Immune response to a hapten of fluorescein isothiocyanate in a single mouse analyzed by two-dimensional affinity electrophoresis.

Immune response to a hapten of fluorescein isothiocyanate (FITC) in a single BALB/c strain mouse was analyzed by two-dimensional affinity electrophoresis (2D-AEP). Anti-FITC antibodies were induced by immunization with FITC-conjugated bovine serum albumin. The antibodies were separated into a large number of spots of IgG due to differences in their isoelectric points(pI) and binding affinities to the FITC ligand. These spots consisted of IgG families which were composed of several spots having an identical affinity to the ligand but a different pI. The spots were not clearly detected in the antiserum taken on day 7 after the primary immunization, but on day 21 the spots of IgG were clearly detected, with a high diversity and specificity for the ligand. The size and number of IgG spots were markedly increased by the secondary immunization; however, the third immunization did not increase the size and number of IgG spots. The IgG spots of each family were specifically stained with an antimouse IgG subclass antibody. Furthermore, a monoclonal antibody (FL-D6) was separated by 2D-AEP into a single family which consisted of seven IgG1 spots having an identical affinity to FITC but different pIs. Therefore, each of the IgG families of anti-FITC antibodies in the antiserum can be generated by a single clone of anti-FITC antibody-producing cells. The substitution of dextran T2000 or lipopolysaccharide for bovine serum albumin as a carrier for FITC induced much smaller amounts of anti-FITC antibodies with a low diversity but high specificity to FITC.

Role of antigen-specific T cell help in the generation of in vivo antibody responses. II. Sustained antigen-specific T cell help is required to induce a specific antibody response.

The injection of mice with a goat or rabbit antibody to mouse IgD stimulates a large polyclonal IgG response, approximately 10% of which is specific for antigenic determinants on the anti-IgD antibody molecule. The large goat IgG (GIgG)-specific antibody response in mice injected with goat antibody to mouse IgD requires that GIgG-specific B cells undergo much greater clonal expansion than B cells specific for other Ag. One possible explanation for the greater clonal expansion of GIgG-specific B cells is that B cells that lack GIgG specificity can only be stimulated with GIgG-specific T help during the relatively short time that anti-IgD binds to, and is processed and presented by, these B cells before they cease to express membrane mIgD. In contrast, GIgG-specific B cells can continue to bind, process, and present GIgG through mIgM after they lose mIgD. To test the hypothesis that extended stimulation with Ag-specific T help is required to generate a specific antibody response, we determined time requirements for Ag-specific T cell help for the development of such a response. Mice were injected with rabbit antibody to mouse IgD plus one or more daily injections of FITC conjugated to a F(ab')2 fragment of rabbit IgG (FITC-(Fab')2), which has a short in vivo half-life, and IgG1 anti-FITC antibody production was analyzed. In this system, each additional injection of FITC-F(ab')2 extends the period during which FITC-specific B cells can process this Ag and present it to rabbit IgG-specific T cells. Each additional injection of FITC-F(ab')2 stimulated a several-fold increase in IgG1 anti-FITC antibody levels, and injections on 5 consecutive days were required to induce a maximal anti-FITC response. These observations provide evidence that sustained Ag-specific T cell help is required to stimulate the degree of B cell clonal expansion that characterizes a specific antibody response.

The effect of ultraviolet B irradiation and urocanic acid isomers on dendritic cell migration.

Irradiation with ultraviolet-B light (UV-B) suppresses some cell-mediated immune responses to a variety of antigens, including contact sensitizers. Following UV irradiation there is modulation of Langerhans' cells' markers and keratinocytes are induced to synthesize and secrete tumour necrosis factor-alpha (TNF-alpha). Cis-urocanic acid (cis-UCA) has been suggested as a photoreceptor for UV and has been demonstrated to suppress immune responses in several experimental systems. UCA is found naturally in the stratum corneum as the trans-isomer and converts to the cis-isomer on irradiation. In the present study the migration of dendritic cells (DC) to lymph nodes following UV-B irradiation or epicutaneous application of UCA isomers was examined in unsensitized mice and mice sensitized with fluorescein isothiocyanate (FITC). It was found that UV-B irradiation alone induced DC migration to draining lymph nodes (DLN) and that UV-B irradiation prior to skin sensitization at the same site enhanced DC migration. A maximum number of DC was present in DLN 48 hr following irradiation. In sensitized mice, the percentage of DC bearing FITC and the quantity of FITC per DC was unaltered by prior UV exposure. In contrast, neither isomer of UCA had any significant effect on DC numbers in sensitized or unsensitized mice. It was concluded that UV-B irradiation induced the migration of DC from the epidermis to draining lymph nodes, an effect possibly mediated by TNF-alpha release, while UCA may act by a different mechanism, perhaps via histamine-like receptors in the epidermis.

AMC-anti-FITC conjugates: novel reagents for amplified immunochemical techniques. Immunofluorescent staining of human fibroblasts.

