Monoclonal antibodies against the human interferon-gamma receptor(s).

Enriched human interferon-gamma (HuIFN-gamma) receptor preparations were obtained by affinity chromatography of non-ionic detergent solubilized COLO 205 cell membranes on immobilized recombinant HuIFN-gamma. The active fractions, identified by a competition ELISA, were used as the immunogen in a BALB/c mouse. Fusion of its splenocytes with myeloma cells yielded several hybrids secreting antibodies that inhibit the antiviral activity of HuIFN-gamma; the two most active ones were selected for further characterization. This blocking activity was restricted to both the human species and the gamma type of IFN. Affinity purification of cell membrane extracts on the immobilized monoclonal antibodies resulted in the visualization of a major protein band with an Mr of 90,000, which is in good agreement with the results obtained by other authors [Aguet M. and Merlin G. (1987) J. exp. Med. 165, 988-999; Novick D., Orchansky P., Revel M. and Rubinstein M. (1987) J. biol. Chem. 262, 8483-8487; Sheehan K. C. F., Calderon J. and Schreiber R. D. (1988) J. Immun. 140, 4231-4237].

Specific responses to two unrelated antigens in mice made orally tolerant to one of them

BDF1 and B10 micr, either normal or exclusively fed egg white dilutd in water, were immunized with ovalbumin (OVA), sheep erythrocytes (SRBC.BDF1, but not B10, mice became tolerant to OVA and presented a 100% increase in the total number of immunoglobulin-secreting cells in the spleen. Immunization with OVA + SRBC markedly increased the response to OVA, except in tolerant BDF1 mice, and had no effect on the anti-SEBC PFC reponse, except in normal (non-tolerant) BDF1 mice

Background (spontaneous) immunoglobulin production in the murine small intestine before and after weaning.

The ontogeny of the murine intestinal B-cell compartment before and after weaning was studied by quantitative analysis of immunoglobulin-secreting cells (Ig-SC) in the small intestine (SI). Before weaning, few Ig-SC were detected in the SI, whereas spleen and bone marrow already contained many Ig-SC. The number of Ig-SC in the SI started to increase immediately after weaning. Comparing early-weaned mice with non-weaned mice of the same age clearly demonstrated that weaning brought on the development of Ig-SC in the SI. The influence of a gut flora on the number of Ig-SC in the SI was examined by comparing the number of Ig-SC in the SI of conventionally housed, specific pathogen free (SPF) and germ-free mice. A bacterial flora was apparently needed for the normal development of Ig-SC in the SI. Comparing mice containing an aerobic Gram-negative bacterial flora with mice containing only an anaerobic Gram-positive bacterial flora demonstrated that the type of bacterial flora is relatively unimportant. No evidence was found that circulating maternal antibodies suppressed the development of the "spontaneous" intestinal and systemic B cell response. The results show that bacterial colonization of the intestine plays a pivotal role in the development of the Ig-SC compartment in the SI.

Incidence of anti-DNA idiotype-positive cells in human peripheral blood.

Human anti-DNA idiotype (Id)-bearing cells in peripheral blood were sought using mouse monoclonal anti-Id antibodies to human monoclonal anti-DNA antibodies. Pretreatment with acid pH or pronase P or preincubation in human serum-free medium markedly decreased the number of anti-Id-reactive cells. Pronase P-treated cells were able to bind to anti-Id once more after incubation for 18 hr at 37 degrees C, indicating the resynthesis of internal idiotypic determinants on the cells. Antiidiotype-reactive cells retained the same idiotypes in their cytoplasma. The cells expressing anti-DNA idiotypes, termed 0-81 and NE-1, were detected in the circulation of most patients with active lupus nephritis but not in those of inactive systemic lupus erythematosus (SLE) and healthy subjects. The idiotype-positive cells occurred in up to 5-10% of B cells from some patients with SLE in the active stage but became undetectable in remission. A limited number of anti-DNA Id-positive cells responsible for anti-DNA production might be preferentially expanded during acute episodes of the disease in some patients with SLE.

Analysis of human IgG and IgA subclass antibody-secreting cells from localized chronic inflammatory tissue.

