Th2 cytokine dependence of IgD production by normal human B cells

Isabelle Levan-Petit, Eric Lelievre, Anne Barra, Anne Limosin, Bruno Gombert, Jean-Louis Preud'homme and Jean-Claude Lecron

ESA CNRS 6031, IBMIG, 40 Avenue du Recteur Pineau, 86022 Poitiers Cedex, France

Correspondence to: J.-C. Lecron


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
IgD is a minor component of serum Ig and the control of IgD secretion is virtually unknown. We measured concentrations of IgD (and IgE and IgM as controls) in culture supernatants of peripheral blood mononuclear cells (PBMC) from 60 normal donors as well as mononuclear cells from 10 tonsils following culture in the absence or presence of CD40 mAb and cytokines. Low levels of IgD were measured in cultures of PBMC, either unstimulated or stimulated by anti-CD40 antibodies. IL-4 and IL-10 significantly increased IgD production by CD40 mAb-stimulated cells in the majority of normal subjects studied, whereas in a limited number of individuals, spontaneous IgD production was either low or high, but with no increase upon stimulation. Spontaneous IgD production by tonsil-derived mononuclear cells was higher than by PBMC and increased after CD40 stimulation and even more in the presence of IL-10, but not IL-4. IL-2 and IFN-{gamma} exerted a dose-dependent inhibition on spontaneous as well as CD40- and cytokine-induced IgD production by PBMC, but not by tonsil mononuclear cells. Activation by IL-4 of CD40-stimulated purified B cells from tonsil and PBMC, and by IL-10 of tonsil B cells increased IgD production, whereas IL-2 and IFN-{gamma} had no detectable inhibitory effect. This suggests that accessory cells indirectly regulate IgD synthesis. IgD production induced in PBMC by IL-4 or IL-10 appeared to result from an active synthesis, and correlated with an increase in the number of IgD-containing plasma cells as demonstrated by immunofluorescence and increased expression of secreted IgD transcripts. These findings suggest that IgD production by normal peripheral blood human B cells is regulated positively by Th2 cytokines and negatively by Th1 cytokines.

Keywords: CD40, IgD, peripheral blood mononuclear cell, Th1, Th2, tonsil


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Secreted IgD (secIgD) is present in small amounts in the human serum, whereas membrane-bound IgD (mIgD) is a major component of the B cell antigen receptor (15). It is co-expressed with IgM on the surface of most mature B lymphocytes, either as a transmembrane protein or through a glycosyl-phosphatidylinositol link (6,7) and both surface Ig share light chains which are predominantly of the {kappa} type. The function of mIgD has been the subject of a large number of studies. Surface IgD and IgM appear to use largely the same signaling route, although engagement of mIgD appears to be more efficient than mIgM in inducing tyrosine phosphorylation, suggesting a different biological outcome (8,9). The phenotype of IgD-deficient mice is virtually normal, except for an impairment of antibody-affinity maturation (10,11). In contrast, the role of secIgD remains largely unknown (12), but results from a recent study of IgM-deficient mice suggest that IgD is able to substitute for IgM in most of its functions (13). It is worth noting that serum levels of polyclonal IgD are increased in various diseases, including the hyper-IgD syndrome and allergic disorders, which raises the question of a possible role of secIgD in such disorders (14,15).

Synthesis of Ig molecules by members of the B cell lineage is a highly regulated process, which involves a series of gene rearrangements occurring during B cell development and alternative splicing of transcripts (16,17). The gene coding for the {delta} chain has several properties that distinguish it from the other heavy chain genes (18,19). The constant {delta} gene (C{delta}) is the only Ig gene in which the secreted C-terminal tail is encoded by a separated exon ({delta}sec), located upstream of the two membrane exons ({delta}m1 and {delta}m2) (19). The expression of secIgD or mIgD is regulated through the use of polyadenylation sites, located 3' of either {delta}sec or {delta}m2 segments, as well as through differential splicing of mRNA (5). Furthermore, the C{delta} gene lacks an authentic switch region, although a Cµ–C{delta} intronic region, functioning as a vestigial switch recombination site, has been found to operate in normal tonsil and neoplasic mIgM mIgD+ B cells (1821). The latter region is involved in µ gene deletion, observed both in mIgM mIgD+ CD38+ germinal center B cells and differentiated IgD plasma cells, most of which predominantly express {lambda}-type light chains (22,23).

Recently, significant progress has been made in understanding the cellular and molecular mechanisms underlying the regulation of Ig synthesis. Interactions between CD40 on B cells and its ligand (CD40L) on activated T cells are critical for the induction of Ig isotype switching and the selection of the switched isotype seems to be subsequently directed by cytokines (2429). The addition of IL-4 to cells stimulated through CD40 induces IgE, IgG1, IgG3 and IgG4 switching and secretion (2932). IL-13 also stimulates IgG4 and IgE switching and secretion (33). In the presence of IL-10, CD40-activated B cells secrete large amounts of IgM, IgG and IgA (34). IL-10 acts as a switch factor for IgG1 and IgG3 (35,36), and, in combination with transforming growth factor (TGF)-ß, for IgA (37). Other cytokines such as IL-2, IL-6 and IFN-{gamma} also regulate Ig secretion (38,39).

Contrasting with this accumulation of data on the regulation of Ig production, the possible involvement of cytokines in IgD synthesis has remained virtually unexplored. Therefore, in the present study we have investigated the effect of CD40-mediated activation and various Th1 and Th2 cytokines on IgD production by human B cells in vitro.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Cytokines
Human recombinant (r) IL-4, rIL-10 and rIL-13 were generous gifts of Dr R de Waal Malefyt (DNAX Research Institute, Palo Alto, CA). IFN-{gamma}, soluble (s) CD23 and TGF-ß were kindly provided by Dr D. Mossalayi (CNRS, Paris, France), rIL-2 by Eurocetus (Amsterdam, The Netherlands), rIL-6 by Dr H Gascan (INSERM, Angers, France), and rIL-1ß by Dr A Shaw (Glaxo, Geneva, Switzerland) respectively. rIL-7 and rIL-8 were purchased from R & D Systems (Minneapolis, MN), and rIL-15 from Tebu (Le Perray en Yvelines, France).

