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The Journal of Immunology, 1999, 162: 78-87.
Copyright © 1999 by The American Association of Immunologists

Mouse IL-13 Enhances Antibody Production In Vivo and Acts Directly on B Cells In Vitro to Increase Survival and Hence Antibody Production1

Yew Hon Lai and Timothy R. Mosmann2

Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Canada


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
IL-13, a Th2 cytokine, exhibits similar functions to IL-4 in stimulating proliferation and class switching of human B cells. Although mouse B cells were reported to be unresponsive to IL-13, we now show that IL-13 directly stimulates mouse B cells, causing extended survival and higher Ab levels. Recombinant mouse IL-13 was administered via osmotic pump during immunization of BALB/c mice with chicken RBCs. IL-13 treatment enhanced not only the plasma levels of total IgG1, IgG2a, and IgG2b but also Ag-specific Ig levels. To examine whether IL-13 acted directly on mouse B cells, B220+ B cells were cultured with fixed, anti-CD3-activated Th2 clones. Production of IgM and IgG1 was enhanced moderately by IL-13 and strongly by IL-4. Anti-CD40-stimulated sIgD+ mouse B cells also responded to IL-13 by producing increased levels of IgM, and to a lesser extent IgG1, IgG2a, IgG2b, and IgG3. No evidence was found for IL-13-induced class switching. Mouse B cells were stimulated directly rather than indirectly via contaminating cells, as IL-13 increased the numbers of both total and Ab-secreting B cells in aliquots of 100 sIgD+ B cells (>99.5% pure) stimulated with anti-CD40 Ab. Stimulation of B cells by IL-13 was unaffected by the addition of anti-IL-4 to the cultures. In contrast to IL-4, IL-13 did not increase CD23 expression or B cell proliferation as measured by dilution of an intracellular fluorescence label. Collectively, these data indicate that IL-13 can enhance mouse B cell Ab production by increasing survival of the B cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Interleukin-13, the latest identified cytokine to join the Th2 cytokine family, was initially identified by screening mouse cDNA libraries for inducible genes (1). Subsequently, human IL-13 (hIL-13)3 cDNA was identified independently by three groups (2, 3, 4). Compared with mouse IL-13 (mIL-13), hIL-13 expression is not as clearly associated with the Th2 cytokine pattern (3, 5). mIL-13 cDNA encodes a 131-amino acid protein, and mIL-13 is secreted as an ~14-kDa protein by transfected COS-7 and BW5147 cells (6). Although they share only limited protein homology (~30%), IL-4 and IL-13 belong to the same {alpha}-helix protein family (5, 7). The IL-4R {alpha}-chain of the IL-4R is a component of some but not all IL-13 receptors (8, 9). In addition, two IL-13-binding receptor chains have been identified: IL-13R{alpha}1 (NR4; Refs. 8 and 10–12) and IL-13R{alpha}2 (13), which share high levels of sequence identity with known cytokine receptors. Four structural models of IL-13R involving different combinations of IL-13R{alpha}1, IL-13R{alpha}2, or IL-4R{alpha} have been proposed (14). The sharing of a receptor component may explain why a mutant IL-4 protein acts as a competitive receptor antagonist to both IL-4 and IL-13 (5, 7). Consistent with their protein homology and partial receptor sharing, the functions of IL-13 partially overlap with those of IL-4.

IL-13 exhibits pleiotropic effects on human peripheral blood monocytes and mouse granulocyte-macrophage CSF-derived bone marrow macrophages. IL-13 (in common with IL-4) induces morphological and surface Ag changes in human monocytes and mouse granulocyte-macrophage CSF-derived bone marrow macrophages, and also inhibits Ab-dependent cellular cytotoxicity activity and production of nitric oxide and proinflammatory cytokines (2, 3, 15, 16) without affecting the phagocytic functions of activated macrophages (16). In addition, IL-13 inhibits the replication of HIV in monocytes (17) and enhances VCAM-1 expression on human endothelial cells in vitro (18). Consistent with the ability of IL-13 to stimulate early hemopoietic precursors in vitro (19), IL-13 administration in vivo also induces extramedullary hemopoiesis and monocytosis in mice (6).

Human IL-13 and IL-4 have similar effects on human B cells, although IL-4 is generally more potent. IL-13 enhances proliferation of anti-IgM- or anti-CD40-activated human B cells (3, 20, 21), modulates their surface Ags (2, 3, 22), and induces class switching to IgG4 and IgE synthesis in human B cells in combination with activated T cells, anti-Ig, anti-CD40 Abs, or CD40 ligand (CD40L; 20–22). However, IL-13 does not have any additive or synergistic effects with IL-4 on induction of IgG4 and IgE (20, 22). In contrast to hIL-13 effects on human B cells, mIL-13 has been reported to have no effects on mouse B cells (5). Although this is consistent with the observation that IL-4-deficient mice do not produce IgE in response to nematode infection (23), these mice produce IgE upon malaria infection (24). This finding suggests that an IL-4-independent mechanism for IgE synthesis also exists in mice and raises the possibility that mIL-13 may also induce IgE synthesis in some circumstances.