The chronic inflammatory diseases in humans have been intensively investigated, however the immune mechanisms underlying diseases such as rheumatoid arthritis (RA), inflammatory bowel disease, and periodontal disease (PD) remain elusive. In this study, we have analyzed the distribution of IgM, IgG, and IgA secreting cells with emphasis on the IgG and IgA subclasses among mononuclear cell populations isolated from gingiva at different stages of PD. Surgically removed tissues were treated with Dispase to gently dissociate cells and the Ficoll-Hypaque gradient centrifugation was used to enrich for viable mononuclear cells rich in lymphocytes, macrophages, and plasma cells. The total numbers of plasma cells increased with the severity of disease. Immunofluorescence analysis showed that most Ig-containing cells were of the IgG isotype; however, significant numbers of IgA-positive cells but few IgM-positive cells were seen. This isolation procedure allowed analysis, at the single cell level, of the distribution of IgG and IgA subclasses of antibody-secreting cells with monoclonal antibodies to human IgG and IgA subclasses. For this, we selected four monoclonal anti-IgG subclass (anti-gamma 1, -gamma 2, -gamma 3, and -gamma 4) antibodies with no subclass cross reactivity for use in the enzyme-linked immunospot assay. Analysis of slight, moderate, and advanced stages of PD showed a progressive increase in spotforming cells (SFC) numbers, and the major isotype of SFC was IgG followed by IgA. The major IgG subclass SFC seen was IgG1 followed by IgG2 whereas similar numbers of IgG3 and IgG4 SFC were observed, a pattern also seen with cells from synovium of RA patients and in mitogen-triggered spleen and PBMC. In terms of the IgA subclass distribution, IgA1 predominated in moderate stages, whereas a selective increase in IgA2 SFC were seen in the more advanced stage of PD. These results show that significant numbers of viable plasma cells/Ig-secreting cells can be isolated from inflamed gingival tissues. Further, careful analysis has shown that IgG subclass responses in gingiva are similar to those found in synovia of RA subjects, and in stimulated PBMC and spleen. However, it should be noted that the number of IgG4- and IgA2-secreting cells increased in the advanced stage of PD.

[Effect of lobenzarit disodium (CCA) on B cell differentiation to antibody secreting cells].

We investigated the effect of the immunomodulator CCA on B cells, using the system of Staphylococcus aureus Cowan 1-stimulated B cells developing into antibody secreting cells in the presence of T cell factors. Results were expressed as the number of plaque-forming cells (PFC), as measured by reverse hemolytic plaque assay. Serially diluted CCA was added to the culture system to evaluate its effect (500-0.005 micrograms/ml). Different results were obtained by adding CCA at a high dose or a low dose. High-dose CCA showed an inhibitory effect on PFC (p less than 0.01). On the other hand, low-dose CCA showed an inhibitory effect on high PFC responses of B cells and a stimulatory effect on low responses. Our data suggest that CCA has a direct effect on B cells and that low-dose CCA has immunomodulatory action on normal variations in immune responses.

Inhibition of direct haemolytic plaques by antistaphylococcal sera.

An in vitro study was carried out to determine the IgM response to staphylococcal antigens (Staphylococcus aureus, serotype 3, ATCC 12600), at different stages of the growth curve. This was in order to observe the number of inhibited haemolytic plaques when immunosera obtained from blood-letting in rabbits previously immunized with antigenic components were used.

A novel two colour ELISPOT assay. I. Simultaneous detection of distinct types of antibody-secreting cells.

A novel assay system has been developed which is based on the ELISPOT methodology and employs a combination of two immunoenzyme visualization systems yielding distinct colour products. This variation permits the simultaneous enumeration of two different types of cell secreting antigenically distinct products. Optimal conditions for the concurrent detection of human mononuclear cells secreting IgG or IgA antibodies are described.

Experimental allergic conjunctivitis: production of different isotypes of antibody by conjunctival-associated lymphoid tissue in culture.

Long-term (greater than 2 years) topical, conjunctival application of fluoresceinyl ovalbumin (FL-OA) induced allergic conjunctivitis-like lesions and hyperplasia of conjunctival-associated lymphoid tissue (CALT) in guinea pigs. Single-cell suspensions of CALT and spleen were prepared by collagenase digestion and cultured with or without FL-OA or lipopolysaccharide; the culture supernatants were assayed for IgG, IgA, IgM, and IgE antibody. Absolute values (ng Ab protein/ml) of anti-FL-OA IgG subclasses (IgG1 and IgG2) were measured using purified preparations of IgG1 and IgG2 anti-FL-OA antibody standards in an enzyme-linked immunosorbent assay. Immunohistochemical studies were performed using frozen sectioned CALT tissues as well as cultured single cells. IgG1, IgG2, IgA, IgM, but not IgE, anti-FL-OA antibodies were detected in the culture supernatants of both CALT and spleen. IgG- and IgA-secreting plasma cells were demonstrated in immunoperoxidase-stained CALT and single-cell cultures. The ratio of IgG1 to IgG2 isotypes produced by CALT in vitro was significantly higher than that produced by spleen and also that found in serum. These findings indicated that a site-specific regulation of antibody isotypes may exist within the hyperplastic CALT induced by the long-term topical exposure to FL-OA.

Detection of IgA antibodies and quantification of IgA antibody-producing cells specific for ovalbumin or Trichinella spiralis in the rat.