Cells and culture conditions
Tonsillar cell suspensions were obtained after gentle tissue teasing through a 100 µm cell strainer. Tonsillar mononuclear cells and peripheral blood mononuclear cells (PBMC) from normal donors were prepared by Ficoll-Hypaque (Nycomed, Oslo, Norway) centrifugation. CD19+ B lymphocytes were isolated using a preparative magnetic cell sorter (VarioMACS; Miltenyi Biotec, Paris, France) according to the experimental procedure recommended by the manufacturer. CD19 was expressed on >98% of the selected CD19+ B cell population as assessed by fluorescence analysis using a FACScan (Becton Dickinson, Mountain View, CA). All cultures were carried out in Yssel's medium (YM) (40), in flat-bottomed 24-well plates for mononuclear cells or 96-well plates for separated B cells (Nunc, Paisley, UK) and incubated at 37°C in 6% CO2. For measurements of Ig production, 1x106 PBMC or tonsil-derived mononuclear cells or 1x105 CD19+ B lymphocytes were cultured in 1 or 0.2 ml of YM respectively in the presence or absence of the murine IgG1 anti-CD40 mAb 89 (1 µg/ml; a kind gift of Dr G. Aversa, DNAX Research Institute) and various cytokines. Supernatants were harvested after 9 days of culture (unless stated otherwise), and IgD, IgE, and IgM levels were determined by ELISA. Viable cells that were not colored with Trypan blue dye were enumerated at day 9. In some experiments, PBMC were harvested after 9 days of culture, washed and incubated for an additional 48 h in the absence or presence of 50 µg/ml of the protein synthesis inhibitor cycloheximide (Sigma, St Louis, MO). For the isolation of RNA, 5x106 PBMC or tonsil-derived mononuclear cells were cultured during 9 days in a final volume of 5 ml, as described above.

ELISA for IgD, IgE and IgM measurements
Flat-bottomed microtiter plates (Maxisorp; Nunc) were coated for 18 h at 4°C in 0.1 M carbonate buffer (pH 9.6) with either polyclonal rabbit anti-human {delta} chain antibody (0.5 µg/ml; Dako, Glostrup, Denmark), polyclonal rabbit anti-human {varepsilon} chain antibody (2 µg/ml; Dako) or polyclonal goat anti-human µ chain antibody (2 µg/ml; Southern Biotechnology, Birmingham, AL). The plates were washed 3 times with 0.05% Tween 20 (Sigma) in PBS, pH 7.3, and saturated for 1 h at 37°C with 200 µl PBS containing 10% FCS (Biowhittaker, Verviers, Belgium). All samples was analyzed in duplicate. Culture supernatants or standards were diluted to the appropriate concentration in YM and 100 µl were added into the wells. For IgD measurements, after a 2 h incubation at 37°C, the wells were washed 3 times and incubated with 0.1 µg/ml of a biotinylated polyclonal goat anti-human {delta} chain antiserum (Sigma) for 2 h at 37°C. After three additional washes, the plates were incubated for 30 min with streptavidin–poly-peroxidase (Tebu). For IgM measurements, samples were incubated overnight at 4°C in the anti-µ coated-wells and the presence of IgM was revealed by incubation with 100 µl of an 1/5000 dilution of peroxidase-tagged polyclonal goat anti-human µ chain antibody (Diagnostic Pasteur, Marne la Coquette, France). For IgE ELISA, the samples were incubated overnight at 4°C, the wells were washed and 40 ng/100 µl of an anti-human {varepsilon} chain mAb (Southern Biotechnology) was added overnight at 4°C. After washing, wells were finally incubated for 1 h at 20°C with an 1/10,000 dilution of a peroxidase-coupled rabbit anti-mouse Ig conjugate (Jackson ImmunoResearch, West Grove, PA). In all experiments, peroxidase activity was assayed using 100 µl/well of 0.05 M citrate buffer, pH 5, containing 0.5 mg/ml o-phenylenediamine (Sigma) and 0.4 µl/ml hydrogen peroxide. The reaction was stopped with 50 µl/well of 12.5% sulfuric acid and OD values were measured at 490 nm. OD values obtained with increasing concentrations of purified IgD (Behring, Marburg, Germany), IgE (Behring) and IgM (human serum pool with a known concentration of IgM) were used to construct the standard curve, allowing Ig measurement in the culture samples. The limits of sensitivity of the assay were 0.3 ng/ml for IgD, 0.15 ng/ml for IgE and 19 ng/ml for IgM. Data were expressed as means ± SD. Ig productions under various culture conditions were compared using the Wilcoxon test.

RNA Isolation and reverse transcription
Cell pellets were harvested, washed with PBS and lysed with TRIzol reagent (Gibco/BRL, Cergy Pontoise, France) according to the manufacturer's instructions. Total RNA (3.5 µg) was reverse transcribed into cDNA using oligo(dT)15 (Boehringer, Mannheim, Germany) and random hexanucleotides (Pharmacia, Uppsala, Sweden) as primers, and SuperScript II RNase H Reverse Transcriptase (Gibco/BRL) following manufacturer's instructions, in the presence of a ribonuclease inhibitor (RNasin; Promega, Madison, WI). The cDNA mixture was diluted 1/5 in water and used as a PCR substrate. Negative controls consisted of RT-PCR performed with the reaction mixtures in the absence of RNA or with RNA extracted from the T cell line Jurkat (data not shown). Positive controls were total RNA extracted from the B cell line Ramos (data not shown).

