Genetic polymorphism of IgG in mink. II. A genetic analysis of allotypes

Population distribution and inheritance pattern were analyzed in mink IgG allotypes: L1 (L chains), H2, H3, H4, H6, H7, and H8 (the constant region of the H chains, i.e. C gamma-allotypes) and conformational allotype 5 with unknown chain localization. Contrary to expectation, neither allelism, nor close linkage were demonstrated for these allotypes. The major feature of the inheritance of H2, H3, and H4 C gamma-allotypes, as well as allotype 5, was significant excess of negative (without these allotypes) progeny in the F1 generation from monohybrid cross. The explanation offered for this departure of the C gamma-allotypes from normal Mendelian genetics suggests widespread latencies of their expression in mink.     

Genetic polymorphism of IgG in the mink. IV. Identification and genetic control of L3 allotype of the light chains

A new allotype of the mink light chains, designated L3, was identified. This allotype is inherited as a Mendelian character at a frequency of 0.46 in the mink population. Data were obtained indicating that L3 is independent of the C gamma-heavy chain allotypes of mink immunoglobulins. The gene L3 is closely linked to a gene encoding L1, another light chain allotype. Alloantigens L1 and L3 are presumably markers of the light chains of two different subtypes. In contrast to L1, which occurs in many mammalian species, L3 is species-specific, i.e., it is a case of light chain polymorphism representative of the whole mink species.     

IgG allotypes of the domestic mink: genetics, expression and evolution.

A brief review summarized the results we obtained with the identification, analysis of population distribution, genetics, expression, and evolution of 12 IgG allotypes in the American mink and several closely related mustelids. The American mink is a unique species with respect to expressed allotypic polymorphism of Ig lambda chains. In contrast to the rabbit, human, mouse and rat, the phenotypic expression of IgC gamma allotypes shows unusual variations which mask their true genetic relationships (linkage of C gamma genes; C gamma = constant region of IgG chains). The allotypic IgG polymorphism in the American mink during mustelid phylogenesis underwent saltatory change. A parallelism between the data on changes in IgG allotype frequencies in man and mink in disease is emphasized. In mink, these changes are provided by allotype-specific activation of the expression of the 2 CH genes (CH = constant region of the Ig heavy chains). The results make apparent the need of including more taxa in investigations of Ig genetics. In addition, the Aleutian disease of the mink is presented as a model of human disease.     

Genetic polymorphism of IgG in the mink. IX. High proportion of allotype-producing lymphocytes in individuals with minor level of allotypes H3 and H4 in serum.

The levels of mink C gamma-allotypes (H3, H4, H6 and H8) were determined in sera, and the proportion of the corresponding allotype-synthesizing B cells was estimated in peripheral blood, spleen and mesenteric lymph nodes. Individual differences in H6 levels and, possibly, those in H8 were entirely dependent on the proliferation degree of the corresponding clone of B cells and also determined by the dosage of the structural gene. There was no correspondence between the great numbers of H3+, H4+ cells and low levels of H3 and H4 allotypes in the sera of the majority of minks with their minor expression. A possible cause of this discrepancy may be a blockade of the secretion of IgG by H3+, H4+ cells. There exists most likely a gene (or genes) controlling the blockade of IgG secretion. The regulation of C gamma-allotype expression is presumably effected in a manner specific to each of the allotypes.     

Genetic polymorphism of IgG in the mink. 7. Expression of the C gamma-allotypes in domestic mink infected with the Aleutian disease virus.

The Aleutian disease (AD), i.e., viral plasmocytosis in mink can be used as a model of the natural development of immune complex pathology in man. The immunogenetic aspect of AD was studied with the help of genetic markers of the constant region of the mink immunoglobulin gamma-heavy chain (the C gamma allotypes H2, H3, H4, H6, H7 and H8). The frequencies of 2 of the 6 allotypes, H3 and H4, were significantly higher in the AD-infected than in normal minks from the same population. This supports and extends the data in the literature indicating that the frequencies of certain human Gm allotypes are significantly higher among patients with multiple sclerosis, some oncological and other diseases compared with normal humans. Individual testing of 110 adult Standard minks before and after artificial inoculation with the AD virus demonstrated that change in allotype frequencies results from the activation of the expression of H3 and/or H4 in many individuals. The obtained results make it possible to consider the regulation of the expression of the two CH genes of immunoglobulins as allotype-specific.     

