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Oxidative stress induces a variety of cellular responses, including apoptosis, and caspase family proteases are known to be involved in apoptosis. Caspase-3(-like) protease activity was examined in Jurkat T cells to investigate the mechanism of apoptosis induced by a thioloxidant, diamide. Caspase-3 was activated when cells were cultured with 200 microM diamide that induced apoptosis, whereas no caspase-3 activation was detected with 500 microM diamide that induced necrosis. When apoptosis was induced in cells with exposure to 200 microM diamide, the intracellular thioredoxin (TRX) levels were maintained and the intracellular generation of reactive oxygen intermediates was marginal. The cytosolic fractions of cytochrome c were increased earlier than the activation of caspase-3. In contrast, when cells were exposed to 500 microM diamide, intracellular reactive oxygen intermediate generation was increased and processing of caspase-3 was not detected despite cytochrome c release, resulting in necrosis. Caspase-3 activity in cell lysate precultured with anti-Fas Ab was suppressed dose dependently by diamide and restored by thiol-reducing agents, DTT or TRX. When cells were precultured with 5 mM of buthionine sulfoximine, an inhibitor of glutathione synthesis, intracellular TRX levels were maintained, and as low as 20 microM diamide could induce apoptosis associated with the increase of cytosolic cytochrome c and the activation of caspase-3. These results indicate that the activation of caspase-3 in diamide-induced apoptosis is mediated, at least partly, by cytochrome c release from mitochondria, and the cellular reducing environment maintained by TRX, as well as glutathione, is required for caspase-3 activity to induce apoptosis.

The CD4 molecule acts as a receptor for class II MHC molecules to stabilize Ag recognition by the TCR and as a high affinity receptor for HIV-1. In this study, we investigated the effect of oxidative stress on the level of CD4 expression on cultured peripheral blood T lymphocytes (PBL blasts). As previously reported, we observed that the surface CD4 was down-regulated by PMA. Unexpectedly, treatment of PBL blasts with hydrogen peroxide (H2O2) or a sulfhydryl oxidative reagent, diamide, almost completely inhibited PMA-induced CD4 down-regulation, although these oxidants per se did not affect the level of CD4 expression. We next assessed the serine phosphorylation of CD4, which is reported to be an indispensable process for PMA-induced CD4 endocytosis. PMA could induce the serine phosphorylation even in the presence of oxidants. We also found that these oxidants had an additive effect on PMA-induced dissociation between CD4 and p56(lck), which is known to be another necessary step for CD4 endocytosis. These results indicate that in T cells, oxidants inhibit protein kinase C-mediated CD4 down-regulation due to perturbing a signaling process other than the above two steps, implying that oxidative stress may tune the functions of CD4+ T cells and their susceptibility to HIV-1 through the control of CD4 expression.

Thiol redox status modulates various aspects of cellular function. We demonstrate that oxidation of cellular sulfhydryl (SH) groups induces apoptosis. In Jurkat T cells and human PBL blasts, the fraction of apoptotic nuclei increased after treatment with an SH-specific oxidant, diamide. Analysis of DNA fragmentation and nuclear morphology also indicated that SH oxidation could induce apoptosis. In the apoptosis induced by SH oxidation, the decrease of cellular glutathione was transient and the increase of glutathione disulfide was observed only after apoptotic changes had occurred. Depletion of cellular glutathione with buthionine sulfoximine failed to induce apoptosis, despite a marked decrease of cellular glutathione, which was greater than that observed in apoptosis induced by diamide. Thus, the changes of cellular glutathione or glutathione disulfide may not be the major cause of apoptosis induced by diamide. Intracellular adult T cell leukemia-derived factor/human thioredoxin, another thiol-related antioxidant protein, was oxidized by incubation with diamide. These results suggest that thiol redox status is one of the key factors of the apoptotic pathway in which thiols other than glutathione may play even more critical roles.

The proliferative response of PBMC to PHA, Con A, OKT3 mAb and IL-2-dependent proliferation of PHA-blasts was examined in a thiol-free environment (cultured in a L-cystine- and GSH-free medium). [3H]TdR incorporation assay and cell cycle analysis revealed that stimulated PBMC could not enter the S phase when deprived of these thiol compounds. In thiol-free cultures, an increase in intracellular free Ca2+ concentration and IL-2R alpha-chain/p 55 (Tac) induction was still observed, whereas transferrin receptor induction was markedly reduced, suggesting that the proliferative response of mitogenically stimulated PBMC was arrested in the late G1 phase in which transferrin receptor is induced. In GSH-depleted cultures, a similar reduction of the proliferative response of PBMC and PHA-blasts was observed when the concentration of L-cystine was lowered, in a dose-dependent manner. Each reduction or loss of proliferative response was partially restored by supplementation of 2-ME or adult T cell leukemia-derived factor (ADF)/human thioredoxin which is considered to be an endogenous dithiol-reducing factor. L-Cystine transport analysis showed that mitogenically stimulated PBMC and PHA blasts incorporated L-cystine, whereas resting PBMC did not. Furthermore, ADF as well as 2-ME exhibited an enhancing activity on the L-cystine transport in PHA blasts. Together with the fact that L-cystine transport is a limiting step in glutathione synthesis, these findings suggest that GSH and ADF might cooperate in the thiol-mediated redox regulation process and might also play key roles in cell cycle (late G1 to S) progression of activated lymphocytes.

Adult T cell leukemia-derived factor (ADF), originally defined as an IL-2 receptor alpha-chain (IL-2R alpha)/p55 (Tac) inducer, is a human thioredoxin homologue and has many cytokine-like activities. In this study, we examined the regulatory effect of ADF on eosinophil migration using human eosinophils and an eosinophilic subline of HL-60 human promyelocytic leukemia cells, YY-1. rADF induced migration of eosinophils from patients with hypereosinophilia, although rADF exhibited little activity on eosinophils from healthy donors. When human eosinophils were incubated with rADF (0.1-10 micrograms/ml) at 37 degrees C for 24 h, both chemotactic and chemokinetic activity of the complement anaphylatoxin peptide C5a on eosinophil migration was markedly enhanced in a dose-dependent manner. Similarly, this enhancing effect of rADF was observed in the migration assay using YY-1 cells. In contrast, rADF showed no modulation of migratory behavior of human eosinophils and YY-1 cells by IL-3, IL-5, nor granulocyte-macrophage colony-stimulating factor. Scatchard analysis of C5a receptors on YY-1 cells using 125I-C5a showed that rADF modulated neither the density nor the affinity of the cell membrane significantly. Furthermore, mutant ADF (mADF), which had no reducing activity, had no enhancing effect on C5a-induced eosinophil migration. These results indicate a possible involvement of ADF in the recruitment of eosinophils through redox regulation by a dithiol reductase activity.

