Immunomodulatory role of IL-4 on the secretion of Ig by human B cells

The effect of IL-4 on the production of Ig by human B cells was examined. Highly purified B cells were stimulated with Staphylococcus aureus (SA) and IL-4 alone or in combination with various other cytokines and the supernatants assayed for Ig by isotype-specific ELISA. IL-4 (10 to 100 U/ml) did not support Ig secretion by SA-stimulated blood, spleen, or lymph node B cells, whereas IL-2 supported the production of all isotypes including IgE. Moreover, IL-4 suppressed the production of all isotypes of Ig by B cells stimulated with SA and IL-2 including IgG1, IgG2, and IgE. IL-4-mediated suppression was partially reversed by IFN-gamma or -alpha and low m.w. B cell growth factor. TNF-alpha and IL-6 did not reverse the IL-4-induced suppression of Ig production. The inhibitory action of IL-4 on Ig production appeared to depend on the polyclonal activator used to stimulate the B cells. Thus, Ig secretion by B cells activated by LPS and supported by IL-2 was not inhibited by IL-4. Whereas IL-4 alone supported minimal Ig production by LPS-activated B cells, it augmented production of all Ig isotypes in cultures stimulated with LPS and supported by IL-2. IFN-gamma further enhanced production of Ig in these cultures. When the effect of IL-4 on the responsiveness of B cells preactivated with SA and IL-2 was examined, it was found not to inhibit but rather to promote Ig production modestly. A direct effect of IL-4 on the terminal differentiation of B cells was demonstrated using B lymphoblastoid cell lines. IL-4 was able to enhance the Ig secreted by an IgA-secreting hybridoma, 219 and by SKW6-CL-4, an IL-6-responsive IgM-secreting EBV transformed B cell line. These results indicate that IL-4 exerts a number of immunoregulatory actions on human B cell differentiation. It interferes with the activation of B cells by SA and IL-2, but promotes the differentiation of preactivated B cells, B cell lines, and B cells activated by LPS without apparent isotype specificity.     

Regulation of isotype production by IL-4 and IL-5. Effects of lymphokines on Ig production depend on the state of activation of the responding B cells

The effects of IL-4 and IL-5 on the production of Ig of different isotypes was investigated. We compared B cells from spleen and from Peyer's patches either stimulated with LPS or without added polyclonal stimulation. We also compared high density (small) and low density (large) B cells. The effect of lymphokines depended on the size and source of the B cells as well as on whether LPS was added. As expected, small B cells from either lymphoid compartment responded to LPS alone and IL-4 suppressed IgM and IgG3 production and enhanced IgG1. In contrast, when large B cells were examined, the suppressive effects of IL-4 were much less apparent but the enhancement of IgG1 was still marked. IL-5 alone had only minimal effects in LPS-stimulated cultures but the combination of IL-4 plus IL-5 appeared to overcome much of the IL-4-mediated suppression of IgM, and IgA production was enhanced. In the absence of LPS, a quite different profile is seen. First, small B cells make little if any response. Second, there is dramatic synergy between IL-4 and IL-5 in the response of large B cells, which is independent of isotype. Third, IL-4 does not suppress any isotype in the absence of LPS. Fourth, IL-4 plus IL-5 stimulate large Peyer's patch B cells to produce 10 times more IgA but three times less IgM than large spleen B cells. Fifth, Th2 cells directly stimulate both large and small B cells.     

Kinetics of interleukin-4 induction and interferon-gamma inhibition of IgE secretion by Epstein-Barr virus-infected human peripheral blood B cells.

