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The Journal of Immunology, 2002, 169: 1696-1704.
Copyright © 2002 by The American Association of Immunologists

IL-4 Promotes Stat6-Dependent Survival of Autoreactive B Cells In Vivo Without Inducing Autoantibody Production1

Suzanne C. Morris2,*, Nanette L. Dragula* and Fred D. Finkelman*,{dagger}

* Cincinnati Veterans Affairs Medical Center, Cincinnati, OH 45220, and Division of Immunology, University of Cincinnati College of Medicine, Cincinnati, OH 45267; and {dagger} Division of Immunology, Children’s Hospital Medical Center, Cincinnati, OH 45229


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Persistent cross-linking of hen egg lysozyme (HEL)-specific B cell membrane Ig (mIg) in double transgenic mice that express soluble HEL as a self Ag (HEL-Ig mice) decreases B cell mIgM expression, responsiveness, and life span. Because in vitro treatment with IL-4 inhibits T cell apoptosis through a Stat6-independent mechanism, increases mIg expression, and suppresses activation-induced B cell death, we studied IL-4 effects on B cell mIg expression, survival, and Ab secretion in Stat6-sufficient and deficient HEL-Ig mice. IL-4 treatment nearly normalized B cell number and greatly increased the percentage of mature B cells in HEL-Ig mice, but failed to normalize mIgM expression or spontaneous LPS-induced IgM secretion. IL-4 effects on B cell survival and maturation were CD4+ T cell independent, but Stat6 dependent, and did not involve receptor editing. IL-4 had to be present while B cells were generated to have a detectable effect on autoreactive B cell survival; however, the survival of B cells generated in the presence of IL-4 was substantially increased even after IL-4 was withdrawn. These observations suggest that: 1) activation-induced B cell death and anergy are independent processes; 2) B cells that survive to maturity develop increased resistance to Ag-induced deletion; and 3) IL-4 promotes B and T cell survival through different mechanisms.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
B cell tolerance that is induced by exposure to Ag in the absence of additional stimuli protects against the development of humoral autoimmunity (1, 2, 3). The characteristics of Ag-induced tolerance depend on the extent of B cell membrane Ig (mIg)3 cross-linking by Ag. Exposure to polyvalent, particulate self Ag strongly cross-links mIg and deletes newly generated Ag-specific B cells before they leave the bone marrow (3, 4, 5, 6, 7), while less intense cross-linking of mIg by a soluble oligovalent Ag allows Ag-specific B cells to mature to the point in which they can migrate from bone marrow to the spleen (1, 8). Most Ag-specific B cells exposed to oligovalent Ag, however, become hyporesponsive (anergic); they secrete less Ig than naive B cells in response to in vitro stimulation with LPS or Th cells, and proliferate poorly in response to anti-Ig Ab stimulation (9). These hyporesponsive B cells lose all or most of their mIgM (8, 10, 11, 12) and are deleted within a few days after migrating to the spleen (12, 13, 14, 15), before they acquire the mature B cell phenotype. The small percentage of Ag-specific B cells that survives to maturity in the presence of oligovalent Ag predominantly does so by further decreasing its responsiveness to Ag, either by undergoing receptor editing (8) or by down-regulating its expression of mIgD as well as mIgM (12).

Ag-induced B cell anergy and deletion are prevented if Ag-exposed B cells are simultaneously stimulated by T cells (16, 17). The help provided by T cells that prevents B cell anergy and deletion includes humoral factors (cytokines) as well as membrane-associated costimulatory molecules, such as CD40 ligand (18). Because one T cell-produced cytokine, IL-4, enhances B cell mIg expression, inhibits B and T cell apoptosis in vitro, and stimulates humoral autoimmunity if overexpressed in vivo (19, 20, 21, 22, 23), it seemed possible that in vivo treatment with IL-4 could prevent Ag-induced B cell tolerance. No prevention of B cell deletion was observed, however, in one study in which B cells in IL-4-overexpressing mice were exposed to a particulate polyvalent self Ag (23). This negative result did not rule out the possibility that IL-4 might inhibit the more subtle form of tolerance that develops when B cells are exposed to an oligovalent, soluble self Ag. To study this issue, we have treated double transgenic (Tgn) mice that express soluble hen egg lysozyme (HEL) and whose B cells express mIgM and mIgD that bind HEL with high affinity (HEL-Ig mice) with a long-acting form of IL-4 (24). Results of these studies demonstrate that IL-4 treatment prevents premature B cell death and allows B cells to acquire a mature phenotype through a Stat6-dependent mechanism, but does not prevent the loss of mIgM or decreased responsiveness to LPS.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

Female C57BL/6 mice, obtained from the Small Animals Division of the National Cancer Institute, National Institutes of Health (Bethesda, MD), were bred in the Cincinnati Veterans Affairs Medical Center animal facility to male C57BL/6 mice that were hemizygous for both the MD4 anti-HEL Ig H and L transgene and the ML5 soluble HEL transgene (a gift of C. Goodnow, Australian National University, Canberra, Australia) to generate mice that carry only the MD4 transgene (Ig Tgn mice), and mice that carry both the MD4 and ML5 transgenes (HEL-Ig mice). C57BL/6-Stat6-deficient and BALB/c-Stat6-deficient mice were originally obtained from M. Grusby (Boston, MA). C57BL/6-Stat6-deficient mice were crossed to C57BL/6-HEL-Ig Tgn mice, and their offspring were backcrossed to C57BL/6-Stat6-deficient mice to generate C57BL/6 Stat6-deficient, Ig Tgn mice and Stat6-deficient, HEL-Ig mice. BALB/c Stat6-deficient mice were bred at the Cincinnati Veterans Affairs Medical Center animal facility, and BALB/c wild-type mice were obtained from the Small Animals Division of the National Cancer Institute, National Institutes of Health. Mice were used at 8–34 wk of age. Mice were age and sex matched in individual experiments.