This report describes procedures to quantify IgA responses in the rat sensitized to ovalbumin or infected with the parasite Trichinella spiralis: an ELISPOT detecting specific IgA antibody-producing cells in lymph nodes, and an ELISA demonstrating IgA antibody in serum and gut mucosal scrapings. For this purpose a mouse monoclonal anti-rat IgA antibody was produced. This IgG1-kappa 1 antibody recognized rat IgA but not rat IgM, IgG, or IgE. It proved very suitable in both assays. Using this reagent we could demonstrate large numbers of IgA anti-ovalbumin-producing cells in the mesenteric lymph nodes 15 days after sensitization to ovalbumin via the Peyer's patches. At 28 days after sensitization the numbers were much lower. IgA antibody titres to ovalbumin in serum were maximal between days 14 and 21 after immunization. Maximal numbers of IgA anti-T. spiralis-producing cells were found in the mesenteric lymph nodes 12 days after infection with muscle larvae, followed by a sharp decrease at 15 days. Maximal IgA anti-T. spiralis antibody titres in serum and mucus scrapings of small intestines were found on days 10 and 12 after oral infection with the parasite.

Comparison of measurements of in vitro production of antithyroid microsomal antibody versus antithyroid peroxidase antibody.

In vitro production of antithyroid microsomal antibody (AMA) and antithyroid peroxidase antibody (APA) by peripheral blood lymphocytes from patients with autoimmune thyroid disease (AITD) has been studied and compared, in view of the evidence for identity of the two differently measured antibodies. Peripheral non-T cells (2 x 10(5)) and autologous CD4 (helper/inducer) cells (2 x 10(5)) from patients with positive serum AMA were cultured for 7 days with pokeweed mitogen (PWM). B cells secreting AMA or APA were detected by the enzyme-linked immunosorbent assay (ELISA) spot assay. AMA or APA in the culture supernatants of these cells was also measured by ELISA. There was a significant correlation between the number of AMA- (IgG class) secreting cells and APA- (IgG class) secreting cells (r = 0.89 p less than 0.001). There was also a significant correlation between AMA- and APA-ELISA indices (r = 0.86, p less than 0.001). Furthermore, the number of AMA- or APA-secreting cells significantly correlated with AMA or APA secreted in the culture supernatants (r = 0.91, r = 0.92), respectively. These data show that peripheral blood lymphocytes from patients with AITD were able to produce antibodies against thyroid peroxidase (TPO) in vitro, as well as antibodies against thyroid microsomal antigen, after PWM stimulation. The significant correlation between in vitro AMA versus APA production, or the number of AMA- versus APA-secreting cells, accords with the evidence that TPO is identical to, or at least the major antigenic protein component of, thyroid microsomal antigen.

Further studies on the ELISA-spot technique. Its application to particulate antigens and a potential improvement in sensitivity.

Applications of the ELISA-spot technique to particulate antigens (sheep erythrocytes and E. coli) are reported; sheep erythrocytes were used to ensure a rigorous comparison of the spot assay and the haemolytic plaque assay. The spot technique was also applied to soluble antigens (dextran and trinitrophenyl-lipopolysaccharide) to assess the putative occurrence of non-haemolytic antibodies. In most instances, the spot assay disclosed higher numbers of antibody-secreting cells than the plaque assay. An examination of the kinetics of spot formation demonstrated that spot development was most rapid during the first and second hours of enzyme activity and slowed thereafter, although the numbers of spots at 16 h were higher than those at 2 h. To shorten the assay time a redox reaction (which yields an insoluble formazan) was coupled to the enzymatic reaction. In duplicate assays, this improved technique gave larger numbers of spots in a shorter time, than the conventional assay.

Avidin-HRP conjugates in biotin-avidin immunoenzyme cytochemistry.

Avidin-HRP conjugates were prepared, analysed and tested for avidin-biotin immunocytochemistry. Suitable biotinylation of enzymes, antigens and antibody was obtained by reacting biotin at equimolar ratio to epsilon aminogroups in proteins. The avidin-biotin interaction was used for immunocytochemical detection of phenomena in the field of immunology, i.e. immune complex trapping, specific antibody forming cells and in serology for the cytochemical detection of human auto-antibodies to basement membrane components. Avidin-HRP conjugation using the two step glutaraldehyde method gave a very small amount of monomeric, low molecular weight conjugate with excellent performance. Avidin-HRP conjugation using the periodate method was modified at two points. The first modification concerns the molar ratio of avidin to HRP in the reaction mixture which was brought to about equimolarity. The second modification concerns the periodate concentration which was decreased five fold, ten fold and twenty fold. Decreasing the periodate concentration decreased the amount of polymeric conjugate. Optimal amounts of monomeric, low molecular weight conjugate were obtained with a ten fold decrease of the periodate concentration. Comparable cytochemical results were obtained with monomeric conjugates obtained using both preparation methods.

