Isolation from individual A/J mice of anti-rho-azophenylarsonate antibodies bearing a cross-reactive idiotype.

Immuization of A/J mice with a KLH-p-azophenylarsonate conjugate induces the formation of antihapten antibodies, some of which share idiotypic specificity common to all recipients. The subpopulation carrying the idiotype generally comprises 20-70% of the total antibody content. Large quantities of antihapten antibody (occasionally over 100 mg) were obtained from individual mice through the induction of an ascites fluid. This facilitated isolation of antibodies with the cross-reactive idiotype by isoelectric focusing. Most of this subpopulation has pI values between 6.65 and 6.95 and essentially all is of the IgG1 subclass. Two peaks, near pI 6.7 and 6.9, were frequently observed. Upon refocusing, the protein artifact of the procedure, but indicates microheterogeneity. The antibodies in the two peaks were found to be idiotypically identical by measurements of cross-inhibition. Preliminary studies have indicated that it is feasible to initiate investigations of primary structure with antibodies from individual inbred mice.

Specificity of antibodies: primary structural basis of hapten binding.

The primiary structure of the 83 residues of the NH(2)-terminus of the V(II), region was determined for each of three different antibodies to hapten which were produced in inbred guinea pigs. Each antibody had a different and distinctive primary structure within each of the two "hypervariable" regions (Hv1 and Hv2) included in the analyzed part of the variable region of the heavy chain. The sequences of Hvl and Hv2 in the three antibodies were either unique or of restricted variability compared with those of "normnal" immunoglobulin G2. Further implication of Hv1 and Hv2 in contributing to ligand-binding specificity of antibodies came from the placement of residues modified by affinity labeling reagents in these hypervariable regions.

In vivo induction of A/J anti-ARS responses with different ranges of affinities: correlation between affinity and CRIA idiotype dominance.

The anti-ARS immune response of A/J mice is characterized by the reproducible and dominant selection of CRIA bearing antibodies. In this report, we have investigated the role of affinity for the antigen in the selection of antibody repertoire during an immune response. A/J anti-ARS responses with different ranges of affinities for arsonate were elicited by the injection of differently arsanylated carrier proteins. The selection of higher affinity A/J anti-ARS responses was shown to be associated with the induction of higher levels of CRIA bearing anti-ARS antibodies. A detailed idiotopic analysis also showed a more precocious selection of the CRIA "canonical combination" in the higher affinity anti-ARS responses. These results strongly suggest an important role for affinity and clonal selection in the dominant expression of the CRIA idiotype in the A/J anti-ARS response.

Evaluation of immunotoxic and immunodisruptive effects of inorganic arsenite on human monocytes/macrophages.

A trivalent inorganic arsenic, arsenite, has been causing chronic inflammation in humans through the consumption of contaminated well water. The total peripheral blood arsenic concentrations of chronic arsenic-exposed patients, who had inflammatory-like immune responses, are less than 1 microM, thus, nM concentrations may be very important regarding the chronic inflammatory effects by arsenite. However, there are few reports about the biological effects of low concentrations of arsenite in mammalian cells, especially in normal immune effector cells. In this study, we examined whether arsenite has any biological and/or toxicological effects on the differentiation of human peripheral blood monocytes into macrophages using the colony-stimulating factor (CSF) in vitro compared with that of other metallic compounds, and found that arsenite sensitively inhibited the CSF-induced in vitro maturation of monocytes into macrophages at nM levels, and it also induced small, nonadhesive and CD14-positive abnormal macrophage generation from monocytes with granulocyte-macrophage CSF (GM-CSF) at 50-500 nM without cell death. The addition of other metallic compounds, including chromium, selenium, mercury, cadmium, nickel, copper, zinc, cobalt, manganese and other human pentavalent arsenic metabolites, such as inorganic arsenate, monomethylarsonic acid and dimethylarsinic acid, could not induce the same abnormal cell generation from monocytes with CSFs at any concentration and any additional time schedules; they showed only simple cytolethality in monocytes and macrophages at n-mM levels accompanied by cell death. This work may have implications in the arsenic-induced chronic inflammation in humans.