Adult T cell leukemia-derived factor (ADF) is a human homologue of thioredoxin with many biologic functions including IL-2R induction, growth promotion, thiol-dependent reducing activity, and radical scavenging activity. The regulatory effect of ADF on the cytotoxic activity of TNF was examined by using a human histiocytic lymphoma cell line, U937. When U937 cells were preincubated with recombinant ADF (rADF) (0.1-100 micrograms/ml) at 37 degrees C for 30 min, TNF-dependent cytotoxicity on U937 cells was markedly inhibited. This inhibitory effect was as high as 95% in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay (rADF 100 micrograms/ml) and 85% in the 51Cr-releasing assay (rADF 10 micrograms/ml). After pretreatment of U937 cells with IFN-gamma to augment the sensitivity to TNF, an inhibitory effect of rADF was also found. When U937 cells were washed after preincubation with rADF, resistance to TNF-dependent cytotoxicity was still observed, indicating that rADF inhibited the sensitivity of U937 to TNF-dependent cytotoxicity rather than modifying TNF molecules. Scatchard analysis of TNF receptors on U937 cells using 125I-TNF showed that rADF modulated neither the density nor the affinity of the cell membrane significantly. rADF also reduced the cytotoxicity induced by anti-Fas IgM mAb which shows cytotoxicity quite similar to TNF. rADF (10 micrograms/ml) reduced 90% of the cytotoxicity by anti-Fas IgM mAb, without a detectable change either in Fas Ag expression (MFI 58.1 vs 53.3) or in the degradation of anti-Fas IgM mAb as determined by flow cytometric analysis. These findings indicated that the rADF-induced resistance to the cytotoxic effect of TNF and anti-Fas mAb was not related to the modulation of the TNF receptor or Fas Ag.

Despite evidence for the expression of low affinity Fc receptor for IgE (Fc epsilon RII)/CD23 in T cell lines and pathologic T cells, Fc epsilon RII/CD23 in normal human T cells is still unclear. We studied the expression of Fc epsilon RII/CD23 on T cells in short-term culture of normal human PBMC stimulated with 15 micrograms/ml PHA. PHA stimulation also resulted in the release of soluble Fc epsilon RII/CD23 (IgE binding factor). Using two-dimensional flow cytometry, more than 10% of the Fc epsilon RII/CD23+ cells were found to co-express CD3 Ag. Both CD4+ and CD8+ T cells expressed Fc epsilon RII/CD23. The induction of Fc epsilon RII/CD23 on PHA-activated T cells was enhanced by IL-2 as well as IL-4. Both IL-2 and IL-4 also augmented PHA-induced production of soluble Fc epsilon RII/CD23. The enhanced expression of Fc epsilon RII/CD23 on T cells by both lymphokines was suppressed by rabbit anti-IL-4 antiserum, suggesting the involvement of an IL-4-dependent process even in the IL-2-dependent Fc epsilon RII/CD23 expression on T cells. The expression of mRNA for Fc epsilon RII/CD23 on PHA and IL-4-stimulated PBMC was examined by Northern blot analysis. Fc epsilon RII/CD23 mRNA was detected in RNA prepared from the T cell fraction depleted of B cells and macrophages (Fc epsilon RII+CD3+ = 6.2%, Fc epsilon RII+CD3- = 0.8%). The expression of the mRNA for Fc epsilon RII/CD23 on CD3+ T cells was also confirmed by in situ hybridization with Fc epsilon RII/CD23 cDNA combined with CD3 rosette formation at the single cell level.

An increase in intracellular cAMP level induced the expression of IL-2R alpha-chain, the 55-kDa component of IL-2R complex, in a human NK-like cell line, YT. We show here that forskolin also induces the expression of IL-2R alpha-chain on mouse large granular lymphocytes (LGL) but not on T cells. In contrast, treatment with a combination of phorbol ester and calcium ionophore, which is a strong inducer of IL-2R alpha-chain on T cells, does not induce the expression of the alpha-chain on LGL cells. Forskolin was shown to activate the transcription of IL-2R alpha-chain gene in YT cells as revealed by the chloramphenicol acetyltransferase assay. Chemical cross-linking experiments using radio-iodinated IL-2 also supported the enhanced expression of IL-2R alpha-chain by treatment with forskolin. In contrast to the alpha-chain, IL-2R beta-chain was not induced by forskolin as revealed by flow cytofluorometry with a mAb against the beta-chain molecule. These results indicate that the activation of adenylate cyclase induces or/and enhance the expression of IL-2R alpha-chain at the transcriptional level in LGL/NK cells including mouse LGL and human YT cell, which leads to the enhanced expression of high affinity IL-2 receptors.

Many human T cell lymphotropic virus-I (HTLV-I) transformed T cells from adult T cell leukemia (ATL) patients continuously produce a humoral factor called ATL-derived factor (ADF) which induces IL-2R/Tac expression on T and NK cells. Using gel filtration, procion red Sepharose, DEAE, and reverse phase chromatography, we have purified ADF protein to homogeneity from 15 liters of serum-free culture supernatant of an HTLV-I(+) T cell line ATL-2. Purified ADF protein had the m.w. of 14,000 by SDS-PAGE and gel filtration, and its isoelectric point is around 5.0. ADF did not react with heteroantibodies against IL-1 alpha and IL-1 beta, which have also IL-2R/Tac-inducing activity on suitable target cells. Partial N-terminal amino acid sequence of ADF is different from other cytokines such as, IFN, BSF-2, and various IL whose cDNA has been cloned. Western blot analysis using rabbit antibodies against N-terminal 10mer synthetic peptide of ADF showed that IL-1 alpha and ADF are different proteins. ADF had its IL-2R/Tac-inducing activity not only on human NK-like cell line YT, but also on HTLV-I(+) T cells, such as ED. In contrast, macrophage-derived IL-1 lacked IL-2R/Tac-inducing activity on ED cells despite their IL-2R/Tac induction on YT, indicating that ADF and IL-1 have their effect via different receptors.

