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Correlation of Gm allotype with the incidence of anti-IgG antibody in coal workers' pneumoconiosis
CLINICAL
lMMUNOLOGY
AND
IMMUNOPATHOLOGY
17, 274-279 (1980)
Correlation of Gm Allotype with the incidence of Anti-IgG Antibody in Coal Workers’ Pneumoconiosisl M. SMENTNECH, Division
D.J. PEARSON,~ J.A. ELLIOTT, P. C. MAJOR
G.TAYLOR,
of Respiratory Disease Studies, Laboratory Investigations Branch, Occupational Safety and Health, Morgantown, West Virginia
National 26505
AND Institute
for
Received January 7, 1980 Three hundred and fifty-four sera from a population at high risk of developing anti-IgG were examined for the presence of this antibody type and for Gm allotypic markers. Significant differences in the incidence of anti-IgG were noted between Gm phenotypes. The data suggest that the risk of anti-IgG development is related to Gm homozygosity rather that the possession of any single common Gm haplotype.
INTRODUCTION
There is now considerable evidence to support the existence of immune response (Ir) genes controlling immunologic reactivity in both animals and man. The majority of work on this subject has concentrated on Ir genes linked to the major histocompatability complex (MHC) of transplantation antigens and a number of human diseases of presumed immunologic etiology have shown significant HLA antigen linkage (1). However, in mice Ir loci unrelated to the MHC are well recognized. In man, X-linked forms of immunodeficiency (2), sex differences in antibody responses to immunization (3), and incidences of autoantibody formation (4) demonstrate the importance of non-MHC-linked genetic factors in immune responsiveness. Since antibody specificity and the biological activity of immunoglobulins are determined by amino acid sequence, an intluence of genes controlling immunoglobulin structure on immunologic reactivity might be expected. In mice antibody responses to dextrans have been linked to distinct immunoglobulin allotypes (5). In man there is also some evidence to suggest that antibody responses are influenced by immunoglobulin allotype. IgG phenotype Gm [ 1;21] is associated with a high antibody response to Salmoneflu adeluide flagellin whereas phenotype Gm [3;5] is associated with a low response to flagellin (6). One follow-up study also showed an increased 6-year survival in Gm [ 1;21]-positive subjects compared to Gm [1;21]-negative subjects (7). In addition Gm [3;5] has been associated with an increased risk of autoimmune thyroid disease (8). Antibodies which agglutinate red cells coated with undenatured human IgG are commonly associated with rheumatoid factor (RF) in conditions such as rheumatoid arthritis but are rare in healthy adults (9). Autoantibodies including I Mention of company names or products does not constitute endorsement by the National Institute for Occupational Safety and Health. * Present Address: Department of Medicine, University Hospital of South Manchester, Manchester M20 8LB, United Kingdom. 274 oo90-1229/80/100274-06$01.00/0
275
Gm AND ANTI-IgG
both RF and non-RF IgG-agglutinating antibodies are found in increased incidence in the absence of connective tissue disease in persons occupationally exposed to various inorganic dusts (10, 11). We therefore examined the association of antiIgG antibodies with Gm allotypes in a group of coal miners, a population known to have a high incidence of such antibodies (unpublished, Pearson et al. 1978). SUBJECTS AND METHODS
Subjecrs. Serum was examined from 354 coal miners, who had been examined and classified by pneumoconiosis status during the U.S. National Coal Study and who volunteered to provide a subsequent blood sample. Caucasian volunteers were selected for inclusion in the study so that subjects with normal chest X rays and subjects with simple or with complicated forms of pneumoconiosis formed three groups of approximately equal size which were matched for age and years of experience in underground mining. All subjects worked in the Appalachian coal area of West Virginia and Pennsylvania. Methods used in the U.S. National Coal Study have been described fully elsewhere (12, 13). Gm typing. The hemagglutination inhibition microtiter technique (14) was used to detect Glm (l-3, 17) and G3m (5, 11, 21) antigens. The reagents are listed in Table 1. Specificity of anti-Gm and anti-Rh coating sera pairs was confirmed using a WHO reference serum panel. Sera causing agglutination of coated red cells were retyped after heating at 65°C. Two sera, in which agglutinating activity was not abolished by heat treatment, were retyped after absorption with coated cells. Anti-ZgG antibodies. Antibodies reactive with native human IgG were detected using human group 0 rhesus DCE/DCE red cells coated with the same human anti-D sera used for Gm typing. The specificity of anti-IgG containing sera was examined by looking for hemagglutination inhibition by a panel of reference sera of known Gm types. All sera were initially screened for anti-IgG activity at a 1 in 30 dilution and none caused agglutination of uncoated human group 0 rhesus DCE/DCE erythrocytes. TABLE 1 Gm ALLOTYPE REAGENT@ Allotype notation
Agglutinator (Anti-Gm)
RBC coat (Anti-D Gm)
Glm 1 2 3 17
Pank Max Staley Pon
Dwinelle Peters Daniels Dwinelle
G3m 5 11 21
Toll’ Toll’ Leh
Hunter Hunter Sullivan
a WHO designations (15). * Obtained Milwaukee Blood Center, Inc. I’ Slitable by dilution.
