IgD antibodies to DNP are specifically induced in ascitic fluid after intraperitoneal immunization.

The induction characteristics of IgD in serum and other body fluids are poorly understood. Here we report that Freund's complete adjuvant (FCA), when used in intraperitoneal (i.p.) immunization, resulted in significantly higher levels of IgD and specific IgD antibodies in ascitic fluid than in the serum of the same animals. This was in sharp contrast to the distribution of IgM and kappa light chain-bearing immunoglobulins (kappa Ig) between these compartments. Female rats were immunized subcutaneously (s.c.) on Day 0 with 1:1 FCA mixed with either 2,4 dinitrophenyl group A streptococcal vaccine (DNP-GASV), 2,4 dinitrophenyl ovalbumin (DNP-OVA), or saline. A week later (and weekly thereafter) they were boosted i.p. with these antigens mixed with FCA 1:9. Eleven of sixteen rats produced some ascites by Day 21, and 15/16 by Day 28. Total immunoglobulin and specific antibodies for the heavy-chain isotypes IgD and IgM and for kappa Ig were measured by ELISA. Between immunization groups no significant differences were observed in total immunoglobulin levels. Subcutaneous immunization with 1:1 FCA caused a significant two- to six-fold increase in serum IgD by Day 7 (P less than 0.01), while no change occurred with serum IgM or kappa Ig. Ascitic fluid collected on Day 21 had significantly high levels of IgD than that of serum IgD (P less than 0.01), with a mean level nine times that of the serum. In contrast, levels of total IgM, kappa Ig and total protein were significantly lower in ascitic fluid than in serum (all P less than 0.05). The high levels of IgD in ascitic fluid disappeared in Day 28 ascitic fluid, while IgM and kappa Ig levels were relatively unchanged. The ratio of ascitic fluid to serum IgD anti-DNP was 9.8 for the DNP-GASV-immunized group on Day 21, and 23.9 for the DNP-OVA-immunized group on day 28. The same ratios for IgM anti-DNP and kappa Ig anti-DNP were much lower, ranging from 0.4 to 2.3 and 0.3 to 3.3, respectively.

Expression of immunoglobulin lambda chains in the laboratory rat.

We immunized a BALB/c mouse with the lambda-bearing rat IgG1 myeloma IR31, fused its spleen cells with the hybridoma parent line P3.X63.Ag8.653, and isolated a monoclonal antibody (G33/11) directed against rat immunoglobulin lambda chains. We used this antibody to classify two existing rat hybridomas as lambda-bearing proteins (D4.37HL.252 and PC61.5), and isolated one new lambda-bearing rat IgM hybridoma, G36/1. All the normal inbred rat sera that were tested contained lambda-bearing Ig as detected by G33/11, at levels ranging from 1.5% to 13% of the total serum Ig, the mean value being 7.9%. This antibody will be valuable for broadening our understanding of the immunogenetics of the rat, and for the characterization of monoclonal antibodies made in this species.

Immunoglobulin D in rat serum, saliva and milk.

Previously, this laboratory has found very high concentrations of IgD in normal rat milk. Using ELISA methods, the relationship between milk, serum and saliva IgD in lactating and suckling rats was examined. Milk IgD appears to be synthesized in the mammary tissue rather than taken up from the blood because (i) serum IgD remains low and is not significantly different from that of non-lactating females, and (ii) serum IgD during lactation is poorly correlated with milk IgD. Serum IgD in suckling rats declines in the first 7 days following birth and remains relatively low during the remainder of lactation (2-4 micrograms/ml). The surprisingly high serum IgD observed at birth (9.3 +/- 3.2 micrograms/ml) is present before suckling begins and is not affected by the onset of suckling. Transient elevations of serum IgD begin to occur following weaning. Rats weaned 10 days earlier than normal (Day 20 vs Day 30) had significantly higher serum IgD on Days 36, 47 and 60. Among 43 adult rats, serum IgD was 5.4 +/- 3.6 micrograms/ml and saliva IgD 2.0 +/- 1.7 micrograms/ml. Serum IgD correlates poorly with saliva IgD. The thymus is not required for IgD synthesis since no significant difference in serum IgD was found between nude rats and their euthymic littermates.

Influence of paternal immunity on idiotype expression of offspring.

The immune response of the rat to group A streptococcal carbohydrate (SACHO) and an associated idiotype, Id-1, was used to examine the effect of paternal immunity on Id-1 and SACHO-specific antibody expression by the offspring. First litters, conceived before immunization of the father, had significantly higher Id-1 levels than litters conceived by the same parental pairs after hyperimmunization of the father (P greater than 0.01). Total anti-SACHO levels were not affected. The effect appeared to be independent of the level of Id-1 expressed by the father or grandfather. No significant difference in Id-1 production was found between offspring of actively immune, neonatally Id-1 suppressed fathers and fathers expressing high levels of Id-1. We suggest that the paternal immunoregulatory influence acts via the maternal immune system to modify the idiotype repertoire expressed in the immune response of the offspring, and is not the result of genetic transmission of a trait acquired by the father. Some possible mechanisms of transmission are discussed.