Interleukin-4 (IL-4) acts directly on purified human peripheral blood B cells cultured in the presence of Epstein-Barr virus (EBV) to induce IgE secretion and to enhance the secretion of IgG and IgM. Interferon-gamma (IFN-gamma) inhibits IgE secretion in this system, without affecting the secretion of the other Ig isotypes. To identify the time period during which EBV-infected B cells can be induced by IL-4 to secrete IgE, we have studied the effects of delayed addition of IL-4, or the termination of IL-4 stimulation by wash out or by neutralization with anti-IL-4 antibodies, on the induction of an IgE response. To induce a maximal IgE response, IL-4 had to be added to cultures of B cells plus EBV no later than 2 days after the initiation of culture, and had to remain present through the tenth day of culture. These two time points correspond to the initiation of detectable DNA synthesis (Days 3 to 4) and the earliest detectable Ig secretion (Days 10 to 12) by EBV-stimulated B cells. No IgE response was induced if the period during which EBV-stimulated B cells were cultured with IL-4 was less than 4 days, or if IL-4 were added later than the tenth day of culture, regardless of how long the culture was continued beyond that time. In contrast, IL-4 considerably enhanced IgG and IgM secretion and B cell CD23 expression, even if it was added after the tenth day of culture. IFN-gamma strongly inhibited the IgE response of B cells cultured with IL-4 plus EBV if added within 6 days of the initiation of culture, but had little effect on the generation of IgM or IgG responses made by these cells, regardless of the time of addition. Neither IL-4 nor IFN-gamma affected ongoing IgE secretion by an established, IgE-secreting, EBV-transformed cell line. These observations suggest that: (i) IL-4 first becomes able to induce EBV-activated B cells to secrete IgE as these cells begin to synthesize DNA, must stimulate B cells for at least 4 days to induce IgE secretion, and loses its ability to induce IgE secretion as these cells differentiate into Ig-secreting cells; (ii) the ability of IFN-gamma to suppress an IgE response is limited to this same time period; and (iii) IL-4 enhancement of CD23 expression and IgM and IgG secretion are independent of IL-4 induction of an IgE response.     

Dual mechanisms of potentiation of murine antigen-specific IgE production by cyclosporin A in vitro.

Concomitant administration of cyclosporin A (CsA) with Ag has been shown to augment the production of Ag-specific IgE in vivo. We demonstrate that addition of CsA also markedly potentiated Ag-specific IgE in vitro. Low doses of CsA (3 and 10 ng/ml) added at the time of culture initiation selectively enhanced Ag-specific IgE but not IgA or IgG1 production, whereas higher doses (30 ng/ml) suppressed production of all the isotypes. Augmented IgE production was found to correlate with enhanced production of IL-4 and diminished production of IFN-gamma. Delayed addition (after 2 days) of low doses of CsA to Ag-stimulated cultures did not potentiate IgE production, even though CsA differentially affected levels of IL-4 and IFN-gamma. CsA enhanced Ag-mediated cognate T/B interaction was not affected by neutralizing doses of anti-IL-4, suggesting Ag-mediated lymphocytic "synapses" may be inaccessible to anti-IL-4. The effect of CsA on Ag presentation was determined by pulsing peritoneal exudate cells, spleen cells, or primed B cells with Ag and low doses of CsA before incubation with primed splenocytes. Enhanced Ag-specific IgE responses were detected with no effect on IL-4 or IFN-gamma levels. Thus, our study indicates that CsA potentiation of Ag-specific IgE response is due to cumulative action of CsA on two independent pathways: first, CsA differentially modulates IL-4 and IFN-gamma levels during the early phase of cognate Th2/B cell interaction; and second, CsA directly affects APC and IgE isotype-specific amplifying cellular components without apparently affecting the secretory levels of IL-4 and IFN-gamma. Dual mechanisms of CsA-potentiated IgE production are consistent with the hypothesis of two-tiered T cell regulation of Ag-specific IgE responses.     

B cell activator. Effects on B cell expression of CD23, proliferation, and antibody secretion.