The effect of interleukin 1 (IL 1) on the expression of interleukin 2 receptor (IL 2R/Tac antigen) on human natural killer (NK) cells and the NK-like cell line, YT was studied with the use of a fluoresceinated anti-IL 2R monoclonal antibody and a Spectrum III flow cytofluorometer. IL 2R was expressed on approximately 10% of NK cells. The expression of IL 2R on NK cells was increased to approximately 25% by the in vitro culture with monocytes or IL 1 and to a less extent by the culture with IL 2 or interferon-gamma (IFN-gamma). IL 2R was expressed on approximately 50% of YT cells without any stimulations. The expression of IL 2R on YT cells was increased up to almost 100% by the culture with IL 1 or monocytes, but not with IL 2, IFN-alpha, IFN-beta, IFN-gamma, or lectins such as concanavalin A and phytohemagglutinin-P. IL 1 absorbed with YT cells or murine thymocytes lost both IL 1 activity detected by the stimulation of murine thymocyte proliferative response and enhancing activity of IL 2R expression on YT cells, suggesting that IL 1 has both activities. However, the assay system of the expression of IL 2R on YT cells is much more sensitive than the stimulation of murine thymocyte proliferative response. By the kinetic study, the enhancement of IL 2R expression was induced by only a 2-hr incubation of YT cells with IL 1. This enhancement did not proceed at 4 degrees C or by the treatment of YT cells with actinomycin D or cycloheximide, suggesting that this enhancement is energy dependent and requires the synthesis of RNA and protein but not DNA. Thus IL 1 plays an important role for the regulation of the expression of IL 2R on NK cells, and IL 1-dependent IL 2R expression on YT cells may give us a good model for the study of the molecular mechanism of the regulation of IL 2R expression.

A continuous cell line (YT cells) with inducible receptor for T cell growth factor (TCGF)/interleukin 2 (IL 2) was established from a 15-yr-old boy with acute lymphoblastic lymphoma and thymoma. YT cells were tetraploid, having 4q+ chromosomal markers, and proliferated continuously in vitro without conditioned medium (CM) or IL 2. They were weakly positive for OKT9, OKT11, and Tac antigen (Ag), a determinant closely associated with the receptor for IL 2 (IL 2-R), and were negative for OKT1, OKT3, OKT4, and OKT8 Ag. YT cells also expressed HNK-1 Ag and Fc receptors for IgG, which are expressed on natural killer (NK) cells. They retained a killing activity against human cell lines, including K562 (myeloid), T, and B cell lines. Unlike Tac Ag/IL 2-R(+) cell lines derived from adult T cell leukemia (ATL), YT cells were negative for HTLV, as proved by Southern blotting with cDNA for viral DNA. The expression of Tac Ag was markedly enhanced in 18 hr, when YT cells were incubated with CM from PHA-stimulated peripheral blood leukocytes (PBL) or spleen cells, as determined by immunofluorescence by using flow cytometry and binding assay with 125I-anti-Tac antibody (Ab). The binding study with 125I-labeled recombinant IL 2 showed 3.2 X 10(4) IL 2 receptor sites on YT cells precultured with CM. PHA-P and Con A neither agglutinate nor enhance the expression of IL 2-R/Tac antigen on these non-T cell line cells. Furthermore, neither recombinant IL 2 nor gamma-interferon could induce IL 2-R on YT cells, suggesting the presence of a unique IL 2-R inducing factor in PBL or spleen CM. Unlike Tac Ag on HTLV(+), ATL-derived cell lines (Hut-102, MT-1, ATL-2), the expression of Tac Ag on YT cells was down-regulated by anti-Tac Ab. The induction of Tac Ag/IL 2-R on YT cells seemed specific, because the enhancement of Tac Ag expression was not associated with that of Ia Ag and T9/transferrin receptor.

Incubation with rat IgE of rat mesenteric lymph node cells obtained 8 days after infection with Nippostrongylus brasiliensis (Nb) resulted in the formation of soluble factors with affinity for IgE, i.e., IgE-binding factors(s). The factors were derived from T lymphocytes and were able to suppress an in vitro IgE response without affecting the IgG response. The minimum concentration of rat IgE for the induction of factor formation was 0.3 to 1.0 microgram/ml. Gel filtration of culture filtrates of the IgE-containing culture identified 2 components with IgE-binding activity; one component had a m.w. of between 10,000 and 20,000, and another component had a m.w. of between 25,000 and 50,000. Both factors could be purified by binding to IgE-Sepharose followed by elution at acid pH. Among the two IgE-binding factors, the lower m.w. component had the ability to suppress selectively the IgE response. In contrast to IgE-potentiating factor, which also had affinity for IgE, the IgE-suppressive factor failed to bind to lentil lectin Sepharose. Formation of IgE-specific suppressive factor was not limited to the lymphocytes obtained 8 days after Nb-infection. Incubation with IgE of lymphocytes obtained 4 wk after infection resulted in the formation of both IgE-specific suppressive factor and IgE-potentiating factor, and the activity of the suppressive factor was greater than that of the potentiating factor.

Polyclonal antibodies against purified IgE-potentiating factor were prepared in guinea pigs, and mouse monoclonal antibodies were prepared against inactive IgE-binding factor from a T cell hybridoma. The polyclonal antibodies and two of the monoclonal antibodies bound the IgE-potentiating factor, the IgE suppressive factor, and the inactive IgE-binding factor. The results indicate that the three IgE-binding factor molecules share a common antigenic determinant. The polyclonal antibodies and the monoclonal antibody bound intracellular IgE-binding factors that did not have affinity for either lentil lectin or peanut agglutinin, which suggests that the antibodies were directed against the peptide portion of IgE-binding factors. Pretreatment of Fc epsilon R+ T hybridoma cells and Fc epsilon R+ B cells with either the monoclonal or the polyclonal anti-IgE-binding factor (anti-IgE-bF) antibody prevented rosette formation of the cells with IgE-coated erythrocytes, whereas the same treatment failed to affect rosette formation of Fc gamma R+ cells with IgG-coated erythrocytes. Specific binding of the guinea pig anti-IgE-bF antibodies to Fc epsilon R on both B and T cells was confirmed by immunofluorescence. The results indicate that Fc epsilon R on B and T cells share a common antigenic determinant with IgE-binding factors. The F(ab')2 fragments of the guinea pig anti-IgE-bF induced the expression of Fc epsilon R on Fc gamma R+ lymphocytes and the formation of IgE-binding factors. The capacities of not only IgE, but also anti-IgE-bF, to induce both Fc epsilon R expression and IgE-binding factor formation indicate that Fc epsilon R on lymphocytes, rather than IgE, transmit the signal for the formation of IgE-binding molecules.

Fc epsilon R-bearing lymphocytes were induced in vitro by the culture of peripheral lymphocytes from ragweed-sensitive patients with antigen E together with, or followed by, human IgE. Neither antigen E alone nor IgE alone induced Fc epsilon R(+) cells. As expected, culture of normal lymphocytes with the antigen and IgE failed to increase Fc epsilon R(+) cells. However, mixed lymphocyte cultures from two normal individuals responsed to human IgE for the induction of Fc epsilon R(+) cells. In both antigen-stimulated and mixed lymphocyte cultures, a portion of Fc epsilon R(+) cells induced in vitro were T lymphocytes. Evidence was obtained that Fc gamma R(+) cells are involved in the induction of Fc epsilon R(+) cells. Depletion of Fc gamma R(+) cells in the lymphocytes from ragweed-sensitive patients, before culture with antigen E and IgE, abolished the ability of the lymphocytes to respond to IgE for an increase in Fc epsilon R(+) cells.

