Suppression of IgE antibody production in SJL mice. IV. Interaction of primed and unprimed T cells.

The mechanism of selective anti-hapten IgE antibody production was studied in SJL mice. Using an adoptive transfer method of spleen cells into syngeneic recipients irradiated with a sublethal dose of 600 rads, it was demonstrated that for the suppression of anti-dinitrophenyl (DNP) IgE antibody production the interaction of two subsets of T cells is necessary. DNP-primed B cells and carrier-primed T helper cells are taken from donors primed with small amounts of DNP-carrier conjugates. Without injection of other cells, high titer and persistent anti-DNP antibodies are produced in the recipients. The two subsets of T cells that are active in suppression of IgE are taken from two types of donors: one donor is immunized (hyperprimed) with larger amounts of carrier protein twice, the other is an unprimed donor. The carrier for hyperpriming the first type of donor may be unrelated to the carrier used for priming the helper T cells. To bring about anti-DNP IgE suppression it is necessary that the animals should be challenged with the same DNP-carrier conjugate used for priming the B and T helper cells. If the hyperprimed donors were immunized with a heterologous, unrelated carrier, then this heterologous unconjugated carrier must also be injected together with the homologous DNP-carrier conjugate. In these conditions, anti-DNP IgE antibody production is suppressed, but the production of anti-DNP IgG1 antibody is not diminished.

Suppression of IgE antibody production in SJL mice. III. Characterization of a suppressor substance extracted from normal SJL spleen cells.

SJL mice were immunized with 1 microng dinitrophenylated keyhole limpet hemocyanin in 1 mg Al(OH)3. The mice were infected 21 days later with 750 third stage larvae of Nippostrongylus brasiliensis. On day 35, 14 days after infection, they were injected with 1 microng DNP-N, brasiliensis extract (Nb) in 1 mg Al(OH)3. In order to obtain high titer and persistent anti-DNP IgE antibody the mice were irradiated (540 R) 1 day after injection of DNP-Nb. Suppression of anti-DNP IgE antibody production was induced by spleen cells from normal SJL mice. Suppression of IgE antibody response is also obtained by an extract from normal SJL spleen cells. The suppressor substance from normal SJL spleen cell extract is a heat-labile protein, and is not absorbed by anti-mouse immunoglobulin. The mol wt of this substance is larger than 300,000 daltons as determined by gel filtration on Sephadex G-200, but after ultracentifugation, the supernate still has suppressive activity on IgE antibody production.

Suppression of IgE antibody production in SJL mice. I. Nonspecific suppressor T cells.

High titer and persistent antihapten IgE production in SJL mice can be obtained using appropriate immunization and radiation. Nonirradiated mice rapidly terminate this antihapten IgE production. Radiation was not necessary to prolong antihapten IgE production in other strains of mice. Termination can be obtained even in irradiated SJL mice by transferring normal SJL spleen cells. That the suppressor cells are T cells is shown by using thymocytes or cells treated with anti-Thy 1.2 and complement. No appreciable suppressive effect by normal spleen cells could be demonstrated on IgG1 production in SJL mice. The characteristic of low and transient IgE antibody response in SJL mice is inherited as a recessive trait controlled by a single Mendelian autosomal gene and is not linked to the H-2-gene complex. This characteristic does not depend on the infectivity of Nippostrongylus brasiliensis, the effect of anticarrier antibody, or the recognition of antigen.

Increased T epsilon cells in BALB/c mice with an IgE-secreting hybridoma.

Since it has been proposed that T cells with receptors for the Fc portion of IgE (T epsilon) in rats, mice and humans are IgE-specific regulatory cells, it was investigated whether mice with an IgE-secreting hybridoma might be a source of large numbers of T epsilon cells. BALB/c mice with ascitic tumors of the IgE hybridoma B53 (epsilon, kappa, anti-DNP) developed high serum concns of monoclonal IgE which was followed by the appearance of large numbers of Lyt1-2+ L3T4- T epsilon cells. In contrast, mice bearing a non-IgE producing variant of B53 failed to develop T epsilon cells. Mice infused with cell-free ascites fluid (containing high levels of monoclonal IgE) from the IgE-secreting B53 hybridoma developed high serum IgE levels and also developed high numbers of T epsilon cells. Mice infused with cell-free ascites obtained from the non-IgE-secreting variant ENP-1 (containing very low amounts of IgE) did not develop either high serum IgE levels or T epsilon cells. These findings suggest that high serum IgE concns induce large numbers of T cells that express phenotypic markers of suppressor cells and have surface IgE-Fc receptors. These studies extend to IgE the principle that hybridomas and plasmacytomas induce large numbers of immunoregulatory T cells that express Fc receptors specific for the heavy chain class of the secreted monoclonal immunoglobulin. Since T epsilon cells are normally present only in small numbers, their marked increase in mice with IgE-secreting hybridomas identifies a ready source of large numbers of T epsilon cells that can be used to investigate their regulatory properties.