Constant region IgG allotypes in hares: Group e allelic polymorphism

Alloantisera prepared against domestic rabbit IgG allotypes were used in hemagglutination-inhibition and direct radioimmune binding tests to detect the Cγ region allotypes in hares from several geographical locations. The d11, d12 and e14 allotypes were absent in all the hare samples tested. The hemagglutination-inhibition and the radioimmune binding tests gave differing results in the classification of hares with respect to the e15 allotype. The apparent enigma was resolved by taking into consideration the concept of multiple antigenic determinants comprising the e15 allotypic system. The results are presented and discussed in terms of possible allelic genetic variants of the Cγ gene in hares.     

Genetic polymorphism of IgG in the mink. V. Two new genetic markers of the lambda light chains of mink immunoglobulins, L4 and L5

Two new allotypes of the light (L) chains IgG, L4 and L5, were identified in the mink with dispecific antiserum produced by immunization with allogenic IgG. By means of hybrid IgG molecules and proteolytic fragments, L4 and L5 were localized on the C region of the L chain. L4 and L5 occurred frequently in the three mink populations studied and L4 and L5 are inherited independently of the known mink C gamma allotypes. L4 and L5 are encoded by closely linked genes. The antigenic specificities of L4 and L5 were not identified in the closely related Mustelidae and in the other mammalian representatives. Consequently, L4 and L5 are species specific to mink. Determination of the phenotype combinations of the five allotypes on the L chains (including the new L4 and L5) demonstrated the existence of seven combinations only with a predominance of L1,2,3; L4,5, and L1,2,3,4,5 phenotypes. Based on the results obtained, it is concluded that the mink C lambda locus has a complex organization. A model for the mink C lambda locus with at least three or possibly five linked genes is suggested.     

Constant region IgG allotypes in cottontail rabbits: Group E allelic polymorphism

Alloantisera prepared against domestic rabbit IgG allotypes were used in hemagglutination-inhibition and direct radioimmune binding tests to detect the C_γ region allotypes in cottontail rabbits. The absence of the group d allotypes (d11 and d12) in cottontail rabbits was confirmed. However, allelic variants of the group e markers (e14 and e15) were found in some of the cottontail rabbits examined. For example, all cottontails exhibited the e15 marker and 6 out of 10 animals exhibited the e14 marker as well. Several experiments were conducted to show that the e14 and e15 antigens of domestic rabbit and cottontail IgG are identical. Other results are consistent with the hypothesis that the e14 and e15 antigens in cottontail rabbits are products of allelic genes.     

Genetic polymorphism of IgG in the mink. III. Instability of expression and the problem of the genetic control of C gamma-allotypes

Quantitative expression of C gamma-allotype H4 of mink immunoglobulins was studied by enzyme-linked immunosorbent assay. The results presented suggest that production of H4 is under specific regulation. the concentration of H4 varies three orders of magnitude (10-10,000 micrograms/ml) from one mink to another. Fifteen percent of the sera of normal minks have the low H4 concentration, undetectable by the standard procedure of double immunodiffusion routinely used to test mink IgG allotypes. However, expression of these 'minor' allotypes may be significantly enhanced by hyperimmunization. Instability of this kind seems to be the main cause of earlier described deviations from Mendelian inheritance of C gamma-allotypes H2, H3 and H4.     

Preparation of IgG subclass allotypes from polyclonal IgG

Two allotypes have been identified for each of the IgG subclasses IgG1, IgG2 and IgG3. These allotypes are referred to as G1m(a) and G1m(f), G2m(n) and G2m(-n), and G3m(g) and G3m(b). Using a pool of normal human serum and a combination of preparative electrophoresis, DEAE ion-exchange and protein A-Sepharose chromatography, it was possible to separate G1m(f) from G1m(a), G2m(-n) from G2m(n) and G3m(g) from G3m(b). Purification of G2m(-n) molecules is of special interest as no genetic marker has been found to identify this allotype.     

Genetic polymorphism of IgG in the mink. VIII. A quantitative study of the expression of C gamma-allotypes (H3, H4, H6, H8) in sera.

