficiency and the presence of milk precipitins. The increased frequency of the HBsAg found in the DS inpatients (15.4 percent) can b e st b e ex p lain ed by the h y p o th esis3,21,22 24 th a t in d iv id u als homozygous for the allele Au(l) are more susceptible to the H B sag w hile those homozygous for the alternate allele Au and heterozygotes are less or transiently susceptible to this antigen. Since the frequency of finding Au(l) is very low in normal individuals and relatively high in certain types of leukemia, Blumberg et al3 have suggested that individuals with this trait may be m ore susceptible to leukem ia than those without it. Approximately 10 to 30 percent of DS inpatients exhibit this antigen in their serum, and there have been many reported cases of DS children developing leukem ia.3,21 22,28 In this study, the fact that increased frequencies of this antigen were found only in inpatients is probably due to the increased risk o f exposure in large institutions. Since, w ithin the four populations studied, seropositivity to the various respiratory agents was not significantly different, this decreases the likelihood that a single virus or group of viruses is responsible for the increased respiratory infections m ost often seen in DS children.

6 2 2 JACOBS, BURDASH, MANOS AND DUNCAN H ow ever, co n tin u in g studies on cell m ediated immunity in these populations may shed further light on the relationship of viral infections to DS. Acknowledgments This work was supported by the South Carolina State Appropriation for Research HR524. References 1. A d in o lfi, M., G a rd n e r, B., and M a rtin, W.: Observations on the levels of yg, ya, and ym globulins, anti-a and anti-b agglutinins, and antibodies to Escherichia coli in Down s anomaly. J. Clin. Path. 20: , Ag a rw a l, D. P., Srivastava, L. M., Ben k m a n n, H. G., and G o e d d e, H. W.: Studies on the polymorphism ofc3, Tfand Bg in D ow n s syndrome and other diseases. Springer-Verlag 29:23-28, Bl u m b e r g, B. S., G e r s t l e y, B. J. S., Hun gerfo rd, D. A., London, W. T., and SUTNICK, A. I.: A serum antigen (Australia antigen) in Down s syndrome, leukemia, and hepatitis. Ann. Intern. Med. 66: , Buckley, R. H. and D ees, S. C.: Correlation of milk precipitins with IgA deficiency. New Eng. J. Med. 281: , D yggve, H. and Clausen, J.: The serum immunoglobulin level in Down s syndrome. Develop. Med. Child Neurol. 72: , F a h e y, J. L. and McKELVEY, E. M.: Quantitative determination of serum immunoglobulins in antibody-agar plates. J. Immunol. 94:84-90, G ordon, M. C., Sinha, S. K., and Carlson, S. D.: Antibody responses to influenza vaccine in patients w ith Down s syndrome. Amer. J. Ment. Def. 75: , Greene, E. L., Shenker, I. R., and Karelitz, S.: Serum protein fractions in patients with Down s syndrome (mongolism). Amer. J. Dis. Child. 215: , G r if f it h s, A. W., Sy l v est er, P. E., and BayliS, E. M.: Serum globulins and infection in mongolism. J. Clin. Path. 22:76-78, H ayashi, H. and LOGRIPPO, G. A.: Humoral immune status of mongoloid children compared with other congenital defects: Quantitative and qualitative aspects of immunoglobulins. Health Lab Science 9: , L o p e z, V. I.: Serum IgE concentration in trisomy 21. J. Ment. Defic. Res. 28: , Mancini, G., Carbonara, A. O., and H e r e- MANS, J. F.: Immunochemical quantitation of antigens by single radial immunodiffusion. Immunochemistry 2 : , M c C r e a, M. G., H e s t o n, J. F., W o o d, H. F., a n d S u l l i v a n, J. E.: M ilk precipitins. J. Amer. M ed. Assoc. 203: , N a t i o n a l B u r e a u o f S t a n d a r d s H a n d b o o k 91: Experimental statistics. Washington, D. C., U.S. Department of Commerce, N a t i o n a l C o m m u n ic a b le D is e a s e C e n t e r : Standardized diagnostic com plem ent fixation method and adaptation to micro test. Atlanta, GA, U.S. Department of Health, Education, and Welfare, Rit t n e r, C. and SCHWINGER, E.: Studies in Down s syndrome: II. Association studies with blood, serum and enzyme groups, and with the Au/SH antigen. Clin. Genet. 4: , R o s n e r, F., K o z in n, P. J., and J e r v i s, G. A.: Leukocyte function and serum immunoglobulins in Down s syndrome. N. Y. State J. Med. 73: , R u n d l e, A. T., C l o t h i e r, B., and S u d e l l, B.: Serum IgD levels and infections in Down s syndrome. Clin. Chim. Acta 35: , SlEGEL, M.: Susceptibility of mongoloids to infection: II. Antibody response to tetanus toxoid and typhoid vaccine. Amer. J. Hyg. 48:63-73, STIEHM, E. R. and F udenberg, H. H.: Serum levels of immune globulins in health and disease: A survey. Pediatrics 37: , SUTNICK, A. I.: Australia antigen and the immune response in human diseases. J. Allergy Clin. Immunol. 53:41-51, Sutnick, A. I., L ondon, W. T., Gerstley, B. J. S., C ronlund, M. M., and Blum berg, B. S.: Anicteric hepatitis associated with Australia antigen. J. Amer. Med. Assoc. 205:80-84, S u t n i c k, A. I., L o n d o n, W. T., and B lu m b e r g, B. S.: Effects of host and environment on immunoglobulins in Down s syndrome. Arch. Intern. Med. 124: , Sutnick, A. I., L ondon, W. T., Blum berg, B. S., and Gerstley, B. J. S.: Susceptibility to leukemia: Immunologic factors in Down s syndrome. J. Nat. Cancer Inst. 47: , Y o k o y a m a, M., B a l l, C., L o u, K., and A le p a, F. P.: Immunogenetic studies on mongolism. Amer. J. Ment. Def. 72 : , 1967.