The regulation of human low affinity FcR for IgE (Fc epsilon R2/CD23) and the soluble Fc epsilon R2 [IgE binding factor (BF)] of monocyte (U937), T (ED), and B (JIJOYE) cell lines was examined by anti-Fc epsilon R2 mAb (H107, Mab176) and the cDNA probe for Fc epsilon R2. The effect of IL-4 and IFN-gamma on Fc epsilon R2 regulation was variable among these three cell lines. IL-4 and IFN-gamma enhanced the Fc epsilon R2 gene expression and the production of Fc epsilon R2 and IgE-BF on U937, whereas IL-4 and IFN-gamma had no significant effect on the Fc epsilon R2 expression on ED. On JIJOYE, IL-4 enhanced the Fc epsilon R2 and IgE-BF production on both protein and mRNA levels. In U937 and JIJOYE cells, there was a marked increase of Fc epsilon R2 mRNA after combined stimulation with IFN-gamma and IL-4. However, in JIJOYE cells, there was a dissociation between the surface expression of Fc epsilon R2 and Fc epsilon R2 mRNA treated with IFN-gamma plus IL-4. In these cells. IFN-gamma even down-regulated the IL-4-induced expression of surface Fc epsilon R2. Stimulation of JIJOYE cells with both IFN-gamma and IL-4 resulted in the increase of the IgE-BF in the supernatant, suggesting that IFN-gamma enhanced the release of IgE-BF from Fc epsilon R2. The results indicated that Fc epsilon R2 and IgE-BF expression is regulated by IFN-gamma at least on two different levels: on transcriptional levels and the levels of cleavage of the surface Fc epsilon R2 to release soluble Fc epsilon R2 (IgE-BF). Ligands binding to the Fc epsilon R2 such as IgE and anti-Fc epsilon R2 mAb enhanced the surface expression of Fc epsilon R2 on these Fc epsilon R2(+) cell lines. This was mainly due to the surface accumulation of the receptors on JIJOYE and U937. However, the stimulation of ED by H107 and anti-Fc epsilon R2 mAb significantly enhanced the mRNA expression, indicating that Fc epsilon R2 synthesis may also be up-regulated by the specific ligands in some cell types.

Attempts were made to induce the formation of soluble factors having affinity for IgE (IgE-binding factors) by activated T cells. Normal rat mesenteric lymph node (MLN) cells were cultured in 1 microgram/ml Con A for 48 hr or in 10 microgram/ml Con A for 72 hr, and Con A-activated lymphocytes were incubated with rat IgE. It was found that IgE induced an increase in the proportion of Fc epsilon R(+) cells in the Con A-activated cells and formation of IgE-binding factors. However, the nature of IgE-binding factors formed by the cells were different depending on the concentration of Con A for activation. Thus, IgE-binding factors formed by 10 microgram/ml Con A-activated cells had a high affinity for lentil lectin and enhanced the IgE-forming cell response of DNP-OA primed cells. In contrast, the majority of IgE-binding factors formed by 1 microgram/ml Con A-activated cells failed to bind to lentil lectin and suppressed the IgE response. Induction of Fc epsilon R by IgE on Con A-activated cells was prevented by tunicamycin, which inhibits protein glycosylation. This antibiotic did not prevent the IgE-induced formation of IgE-binding factors but changed the nature of the factors formed by 10 microgram/ml Con A-activated cells. The majority of IgE-binding factors formed in the presence of tunicamycin lacked affinity for lentil lectin and Con A, and suppressed, rather than enhanced, the IgE response. The nature and amount of IgE-binding factors formed by 1 microgram/ml Con A-activated cells was not affected by tunicamycin. The results suggest that glycosylation of IgE-binding factors during their biosynthesis is an important step in determining their biologic function.

Peripheral lymphocytes stimulated with phytohemagglutinin (PHA-blasts) were examined for their responsiveness to exogenous interleukin 2 (IL-2). The proliferative response of PHA-blasts to IL-2 was significantly lower in patients with systemic lupus erythematosus (SLE) than in normal subjects. To clarify the reason for this defect, the expression of IL-2 receptor (IL-2R) on PHA-blasts was investigated using anti-Tac antibody and purified IL-2. Cytofluorometric analysis showed no statistical differences in the Tac positivity of PHA-blasts among normal subjects and patients with active and inactive SLE. Scatchard analysis using 125I-labeled anti-Tac monoclonal antibody revealed that the number of Tac epitopes on PHA-blasts was also not different among them. Next, the affinity of IL-2R expressed on PHA-blasts was determined by Scatchard analysis using radiolabeled IL-2 as a ligand. The number of high affinity IL-2R on the PHA-blasts was significantly decreased in patients with active and inactive SLE, as compared with normal subjects. The responsiveness of PHA-blasts to exogenous IL-2 was well correlated to the number of high affinity IL-2R, but not to the number of Tac epitopes or total IL-2R. Inasmuch as high affinity components of IL-2R are functionally active, the defective expression of high affinity IL-2R may be responsible for the T cell dysfunctions in SLE.

T2D4 murine T hybridoma cells have previously been shown to express Fc receptors (FcR) for IgG (Fc gamma R) and for IgA (Fc alpha R) and to produce an IgG binding factor (IgGBF) that suppresses IgG and IgM responses. In the present work we report on the behavior of IgA bound to T2D4 cells and on the production of IgA binding factor (IgABF) and its ability to suppress IgA antibody production. A dose-dependent binding of MOPC315 IgA with anti-TNP activity by T2D4 cells was demonstrated by rosette formation with trinitrophenylated ox red blood cells (TNP-ORBC) and fixation of iodinated DNP-BSA. IgA bound to the cells disappeared after a short-term culture of 3 hr at 37 degrees C, but not at 4 degrees C. Because this phenomenon was inhibited by 0.1% sodium azide and 100 microM dansylcadaverine, a transglutaminase inhibitor, Fc alpha R-IgA complexes seemed to be released by an active process involving receptor movement. In the culture supernatant of IgA-treated T2D4 cells, we detected a factor(s) that binds to IgA-Sepharose and competitively inhibits the binding of IgA to T2D4 cells. The factor (IgABF) failed to inhibit the rosette formation of Fc gamma R(+) cells with IgG-sensitized ORBC (EAox gamma), indicating that it binds specifically to IgA. IgABF was undetectable in the culture supernatants of untreated T2D4 cells of Fc alpha R(-) BW5147 T lymphoma cells used as parent cells for the establishment of the hybridoma. To study the effect of IgABF on antibody formation, culture filtrates of IgA-treated or untreated T2D4 cells were fractionated on IgA-Sepharose beads and were added to BALB/c spleen cells cultured with pokeweed mitogen. By use of a reverse plaque assay, it was shown that the IgA plaque-forming cell (PFC) response was suppressed by the acid eluate but not by the effluent of IgA-Sepharose beads incubated with the filtrates of IgA-treated T2D4 cell cultures. The suppression was IgA specific, because neither IgG nor IgM responses were suppressed by the eluate. As expected, there was no significant IgA suppressive activity in the acid eluates of the beads incubated with the culture filtrate of untreated T2D4 cells or IgA-treated BW5147 cells. IgA-specific suppressive activity proved to be due to IgA binding factor(s), because suppressive activity in the eluate was completely adsorbed by IgA-Sepharose but not by IgG- nor BSA-Sepharose.