As IL-13 is associated with the Th2 response in mice, which includes strong Ab production, and hIL-13 stimulates Ab production by human B cells, we have re-examined the potential effects of mIL-13 on mouse Ab production. We found that IL-13 stimulated Ab production in vivo during a strong immune response. Analysis of mouse B cells in vitro also showed that IL-13 stimulated Ab production by B cells that were activated by various stimuli, including T cells or anti-CD40 Ab. IL-13 was less effective than IL-4 and did not show the strong selectivity for IgG1 and IgE that is characteristic of IL-4. Nevertheless, in the presence of low doses of anti-CD40, IL-13 directly stimulated sIgD+ B cells, resulting in increased numbers of Ab-producing B cells as measured by enzyme-linked immunospot (ELISPOT) assay. Similar findings were obtained in the presence of neutralizing IL-4 mAb, suggesting that the effect of IL-13 was independent of IL-4. This enhancement appears to be due to increased survival, but not proliferation, of mouse B cells. Thus, IL-13 can also contribute to B cell Ab production in mice, consistent with some of the known effects of IL-13 on human B cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Purification of IL-13

Recombinant mIL-13 was purified using Mono Q 16/10 anion exchange column (Pharmacia Biotechnology, Uppsala, Sweden), as previously described, from the supernatant of mouse BW5147 cells stably transfected with mIL-13 (6). Fractions from the peak two were then pooled, equilibrated in 0.1% trifluoroacetic acid-H2O, and further purified using a RESOURCE RPC reverse-phase column (Pharmacia Biotechnology) eluting with a gradient of 0–70% acetronitrile-0.1% trifluoroacetic acid. Fractions were screened for IL-13 bioactivity on TF-1 cells, and positive fractions were equilibrated in PBS as described (6). The purity of IL-13 was assessed by 4 M urea urea-15% SDS-PAGE. Both Coomassie blue staining and Western blot analysis using anti-mouse-IL-13 (RAMP1) Ab demonstrated that the purified IL-13 migrated as an ~14 kDa doublet, similar to the native IL-13 secreted by D10.G4.1, a Th2 cell line. In addition to the doublet, Western blot analysis and Coomassie staining also recognized small amounts of heterogenous material at higher m.w., which may be due to carbohydrate heterogeneity on IL-13. Subsequent experiments were performed with this purified IL-13 with sp. act. of 3.6 U/ng (6). Purified IL-13 contained <0.1 ng of endotoxin per 400 µg of IL-13, as determined by the Limulus assay (Sigma, St. Louis, MO).

In vivo IL-13 treatment

BALB/c female mice (6–8 wk old) were obtained from Health Sciences Laboratory Animal Services (HSLAS, Edmonton, Alberta, Canada). PBS controls or IL-13 were coded and loaded into Alzet microosmotic pumps (Alza, Palo Alto, CA), which were then implanted into the peritoneal cavity through a small dorsal incision as previously described (6). Three or four mice were implanted with pumps containing PBS or each dose of IL-13. Four hours later, each mouse was immunized i.p. with 0.2 ml of 50% chicken RBCs (CRBCs) resuspended in saline. The mice were maintained in the HSLAS animal facility according to the guidelines of the Canadian Council on Animal Care and monitored daily for any abnormality. On day 7, the mice were anesthetized, and blood was collected by cardiac puncture in uncoated or heparin-coated syringes. The mice were sacrificed, and their spleens were collected and weighed.

Hemagglutination assay

Direct and indirect hemagglutination assays were performed as described (25, 26). Briefly, wells of round-bottom 96-well plates containing 100 µl of doubling dilutions of plasma in PNS buffer (0.05 M phosphate, 0.1 M NaCl, 1% FBS) were incubated with 100 µl of 0.5% CRBCs for a minimum of 45 min, after which the wells were scored for direct hemagglutination titer. To obtain indirect hemagglutination titers, the cells were washed three times with PNS and 200 µl of a 1:1000 dilution of anti-mouse IgG1, IgG2a, IgG2b, IgG3 (Sigma), or Ig pan-specific (PharMingen, San Diego, CA) Abs were added. Titers were expressed as the reciprocal of the last dilution that gave positive CRBCs agglutination.

B cell purification

B220+ or sIgD+ B cells were obtained as previously described (27). Briefly, nonadherent spleen cells were obtained from 8- to 12-wk-old female BALB/c mice (HSLAS), stained with phycoerythrin-conjugated anti-CD4 and anti-CD8 and FITC-conjugated anti-B220 or anti-IgD (PharMingen) on ice for 30 min, and washed. After purification by Lympholyte-M (Cedarlane Laboratories, Hornby, Ontario, Canada), the lymphocytes were sorted for B220+ or sIgD+ cells using a Coulter EPICS Elite ESP cell sorter (Beckman Coulter, Hialeah, FL). The purity of the sorted cells was verified to be >99.5% by FACScan analysis (Becton Dickinson, San Jose, CA).

B cell stimulation by fixed T cells

T cell lines were maintained as described previously (28). After being coated overnight at 4°C or room temperature for 4 h with purified anti-CD3 (145-2C11, provided by J. Bluestone, University of Chicago, Chicago, IL) in PBS, polystyrene plates were rinsed with PBS, and T cells were added at a density of 1 x 106 cells/ml for 24-h activation. Anti-CD3-activated T cells were collected, washed three times with PBS, and fixed with 0.4% paraformaldehyde (PFA) for 5 min (29, 30). The fixed anti-CD3-activated T cells were washed with PBS and resuspended in RPMI 1640 (Life Technologies, Burlington, Ontario, Canada) before being added to cultures. Sorted B cells (3 to 5 x 104 cells/well) with at least 99% purity were cultured for 7 days with different ratios of 0.4% PFA-fixed anti-CD3-activated M264-15 or D10.G4.1 cells. Ab levels were measured by ELISA as described below.

