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IgE Versus IgG4 Production Can Be Differentially Regulated by IL-10

Geneva Biomedical Research Institute, Immunology Department, Glaxo Wellcome Research and Development SA, Geneva, Switzerland

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Allergen-specific IgE plays a key role in the physiopathology of allergic disorders. This IgE response is usually accompanied by a production of IgG4. Indirect evidence suggests that IgG4 may not be a sensitizing Ab but, in contrast, could be protective. As such, it may be of potential therapeutic interest to selectively modulate IgE vs IgG4 production. To date, IgE and IgG4 switching seems to be controlled by common mechanisms. We report here that IL-10 has a differential effect on IgE vs IgG4 production by PBMC. IL-10 decreases transcript expression and IgE production induced by IL-4 when added during the first 3 days of in vitro culture, suggesting that IL-10 decreases IL-4-induced IgE switching. In contrast, if added later on B cells that are already IgE switched, IL-10 potentiates IgE production. Interestingly, whatever the time of addition, IL-10 augments IL-4-induced 4 transcript expression and IgG4 production, with a maximal effect when added during the first 3 days. As IL-10 is not a switch factor for IgG4, it is likely that IL-10 enhances IgG4 production by potentiating IL-4-induced IgG4 switching. However, IL-10 may also act by enhancing the growth and/or differentiation of cells that are already IgG4 committed. Finally, CD40 ligation reverses the early down-regulating effect of IL-10 on IgE production. These results are the first evidence of a molecule that differentially regulates IgE vs IgG4 production, thereby suggesting the existence of a pathway(s) selectively controlling their production.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Induction of IgE switching in vitro requires two primary signals. The first one, given by IL-4 or IL-13, induces the expression of the sterile transcript (1, 2, 3). The second one, provided by the triggering of CD40 with anti-CD40 mAb, recombinant CD40 ligand (CD40L),² or membrane CD40L expressed on different cell types including activated T cells, induces the expression of the mature transcript encoding for IgE (4, 5, 6). The production of IgE is controlled by additional secondary signals provided by soluble mediators or by direct T:B cell contact. The cytokines IL-2, IL-5, IL-6, IL-9, and TNF- (7, 8, 9, 10, 11); some chemokines (12, 13); histamine (14); and the ligation of the B cell accessory molecules CD21, CD54, CD58 (15, 16, 17), CD44, and CD86 (18) enhance IgE production. Conversely, the soluble factors PGE-2, IFN-, IFN-, IL-12, and TGFß down-regulate this production (10, 19, 20). To date, all the signals recognized to be involved in IgE switching also control IgG4, suggesting that IgE and IgG4 synthesis are regulated by common mechanisms (6, 12, 13, 14, 21).

In contrast to healthy subjects, allergic patients develop specific IgE directed against sensitizing allergens that play a key role in the physiopathology of allergic diseases (22). In parallel to the IgE response, allergic patients usually produce high levels of total and allergen-specific IgG4 Ab (23, 24, 25). However, a potential role for IgG4 Ab in allergic diseases has not yet been clearly established. In allergic patients, the finding of seric IgG4 Ab directed against allergens to which the patients were not sensitive suggests that they are not sensitizing Ab (26). On the contrary, the existence of a positive correlation between a successful Hymenoptera venom desensitization and the high levels of specific IgG4 Ab generated suggests that IgG4 may be protective (25, 27, 28). Therefore, it may be of potential therapeutic interest to be able to modulate selectively the production of IgE vs IgG4.

IL-10 is a lymphokine produced by numerous cell types including activated T cells, mast cells, and macrophages. By blocking cytokine production and APC functions of monocytes and/or macrophages, IL-10 plays a major role in suppressing immune and inflammatory responses (reviewed in 29 . IL-10 also acts on human B cells activated with an anti-CD40 mAb by enhancing: 1) switching to IgA, IgG1, and IgG3 isotypes (30, 31, 32); 2) short-term proliferation (33, 34); and 3) differentiation into Ig-secreting plasma cells (33, 35). Interestingly, IL-10 decreases IgE production by IL-4-stimulated PBMC (36). The aim of this work was to evaluate the effect of IL-10 on the production of the other isotypes by IL-4-stimulated PBMC. Results showed that IL-10 has opposite effects on IgE vs IgG4 synthesis according to the nature of the stimulus and the kinetic of addition of IL-10. These data suggest the existence of a pathway selectively involved in the control of IgE and IgG4 production.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Subjects

Five patients sensitive to the house dust mite Dermatophagoides pteronyssinus were selected on the basis of the following criteria: history of allergic symptoms (asthma and/or perennial rhinitis), positive cutaneous prick tests toward house dust mite allergens (diameter >9 mm), and specific anti-D. pteronyssinus IgE. The healthy subjects (n = 7) had no history of allergic disorders.