Low IgG response of the mouse strain C57BL/10ScSn after immunization with protein antigens.

The low IgG response of the strain C57BL/10ScSn is not restricted to the reaction to sheep red blood cells; but it can be demonstrated even after immunization with ARS, DNP or FITC haptens, coupled to various heterologous (BGG, RSA) or autologous (MGG) protein carriers. The level of the IgG response is - using the same immunization schedule - influenced both by the bound hapten and the carrier. In both strains tested (i.e. in the high-responding A/J and the low-responding C57BL/10ScSn), the highest IgG response is elicited by FITC-BGG. The response of the C57BL/10ScSn strain is, similarly as after immunization with SRBC, approximately ten times lower. The IgG response to other antigens tested was lower in both strains and therefore the quantitative differences were less pronounced. The affinity of antibodies against the ARS and TNP determinant, detected by inhibition of plaque-forming cells, is similar in the two strains. Thus the low reactivity of the strain C57BL/10ScSn is not caused by the absence of suitable VH and VL genes, but it rather indicates a defect of some general regulatory mechanism, involved in the synthesis of IgG antibodies. After repeated administration of ARS-BGG, the antigen and the antigen - antibody complexes accumulate in high concentrations primarily in the liver of mouse strain A/J. The amount of antigen accumulated in the liver of strain C57BL/10ScSn is significantly lower.

Structural studies on induced antibodies with defined idiotypic specificities. II. The light chains of anti-p-azophenylarsonate antibodies from A/J mice bearing a cross-reactive idiotype.

N-terminal amino acid sequence analyses have been performed on three preparations of light chains of A/J mice. Light chains derived from the IgG of unimmunized animals were compared to light chains of anti-p-azo-phenylarsonate (anti-Ar) antibodies possessing a cross-reacting idiotype (CRI); the latter were derived from the ascites fluid of a single A/J mouse, or from the pooled ascites fluids of 18 A/J mice. The heavy chains of these same two antibody preparations had previously been shown to comprise a single, homogeneous sequence to position 40. With few exceptions, the first 26 positions of light chains derived from unimmunized animals were extremely heterogeneous; the heterogeneity is comparable to that observed in a composite of sequence data on light chains of BALB/c myeloma proteins. Although the light chains obtained from anti-Ar antibodies possessing the CRI (whether from the pool of 18 A/J mice or from a single mouse) were more restricted in their sequence, at several positions as many as four alternative amino acids were detected. These studies indicate that an antibody population with defined idiotypic specificity, and very possibly identical heavy chain sequences, may contain at least four distinct light chains. The feasibility of structural studies on antibodies induced in individual mice is further demonstrated.

Humoral and cell-mediated immunity in experimental progressive thyroiditis in rabbits.

Rabbits immunized over a long period of time with serial injections of aqueous preparations of either bovine thyroglobulin or chemically altered rabbit thyroglobulin develop progressive thyroiditis. As is short-term thyroiditis in rabbits and mice, this thyroiditis is characterized by lesions and cellular infiltration similar to that observed in Arthus reactions. Once the progressive thyroiditis is established, the rabbits respond readily to subsequent injections of native rabbit thyroglobulin. No significant reduction of lesions or circulating antibody is observed when injections of native rabbit thyroglobulin are substituted for the preparations used to induce the disease. Cell-mediated hypersensitivity to rabbit thyroglobulin, as evidenced by MIF activity, develops in rabbits after prolonged immunization with altered or cross-reacting thyroglobulin. It is suggested that this activity develops as a result of a loss in the unresponsive state in T lymphocytes. The data indicate that it is the persistence of circulating antibody to autologous thyroglobulin which sequesters autologous thyroglobulin from peripheral lymphoid tissue, and thus, results in the loss of the unresponsive state in lymphocytes of these tissues. It is suggested that similar events may be involved in the development of cell-mediated hypersensitivity in thyroiditis in humans.