B cell stimulation by anti-CD40 Ab

Sorted sIgD+ B cells (5 x 104 cells/well) with >98% purity were cultured with various concentrations of anti-CD40 Ab (PharMingen), with or without purified recombinant mIL-4 (a generous gift from Schering-Plough, Kenilworth, NJ), IL-13, or anti-IL-4 mAb (11B11). Iscove’s modified Dulbecco’s medium (IMDM, Life Technologies) containing 8% FCS (Hy-Clone, Logan, UT), 5 µg/ml of gentamicin sulfate (Life Technologies), and 50 µM of 2-ME (Sigma) was used in all anti-CD40 cultures. Supernatants were harvested after 6–7 days of culturing, and Ig levels were quantitated as described below. For microcultures, the bulk cultures were serially diluted, and 10 µl of the cultures with various concentrations of anti-CD40 Ab, with or without purified IL-4, IL-13, or anti-IL-4 mAb (11B11), containing 90–100 sorted sIgD+ or B220+ B cells were cultured in Terasaki 96-well trays (Robbins Scientific, Sunnyvale, CA). The Terasaki trays were incubated in moist containers, and B cells in each well were enumerated after culturing for 2 days.

Determination of cell surface molecules

Sorted sIgD+ B cells (5 x 104 cells/ml) were cultured with 1 or 3 ng/ml of anti-CD40 Ab, with or without purified mouse IL-4 or IL-13, for 2 days. Cells were harvested, enumerated, and stained with Abs to MHC class II, MHC class I, CD23, and IgM (all from PharMingen). Cells (25,000/population) were analyzed by a FACScan using CellQuest software (Becton Dickinson).

ELISA assay for Igs

All Abs were used at 1:1000 dilution unless stated otherwise. Affinity-purified goat anti-mouse IgG1, IgG2a, IgG2b, IgG3, IgA, or IgM Abs (Sigma) in PBS were used to coat Falcon 3911 microtiter plates (Becton Dickinson Labware, Oxnard, CA) at 4°C overnight. Each coating Ab exhibited minimal cross-reactivity with other Ig subclasses. After washing the plates, dilutions of plasma or culture supernatants were added to the wells and incubated for at least 30 min at room temperature. Peroxidase-conjugated polyvalent anti-mouse Ig was used for detection (Sigma). Purified mouse IgG1, IgG2a, IgG2b, IgG3, and IgM (Sigma) were used as standards. Mouse IgE levels were quantitated in comparison to purified mouse IgE{kappa} by two-site sandwich ELISA (PharMingen), as previously described (6) and according to the manufacturer’s recommendations.

ELISPOT assay for mouse IgM production

B cells from each Terasaki well were transferred to MultiScreen 96-well plates (Millipore, Bedford, MA) that were coated with affinity-purified goat anti-mouse IgM Ab at 1:1000 dilution (Sigma). After overnight incubation, the plates were developed for Ab spots. After each step, the incubation buffers were filtered and washed with 0.05% Tween 20 (Sigma) in PBS. Each well was incubated with biotinylated affinity-purified goat anti-mouse Ig resuspended in PBS buffer containing 1% FCS and 1% Tween 20 for 1 h. After being incubated with peroxidase-conjugated strepavidin (Jackson ImmunoResearch Laboratories, West Grove, PA), the plates were washed and developed with AEC substrate (Vector Laboratories, Burlingame, CA) according to the manufacturer’s instructions. The plates were dried, and Ab spots were enumerated.

B cell proliferation assay

Sorted sIgD+ B cells (5 x 104 or 1 x 104 cells/well) were cultured with various concentrations of anti-CD40 Ab, with or without purified IL-13 or IL-4, for 3 days. A total of 1 µCi/well of [3H] (Amersham, Arlington Heights, IL) was added during the final 18 h of culture. Cells were harvested using a MicroMate 196 cell harvester (Canberra Packard, Meriden, CT), and radioactive incorporation was determined on a Matrix 96 (Canberra Packard).

Determination of B cell division

Sorted sIgD+ B cells were stained with 5-(and 6-)carboxyfluorescein diacetate, succinimidyl ester (CFSE, Molecular Probes, Eugene, OR) as described (31). Briefly, the cells were washed with IMDM and stained with 0.1–1 µM CFSE for 10 min. At the end of the incubation, the cells were washed three times with ice-cold IMDM containing 8% FCS. The stained cells (at a density of 1.25 x 105 c/ml) were cultured in a 24-well Falcon plate (Becton Dickinson Labware) in the presence of 3 ng/ml of anti-CD40, with or without 5–20 ng/ml of IL-13 or 10 ng/ml of IL-4. On day 3, the cells were harvested and washed, and viable cells were counted using Trypan Blue. Cell division was assessed by flow cytometry analysis of 25,000 cells/population using a FACScan and CellQuest software (Becton Dickinson). Dead cells were excluded based on their light scattering properties.