The studies herein describe a B cell hybridoma-derived, low m.w. (less than 1000 Da), hydrophilic mediator denoted B cell activator (BCA). BCA stimulates B cell expression of IgE-specific FcR (Fc epsilon RII or CD23) in a manner similar to IL-4. However, BCA can be readily distinguished from IL-4 because it does not 1) enhance B cell Ia expression; 2) bind 11B11 anti-IL-4 mAb; or 3) elicit superinduction of Fc epsilon RII expression or IgE production in cultures of LPS-activated B cells. Moreover, BCA is considerably more mitogenic than IL-4 for LPS-activated B cells and, in contrast to IL-4, lacks mitogenicity for anti-mu-activated B cells. BCA can enhance IgG2b and IgG3 production by LPS-activated B cells, responses that are suppressed by IL-4. BCA alone did not stimulate IgE and IgG1 production by LPS-activated B cells, but exerted synergistic activity when combined with IL-4 in stimulating secretion of these antibody isotypes. Finally, secondary Ag-driven IgG1, IgE, and IgA antibody responses can be stimulated by BCA in vitro. Thus, BCA appears to be a novel mediator with broad B cell activation properties.     

Antibodies to IL-3 and IL-4 suppress helminth-induced intestinal mastocytosis

Rodents infected with the nematode parasite Nippostrongylus brasiliensis (Nb) develop intestinal mastocytosis, eosinophilia, and elevated serum IgE levels. Although IL-4 and IL-5 are necessary for stimulation of IgE synthesis and eosinophilia, respectively, the cytokines that regulate gut mast cell hyperplasia have not been identified. To address this question, 6- to 8-wk-old BALB/c mice were injected on day 0 and day 7 of Nb infection with a rat anti-mouse IL-4 mAb, and with polyclonal sheep (day 0) and rabbit (day 7) anti-mouse IL-3 IgG antibodies. Additional Nb-infected mice received equal doses of isotype- and species-matched control antibodies. Mice were sacrificed on days 12 or 13 post-infection, and mucosal mast cells (MMC) in sections of the small intestine were enumerated. Nb infection induced a 25- to 40-fold increase in MMC over that observed in uninfected controls. Anti-IL-3 or anti-IL-4 alone suppressed the Nb-induced MMC response by 40 to 50%, whereas both antibodies combined suppressed the MMC response by 85 to 90%. Anti-IL-3 alone had no effect on the serum IgE levels, which were essentially abrogated in the Nb-infected mice treated with anti-IL-4. Blood eosinophilia was not affected by treatment with anti-IL-3 and/or anti-IL-4. These studies demonstrate that IL-3 and IL-4 are physiologically important stimuli of mastocytosis in vivo, and suggest therapeutic interventions that may counteract adverse host responses to allergens as well as to parasites.     

IL-4 and IL-10 are essential for immunosuppression induced by high molecular weight proteins from Ascaris suum

The extract from Ascaris suum worms (Asc) impairs Th1 and Th2 responses to a non-related antigen, i.e. ovalbumin (OVA). Its suppressive capacity is due to high molecular weight components present in a gel filtration fraction (PI). This fraction is able to elicit IL-4 and IL-10 secretion. Interestingly enough, it induces anti-PI non-anaphylactic IgG1 synthesis through the action of IL-12/IFN-gamma. Here, we investigated the down-regulation of the immune response to OVA by PI in IL-12, IFN-gamma, IL-4 or IL-10 C57BL/6 knockout mice immunized with OVA+PI in adjuvant. OVA-induced delayed-type hypersensitivity (DTH) reactions, secretion of IL-2 and IFN-gamma, and IgG1, IgG2c and IgE antibody production were suppressed by PI in wild-type mice, as well as in IL-12- or IFN-gamma-deficient mice. In contrast, PI had no effect on anti-OVA IgE production and DTH, and induced only a partial suppression of IgG1 and IFN-gamma in IL-10(-/-) mice. The experiments also showed that IL-4 was involved in the PI-induced suppression of IgG2c antibodies and IL-2 secretion. Finally, down-regulation of IFN-gamma was not seen in mice lacking both IL-4 and IL-10, i.e. IL-4(-/-) mice treated with anti-IL-10 antibodies before immunization. These results exclude the participation of IL-12 and IFN-gamma in PI-induced immunosuppression, and highlight the essential role of IL-10 in the suppression of OVA-specific Th2-related parameters, as well as the cooperation between IL-10 and IL-4 in the suppression of Th1-related parameters.     