Regulation of the expression of a mammalian idiotype in chickens.

Id-1, an idiotype associated with rat anti-Group A streptococcal carbohydrate (anti-SACHO) antibodies has not been detected in any other tested species immunized with Group A streptococcal vaccine (GASV). The immunization of chickens with rat Id-1 prior to challenge with GASV, however, induced the production of Id-1 and anti-Id-1 in some chickens. The concentrations of both idiotype and anti-idiotype were regulated by challenges with GASV. Thus, it appears that this non-mammalian species has the genetic potential to express the mammalian Id-1 network but such a potential is normally masked by regulatory pressures.

Allotypic determinants (Igh-3) associated with the IgG2c subclass of rat immunoglobulins.

Rat antibodies to group A streptococcal carbohydrate are primarily of the IgM and IgG isotypes. Hyperimmunization often results in antibodies of a restricted molecular heterogeneity of the IgG2c isotype. An allotypic marker, designated Igh-3a, was detected on the heavy chains of the IgG2c isotype from Copenhagen rats. Igh-3b was detected on the heavy chains of IgG2c from SHR rats. A strain distribution of the Igh-3 determinants as well as inheritance patterns for their expression are presented.

The assessment of anti-idiopathic antibodies as effective immunoregulatory probes in vivo.

In order to increase our understanding of the potential to use anti-idiotypic antibodies as immune modulators in vivo, we extensively analysed influences induced by one such antibody (anti-Id-l) following its administration to animals of different ages, genetic backgrounds, and immunological histories. Id-l is an inter-strain idiotype associated with rat anti-Group A streptococcal carbohydrate antibodies. The intraperitoneal (i.p.) injection of anti-Id-l antibodies, prepared against Id-l+ antibodies from an HPR rat could effectively induce long-term idiotype suppression in all tested strains of rats, regardless of the age at the time of treatment, the RTl haplotype or IgG2c or k-chain allotype. Total anti-streptococcal antibodies were not suppressed by this treatment. Although long-term suppression could be induced at any age, the percentage of animals suppressed following neonatal injections was consistently less than that following adult injections of anti-idiotypic antibodies. In addition, neonatal injections of anti-Id-l or Id-l with anti-Id-l appeared to enhance Id-l production in a minority of the animals. Similar treatment of adult animals never increased Id-l synthesis, suggesting that cells associated with enhanced Id-l production in older animals are either refractory to activation-differentiation signals and/or are sequestered and no longer accessible by i.v. or i.p. routes of administration of the probe. Auto-anti-Id-l immunity induced by immunizing adult rats with heavy, light, F(ab')2 fragments or whole IgG molecules could also induce an Id-l suppressed state. We were not able to induce significant Id-l suppression if animals were immunized with antigen prior to the injection of anti-Id-l. There was evidence, however, that such treatment might lead in time to the development of some idiotype specific suppression.

Phylogeny of immunoglobulin structure and function. XIV. Peptide map and amino acid composition studies of shark antibody light chains.

Light chains from antibodies to the streptococcal A-variant carbohydrate from individual nurse sharks were compared by peptide maps of tryptic digests and amino acid compositions. Although the amino acid compositions of the different chains were quite similar, considerable differences as well as similarities were demonstrable on peptide maps. The peptide maps were interpreted as indicating that shark L chains likely have constant and variable regions as seen in the immunoglobulins of higher animals. Furthermore the unique peptides characteristic of different L chains support the hypothesis that nurse sharks, as a species, possess a relatively large number of different L chain amino acid sequences which are compatible with antibody binding sites to the streptococcal antigen. Hence the repetoire of nurse shark antibody combining sites to this antigen appears to be quite extensive.

Phylogeny of immunoglobulin structure and function XV. Idiotypic analysis of shark antibodies.

Nurse shark antibodies to the streptococcal A-variant carbohydrate were specifically purified from the sera of four individual animals and used as immunogens in guinea pigs. The resultant guinea pig antisera contained antibodies with apparent idiotypic specificities for the homologous shark proteins. The shark idiotypic sites were located on the Fab fragments and appeared to require the participation of H and L chains for full expression. Tests for cross reactivity employing the four guinea pig anti-idiotypic sera and antibodies from 13 immunized sharks were positive in only two cases (heterologous inhibition). These findings indicate that the idiotypic library (and by inference the antibody combining site repertoire) of nurse sharks to the streptococcal A-variant antigen is probably quite extensive.

Inheritance patterns of idiotype expression: maternal-fetal immune regulatory networks.