Statistical analyses

The unpaired Student’s two-tailed t test was used to calculate p values. Alternatively, the Kruskal-Wallis test was used to calculate p values for serum CRBCs agglutination titers.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
IL-13 increased Ab production levels in vivo

To study the effects of IL-13 during a strong immune response, IL-13 or PBS was administered via osmotic pump, implanted into the peritoneal cavity of BALB/c female mice, over a period of 7 days. Dosages of IL-13 ranging from 0.5 to 6.5 µg/mouse/day were administered. After the pump implantation, the mice were immunized with CRBCs in the peritoneal cavity. Consistent with our previous observations (6), in vivo IL-13 administration induced splenomegaly (Expt. 2: 2-fold, p < 0.03; and Expt. 3: 1.5-fold, p < 0.04) due to increased splenocyte numbers, even during the strong immune response to CRBCs. PBS-treated mice immunized with CRBCs showed an approximate 2-fold increase of total plasma IgG1, IgG2a, IgG2b, IgG3, and IgM compared with untreated and nonimmunized littermates (data not shown).

When CRBC-immunized mice were treated continuously with different doses of IL-13 in three separate experiments, significant further increases in total Ig levels were observed for IgG1, IgG2a, and IgG2b (Fig. 1Go). IgM and IgG3 levels showed a significant increase in only one of the three experiments, and IgA did not show any significant changes. In one experiment (Expt. 3), IgE levels (IL-13, 137 ± 82.3 vs PBS, 176.6 ± 93.5) were not significantly different between IL-13 and control groups. Thus, in vivo administration of IL-13 enhanced total serum or plasma levels of at least three IgG subclasses during a strong immune response to CRBCs.



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  		FIGURE 1. In vivo IL-13 administration increased the levels of three IgG isotypes. Mice that were immunized with CRBCs were continuously administered with PBS or IL-13 (Expt. 1, 3.5 µg/mouse; Expt. 2, 4.5 ({blacktriangledown}) or 45 ({blacktriangleup}) µg/mouse, or Expt. 3, 25 µg/mouse) by osmotic pumps for 7 days in the peritoneal cavity. Serum or plasma from three PBS- ({circ}) or three IL-13-treated ({blacktriangleup} or {blacktriangledown}) mice from each experiment were quantitated for IgM, IgG1, IgG2a, IgG2b, IgG3, and IgA levels by sandwich ELISA. Each symbol represents data obtained from an individual mouse, and the error bars indicate the SD of quadruplicate or triplicate assay determinations from each mouse sample. The average Ig level in each group is indicated by a solid horizontal bar. The symbols * and ** indicate Ig levels from IL-13-treated mice that are significantly higher than those from mice treated with PBS, with p < 0.05 or p < 0.01, respectively.

 
IL-13 enhanced hemagglutination titers

The indirect and direct hemagglutination titers in the plasma of these mice were assayed to quantitate Ag-specific Ab production in vivo. The IL-13-treated groups consistently showed increased direct and indirect CRBCs agglutination titers compared with the PBS-treated groups (Fig. 2GoA). Isotype-specific indirect CRBCs agglutination titers of IgG1, IgG2a, and IgG2b were also elevated in the IL-13-treated group (Fig. 2GoB). Hence, in vivo treatment with IL-13 enhanced the Ag-specific Ab response.



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  		FIGURE 2. IL-13 enhanced direct and indirect CRBCs hemagglutination titers. Doubling dilutions of plasma from PBS- ({circ}) or IL-13-treated ({blacktriangleup}) mice from Fig. 1Go were tested for the ability to agglutinate CRBCs directly (A), or using a pan-specific anti-Ig antiserum, or isotype-specific antisera (B) for IgG1, IgG2a, IgG2b, or IgG3. Titers are expressed as the reciprocal of the last dilution that gave positive CRBCs agglutination. Each symbol represents data obtained from an individual mouse. The average hemagglutination titer in each group is indicated by a solid horizontal bar. Plasma from unimmunized mice had direct or indirect CRBCs agglutination titers of 1:200 or less. *, Data significantly greater (p < 0.05) than data from PBS-treated controls.

 
IL-13 enhanced Ig levels in vitro

Collectively, these data indicate that in vivo IL-13 treatment induced higher levels of IgG1, IgG2a, and IgG2b in vivo. These in vivo effects could have been due to direct or indirect effects of IL-13. As IL-13 treatment in vivo did not alter the levels of cytokines secreted by Con A-stimulated spleen cells in vitro (data not shown), we hypothesized that mouse B cells might respond directly to mIL-13, analogous to the direct effects of hIL-13 on human B cells (2, 3, 20, 21, 22). To investigate the effects of IL-13 on mouse B cells, different numbers of PFA-fixed anti-CD3-activated D10.G4.1 (Th2) T cells were cultured with B220+ splenic B cells in the absence or presence of varying doses of IL-13 or IL-4 for 7 days. T cells fixed after activation provide a source of CD40L, which is expressed on activation and is the major cell-surface activating signal delivered by T cells to B cells (29, 30, 32, 33, 34, 35, 36, 37, 38, 39).