IL-4 induces co-expression of intrinsic membrane IgG1 and IgE by murine B cells stimulated with lipopolysaccharide

IL-4 promotes IgG1 and IgE secretion by murine B cells stimulated with bacterial LPS. We show that stimulation of unprimed resting splenic B cells with LPS and 10(4) U/ml rIL-4 results in the expression of membrane (m) IgG1 and mIgE on 40 to 50% and 15 to 25% of the total B cell population, respectively, on day 4 of culture. The possibility of a significant contribution to cell surface staining by cytophilic, secreted Ig isotypes was eliminated by either the addition of anti-Fc gamma or anti-Fc epsilon R mAb during the culture or by acid treatment before staining. A similar proportion of IgE-expressing B cells are also found, after stimulation with LPS and 10(4) U/ml IL-4, by cytoplasmic staining using fluorescence microscopy. Cell sorting analysis further indicates that B cell populations that express mIgG1 and mIgE secrete these respective Ig isotypes. In addition, such cells show striking diminution in IgM secretion compared to mIgG1- or mIgE- sorted B cells. Stimulation with LPS and IL-4 (10(4) U/ml) induces co-expression of mIgG1 and mIgE on LPS-stimulated B cells; up to 75% of mIgE+ B cells co-express mIgG1 and up to 19% of mIgG1+ B cells express mIgE. This striking co-expression of mIgG1 and mIgE is mirrored by the co-expression of mIgG1 with mIgG3 and mIgG2b by B cells stimulated with LPS and 200 U/ml IL-4. Cell sorting analysis demonstrates that the B cell population that co-expresses mIgG1 and mIgE secretes both IgG1 and IgE. However, "two-color" cytoplasmic staining fails to demonstrate any B cells that simultaneously secrete both IgG1 and IgE.     

Human recombinant IL-4 induces activated B lymphocytes to produce IgG and IgM

In this report, we describe a novel biologic activity of IL-4 namely, its ability to induce activated human B cells to produce IgM. Staphylococcus aureus Cowan I-activated blasts prepared from high density tonsil B cells were found to secrete IgG and IgM, but no IgE, when cultured in the presence of rIL-4. The differentiating activity of rIL-4 was totally blocked by a neutralizing anti-IL-4 antiserum, therefore demonstrating that the IgG/IgM-inducing activity of rIL-4 was an intrinsic property of IL-4. rIL-4 was only minimally inducing Ig production of blasts prepared from low density B cells, whereas it induced B cell blasts prepared from high density B cells to secrete a high amount of Ig. Delayed additions of the neutralizing anti-IL-4 antiserum demonstrated that a 48-h contact between IL-4 and B cell blasts was required for optimal Ig production. The IL-4-mediated IgG and IgM production was neither suppressed by IFN-gamma nor by anti-CD23 mAb 25, whereas these agents have been shown earlier to inhibit IgE production of enriched B cells cultured in the presence of IL-4. These data indicate that the IgG/IgM-inducing activity of IL-4 is not regulated like the IL-4-induced IgE production by enriched B cells.     

Humoral immune functions in IL-4 transgenic mice

We have analyzed mice expressing IL-4 as a transgene, and found that expression of this lymphokine has profound effects on B cell function. B cells from transgenic mice exhibit phenotypic changes, including an increase in size and elevated expression of class II MHC. IL-4 increases the quantity of IgE produced by transgenic-derived B cells in response to LPS stimulation. In vivo, IL-4 markedly affects the serum Ig isotype repertoire. Serum levels of IgG1 and IgE are elevated, and levels of IgG2a, IgG2b, and IgG3 are depressed in IL-4 transgenic mice. Ag-specific antibody responses to immunization with hapten-carrier conjugates are also affected by IL-4. Transgenic mice show increased anti-hapten IgE and IgG1 and reduced anti-hapten IgG2a, IgG2b, and IgG3, compared with wild-type mice. Ag-specific IgE is substantially induced by T cell-dependent Ag, but not T cell-independent Ag, suggesting that cognate T-B interactions in addition to IL-4 are required for generating IgE responses in vivo. In vivo treatment with the anti-IL-4 mAb 11B11 reverses many of the isotype alterations in the transgenic mice, indicating that these changes arise as a direct consequence of IL-4 secretion.     

