Heterozygous Rabbits Research Articles

The idiotypy of auto anti-allotype antibody induced in immunoglobulin allotype suppressed rabbits

Rabbit anti-rabbit idiotype antibody was raised to both clonally heterogeneous and restricted auto anti-b6 antibodies induced in b6 allotype-suppressed (b6)/(b6) homozygous and b4/(b6) heterozygous rabbits. In every case the anti-idiotypic antibodies were specific only for the inducing antibody as shown by direct binding solid-phase RIA. Anti-idiotypes directed to the same antibody preparation had a similar but not identical specificity. It was demonstrated by IEF that the same idiotype specificity (spectrotype) was present throughout the anti-b6 response in individual rabbits.

Double immunocytochemical staining for the in situ study of allotype distribution during an anti-trinitrophenyl immune response in chimeric rabbits.

After incubation of tissue sections with anti-allotype-enzyme conjugates, the localization of immunoglobulin-allotype-bearing cells in the lymphoid tissues of conventional and chimeric rabbits could be established. The use of anti-allotype sera bearing distinct enzyme labels allowed simultaneous recognition of B cells producing immunoglobulin of one or the other parental types in heterozygous rabbits, or of B cells from the donor and recipient in chimeras. After immunization of chimeric rabbits with trinitrophenyl-keyhole limpet hemocyanin, anti-trinitrophenyl antibody-forming cells could be demonstrated through the use of a trinitrophenyl-alkaline phosphatase conjugate. Simultaneous incubation of sections with this reagent and with horseradish peroxidase coupled to (donor or recipient) anti-allotype sera made possible the determination of the origin (donor or recipient) of the antibody-forming cells. In agreement with the results of plaque assays and analyses of serum antibodies, all the anti-TNP producing cells were of donor origin when the chimeras had been created through injection of spleen or lymph node cells from trinitrophenyl primed donors. With this study we introduce a simple, direct method for the simultaneous identification of cells that produce antibody of a given allotype and a given specificity, applicable to appropriate studies in heterozygous or chimeric rabbits. The procedure has various advantages over previously reported methods.

Evolution of the immunoglobulin kappa light chain locus in the rabbit: evidence for differential gene conversion events.

The rabbit kappa light chain gene family is characterized by the presence of two constant region (C kappa) genes; the C kappa 1 gene encodes the constant region of the principal rabbit immunoglobulin light chain, the C kappa 2 gene being not or very poorly expressed in domestic rabbits. There exist four major K1 alleles (b4, b5, b6, and b9), which are unequally expressed in heterozygous rabbits at the K1 locus. Here, we compare the nucleotide sequences of the joining (J) clusters of the kappa light chain gene (J kappa) linked to the b4K2 locus and to the b4 and b9 alleles at the K1 locus. As for C kappa genes, there is evidence for intergenic conversion between the J kappa 1 and J kappa 2 clusters as well as maximum divergence in the expressed J segments. The b9 J kappa 1 cluster differs from its b4 counterpart in that two out of the five J kappa segments (J1 and J2) are expressed instead of only one. This implies that preferential expression of the b4 allele as compared to the b9 allele is not only correlated to the number of available J kappa pieces. The b9 J2 segment is functional in spite of the presence of a termination codon immediately upstream of its coding region. Two major structural differences were observed between the J-C intron sequences of the b9 and b4 alleles; namely a 160-base-pair deletion of an A + T-rich sequence in b9 (which also occurs in the K2 locus) and a 10-base-pair deletion plus some substitutions in the region corresponding to the mouse kappa intron activating element. These differences could underlie the lower transcriptional rate of the b9 allele.