Amplification and analysis of {delta} cDNA
Pairs of PCR primers were designed to discriminate between {delta} chain mRNA containing secretory {delta} chain exon sequence {delta}sec or membrane exon sequence ({delta}m1–{delta}m2). The size was deduced from C{delta} chain sequence data available from EMBL/GenBank/DDBJ under accession no. X57331 HSIGCMUDE. The following primers were used: sense C{delta}3 5'-CCTCCCGAGGCGGCCTCGTGGC-3' (nucleotides 15777–15798); antisense {delta}sec 5'-CGGCCAGAGG-GCTGCTGAGTGGCG-3' (nucleotides 18062–18083); antisense {delta}m 5'-GTGGCGGACAGAGGGGAGCCGG-3' (nucleotides 19417–19438). Specificity of the PCR was confirmed by the comparison of the sequences of the amplification products (data not shown) with previously reported sequences (19,20). The specific primers for ß-actin transcript are sense 5'-GTCGGGCGCCCCAGGCACCA-3' (nucleotides 1–20); antisense 5'-CTCCTTAATGTCACGCACGATTTC-3' (nucleotides 525–548). Primer oligonucleotides were synthesized by Eurogentec (Angers, France). PCR was performed using standard methods with 5 µl of the cDNA preparations, 0.1 mM of each dNTP (Promega), 0.1 µg of each primer, 1.5 mM MgCl2 and 2.5 U of Taq DNA polymerase (Promega) in 50 µl PCR buffer (Promega). Forty PCR cycles were performed for amplification of cDNA encoding {delta}sec or {delta}m and 35 cycles for amplification of cDNA encoding ß-actin. Following a hot start at 94°C during 3 min, the reactions were carried out during 1 min at 94°C for denaturation, 45 s at 69°C for primer annealing and 1 min at 72°C for primer extension. Annealing of ß-actin primers was performed at 55°C. Thereafter, PCR products were subjected to electrophoresis on a 2% agarose gel containing ethidium bromide.

Sequencing of PCR products were performed using the ABI Prism dye terminator cycle sequencing ready reaction kit with ampliTaq DNA polymerase FS (PE Applied Biosystems, les Ulis, France) and {delta}sec, {delta}m or C{delta}3 primers, and analyzed on a 310 Applied Biosystems automatic gene analyser.

Immunofluorescence studies
The presence of intracytoplasmic Ig was analyzed by direct immunofluorescence using polyclonal FITC-labeled rabbit anti-human {delta} chain antibody (Dako) and polyclonal rhodamine-labeled goat anti-human IgA, IgG and IgM (heavy + light chains) F(ab')2 fragments (Jackson ImmunoResearch) as previously described (41). The specificity of the anti-human {delta} chain antibody was controlled using ELISA plates coated with purified monoclonal Ig of the various isotypes and peroxidase-coupled goat anti-rabbit IgG as revealing antibody (Immunotech, Marseilles, France). The presence of mIgD was analyzed on a FACScan flow cytometer (Becton Dickinson) using CellQuest software. After washing, cells were incubated for 20 min at 4°C with polyclonal FITC-labeled rabbit anti-human {delta} chain antibody (Dako). Control was FITC-labeled rabbit F(ab')2. The lymphocyte gate was set according to forward and sideward light scatter; dead cells were stained with propidium iodide and excluded from the analysis. Analysis of mean fluorescence intensity (MFI) of IgD staining was performed.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
IL-4 and IL-10 enhance IgD synthesis by CD40-activated mononuclear cells
In preliminary experiments, detectable amounts of IgD were measured in supernatants of PBMC (Fig. 1AGo) from day 7 of culture and in supernatants of tonsil mononuclear cells (Fig. 1BGo) from day 3, either in medium alone or in the presence of anti-CD40 mAb. IgD levels in supernatants increased until the last day of culture (day 13). IL-4 or IL-10 increased IgD production by CD40-stimulated PBMC (Fig. 1CGo) or tonsil mononuclear cells (Fig. 1DGo) in a dose-dependent manner with an optimal effect at 400 U/ml IL-4 and 100 U/ml IL-10. No additive effect of the two cytokines on anti-CD40-induced production of IgD was observed (data not shown).



View larger version (14K):
[in this window]
[in a new window]
 
Fig. 1. Kinetics of IgD production and dose–response effects of IL-4 and IL-10 by normal PBMC (A and C) or tonsil mononuclear cells (B and D). (A and B) Aliquots of 106 cells/well in 1 ml YM were seeded in flat-bottomed 24-well plates in medium alone ({blacksquare}), or stimulated with 1 µg/ml anti-CD40 mAb alone (•) or in the presence of 400 U/ml IL-4 ({blacktriangledown}) or 100 U/ml IL-10 ({blacklozenge}). Supernatants were harvested at the times indicated. (C and D) PBMC from one healthy donor (C) or mononuclear cells from a tonsil (D) were cultured for 9 days in flat-bottomed 24-well plates at 106 cells/well in 1 ml YM in the presence of anti-CD40 mAb (1 µg/ml) in combination with increasing concentrations of IL-4 ({blacktriangledown}) or IL-10 ({blacklozenge}) (0–4000 and 0–1000 U/ml respectively). Concentrations of IgD were measured by ELISA. One experiment out of three (similar results were obtained with PBMC from two other donors or mononuclear cells from two other tonsils).

 
We further analyzed IgD concentrations in 9-day culture supernatants of PBMC from 60 normal donors. No differences were observed for IgD production between PBMC incubated in medium alone or in the presence of CD40 mAb (Fig. 2AGo). In contrast, IL-4 or IL-10 significantly enhanced IgD synthesis (P < 0.0001 for both cytokines in comparison with PBMC cultured in medium or with the CD40 mAb alone), and a correlation was found between IgD production induced by IL-4 and IL-10 (r = 0.78, P < 0.0001). Mean IgD production was slightly higher in IL-4- than IL-10-stimulated cultures (P < 0.0001), in spite of the heterogeneity of individual responses, the highest IgD levels being variably induced by either IL-4 or IL-10. Contrasting with PBMC, IgD production by cultured tonsil mononuclear cells was enhanced in the presence of the CD40 mAb alone (P < 0.01) (Fig. 2DGo). The addition of IL-10, or to a lesser extent IL-4, further increased IgD production in comparison with tonsil cells cultured in medium (P < 0.01) or with the CD40 mAb alone (P < 0.01).