Both IL-13 and IL-4 consistently enhanced the levels of IgM and IgG1 in the supernatant (Fig. 3Go). However, compared with IL-4, IL-13 was consistently at least 2-fold less effective in enhancing IgM production and more than 10-fold less effective on IgG1 synthesis. In most experiments, IL-13 also showed at least additive effects in stimulating Ab production by B cells stimulated with Th1 clones, irradiated primary alloreactive Th1 cells, or CD40L-transfected cells (data not shown). IL-13 effects on the production of IgG2a, IgG2b, and IgG3 varied between experiments, possibly due to the release of endogenous cytokines such as IFN-{gamma}, IL-4, or IL-13 from the fixed T cells.



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  		FIGURE 3. IL-13 enhanced Ig levels in vitro in the presence of fixed, activated Th2 cells. Sorted B220+ mouse splenic B cells (3 x 104 cells/well) were cultured alone ({circ}) or with 5 x 104 ({blacktriangleup}) or 1 x 105 ({blacktriangledown}) PFA-fixed anti-CD3 activated D10.G4.1 cells, in the absence or presence of IL-13 (3 or 30 ng/ml) or IL-4 (3 or 30 ng/ml) in triplicate for 7 days. IgM and IgG1 levels were quantitated, and the mean and SD of replicate cultures are shown. The inset in the IL-13 panel for IgG1 production shows an expanded scale.

 
The above results, particularly with PFA-fixed cells, strongly suggested that IL-13 stimulated naive mouse B cells. However, as activated T cells produce large amounts of cytokines, leakage of even a small fraction of cytokines from fixed cells might have influenced the result. To exclude this possibility, we stimulated B cells in the absence of T cells, by culturing sIgD+ splenic mouse B cells with different doses of anti-CD40 Ab in the absence or presence of various doses of IL-13 or IL-4 for 6–7 days. In the presence of suboptimal doses of anti-CD40 Ab, IL-13 significantly enhanced IgM, IgG1, IgG2b, and IgG3 production by 3- to 8-fold (Fig. 4Go). By comparison, IL-4 induced similar levels of IgM but higher levels of the IgG isotypes, particularly IgG1. In this mouse B cell system, IL-4 induced at least 100-fold more IgG1 production than IL-13. In the presence of anti-CD40 Ab and up to 100 ng/ml of IL-13, IgE levels in the cultures remained below the detection limit (6.25 ng/ml), in contrast to the induction of nearly 280 ng/ml IgE by anti-CD40 and 20 ng/ml of IL-4. Thus, low concentrations of anti-CD40 and IL-13 synergized in stimulating a moderate level of Ab production. Similar results were obtained in three other experiments, except that IL-4 often induced higher levels of IgM than IL-13.



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  		FIGURE 4. IL-13 enhanced Ig production from anti-CD40-stimulated sIgD+ B cells. Sorted sIgD+ mouse splenic B cells (5 x 104 cells/well) were cultured without ({circ}), or with 3 ({blacktriangleup}) or 10 ({blacktriangledown}) ng/ml anti-CD40 Ab, in the absence or presence of IL-13 or IL-4 for 7 days. IgM, IgG1, IgG2a, IgG2b, and IgG3 levels were quantitated. These data are representative of three other experiments. The mean and SD of quadruplicate or triplicate cultures are shown. The insets in the IL-13/IgG1 and IL-13/IgG2b panels show expanded scales.

 
As IL-4 enhances the expression of CD23 and MHC class II on mouse B cells, the effects of IL-13 on these two surface molecules were also tested. In contrast to IL-4, IL-13 did not up-regulate levels of CD23 (Fig. 5Go) or MHC class II (results not shown).



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  		FIGURE 5. IL-13 did not enhance surface CD23 expression on anti-CD40-activated B cells. Sorted IgD+ B cells were cultured with 3 ng/ml of anti-CD40 mAb in the absence or presence of IL-13 (20 ng/ml) or IL-4 (10 ng/ml) for 40–48 h. Cells were harvested, counted, blocked with anti-Fc receptor mAb (2–4G2), and stained with phycoerythrin-conjugated anti-CD23 mAb. At least 15,000 cells were analyzed by FACScan. Dotted or solid lines represent cells that were stained with isotype control or anti-CD23 mAb, respectively. Similar results were obtained in three other experiments with different doses (1–3 ng/ml) of anti-CD40 mAb.

 
IL-13 directly stimulated Ig production by mouse B cells

Although IL-13 enhanced Ig production in bulk culture, and the sIgD+ sorted mouse B cells were >99.5% pure, each well contained 50,000 cells. Thus, up to 250 contaminating cells could have been present per well, and indirect effects of IL-13 were theoretically possible. To exclude this possibility, sIgD+ sorted mouse B cells were titrated to 90–100 cells per well and cultured in various combinations of anti-CD40 mAb, IL-13, and IL-4. Under these conditions, at least some wells must have contained only B cells, allowing a stringent test of the ability of IL-13 to enhance Ab production in the absence of any other cell type. After 2 days, the cells in each well were counted and transferred to affinity-purified goat anti-mouse IgM-coated nitrocellulose plates for an ELISPOT assay. In the presence of anti-CD40 mAb, different doses of IL-13 not only enhanced the number of B cells that survived after 2 days of culture but also increased the number of IgM-secreting B cells (Fig. 6Go). Similar data were obtained in eight separate experiments (three are shown in Fig. 6Go). Unlike IL-13-treated cells, IL-4 and anti-CD40-responsive sIgD+ B cells formed tight clusters, and the number of Ab spots could not be accurately enumerated, supporting the idea that IL-4 and IL-13 have different effects on mouse B cells.