CD40 stimulation provides an IFN-gamma-independent and IL-4-dependent differentiation signal directly to human B cells for IgE production.

IgE induction from human cells has generally been considered to be T cell dependent and to require at least two signals: IL-4 stimulation and T cell/B cell interaction. In the present study we report a human system of T cell-independent IgE production from highly purified B cells. When human cells were co-stimulated with a mAb directed against CD40 (mAb G28-5), there was induction of IgE secretion from purified blood and tonsil B cells as well as unfractionated lymphocytes. Anti-CD40 alone failed to induce IgE from blood mononuclear cells or purified B cells. The effect of the combination of anti-CD40 and IL-4 on IgE production was very IgE isotype specific as IgG, IgM, and IgA were not increased. Furthermore, anti-CD40 with IL-5 or PWM did not co-stimulate IgG, IgM, or IgA and in fact strongly inhibited PWM-stimulated IgG, IgM and IgA production from blood or tonsil cells. IgE synthesis induced by anti-CD40 plus IL-4 was IFN-gamma independent as is the in vivo production of IgE in humans; the doses of IFN-gamma that profoundly suppressed IgG synthesis induced by IL-4, or IL-4 plus IL-6, had no inhibitory effect on anti-CD40-induced IgE production. Anti-CD23 and anti-IL-6 also could not block anti-CD40 plus IL-4-induced IgE production, but anti-IL-4 totally blocked their effect. IgE production via CD40 was not due to IL-5, IL-6 or nerve growth factor as none of these synergized with IL-4 to induce IgE synthesis by purified B cells. Finally, we observed that CD40 stimulation alone could enhance IgE production from in vivo-driven IgE-producing cells from patients with very high IgE levels; cells that did not increase IgE production in response to IL-4. Taken together, our data suggest that the signals delivered for IgE production by IL-4 and CD40 stimulation may mimic the pathway for IgE production seen in vivo in human allergic disease.     

Distinct modulatory effects of LPS and CpG on IL-18-dependent IFN-gamma synthesis

Innate cellular production of IFN-gamma is suppressed after repeated exposure to LPS, whereas CpG-containing DNA potentiates IFN-gamma production. We compared the modulatory effects of LPS and CpG on specific cellular and cytokine responses necessary for NK-cell dependent IFN-gamma synthesis. C3H/HeN mice pretreated with LPS for 2 days generated 5-fold less circulating IL-12 p70 and IFN-gamma in response to subsequent LPS challenge than did challenged control mice. In contrast, CpG-pretreated mice produced 10-fold more circulating IFN-gamma without similar changes in IL-12 p70 levels, but with 10-fold increases in serum IL-18 relative to LPS-challenged control or endotoxin-tolerant mice. The role of IL-18 in CpG-induced immune potentiation was studied in splenocyte cultures from control, LPS-conditioned, or CpG-conditioned mice. These cultures produced similar amounts of IFN-gamma in response to rIL-12 and rIL-18. However, only CpG-conditioned cells produced IFN-gamma when cultured with LPS or CpG, and production was ablated in the presence of anti-IL-18R Ab. Anti-IL-18R Ab also reduced in vivo IFN-gamma production by >2-fold in CpG-pretreated mice. Finally, combined pretreatment of mice with LPS and CpG suppressed the production of circulating IFN-gamma, IL-12 p70, and IL-18 after subsequent LPS challenge. We conclude that CpG potentiates innate IFN-gamma production from NK cells by increasing IL-18 availability, but that the suppressive effects of LPS on innate cellular immunity dominate during combined LPS and CpG pretreatment. Multiple Toll-like receptor engagement in vivo during infection can result in functional polarization of innate immunity dominated by a specific Toll-like receptor response.     