View larger version (23K):
[in this window]
[in a new window]
 
Fig. 2. IgD, IgE and IgM production by anti-CD40-activated PBMC (A–C) or anti-CD40-activated tonsil mononuclear cells (D–F) with or without 400 U/ml IL-4 or 100 U/ml IL-10. PBMC from 60 healthy donors or mononuclear cells from 10 tonsils were cultured for 9 days in flat-bottomed 24-well plates at 106 cells/well in 1 ml YM. IgD (A and D), IgE (B and E) and IgM (C and F) levels in supernatants were measured by ELISA. Results are expressed as means ± SD. Only significant differences are shown by horizontal bars. Scales are worth noting (ng/ml for IgD and IgE; µg/ml for IgM).

 
As a control, IgE and IgM levels were measured in culture supernatants. As expected (24,25), IL-4 stimulated IgE production by CD40-activated blood and tonsil B lymphocytes (Fig. 2B and EGo) and IL-10 induced IgM production (Fig. 2C and FGo). IgD, IgE and IgM concentrations were similar in supernatants of unstimulated cultures, and of cultures stimulated by IL-4 or IL-10 but without CD40 mAb (data not shown).

The analysis in more detail of IgD production by PBMC of individual subjects showed a marked heterogeneity of spontaneous or inducible IgD production. Three groups could be distinguished. In the majority (42 subjects) of the normal donors studied (Fig. 3AGo), mean basal IgD secretion was ~5 ng/ml (range 0.41–18 ng/ml) with clear-cut stimulating effects of IL-4 or IL-10 on CD40-activated cells. In contrast, in 11 (Fig. 3BGo) and seven (Fig. 3CGo) subjects, spontaneous IgD production was low or high, respectively, but was not affected by stimulation. No correlation was observed between serum IgD level and the capacity of PBMC to secrete IgD in vitro (data not shown). This individual heterogeneity of Ig production led us to search for correlations between IgD and IgE, and IgD and IgM levels in supernatants of IL-4- or IL-10- and CD40-activated PBMC. Under IL-10 + aCD40-stimulation, a group of subjects with high IgM and low IgD, a group with low IgD and high IgM, and a group with both low IgD and IgM productions could be distinguished. Similar groups could not be identified after IL-4 + aCD40 stimulation (data not shown).



View larger version (15K):
[in this window]
[in a new window]
 
Fig. 3. IgD production by PBMC from 60 donors classified in three groups. Group B (n = 11) and group C (n = 7) were distinguished by spontaneous IgD secretion as either low (<5 ng/ml) or very high (>20 ng/ml) respectively with no increase after stimulation. Group A (n = 42) is characterized by an enhancement of IgD production by anti-CD40 mAb and IL-4 or IL-10. Results are expressed as means ± SD. Only significant differences are shown by horizontal bars (P < 0.0001).

 
When PBMC from three group A donors were cultured for 9 days, treated with cycloheximide and further incubated for 2 days in medium alone, IgD production was partially inhibited (~50%) under every condition tested. Cycloheximide induced a similar level of inhibition on IL-4-induced IgE synthesis (data not shown).

Th1 cytokines inhibit IgD synthesis by CD40-activated PBMC
The effect of various cytokines known to regulate Ig synthesis on IgD production by CD40-stimulated cells was examined. The Th1 cytokines IL-2 and IFN-{gamma} inhibited IgD production by PBMC (10 experiments) but not by tonsil-derived mononuclear cells (three experiments). The inhibitory effects of IL-2 and IFN-{gamma} on IgD production by CD40-stimulated PBMC (Fig. 4A and BGo) were dose-dependent, whereas no inhibitory effect was observed on tonsil cells at any concentrations tested (Fig. 4C and DGo). IL-2 and IFN-{gamma} also inhibited in a dose-dependent way IgD production by CD40- and IL-4- or IL-10- stimulated PBMC (Fig. 4A and BGo) but not stimulated tonsil cells (Fig. 4C and DGo). High concentrations of IL-2, but not IFN-{gamma}, enhanced CD40 + IL-10-induced IgD production by tonsil cells (Fig. 4C and DGo). The number of viable cells in the same experiments remained virtually unchanged whatever the concentrations of IFN-{gamma} used alone or in combination with IL-4 or IL-10 (Fig. 4F and HGo). The number of viable cells recovered from cultures performed with high IL-2 concentrations was markedly increased (Fig. 4E and GGo).



View larger version (21K):
[in this window]
[in a new window]
 
Fig. 4. Effects of increasing concentrations of IL-2 and IFN-{gamma} on IgD production and cell proliferation by anti-CD40-activated mononuclear cells. PBMC from one healthy donor belonging to group A (A, B, E and F) or tonsil mononuclear cells (C, D, G and H) were cultured for 9 days in flat-bottomed 24-well plates at 106 cells/well in 1 ml YM in the presence of CD40 mAb (1 µg/ml) and of various concentrations of either IL-2 or IFN-{gamma} (0–100 ng/ml and 0–2000 U/ml respectively). IgD concentrations in supernatants were measured by ELISA (A–D). Viable cells (E–H) that were not colored with Trypan blue dye were enumerated on day 9. •, IL-2 alone; {blacktriangledown}, IL-2 and IL-4 (400 U/ml); {blacklozenge}, IL-2 and IL-10 (100 U/ml); {circ}, IFN-{gamma} alone; {triangledown}, IFN-{gamma} and IL-4; {lozenge}, IFN-{gamma} and IL-10. Results from one experiment out of three which yielded similar results.

 
IL-13 displayed the same enhancing effect on IgD production as IL-4 and IL-10, and IL-15 inhibited IgD production by PBMC, while IL-1ß, IL-6, IL-7, IL-8, sCD23 and TGF-ß appeared to be inactive (data not shown).

IgD synthesis by purified B cells
IgD concentrations were measured in 9-day culture supernatants of CD19+ B cells purified from tonsil or peripheral blood samples. Spontaneous IgD production was lower in the latter cultures than in those of unseparated cells. It was increased by stimulation by CD40 mAb and further by IL-4 for blood B cells (Fig. 5AGo) and either IL-4 or, to a less extend, IL-10 for tonsil B cells (Fig. 5CGo). No inhibitory effect of the addition of IL-2 or IFN-{gamma} on CD40 mAb-induced IgD secretion by purified B cells was observed. Altogether, the results obtained with B cells were clearly different from those observed with unseparated cells (Fig. 5B and DGo).