A novel method to investigate the heterocliticity of antibodies.

Monoclonal and anti-dinitrophenyl and anti-trinitrophenyl IgE antibodies were used to measure heterocliticity using competitive inhibition assays with homologous and heterologous haptens. The antibodies or antibody-containing ascites fluids were diluted to give 50% of the maximum binding to wells of antigen-coated microtiter plates. The % inhibition of binding of the antibody to the antigen by various concentrations of homologous and heterologous haptens at a standard dilution of antibody can then be compared. The advantages of this method of determination of heterocliticity are that it is fast, simple, quantitative and does not need radiolabeled reagents.

Correlation of murine anti-dinitrophenyl antibody content as determined by ELISA, passive cutaneous anaphylaxis and passive hemolysis.

Antibody contents of IgG1, IgG2a, IgG2b and IgE were measured by enzyme-linked immunosorbent assay (ELISA) in ascites and sera obtained from mice injected with hybridomas producing monoclonal anti-DNP antibodies. In addition, IgG1 and IgE antibodies from sera of immunized mice were also measured by ELISA. Concomitantly, antibody contents were also determined by passive cutaneous anaphylaxis (PCA) in mice for IgG1, IgG2a, IgG2b, by PCA in guinea pigs for IgG2a, by PCA in rats for IgE and by passive hemolysis (PL) for IgG2a and IgG2b. Good correlations were found in the investigated samples between ELISA and the biological determinations.

On the stability of IgE antibodies.

Mouse and rabbit IgE antibody-containing sera do not lose sensitivity capacity when stored at -20 degrees C even for 2 years or when lyophilized.

Detection of IgE antibody-forming cells by passive cutaneous anaphylaxis using cell extract from lymphoid organs.

IgE and IgG2a antibody-forming cells in the lymphoid organs of rats were detected by passive cutaneous anaphylaxis (PCA) reaction using the extract from the tissue. The amount of antigen-specific IgE and IgG2a antibodies in the tissue extract could be expressed as PCA titers in which extract from 10(8) cells/ml was used as starting dilution. The total amount of antigen-specific IgE antibody produced in mouse IgE-producing hybridoma cells was calculated by maximum dilution of the extract to the minimal amount of IgE antibody inducing PCA reaction, i.e., 4 ng/ml. In the case of the extract from mouse IgE antibody-forming hybridomas, the minimal number of hybridoma cells to be detected by this method proved to be 2-3 X 10(4) cells/ml.

Anaphylactic IgE and IgG1 production for hapten can be enhanced by contrast media.

Various side effects have been associated with the clinical use of contrast media. Immunological mechanisms have been proposed but there have been very few experimental studies with animal models. We have attempted to develop murine models to determine whether or not anaphylactic antibodies such as IgE and IgG1 against hapten (DNP) were enhanced with contrast medium (iopamidol) as an adjuvant or if the contrast medium itself produced antibodies of the IgE class. The results showed that anti-hapten IgE and IgG1 production was greatly enhanced with immunogen plus contrast medium. Anti-contrast medium antibodies of the IgE class could not be detected by PCA reactions. The enhancement of IgE and IgG1 production for hapten was associated with IL-4 release by the neutralization test used by monoclonal anti-IL-4 antibodies. This is the first observation to show that contrast media may have a strong adjuvant effect for the production of IgE and IgG1. This murine model demonstrates a possible immunological function of contrast media in vivo.

Effects of ethanol intake on the immune system of guinea pigs.

Groups of guinea pigs were maintained with alcohol solutions not sufficient to result in liver dysfunction prior to and during investigations designed to determine whether ethanol would nonetheless impair cell-mediated or humoral immunity. No impairment of either was observed in any of the experimental groups.

Immediate hypersensitivity. A brief, personal history.

The discovery of anaphylaxis by Portier and Richet that reinjection of a substance caused disease instead of immunity was sensational as it was against the prevailing DOGMA. Passive transmission of hypersensitivity with human antibody by Prausnitz (the P-K reaction, 1921) was an important step in the study of human hypersensitivity. Anaphylaxis was shown to be the consequence of liberation of vasoactive substances (histamine and SRS-A) from mast cells when the allergen crosslinks two IgE molecules fixed to mast cell Ig receptors (Ovary, 1961). The use of smooth muscle contraction (Dale, 1913) and vascular permeability increase (PCA, Ovary, 1948) became important for experimental studies. The clonal selection of antibody formation (Burnet, 1929) opened a new era in immunological concepts. The demonstration of the Fc receptor on mast cells (Ovary, 1961) called attention to the importance of cellular receptors. The carrier effect (Ovary & Benacerraf, 1963) was explained by recognition by T cell receptors of a processed carrier fragment complexed to Ia molecules (Unanue, Grey, 1981). Human IgE responsible for allergies was discovered in 1965 by K. & T. Ishizaka. Tonegawa in 1973 destroyed the "one gene-one protein" DOGMA, showing that the immunoglobulin, germline gene is discontinuous: i.e., composed of exons (which will form the Ig molecule) separated by introns. The CD4 cells were subdivided into Th1 and Th2 cells (Mosmann & Coffman, late 1980's). The Th2 secretes IL-4 necessary for IgE production (Paul, Vitetta, & others, early 1980's). B cells multiply before antibody production or become memory B cells, but what causes a B cell to become a memory cell is not known. The B cell does not change specificity but can switch the isotype using "switch recombinase" and the s segment of the Ig molecules (Honjo, early 1980's). IgE production was shown to be suppressed by lymphokines, such as IFN-gamma and IL-2. A great progress in understanding the mechanism of allergic reaction has been the result of intense investigations by many scientists. A more complete understanding, better prophylaxis and an improved treatment are the goals of the near future.