Typing of Tgn mice

Mice that expressed the HEL transgene and/or the anti-HEL transgene were identified by PCR (25). DNA was isolated with QIAamp tissue kits for DNA isolation (Qiagen, Santa Clarita, CA). PCRs were performed as described (12). Stat6 deficiency was determined by PCR. The following three oligonucleotides were used in Stat6 PCR: Stat6 upper, 5'-TGAGGTGGGGACCAGCCGG-3'; Stat6 lower, 5'-GTGACCAGGACACACAGCGG-3'; and Neo, 5'-GCTACCCGTGATATTGCTGAAGAG-3'. PCR amplification of cells from Stat6-deficient mice yields a 225-bp product; PCR amplification of cells from wild-type mice yields a 100-bp product. Oligonucleotide primers were produced by the BIC synthesis center at the Uniformed Services University of the Health Science (Bethesda, MD).

Experimental conditions

All mice, except for BALB/c wild-type and Stat6-deficient mice, were maintained on drinking water that contained 25 mM ZnCl2 for at least 3 days before the initiation of other treatments and for the duration of each experiment to maximize serum HEL levels in HEL-Ig mice (15). In experiments in which newly generated B cells were identified by 5-bromo-2'-deoxyuridine (BrdU) incorporation (7, 13), 0.8 mg/ml BrdU (Sigma-Aldrich, St. Louis, MO) was also added to drinking water for a defined period of time. BrdU-containing water also contained 2 mg/ml glucose (except for the experiment in Fig. 7GoD) and was shielded from light and changed every third day.



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  		FIGURE 7. IL-4 increases the survival of HEL-Ig B cells in HEL-Ig Tgn mice that are generated in its presence. Six separate experiments were performed in which Ig Tgn and HEL-Ig mice (3–5/group) were left untreated, were injected three times per week i.p. with IL-4C that contained 0.5 µg mouse IL-4, or received a combination of both IL-4C and anti-IL-7 mAb (3 mg, three times per week i.p.). Mice were administered drinking water that contained BrdU for the indicated periods. Mice were sacrificed in each experiment at the end of the treatment period or 12 days after the last IL-4C injection (F). Spleen cell suspensions were prepared, counted, stained with biotin-anti-HSA and Cy5-anti-B220 mAbs, followed by S-PE, then fixed, permeabilized, and stained with FITC-anti-BrdU mAb. Percentages of BrdU+ and BrdU- HSAbright (immature) and HSAdull (mature) B220+ cells were determined by flow cytometry and multiplied by total spleen cell numbers to calculate numbers of cells of each phenotype. Information on immature and mature splenic B cell numbers is shown, except for B and E, which depict total splenic B cells.

 
Abs and immunological reagents

The following hybridomas were obtained and grown as ascites in either pristane-primed athymic nude, BALB/c, or CB20 mice: RA3-6B2 (rat IgG2a anti-mouse CD45R/B220), DS-1 (mouse IgG1 of the b allotype specific for mouse IgM of the a allotype), H{delta}a/1 (mouse IgG2b of the b allotype specific for mouse IgD of the a allotype), AF3.33 (mouse IgG2a of the a allotype specific for mouse IgD of the b allotype), 2D1 (mouse IgG1 anti-HEL) (a generous gift of D. Metzger, Albany, NY) (26), HyHEL10 (mouse IgG1 anti-HEL) (a generous gift of S. Smith-Gill, Bethesda, MD) (27), BVD4-1D11.2 (rat IgG2b anti-mouse IL-4), 24G2 (rat IgG2b anti-mouse Fc{gamma}RII/III), m25 (mouse IgG1 anti-IL-7) (a generous gift of K. Grabstein, Immunex, Seattle, WA) (28), and GK1.5 (rat IgG2b anti-mouse CD4). mAbs were purified from ascites by (NH4)2SO4 precipitation and DE-52 (Whatman, Clifton, NJ) cation exchange column chromatography, unless otherwise stated. Some of these mAbs were labeled with FITC (Calbiochem-Behring, La Jolla, CA), biotin-N-hydroxysuccinimide (Calbiochem-Behring), or Cy5-reactive dye (Research Organics, Cleveland, OH), as suggested by the manufacturer. Biotin or FITC-labeled M1/69 (anti-heat-stable Ag (HSA)), PE-labeled 1D3 (anti-CD19), and PerCP-labeled RA3-6B2 (anti-B220) were purchased from BD PharMingen (San Diego, CA). FITC anti-BrdU was purchased from BD Immunocytometry Systems (San Jose, CA). HB{delta}7, also known as LO-MD-7 (rat IgG2a anti-mouse IgD) (29, 30), was provided by H. Bazin (Brussels, Belgium). Alkaline phosphatase conjugated to streptavidin was purchased from Jackson ImmunoResearch (West Grove, PA). HEL, BrdU, LPS (Escherichia coli 0111:B4), and glucose were purchased from Sigma-Aldrich. Purified mouse rIL-4 was purchased from PeproTech (Rocky Hill, NJ).

