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* Cincinnati Veterans Affairs Medical Center, Cincinnati, OH 45220, and Division of Immunology, University of Cincinnati College of Medicine, Cincinnati, OH 45267; and
Division of Immunology, Children’s Hospital Medical Center, Cincinnati, OH 45229
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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. 7
D) and was shielded from light and changed every third
day.
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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
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
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). HB7, 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-FcRII/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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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. 1A) and fully
corrected splenic B cell number in HEL-Ig mice after 14 days (Fig. 1B). This effect of IL-4 was CD4+ T
cell independent, as it was not blocked by anti-CD4 mAb (Fig. 1C). 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. 1B) was not observed in most experiments (see,
for example, Figs. 5 and 7
B, below).
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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. 2) 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. 2, bottom panel).
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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. 3).
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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. 4). 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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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. 5, top panel), IL-4C causes a
large increase in the low number of mature splenic B cells in these
mice (Fig. 5, 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. 5, 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. 6, 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. 6, 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. 6).
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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. 7A),
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. 7B; 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. 7C). The same was true for mature
B cells generated during the first 3 days of a 14-day period of IL-4C
treatment (Fig. 7D; 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. 7E) 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. 7F, 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. 7, 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. 8). Thus, IL-4C treatment does not
increase HEL-Ig B cell survival by increasing receptor editing.
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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. 9). 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. 9).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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. 7F). 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. 7D). 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.
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Acknowledgments |
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Footnotes |
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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
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.
Received for publication August 7, 2001. Accepted for publication June 3, 2002.
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antibodies induce apoptosis in mature murine B lymphocytes. Eur. J. Immunol. 24:974.[Medline]
1 genes on the expressed and unexpressed chromosomes of lipopolysaccharide-activated normal murine B cells. J. Immunol. 143:334.[Abstract]This article has been cited by other articles:
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