View larger version (38K):
[in this window]
[in a new window]
 
Fig. 5. Effects of IL-2, IL-4, IL-10 and IFN-{gamma} on IgD secretion by blood and tonsil-derived CD19+ B lymphocytes (A and C) in comparison with unseparated PBMC and tonsil mononuclear cells (B and D). Purified CD19+ B lymphocytes derived from PBMC from two healthy donors (A) and from three tonsils (C) were cultured for 9 days in flat-bottomed 96-well plates at 105 cells/well in 0.2 ml YM. PBMC from 10 donors (B) and unseparated mononuclear cells from three tonsils (D) were cultured in flat-bottomed 24-well plates at 106 cells/well in 1 ml YM. Cells were seeded in medium alone, or stimulated with 1 µg/ml anti-CD40 mAb alone, or in the presence of 100 ng/ml IL-2, 200 U/ml IFN-{gamma}, 400 U/ml IL-4 or 100 U/ml IL-10. Concentrations of IgD were measured by ELISA. Results are expressed as means ± SD.

 
Detection of intracytoplasmic Ig
In cultured PBMC and tonsil mononuclear cells, the percentage of morphologically typical plasma cells containing IgG, IgA or IgM increased in the presence of CD40 mAb in comparison with cells incubated in medium alone. IL-10 further increased the percentage of these plasma cells, whereas IL-4 had no such clear effect (Table 1Go). The total number of IgD-containing morphologically typical plasma cells per slide was very low but clearly increased after CD40 and IL-4 or IL-10 stimulation (Table 1Go).


View this table:
[in this window]
[in a new window]
 
Table 1. Intracytoplasmic immunofluorescence detection of Ig-containing plasma cells
 
RT-PCR analysis of {delta} transcripts and intensity of surface IgD staining
RT-PCR was carried out using primers designed to search for the two forms of {delta} chain mRNA encoding secIgD and mIgD (Fig. 6AGo) in 9-day cultured PBMC (Fig. 6 BGo) and tonsil mononuclear cells (Fig. 6CGo). A 594 bp fragment corresponding to mIgD and a 348 bp fragment corresponding to secIgD were detected under all conditions of stimulation tested. In comparison with PBMC activated by CD40 mAb alone, the addition of IL-4 or IL-10 appeared to increase the levels of secIgD transcripts, whereas IL-2 appeared to decrease it in PBMC. The expression of the transcripts specific for mIgD by PBMC was also apparently enhanced in the presence of CD40 mAb and IL-4 or IL-10 (Fig. 6 BGo). The secIgD transcripts from tonsil mononuclear cells appeared to be enhanced by CD40 mAb and the addition of IL-2, IL-4 or IL-10 appeared to further increase them. mIgD transcripts also increased in the presence of CD40 mAb alone, or in combination with IL-2, IL-4 or IL-10 (Fig. 6CGo).



View larger version (30K):
[in this window]
[in a new window]
 
Fig. 6. Effects of IL-2, IL-4 and IL-10 on {delta} transcript expression by anti-CD40 activated PBMC or tonsil mononuclear cells. The expression of {delta}sec, {delta}m and ß-actin transcripts was evaluated by RT-PCR. The products of two primers pairs are shown, along with a map that diagrams the position of the pairs with respect to the exons. C{delta}3–{delta}sec was designed to detect a product of secIgD mRNA and C{delta}3–{delta}m a product of mIgD mRNA (A). Primers for ß-actin were used as positive controls. RT-PCR bands derived from total RNA of PBMC (B) or tonsil mononuclear cells (C) (5x106 cells per condition) cultured in YM alone, or activated by CD40 mAb alone or by anti-CD40 mAb and IL-2 (100 ng/ml), IL-4 (400 U/ml) or IL-10 (100 U/ml) and collected on day 9. The size of the PCR products are 348 bp for sIgD, 594 bp for mIgD and 548 bp for ß-actin. Total RNA concentration used for amplification was the same in all experiments. Results from one experiment out of three which yielded similar results.

 
FACS analysis of MFI of cultured cells after staining of living cells with anti-{delta} antibody showed a minor (35%) increase in CD40-activated PBMC cultured with IL-4 or IL-10 in comparison with cells incubated in medium alone or only with CD40 mAb, with no detectable effect of IL-2. Compared to tonsil cells cultured in medium alone, MFI increase was 94% for cells incubated with CD40 mAb alone, 123% with IL-2 and CD40 mAb, 109% with IL-4 and CD40 mAb, and 300% with IL-10 and CD40 stimulation.


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Whereas factors involved in the regulation of the production of IgG1, IgG3, IgG4, IgE and IgA are relatively well known (2439), mechanisms underlying the regulation of IgD production remain almost unexplored. In the present study, a spontaneous production of IgD by PBMC and tonsil mononuclear cells was detected, as previously reported by Litwin and Zehr (42,43). IgD levels measured by these authors in tonsil cell supernatants were very similar to those reported herein. Here, we demonstrate that stimulation of PBMC by triggering the CD40 pathway with an anti-CD40 mAb in the presence of the Th2 cytokines IL-4 or IL-10 significantly enhances the production of IgD in vitro in comparison with unstimulated cells or cells stimulated with IL-4, IL-10 or CD40 mAb alone. In comparison with unstimulated cultures, IgD levels were significantly higher after activation of tonsil mononuclear cells by CD40 mAb and IL-10. Interestingly, we detected IgD earlier in cultures of tonsil mononuclear cells than of PBMC. The presence of differentiated B cells and IgD plasma cells in tonsil (23) could account for this phenomenon. Measurements of IgM and IgE levels performed as positive controls yielded the expected results (24,25).

Results obtained with cultured tonsil mononuclear cells are homogeneous, but the number of tonsils studied is relatively small. In contrast, in vitro IgD production by PBMC greatly varied between individual normal donors. Three groups could be distinguished on the basis of the ability of PBMC to spontaneously produce low or high IgD levels and of the enhancement of IgD production by CD40 and IL-4 or IL-10 activation. It remains unclear how IL-4 and IL-10 independently induce IgD synthesis. As previously described, these two cytokines also induce synthesis and/or switch recombination toward IgG1 and IgG3 (32,35,36), whereas only IL-4 induces IgE switching and synthesis (31).