The results of a quantitative study of the expression of mink C gamma-allotypes (H3, H4, H6, and H8) in sera are presented. H6 and H8 were found to be stably expressed, and the individual concentrations of the allotypes varied within one order of magnitude. Gene dosage effects were observed for H6 and H8: average sera allotype concentrations in homozygotes were twice those in heterozygotes. In contrast, the serum concentrations of H3 and H4 varied by three orders of magnitude, ranging from minor (2-200 micrograms/ml) to high (1-10 mg/ml). No gene dosage effects were observed for the expression of H3 and H4. Histograms for the population of H3 concentrations showed three peaks, sharply differing from those of H4, H6, and H8. There was no association between the minor expression of H3 and H4. The data obtained indicate that the expression of mink C gamma-allotypes is regulated by different allotype-specific mechanisms.     

Primary hepatocellular carcinoma and IgG heavy chain allotypes.

We studied Gm typing of serum samples from 838 donors; 177 had chronic hepatitis, 166 liver cirrhosis, 113 primary hepatocellular carcinoma, 21 alcoholic hepatitis, 18 fatty liver and 343 were unrelated normal blood donors. The distribution of Gm phenotypes and haplotypes in sera from patients with primary hepatocellular carcinoma differed from that in the normal controls; the Gm phenotype (1,2,21,13,15,16) and the haplotype Gm1,2,21 were significantly more common in this patient group (X2 = 18.56, corrected P less than 0.01, relative risk = 3.12; X2 = 25.52, corrected P less than 0.005, respectively). Overall, in the other liver diseases, we observed no significant Gm phenotype or haplotype association. The commitment to progression to primary liver carcinoma seems to be ascribable to a gene or polygenes close to the IgG heavy chain loci.     

IgG heavy chain allotypes (Gm) in autoimmune diseases.

Serum samples from 100 patients with myasthenia gravis, 322 with Graves' disease, 113 with Hashimoto's disease, 132 with systemic lupus erythematosus (SLE), 192 with insulin-dependent juvenile diabetes mellitus, 83 with Behçet's syndrome, 73 with psoriasis vulgaris, 258 with leprosy, 112 with Duchenne progressive muscular dystrophy and 343 non-related normal controls were studied for Gm allotypes. The incidence of Gm phenotypes with Gm(2) was significantly increased in patients with myasthenia gravis. Graves' disease, Hashimoto's disease, and high in SLE patients. The Gm1,2,21 haplotype was increased in patients with myasthenia gravis (chi 2 = 34 . 08, corrected P less than 0 . 001), Hashimoto's disease (chi 2 = 12 . 39, corrected P less than 0 . 05), Graves' disease (chi 2 = 8 . 65, corrected P less than 0 . 05), and SLE (chi 2 = 6 . 41, 0 . 1 greater than corrected P greater than 0 . 05). The total chi-square for the four different Gm haplotypes was significantly increased in patients with myasthenia gravis (chi 2 = 44 . 46, corrected P less than 0 . 001), SLE (chi 2 = 20 . 70, corrected P less than 0 . 005), Hashimoto's disease (chi 2 = 17 . 03, corrected P less than 0 . 025), and Graves' disease (chi 2 = 11 . 87, corrected P less than 0 . 025). Our data suggest the presence of Gm-associated pathogenic polygenes in certain autoimmune disorders.     

Genetic polymorphism of immunoglobulin G in the mink. VI. A regulatory gene controlling the expression of the gamma-chain constant region allotype of mink immunoglobulin

We describe here the inheritance of H6, one of the six known allotypes of the gamma-chain constant region of mink immunoglobulin (IgG). H6 is not present in minor concentrations in the serum, and its phenotypic expression is stable. However, in offspring of some of (H6-)X(H6-) crosses. H6 appeared unexpectedly and, by contrast, it disappeared in some H6+/H6+ homozygote offspring. Based on pedigree analysis, a transregulation of H6 expression in the serum by an autosomal recessive gene not linked to the structural allotype gene is postulated.     

The heterogeneity of bovine IgG2--V. Differences in the primary structure of bovine IgG2 allotypes.