Differential inhibition of T and B cell function in IL-4-dependent IgE production by cyclosporin A and methylprednisolone

The present study examines the role of the immunosuppressive agents methylprednisolone (MPN) and cyclosporin (Cs)A on IL-4-dependent IgE and IgG production. Addition of optimal amounts of IL-4 (100 U/ml) to cultures of tonsil mononuclear cells resulted in a mean increase in IgG production of 175% and in IgE production of 2460%. Frequency analysis of IgE- and IgG-producing B cells, using an ELISA spot assay, showed parallel increases in both Ig production and numbers of Ig-secreting B cells. IgE production was also enhanced by addition of IL-2 (10 U/ml) and maximal IgE production was obtained with a combination of IL-4 and IL-2. MPN (10(-7) M) and CsA (1 microgram/ml) markedly reduced IL4-induced IgE and IgG production as well as numbers of Ig-secreting cells in a dose-dependent fashion. The suppression of Ig production by the cyclosporins was restricted to the immunosuppressive compounds CsA, CsG, and dihydro-CsD, but not the nonimmunosuppressive drug CsH. Delayed addition of CsA revealed that inhibition was maximal when the drug was added during the first 48 h after addition of IL-4 to the culture. Addition of IL-2 (10 U/ml) partially overcame the inhibition induced by CsA. In coculture experiments, in which separated T or B cells were precultured with the drugs and the cells were then combined and further incubated in the presence of IL-4, the suppressive effects of CsA on IgE production were related to pretreatment of the T but not B cells. The maximum inhibiting effects of MPN were similarly observed when the drug was present in the cultures from the beginning, and addition of IL-2 also partially reversed this inhibition. In contrast to the results with CsA, pretreatment of the B but not T cells with MPN-reduced IgE production. These studies demonstrate that IL-4 increases both numbers of IgE-secreting cells as well as IgE production and CsA and MPN differentially affect the responding T and B cells, resulting in inhibition of Ig production.     

Cellular control of IgE induction by a polyphenol-rich compound. Preferential activation of Th2 cells

The polyphenol group rutin (R) appears to influence isotype expression, because R-BSA conjugates induce anti-BSA responses in mice that show a significant decrease in hemagglutinating antibodies (HA) to BSA, as compared to mice immunized with BSA. However, the level of IgE antibodies to BSA is unaltered. To determine if suppressor cells for isotypes other than IgE are induced by R-BSA, cell transfers were performed. The results were consistent with the view that the decrease in HA titer to BSA in R-BSA immunized mice is not due to the activation of suppressor cells for isotypes other than IgE. Inasmuch as the IgE response in mice is associated with the production of IL-4 by Th2 cells, we analyzed the factors produced by spleen cells cultured with R-BSA. We found that supernatant from spleen cells cultured with R-BSA contained IL-4 as determined by the enhanced expression of Fc epsilon R (CD23) on B cells. This enhancement was inhibited by 11B11, the anti-IL-4 mAb. IL-2, a product of Th1 cells, was not detected in these supernatants. Moreover, IL-4 mRNA, but not IL-2 mRNA, was detected by Northern blot analysis of RNA from spleen cells cultured with R-BSA. Taken together the data suggest that the polyphenol containing compounds preferentially activate Th2 cells, thereby favoring IgE production.     