T lymphocytes in the mesenteric lymph nodes of rats infected with Nippostrongylus brasiliensis spontaneously released a soluble factor that selectively potentiated the IgE-forming cell response of antigen-primed cells to homologous antigen. The factor could enhance the IgE response of DNP-OA-primed cells to DNP-HSA and T cell-replacing factor. In contrast, the treatment of OA-primed T cells with the factor failed to enhance either the IgE or IgG response of the mixture of DNP-KLH primed cells and OA-primed T cells to DNP-OA. The results collectively suggested that the target cells of the IgE-potentiating factor are B cells. Indeed, IgE potentiating factor was absorbed by B cells rather than T cells or thymocytes. Evidence was obtained that IgE-potentiating factor could be absorbed by IgE-bearing B cells or IgE-coupled Sepharose, indicating that the factor had affinity for IgE. It appeared that the potentiating factor bound to IgE-bearing B cells and selectively enhanced the differentiation of IgE-B cells to IgE-forming cells. It was also found that the major source of the factor was Fc epsilon R-bearing T cells.

A monoclonal antibody termed anti-Tac antibody is reactive with activated and functionally mature human T cells, but not reactive with resting T cells or B cells. We found that the expression of Tac antigen on activated T cells was inhibited by the addition of anti-Tac antibody in the culture of T cells activated with Con A or alloantigen. In the mixed lymphocyte culture, the expression of Ia-like antigen on allo-activated T cells was also inhibited by anti-Tac antibody, although the antibody does not recognize Ia-like antigen. However, anti-Ia monoclonal antibody did not suppress the expression of either Tac or Ia-like antigen. In addition, Tac antigen expressed on activated T cells was modulated by anti-Tac antibody cultured with activated T cells at 37 degrees C for 48 hr. Antigenic modulation, which means a specific loss of Tac antigen from the cell surface of T cells, was detected by the indirect immunofluorescence method. Tac antigen was reexpressed on T cells after restimulation when the anti-Tac antibody was removed, whereas Ia-like antigen already expressed on allo-activated T cells was not modulated by anti-Tac antibody. These results suggest that Tac and Ia-like antigens are carried on different molecules, both of which are closely linked in expression on activated T cells. The expression of Tac antigen may be essential for the following expression of Ia-like antigen.

Spleen cells obtained from Lewis rats were cultured with 4 micrograms/ml Con A for 96 hr, and activated cells were fused with BW5147 mouse T lymphoma cells. Seven clones obtained by fusion expressed rat IL 2R. The expression of rat IL 2R on those hybrid cells could be up-regulated by IL 2 itself, ATL-derived factor, and TPA and CA++ ionophore. Those IL 2R could be down-regulated by murine MoAb ART-18 that detects rat IL 2R. All hybrid clones produced IL 2 constitutively. IL 2 produced by hybrid cells bound to its receptor and promoted the proliferation of hybrid cells in some clones. Incubation of cells with exogenous IL 2 resulted in the proliferation of hybrid cells, whereas the proliferation of some clones was inhibited by exogenous IL 2, indicating that IL 2 had bifunctional properties on cell growth. Rat IL 2R from 6H2-F9 hybrid cells was studied by both one- and two-dimensional SDS-PAGE with ART-18. The IL 2R derived from 6H2-F9 cells had 72,000 to 77,000 and 40,000 to 48,000 m.w. major components under nonreducing conditions, and had 50,000 to 56,000 m.w. major and 35,000 to 38,000 m.w. minor components under reducing conditions. The 110,000 m.w. component, the third component of IL 2R, was constantly observed in 6H2-F9 hybrid cells.

The proportion of lymphocytes bearing receptors for IgE (FcepsilonR) markedly increased after infection of rats with Nippostrongylus brasiliensis (Nb). The FcepsilonR-bearing lymphocytes from the infected animals bound more IgE-coated erythrocytes in rosette assay than FcepsilonR-bearing cells from normal rats, suggesting that the number of FcepsilonR per cell may also increase following the infection. In contrast, the number of IgE-receptors on peritoneal mast cells did not change after Nb infection. The increase in the proportion of FcepsilonR-bearing lymphocytes in Nb-infected rats is probably due to an increased concentration of IgE in the environment. The proportion of FcepsilonR-bearing cells in normal rat lymphocyte suspensions increased by culture of the cells with rat IgE of 1 microgram/ml or higher concentration. Other immunoglobulins such as rat IgG, human IgE, or rabbit IgG failed to induce either FcepsilonR-bearing cells or FcgammaR-bearing cells. It was also found that induction of Fc receptors by rat IgE is confined to FcepsilonR. Kinetic studies on the induction of FcepsilonR-bearing lymphocytes in vitro showed that the proportion of these cells in lymphocyte suspensions increased within 8 hr incubation with rat IgE but not within 4 hr. Evidence was obtained that both RNA synthesis and protein synthesis, but no DNA synthesis, are required for the induction of FcepsilonR-bearing cells or the expression of the receptors on the cell surface.

Chemical and functional properties of IgA binding factor(s) (IgA-BF) from both murine Con A-activated spleen cells and Fc gamma R+, Fc alpha R+ T hybridoma cells (T2D4) were studied. IgA-BF produced from the cells after preculture with IgA were purified with IgA-Sepharose. Purified IgA-BF inhibited the binding of IgA to Fc alpha R+ L5178Y T lymphoma cells, and class-specifically suppressed in vitro IgA synthesis of the pokeweed mitogen (PWM)-stimulated murine spleen cells. Both IgA-specific suppressive activity and IgA binding activity of the factor(s) were co-fractionated between BSA and OVA in gel filtration analysis. SDS-PAGE analysis of IgA-BF biosynthetically labeled with [35S]methionine showed a specific band on 56,000. Suppressive activity of IgA-BF was absorbed with lentil-lectin-Sepharose and was eluted with 0.2 M alpha-methyl-D-mannoside. The suppressive activity obtained from T2D4 cells (H-2k) and BALB/c Con A blasts (H-2d) was absorbed with the corresponding anti-H-2 and anti-I-A column and recovered in the acid-eluate. The activity was not absorbed with the unrelated anti-H-2 column. Despite the presence of MHC products, IgA-BF from both cell sources equally suppressed IgA-specific responses of BALB/c (H-2d), C3H/He (H-2k), and C57BL/10 (H-2b) spleen cells. They also suppressed IgA production as well as IgA synthesis of PWM-stimulated culture of human peripheral blood lymphocytes without affecting IgM and IgG responses. Suppression of murine and human IgA responses both in mouse and human were mediated by the molecules having the same Ia products, suggesting that there is no MHC, as well as species restriction, for the interaction between IgA-BF and their target cells. IgA-specific suppressive activity was absorbed with human B blastoid cells bearing surface IgA (Dakiki) but not with those bearing surface IgG (CESS) or murine and human T cell line cells (BW5147, L5178Y, HPB-ALL, and MOLT4), indicating that IgA-BF interact with B cells bearing IgA to suppress their differentiation.