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  		FIGURE 6. IL-13 directly stimulated mouse IgD+ B cells. Sorted sIgD+ mouse B cells (>99.5% pure, 100/well, Expt. 1 and 2; or 90/well, Expt. 3) were cultured in Terasaki wells for 2 days in the absence ({circ}) or presence of 3 ({blacktriangleup}) or 10 ({blacktriangledown}) ng/ml anti-CD40 mAb with different doses of IL-13. The cells in each well were counted and transferred to affinity-purified goat anti-mouse IgM Ab-coated nitrocellulose plates for ELISPOT assay. The mean and SD of three (Expt. 1) or six (Expt. 2 and 3) replicate cultures are shown.

 
When sIgD+ (naive) B cells were compared with B220+ (naive plus memory) B cells, IL-13 and anti-CD40 induced similar levels of Ab-secreting cells from both populations (Fig. 7Go), indicating that the response of the naive population was not due to a stronger response of small numbers of contaminating IgD- memory B cells. In addition, the biological effects of IL-13 on mouse B cells were independent of IL-4 as anti-IL-4 mAb failed to inhibit the response (Fig. 7Go). Collectively these data indicate that IL-13 directly stimulated naive mouse B cells by an IL-4-independent pathway.



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  		FIGURE 7. IL-13 directly stimulated mouse IgD+ or B220+ B cells, independent of IL-4. Sorted mouse IgD+ or B220+ B cells from the same pool of spleen cells were cultured in Terasaki wells for 2 days at 90/well in the absence ({circ}) or presence of 1 ({blacktriangleup}) or 3 ({blacktriangledown}) ng/ml anti-CD40 mAb with different doses of IL-13. Anti-IL-4-blocking mAb (10 µg/ml, dotted lines) was used in the corresponding cultures. ELISPOT assays were performed as described in Fig. 6Go. The mean and SD of quadruplicate cultures are shown.

 
IL-13 effects on B cell survival

The increased numbers of B cells in the microwell experiments indicated that IL-13 enhanced either the survival or proliferation of mouse B cells. To evaluate the effects of IL-13 on proliferation, sIgD+ splenic mouse B cells were stimulated with anti-CD40 Ab in the absence or presence of IL-13 or IL-4, and thymidine incorporation was measured on day 3. IL-4 strongly enhanced proliferation of mouse B cells, particularly in the presence of anti-CD40 Ab (Fig. 8Go). However, IL-13 did not consistently enhance the proliferation of anti-CD40-stimulated sIgD+ B cells, although slight effects were seen in some experiments (e.g., Fig. 8Go).



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  		FIGURE 8. Effect of IL-13 on thymidine incorporation by sIgD+ B cells in the presence of suboptimal concentrations of anti-CD40. Sorted sIgD+ mouse splenic B cells were cultured at 1 x 104/ml without ({circ}) or with 10 ({blacktriangleup}) or 100 ({blacktriangledown}) ng/ml of anti-CD40 mAb and in the absence or presence of IL-13 (10 or 50 ng/ml) or IL-4 (10 ng/ml) in triplicate for 3 days. Thymidine was added during the final 18 h. The inset shows an expanded scale for the IL-13 results. An asterisk (*) indicates data significantly greater (p < 0.05) than cultures in the absence of exogenous cytokines. The inset in the IL-13 panel shows an expanded scale.

 
To clearly distinguish whether IL-13 affected mouse B cell survival or proliferation, mouse sIgD+ B cells were stained with CFSE and stimulated with 3 ng/ml of anti-CD40 Ab in the presence or absence of IL-13 or IL-4 for 3 days. Viable B cells in bulk cultures were counted and subjected to flow cytometric analysis (Fig. 9Go). Consistent with the microculture observations, in the presence of a suboptimal dose of anti-CD40 Ab, cultures with IL-13 and IL-4 resulted in increased numbers of B cells. The fluorescent intracellular stain CFSE allows tracking of the proliferation or division history of individual cells (31). After 2 days in anti-CD40 alone, the majority of the B cells were still small, nonproliferating lymphocytes as assessed by forward scatter (Fig. 9Goa) and high CFSE content (Fig. 9Goe), whereas a smaller subpopulation were larger and appeared to have divided once, as indicated by halving of the CFSE intensity. Incubation in anti-CD40 plus IL-13 (5 or 20 ng/ml) resulted in similar forward scatter and CFSE profiles to the cultures stimulated with anti-CD40 alone (Fig. 9Go, b, c, f, and g). This indicated that IL-13 did not induce any additional proliferation, and thus the increase in viable cell number induced by IL-13 (Fig. 9Go, j and k) was due to increased survival. In contrast, anti-CD40 plus IL-4 induced further proliferation, as shown by a marked increase in the forward scatter of the replicating cells (Fig. 9God) and the appearance of CFSE fluorescence peaks indicating two and three divisions (Fig. 9Goh). Collectively, the above proliferation and Ab data indicate that IL-13 enhanced Ab synthesis by increasing survival rather than inducing proliferation of anti-CD40-activated B cells.