IgE production by normal human B cells induced by alloreactive T cell clones is mediated by IL-4 and suppressed by IFN-gamma

Seven T cell clones were established from mixed leukocyte cultures in which PBMC from two healthy donors and from one patient suffering from the hyper-IgE syndrome were stimulated by the irradiated EBV-transformed B cell lines JY or UD53. Five of seven T cell clones, after activation by co-cultivation with JY or UD53 cells, induced a low degree of IgE production by normal blood B cells. In one experiment in which the normal B cells could activate the T cell clones directly, IgE production was also observed in the absence of the specific stimulator cells. IgE production was also obtained with supernatants of the T cell clones collected 4 to 5 days after activation by their specific stimulator cells. In addition, the supernatants induced IgG, IgA, and IgM synthesis. All seven clones produced variable concentrations of IL-4 and IFN-gamma. The clones FA-28 and BG-39, which failed to induce IgE synthesis, produced, compared with the other clones tested, relatively high quantities of IFN-gamma (4700 and 2500 pg/ml, respectively). These high levels of IFN-gamma accounted for the lack of induction of IgE synthesis, because in the presence of a polyclonal anti-IFN-gamma antiserum, supernatants of FA-10 and BG-39 induced significant IgE production. In addition, the low degree of IgE production induced by supernatants of two other T cell clones (FA28 and BG24) was 15- and 3-fold enhanced, respectively, in the presence of the anti-IFN-gamma antiserum. IgE synthesis by normal B cells was also induced by rIL-4, indicating that IL-4 present in T cell clone supernatants was responsible for induction of IgE production. This notion was supported by the finding that IgE production induced by supernatant of BG-24 was strongly inhibited by a polyclonal anti-IL-4 antiserum. In contrast, IgG and IgA production induced by supernatant of BG-24 were not significantly affected by the anti-IL-4 antiserum. Only a slight inhibition of IgM synthesis was observed. Collectively, our results indicate that both recombinant and naturally produced IL-4 induce normal human B cells to synthesize IgE. However, final IgE production induced by T cell clone supernatants is the net result of the inducing and suppressive effects of IL-4 and IFN-gamma respectively, that are secreted simultaneously by the T cell clones upon activation.     

IL-21 effects on human IgE production in response to IL-4 or IL-13

In human atopic disease, IgE sensitizes the allergic response, while IgG4 is protective. Because IL-4 and IL-13 trigger switch recombination to both IgE and IgG4, additional agents must regulate the balance between these isotypes to influence susceptibility or tolerance to atopy. In this report, we define in vitro conditions leading to activation or inhibition of human IgE and IgG4 production by IL-21. IL-21 reduced IL-4-driven IgE synthesis by mitogen-stimulated human PBMC. IL-21 inhibition of human IgE production was not a direct effect on B cells, was not seen following B cell activation with IL-13, and was overcome by CD40 ligation. Neither IFN-gamma, IL-10, IL-12, CD40L expression, nor apoptosis was responsible for the inhibitory effect. In contrast, IL-21-stimulated secretion of IgG4 from PBMC. Our findings indicate that IL-21 may influence the production of both human IgE and IgG4, and thus contribute to the regulation of atopic reactions.     

Modulation of IL-4-induced human IgE production in vitro by IFN-gamma and IL-5: the role of soluble CD23 (s-CD23)

IL-4 specifically induced IgE production by peripheral blood lymphocytes or by tonsil or spleen cells from healthy donors. IL-4-induced IgE synthesis was dependent on CD4+ T cells and monocytes and was blocked by IFN-gamma, IFN-alpha, and prostaglandin E-2 (PGE-2). These substances also inhibited IL-4-induced CD23 expression and subsequent release of soluble CD23 (s-CD23). In addition, IgE production was blocked by F(ab')2 fragments of an mAb against CD23. In contrast, IL-5 enhanced IL-4-induced IgE production, provided IL-4 was added at nonsaturating concentrations. This increase in IgE production correlated quantitatively with an enhanced release of s-CD23. Collectively, these results indicate that there is a correlation between s-CD23 release and IgE production. However, s-CD23 fractionated from supernatants of the lymphoblastoid cell line RPMI-8866 was ineffective in inducing IgE production in the absence of IL-4, but acted synergistically with suboptimal concentrations of IL-4. In addition, it is demonstrated that alloreactive T-cell clones produced varying concentrations of IL-4, IL-2, or IFN-gamma upon stimulation. Only supernatants of 2/4 of these T-cell clones induced a low degree of IgE synthesis, but in the presence of anti-IFN-gamma antibodies, all four supernatants induced a strong induction of IgE production. This IgE synthesis was blocked specifically by anti-IL-4 antibodies, indicating that IL-4 is the sole inducer of IgE synthesis. Our findings demonstrate that IL-4-induced IgE production involves complex interactions of T cells, B cells, and monocytes and is positively modulated by IL-5 and s-CD23 but down-regulated by IFN-gamma, IFN-alpha, and PGE-2, respectively.     