276
MENTNECH
ET
AL.
Rheumatoid factor. Classical rheumatoid factor (RF) was detected by the latex agglutination technique. A titer of 1 in 80 was accepted as positive. Data were evaluated using x2 contingency testing. Reported P values reflect independent comparisons, all having been preselected before analysis. Relative risk was calculated using the equation (16)
relative
risk = e
PI
,
where LI represents individuals with a trait but negative for antibody and b represents individuals negative for trait in a and also negative for antibody; c represents individuals with trait in a and positive for antibody, and d represents individuals negative for trait in a, but positive for antibody. These analyses were conducted on the following groupings: allotypes, haplotypes, homozygosites, and heterozygosites. RESULTS
The frequency of Gm phenotypes in the three groups of coal miners divided according to presence and category of pneumoconiosis is shown in Table 2. By x2 analysis no significant differences were observed in the frequency of Gm phenotypes or in the frequency of any single Gm type between the three groups. Although antibodies reactive with native human IgG were significantly more prevalent (P < 0.05) in simple (28%) and complicated pneumoconiosis (26%) groups than in the normal control miner group (12%) the antibody-positive individuals within each group were evenly distributed according to Gm phenotype (P > 0.10). Therefore the three coal workers’ pneumoconiosis category groups were combined for the purposes of further analysis with regard to anti-immunoglobulin antibody. The specificity of the anti-immunoglobulin antibodies in sera causing agglutination of IgG-coated erythrocytes was examined in hemagglutination-inhibition experiments. By this means only 3 of 75 anti-IgG-positive sera could be demonstrated to have specificity for a single Gm allotype. Of the other 72 sera, 26 contained classical rheumatoid factor as detected in the latex agglutination text. The remainder either reacted with multiple-coating anti-sera, did not inhibit with normal sera, or gave complex inhibition patterns. We did not feel that it was TABLE
Cm PHENOTYPES
SEGREGATED
BY COAL
2
WORKERS’
PNEUMOCONIOSIS
(CWP)
STATUS
Gm phenotype Classification NOrId Simple CWP” PMF’
(3;5,11) 65 54 57
(1,17;21) 5
1 8
(1,2,17;21) 2 7 2
Total 0 Classification by chest X ray and other related data (12). b Simple pneumoconiosis. c Progressive massive fibrosis or complicated pneumoconiosis.