The production of Id-1, a cross-reactive idiotype associated with rat anti-group. A streptococcal carbohydrate antibodies, by 11 strains of rats indicates that genes coding for Id-1 are in the germline. Its expression, however, follows a complex inheritance pattern. It was our intent in these studies to determine if immune responsiveness of streptococcus (GASV) immunized females could alter Id-1 expression of GASV-immunized progeny, and, in turn, introduce complications in Id-1 inheritance patterns. - We observed that Id-1-specific immune reactivity of GASV-immunized females could induce significant alterations in Id-1 production by progeny. The relationship between maternal and progeny Id-1 was complex, reflecting the complexity of autologous regulation of Id-1 production, and could be the opposite of what one would predict based upon parental transfer of Id-1 regulatory genes. The nongenetic nature of the maternal regulatory influence was confirmed by foster-mother studies. - We conclude that antigen-induced maternal immune responsiveness can exert a permanent regulatory influence on idiotype expression by progeny and consequently introduce error in to the interpretations of idiotype inheritance patterns. The recognition of this maternal regulatory influence also lends further support to Jerne's hypothesis that idiotype-specific immune networks play a significant role in the regulation of immune responsiveness in vivo.

Gut-associated lymphoid tissue in the chicken. I. Morphology, ontogeny, and some functional characteristics of Peyer's patches.

Lymphoid aggregates with many of the characteristics of mammalian Peyer's patches (PP) were identified in the chicken intestine. These chicken PP could not be seen with the naked eye at the time of hatching, but by 10 days of age, 1 or 2 PP were identified in 50% of the birds. Up to 16 wk of age, the numbers of PP increased to a maximum of 5 per animal and they were widely scattered in the intestine, apart from one that was regularly found about 5 to 10 cm anterior to the ileocecal junction. As the birds aged, the number of PP declined so that in older birds (58 wk) only a single PP was evident near the ileocecal junction. Chicken PP possess: a distinct lymphoepithelium with M cells and strong pinocytotic activity; follicles and a subepithelium zone and central zone similar to those of the cecal tonsil (CT). Studies with in ovo hormonal bursectomy and age-associated involution of PP suggested that the subepithelial zone is a B-dependent area and the central zone a T, or thymus-dependent region. These observations extend our knowledge of gut-associated lymphoid tissue (GALT) in the bird to include the bursa of Fabricius, CT, PP, and aggregates in the urodeum and proctodeum. They raise issues about the roles of chicken PP that may aid our understanding of the functions of mammalian GALT.

Cross-reactivity of rat, mouse, and human IgD.

Highly purified sheep anti-rat lymphocyte membrane IgD (mIgD) was used to detect cross-reactivity with the putative murine-delta chain on mouse lymphocytes. Cross-reactivity is demonstrated by indirect immunofluorescent staining and by immunoprecipitation of 125I-labeled lymphocyte membrane extracts followed by electrophoresis on 10% polyacrylamide gels. In addition, cross-reactivity of anti-rat-delta with human IgD is shown by gel diffusion analysis. The anti-rat-delta reagent stained both Ig5a+ and Ig5b+ lymphocytes. Preincubation of Ig5b+ (but not Ig5a+) cells with monoclonal allotype-specific antibodies (anti-Ig5b) under capping conditions caused inhibition of staining by the sheep anti-rat-delta reagent, indicating that it is the delta-chain that is recognized on mouse lymphocytes and that the anti-rat-delta reagents does not distinguish between mouse-delta allotypes. Furthermore, absorption of the sheep anti-rat-delta serum with purified human IgD reduced subsequent staining of mouse lymphocytes by approximately 50%; staining was not affected by absorption with human IgM. This xenogeneic anti-delta antiserum appears to detect determinants on the delta-heavy chain, which are shared by at least three species of mammals, suggesting that these determinants represent important molecular features conserved during evolution.

In vivo effects of antiserum to IgD on surface immunoglobulins, serum immunoglobulins and lymphocyte blastogenesis in rhesus monkeys.

The effects of injecting monkeys with goat antiserum to IgD, the IgG fraction of that antiserum or normal goat serum (NGS) were compared. The subcutaneous injection of 4 ml/kg of the whole antiserum resulted in decreased percentages of lymphocytes with surface IgD or IgM lasting from day 1 through day 7 post-injection followed by substantial recovery on day 10. Lymphocytes from these animals were stimulated as indicated by the increased incorporation of 3H-TdR by cells placed in culture on days 7-21 post-injection. The increased blastogenesis occurred in rosette-depleted (B cell) populations and did not occur in rosette-enriched (T cell) preparations. Hypergammaglobulinaemia and increased concentration of serum IgG were first detected on day 10 postinjection, maximal on day 14 and were in decline by day 18. Injection of 4 ml/kg NGS did not alter the percentages of lymphocytes with surface immunoglobulins, result in hypergammaglobulinaemia, or stimulate the degree of blastogenesis observed after anti-IgD. Injection of the IgG fraction of the antiserum resulted in decreased lymphocytes with surface immunoglobulins but did not stimulate hypergammaglobulinaemia or increase blastogenesis. Injection of one monkey with the IgG fraction of anti-IgD combined with NGS resulted in increased serum IgG and increased blastogenesis. Both antibody to IgD and multiple antigenic challenge appear to be required for these responses.