Impaired lymphocyte functions in heterozygous rabbits recovering from neonatal allotype suppression

Lymphocytes from heterozygous rabbits suppressed for an allotypic determinant on kappa light chains by exposure to maternally derived antibodies specific for the paternal gene product were analyzed for their capacity to express membrane-bound and secreted immunoglobulin (Ig). Individual cells displaying allotypic membrane Ig (mIg) were enumerated by a rosette test, while Ig-secreting cells were assessed by means of a hemolytic plaque assay. In a group of suppressed rabbits varying in age from 3 to 19 months, the proportion of cells with mIg of the paternal type was markedly higher than that of cells secreting that type of Ig. The same high proportion of lymphocytes displaying mIg of the suppressed type was observed whether lymphocytes from blood, spleen, or lymph nodes of suppressed rabbits were examined. In contrast, similar analyses performed with cells of normal heterozygous rabbits showed no discrepancy between mIg expression and secretion of either allotype. Lymphocytes synthesizing Ig of the paternal type were also defective in responses to lipopolysaccharide (LPS), which stimulates differentiation to Ig secretion in normal B lymphocytes. These results support the idea that B lymphocytes capable of synthesizing the suppressed type of Ig have functional impairments affecting secretion and responses to environmental stimuli.

A bi-directional immune network mechanism: Simultaneous induction of idiotype and anti-anti-idiotype in rabbits immunized with antibody to a common idiotypic specificity of anti-VHa2-allotype antibodies

Recently, we identified a common idiotypic specificity ( IdC) present on all of the allo and auto anti-VHa2-allotype antibodies tested. In this report, we immunized allotype-matched rabbits with anti- IdC Ab. When an a 1 a 1 homozygote was immunized with anti- IdC Ab (also from an a 1 a 1 rabbit), two distinct Ab populations were induced which were isolated through the use of immunoadsorbent column chromatography. One of these Ab populations, adsorbed to and eluted from an insolubilized a2 IgG column, was anti-VHa2-allotype Ab (Ab 1), which reacted with a2 Ig and inhibited the reaction between the a2 allotype and anti-VHa2-allotype Ab. The other Ab population, adsorbed to and eluted from an insolubilized immunogen anti- IdC column, was anti-anti- IdC Ab (Ab 3), which was specific for the immunogen anti- IdC Ab. In another experiment, when an a 1 a 1 heterozygous rabbit was immunized with anti- IdC Ab, only the Ab 3 was induced, which appeared to be identical to the Ab 3 induced in the a 1 a 1 rabbit. However, when the expression of the a2 allotype in an a 1 a 2 heterozygous rabbit was suppressed and then this rabbit was immunized with anti- IdC Ab, both Ab 1 and Ab 3 were induced. These data indicate the occurrence of a bi-directional immune response within our immune network system. According to Jerne's network theory, Ab 3 appeared to be induced through a stimulation by the idiotope of the immunogen Ab 2, whereas Ab 1 appeared to be induced through a reverse stimulation by the paratope of the immunogen Ab 2. Furthermore, the production of Ab 1 in these rabbits exhibiting the bi-directional immune response was at least 3–5 times greater than that of Ab 3 production indicating that the response through the reverse stimulation appeared to be dominant. The importance of this bi-directional immune response is discussed in this paper.

Biochemical characterization of skin fibroblasts derived from WHHL-rabbit, a notable animal model for familial hypercholesterolemia.