(1,3,17;5,11,21) 41 38 33
(1,2,3,17;5,11,12) 17 8 16 354
Gm AND
277
ANTI-IgG
possible, with confidence, to distinguish in the third group between multiple anti-am specificities, antibodies specific for as yet unrecognized Gm allotypes, and antibodies directed at other than Gm immunoglobulin antigenic specificities. As far as group sizes would allow analysis, there did not appear to be any significant differences between anti-IgG positive sera of each Gm allotype, in the relative frequencies of rheumatoid factor, or any other recognizable subset of hemagglutinating antibodies. Therefore, in further analyses all antibodies causing agglutination of native IgG-coated erythrocytes were considered as one group. The distribution of Gm phenotypes in anti-immunoglobulin antibody-positive and -negative subjects is shown in Table 3. Subjects with the phenotype Gm [3;5,11] had an incidence of anti-immunoglobulin antibody of 26%, significantly higher than that in the rest of the study population combined (P < 0.05). The lowest antibody incidence of 7% was observed in Gm [1,2,3,17;5,11,21] subjects (P < 0.025). When the presence or absence of individual Gm allotypes are considered Glm (l), Glm (17), and G3m (21) appear to be associated with a reduced frequency of anti-immunoglobulin (P < 0.05). The antibody incidence between Gm1J7;21 and Gm1,2*17;21is not statistically significant. The relative risk for the presence of anti-immunoglobulin is 0.59 for all Glm (1)-positive subjects. The differences in incidences of anti-immunoglobulin between Gm3G5,11-positive and -negative subjects are not statistically significant and also, surprisingly, the relative risk for antibody in all Gm3;j,” subjects is also less than 1, at 0.84. These data strongly suggest that some factor other than the simple presence or absence of a single haplotype is responsible for the differences observed in the incidences of antibody in the phenotype groups. If individuals who may be presumed homozygous for one of the two most common Gm haplotypes, Gm3;5,11 and Gm1,17;21, and those who may be presumed heterozygous, Gm1,3*17;5*11*21and Gm1,2,3,17;5J1*21,are compared [with the group about which no such assumption can be made, Gm 1,2,17;21 being excluded], a difference which is significant (P < 0.04) level is observed. The former group has an antibody incidence of 25% and a relative risk of 1.77, the latter, an incidence of 16% and a relative risk of 0.57. It seems unlikely that the lower incidence of anti-immunoglobulin antibody in heterozygotes is due to the presence of Glm (1) or Glm (17), since the incidence of anti-immunoglobulin in all Gm1J7;21 -positive, Gm3i5,11 -negative subjects was 24%.
DISTRIBUTION
OF DETECTED
TABLE ANTIBODIES
3 ACCORDING
TO Cm
PHENOTYPE
Gm phenotype Antibody
(3;5,11)
(1,17;21)
Positive Negative
45 131
2 12
4 7
21 91
3 38
75 279
176
14
11
112
41
354
(1,2,17;21)
(1,3,17;5,11,21)
(1,2,3,17;5,11,21)
Total
278
MENTNECH
ET
AL.
DISCUSSION
The evidence presented here indicates that among a population environmentally predisposed to the development of autoantibodies, the risk of anti-IgG occurrence is related to Gm phenotype. Such an association is subject to a number of possible explanations: either a Gm-linked Ir gene or the structure of the Gm region itself may control immunological reactivity. If particular Gm allotypes are associated with a restricted range of antibody specificities, Gm-heterozygotes might be expected to have the capacity to react to a wider range of antigenic determinants. Since Gm-bearing molecules were the antigens in the system studied here, Gm antigens might influence anti-IgG production by their own potential to act as immunogens or tolerogens. Our results demonstrated a high risk of anti-IgG antibodies in phenotype Gm [3;5,1 l] subjects; a significantly lower antibody incidence being observed in subjects possessing Glm [ 11, Glm [ 171, and G3m [21] antigens. This might be taken to imply a linkage between anti-IgG response and Gm haplotype. However, whether protection from anti-IgG production is truly a direct effect of Glm [l], Glm[17], G3m [21], or heterozygosity is not certain. The number of phenotype Gm [ 1,17;21] and Gm [ 1,2,17;21] subjects formed a small proportion of the total study population (4 and 5%, respectively) and Glm [II-positive, Glm [3]-negative