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  		FIGURE 9. IL-13 enhanced survival but not proliferation of anti-CD40-stimulated sIgD+ B cells. Sorted sIgD+ mouse splenic B cells were stained with CFSE and cultured in triplicate at 1.25 x 105 cells/ml with 3 ng/ml anti-CD40 mAb in the absence (a, e, and i) or presence of 5 ng/ml IL-13 (b, f, and j) or 20 ng/ml IL-13 (c, g, and k), or 10 ng/ml IL-4 (d, h, and l) for 3 days. Viable B cells were counted, and the number was expressed as a percentage of the starting population (i–l). Forward light scatter (a–d) and CFSE levels (e–h) were analyzed by flow cytometry. Similar data were obtained in two other experiments. An asterisk (*) indicates data significantly greater (p < 0.01) than the anti-CD40 cultures in the absence of exogenous cytokines (i).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
After continuous administration by osmotic pumps for 7 days, mIL-13 enhanced Ag-specific Ab levels in the plasma, indicating that IL-13 can play a role in enhancing Ab production in vivo. At least part of this effect may be due to direct stimulation of B cells, as IL-13 induced B cells in vitro to secrete higher Ig levels in response to a number of stimuli: PFA-fixed anti-CD3-activated T cells, irradiated alloreactive primary Th1 cells, and most significantly, anti-CD40 Ab acting on sIgD+ or B220+ B cells. The magnitude of the IL-13 effect was generally lower than that induced by IL-4, even with saturating amounts of IL-13. Higher levels of IL-13 were also required before saturation was observed. These observations are consistent with the weak effects of IL-13 on human B cells compared with those of IL-4 (21). Unlike IL-4, IL-13 did not up-regulate surface MHC class II or CD23 expression on anti-CD40-stimulated mouse B cells. However, mIL-13 induced parallel increases in the numbers of total B cells and Ab-secreting cells in microcultures containing only B cells, confirming the direct and IL-4-independent biological effects of IL-13 on mouse B cells. Interestingly, treatment of mice with anti-IL-13 polyclonal Ab reduced humoral immune responses at mucosal surfaces, further supporting a role for IL-13 during humoral responses (40). As IL-13 did not increase cell division, it is thus concluded that mIL-13 increases Ab production by enhancing B cell survival.

In agreement with previous reports (5, 41), we also found that IL-13 had no detectable effect on LPS-stimulated mouse B cells, and B cells stimulated strongly by anti-CD40 Ab (>100 ng/ml) also showed no additional response to IL-13 (data not shown). Hence, the biological effects of IL-13 on mouse B cells may not be revealed during potent stimulation by either anti-CD40 Ab or LPS. We have now shown that IL-13 has biological effects on mouse B cells at suboptimal doses of anti-CD40 Ab, which may represent more physiological stimulation levels. Thus, effects of IL-13 may not be observed in all B cell stimulatory systems, which may account for a previous report that IL-13 failed to exhibit any biological functions on mouse B cells (5).

IL-4 enhances proliferation of mouse and human B cells and moderately stimulates synthesis of most Ig subclasses. In addition, IL-4 strongly stimulates synthesis of IgG1 (mouse) or IgG4 (human) and IgE (mouse and human) by the induction of specific class switching (42, 43, 44, 45). hIL-13 stimulates sIgD+ B cells to produce IgG4 and IgE (20, 21, 22). However, our results with mIL-13 suggested that although this cytokine may share some of the general B cell stimulatory effects of IL-4, there is no strong isotype selectivity. IgG1 levels were enhanced only to a similar extent as other isotypes, and we were unable to detect significant levels of IgE production when B cells were stimulated by IL-13 and fixed, activated T cells or anti-CD40 Ab, although IL-4 induced high levels of IgE in both conditions. If IgE production is stimulated by IL-13, the levels are below our detection limit (6.25 ng/ml), or at least 50-fold below the levels induced by IL-4. The difference between IL-4- and IL-13-induced levels is more than 10-fold for IgG1, thus it remains possible that IL-13 may indirectly enhance IgE levels to a low extent, by increasing B cell survival, for example.

Interestingly, IgE was produced in IL-4-deficient mice infected with malaria (24) or Leishmania major (46) or during the course of retrovirus-induced immunodeficiency syndrome or anti-IgD treatment (47), but not during nematode infection (23, 47, 48), suggesting that there is an IL-4-independent mechanism that can also stimulate IgE production in some circumstances. Although our results do not provide any evidence for a selective role of IL-13 in IgE production, we cannot yet exclude the possibility that IL-13 may enhance IgE levels under certain conditions that we have not yet tested.

The enhancement of IgE synthesis by IL-13 in human but not mouse B cells suggests a species difference in the requirements for IgE class switching. Although many cytokine effects are conserved between mice and humans, some differences have been reported. Mouse bone marrow cells cultured with mIL-3 for >4 wk produce almost pure mouse-mast cell populations (49), whereas hIL-3 induces production of basophils and eosinophils but not mast cells from human cord blood or bone marrow mononuclear cells (50). Moreover, mIL-5 is a potent B cell stimulator, but hIL-5 does not appear to be a regulatory factor for human B cells except in the presence of mitogenic stimuli such as Moraxella catarrhalis (51) or Staphylococcus aureus Cowan I strain (SAC) under particular conditions (52). Also, IL-10 stimulates proliferation of SAC-stimulated human B cells (53), but inhibits proliferation of LPS-stimulated mouse B cells (54).