Ig assays

PBMC were isolated by centrifugation on Ficoll-Hypaque (Pharmacia, Uppsala, Sweden). Human tonsillar B cells were purified as described (21). Surface IgD⁺ B cells were purified by cell sorting using a FACS Vantage (Becton Dickinson, Erembodegem, Belgium) after labeling with a FITC-labeled anti-human IgD mAb (Dako, Glostrup, Denmark). Purity was routinely >98%. Cells (2 x 10⁵/200 µl/well) were cultured in enriched Iscove’s medium in 96-well plates (Nunc, Roskilde, Denmark) and stimulated, in quintuplicate, with 200 U/ml of IL-4 (Geneva Biomedical Research Institute (GBRI), Geneva, Switzerland), 0.1 µg/ml of anti-CD40 mAb (Serotec, Oxford, U.K.), or combinations of IL-4 plus anti-CD40 mAb in the presence or absence of 0.4 to 50 ng/ml of IL-10 (R&D Systems, Abingdon, U.K.). In some experiments, anti-CD40 mAb was replaced by 5 µg/ml of rCD40L (GBRI). PBMC from allergic patients were stimulated as described above in the presence or absence of 1 IR/ml (biologic units used by the Laboratoire des Stallergènes, Fresnes, France) of D. pteronyssinus crude extract (Laboratoire des Stallergènes). In some sets of experiments, IL-10 was added at different time points following stimulation of PBMC with IL-4. In other sets of experiments, PBMC were stimulated with IL-4 in the presence of IL-1, IL-2, IL-3, IL-5 (all from GBRI), IL-8, IL-9, IL-11, IFN- (all from R&D Systems), IL-6, TNF- (both from AMS Biotechnology, Lugano, Switzerland), or TGFß (Sigma, St. Louis, MO). Supernatants were collected at day 12 to quantify Ig.

Ig quantification

The total Ig were quantified by ELISA. IgE and IgG4 ELISA were performed as described (21). IgA1, IgA2, IgG1, IgG2, and IgG3 were quantified as follows: 96-well plates (Nunc) were coated overnight at 4°C with 0.1 µg/well of anti-IgA1 mAb (Sigma), 0.2 µg/well of anti-IgA2 mAb (Nordic Immunological Laboratories, Tilburg, The Netherlands), 1 µg/well of anti-IgG1 mAb (clone NL16) (Oxoid Unipath, Hampshire, U.K.), 0.2 µg/well of anti-IgG2 mAb (clone HP6002), or 0.2 µg/well of anti-IgG3 mAb (clone HP6050) (both from Calbiochem, La Jolla, CA) in 0.1 M carbonate-bicarbonate buffer, pH 9.6, and then saturated with 1% (w/v) BSA in PBS. Cell culture supernatants, diluted in PBS/BSA containing 0.1% (v/v) Tween-20, were incubated for 4 h at room temperature. After washing, peroxidase-conjugated anti-human - or -chain immune sera (Sigma) were incubated at room temperature for 3 h, for quantification of IgA and IgG, respectively. After washing, bound Abs were detected using o-phenylenediamine substrate, and the colorimetric reaction was stopped with 2 M H₂SO₄. OD values were determined at 492 nm. The standard curves were done using purified human Ig (Sigma). The sensitivity of the ELISA was higher than 5 ng/ml. The specificity was verified using purified IgA1-2, IgG1-4 (Sigma), and IgE (The Binding Site, Birmingham, U.K.). Results are expressed in ng/ml, or as a percentage of increase or decrease of Ig production defined as (A - O ÷ O) x 100 or (O - A ÷ O) x 100, where A and O are the concentrations of Ig produced in the presence or absence of IL-10, respectively.

Proliferation assays

PBMC or B cells (2 x 10⁵/200 µl/well) were stimulated in triplicate in 96-well plates with IL-4 and/or anti-CD40 mAb in the presence of 50 ng/ml of IL-10. After 48 h of incubation, cells were pulsed with 0.25 µCi/well of [³H]TdR (Amersham International, Amersham, U.K.) for 6 h. Radioactive incorporation was measured by standard liquid scintillation counting. Results are given as proliferation index (PI) calculated as follows: A ÷ O where A and O were the cpm values obtained with cells stimulated or not with IL-10, respectively.

Analysis of IgE mRNA transcription by Northern blotting

PBMC were either unstimulated or stimulated for 10 days with 200 U/ml of IL-4, 50 ng/ml of IL-10, or IL-4 plus IL-10. In this last condition, IL-10 was added together with IL-4 (at day 0) or 4 days later. Total RNA extraction and hybridizations were performed with cRNA probes complementary to C and ß-actin mRNA (2). IgE protein was quantified in the supernatants by ELISA.

Analysis of mature 4 mRNA expression by PCR

The single-strand cDNA was synthesized using 5 µg of total RNA, prepared as described above, by reverse transcription using an oligo(dT) primer (Pharmacia). cDNA corresponding to 0.5 µg of total RNA were used for the amplification reactions. Each PCR tube contained 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2 mM MgCl₂, 0.01% gelatin, 2 U of AmpliTaq DNA polymerase (Perkin-Elmer Cetus, Norwalk, CT), 0.2 mM concentrations of each deoxynucleotide (Pharmacia), and 10 µM of each primer. Sequences of the oligonucleotides for the 4 mature transcripts are as follows: 5'J_H consensus 5'-ACC^C/_ATGGTCACCGTCTCCTCA-3' and 3' C4 5'-GGGACCATATTTGGACTC-3' (37, 38). Control consisted of amplifying ß-actin transcripts using the following primers: 5'-CGATTTCCCGCTCGGCCGTGGTGGTGAAGC-3' and 5'-GGCGACGAGGCCCAGAGCAAGAGAGGCATC-3'. The amplification consisted of 1 cycle for 4 min at 94°C followed by 35 cycles for 30 s at 94°C, 60°C for 1 min, and 72°C for 1 min. The final extension cycle was for 5 min at 72°C. PCR products were size separated on a 1% agarose gel and visualized by ethidium bromide staining.