The partial amino acid sequences of the gamma chains of the bovine IgG2a(A1) and IgG2a(A2) allotypes were determined. Sequence differences were found in the CH1 domain, the hinge region, and the CH3 domain. The hinge regions displayed only 71.4% similarity and all of the differences were of a radical nature. The A2 hinge has isoleucine instead of serine at 229, histidine for asparagine at 235, proline for histidine at 238, and cysteine instead of proline in position 234; the latter has the potential for forming an additional interheavy chain disulphide bridge. The occurrence of such a bridge could explain the presence of a pepsin fragment consisting of the hinge region and the Fc. A corresponding fragment is not obtained with the A1 allotype. Both allotypes have a shortened hinge region and a truncated CH2 domain. This feature is characteristic of all reported sequences of IgG2 proteins but not IgG1 in cattle and the goat. This structural feature may be important in subclass-specific recognition by Fc gamma receptors in ruminants. A surprising discovery was the occurrence of five substitutions in the CH3 domain of the IgG2a(A2) in comparison with the A1, which are shared with the CH3 of IgG1. These permit the occurrence of isoallotypic determinants and can explain the difficulty encountered in preparing A2-specific antisera during which adsorption with IgG1 is a routine procedure. The primary sequence data we report confirm the presence of major structural differences between the A allotypes of cattle that was suggested by previous work. The sequence of the A1 allotype most closely agrees with the two IgG2 sequences deduced from their nucleotide sequences whereas the sequence differences in the hinge and C-terminal CH3 make IgG2a(A2) unique. The structural differences between allotypes could have major consequences for such biological activities as phagocytosis, transepithelial transport, lymphocyte and complement activation.     

L chain peptide maps of rabbit IgG of different allotypes

Light (L) chains of IgG from rabbits homozygous and heterozygous for allotypic specificities (As4, 5 and 6) at the b locus were examined by the technique of peptide mapping.

They have between twenty-six and thirty dark peptides, 40 per cent of them being common to all three allotypic specificities. About 60 per cent peptides are shared by any two specificities (As4—5, As4—6 and As5—6). Amongst the peptides unique for each specificity there was always one most prominent peptide present by which the specificity could be identified by peptide maps.

The L chains from IgG of heterozygous rabbits have in general all the peptides characteristic of both specificities of the homozygous animal, although As4 peptides are always most prominent. Thus multiple peptide differences between L chains of three allotypic specificities were found.

     

Monoclonal antibodies against heavy and light chains of domestic mink IgG.

A panel of 26 monoclonal antibodies (MAbs) specific to mink IgG was produced and analyzed by ELISA, immunodiffusion assay (IDA) and immunoblotting assay. All the raised MAbs were directed against the isotypic IgG epitopes. Immunoblotting assay demonstrated that 11 MAbs reacted only with the Fc-fragments of IgG and 7 only with the light chains. Four antibodies bound to the Fab-containing fragments and failed to react with the Fc-fragments or isolated L-chains. Three MAbs did not react with IgG in IDA. Based on the results of IDA and cross-blocking assays, the MAbs were divided into 10 groups, with the MAbs of each group recognizing the same epitope. In IDA some MAbs were able to react with the epitopes which are common to the IgGs of some other representatives of Mustelidae family and also to some mammalian species remote from mink (dog, horse, pig, fox and rabbit).     

Gm allotypes influence the production of IgG3 but the effect is age-dependent.

Serum concentrations of IgG3 were found to be higher in Gm-f-positive (= b-positive) than in f-negative individuals except in young children. Young children aged 3-4 months had a mean concentration of 0.24 g/l of IgG3 regardless of allotype. The concentration gradually rose with age in f-positive individuals to a geometric mean of 0.56 g/l in adults but it remained essentially unchanged in f-negative people. A corresponding allotype effect was seen in influenza-specific antibody responses. While the total IgG response (mainly IgG1) was equally strong in f-positive and in f-negative patients, f-positive (= b-positive) patients produced more IgG3 antibodies than f-negative patients. The difference between geometric mean values of opposite homozygotes (f/f versus f-negative) was 2.3-fold (p = 0.0113). This finding indicates that the b-positive gamma-3 allele is more productive than the g-positive allele.     

Double line phenotypes in rabbit IgG allotypes and their relation to the cis/trans configuration of the IgG markers.

Rabbit antiallotype sera raised against heavy chain markers sometimes show double precipitin lines with all or some of the corresponding antigens (double and single line phenotypes). In a number of cases the double line phenotypes behave as alleles of the single line phenotypes and this feature allows a genetic and immunochemical analysis of these systems. In three cases that have been analysed, the double line phenotype arise when a precipitating a locus allotype and a non-precipitating d or e locus allotype are present on the same molecule (a1 and d14), (a1 and d11), (a3 and d11). This only happens when the corresponding genes are present on the same chromosome (cis configuration) of the diploid pair. These sera are therefore useful for determining directly the genotype of the animals.