Enzyme-linked immunosorbent assays

To determine IgMa anti-HEL in culture supernatants, microtiter plate wells were coated with 100 µl/well DS-1 anti-IgMa mAb (10 µg/ml) and blocked with skim milk. Serial 4-fold dilutions of supernatants were then added, in duplicate, to wells, followed sequentially by HEL (100 ng/ml), biotin-labeled 2D1 anti-HEL mAb (2 µg/ml), alkaline phosphatase-labeled streptavidin (1/2000), and substrate (p-nitrophenylphosphate at 1 mg/ml (Calbiochem-Behring)). OD405 values of individual microtiter plate wells were determined with a Multiskan MS ELISA plate reader (Labsystems, Franklin, MA). Titers are the reciprocals of the dilutions that generated a specific OD405 value on the linear part of the titration curve. Titers were corrected for the percentage of B cells in a given culture.

To determine serum HEL levels, microtiter wells were coated with the anti-HEL mAb HyHEL10 (25 µg/ml) and blocked with skim milk. Serial 4-fold dilutions of serum or HEL (standard) were then added, in duplicate, to wells, followed sequentially by a mAb to a second HEL epitope (biotin-labeled 2D1 at 200 ng/ml), alkaline phosphatase-labeled streptavidin, and substrate, as above. Concentrations of HEL were determined by comparing the titers for the HEL standard with the titers obtained for the serum.

Preparation of cytokine/anti-cytokine Ab complexes

IL-4 (200–1000 µg/ml) was mixed at a 2:1 molar ratio (1:6 weight ratio) with neutralizing anti-IL-4 mAb (BVD4-1D11.2) to prepare IL-4/anti-IL-4 mAb complexes (IL-4C), which greatly increase the in vivo t1/2 and activity of IL-4 (24). After 2 min at room temperature, complexes were diluted with 1% C57BL/6 serum or 1% BALB/c serum to a concentration of 2.5 or 10 µg IL-4/ml, for injection into mice. Complexes were always freshly prepared before use.

Immunofluorescence staining

Spleen cells were depleted of erythrocytes, filtered through nylon gauze, and suspended at 20 x 106 cells/ml in HBSS with 10% newborn bovine serum and 0.2% NaN3 (HNA). A total of 100 µl cell suspension was stained for 30 min on ice with 1 µg each of appropriately labeled Abs. Cells were washed twice with HNA, then, if appropriate, exposed to streptavidin-R-PE (S-PE, purchased from BD Immunocytometry Systems) for 30 min on ice. All staining was performed in the presence of 10 µg/ml unlabeled anti-Fc{gamma}RII/III mAb (24G2). After washing once with HNA, all samples, except those that required staining for BrdU incorporation, were washed once with HBSS/0.2% sodium azide, then fixed in PBS/2% paraformaldehyde. Staining for BrdU was performed as previously described (12). All samples were analyzed with either a FACScan or a FACSCalibur Analyzer equipped with a red diode laser (BD Biosciences, Mountain View, CA). Data were analyzed with Lysis II or CellQuest software. Light scatter gates were set to exclude most nonlymphoid cells and cells that had died before fixation. Cells that had been stained with a single fluorochrome-labeled Ab were used to determine compensation for overlap between emission spectra. Percentages of specifically stained cells and the mean and/or median fluorescence intensities of specifically stained cells were determined.

Cultures

Spleen cells were depleted of erythrocytes, filtered through nylon gauze, and suspended to a concentration of 5 x 105 cells/ml in RPMI 1640 without phenol red (BioWhittaker, Walkersville, MD) that was supplemented with 2 mM L-glutamine, 10% FBS (Life Technologies, Gaithersburg, MD), 0.05 mM 2-ME, 50 µg/ml penicillin, 50 µg/ml streptomycin, nonessential amino acids, 1 mM sodium pyruvate, 25 mM HEPES, and 20 µg/ml LPS. A total of 200 µl each cell suspension was plated in six wells of 96-well flat-bottom polystyrene tissue culture plates (Corning, Corning, NY). Plates were incubated for 72 h at 37°C in a humidified 5.5% CO2 atmosphere. Cultures were harvested and samples centrifuged at 18,000 x g for 2 min to obtain cell-free supernatants. Supernatants were kept frozen before assay.

Cell counts

Nucleated cells were counted with a Coulter Counter (Coulter, Miami, FL) that was set to exclude dead cells. Absolute numbers of cells that had a defined phenotype were determined by multiplying the percentage of cells that expressed that phenotype by total cell number.

Statistical methodology

Differences in means of continuous numeric variables were tested for statistical significance using the two-sided independent t test when normally distributed populations were examined, or the Mann-Whitney rank sum test when the values within a group were not normally distributed. Values of p <= 0.05 were considered significant. SigmaStat software was used to perform the analysis.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
IL-4 increases splenic B cell number in HEL-Ig mice

To determine whether treatment with a long-acting formulation of IL-4 would increase the low splenic B cell number in HEL-Ig mice, Ig Tgn and HEL-Ig mice were left untreated or were treated with IL-4C (0.5 µg IL-4 + 3 µg anti-IL-4 mAb) three times per week for 5 or 14 days. IL-4C treatment had little effect on splenic B cell number in Ig Tgn mice, but caused a doubling of splenic B cell number in HEL-Ig mice in 5 days (Fig. 1GoA) and fully corrected splenic B cell number in HEL-Ig mice after 14 days (Fig. 1GoB). This effect of IL-4 was CD4+ T cell independent, as it was not blocked by anti-CD4 mAb (Fig. 1GoC). Note that splenic B cell number in untreated Ig Tgn and HEL-Ig mice can vary considerably from one experiment to another; for this reason, we are only able to compare age- and sex-matched groups of mice that are studied simultaneously. Note also that an apparent decrease in splenic B cell number in Ig Tgn mice treated with IL-4C for 14 days (Fig. 1GoB) was not observed in most experiments (see, for example, Figs. 5Go and 7GoB, below).