subjects were outnumbered by Glm [l], Glm [3]-positive subjects by 5 to 1. While the serum from only 2 of 14 subjects with phenotype Gm [ 1,17;21] contained anti-IgG, if phenotypes Gm [ 1,17;21] and Gm [ 1,2,17;21] are considered homozygous for Glm [l] and Glm [17] and combined to form a group of more reasonable size the incidence of antibody is similar to that in the presumably homozygous Gm [3;5,1 l] subjects. The observation that the relative risk for the presence of anti-IgG antibody for each Gm haplotype was less than one also seems to support the view that it is Gm-heterozygosity rather than possession of Glm [ 11, Glm [ 171, or G3m [21] itself that is associated with the lower incidence of anti-IgG. In subjects without rheumatoid factor, anti-IgG antibodies are most frequently observed to be directed against immunoglobulins of Gm types not found in that individual’s serum. It seems reasonable to ascribe this to self-tolerance and the development of anti-Gm antibodies to exposure to foreign immunoglobulin or similar determinants on other proteins. Antibodies reactive with IgG of other than the patient’s Gm type have been detected following multiple blood transfusions (17). Non-RF, IgG-agglutinating antibodies are more common in children than adults and probably follow transplacental transfer of IgG, since they are usually directed against maternal Gm types (18). Maternal antibody production may also result from matemofetal Gm incompatability (19). All our subjects were male and only five antibody-positive subjects gave a history of blood transfusion. In only three sera could antibodies against a single Gm specificity be identified, and in each case this was directed against an antigen not present on the subjects’ own IgG. The concept of self-tolerance might be used to explain the lower incidence of anti-IgG antibodies observed in presumably heterozygous subjects observed in our series. One would expect that individuals heterozygous for Gm haplotypes
Gm AND ANTI-IgG
279
would be tolerant of more Gm antigens than homozygotes, and, it would be reasonable to assume, to other allotypic determinants even though the identity of the latter cannot be determined using available antisera. However, the observation that the relative frequency of subsets of anti-IgG antibody, as identified by hemagglutination-inhibition experiments, did not differ between Gm groups, would militate against the concept that “self-tolerance” alone is responsible for the lower frequency of anti-IgG in Gm-heterozygous subjects. Also, as stated earlier, the number of sera with readily identifiable anti-Gm specificities was very low and over one-third of all sera with anti-IgG antibodies contained activity against denatured IgG (rheumatoid factor). In conclusion, we emphasize that the evidence presented may suggest an influence of Gm allotype or a Gm-linked Ir gene on anti-IgG response. An alternative, and more probable interpretation, links anti-IgG antibody production to Gmhomozygosity/heterozygosity. However, the mechanisms involved in these phenomena remain obscure. Our data, none-the-less, support a correlation of Gm allotype with the incidence of IgG antibody in coal workers’ pneumoconiosis. ACKNOWLEDGMENT We wish to thank Debra Spurgeon for her secretarial assistance.
REFERENCES 1. Dausset, J., In “HLA and Disease” (J. Dausset and A. Svegarrd, Eds.), pp. 46-71, Williams & Wilkins, Baltimore, 1977. 2. Burton, 0. C., Pediatrics 9, 722, 1972. 3. Edsall, G., Belsey, M. A., LeBlanc, D. R., and Levine, L., Prog. Drug Res. 19, 263, 1975. 4. Hooper, B., Whittingham, S., Mathews, J. D., Mackay, I. R., and Curnow, D. H., C/in. Exp. Immunol. 12, 79. 1972. 5. Blomberg, B., Geckelar, W. R., and Weigert, M., Science 177, 178, 1972. 6. Wells, J. V., Fudenberg, H. H., and Mackay, I. R., J. Immunol. 107, 1505, 1971. 7. Mackay, I. R., Wells, .I. V., and Fudenberg, H. H., Ctin. Immunoi. Immunopathol. 3, 408, 1975. 8. Farid, N. R., Newton, R. M., Noel, E. P., and Marshall, W. H., J. Immunogen. 4, 429, 1977. 9. Grubb, R., “The Genetic Markers of Human Immunoglobulins”, Springer-Verlag, New York, 1970. 10. Soutar, C. A., Turner-Warwick, M., and Parkes, W. R., Brit. Med. .I. 3, 145, 1974. 11. Lippman, M., Eckert, H. L., Hahon, N., and Morgan, W. K. C., Ann. Intern. Med. 79,807,1973. 