WHHL-rabbit (Watanabe-heritable hyperlipidemic rabbit), a strain of animal with spontaneous hyperlipidemia, is characterized by the remarkable resemblance of the pathology to type II hyperlipoproteinemia in man. In order to understand the biological basis of the disorder in the WHHL-rabbit, the lipid metabolism of cultured skin fibroblasts from the mutant rabbits was studied. Skin fibroblasts from WHHL-rabbits grown in the presence of whole serum showed 12.3-times and 3.5-times higher activities of sterol and fatty acid syntheses from [14C]acetate, respectively, when compared with fibroblasts from normal rabbits. The cholesterol esterification activity, which was measured by the incorporation of [14C]oleate into cholesterol esters, however, was 9-times lower in the mutant cells than the normal cells. The higher sterol-synthesizing capacity of the WHHL-rabbit fibroblasts was attributed to the 5-times-enhanced activity of 3-hydroxy-3-methylglutaryl-CoA reductase, the rate-limiting enzyme in cholesterol synthesis, compared with that in normal cells. In normal cells, the removal of lipoproteins from the culture medium caused a marked enhancement of 3-hydroxy-3-methylglutaryl-CoA reductase activity with a concomitant decrease of cholesterol esterification activity. The incubation of normal cells with low density lipoprotein (LDL) led to a reciprocal decrease in 3-hydroxy-3-methylglutaryl-CoA reductase activity and increase in the esterification activity. In contrast, the high level of 3-hydroxy-3-methylglutaryl-CoA reductase, which was associated with low cholesterol-esterification activity, in the WHHL-rabbit cells was essentially unchanged by removal or addition of LDL, indicating the absence of the functional LDL-mediated regulation system in the mutant cells. The studies using 125I-labeled LDL showed that normal cells possessed the specific receptor for LDL; LDL was bound, internalized by the receptor and degraded within cells. On the other hand, the finding that only 6 % of the receptor activity was present in the mutant cells when compared with normal cells, revealed that the mutant cells were the receptor-defective type. Thus, the primary defect in WHHL-rabbit fibroblasts is the lack of LDL receptor which regulates both 3-hydroxy-3-methylglutaryl-CoA reductase and cholesterol esterification activities. The mode of inheritance of LDL receptor-deficiency in WHHL-rabbit cells was found to be recessive, since all cell lines from the heterozygous rabbits possessed almost the same LDL receptor activities as normal cells. WHHL-rabbit, the first LDL receptor-deficient animal. can be a suitable model for familial hypercholesterolemia in man.

Kinetics of Ig VH region gene expression: Allotypic and idiotypic specificities of anti- p-azophenylarsonate antibodies produced by al-suppressed a1a3 rabbits

The amount of anti- p-azophenylarsonate (anti-Ars‡) antibodies produced by al-suppressed a 1 a 3 heterozygous rabbits was similar to the amount produced by nonsuppressed a 1 a 3 rabbits. However, when the al allotype emerged from suppression, the previously observed preferential expression of the al allotype for these antibodies was not restored: the al anti-Ars Ab produced in these rabbits remained very low. These observations suggest that the establishment of clones of the nonsuppressed-a3 allotype early in the immune response prevented the preferential expression of al anti-Ars Ab later on. By radioimmunoprecipitation, the kinetics of the idiotypic specificities of the anti-Ars antibodies were studied, along with the VH a locus allotypic specificities, from two rabbits. The idiotype of the al anti-Ars Ab produced, after the emergence of the al allotype from suppression, appeared to be different from the a3 anti-Ars Ab produced in the same rabbit, rabbit R2-3. In a second rabbit, R3, producing exclusively anti-Ars Ab of the a3 allotype, however, two transitions in idiotype expression were observed; the first idiotype disappeared for a period of about 30 weeks, and then reappeared. This transition in idiotypes may reflect a mechanism of autoregulation, that requires further investigation.

Genetic control of the expression of allelic Ig genes at the VH a locus in a1/a2 heterozygous rabbits.

Heterozygous rabbits representing 9 of 15 possible a1 and a2 heavy chain haplotype gene combinations among rabbits in the University of Illinois colony were analyzed for ratios of a1 to a2 in serum immunoglobulin (Ig). The Ig from rabbits of the a1x-y-n81f73g74de12,15 heavy chain haplotype in combination with any of three a2-associated heavy chain haplotypes have higher ratios of a1 to a2 than Ig from rabbits in which a1 is encoded by 4 other heavy haplotypes. For example, the mean a1:a2 ratio for adult a1x-y-n81f73g74de12,15/a2x32y33,- n82,f71g75de12,15 rabbits was 12:1 compared to 5:1 for a1x-y33,30n83,f71g75de12,15/a2x32y33,- n82,f71g75de12,15 heterozygous rabbits. Family studies indicated that the a1:a2 ratio was under the control of the heavy chain chromosomal region or a locus closely linked to it. Whether the regulation is due to varying numbers of VH genes and/or J gene segments, a separate regulator gene, or more efficient joining of certain gene segments, has yet to be determined.