The capacity of IL-4 and IL-13 to enhance DNA synthesis and proliferation of human B cells stimulated with anti-IgM (55) or anti-CD40 (3, 20, 21, 39, 56, 57) is widely established. mIL-4 also enhances mouse B cell proliferation and survival (55, 58). The results in Fig. 9Go also show that IL-4 induced additional divisions of mouse B cells stimulated with anti-CD40 Abs. Under similar conditions, IL-13 did not significantly induce proliferation of mouse B cells beyond that induced by anti-CD40 alone. In contrast, mIL-13 enhanced proliferation of the B9 mouse plasmacytoma cell line (5, 59), suggesting that IL-13 might enhance normal B cell proliferation under other circumstances. The effects of IL-13 on mouse B cells were consistent with the known effects of hIL-13 in providing survival signals to anti-CD40-stimulated human peripheral blood B cells (60), B-chronic lymphocytic leukemia cells (61), and non-Hodgkin’s lymphoma B cells (62). Similar to IL-13, IL-4 prolongs the viability of not only human and mouse B cells (58, 63, 64, 65, 66) but also malignant human B cells (67, 68).

Interestingly, in some experiments, high concentrations of anti-CD40 Ab induced purified B cells to produce Abs in the absence of any exogenous cytokine. This effect could not be inhibited by anti-IL-4 mAb, indicating an IL-4-independent stimulation pathway. Furthermore, in the presence of IL-4, anti-CD40-stimulated B cells formed tight clusters, in sharp contrast to anti-CD40 plus IL-13 cultures. As the EBV-transformed human B cell lines express IL-13 mRNA and produce a small amount of IL-13 (69, 70, 71), it is conceivable that mouse splenic B cells can produce IL-13 upon strong anti-CD40 activation and thus function in an autocrine fashion. This would be consistent with the inability of exogenous IL-13 to further enhance Ab production at high anti-CD40 concentrations.

In addition to the direct effect of IL-13 on B cells that we have demonstrated, other factors may also be involved in up-regulating Ab production during in vivo IL-13 administration and CRBCs immunization. IL-13 induces extramedullary hemopoiesis and monocytosis in mice (6). It is conceivable that the increased numbers of macrophages may secrete soluble mediators that enhance the Ab response, or that enhanced Ag presentation to T cells may provide more B cell help. This may also explain the isotype differences between the enhancement mainly of IgM levels in vitro compared with the enhancement of IgG levels in vivo. Unlike IL-4, which plays an important role in T cell differentiation (27, 72, 73, 74), IL-13 does not appear to bind to or exhibit such biological function on either human or mouse T cells (5, 7, 69). Thus, IL-13 is unlikely to affect B cells by directly altering T cell differentiation in vivo.

In comparison to IL-4, our results suggest that IL-13 may play a more minor role during Ab responses in vivo. However, there is potential for a more substantial role in circumstances where other B cell stimulatory cytokines, such as IL-4, are absent or ineffective. The IL-4R {alpha}-chain is also a component of some IL-13 receptors (8, 9, 14, 75, 76, 77, 78, 79). IL-4R {alpha}-chain-deficient mice fail to produce Ag specific IgG1 responses (80), whereas IL-4-deficient mice generate lower Ag specific IgG1 levels (23, 81), suggesting a possible role for IL-13 in Ab production in vivo. In addition, IL-13 is not produced in complete concordance with IL-4, and so there may be circumstances in which IL-13 but not IL-4 is expressed. hIL-13 is expressed within 2 h of activation and hIL-13 mRNA can still be detected after 72 h. In contrast to the early, yet sustained hIL-13 mRNA expression, hIL-4 mRNA expression in T cells is transient and can only be detected 24 h after activation (5). If mIL-13 has similar expression kinetics, it may play an important role in stimulating an initial Ab response before the expression of IL-4, or sustaining Ab production at later times.


   Acknowledgments
 
We thank the excellent technical assistance of R. Marcotte, useful discussions with D. C. Parker, L. J. Guilbert, and D. Kunimoto, and statistical analyses by R. C. Sinclair. We also thank Dorothy Rutkowski and Juanita Wizniak for operating the cell sorters.


   Footnotes
 
1 This work was supported by the Medical Research Council of Canada and the Howard Hughes Medical Institute. Back

2 Address correspondence and reprint requests to Dr. Timothy R. Mosmann, Center for Vaccine Biology and Immunology, Institute for Biomedical Sciences, University of Rochester Medical Center, 601 Elmwood Ave., Box 609, Rochester, NY 14642. E-mail address: Back

3 Abbreviations used in this paper: h, human; m, mouse; CD40L, CD40 ligand; ELISPOT, enzyme-linked immunospot; HSLAS, Health Sciences Laboratory Animal Services; CRBCs, chicken RBCs; PFA, paraformaldehyde; IMDM, Iscove’s modified Dulbecco’s medium; CFSE, 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester. Back

Received for publication January 15, 1998. Accepted for publication September 2, 1998.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
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