An ELISA assay efficiently detects clonal antibody formation by single, hapten-specific B lymphocytes.

Spleen cells from non-immunized adult mice were fractionated on thin layers of fluorescein (FLU)-gelatin to yield FLU-specific B cells. These B cells were cultured either singly or in very small numbers in 10 microliter microcultures with 0.1 microgram/ml of the T cell-independent (TI) antigen FLU-E. coli lipopolysaccharide (FLU-LPS). Cultures were either filler cell-free, or supported by the addition of 10(5) CBA/N thymus cells per well. At 4-6 days, culture supernatants were assayed for the presence of anti-FLU antibody either by an enzyme-linked immunosorbent assay (ELISA) or a radioimmunoassay (RIA). With the filler cell-free cultures, B cell proliferation was scored microscopically before removal of culture supernatant. The cultured cells from each well were assayed for their capacity to form directly hemolytic FLU-specific plaques. In the filler cell-free system, the ELISA was much more sensitive than the plaque assay in identifying antibody-forming cell (AFC) clones, with over 10% of fractionated B cells yielding clones secreting detectable antibody, though with a low mean optical density (OD). This value represented over 80% of the proliferating clones. In the more efficient, filler cell-supported system, the difference between the 2 read-out methods was smaller. Here, one half of the hapten-specific B cells formed AFC clones, the highest cloning efficiency yet reported for an antigen-driven system. Comparative studies showed the RIA to be only marginally more sensitive than the ELISA, and not nearly as convenient for routine use.

T cell involvement in production of tumor necrosis factor: reconstitution experiments with nude mice.

In order to investigate the role of T cells in the production of tumor necrosis factor (TNF), a reconstitution experiment was performed with nude mice (Balb/c, nu/nu). The results obtained were as follows: 1) The cytotoxic activity of tumor necrosis serum (TNS) from Balb/c, nu/nu mice treated with Propionibacterium acnes-LPS was 1/22 of that from Balb/c, nu/+ mice. 2) TNF activity increased 14 times in reconstituted nude mice as compared to Balb/c, nu/nu mice. 3) The production of the cytotoxic activity per cell was investigated using T cell and macrophage fractions separated from the spleens of both Balb/c, nu/nu and Balb/c, nu/+ mice treated with P. acnes as a priming agent. Elicitation with LPS was done in vitro. Release of cytotoxic activity into the culture medium was observed in the macrophage fraction, but not in the T cell fraction. However, no significant species difference was found. 4) With P. acnes treatment, the population of macrophages in the spleens from Balb/c, nu/+ mice increased 25.5 times, whereas that from Balb/c, nu/nu mice only increased 6.8 times. The above results suggest that the mechanism of the incremental effect of T cells on TNF production was due to the promotion of macrophage proliferation during the priming period after injection of P. acnes.

Specific antibody-containing cells in the mammary gland of non-lactating sheep after intraperitoneal and intramammary immunisation.

Ewes were immunised intraperitoneally with ovalbumin and Brucella abortus in Freund's complete adjuvant, followed seven days later by intramammary immunisation in which ovalbumin was presented to one mammary gland and Brucella abortus to the other. Mammary tissue taken after a further seven days contained more antigen-specific plasma cells than ewes given intraperitoneal or intramammary immunisation alone. These cells were found predominantly in the specifically immunised gland and only a few were found in the contralateral gland. Most of these cells were of the IgG1 isotype. There was also an increase in the total number of IgG1- and IgG2-containing cells in mammary gland tissues of these ewes, indicating a non-specific response to immunisation. Following either intraperitoneal or intramammary immunisation there was also a significant increase in the number of antigen-specific IgA cells in the lamina propria of the jejunum. The gut response following intramammary immunisation alone was abrogated by chronic drainage of intestinal lymph but not mammary lymph. This suggests that antigen may relocate from the mammary gland to the intestine where an IgA response is generated from gut associated lymphoid tissue. These data provide evidence for interaction between the gut and mammary gland of sheep in response to antigen.

TNP-enzyme conjugates for the detection of anti-TNP antibody producing cells in vivo.

After injection of TNP-KLH in mice and TNP-BGG-PEN in rabbits, anti-TNP antibody-forming cells were observed in the spleen. When cryostat sections of the stimulated spleen were incubated with TNP-HRP conjugate, and then treated for HRP cytochemistry, the cytoplasm of anti-TNP-forming cells was stained red. When similar sections were incubated with TNP-AP conjugate, and then treated for AP cytochemistry, the cytoplasm of anti-TNP-forming cells was stained blue. After simultaneous incubation with TNP-AP conjugate and PEN-HSA-HRP conjugate, followed by treatment for HRP cytochemistry and AP cytochemistry, anti-TNP-forming cells with a blue cytoplasm and anti-PEN-forming cells with a red cytoplasm could be distinguished in the same spleen section.