Immunofluorescence studies on the expression of VH a allotypes by pre-B and B cells of homozygous and heterozygous rabbits.

Fluorochrome-conjugated antibodies specific for C mu determinants and VH a allotypes were used to examine pre-B cells and B lymphocytes for expression of these markers. The majority of mu+ bone marrow pre-B cells were shown to express a allotypic determinants in conjunction with C mu. Both pre-B and B cells from a2 a3 heterozygous rabbits showed allelic exclusion of these allotypes. Pre-B cells expressing the a2 or a3 specificities appeared to be generated in approximately equal numbers in heterozygotes, while B lymphocytes expressing a3 appeared to undergo preferential clonal expansion very early in development. It was also observed that rabbit bone marrow and blood contained a population of myeloid cells which, in a2 a3 heterozygotes, stained for both a2 and a3 determinants. The frequencies of this cell type, which exhibited bright immunofluorescence staining for a allotypes, could not be reduced by prolonged incubation at 37 degrees C but were readily reduced after cell suspensions were treated with low pH buffer. It is concluded that these myeloid cells bear high avidity Fc receptors for serum immunoglobulin and may be the culprits in studies which have found production of two a or b allotypes by individual B lymphocytes.

Identification of basophils as the cells bearing both allelic immunoglobulin allotypes among white blood cells from the peripheral blood of heterozygous rabbits.

Identification of basophils as the cells bearing both allelic immunoglobulin allotypes among white blood cells from the peripheral blood of heterozygous rabbits.

A reverse hemolytic plaque assay for the detection and the enumeration of immunoglobulin allotype-secreting cells

We describe a reverse hemolytic plaque assay to enumerate rabbit immunoglobulin allotype-secreting cells. This technique makes use of sheep red blood cells (SRBC) sensitized with goat anti-rabbit IgG and rabbit anti-allotypic sera as revealing antisera. We have used the assay to compare at the IgG molecule level and at the immunoglobulin allotype-secreting cell level, the preferential expression (pecking order), in heterozygous rabbits, of one of the two alleles either at the a or the b locus, respectively, governing the a series allotypic specificities carried by the variable region of the heavy chains and the b series allotypic specificities essentially carried by the constant region of the K light chains.

Functional diploidy for the secretion of the a allotypes by the lymphoid cell of heterozygous rabbits

Can a given heterozygous lymphoid cell, for the ‘a’ system of allotypes in the rabbit, secrete immunoglobulin molecules of the two types (functional diploidy) with the same antibody specificity? This problem was investigated by two methods (applied to immunized lymph node or spleen cells): enhancement of plaques of hemolysis by separate action of two anti-allotype antibodies vs the effect of a mixture of antisera, or by micromanipulation. Both methods lead to the same results: many cells manifest functional diploidy. In addition, it was found that only two populations of cells existed: cells secreting only al and cells secreting a1 and a2 or a1 and a3.

Exclusion of VHa and VHy loci expression on individual B cells from normal and VH allotype-suppressed rabbits.

The distribution of two heavy chain subgroups, VHa and VHy, on rabbit peripheral blood lymphocytes was examined by double membrane immunofluorescence. Fluroescent anti-a1 and anti-y33 were found to react with separate B cell populations; no doubly stained cells were observed. Further evidence for the independent expression of genes controlling the VHa and VHy subgroups were obtained by neonatal suppression of a 2 or y33 in a2y33/a3y- heterozygous rabbits. Suppression of VHa did not affect the expression of VHy, nor did the suppression of VHy affect the expression of VHa. The expression of a single VH gene per B cell is in marked contrast to the simultaneous expression of multiple CH genes.