Fluorescein antibodies were labelled with 7-aminocoumarin (AMC) derivatives, the 3-acetic acid and the 3-propionic acid N-hydroxysuccinimide esters. The labelled antibodies were used in conjunction with fluorescein isothiocyanate (FITC) and carboxyfluorescein-conjugated primary and secondary antibodies to develop novel immunofluorescent staining procedures. These methods combine the advantages of the fluorescence properties of AMC and the ready availability of FITC-labelled antisera to provide an amplified fluorescence signal as well as overcoming the photobleaching problems in FITC staining. The method is easy to perform and is expected to make an important contribution to the improvement of the quality of staining achieved with immunofluorescence. Details of the procedure used to stain human fibroblasts with antifibronectin antibodies are reported in order to illustrate the method.

Reduction of the rate of fluorescence decay of FITC- and carboxyfluorescein-stained cells by anti-FITC antibodies.

Anti-fluorescein antibodies were found to prevent the fading of emitted fluorescence from fibroblasts stained with fluorescein-labelled fibronectin antibodies. The prevention of fading is the result of specific binding of the fluorochromes present on the stained cells by the anti-fluorescein antibodies. The sheep anti-FITC antibody used in this study was equally effective in preventing the fading of both FITC- and carboxyfluorescein-labelled fibronectin antibodies. The method is simple, effective, does not interfere with the primary immune reaction, and in addition to preventing the fading of fluorescence it reduced the background fluorescence of the specimens. The procedure is expected to make an important contribution to improving the quality of fluorescence immunohistochemical techniques used in diagnosis.

Autologous anti-metatype immune response in rabbits.

Rabbits hyperimmunized with fluorescyl-conjugated KLH exhibited bound ligand associated with a high affinity circulating IgG anti-fluorescein population. After cessation of immunogen administration the liganded complexes were eventually spontaneously cleared from the circulation. Individual rabbits synthesized autologous anti-metatype antibodies specific for ligand-antibody complexes. Autologous anti-metatype antibodies reacted optimally with autologous liganded anti-fluorescein antibodies. However, cross reactivity was noted with allogenic rabbit liganded antibodies from three affinity-purified pools. An autologous anti-metatype response, reminiscent of autoanti-idiotype responses, has important implications concerning in vivo clearance of antigen-antibody complexes and may serve as a model to study immune complex diseases.

Profound specific suppression by antigen of persistent IgM, IgG, and IgE antibody production.

Ongoing, high-titer T-cell-dependent immune responses in adult mice, consisting of IgM, IgG, and IgE anti-fluorescein antibodies, can be specifically and substantially reduced (90-99%) when the mice are injected with appropriate doses of fluoresceinated dextran of defined molecular weight and hapten valence. This suppressive form of the antigen is nontoxic and specific, as responses to other antigens are unaffected. The suppression is long lasting and reduces high-affinity antibodies most markedly. Moreover, plasma cell secretion of specific antibody is virtually eliminated. This demonstrates that the reduction in antibody titer is not simply due to masking of serum antibody by the suppressive polymer. The results are discussed with reference to proposed models of B-cell and T-cell tolerance. Extension of these findings to disease-related immunogens may yield effective antigen-specific treatments of human allergy and autoimmune diseases.

Temporal variations in the fine specificity of IgM anti-fluorescyl antibodies.

This study compares the fine specificities of the primary and secondary fluorescein (FITC)-specific immunoglobulin M (IgM) repertoires in BALB/c mouse serum and monoclonal antibodies (MoAb) and has found reproducible, immunization-dependent differences. FITC and four of its homologues; iodoacetamido fluorescein (IAF), dichlorotriazinyl aminofluorescein (DTAF), substituted rhodamine isothiocyanate (XRITC) and tetramethyl rhodamine isothiocyanate (TRITC), each conjugated to bovine serum albumin (BSA), were used to determine reactivity patterns of serum IgM from mice immunized once or twice with FITC-haemocyanin (FITC-Hy). Reactivity patterns were also obtained for 20 IgM MoAb, eight of which were produced by fusions of SP2/0 myeloma cells with splenocytes from mice immunized once (primary) and 12 from mice immunized twice (secondary) with FITC-Hy. Each MoAb exhibited a unique fine specificity pattern, evidence of extensive heterogeneity in the FITC-specific repertoire. Reactivities of IgM MoAb with certain homologues were found to be more characteristic of either the primary or secondary response. Polyclonal serum IgM also showed reproducible immunization-dependent variations in fine specificity. Such a pattern could result from idiotypic suppression of primary antibodies, from the expansion of subsets of IgM memory cells utilizing novel genes and/or from somatic mutation absent in primary IgM antibodies.

Regulation of isotype immunoglobulin and interleukin production by adjuvants in vitro.

An in vitro system has been developed in which antibodies to fluorescein isothiocyanate (FITC)-labelled human gamma globulin (HGG) or dextran sulfate (DXS), are produced in the presence or absence of different adjuvants. The antibody response of in vitro cultures was measured by assaying the total Ig-secreting cells and FITC-specific plaque-forming cells (PFC). The presence of low levels of antigen and various cytokines were necessary for the production of isotypes other than IgM. Our results indicate that the regulation of isotype switching in vitro is dependent upon the non-specific stimuli-adjuvants, which probably activate different types of cells for cytokine production. The pre-activation of spleen cells, in our system, by antigen, seems to play a decisive role in the subclass of IgG produced. The adjuvant may still be able to influence the precommitted cell isotype to switch to another subclass but only in a "down-stream" direction i.e., IgG3----IgG1----IgG2b----IgG2a.