Statistical analysis

Statistical significance was determined using Student’s t test.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Differential effect of IL-10 on IgE and IgG4 production by IL-4- or IL-13-stimulated PBMC from healthy subjects

Stimulation of PBMC from healthy subjects with IL-4 (Fig. 1A) or IL-13 (data not shown) induces IgE and IgG4 production (1, 3, 5). In agreement with others (36), IL-10 when added together with IL-4 (at day 0) decreased IL-4-induced germ line and productive transcript expression and IgE production (decrease of 82 ± 11%, mean ± SD, n = 6) (Fig. 1A, top, and 2A). Surprisingly, IL-10 potentiated IL-4-induced productive 4 transcript expression and IgG4 production (increase of 902 ± 136% at day 0) (Fig. 1A, bottom, and 2B). These effects of IL-10 on IgE and IgG4 production were dose dependent, in the range of 1 to 50 ng/ml (Fig. 1B). IL-10 also modulated IL-13-induced IgE and IgG4 production (decrease of 72 ± 15% and increase of 1035 ± 322%, respectively, mean ± SD, n = 3) (data not shown). Among the different cytokines tested, IL-10 was the only one that had an opposite effect on IgE vs IgG4 synthesis by IL-4-stimulated PBMC (Fig. 3). IL-10 also enhanced IgA1-2 and IgG1-3 production by IL-4- or IL-13-stimulated PBMC (increase of 157 ± 34%, 228 ± 15%, 380 ± 36%, 272 ± 96%, and 214 ± 75%, respectively, mean ± SD, n = 3) (Fig. 1C).

View larger version (31K): [in this window] [in a new window]   FIGURE 1. Effect of IL-10 on Ig production by IL-4-stimulated PBMC. A, IL-10 decreases IgE and increases IgG4 production. PBMC from healthy subjects were stimulated or not with IL-4 in the absence () or presence () of 50 ng/ml of IL-10. IgE (top) and IgG4 (bottom) were quantified in 12-day supernatants. B, IL-10 affects IgE and IgG4 production in a dose-dependent manner. PBMC were stimulated with IL-4 in the absence or presence of 0.1 to 50 ng/ml of IL-10. IgE and IgG4 (•) were quantified in 12-day supernatants. C, IL-10 increases IgA1, IgA2, and IgG1-3 production. PBMC were stimulated by IL-4 without () or with () 50 ng/ml of IL-10. IgA1, IgA2, and IgG1-3 were quantified in 12-day supernatants. A, B, and C, Results are expressed in ng/ml or in µg/ml (mean ± SD of quintuplicate values) and are representative of one of six (A) or three (B and C) experiments.

View larger version (29K): [in this window] [in a new window]   FIGURE 3. Effect of different cytokines on IgE and IgG4 production by IL-4-stimulated PBMC. PBMC were stimulated with IL-4 in the absence or presence of 50 ng/ml of different cytokines. IgE (top) and IgG4 (bottom) were quantified in 12-day supernatants. Results are expressed in ng/ml or µg/ml (mean ± SD of quintuplicate values) and are representative of one out three separate experiments.

Thus, IL-10 has a dual effect on IgE vs IgG4 production since it prevents IgE but potentiates IgG4 synthesis by IL-4- or IL-13-stimulated PBMC.

IL-10 potentiates IL-4 plus anti-CD40 mAb-induced IgE and IgG4 production by PBMC from healthy subjects

IgE synthesis by B cells requires a signaling through IL-4 and/or IL-13 receptors and CD40 (4, 5). As such, the stimulation of PBMC with both IL-4 plus anti-CD40 mAb results in a superinduction of IgE and IgG4 production. In this condition of stimulation, IL-10 increased both IgE and IgG4 production (increase of 715 ± 144% and 878 ± 122%, respectively with 50 ng/ml IL-10, mean ± SD, n = 6) (Fig. 4, A and B). IL-10 also enhanced IL-4 plus rCD40L-induced IgE and IgG4 production by PBMC (increase of 624 ± 127% and 792 ± 125%, respectively, mean ± SD, n = 3). This effect was dose dependent in the range of 1 to 50 ng/ml of IL-10 (data not shown). IL-10 also increased IgA1-2 and IgG1-3 production (increase of 611 ± 221%, 914 ± 215%, 545 ± 156%, 879 ± 211%, and 541 ± 108%, respectively, mean ± SD, n = 3) (Fig. 4C).

View larger version (21K): [in this window] [in a new window]   FIGURE 4. Effect of IL-10 on IgE and IgG4 production by IL-4 plus anti-CD40 mAb-stimulated PBMC. PBMC from healthy subjects were stimulated with an anti-CD40 mAb without or with IL-4 in the absence () or presence () of 50 ng/ml of IL-10. IgE (A), IgG4 (B), IgA1, IgA2, and IgG1-3 (C) were quantified in 12-day supernatants. Results are expressed in µg/ml (mean ± SD of quintuplicate values) and are representative of one of six (A and B) or three (C) experiments.