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  		FIGURE 1. IL-4 increases splenic B cell number in HEL-Ig mice by a CD4+ T cell-independent mechanism. In three separate experiments, Ig Tgn and HEL-Ig mice (2–4/group) were left untreated or were injected i.p three times per week with IL-4C that contained 0.5 µg mouse rIL-4. Mice were sacrificed either 5 days (A) or 14 days (B) after the initiation of IL-4C treatment. Spleen cells from individual mice were counted and stained with biotin-anti-B220 and FITC-anti-HSA mAbs, followed by S-PE, and analyzed by flow cytometry to determine percentages of B220+HSA+ cells. Numbers of splenic B cells were calculated by multiplying percentages of B220+HSA+ cells by total spleen cell number. A third experiment (C) was performed identically to that shown in B, with the exception that all mice were treated i.v. with 1 mg anti-CD4 mAb on days 0 and 7. Means and SEs are shown in each panel and in all subsequent figures.

 


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  		FIGURE 5. Stat6 is required for IL-4-induced normalization of mature splenic B cell number in HEL-Ig Tgn mice. Wild-type and Stat6-deficient Ig Tgn and HEL-Ig mice (10–12/group) were left untreated or were injected i.p. three times per week for 14 days with IL-4C that contained 0.5 µg mouse IL-4. Mice were sacrificed 14 days after initiation of IL-4C treatment. Spleen cells from individual mice were counted and stained with anti-B220 (either Cy5 or PerCP labeled), FITC-anti-HSA, and PE-anti-CD19 mAbs and analyzed by flow cytometry to determine the percentage of B220+CD19+ cells that were B220brightHSAdull (mature) or B220dullHSAbright (immature). Numbers of mature and immature splenic B cells were determined as in Fig. 1Go. HSA expression (MFI) was determined for B220+CD19+ splenic B cells.

 
IL-4 matures splenic B cells in HEL-Ig mice

Most splenic B cells in HEL-Ig mice have an immature (HSAbright) phenotype, which reflects their relatively normal rate of production and rapid rate of elimination (12). To determine whether IL-4C treatment normalizes splenic B cell number by increasing B cell survival (which would lower mean HSA expression) or by increasing B cell production (which would increase mean HSA expression), we evaluated splenic B cell HSA expression in untreated Ig Tgn and HEL-Ig mice and in mice of the same strains that had been treated with IL-4C for 1, 5, or 14 days. IL-4C treatment had only a slight effect on splenic B cell HSA expression in Ig Tgn mice, but fully normalized splenic B cell HSA expression in HEL-Ig mice within 5 days of the initiation of treatment. Although we cannot completely rule out the possibility that IL-4 decreases B cell HSA expression through effects not related to maturation or prolongation of survival, the failure of IL-4 to affect in vivo B cell HSA expression 1 day after the initiation of treatment (Fig. 2Go) or to specifically decrease HSA expression by HEL-Ig B cells cultured in vitro (data not shown) makes this unlikely. IL-4C effects on HSA expression, like IL-4C effects on splenic B cell number, were CD4+ T cell independent (Fig. 2Go, bottom panel).



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  		FIGURE 2. IL-4 treatment selectively decreases HSA expression by splenic B cells in HEL-Ig mice. HSA expression (mean fluorescence intensity (MFI)) was determined for splenic B cells in the same experiments illustrated in Fig. 1Go and in a fourth experiment in which mice were sacrificed 1 day after the initiation of IL-4C treatment.

 
IL-4C fails to correct defective mIgM expression in HEL-Ig mice

The ability of IL-4 to increase splenic B cell number and level of maturity in HEL-Ig mice suggested that it might also correct other defects in these B cells that result from chronic cross-linking of B cell mIg, including the selective decrease in expression of B cell mIgM (but not mIgD) that results primarily from a block in the terminal glycosylation and membrane insertion of µ-chain (11). This was not the case: although IL-4C enhanced both mIgM and mIgD expression by Ig Tgn B cells and enhanced mIgD expression by HEL-Ig B cells, it failed to increase the very low expression level of HEL-Ig B cell mIgM (Fig. 3Go).



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  		FIGURE 3. IL-4 fails to increase B cell mIgM expression in HEL-Ig mice. Ig Tgn and HEL-Ig mice (6–7/group) were left untreated or were injected i.p. three times per week for 2 wk with IL-4C that contained 0.5 µg mouse IL-4, after which mice were sacrificed and spleen cell suspensions were prepared, stained with biotin-anti-IgDa and FITC-anti-IgMa mAbs, followed by S-PE, and analyzed by flow cytometry for mIgMa and MFI.