The regulation of interleukin 1 (IL 1) receptor expression on a human large granular lymphocyte cell line, YT, and fate of internalized 125I-labeled IL 1 beta (125I-IL 1 beta) were studied. YT cells were selected for this study, because this cell line expresses a large number of specific high-affinity receptor for IL 1, responds biologically to exogenously added IL 1 by expressing high-affinity IL 2 receptors, and does not produce IL 1. YT cells constitutively express approximately 7 X 10(3) IL 1 receptors/cell with a Kd approximately 10(-10) M. Neither IL 2, phorbol myristic acid, nor lipopolysaccharide affected the total binding of 125I-IL 1 beta by YT cells. In contrast, the capacity of YT cells to bind 125I-IL 1 beta when incubated at 37 degrees C for 3 to 16 hr with a low dose of purified IL 1 beta (approximately 6 U/ml) was reduced by greater than 80%. The loss of binding capability gradually recovered by 16 hr after removal of IL 1 beta from cultured YT cells. The apparent loss of IL 1 receptor expression was accompanied by the internalization of 125I-IL 1 beta into cells. Acid treatment of YT cells to remove bound 125I-IL 1 beta at 4 degrees C showed that 50% of the 125I-IL 1 beta bound to cells could no longer be recovered after 30 min at 37 degrees C, and this increased to 80% after 3 hr at 37 degrees C. Fractionation of cell extracts on Percoll gradient additionally showed 125I-IL 1 beta to appear intracellularly after receptor binding on plasma membranes, and to be successively transferred to some membranous organelles (d approximately equal to 1.037) through an intermediate density organelle (d approximately equal to 1.050), and to finally end up in lysosomal cell fractions (d approximately equal to 1.05 to 1.08) after approximately 3 hr at 37 degrees C. Only approximately 5% of internalized 125I-IL 1 beta was released into culture media by 6 hr of incubation at 37 degrees C. However, the radioactivity in the TCA soluble fraction of the culture media increased gradually by 6 hr and a lysosomotropic enzyme, ethylamine, significantly inhibited both the transfer of internalized 125I-IL 1 beta to the lysosomal fraction and the degradation of 125I-IL 1 beta. This study represents the first evidence of autoregulation of IL 1 receptors by IL 1 and internalization of IL 1 molecules after binding to receptors.(ABSTRACT TRUNCATED AT 400 WORDS)

We used two human cell lines, NK-like YT-C3 and an EBV-containing B cell line, 3B6, as models to study the receptor(s) for IL-1. Two distinct types of saturable binding sites were found on both cell lines at 37 degrees C. Between 1 pM and 100 pM of 125I-IL-1-alpha concentration, saturable binding sites were detected on the YT-C3 cells with a K of 4 x 10(-11) M. The K found for the IL-1-alpha binding sites on 3B6 cells was 7.5 x 10(-11) M. An additional binding curve was detected above 100 pM on YT-C3 cells with a K of 7 x 10(-9) M and on 3B6 cells with a K of 5 x 10(-9) M. Scatchard plot analysis revealed 600 sites/cell with high affinity binding and 7000 sites/cell with low affinity for YT-C3 cells and 300 sites/cell with high affinity binding and 6000 sites/cell with low affinity for 3B6 cells. At 37 degrees C, the internalization of 125I-labeled IL-1 occurred via both high and low affinity IL-1R on both YT-C3 and 3B6 cells, whereas the rates of internalization for high affinity binding sites on YT-C3 cells were predominant in comparison to that of low affinity binding sites. In chemical cross-linking studies of 125I-IL-1-alpha to 3B6 and YT-C3 cells, two protein bands were immunoprecipitated with Mr around 85 to 90 kDa leading to an estimation of the Mr of the IL-1R around 68 to 72 kDa. In similar experiments, the Mr found for the IL-1R expressed on the murine T cell line EL4 was slightly higher (around 80 kDa). Whether these distinct affinity binding sites are shared by a single molecule or by various chains remains to be elucidated.

A hybridoma-producing monoclonal antibody blocking the binding of human IgE to lymphocytes Fc receptor (Fc epsilon R) was established by the fusion of murine myeloma cells. P3X63.653.Ag8, with BALB/c spleen cells immunized with Fc epsilon R(+) human B lymphoblastoid cell line cells, RPMI1788. A clone of the hybridoma (H107) produced a monoclonal IgG2b antibody that inhibited the rosette formation of Fc epsilon R(+) human B lymphoblastoid cell line cells (RPMI1788, RPMI8866, CESS, Dakiki, and IM9) with fixed ox red blood cells (ORBC) conjugated with human IgE (IgE-ORBC). In contrast, the rosette formation with IgG-conjugated ORBC (IgG-ORBC) on Fc gamma R(+), Fc epsilon R(-) Daudi cells were not affected by the H107 antibodies. A close association of Fc epsilon R and the antigenic determinant recognized by H107 antibody was suggested by the following results. First, the bindings of 125I-labeled IgE (125I-IgE) or 125I-labeled H107 IgG2b antibody (125I-H107) to RPMI8866 cells were inhibited by cold human IgE and H107 IgG2b but not by other classes of human Ig (IgA and IgG), MPC11 IgG2b, or unrelated monoclonal antibodies. Second, H107 antibody reacted with Fc epsilon R(+) B cell lines but not with Fc epsilon R(-) B cell lines as determined by an indirect immunofluorescence. Third, Fc epsilon R(+) cells were depleted by the incubation in the dish coated with H107 antibody or IgE but not in the dish coated with unrelated antibodies. Finally, there was a correlation between the increase of Fc epsilon R(+) cells and that of H107(+) cells in the peripheral blood lymphocytes of the patients with atopic dermatitis. The surface antigens on Fc epsilon R(+) RPMI8866 cells recognized by H107 antibodies had the molecular size of 45,000 as determined by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis.