Differential effect of IL-10 on IgE and IgG4 production by PBMC from allergic patients stimulated by the sensitizing allergen

We have evaluated the clinical relevance of the above results on PBMC from D. pteronyssinus-sensitive patients. Resting and D. pteronyssinus-stimulated PBMC from allergic patients produced undetectable or low levels of IgE (4.5 ± 2 and 6 ± 3 ng/ml, respectively, mean ± SD, n = 5) and IgG4 (12 ± 3 and 15 ± 4 ng/ml, respectively) (Fig. 5, A and B). Addition of IL-10 significantly enhanced both IgE (22 ± 6 and 24 ± 5 ng/ml, respectively) and IgG4 production (389 ± 96 and 332 ± 72 ng/ml, respectively) (p < 0.01) (Fig. 5, A and B).

View larger version (30K): [in this window] [in a new window]   FIGURE 5. Effect of IL-10 on IgE and IgG4 production by allergen-stimulated PBMC from a D. pteronyssinus-sensitive patient. PBMC from a D. pteronyssinus-sensitive patient either unstimulated (A), stimulated with D. pteronyssinus (B), or with an anti-CD40 mAb (C) were or were not incubated with IL-4 in the absence () or presence () of 50 ng/ml of IL-10. IgE and IgG4 were quantified in 12-day supernatants. Results are expressed in µg/ml (mean ± SD of quintuplicate values) and are representative of one of five experiments.

These data indicate that in the absence of CD40 ligation, IL-10 decreases IgE and increases IgG4 production by PBMC from allergic patients specifically stimulated in the presence of IL-4.

Time-dependent effect of IL-10 on IL-4-induced IgE synthesis by PBMC

To investigate the mechanism underlying the dual effect of IL-10 on IgE synthesis, IL-10 was added at different time points to IL-4-stimulated PBMC. When added during the first 2 days following IL-4 stimulation, IL-10 prevents IgE production with a maximal effect when added at day 0 (decrease of 82 ± 12% and 35 ± 8% at days 0 and 2, respectively, n = 4) (Fig. 6A). When added later, IL-10 potentiates IgE production with a maximum occurring at days 4 or 6, according to the subject (maximal increase of 302 ± 36%) (Fig. 6A). Furthermore, IL-4-induced transcript expression was decreased when IL-10 was added at day 0, whereas it was weakly increased when added at day 4 (Fig. 2A). Thus, according to the time of addition, IL-10 presents an opposite effect on IgE production by IL-4-stimulated PBMC. In contrast, whatever the time of addition, IL-10 increased IgG4 production with a maximal effect when added at day 0 (increase of 895 ± 136% and 372 ± 57% when added at days 0 and 4, respectively) (Fig. 6B). In parallel, IL-4-induced productive 4 transcript expression was enhanced to a higher extent when IL-10 was added at day 0 rather than at day 4 (Fig. 2B).

View larger version (12K): [in this window] [in a new window]   FIGURE 6. Kinetics for the addition of IL-10 on IL-4-stimulated PBMC. PBMC from a healthy subject were stimulated at day 0 with IL-4, and IL-10 (50 ng/ml) was added at different time points. IgE (A) and IgG4 (B) were quantified in day-12 supernatants. The levels of IgE and IgG4 obtained in the absence of IL-10 are represented as horizontal hatched bars. Results are expressed in ng/ml or µg/ml (mean ± SD of quintuplicate values) and are representative of one of four experiments.

View larger version (48K): [in this window] [in a new window]   FIGURE 2. Effect of IL-10 on and 4 transcript expression by IL-4-stimulated PBMC. PBMC were either unstimulated or stimulated with IL-4, IL-10, or IL-4 plus IL-10; in this last condition, IL-10 was added together with IL-4 (day 0) or 4 days later. RNA were collected at day 10. A, The expression of germline and productive transcripts (top) and of ß-actin (bottom), used as control, was evaluated by Northern blotting. B, The expression of productive 4 and ß-actin transcripts was evaluated by reverse transcriptase-PCR. IgE (A) and IgG4 (B) were quantified in the 10-day supernatants.

IL-10 is not a switch factor for IgG4

On the basis of the above results, it was tempting to speculate that IL-10 may differentially affect IgE and IgG4 switching. As such, we have first evaluated the effect of IL-10 on IgE and IgG4 production by purified tonsillar B cells, in the absence of signals provided by T cells (Table I). B cells either unstimulated or stimulated with IL-4 or anti-CD40 mAb did not produce IgE or IgG4. In these conditions of stimulation, IL-10 induced a weak production of IgG4 (<200 ng/ml) but no IgE. However, IL-10 potentiated IL-4 plus anti-CD40 mAb-induced IgE and IgG4 production (increase of 703 ± 105% and 948 ± 203%, respectively, mean ± SD, n = 4). In parallel, in agreement with previous data, IL-10 enhanced anti-CD40 mAb and anti-CD40 mAb plus IL-4-induced B cell proliferation (SI = 2.2 ± 0.4 and 4.5 ± 0.6, respectively, mean ± SD, n = 4) (Table I) (31, 32, 33). Thus, we have determined the effect of IL-10 on IgE and IgG4 production by naive surface IgD⁺ B cells. IL-10 did not induce IgE or IgG4 production by non-isotype-committed B cells that were either unstimulated, or stimulated with IL-4, anti-CD40 mAb (Table I), or rCD40L alone (data not shown). However, IL-10 potentiated IgE and IgG4 production induced by IL-4 plus anti-CD40 mAb (Table I).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The molecules involved in the induction (IL-4, IL-13, and CD40L) and regulation (see Fig. 3) of IgE production also control IgG4 synthesis (5, 12, 13, 14, 21). We report here that IL-10 inhibits IgE and increases IgG4 synthesis by IL-4-stimulated PBMC. Thus, IL-10 is, to our knowledge, the only molecule that differentially affects IgE vs IgG4 production, thereby suggesting the existence of a pathway(s) selectively involved in the control of their production.