 
IL-4 does not affect serum HEL levels in HEL-Ig mice

IL-4 enhancement of B cell survival and mIgD expression in HEL-Ig mice might reflect either increased B cell resistance to mIg cross-linking-induced cell death or suppression of HEL synthesis, which would decrease B cell mIg cross-linking. To differentiate between these possibilities, we measured serum HEL levels in untreated and IL-4C-treated HEL-Ig mice (Fig. 4Go). IL-4C treatment had no detectable effect on serum HEL levels (p = 0.52), indicating that decreased Ag-induced mIg cross-linking is not responsible for the effects of IL-4 on B cell survival.



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  		FIGURE 4. IL-4 treatment has little affect on serum HEL levels in HEL-Ig mice. Serum HEL levels were determined by ELISA for Ig Tgn and HEL-Ig mice (3–5/group) that were left untreated or were injected i.p. three times per week with IL-4C that contained 0.5 µg mouse IL-4 for 16 days before bleeding. Differences in serum HEL levels in untreated and IL-4C-treated HEL-Ig Tgn mice were not significant (p = 0.52).

 
IL-4 selectively increases the number of mature B cells in HEL-Ig mice through a Stat6-dependent mechanism

Our observation that IL-4 increases B cell survival in HEL-Ig mice suggested that this cytokine selectively increases mature splenic B cell number in these mice. Furthermore, observations that IL-4 increases splenic T cell survival through a Stat6-independent mechanism (21) and enhances anti-Ig Ab-induced B cell proliferation in the absence of Stat6 (31) suggested that the effects of IL-4 on splenic B cell survival and maturation would also be Stat6 independent. To test these hypotheses, we bred HEL-Ig mice and Ig Tgn mice with Stat6-deficient mice (all on a C57BL/6 background) and compared the responses of Stat6-deficient and Stat6-sufficient HEL-Ig and Ig Tgn mice to IL-4. Results of these studies confirmed the first hypothesis, but refuted the second. Although splenic B cells that have an immature (B220dullHSAbright) phenotype are normal in number in Stat6-sufficient HEL-Ig Tgn mice and do not appreciably increase in number following IL-4C treatment (Fig. 5Go, top panel), IL-4C causes a large increase in the low number of mature splenic B cells in these mice (Fig. 5Go, middle panel). Contrary to our expectations, both the IL-4-induced increase in the number of mature HEL-Ig splenic B cells and the IL-4-induced decrease in their mean splenic B cell HSA expression were completely Stat6 dependent (Fig. 5Go, middle and bottom panels).

IL-4 inhibits in vivo deletion of mature B cells by anti-IgD mAb

To determine whether the B cell-sparing effect of IL-4 could be observed with normal, as well as Ig Tgn B cells that were activated by mIg cross-linking, we studied a system in which treatment of BALB/c mice with an anti-IgD mAb causes deletion of mature B cells (32). Our initial experiment (Fig. 6Go, left panels), which used our standard dose of IL-4C, showed that anti-IgD mAb had its expected effect on mature splenic B cell number in wild-type BALB/c mice. However, IL-4C treatment did not increase mature splenic B cell number significantly more in anti-IgD Ab-treated mice than in mice that did not receive anti-IgD Ab (p = 0.091). Because anti-IgD mAb cross-links mIgD on normal B cells to a much greater extent than HEL cross-links mIg on HEL-Ig B cells (18), we reasoned that more IL-4 signaling might be required to rescue anti-IgD mAb-ligated wild-type B cells than to rescue HEL-ligated B cells in HEL-Ig mice. For this reason, we increased the quantity of IL-4C used to treat anti-IgD mAb-injected mice 4-fold. Although this treatment still did not fully negate anti-IgD mAb depletion of mature splenic B cells (Fig. 6Go, right panels), depletion was significantly inhibited (p = 0.029). Furthermore, this higher dose of IL-4C increased the number of mature splenic B cells in anti-IgD mAb-treated mice more than in otherwise untreated mice (p = 0.001). Thus, there appears to be a relationship between the intensity of mIg cross-linking and the concentration of IL-4C that is required to inhibit mIg cross-linking-induced B cell deletion. Studies with anti-IgD mAb also provided confirmation that IL-4 prevents mIg cross-linking-induced B cell deletion through a Stat6-dependent mechanism (Fig. 6Go).



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  		FIGURE 6. Prevention of in vivo deletion of B cells in anti-IgD mAb-treated mice requires an increased amount of IL-4 and is Stat6 dependent. Wild-type and Stat6-deficient BALB/c mice (4/group) were treated with anti-CD4 mAb (1 mg i.v./wk) and anti-IL-7 mAb (3 mg i.p. three times per week), starting 1 wk before other treatments. All mice received anti-Fc{gamma}RII mAb (0.5 mg i.p.) on the same day that additional treatments were begun. Mice received no additional treatment (untreated), 200 µg HB{delta}7 (anti-{delta} mAb) i.v., IL-4C that contained 0.5 or 2 µg IL-4 i.p. every other day, or both HB{delta}7 and IL-4C. All mice were sacrificed 5 days after the initiation of either HB{delta}7 or IL-4C treatment. Spleen cells from individual mice were counted and stained with PerCP-anti-B220, FITC-anti-HSA, and PE-anti-CD19 mAbs and analyzed by flow cytometry to determine the percentage of B220+CD19+ cells that were B220brightHSAdull (mature) or B220dullHSAbright (immature). Numbers of mature and immature splenic B cells were determined as in Fig. 1Go. Treatment with IL-4C that contained 2.0 µg IL-4 increased mature B cell number in anti-{delta}-treated wild-type mice significantly more than in mice that did not receive anti-{delta} (p = 0.001); no significant difference was seen with IL-4C that contained 0.5 µg IL-4 (p = 0.091). IL-4C that contained 2 µg IL-4 significantly blocked anti-{delta} depletion of mature splenic B cells (p = 0.029).