Genetic Characterization of a new allele of the rabbit group b Ck allotypes

An inherited variant (b4(v)) of the rabbit kappa-chain allotype b4 is characterized by the presence of serine in place of alanine at position 121 and leucine in place of glutamine at position 124. The variant was traced through eight generations of a pedigreed rabbit family. Genetic analysis of this trait demonstrated that it is an allele of the other group b allotypes, and recent breedings have produced rabbits homozygous for this light-chain type. Two findings, other than the amino acid sequence differences, distinguish b4(v) from b4 in our colony. First, the level of expression of b4(v) in heterozygous rabbits is less than that of b4. For example, in b4b4(v) rabbits, approximately 30% of the preimmune IgG carries b4(v). In b4(v)b5 animals, 46% of the IgG carries the allotype b5, although in animals of allotype b4b5, 38% of the IgG is b5. Second, retrospective analysis of some litters revealed an abnormally low frequency of b4(v) in male heterozygotes. However, male b4(v)b4(v) homozygotes were found at the expected frequency in prospective crosses between b4(v)b5 rabbits.

Pre-B and B cells in rabbits. Ontogeny and allelic exclusion of kappa light chain genes.

Pre-B cells in developing rabbits were identified by immunofluorescence as cells containing small amounts of cytoplasmic IgM (cIgM) but lacking surface immunoglobulin (sIg). During ontogeny the first pre-B cells appeared in fetal liver at 23 days gestation, 2 days before the appearance of sIgM+ B lymphocytes. Pre-B cells were relatively frequent in fetal and adult bone marrow, but were not found in other tissues except rarely in fetal spleen. Allelic exclusion is apparently established at this early stage of development, because individual pre-B cells and B lymphocytes from heterozygous rabbits expressed only one of the alternative alleles in amounts sufficient for detection. Development of isotype diversity among rabbit B lymphocytes followed the general pattern seen in mouse and man. sIgM+ cells were detected before birth. Expression of sIgG was detected in neonatal rabbits on cells which were also sIgM+ but in older animals most sIgG+ cells lacked sIgM. Cells bearing sIgA were not found until 5-6 days of age, and had no other isotype on their surface.

Allelic allotype inclusion and exclusion among rabbit Ig-bearing lymphocytes of peripheral blood and lymphoid organs

Mononuclear cells isolated from peripheral blood, appendix, sacculus rotundus, mesenteric lymph nodes, and spleen of b 4 b 5 heterozygous rabbits were examined for surface Ig allotypes of the b locus. Ig allotype-bearing cells were detected as cells binding erythrocytes or bacteria coated with monospecific anti-b4 or anti-b5 antibody (Ab). Rosetting the cells with Ab-coated erythrocytes indicated that many peripheral blood lymphocytes, but relatively few appendix cells, bore both the b4 and b5 allotypes. Lymphocytes bearing both the b4 and b5 allotypes were also detected by incubating the cells with a mixture of Escherichia coli coated with anti-b4 Ab and Gaffkya tetragena coated with anti-b5 Ab. The percentage of Ig-positive lymphocytes binding both bacteria was 22–31% in the peripheral blood, 4–6% in the appendix, 3–5% in the sacculus rotundus, 4–10% in the mesenteric lymph nodes, and 5% in the spleen. Thus, the percentage of double-bearing lymphocytes was higher in the blood than in the appendix, sacculus rotundus, mesenteric lymph nodes, or spleen. The b4b5-bearing cells in the blood were not cells with adsorbed cytophilic Ab, since these cells still bore both the b4 and b5 allotypes after pronase digestion and Ig regeneration. These double-bearing lymphocytes, i.e., cells exhibiting allelic allotype inclusion, are probably less differentiated cells.

Regulation of allotype expression in heterozygous rabbits III. Concomitant modulation and concomitant suppression of a2 and a3 allotypes on individual peripheral blood lymphocytes

Sensitization of peripheral blood lymphocytes from heterozygous a2/a3 rabbits with purified, monospecific anti-a3 antibodies, raised in a1/a1 rabbits, resulted in the disappearance of surface a2 and a3 allotypes (concomitant modulation) after subsequent incubation at 37 degrees C, as determined by the mixed antiglobulin (rosette) test. Similar results were obtained when anti-a2 antibodies were used. The dose dependence of modulation and comodulation were also studied. Testing of mixtures of homozygous a2/a2 plus a3/a3 cells never led to comodulation. Blocking studies, performed to determine the surface contiguity of a2 and a3 determinants, indicated that both allotypes are situated close together in the membrane on cells exhibiting allotype inclusion. Overnight culture in serum-free medium revealed that cells which underwent extensive modulation and comodulation were often suppressed for both homologous and alternate allotypes (concomitant suppression). These and other data suggest that a single modulation event, in which extensive removal of cell membrane Ig occurred, could serve to inhibit the re-expression of Ig. This may, in part, reflect interactions with membrane receptors involved in the regulation of expression of VH gene products. Implications of VH allotype inclusion are discussed.