In agreement with others (36), we have observed that IL-10 decreases transcript expression and IgE production by IL-4- or IL-13-stimulated PBMC. This effect was extended to PBMC from allergic patients specifically stimulated with the sensitizing allergen. More precisely, we found that IL-10 inhibited transcript expression and IgE synthesis only when added during the first 2 days after addition of IL-4, at the time when the switch process takes place. These results suggest that IL-10 inhibits IL-4-induced IgE switching. Previous data showing that IL-10 decreases the frequency of IgE-secreting cells in IL-4-stimulated PBMC from allergic patients reinforces this observation (39).

On the contrary, IL-10 potentiated 4 transcript expression and IgG4 production by IL-4-stimulated PBMC. This effect could result from a positive effect of IL-10 on IgG4 switching and/or on the growth and differentiation of IgG4-secreting B cells. IL-10 is a switch factor for IgA, IgG1, and IgG3 (30, 31, 32). Moreover, we have observed that IL-10 enhanced IgG4 production by PBMC even in the absence of IL-4. In view of these effects, it was tempting to speculate that IL-10 may induce IgG4 switching. However, in agreement with others (31), we found that IL-10 was unable to replace IL-4 or CD40 triggering in inducing IgG4 switching in noncommitted IgD⁺ B cells, thereby confirming that IL-10 is not a switch factor for IgG4 (31). Nevertheless, IL-10 has been shown to increase the growth of CD40-activated B cells and to be a potent plasma cell differentiation factor (33, 34, 35). In accordance with these properties, we have observed that IL-10 acted on PBMC by enhancing the production of 1) IgE by B cells that were already IgE switched, 2) IgG4 in the absence of IL-4, and 3) IgA and IgG1-3 in the absence (data not shown) or presence of IL-4. In addition, we report that IL-10 up-regulates 4 transcript expression with a maximal effect when added together with IL-4. Thus, IL-10 may increase IgG4 synthesis both by enhancing the growth and/or differentiation of the IL-4-induced IgG4-producing cells and by potentiating IL-4-induced IgG4 switching.

In summary, when added together with IL-4, IL-10 decreases IgE synthesis by IL-4-stimulated PBMC. This decrease seems to result from a dual effect: an inhibition of IgE switching and a potentiation of the proliferation and/or differentiation of IgE-secreting cells. In parallel, IL-10 potentiates IL-4-induced IgG4 by PBMC. As IL-10 is not a switch factor for IgG4, this effect seems to result from the combined effects of the two lymphokines: 1) IL-4 induces IgG4-switching and 2) IL-10 potentiates IL-4-induced IgG4 switching and/or favors the growth and differentiation of IgG4-producing cells.

It has been suggested that IL-10 inhibited IgE synthesis of PBMC by acting on monocytes (36). However, we have observed that IL-10-decreased IgE production required the presence of T cells and could occur in the absence of monocytes (data not shown). This result suggests that IL-10 interferes with signal(s) provided by T cells and involved in the control of IgE synthesis. It is unlikely that IL-10 decreases IgE synthesis by modulating IL-4 production, since the experiments were performed with optimal concentrations of IL-4. Interestingly, CD40 triggering bypasses the down-regulating effect of IL-10 on IgE production, suggesting that IL-10 may prevent IgE production by decreasing CD40L expression on T cells. However, CD40-CD40L interaction also participates to the regulation of IgG4 production (5) and different concentrations of anti-CD40 mAb or rCD40L modulate in parallel, and in a dose-dependent manner, IgE and IgG4 production (our unpublished observations). Moreover, we found that IL-10 does not affect CD40L expression by IL-4-stimulated PBMC (data not shown). As such, a modulation of this pathway cannot explain the differential effect of IL-10. Thus, these data suggest the existence of a T cell-derived signal(s), different from CD40L and selectively involved in the control of IgE synthesis. In line with this hypothesis, it has been reported that T cells from hyper-IgM patients, which express a nonfunctional CD40L, were still able to provide help for IgE synthesis (40). IL-10 may also interfere with costimulatory signals involved in IgE synthesis. IL-10 has been shown to decrease IL-4-induced CD23 expression on monocytes and IL-6 production by T cells and monocytes (41, 42). Nevertheless, a potential modulation of CD23 expression and IL-6 production is not sufficient to explain the selective effect of IL-10 on IgE vs IgG4 synthesis. Indeed, IgG4 production also requires CD23-CD21 interaction (21) and is also potentiated by IL-6 (Table I). Thus, until now, the mechanism(s) responsible for the selective effect of IL-10 on IgE vs IgG4 production by IL-4-stimulated PBMC remained undetermined. Finally, an important finding is that CD40 triggering bypasses the inhibitory activity of IL-10. This observation underlines that using anti-CD40 mAb or rCD40L in vitro may bypass signals provided by different cell types and involved in the tight regulation of IgE synthesis.