12. Morgan, W. K. C., Burgess, D. B., Jacobson, G., O’Brien, R. J., Pendergrass, E. P., Reger, R. B., and Shoub, E. P., Arch. Environ. Health 27, 221, 1973. 13. Heise, E. R., Mentnech, M. S., Olenchock, S. A., Kutz, S. A., Morgan, W. K, C., Merchant, J. A., and Major, P. C., Amer. Rev. Resp. Dis. 119, 903, 1979. 14. Schantield, M. S., and Gershowitz, H., Hum. Hered. 21, 168, 1971. 15. WHO meeting on Human Immunoglobulin Allotypic Markers, held July 16-19, 1974, Roven, France. Report amended June 1976, J. Immunogenetics 3, 357, 1976. 16. Heise, E. R., Major, P. C., Mentnech, M.S., Parrish, E. J., Jordon, A. L., and Morgan, W. K. C., In “Inhaled Particles IV” (W. H. Walton, Ed.), pp. 495-508. Pergamon, New York, 1977. 17. Allen, J. C., and Kundel, H. G., Science 139, 418, 1963. 18. Steinberg, A. G., and Wilson, J. A., Science 140, 303, 1963. 19. Fudenberg, H. H., and Fudenberg, B. R., Science 145, 170, 1964.
lMMUNOLOGY
AND
IMMUNOPATHOLOGY
17, 274-279 (1980)
Correlation of Gm Allotype with the incidence of Anti-IgG Antibody in Coal Workers’ Pneumoconiosisl M. SMENTNECH, Division
D.J. PEARSON,~ J.A. ELLIOTT, P. C. MAJOR
G.TAYLOR,
of Respiratory Disease Studies, Laboratory Investigations Branch, Occupational Safety and Health, Morgantown, West Virginia
National 26505
AND Institute
for
Received January 7, 1980 Three hundred and fifty-four sera from a population at high risk of developing anti-IgG were examined for the presence of this antibody type and for Gm allotypic markers. Significant differences in the incidence of anti-IgG were noted between Gm phenotypes. The data suggest that the risk of anti-IgG development is related to Gm homozygosity rather that the possession of any single common Gm haplotype.
INTRODUCTION
There is now considerable evidence to support the existence of immune response (Ir) genes controlling immunologic reactivity in both animals and man. The majority of work on this subject has concentrated on Ir genes linked to the major histocompatability complex (MHC) of transplantation antigens and a number of human diseases of presumed immunologic etiology have shown significant HLA antigen linkage (1). However, in mice Ir loci unrelated to the MHC are well recognized. In man, X-linked forms of immunodeficiency (2), sex differences in antibody responses to immunization (3), and incidences of autoantibody formation (4) demonstrate the importance of non-MHC-linked genetic factors in immune responsiveness. Since antibody specificity and the biological activity of immunoglobulins are determined by amino acid sequence, an intluence of genes controlling immunoglobulin structure on immunologic reactivity might be expected. In mice antibody responses to dextrans have been linked to distinct immunoglobulin allotypes (5). In man there is also some evidence to suggest that antibody responses are influenced by immunoglobulin allotype. IgG phenotype Gm [ 1;21] is associated with a high antibody response to Salmoneflu adeluide flagellin whereas phenotype Gm [3;5] is associated with a low response to flagellin (6). One follow-up study also showed an increased 6-year survival in Gm [ 1;21]-positive subjects compared to Gm [1;21]-negative subjects (7). In addition Gm [3;5] has been associated with an increased risk of autoimmune thyroid disease (8). Antibodies which agglutinate red cells coated with undenatured human IgG are commonly associated with rheumatoid factor (RF) in conditions such as rheumatoid arthritis but are rare in healthy adults (9). Autoantibodies including I Mention of company names or products does not constitute endorsement by the National Institute for Occupational Safety and Health. * Present Address: Department of Medicine, University Hospital of South Manchester, Manchester M20 8LB, United Kingdom. 274 oo90-1229/80/100274-06$01.00/0
275
Gm AND ANTI-IgG
both RF and non-RF IgG-agglutinating antibodies are found in increased incidence in the absence of connective tissue disease in persons occupationally exposed to various inorganic dusts (10, 11). We therefore examined the association of antiIgG antibodies with Gm allotypes in a group of coal miners, a population known to have a high incidence of such antibodies (unpublished, Pearson et al. 1978). SUBJECTS AND METHODS