 
IL-4 selectively prolongs the survival of Ag-activated B cells generated in its presence

Six experiments were performed to determine the kinetics of the relationship between IL-4C treatment and increased survival of Ag-activated B cells. These studies used BrdU labeling to identify cells that had divided while BrdU was being administered (predominantly newly generated B cells) (33) and anti-IL-7 mAb to inhibit B lymphopoiesis in the bone marrow (28). An initial study, in which Ig Tgn and HEL-Ig mice were treated with BrdU ± IL-4C ± anti-IL-7 mAb for the 14 days before sacrifice (Fig. 7GoA), demonstrated that IL-4C treatment selectively increased the number of mature HEL-Ig splenic B cells that were generated while IL-4C was being administered (BrdU+ B cells). Treatment with anti-IL-7 mAb blocked this effect completely, presumably by blocking B lymphopoiesis. In contrast, IL-4C treatment, initiated 1–2 wk after HEL-Ig B cells were generated, had no significant effect (p = 0.314) on splenic B cell survival (Fig. 7GoB; note log scale on abscissa). When IL-4C was administered to HEL-Ig mice for 16 days, mature splenic B cells generated during the first 7 days or the last 9 days of this period were both considerably increased in number (Fig. 7GoC). The same was true for mature B cells generated during the first 3 days of a 14-day period of IL-4C treatment (Fig. 7GoD; note log scale on abscissa). Increased survival of HEL-Ig Tgn B cells generated in the presence of IL-4C was more directly demonstrated by an additional experiment (Fig. 7GoE) that measured the percentages of splenic B cells that were BrdU+ 4 and 11 days after a 3-day BrdU pulse in mice that did or did not receive IL-4C for the entire experiment. Although the percentage of splenic B cells that were BrdU+ was higher in untreated than in IL-4C-treated mice 4 days after the pulse (34 vs 25%, respectively), the percentage of BrdU-labeled splenic B cells declined 11 days after the pulse to 3% in untreated mice vs 6.5% in IL-4C-treated mice. Thus, survival of BrdU-labeled B cells over this time period increased ~3-fold as a result of IL-4C treatment. Similar results were obtained when calculations were based on absolute numbers of splenic BrdU+ B cells, rather than percentages of splenic B cells that were BrdU+ (data not shown).

To determine whether the continuing survival of Ag-stimulated B cells generated during a period of IL-4C treatment depends upon continuing IL-4C stimulation, Ig Tgn and HEL-Ig mice were treated with BrdU and IL-4C for 5 days and sacrificed 11 days after termination of BrdU treatment (Fig. 7GoF, note log scale on abscissa). Even though this short period of IL-4C treatment had no effect on total splenic B cell number in either mouse strain, it significantly increased the number of mature, BrdU+ splenic B cells in HEL-Ig mice (p = 0.039); this increase (2.1-fold) was not as large (3.6-fold) as that observed when IL-4C treatment was continued until the time of sacrifice (Fig. 7Go, compare D and F); however, the magnitudes of these increases were not significantly different (p = 0.21). Thus, if newly generated, Ag-stimulated B cells are induced by IL-4 to survive and mature, they develop increased ability to survive continuing Ag activation. This ability to survive may be increased further, however, if Ag-activated B cells continue to be stimulated by IL-4 after they have matured.

Increased survival of IL-4-stimulated, Ag-activated B cells is not a result of increased receptor editing

Receptor editing (replacement of an Ig L chain that allows for autoreactivity with one that does not) has been demonstrated in mice that have autoreactive, Tgn mIg (34, 35). Receptor-edited B cells can escape deletion by self Ag, because their mIg no longer reacts (or reacts less avidly) with self Ag. To determine whether IL-4 enhances B cell survival by increasing receptor editing, we evaluated the ratio of HEL-binding capacity to mIg expression in mature splenic B cells from HEL-Ig mice. Previous studies have established that non-receptor-edited B cells in these mice demonstrate a linear relationship between HEL binding and mIg expression, while receptor-edited B cells express a lower ratio of HEL binding to mIg expression (12). Using this technique, we found that IL-4C treatment selectively increases the number of mature, non-receptor-edited splenic B cells as well as the percentage of mature splenic B cells that is not receptor edited in HEL-Ig mice, and that the IL-4-induced increases in the non-receptor-edited B cell population is selectively suppressed by anti-IL-7 mAb treatment (Fig. 8Go). Thus, IL-4C treatment does not increase HEL-Ig B cell survival by increasing receptor editing.



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  		FIGURE 8. IL-4 increases the survival of non-receptor-edited B cells in HEL-Ig Tgn mice. HEL-Ig Tgn mice (4–5/group) were left untreated, were injected three times per week i.p. for 16 days with IL-4C that contained 0.5 µg mouse IL-4, or received a combination of both IL-4C and anti-IL-7 mAb (3 mg three times per week i.p.). Mice were sacrificed 16 days after the initiation of treatment. Spleen cells from individual mice were counted and incubated for 1 h with 100 ng/ml HEL at 4°C, then stained with PerCP-anti-B220 mAb, biotin-anti-HSA mAb, FITC-anti-HEL mAb, and a mixture of Cy5-labeled anti-IgMa and anti-IgDa mAbs. mIg (Da + Ma) and HEL staining of mature (B220brightHSAdull) spleen cells was determined. Upper contour plots, B220 and HSA staining that were used to differentiate mature from immature B cells in representative mice; lower contour plots, mIg (Da + Ma) and HEL staining of mature (B220bright, HSAdull) spleen cells from these same mice. Splenic B cells that stain less brightly for HEL than would be expected from their staining for mIg (Da + Ma) are considered to have undergone receptor editing. Bar graph indicates numbers of mature mIg+ splenic B cells that lack evidence of receptor editing and mIg+ splenic B cells that show evidence of receptor editing. Percentages were determined by flow cytometry and multiplied by total spleen cell numbers to determine numbers of spleen cells of each type.