Competition for antigen by cell populations having receptors with the same specificity but of different idiotype.

All normal A/J mice immunized with keyhole limpet hemocyanin-p-azophenylarsonate (KLH-Ar) produce anti-Ar antiboides, some of which share a cross-reactive idiotype. The present results indicate that the biosynthesis of antibody molecules bearing this idiotype does not occur in mice having an excess of lymphoid cells with receptors for Ar that lack the idiotype. This was shown, first, by introducing into normal, nonirradiated mice lymphoid cells from mice which had been suppressed with respect to production of the idiotype and then immunized with KLH-Ar. The recipients failed to express the idiotype upon immunization. Alternatively, cells from suppressed, mice were transferred into lethally irradiated syngeneic recipients, followed 17 days later by normal cells. All recipients expressed the idiotype upon immunization. If, however, the recipients were challenged with antigen between the two cell transfers, antibodies bearing the idiotype were not produced during subsequent immunization. Arguing against active, cell-mediated suppression was the production of the idiotype by normal cells in the presence of cells from a suppressed animal. However, the possibility of active suppression by hyperimmune suppressed cells was not ruled out. On the basis of the present data, the simplest interpretation is that cells with anti-Ar receptors from immune, suppressed animals, being present in larger numbers, compete successfully for antigen with nonimmune cells and thus prevent the expression of the idiotype. The system may provide a basis for quantitative studies of competition among cells for a limited supply of antigen, particularly if B cell populations are utilized.

Adsorption-desorption of antigen to polystyrene plates used in ELISA.

Since ELISA reliability depends to a great extent upon solid-phase reagent concentration and stability, we sought to analyse the influence of experimental conditions during ELISA performance on the adsorption/desorption of proteins to microplates. The effect upon desorption of several experimental parameters (antigen concentration, antibody concentration and affinity, washings, conjugate and inhibitor incubations) and quantitative treatment of protein-polystyrene adsorption were analysed. The adsorption to polystyrene microplates was studied with a hapten-conjugated protein (BSA-Ar36) in order to facilitate the analysis of the influence of antibody affinity on desorption during ELISA. Our results show that polystyrene plates adsorb BSA-Ar36 according to the Langmuir isotherm. The adsorption constant was 2.1 X 10(8) L/mol and maximal surface concentration of protein on solid phase was 1.8 X 10(-7) g/cm2. Although desorption was present, we observed that it did not affect the reliability of results of either direct or inhibition ELISA, because it was not dependent on the composition of the sample.

Germline transcription and switch recombination of a transgene containing the entire H chain constant region locus: effect of a mutation in a STAT6 binding site in the gamma 1 promoter.

The switch (S) in H chain class is preceded by germline transcription and then mediated by a DNA recombination event. One of the impediments toward understanding the mechanism is the lack of a system in which a recombinant DNA molecule undergoes cytokine-regulated class S recombination. To study class S recombination, we used transgenic mice with a 230-kb bacterial artificial chromosome that included a rearranged VDJ gene and the entire murine H chain constant region locus. We found that both germline transcription and S recombination to the transgenic gamma1 H chain gene were regulated by IL-4 like that of the endogenous genes. In mice with two or more copies of the H chain locus transgene, both germline transcripts and S recombination took place at levels comparable to those from the endogenous loci. We also prepared a version of the transgene with a 4-bp mutation in a STAT6 binding site in the gamma1 promoter region. On the average, this mutation reduced germline transcription by 80%, but did not change the amount of S recombination in vitro. Among both the wild-type and mutant transgenes, we found no significant correlation between the amount of germline transcripts and the amount of S recombination. We infer that the physiologic level of germline transcription of the gamma1 gene is in excess over the amount required for efficient S recombination.