Auto-Antibody to an Ig VH Region Allotype: Induction of Anti-a1 Antibody in an a1-Suppressed a1a2 Heterozygous Rabbit

Abstract Two a1a2 heterozygous sibling rabbits were first suppressed for the paternally inherited a1 VH region allotype and then immunized with a1 IgG. Anti-a1 antibody was detected in the serum of one of the rabbits. The anti-a1 auto-antibody reacted with the same amount of a1 IgG as did a conventional anti-a1 allo-antibody. Most of the IgG and IgM of this rabbit was of the a2 allotype and no significant amount of the a1 allotype was detected as would be expected for an a1 suppressed a1a2 heterozygous rabbit. However, allotype suppression in this rabbit is maintained by endogenous anti-allotype antibody. Rabbits with anti-allotype auto-antibody may be exploited to produce litters of heterozygous and homozygous rabbits efficiently suppressed for selected allotypes.

Antibody Avidity and Allelic Selection for L Chain Allotypes in Rabbits Hyperimmunized with Streptococcal Vaccine

Abstract An almost absolute selection in favor of allotype b4 occurs in the antibody response of heterozygous b4b9 rabbits immunized with Group C streptococcal vaccine. A possible explanation for this “allelic selection” in favor of b4 is that during the antistreptococcal response the favored allotype (b4) is associated with V region binding sites yielding higher avidity anti-streptococcal antibodies. In order to test this possibility, IgG from heterozygous, hyperimmune rabbits was fractionated by avidity on a Group C immunoadsorbent column and allotypes of the various fractions were quantitatively determined. Contrary to the predicted result, when secondary immunization sera were examined the fractions of highest avidity were enriched in the nonselected allotype. Antibodies from two primary immunization sera lacked the selection for the non-b4 allotype in high avidity fractions observed for the later sera. Thus, the uneven allotype ratios observed in fractions of different avidity from secondary immunization sera can be interpreted as the result of a secondary maturation effect. The observations of allelic selection in heterozygous rabbits were extended to rabbits with allotypes b4b5 and b4b6. Although b4 predominated in immunoglobulin from these rabbits, the allotype ratios in anti-streptococcal antibody were the same as those in total IgG before immunization.

The inheritance of antibody V regions in the rabbit: linkage of an H-chain-specific idiotype to immunoglobulin allotypes.

Anti-idiotype antibodies specific for the H chain of an homogeneous antistreptococcal antibody (4135 Ab) were prepared by injection of recombinant molecules consisting of the H chains from 4135 Ab and L chains isolated from the injected rabbit. The antibodies prepared in this fashion (anti-HId) were specific for the VH region of 4135 Ab. Using this preparation in an inhibition of binding assay, sera from rabbits related and unrelated to 4135 were screened for the presence of the 4135 HId. It was found that about 45% of the related rabbits, when immunized with streptococcal Group C vaccine, produced antibodies with a cross-reactive idiotype, while less than 10% of similarly immunized unrelated rabbits produced molecules bearing HId. The expression of HId was linked to the a3 allotype present in the H chain allogroup J. Antibody molecules bearing the HId determinant were isolated from heterozygous (a1a3 and a2a3) rabbits and shown to express the a3 allotype. One rabbit lacking the a3 allotype produced significant amounts of antibodies expressing HId. These antibodies were found to express both VH and CH allo-types characteristic of the J allogroup, although neither allotype was found in a preimmune IgG sample from this rabbit.