While IgE and IgG4 synthesis in vitro require identical signals, in vivo observations suggested that these isotypes can be produced independently. Allergic patients have IgG4 Ab directed against allergens to which they are not sensitive (i.e., in the absence of a specific IgE Ab) (26). Moreover, in response to an immunization with keyhole limpet hemocyanin, atopics develop a specific IgG4 response but no IgE (23). Both of these observations suggest that IgG4 Ab, in contrast to IgE, are not sensitizing Ab. Moreover, the development of a potent and specific IgG4 Ab response during hymenoptera venom desensitization has been associated with a positive outcome (25, 27, 28). Consequently, in IgE-mediated diseases such as allergic disorders, it could be of clinical interest to prevent IgE production (43) without affecting IgG4 response. Due to its ability to inhibit IgE and to potentiate IgG4 production by allergen-stimulated PBMC from allergic individuals, IL-10 would appear to be a potential candidate. However, different data suggest the contrary: 1) IgE-producing B cells already exist in allergic individuals and IL-10 may increase their proliferation and/or differentiation and consequently favor IgE synthesis, and 2) in response to an allergenic stimulation, IL-10 could be produced later than IL-4 and then may favor the differentiation of IgE-producing B cells generated by IL-4 (44).

Altogether, our results show that, in addition to common pathways such as signaling through IL-4 or IL-13 receptors and CD40, IgE and IgG4 switching can be induced through different mechanisms, as evidenced by the ability of IL-10 to inhibit IgE and potentiate IgG4 synthesis by IL-4-stimulated PBMC.

	Acknowledgments

 
We thank Dr. M. H. Kosco-Vilbois and D. Gretener for critical reading of the manuscript.

	Footnotes

 
¹ Address correspondence and reprint requests to Dr. Pascale Jeannin, Centre d’Immunologie Pierre Fabre, 5, Avenue Napoléon III, B.P. 97, F-74164 Saint-Julien en Genevois, France.

² Abbreviations used in this paper: CD40L, CD40 ligand; PI, proliferation index.