 
IL-4 does not increase spontaneous IgM secretion or the IgM secretory response of HEL-Ig splenic B cells to LPS

The ability of IL-4 treatment to enhance maturation and survival of autoreactive B cells suggested that this cytokine might also prevent autoantigen-induced B cell unresponsiveness. Three similar experiments addressed this issue by determining whether IL-4C treatment would increase spontaneous IgMa secretion or the B cell secretory response to LPS by cultured spleen cells from HEL-Ig mice. IL-4C treatment failed to increase the negligible levels of IgM anti-HEL Ab in HEL-Ig serum or in 1- or 3-day culture supernatants of unstimulated HEL-Ig spleen cells (data not shown) and failed to increase LPS-induced IgM secretion by cultured HEL-Ig splenic B cells in two of three experiments (Fig. 9Go). In contrast, Ig Tgn B cells consistently responded to LPS, although IL-4 treatment decreased IgM secretion by LPS-stimulated Ig Tgn B cells through a Stat6-dependent mechanism (Fig. 9Go).



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  		FIGURE 9. IL-4 treatment fails to markedly enhance Ab production by LPS-stimulated HEL-Ig Tgn B cells. In three separate experiments, wild-type and Stat6-deficient Ig Tgn and HEL-Ig Tgn mice (3–4/group) were left untreated or were injected i.p. three times per week for 14 days with IL-4C that contained 0.5 µg mouse IL-4. Mice were sacrificed 14 days after initiation of IL-4C treatment. Spleen cells from individual mice were counted and cultured at a concentration of 5 x 105 cells/ml (1 x 105 cells/well) in medium that contained 20 µg LPS/ml. Supernatants were harvested 3 days later, and IgMa anti-HEL titers were determined by ELISA. Titers for each sample were corrected for percentages of B cells in cultures.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our studies support five conclusions: 1) IL-4 inhibits the early death and promotes the maturation of Ag-specific B cells that are chronically exposed to a soluble, oligovalent Ag; 2) these effects are Stat6 dependent; 3) enhanced survival is achieved primarily by allowing immature B cells to mature to a developmental stage that has increased resistance to Ag-induced cell death; however, IL-4 also enhances the survival of B cell receptor-activated mature B cells; 4) higher concentrations of IL-4 are required to prevent in vivo deletion of B cells by stronger mIg cross-linking; and 5) IL-4 enhancement of B cell survival and maturation is not accompanied by normalization of mIg expression, spontaneous Ig secretion, or responsiveness to LPS. These observations will be discussed in turn.

IL-4 promotion of autoreactive B cell survival and maturation

Previous studies that demonstrated humoral autoimmunity in Tgn mice that overproduce IL-4 (22, 23) and a decrease in autoantibody production in IL-4-deficient MRL/Mp-lpr/lpr mice (36) hypothesized that IL-4 might contribute to autoimmunity by inhibiting Ag-induced deletion of autoreactive B cells. The only published study that examined IL-4 effects on the deletion of autoreactive B cells (23), however, concluded that even a high level of autologously produced IL-4 did not prevent such deletion. One important difference between that study and ours is the nature of the autoantigen, which was particulate and multivalent in the previous study, but soluble and oligovalent in ours. Comparison of the results in these two studies suggests that the ability of IL-4 to prevent Ag-induced B cell deletion decreases as the intensity of Ag-induced mIg cross-linking increases. Thus, IL-4 might be expected to inhibit the deletion of B cells that are specific for soluble, oligovalent self Ags that are present at relatively low concentration, but not to prevent the deletion of B cells specific for polyvalent cell membrane Ags. This expectation is consistent with the relatively mild autoimmunity that develops in IL-4 Tgn mice (22, 23) and the relatively modest effect that deleting the IL-4 gene has on lpr-associated autoimmunity (36).

Stat6 dependence of IL-4 effects on autoreactive B cell survival and maturation

Although in vitro effects of IL-4 on T cell survival have been Stat6 independent (21), our studies demonstrate that IL-4 rescue of Ag-activated B cells is Stat6 dependent. Thus, although IL-4 promotes the survival of both Ag-stimulated B and T cells, our observations suggest that this is accomplished though different molecular mechanisms. Consistent with this, we have observed that splenic T cell numbers are increased to a much greater extent than splenic B cell numbers in IL-4 Tgn Stat6-deficient mice (S. C. Morris and F. D. Finkelman, unpublished observations).