Subjecrs. Serum was examined from 354 coal miners, who had been examined and classified by pneumoconiosis status during the U.S. National Coal Study and who volunteered to provide a subsequent blood sample. Caucasian volunteers were selected for inclusion in the study so that subjects with normal chest X rays and subjects with simple or with complicated forms of pneumoconiosis formed three groups of approximately equal size which were matched for age and years of experience in underground mining. All subjects worked in the Appalachian coal area of West Virginia and Pennsylvania. Methods used in the U.S. National Coal Study have been described fully elsewhere (12, 13). Gm typing. The hemagglutination inhibition microtiter technique (14) was used to detect Glm (l-3, 17) and G3m (5, 11, 21) antigens. The reagents are listed in Table 1. Specificity of anti-Gm and anti-Rh coating sera pairs was confirmed using a WHO reference serum panel. Sera causing agglutination of coated red cells were retyped after heating at 65°C. Two sera, in which agglutinating activity was not abolished by heat treatment, were retyped after absorption with coated cells. Anti-ZgG antibodies. Antibodies reactive with native human IgG were detected using human group 0 rhesus DCE/DCE red cells coated with the same human anti-D sera used for Gm typing. The specificity of anti-IgG containing sera was examined by looking for hemagglutination inhibition by a panel of reference sera of known Gm types. All sera were initially screened for anti-IgG activity at a 1 in 30 dilution and none caused agglutination of uncoated human group 0 rhesus DCE/DCE erythrocytes. TABLE 1 Gm ALLOTYPE REAGENT@ Allotype notation
Agglutinator (Anti-Gm)
RBC coat (Anti-D Gm)
Glm 1 2 3 17
Pank Max Staley Pon
Dwinelle Peters Daniels Dwinelle
G3m 5 11 21
Toll’ Toll’ Leh
Hunter Hunter Sullivan
a WHO designations (15). * Obtained Milwaukee Blood Center, Inc. I’ Slitable by dilution.
276
MENTNECH
ET
AL.
Rheumatoid factor. Classical rheumatoid factor (RF) was detected by the latex agglutination technique. A titer of 1 in 80 was accepted as positive. Data were evaluated using x2 contingency testing. Reported P values reflect independent comparisons, all having been preselected before analysis. Relative risk was calculated using the equation (16)
relative
risk = e
PI
,
where LI represents individuals with a trait but negative for antibody and b represents individuals negative for trait in a and also negative for antibody; c represents individuals with trait in a and positive for antibody, and d represents individuals negative for trait in a, but positive for antibody. These analyses were conducted on the following groupings: allotypes, haplotypes, homozygosites, and heterozygosites. RESULTS
The frequency of Gm phenotypes in the three groups of coal miners divided according to presence and category of pneumoconiosis is shown in Table 2. By x2 analysis no significant differences were observed in the frequency of Gm phenotypes or in the frequency of any single Gm type between the three groups. Although antibodies reactive with native human IgG were significantly more prevalent (P < 0.05) in simple (28%) and complicated pneumoconiosis (26%) groups than in the normal control miner group (12%) the antibody-positive individuals within each group were evenly distributed according to Gm phenotype (P > 0.10). Therefore the three coal workers’ pneumoconiosis category groups were combined for the purposes of further analysis with regard to anti-immunoglobulin antibody. The specificity of the anti-immunoglobulin antibodies in sera causing agglutination of IgG-coated erythrocytes was examined in hemagglutination-inhibition experiments. By this means only 3 of 75 anti-IgG-positive sera could be demonstrated to have specificity for a single Gm allotype. Of the other 72 sera, 26 contained classical rheumatoid factor as detected in the latex agglutination text. The remainder either reacted with multiple-coating anti-sera, did not inhibit with normal sera, or gave complex inhibition patterns. We did not feel that it was TABLE
Cm PHENOTYPES
SEGREGATED
BY COAL
2
WORKERS’
PNEUMOCONIOSIS
(CWP)
STATUS
Gm phenotype Classification NOrId Simple CWP” PMF’
(3;5,11) 65 54 57
(1,17;21) 5
1 8
(1,2,17;21) 2 7 2
Total 0 Classification by chest X ray and other related data (12). b Simple pneumoconiosis. c Progressive massive fibrosis or complicated pneumoconiosis.