Received for publication March 24, 1997. Accepted for publication December 9, 1997.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Lebman, D. A., R. L. Coffman. 1988. Interleukin 4 causes isotype switching to IgE in T cell-stimulated clonal B cell cultures. J. Exp. Med. 168:853.[Abstract/Free Full Text]
2. Gauchat, J. F., D. A. Lebman, R. L. Coffman, H. Gascan, J. E. de Vries. 1990. Structure and expression of germline epsilon transcripts in human B cells induced by interleukin 4 to switch to IgE production. J. Exp. Med. 172:463.[Abstract/Free Full Text]
3. Punnonen, J., G. Aversa, B. G. Cocks, J. E. de Vries. 1994. Role of interleukin-4 and interleukin-13 in synthesis of IgE and expression of CD23 by human B cells. Allergy 49:576.[Medline]
4. Jabarah, H. H., S. M. Fu, R. S. Geha, D. Vercelli. 1990. CD40 and IgE: synergy between anti-CD40 monoclonal antibody and interleukin 4 in the induction of IgE synthesis by highly purified human B cells. J. Exp. Med. 172:1861.[Abstract/Free Full Text]
5. Gascan, H., J. F. Gauchat, G. Aversa, P. Van Vlasselear, J. E. de Vries. 1991. Anti-CD40 monoclonal antibodies or CD4 T cell clones and IL-4 induce IgG4 and IgE switching in purified human B cells via different signaling pathways. J. Immunol. 147:8.[Abstract]
6. Spriggs, M. K., R. J. Armitage, L. Strockbine, K. N. Clifford, B. M. Macduff, T. A. Sato, C. R. Maliszewski, W. C. Fanslow. 1992. Recombinant human CD40 ligand stimulates B cell proliferation and immunoglobulin E secretion. J. Exp. Med. 176:1543.[Abstract/Free Full Text]
7. Muraguchi, A., T. Hirano, B. Tang, T. Matsuda, Y. Horii, K. Nakajima, T. Kishimoto. 1988. The essential role of B cell stimulatory factor 2 (BSF-2/IL-6) for the terminal differentiation of B cells. J. Exp. Med. 167:332.[Abstract/Free Full Text]
8. Pene, J., F. Rousset, I. Chrétien, J. Wideman, J. Y. Bonnefoy, J. E. De Vries. 1988. Interleukin-5 enhances interleukin-4-induced IgE production by normal human B cells. Eur. J. Immunol. 18:929.[Medline]
9. Maggi, E., G. F. Del Prete, P. Parronchi, A. Tiri, D. Macchia, C. Simonelli, M. Ricci, S. Romagnani. 1989. Role for T cells, IL-2 and IL-6 in the IL-4-dependent in vitro human IgE synthesis. Immunology 68:300.[Medline]
10. Gauchat, J. F., G. Aversa, H. Gascon, J. E. de Vries. 1992. Modulation of IL-4 induced germline epsilon RNA synthesis in human B cells by tumor necrosis factor-alpha, anti-CD40 monoclonal antibodies or transforming growth factor-beta correlates with levels of IgE production. Int. Immunol. 4:397.[Abstract/Free Full Text]
11. Dugas, B., J. C. Renault, J. Pène, J. Y. Bonnefoy, C. Petit-frère, P. Braquet, J. Bousquet, J. Van Snick, J. M. Mencia-Huerta. 1993. IL-9 potentiates the IL-4-induced immunoglobulin (IgG, IgM and IgE) production by normal human B lymphocytes. Eur. J. Immunol. 23:1687.[Medline]
12. Kimata, H., I. Lindley, K. Furusho. 1995. Selective inhibition of spontaneous IgE and IgG4 production by interleukin-8 in atopic patients. Blood 85:3191.[Abstract/Free Full Text]
13. Kimata, H., A. Yoshida, C. Ishioka, M. Fujimoto, I. Lindley, K. Furusho. 1996. RANTES and macrophage inflammatory protein 1-alpha selectively enhance immunoglobulin (IgE) and IgG4 production by human B cells. J. Exp. Med. 183:2397.[Abstract/Free Full Text]
14. Kimata, H., M. Fujimoto, C. Ishioka, A. Yoshida. 1996. Histamine selectively enhances human immunoglobulin E (IgE) and IgG4 production induced by anti-CD58 monoclonal antibody. J. Exp. Med. 184:357.[Abstract/Free Full Text]
15. Aubry, J. P., S. Pochon, P. Graber, K. U. Jansen, J. Y. Bonnefoy. 1992. CD21 is a ligand for CD23 and regulates IgE production. Nature 358:505.[Medline]
16. Diaz-Sanchez, D., S. Chegini, K. Zhang, A. Saxon. 1994. CD58 (LFA-3) stimulation provides a signal for human isotype switching and IgE production distinct from CD40. J. Immunol. 153:10.[Abstract]
17. Yoshinori, K., T. Tanaka, H. Ochi, M. Aitani, A. Yokota, H. Kikutani, S. Masaki, T. Kishimoto. 1996. B cell-B cell interaction through intercellular adhesion molecule-1 and lymphocyte functional antigen-1 regulates immunoglobulin E synthesis by B cells stimulated with interleukin-4 and anti-CD40 antibody. Eur. J. Immunol. 26:192.[Medline]
18. Jeannin, P., Y. Delneste, S. Lecoanet-Henchoz, J. F. Gauchat, J. Ellis, J. Y. Bonnefoy. 1997. CD86 (B7-2) on human B cells: a functional role in proliferation and selective differentiation into IgE and IgG4-producing cells. J. Biol. Chem. 272:15613.[Abstract/Free Full Text]
19. Pène, J., F. Rousset, F. Briere, I. Chretien, J. Y. Bonnefoy, H. Spits, T. Yokota, N. Arai, K. I. Arai, J. Banchereau. 1988. IgE production by normal human lymphocytes is induced by interleukin 4 and suppressed by interferons and and prostaglandin E2. Proc. Natl. Acad. Sci. USA 85:6880.[Abstract/Free Full Text]
20. Kiniwa, M., M. Gately, U. Gubler, R. Chizzonite, C. Fargeas, G. Delespesse. 1992. Recombinant interleukin-12 suppresses the synthesis of immunoglobulin E by interleukin-4 stimulated human lymphocytes. J. Clin. Invest. 90:262.
21. Henchoz-Lecoanet, S., P. Jeannin, J. P. Aubry, P. Graber, C. G. Bradshaw, S. Pochon, J. Y. Bonnefoy. 1996. The Epstein Barr virus-binding site on CD21 is involved in CD23 binding and IL-4-induced IgE and IgG4 production by human B cells. Immunology 88:35.[Medline]