Relationship between B cell survival and maturity

Several studies have suggested that exposure to Ag deletes newly generated B cells more readily than mature B cells (37, 38, 39, 40); however, previous studies with Ig Tgn and HEL-Ig mice and with mice injected with anti-IgD mAb have demonstrated that mIg cross-linking in the absence of T cell help also deletes mature B cells (12, 32, 41). Our studies with IL-4 suggest that the difference between the behavior of immature and mature B cells is quantitative rather than qualitative. Treatment of HEL-Ig mice with IL-4 for a few days doubled the number, and presumably, increased the survival, of B cells generated while IL-4 was being administered, even 12 days after the cessation of IL-4 administration (Fig. 7GoF). The survival of B cells generated during the same period appeared to be somewhat greater when IL-4 treatment was continued until the time of sacrifice (Fig. 7GoD). Thus, while IL-4 treatment of nascent, Ag-stimulated B cells allows these cells to survive to maturity and makes them somewhat resistant to deletion by continuing exposure to Ag, even without further IL-4 stimulation, optimal survival of mature, Ag-stimulated B cells may require continued exposure to IL-4. The latter possibility is supported by experiments in which IL-4 inhibited anti-IgD mAb-induced deletion of wild-type B cells that had a mature phenotype.

Relationship between intensity of mIg cross-linking and IL-4 requirement to inhibit B cell deletion

The intensity of mIg cross-linking is greater in wild-type mice injected with 200 µg anti-IgD mAb than in HEL-Ig mice. This is demonstrated by the almost total modulation of B cell mIgD in mice treated with anti-IgD mAb, as compared with little or no loss of mIgD by B cells in HEL-Ig mice. This difference may reflect either the high serum concentration of anti-IgD mAb in mice injected with 200 µg of this mAb, relative to the concentration of serum HEL in zinc-treated HEL-Ig mice, or the bivalency of the anti-IgD mAb injected, in contrast to HEL, which is structurally univalent and becomes oligovalent only when it associates with itself or with other molecules (8). Concentrations of IL-4 that normalized B cell number and HSA expression in HEL-Ig mice had relatively little effect on B cell deletion in anti-IgD mAb-treated mice; however, increasing the quantity of IL-4 administered clearly inhibited anti-IgD mAb-induced B cell deletion. Thus, it does not appear that a fixed amount of IL-4 signaling is required to prevent B cell deletion; instead, there appears to be a relationship between the intensity of the stimulus that leads to B cell deletion and the quantity of IL-4 required to inhibit deletion.

Lack of effect of IL-4 on B cell anergy

In contrast to its potent effect on B cell survival and maturation in HEL-Ig mice, IL-4 has little effect on B cell mIgM expression in these mice, but enhanced mIgD expression in both Ig and HEL-Ig Tgn mice. Additionally, IL-4 treatment does not consistently overcome the decreased responsiveness of HEL-Ig B cells to LPS, as measured by IgM secretion. This latter observation is more difficult to interpret than the former, because IL-4 has an inhibitory effect on LPS-induced IgM secretion by B cells from Ig Tgn mice and because a modest stimulatory effect of IL-4 on LPS induction of IgM secretion by HEL-Ig B cells was observed in one of three experiments. It is impossible to rule out the possibility that IL-4 might enhance differentiation to IgG1 secretion by wild-type B cells that have been exposed to self Ag, because, while IL-4 stimulates normal, LPS-activated B cells to switch to IgG1 secretion (42), the structure of the Ig transgene prevents such switching. Similarly, it is possible that IL-4 may increase the ability of self Ag-exposed B cells to respond to other stimuli that promote Ig secretion, such as type 2 T-independent Ags or T cell-associated CD40 ligand. Regardless of these issues, the greater ability of IL-4 to prevent the deletion of self Ag-exposed B cells than to promote their differentiation to Ab-secreting cells suggests that deletion and anergy are different processes and may account for the limited nature of the autoimmunity that develops in IL-4 Tgn mice.

Although our results indicate that IL-4 can prevent the deletion of autoreactive B cells, we cannot be certain of the physiological importance of this effect because we do not know how levels of IL-4 in IL-4C-treated mice or IL-4 Tgn mice compare with those generated in the immediate vicinity of a B cell that may be exposed to self Ag. Although the decreased autoreactivity that is observed in IL-4-deficient MRL/Mp-lpr/lpr mice (36) suggests that IL-4 has a physiological role in autoimmunity, further studies in autoimmune disease models are required to test the possibility that the deletion-inhibiting effect of IL-4 is of general importance in autoimmune disease and, if so, to determine the relative importance in autoimmunity of the Stat6-dependent effects of IL-4 on B cells and the Stat6-independent effects of IL-4 on T cells.


   Acknowledgments
 
We thank Dr. Edward Giannini for assistance with statistical analysis, and Tatyana Orekhova and Kathryn Lang for technical assistance.


   Footnotes
 
1 This work was supported by a Merit Review Entry Program Award (to S.C.M.) and a Merit Award (to F.D.F.) from the Veteran’s Administration, National Institutes of Health Grant P60-AR-44059, and a Biomedical Science Award from the Arthritis Foundation. Back

2 Address correspondence and reprint requests to Dr. Suzanne C. Morris, Department of Veterans Affairs Medical Center, Research Service (151), 3200 Vine Street, Cincinnati, OH 45220. E-mail address: morrissc{at}email.uc.edu Back

3 Abbreviations used in this paper: mIg, cell membrane Ig; BrdU, 5-bromo-2'-deoxyuridine; HEL, hen egg lysozyme; HSA, heat-stable Ag; MFI, mean fluorescence intensity; S-PE, streptavidin-R-PE; Tgn, transgenic. Back

Received for publication August 7, 2001. Accepted for publication June 3, 2002.


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 Materials and Methods
 Results
 Discussion
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