Culture of mesenteric lymph node cells from rats infected with Nippostrongylus brasiliensis resulted in the release of a soluble factor (IgE-binding factor) that can inhibit rosette formation of Fc epsilon R(+) lymphocytes with IgE-coated red cells. The factor is specifically absorbed with IgE-coated Sepharose. It has a m.w. of between 10,000 and 20,000. The major source of IgE-binding factor appears to be Fc epsilon R(+) T cells. The formation of IgE-binding factor by the lymphocytes was enhanced by IgE added to the culture medium. Evidence was obtained that Fc epsilon R(+) cells are involved in the induction of IgE-induced factor formation. Normal rat lymphocytes cultured alone failed to release IgE-binding factor, but incubation of normal lymphocytes with rat IgE resulted in the formation of IgE-binding factor and an increase in the proportion of Fc epsilon R(+) cells. It was also found that normal T cells formed the soluble factor upon incubation with IgE. In the induction of factor formation by normal lymphocytes, Fc gamma R(+) cells are essential; an Fc gamma R-depleted fraction failed to form IgE-binding factor upon incubation with IgE. The results suggest that interaction of IgE with Fc epsilon R(+) T cells and Fc gamma R(+) T cells induces the formation of IgE-binding factor(s).

An i.p. injection of Bordetella pertussis vaccine (BP) into rats induced the formation of soluble factors that had affinity for IgE (IgE-binding factors). The factor was detected in the serum of BP-treated animals 5 to 7 days after the treatment. Their circulating lymphocytes as well as spleen cells spontaneously released IgE-binding factors in the serum of BP-treated rats and those released from their circulating lymphocytes had affinity for lentil lectin, and the ability to selectively potentiate an in vitro IgE response of DNP-OA primed cells to homologous antigen. The molecular size of IgE-potentiating factor was between 10,000 and 20,000, and was comparable to that formed by lymphocytes of rats infected with Nippostrongylus brasiliensis. Evidence was obtained that IgE-potentiating factor was derived from Fc epsilon R(+) T cells, with a T cell marker identified by monoclonal antibody W 3/25. Their production of IgE-potentiating factor may be the basis of the adjuvant effect of BP on the IgE response.

A B cell hybridoma secreting monoclonal antibody against human IgE binding factors was obtained by immunization of BALB/c mice with partially purified IgE binding factors, and fusion of their spleen cells with SP-2/0-AG14 cells. The monoclonal antibody bound all of the 60,000, 30,000, and 15,000 dalton IgE binding factors from two T cell hybridomas and those from activated T cells of a normal individual. The antibody bound both IgE-potentiating factors, which had affinity for lentil lectin, and IgE-suppressive factors, which had affinity for peanut agglutinin. However, the monoclonal anti-IgE-binding factor bound neither Fc epsilon R on RPMI 8866 cells nor IgE binding factors from the B lymphoblastoid cells. A monoclonal antibody against Fc epsilon R on B cells (H107) bound the 60,000 and 30,000 dalton IgE binding factors from both T cell hybridomas and RPMI 8866 cells but did not bind the 15,000 dalton IgE binding factors from either T cells or B cells. The results indicate that T cell-derived IgE binding factors have a unique antigenic determinant that is lacking in both Fc epsilon R on B cells and B cell-derived IgE binding factors. The anti-IgE binding factor and anti-Fc epsilon R monoclonal antibodies both failed to stain cell surface components of IgE binding factor-producing T cell hybridomas. However, both antibodies induced the T cell hybridoma to form IgE binding factors. The results suggest that the T cell hybridomas bear low numbers of Fc epsilon R that share antigenic determinants with IgE binding factors secreted from the cells.

An NK-like cell line (YTN10 cells) was stimulated with IL-1, and the IL-2R, as detected by flow cytometry analysis using fluorescein-labeled anti-IL-2R (Tac) antibody, increased displaying a dose-response curve in the shape of an inverted "U." This was associated with increases in 1) the number of high affinity binding sites for IL-2 (Kd approximately 45 pM) which increased from 1270 to 4220 sites per cell, and 2) the number of binding sites for anti-Tac antibody, which increased from 999 to 6763 sites per cell. They were both detected by using radiolabeled ligand-binding methods which are one to three orders of magnitude more sensitive than flow cytometry analysis. No binding sites with low affinity for IL-2 could be detected on untreated or treated cells. However, a change was noted in the number of IL-2 binding sites with intermediate affinity (Kd approximately 1000 pM), which increased from 54,000 to 67,000 sites per cell. This binding affinity is equivalent to that of the p70 IL-2-binding protein detected on YT cells and on large granular lymphocytes by other investigators. This suggests that the increase in the Tac peptide was the limiting factor in the combination of the Tac and p70 components to form the high affinity IL-2R. The corollary is that all new Tac molecules were incorporated into these R. The IL-1 treatment also induced an increase in production of Tac mRNA as detected by Northern blots. Comparison of the relative amounts of Tac mRNA by dot blots verified the quantitative difference between treated and untreated cells. These data demonstrate that the IL-1-induced positive regulation of the Tac protein and high affinity IL-2R on YTN10 cells may operate at the level of gene transcription.

Human interleukin 2 receptors ( IL2R ) from various cell sources such as mitogen-activated normal human T cells, adult T cell leukemia (ATL)-derived cell line cells (MT-1, ATL-6, Hut-102), and a natural killer-like cell line YT cells, were studied both by one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), with a monoclonal anti- IL2R antibody (anti-Tac). In synthetic labeling with [35S]methionine, anti-Tac specifically precipitated two glycoproteins, one with m.w. of 60,000 to 65,000 and pI 4.2 to 4.7, and the other with m.w. about 40,000 and pI 6.2 to 6.5. A study of surface iodination revealed that the former component was membrane-associated mature IL2R , and a pulse-chase study showed that the latter component was a precursor for IL2R that already possessed tunicamycin-sensitive N-linked sugar side chain(s). This precursor was found to be posttranslationally processed into mature IL2R by further glycosylation within 240 min. Among the cells studied, a human T cell leukemia virus-positive leukemic cell line, Hut-102, was distinctive in that it possessed an additional membrane glycoprotein (m.w. 55,000 to 60,000, pI 4.2 to 5.0) also defined by anti-Tac. This unique glycoprotein of Hut-102 cells appeared to be an aberrant IL2R .