(1,3,17;5,11,21) 41 38 33
(1,2,3,17;5,11,12) 17 8 16 354
Gm AND
277
ANTI-IgG
possible, with confidence, to distinguish in the third group between multiple anti-am specificities, antibodies specific for as yet unrecognized Gm allotypes, and antibodies directed at other than Gm immunoglobulin antigenic specificities. As far as group sizes would allow analysis, there did not appear to be any significant differences between anti-IgG positive sera of each Gm allotype, in the relative frequencies of rheumatoid factor, or any other recognizable subset of hemagglutinating antibodies. Therefore, in further analyses all antibodies causing agglutination of native IgG-coated erythrocytes were considered as one group. The distribution of Gm phenotypes in anti-immunoglobulin antibody-positive and -negative subjects is shown in Table 3. Subjects with the phenotype Gm [3;5,11] had an incidence of anti-immunoglobulin antibody of 26%, significantly higher than that in the rest of the study population combined (P < 0.05). The lowest antibody incidence of 7% was observed in Gm [1,2,3,17;5,11,21] subjects (P < 0.025). When the presence or absence of individual Gm allotypes are considered Glm (l), Glm (17), and G3m (21) appear to be associated with a reduced frequency of anti-immunoglobulin (P < 0.05). The antibody incidence between Gm1J7;21 and Gm1,2*17;21is not statistically significant. The relative risk for the presence of anti-immunoglobulin is 0.59 for all Glm (1)-positive subjects. The differences in incidences of anti-immunoglobulin between Gm3G5,11-positive and -negative subjects are not statistically significant and also, surprisingly, the relative risk for antibody in all Gm3;j,” subjects is also less than 1, at 0.84. These data strongly suggest that some factor other than the simple presence or absence of a single haplotype is responsible for the differences observed in the incidences of antibody in the phenotype groups. If individuals who may be presumed homozygous for one of the two most common Gm haplotypes, Gm3;5,11 and Gm1,17;21, and those who may be presumed heterozygous, Gm1,3*17;5*11*21and Gm1,2,3,17;5J1*21,are compared [with the group about which no such assumption can be made, Gm 1,2,17;21 being excluded], a difference which is significant (P < 0.04) level is observed. The former group has an antibody incidence of 25% and a relative risk of 1.77, the latter, an incidence of 16% and a relative risk of 0.57. It seems unlikely that the lower incidence of anti-immunoglobulin antibody in heterozygotes is due to the presence of Glm (1) or Glm (17), since the incidence of anti-immunoglobulin in all Gm1J7;21 -positive, Gm3i5,11 -negative subjects was 24%.
DISTRIBUTION
OF DETECTED
TABLE ANTIBODIES
3 ACCORDING
TO Cm
PHENOTYPE
Gm phenotype Antibody
(3;5,11)
(1,17;21)
Positive Negative
45 131
2 12
4 7
21 91
3 38
75 279
176
14
11
112
41
354
(1,2,17;21)
(1,3,17;5,11,21)
(1,2,3,17;5,11,21)
Total
278
MENTNECH
ET
AL.