IL-4 is an essential factor for the IgE synthesis induced in vitro by human T cell clones and their supernatants

The property of 109 CD4+ T cell clones (TCC) to induce IgE synthesis in vitro in human B cells was compared with their ability to produce IL-2, IL-4, and IFN-gamma in their supernatants (SUP) after 24-h stimulation with PHA. A significant positive correlation was found between the property of TCC to induce or enhance spontaneous IgE synthesis and their ability to release IL-4. In contrast, there was an inverse relationship between the IgE helper activity of TCC and their ability to release IFN-gamma, whereas no statistical correlation between the property to induce IgE synthesis and to produce IL-2 was observed. The ability of PHA-SUP from 71 CD4+ TCC to induce IgE synthesis in B cells was also investigated. Twenty-nine SUP (all derived from TCC active on IgE synthesis) induced production of substantial amounts of IgE in target B cells. There was a correlation between the amount of IgE synthesized by B cells in response to these SUP and their IL-4 content. An even higher correlation was found between the IgE synthesis induced by these SUP and the ratio between the amount of IL-4 and IFN-gamma present in the same SUP. Like IL-4-containing SUP, rIL-4 also showed the ability to induce IgE production in B cells from both atopic and nonatopic donors. The addition to B cell cultures of anti-IL-4 antibody virtually abolished not only the IgE synthesis induced by rIL-4, but also that stimulated by TCC and their SUP. In contrast, the IgG synthesis induced by TCC SUP was not or only slightly inhibited by the anti-IL-4 antibody. These data indicate that IL-4 is an essential mediator for the IgE synthesis induced in vitro by human TCC and their SUP in the absence of a polyclonal activator, whereas IFN-gamma seems to exert a negative regulatory effect on the production of IgE.     

Two distinct T cell subsets, CD4+ and CD8+CD60+, and their cytokines are required for in vitro induction of human ragweed-specific memory IgE responses

CD8(+)CD60(+) T cells (80-98% CD45RO(+); 20% CD23(+)) are significantly increased in the blood of serum IgE(+) ragweed-sensitized (RS) compared with serum IgE-nonatopic humans (p = 0.001). CD8(+)CD60(+) T cells of the RS patients produced IL-2, IL-4, IL-10, IL-12, IFN-alpha. and IFN-gamma, but not IL-6 or IL-13. When their PBMC were cultured with ragweed Ag (RA), peak IgE responses occurred on day 10; none was induced with non-cross-reacting or without Ag; nonatopic PBMC did not respond to any stimulant. When either CD4(+) or CD8(+)CD60(+) T cells were depleted from RS PBMC before culture with RA, no IgE responses were induced. If purified CD4(+) T cells or low numbers of CD8(+)CD60(+) T cells were added back to the depleted PBMC, IgE responses were restored. However, higher numbers of CD8(+)CD60(+) T cells totally suppressed IgE responses. Total suppression also was obtained when RS PBMC were cultured with RA and either anti-IL-2, IL-4, IL-10, IL-12, IFN-gamma (all concentrations), or IFN-alpha (low concentrations), but not anti-IL-6 or IL-13. Higher concentrations of anti-IFN-alpha potentiated IgE responses.