Previous experiments have shown that Fc epsilon receptor-bearing (Fc epsilon R(+)) T lymphocytes in mesenteric lymph nodes (MLN) of rats infected with Nippostrongylus brasiliensis (Nb) release a soluble factor that selectively potentiates the IgE response. The IgE-potentiating factor has affinity for IgE, and can be detected by the ability to inhibit rosette formation of Fc epsilon R(+) cells with IgE-coated erythrocytes. The factor was bound to IgE-coated Sepharose and was eluted from the beads at acid pH. It was also found that the IgE-potentiating factor binds to lentil lectin-Sepharose, indicating that the factor is a glycoprotein. The affinity of the factor for IgE was lost after treatment with trypsin but was maintained after treatment with neuraminidase. However, the ability of the factor to potentiate the IgE response was lost after neuraminidase treatment. The results suggested that the factor's binding site for IgE is associated with a protein (peptide) moiety but that its carbohydrate moiety is essential for its biologic activity. When MLN cells were incubated at 37 degrees C, a substantial amount of the IgE-potentiating factor was released into culture medium within 4 hr even in the presence of cycloheximide. Pretreatment of the cells with trypsin, which removed Fc epsilon R, markedly diminished the release of IgE-potentiating factor, suggesting that the factor is derived from Fc epsilon R on the cell surface.

Several mouse IL 3-dependent cell lines, IC2, LT4, FDC-P2, and PB-3C, derived from spleen or bone marrow cells were shown to express low affinity receptors for IL 2 (Kd; 0.5 to 8 X 10(-8) M). High affinity receptors for IL 2 were not detected on the IL 3-dependent cells within the experimental limitation of this study. The clones did not respond to IL 2 at all at the concentration as high as 25 micrograms/ml. The number of the receptors expressed on those clones was estimated to be 0.2 to 2 X 10(5)/cell, which is comparable with the number of those on IL 2-dependent T cell clones. Expression of IL 2 receptor was confirmed in mRNA levels for both IC2 and LT4 cells. A relatively low level expression of one (4.5 Kb) of four IL 2 receptor mRNA species was observed with those IL 3-dependent clones compared with IL 2-dependent T cells. It seems that these low affinity receptors may be expressed on IL 3-dependent cells that undergo differentiation or maturation in mast cell and some myeloid cell lineages.

Incubation of rat lymphocytes with homologous IgE induced an increase in Fc epsilon R(+) lymphocytes and the formation of IgE-binding factors. Pretreatment of rat lymphocytes with 1 to 5 microM dexamethasone, however, prevented the IgE-induced expression of Fc epsilon R on both B and T lymphocytes. Upon incubation with IgE, T cells activated with 10 micrograms/ml Con A produced IgE-potentiating factors that had affinity for lentil lectin and selectively enhanced the IgE response. Pretreatment of the Con A-activated T cells with dexamethasone, before incubation with IgE, changed the nature of IgE-binding factors formed by the cells. The majority of IgE-binding factors formed by the dexamethasone-treated, Con A-activated cells failed to bind lentil lectin Sepharose and selectively suppressed the IgE response. An injection of 0.2 mg dexamethasone into rats infected with Nippostrongylus brasiliensis markedly diminished the proportion of Fc epsilon R(+) lymphocytes in their mesenteric lymph nodes. The lymph node cells from the infected animals spontaneously released IgE-potentiating factors in vitro. However, the majority of IgE-binding factors formed by the mesenteric lymph node cells from the dexamethasone-treated, Nb-infected animals lacked affinity for lentil lectin and selectively suppressed the IgE response. The results indicate that glucocorticoid treatment prevented the glycosylation of IgE-binding factors and thereby changed the biologic activities of the factors.

To study the expression of FcR specific for IgA (Fc alpha R) on human peripheral lymphocytes (PBL), PBL from normal donors were incubated with 300 to 500 micrograms/ml MOPC 315 IgA having anti-trinitrophenyl (TNP) antibody activity at 4 degrees C or 37 degrees C for 60 min. Under this condition, less than 2% of total cells could form rosettes with TNP-coated ox red blood cells (TNP-ORBC). When cultured with MOPC 315 IgA at 37 degrees C for 18 hr, however, there was a dose-dependent increase of the rosette-forming cells (RFC) binding TNP-ORBC. Because 15 to 20% of the total cells bound TNP-ORBC but not unsensitized ORBC, the rosette formation appeared to be due to the cytophilic binding of IgA to the cells. The binding of MOPC 315 IgA was competed by TEPC 15 IgA and human myeloma IgA, but not by murine myeloma proteins of other classes, indicating that the receptor is specific for IgA. Fc alpha R was induced on 15 to 20% of fractionated T and B cells, as well as on 15 to 18% of concanavalin A-(Con A) activated lymphocytes when cultured with IgA. The induction of the receptor was dependent on protein and RNA synthesis, but not on DNA synthesis as suggested by the sensitivity to metabolic inhibitors, such as mitomycin C, actinomycin D, puromycin, and cycloheximide. In five patients with selective IgA deficiency (serum IgA, 0 to 4 mg/dl), only 5.1% +/- 1.7 of PBL formed rosettes with TNP-ORBC after culture with MOPC 315 IgA, whereas 12.5% +/- 2.5 of PBL from normal donors (serum IgA, 90 to 330 mg/dl) formed rosettes. Fc alpha R was induced on more than 15% of the cells from these patients, however, when cultured with IgA in the presence of a conditioned medium obtained from mixed lymphocyte culture from two normal donors. The results suggested that the abnormality in the patients' PBL might be in the induction mechanism rather than in the number of precursor cells that could express Fc alpha R in the presence of IgA. On the other hand, Fc alpha R was induced on 10.4% +/- 1.5 of PBL from the patients with IgA nephropathy (serum IgA, 382 +/- 11 mg/dl) when they were incubated with IgA for 1 hr at 37 degrees C. Because Fc alpha R on normal PBL was not induced by 1 hr of incubation with IgA, it appeared that the receptor was already expressed in vivo on the cells of these patients.

Mesenteric lymph node (MLN) cells from rats treated with complete Freund's adjuvant (CFA) spontaneously released soluble factors, which have affinity for IgE (IgE-binding factors). The factors were purified by absorption of culture filtrates of the lymphocytes with IgE-Sepharose followed by elution at acid pH. Purified IgE-binding factors selectively suppressed IgE-forming cell response of DNP-ovalbumin-primed MLN cells to homologous antigen without affecting the IgG2 response. Fractionation of purified IgE-binding factors on lentil lectin-Sepharose showed that the majority of IgE-binding factors did not have affinity for lentil lectin, whereas a minor fraction was bound to the lectin-coupled beads. It was found that IgE-binding factors lacking affinity for lentil lectin suppressed IgE response, whereas those eluted from lentil lectin-Sepharose selectively enhanced IgE response. Serum of CFA-treated animals also contained both IgE-suppressive factor and IgE-potentiating factor, and the amount of the former factor was more than the latter. The source of IgE-suppressive factor is non-B lymphocytes. Depletion of Fc, receptor(+) lymphocytes in MLN cells from CFA-treated animals did not affect the formation of IgE-suppressive factor, indicating that Fc epsilon R-bearing cells are not the source of the factor.