Litwin and Zehr reported that high serum IgD levels negatively correlated with IgM levels (43). Interestingly, we observed an inverse correlation between IgD and IgM in vitro production by CD40 + IL-10-activated cells. Hence, IL-10 might be involved in the dichotomy leading to the preferential expression of IgD or IgM. IgM-deficient mice produce high IgD level, which largely substitute for loss of IgM functions (13). To explain this phenomenon, it has been suggested that the DNA fragment deleted by homologous recombination in these knockout mice contains regulatory sequences attenuating IgD expression (44). The presence of such sequences could also explain the heterogeneity of human serum IgD levels distribution (45) and in vitro IgD production remains an open question.

IL-1ß, IL-6, IL-7, IL-8, sCD23 and TGF-ß did not modulate IgD production by unactivated and CD40-activated PBMC or tonsil mononuclear cells, whereas IL-2 and IFN-{gamma} exerted a dose-dependent inhibition on spontaneous and IL-4- or IL-10-induced IgD production by PBMC, but not by tonsil mononuclear cells. In vitro, IFN-{gamma} enhances or inhibits B cell responses depending on the presence or absence of IL-4 and on the nature of the co-stimulatory signal. However, IFN-{gamma} appears to have negligible effect on IL-4-induced IgE synthesis (24,25), and to increase IgG1 and IgA (25) production by CD40-activated B cells. Nevertheless, in another study, IFN-{gamma} was shown to inhibit IgG4 and IgE synthesis by IL-4-activated PBMC (46). IL-2 enhances IgG1, IgA and IgM synthesis by CD40-activated B cells (25). Hence, IgD production appears to be regulated positively by Th2 cytokines and negatively by Th1 cytokines in a pattern that seems to differ from the known cytokine regulation of other Ig isotype production.

Interestingly, IL-4 increased IgD secretion by CD40- activated B cells purified from PBMC, and both IL-4 and, to a lesser extent, IL-10 showed the same enhancing effect on tonsil B cells, whereas IL-2 and IFN-{gamma} had no detectable inhibitory effect, in contrast to unfractionated PBMC. These results suggest that IL-4 and IL-10 directly stimulate B cells, and that the inhibition by IL-2 and IFN-{gamma} is mediated by accessory cells. Variability of blood accessory cells might contribute to the heterogeneity of individual responses. Furthermore, the addition of CD40 mAb to blood purified CD19+ B cells enhanced their capacity to produce IgD to a level similar to that of total PBMC cultured alone, which might suggest that cells other than B lymphocytes play a role by activating the CD40 pathway. In preliminary experiments, we had compared CD40 activation by CD40 mAb and irradiated CD40L-transfected cells (a gift of Dr F. Brière, Shering Plough, Dardilly, France). Both activation procedures yielded very similar results regarding IgD production, which led us to decide to use the (more convenient) mAb. It is worth noting that IL-4 was more effective than IL-10 with respect to the increase of IgD production by purified B cells. The main B cell targets of IL-10 are the more differentiated cells, i.e. plasma cells (47) which do not express CD19, and B cells were purified on the basis of CD19 expression. This could explain the weak effect of IL-10 on purified B cells.

In spite of their very small number, the increase of IgD production induced by IL-4 or IL-10 appeared to correlate with an increase of the number of IgD-secreting plasma cells. Cycloheximide added 48 h before the end of the culture led to an inhibition of IgD synthesis to the same extent as for IgE production, suggesting active IgD synthesis. Although the RT-PCR used in the present study was not really quantitative, results about {delta}sec transcripts appeared to be well correlated with measurement of IgD in culture supernatants and cytoplasmic immunofluorescence data (increase of secIgD transcripts after CD40 and IL-4 or IL-10 activation, decrease in the presence of IL-2). These data are in keeping with the previous demonstration that the bulk of IgD present in culture supernatants is the result of de novo synthesis of secIgD (42). Regarding {delta}m transcripts, RT-PCR results closely correlated with the analysis of MFI of cells staining with anti-{delta} antibody.

The inhibitory effect of Th1 cytokines on IgD production in the present study is not linked to decreased cell proliferation or survival. Indeed, about twice as many cells were recovered at the end of culture when IL-2 was added in CD40- and CD40 + IL-10-activated PBMC cultures, i.e. conditions that led to an inhibition of IgD production. In contrast, IL-2 increased both cell recovery and IgD production in tonsil cell cultures. Previous studies suggested that IL-2 and IL-10 may act in synergy to induce the growth and differentiation of CD40-activated normal tonsil B lymphocytes (48). In agreement with this report, Arpin et al. recently demonstrated that IgM IgD+ CD38+ germinal center B cells displaying a Cµ–C{delta} molecular switch could differentiate into IgD-secreting cells in vitro in the presence of IL-10 and IL-2 (23).

Altogether, the present data show the possibility to regulate in vitro production of IgD by PBMC or tonsil mononuclear cells. We are presently studying subpopulations of purified naive B cells in order to dissect the molecular mechanisms leading to IgD synthesis after cytokine and CD40 triggering. Finally, similar experiments could be helpful in studying the in vitro regulation of IgD synthesis by PBMC of patients suffering of the hyper-IgD syndrome.


    Acknowledgments
 
We are endebted to Eurocetus and Drs R. de Waal Malefyt, G. Aversa and D. Mossalayi for the kind gift of antibody and reagents. We thank Drs F. and A. Brizard for their useful help with cytology, and Dr Fontanel for providing tonsils. We gratefully acknowledge Dr H. Yssel for helpful comments and critical reading of the manuscript. I. L.-P. was supported by MENRT, and E. L. by Région Poitou-Charentes and Fondation pour la Recherche Médicale. This work was supported by a Clinical Research Grant from Poitiers Hospital.