22. Kapsenberg, M. L., H. M. Jansen, J. D. Bos, E. A. Wierenga. 1992. Role of type 1 and type 2 helper cells in allergic diseases. Curr. Opin. Immunol. 4:788.[Medline]
23. Falconer, A. E., P. S. Friedmann, P. Bird, J. E. Calvet. 1992. Abnormal immunoglobulin G subclass production in response to keyhole limpet haemocyanin in atopic patients. Clin. Exp. Immunol. 89:495.[Medline]
24. Merret, J., R. Barnetson, M. L. Burr, T. G. Merret. 1984. Total and specific IgG4 antibody levels in atopic eczema. Clin. Exp. Immunol. 56:645.[Medline]
25. Aalberse, R. C., F. Van Milligen, K. Y. Tan, S. O. Stapel. 1993. Allergen-specific IgG4 in atopic disease. Allergy 48:559.[Medline]
26. Jeannin, P., Y. Delneste, I. Tillie-Leblond, B. Wallaert, A. Carlier, J. Pestel, A. B. Tonnel. 1994. Abnormal IgG4 antibody response to aeroallergens in allergic patients. Int. Arch. Allergy Immunol. 104:191.[Medline]
27. Jarolim, E., L. K. Poulsen, B. M. Stadler, H. Mosbech, O. Oesterballe, D. Kraft, B. Weeke. 1990. A long-term follow-up study of hyposensitization with immunoblotting. J. Allergy Clin. Immunol. 85:996.[Medline]
28. Lesourd, B., J. Paupe, M. Thiollet, R. Moulias, J. Sainte Laudy, P. Scheinmann. 1989. Hymenoptera venom immunotherapy. I: Induction of T-cell-mediated immunity by honeybee venom immunotherapy: relationships with specific antibody responses. J. Allergy Clin. Immunol. 83:563.[Medline]
29. Moore, K. W., A. O’Garra, R. de Waal Malefyt, P. Vieira, T. R. Mosmann. 1993. Interleukin-10. Annu. Rev. Immunol. 11:165.[Medline]
30. Defrance, T., B. Vanbervliet, F. Brière, I. Durand, F. Rousset, J. Banchereau. 1992. Interleukin 10 and transforming growth factor ß cooperate to induce anti-CD40-activated naive human B cells to secrete immunoglobulin A. J. Exp. Med. 175:671.[Abstract/Free Full Text]
31. Brière, F., C. Servet-Delprat, J.-M. Bridon, J.-M. Samy, J. Banchereau. 1994. Human interleukin 10 induces naive surface immunoglobulin D⁺ (sIgD⁺) B cells to secrete IgG1 and IgG3. J. Exp. Med. 179:757.[Abstract/Free Full Text]
32. Malisan, F., F. Brière, J.-M. Bridon, N. Harindranath, F. C. Mills, E. E. Max, J. Banchereau, H. Martinez-Valdez. 1996. Interleukin-10 induced immunoglobulin G isotype switch recombination in human CD40-activated naive B lymphocytes. J. Exp. Med. 183:937.[Abstract/Free Full Text]
33. Rousset, F., E. Garcia, T. Defrance, C. Peronne, N. Vezzio, D.-H. Hsu, R. Kastelein, K. W. Moore, J. Banchereau. 1992. Interleukin 10 is a potent growth and differentiation factor for activated human B lymphocytes. Proc. Natl. Acad. Sci. USA 89:1890.[Abstract/Free Full Text]
34. Armitage, J. A., B. M. Macduff, M. K. Spriggs, W. C. Fanslow. 1993. Human B cell proliferation and Ig secretion induced by recombinant CD40 ligand are modulated by soluble cytokines. J. Immunol. 150:3671.[Abstract]
35. Rousset, F., S. Peyrol, E. Garcia, N. Vezzio, M. Andujar, J.-A. Grimaud, J. Banchereau. 1995. Long-term cultured CD40-activated B lymphocytes differentiate into plasma cells in response to IL-10 but not IL-4. Int. Immunol. 7:1243.[Abstract/Free Full Text]
36. Punnonen, J., R. de Waal Malefyt, P. van Vlasselaer, J.-F. Gauchat, J. E. de Vries. 1993. IL-10 and viral IL-10 prevent IL-4-induced IgE synthesis by inhibiting the accessory cell function of monocytes. J. Immunol. 151:1280.[Abstract]
37. Jumper, M. D., J. B. Splawski, P. E. Lipsky, K. Meek. 1994. Ligation of CD40 induces sterile transcripts of multiple Ig H chain isotypes in human B cells. J. Immunol. 152:438.[Abstract]
38. Kitani, A., W. Strober. 1993. Regulation of C subclass germ-line transcripts in human peripheral blood B cells. J. Immunol. 151:3478.[Abstract]
39. Bober, L. A., M. J. Grace, C. Plugliese-Sivo, T. A. Waters, L. M. Sullivan, S. K. Narula. 1994. Human IL-10 reduces the number of IL-4-induced IgE B cells in cultures of atopic mononuclear cells. Int. Arch. Allergy Immunol. 105:26.[Medline]
40. Life, P., J. F. Gauchat, V. Schnuriger, S. Estoppey, G. Mazzei, A. Durandy, A. Fischer, J. Y. Bonnefoy. 1994. T cell clones from an X-linked hyper-immunoglobulin (IgM) patient induce IgE synthesis in vitro despite expression of nonfunctional CD40 ligand. J. Exp. Med. 180:1775.[Abstract/Free Full Text]
41. Hampel, L., D. Korholz, H. Bonig, M. Schneider, A. Klein-Vehne, J. Packeisen, C. Mauz-Korholz, S. Burdach. 1995. Interleukin-10 directly inhibits the interleukin-6 production in T cells. Scand. J. Immunol. 41:462.[Medline]
42. Spittler, A., C. Schiiler, M. Wilheim, C. Tempfer, S. Winkler, G. Boltz-Nitulescu. 1995. IL-10 augments CD23 expression on U937 cells and down-regulates IL-4-driven CD23 expression on cultured human blood monocytes: effects of IL-10 and other cytokines on cell phenotype and phagocytosis. Immunology 85:311.[Medline]
43. Yssel, H., R. de Waal Malefyt, M. G. Roncarolo, J. S. Abrams, R. Lahesmaa, J. E. de Vries. 1992. IL-10 is produced by subsets of human CD4⁺ T cell clones and peripheral blood T cells. J. Immunol. 149:2378.[Abstract]
44. De Vries, J. E.. 1994. Novel fundamental approaches to intervening in IgE-mediated allergic diseases. J. Invest. Dermatol. 102:141.[Medline]

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