Recent insight into class-specific properties of murine immunoglobulins obtained with the help of monoclonal antibodies.

BALB/c and C57BL/6 mice were immunized with monoclonal anti-DNP IgE, obtained by fusion of PX63AG8-6-5-3 cells with spleen cells from immunized C57BL/6 or BALB/c mice, respectively. With the antiallotype sera thus produced the allotype of IgE (i.e. 7) could be defined since BALB/c is of the Igh-a and C57BL/6 of the Igh-b allotype. The antiallotype 7b does not cross-react with other allotypes. Antiallotype 7a recognizes in addition to allotype 7a also allotypes 7j, 7d, and 7e as expected. Both the complement fixing and the sensitizing activities of murine anti-TNP and anti-DNP monoclonal antibodies were examined. The minimal amount/ml for complement fixation was 0.46 micrograms from one of the IgG1 antibodies, 0.46 from IgG2a, 12.5 ng from IgM, 0.7 micrograms from IgG3. Complement fixation by IgG1 was unexpected. The minimal amount/ml for mouse PCA was 0.5-0.7 micrograms from IgG1, 1.44-2.3 micrograms from IgG2a, 5.6 micrograms from purified IgG2b and about 15 ng from IgE. Sensitization by IgG2a and IgG2b were new findings. Minimal amount/ml for guinea pig PCA was 0.23 micrograms from IgG2a. Minimal amount/ml for rat PCA was 2 ng from IgE.

Heteroclitic antibodies: differences in fine specificities between monoclonal antibodies directed against dinitrophenyl and trinitrophenyl haptens.

The inhibition of binding of monoclonal antibodies by different haptens was studied using the 50% antibody binding assay. The binding of antidinitrophenyl and antitrinitrophenyl antibodies to dinitrophenylated or trinitrophenylated bovine serum albumin could be inhibited by monovalent dinitrophenyl or trinitrophenyl epsilon-aminocaproic acid. Some of the antibodies could be inhibited to a greater degree with the cross-reacting haptens than with the haptens homologous to the immunizing antigen, therefore these antibodies were heteroclitic.

Suppression of IgE antibody response by irradiated reticulum cell sarcoma cells in SJL mice.

A low number of highly irradiated spleen or lymph node cells from reticulum cell sarcoma (RCS)-bearing SJL mice brings about suppression of IgE antibody production in adequately immunized and sublethally irradiated recipients, probably by known proliferative action of RCS cells on the few Ly-1 T cells which escaped from the irradiation.

Suppression of IgE antibody production in SJL mice. V. Effect of irradiation and adult thymectomy on the suppression of IgE antibody production in SJL mice.

Anti-DNP IgE antibody production was low and transient in SJL mice which were immunized with 1 microgram DNP-Nb and 1 mg A1(OH)3. The immunized SJL mice were irradiated (60-540 R) 1 day after challenge. A dose higher than 180 R induced enhancement of anti-DNP IgE antibody production as compared to nonirradiated control mice, suggesting the existence of irradiation-sensitive suppressor cells. Anti-DNP IgE antibody production was suppressed when immunized and irradiated SJL mice were injected with spleen cells from adult-thymectomized SJL mice. The donors of the spleen cells were thymectomized 2 or 4 months previously, and this suggests that the suppressor cells from unprimed mice are long-living T cells.

Adjuvant effects of amorphous silica and of aluminium hydroxide on IgE and IgG1 antibody production in different inbred mouse strains.

The adjuvant effects of an amorphous silica (Aerosil) and of A1(OH)3 on primary and secondary IgE and IgG1 immune responses to small doses of OA were studied comparatively in BALB/c, C57BL, DBA/1, AKR, DBA/2 and SJL inbred mouse strains. During the primary response, all mouse strains produced both IgG1 and IgE antibodies when antigen was given with A1(OH)3, whereas only DBA/1, BALB/c and DBA/2 mice produced IgG1 and/or IgE antibodies when Aerosil was used as the adjuvant. A booster effect, higher than that obtained by A1(OH)3, was induced by Aerosil in all mouse strains. The adjuvant effect of Aerosil was more selectively directed to the production of IgE antibody.