    Abbreviations
 
CD40L CD40 ligand
MFI mean fluorescence intensity
mIgD membrane IgD
PBMC peripheral blood mononuclear cell
secIgD secreted IgD
TGF transforming growth factor
YM Yssel's medium

    Notes
 
Transmitting editor: J. Borst

Received 23 February 1999, accepted 27 July 1999.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Rowe, D. S. and Fahey, J. L. 1965. A new class of human immunoglobulin. J. Exp. Med. 121:171.[ISI]
  2. Van Boxel, J. A., Paul, W. E., Terry, W. D. and Green, I. 1972. IgD-bearing human lymphocytes. J. Immunol. 109:648.[ISI][Medline]
  3. Abney, E. R. and Parkhouse, M. E. 1974. Candidate for immunoglobulin D present on murine lymphocytes. Nature 252:600.[ISI][Medline]
  4. Finkelman, D. F., Woods, V. L., Berning, A. and Scher, I. 1979. Demonstration of mouse serum IgD. J. Immunol. 123:1253.[ISI][Medline]
  5. Blattner, F. R. and Tucker, P. W. 1984. The molecular biology of immunoglobulin D. Nature 307:417.[ISI][Medline]
  6. Preud'homme, J. L., Brouet, J. C. and Seligmann, M. 1977. Membrane-bound on human lymphoid cells, with special reference to immunodeficiency and immunoproliferative diseases. Immunol. Rev. 37:127.[ISI][Medline]
  7. Wienands, J. and Reth, M. 1992. Glycosyl-phosphatidylinositol linkage as a mechanism for cell-surface expression of immunoglobulin D. Nature 356:246.[ISI][Medline]
  8. Kim, K. M. and Reth, M. 1995. The B cell antigen receptor of class IgD induces a stronger and more prolonged protein tyrosine phosphorylation than that of class IgM. J. Exp. Med. 181:1005.[Abstract]
  9. Kim, K. M. and Reth, M. 1995. Function of B-cell antigen receptor of different classes. Immunol. Lett. 44:81.[ISI][Medline]
  10. Nitschke, L., Kosco, M. H., Köhler, G. and Lamers, M. C. 1993. Immunoglobulin D-deficient mice can mount normal immune responses to thymus-independent and -dependent antigens. Proc. Natl Acad. Sci. USA 90:1887.[Abstract]
  11. Roes, J. and Rajewsky, K. 1993. Immunoglobulin D (IgD)-deficient mice reveal an auxiliary receptor function for IgD in antigen-mediated recruitment of B cells. J. Exp. Med. 177:45.[Abstract]
  12. Thorbeck, G. J. and Leslie, G. A. 1982. Immunoglobulin D: structure and function. Ann. NY Acad. Sci. 399.
  13. Lutz, C., Lederman, B., Kosco-Vilbois, M. H., Ochsenbein, A. F., Zingernagel, R. M., Kölher, G. and Brombacher, F. 1988. IgD can largely substitute for loss of IgM functions in B cells. Nature 386:797.
  14. Peng, Z., Fisher, R. and Adkinson, N. F. 1991. Total serum IgD increased in atopic subjects. Allergy 46:436.[ISI][Medline]
  15. Drenth, J. P. H., Haasgsma, C. J. and Van Der Meer, J. W. M. 1994. The international hyper-IgD study group: hyperimmunoglobulinemia D and periodic fever syndrome. Medicine 73:133.[ISI][Medline]
  16. Tonegawa, S. 1983. Somatic generation of antibody diversity. Nature 302:575.[ISI][Medline]
  17. Kerr, W. G., Hendershot, L. M. and Burrows, P. D. 1991. Regulation of IgM and IgD expression in human B-lineage cells. J. Immunol. 146:3314.[Abstract/Free Full Text]
  18. Milstein, C. P., Deverson, E. V. and Rabbitts, T. H. 1984. The sequence of the human immunoglobulin mu–delta intron reveals possible vestigial switch segments. Nucleic Acids. Res. 12:6523.[Abstract]
  19. White, M. B., Shen, A. L., Word, C., Tucker, P. W. and Blattner, F. R. 1985. Human immunoglobulin D: genomic sequence of delta heavy chain. Science 228:733.[ISI][Medline]
  20. Word, C. J., White M. B., Kuziel, W. A., Shen, A. L., Blattner, F. R. and Tucker, P. W. 1990. The human immunoglobulin Cµ–C{delta} locus: complete nucleotide sequence and structural analysis. Int. Immunol. 1:296.
  21. Kluin, P. M., Kayano, H., Zani, V. J., Kluin-Nelemans, H. C., Tucker, P. W., Satterwhite, E. and Dyer, M. J. S. 1995. IgD class switching: identification of a novel recombination site in neoplastic and normal B cells. Eur. J. Immunol. 25:3504.[ISI][Medline]
  22. Arpin, C., de Bouteiller, O. Razanajaona, D., Brière, F., Banchereau, J., Lebecque, S. and Liu, Y. J. 1997. SIgMIgD+CD38+ hypermutated germinal center centroblasts preferentially express Ig{lambda} light chain and have undergone µ-to-{delta} switch. Ann. NY Acad. Sci. 815:193.[ISI][Medline]
  23. Arpin, C., de Bouteiller, O., Razanajaona, D., Fugier-Vivie, I., Brière, F., Banchereau, J., Lebecque, S. and Liu, Y. J. 1998. The normal counterpart of IgD myeloma cells in germinal centers displays extensively mutated IgVH gene, Cµ–C{delta} switch, and delta light chain expression. J. Exp. Med. 187:1169.[Abstract/Free Full Text]
  24. Rousset, F., Garcia, E. and Banchereau, J. 1991. Cytokine-induced proliferation and immunoglobulin production of human B lymphocytes triggered through their CD40 antigen. J. Exp. Med. 173:705.[Abstract]
  25. Armitage, R. J., Macduff, B. M., Spriggs, M. K. and Fanslow, W. C. 1993. Human B cell proliferation and Ig secretion induced by recombinant CD40 ligand are modulated by soluble cytokines. J. Immunol. 150:3671.[Abstract/Free Full Text]
  26. Nishioka, Y. and Lipsky, P. E. 1994. The role of CD40–CD40 ligand interaction in human T cell–B cell collaboration. J. Immunol. 153:1027.[Abstract/Free Full Text]