DISCUSSION
The evidence presented here indicates that among a population environmentally predisposed to the development of autoantibodies, the risk of anti-IgG occurrence is related to Gm phenotype. Such an association is subject to a number of possible explanations: either a Gm-linked Ir gene or the structure of the Gm region itself may control immunological reactivity. If particular Gm allotypes are associated with a restricted range of antibody specificities, Gm-heterozygotes might be expected to have the capacity to react to a wider range of antigenic determinants. Since Gm-bearing molecules were the antigens in the system studied here, Gm antigens might influence anti-IgG production by their own potential to act as immunogens or tolerogens. Our results demonstrated a high risk of anti-IgG antibodies in phenotype Gm [3;5,1 l] subjects; a significantly lower antibody incidence being observed in subjects possessing Glm [ 11, Glm [ 171, and G3m [21] antigens. This might be taken to imply a linkage between anti-IgG response and Gm haplotype. However, whether protection from anti-IgG production is truly a direct effect of Glm [l], Glm[17], G3m [21], or heterozygosity is not certain. The number of phenotype Gm [ 1,17;21] and Gm [ 1,2,17;21] subjects formed a small proportion of the total study population (4 and 5%, respectively) and Glm [II-positive, Glm [3]-negative subjects were outnumbered by Glm [l], Glm [3]-positive subjects by 5 to 1. While the serum from only 2 of 14 subjects with phenotype Gm [ 1,17;21] contained anti-IgG, if phenotypes Gm [ 1,17;21] and Gm [ 1,2,17;21] are considered homozygous for Glm [l] and Glm [17] and combined to form a group of more reasonable size the incidence of antibody is similar to that in the presumably homozygous Gm [3;5,1 l] subjects. The observation that the relative risk for the presence of anti-IgG antibody for each Gm haplotype was less than one also seems to support the view that it is Gm-heterozygosity rather than possession of Glm [ 11, Glm [ 171, or G3m [21] itself that is associated with the lower incidence of anti-IgG. In subjects without rheumatoid factor, anti-IgG antibodies are most frequently observed to be directed against immunoglobulins of Gm types not found in that individual’s serum. It seems reasonable to ascribe this to self-tolerance and the development of anti-Gm antibodies to exposure to foreign immunoglobulin or similar determinants on other proteins. Antibodies reactive with IgG of other than the patient’s Gm type have been detected following multiple blood transfusions (17). Non-RF, IgG-agglutinating antibodies are more common in children than adults and probably follow transplacental transfer of IgG, since they are usually directed against maternal Gm types (18). Maternal antibody production may also result from matemofetal Gm incompatability (19). All our subjects were male and only five antibody-positive subjects gave a history of blood transfusion. In only three sera could antibodies against a single Gm specificity be identified, and in each case this was directed against an antigen not present on the subjects’ own IgG. The concept of self-tolerance might be used to explain the lower incidence of anti-IgG antibodies observed in presumably heterozygous subjects observed in our series. One would expect that individuals heterozygous for Gm haplotypes
Gm AND ANTI-IgG
279
would be tolerant of more Gm antigens than homozygotes, and, it would be reasonable to assume, to other allotypic determinants even though the identity of the latter cannot be determined using available antisera. However, the observation that the relative frequency of subsets of anti-IgG antibody, as identified by hemagglutination-inhibition experiments, did not differ between Gm groups, would militate against the concept that “self-tolerance” alone is responsible for the lower frequency of anti-IgG in Gm-heterozygous subjects. Also, as stated earlier, the number of sera with readily identifiable anti-Gm specificities was very low and over one-third of all sera with anti-IgG antibodies contained activity against denatured IgG (rheumatoid factor). In conclusion, we emphasize that the evidence presented may suggest an influence of Gm allotype or a Gm-linked Ir gene on anti-IgG response. An alternative, and more probable interpretation, links anti-IgG antibody production to Gmhomozygosity/heterozygosity. However, the mechanisms involved in these phenomena remain obscure. Our data, none-the-less, support a correlation of Gm allotype with the incidence of IgG antibody in coal workers’ pneumoconiosis. ACKNOWLEDGMENT We wish to thank Debra Spurgeon for her secretarial assistance.
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