  27. Banchereau, J., Bazan, F., Blanchard, D., Briere, F., Galizzi, J. P., Van Kooten, C., Liu, Y. J., Rousset, F. and Saeland, S. 1994. The CD40 antigen and its ligand. Annu. Rev. Immunol. 12:881.[ISI][Medline]
  28. Jumper, M. D., Splawski J. B., Lipsky, P. E. and Meek, K. 1994. Ligation of CD40 induces sterile transcripts of multiple IgH chain isotypes in human B cells. J. Immunol. 152:438.[Abstract/Free Full Text]
  29. Ishizaka, A., Sakiyama, Y., Nakanishi, M., Tomizawa, K., Oshika, E., Kojima, K., Taguchi, Y., Kandil, E. and Matsumoto, S. 1990. The inductive effect of interleukin-4 on IgG4 and IgE synthesis in human peripheral blood lymphocytes. Clin. Exp. Immunol. 79:392.
  30. Jabara, H., Man Fu Shu, Geha, R. S. and Vercelli, D. 1990. CD40 and IgE: synergism 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]
  31. Gascan, H., Gauchat, J.-F., Roncarolo, M-G., Yssel, H., Spits, H. and de Vries, J. E. 1991. Human B cell clones can be induced to proliferate and to switch to IgE and IgG4 synthesis by interleukin 4 and a signal provided by activated CD4+ T cell clones. J. Exp. Med. 173:747.[Abstract]
  32. Fujieda, S., Zhang, K. and Saxon, A. 1995. IL-4 plus CD40 monoclonal antibody induces human B cells {gamma} subclass-specific isotype switch: switching to {gamma}1, {gamma}3, and {gamma}4, but not {gamma}2. J. Immunol. 155:2318.[Abstract]
  33. Punnonen, J., Aversa, G. G., Cocks, B. G., McKenzie, A. N. J., Menon, S., Zurawski, G., de Wall Malefyt, R. and de Vries, J. E. 1993. Interleukin-13 induces interleukin-4 IgG4 and IgE synthesis and CD23 expression by human B cells. Proc. Natl Acad. Sci. USA 90:3730.[Abstract]
  34. Rousset, F., Garcia, E., Defrance, T., Peronne, C., Vezzio, N., Hsu, D. H., Kastelein, R., Moore, K. W. and Banchereau, J. 1992. Interleukin 10 is a potent growth and differentiation factor for activated human B lymphocytes. Proc. Natl Acad. Sci. USA 89:1890.[Abstract]
  35. Malisan, F., Brière, F., Bridon, J. M., Harindranath, N., Mills, F. C., Max, E. E. and Banchereau, J. 1996. Interleukin-10 induces immunoglobulin G isotype switch recombination in human CD40-activated naive B lymphocytes. J. Exp. Med. 183:289.[Abstract]
  36. Brière, F., Servet-Delprat, C., Bridon, J. M., Saint-Rémy, J. M. and Banchereau, J. 1994. Human interleukin 10 induces naive surface immunoglobulin D+ (sIgD+) B cells to secrete IgG1 and IgG3. J. Exp. Med. 179:757.[Abstract]
  37. Defrance, T., Vanbervliet, B., Briere, F., Durand, I., Rousset, F. and Banchereau, J. 1992. Interleukin 10 and transforming growth factor cooperate to induce anti-CD40-activated naive human B cells to secrete immunoglobulin A. J. Exp. Med. 175:671.[Abstract]
  38. Kawano, Y., Noma, T. and Yata, J. 1994. IFN{gamma} and IL-6 act antagonistically in the induction of human IgG1 but additively in the induction of IgG2. J. Immunol. 153:4948.[Abstract/Free Full Text]
  39. Garraud, O. and Nutman, T. B. 1996. The role of cytokines in human B-cell differentiation into immunoglobulin-secreting cells. Bull. Inst. Pasteur 94:285.[ISI]
  40. Yssel, H., de Vries, J. E., Koken, M., Van Blitterswiijk, W. and Spits, H. 1984. Serum-free medium for the generation and the propagation of human cytotoxic and helper T-cells clones. J. Immunol. Methods 74:219.
  41. Preud'homme, J. L., Hurez, D. and Seligmann, M. 1970. Immunofluorescence studies in Waldenström's macroglobulinemia. Rev. Eur. Et. Clin. Biol. 15:1127.
  42. Litwin, S. D. and Zehr, B. D. 1987. In vitro studies on human IgD: I. Sources and characteristics of `externalized' IgD in tonsil lymphocyte cultures. Eur. J. Immunol. 17:483.[ISI][Medline]
  43. Litwin, S. D. and Zehr, B. D. 1988. In vitro studies on human IgD: III. Immunologic features of individuals with high sera IgD and spontaneous IgD biosynthesis. Clin. Immunol. Immunopathol. 47:75.[ISI][Medline]
  44. Yuan, D., Witte, P. L., Tan, J. and Hawley, J. 1996. Regulation of IgM and IgD heavy chain gene expression: effect of abrogation of intergenic transcriptional termination. J. Immunol. 157:2073.[Abstract]
  45. Litwin, S. D. and Zehr, B. D. 1987. Membrane IgD-positive B cells of `low-IgD serum phenotype' individuals fail to secrete IgD and fail to shift to preferential lambda light-chain expression in vitro. J. Clin. Immunol. 7:114.[ISI][Medline]
  46. Pène, J., Rousset, F., Brière, F., Chrétien, I., Pailard, X., Banchereau, J., Spits, H. and De Vries, J. A. 1988. IgE production by normal human B cells induced by alloreactive T cell clones is mediated by IL-4 and suppressed by IFN-{gamma}. J. Immunol. 141:1218.[Abstract/Free Full Text]
  47. Rousset, F., Peyrol, S., Garcia, E., Vezzio, N., Andujar, M., Grimaud, J. A. and Banchereau, J. 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]
  48. Fluckiger, A. C., Garrone, P., Durand, I., Galizzi, J. P. and Banchereau, J. 1993. Interleukin 10 (IL-10) upregulates functional high affinity IL-2 receptor on normal and leukemic B lymphocytes. J. Exp. Med. 178:1473.[Abstract]