Th1 versus Th2 cytokine profile determines the modulation of in vitro T cell-independent type 2 responses by IL-4
Quirijn Vos,
Clifford M. Snapper and
James J. Mond
1 Departments of Medicine and Pathology, Uniformed Services University of the Health Sciences, Bethesda, MD 20814-4799, USA
Correspondence to:
Q. Vos
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Abstract
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We have previously demonstrated that stimulation of B cells by multivalent membrane Ig cross-linking, using dextran-conjugated anti-IgD mAb (
-dex), in the presence of cytokines, is an in vitro model for T cell-independent type 2 (TI-2) Ig secretory responses. Earlier studies have shown that IL-4 enhances IgM secretion upon stimulation with 
-dex plus IL-5 and induces IgG1 isotype-switching, without altering the proliferative response to 
-dex. Here we show that IL-4 can have both stimulatory and inhibitory effects on 
-dex-induced Ig secretion. Both the kinetics and time of exposure to IL-4, and the nature of the cytokine additions, Th1 versus Th2, determine whether stimulation or inhibition is observed. Preincubation of sort-purified B cells with IL-4 caused a 6- to 8-fold increase in Ig secretory responses to subsequent stimulation with 
-dex plus IL-1, IL-2 or a combination of both. However, the continued presence of IL-4 during B cell stimulation suppressed responses to all cytokine combinations tested, except for those which included IL-5. Of 11 cytokines tested, only IL-4 showed this dual effect of enhancement and suppression. The stimulatory effect of IL-4 required a minimum of 4 h of preincubation and could be inhibited by the addition of IFN-
. Thus stimulation of non-MHC class II-dependent T or non-T cells by multivalent antigens to secrete IL-4 may regulate the response to these antigens, such that early and brief exposure of B cells to IL-4 will enhance a subsequent TI-2 response in the presence of Th1-dependent cytokines, while continuous exposure will result in inhibition of the response.
Keywords: B lymphocytes, cellular differentiation, cytokines, Ig secretion
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Introduction
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Based on the requirement of T cell-mediated MHC class II-restricted help to the B cell, humoral immune responses can be divided in T cell-dependent (TD) (1) and T cell-independent (TI) (25) responses. Among TI antigens, two types can be distinguished: TI type 1 antigens that induce polyclonal B cell activation and TI type 2 (TI-2) antigens that fail to induce responses in neonatal B cells and in B cells of mice that lack expression of functional Bruton's tyrosine kinase (610).
We have previously shown that activation of purified B cells with a multivalent dextran-conjugated anti-IgD mAb (
-dex) provides an in vitro polyclonal TI-2 model antigen that induces sustained mobilization of intracellular calcium (11) and proliferation (12) in B cells at concentrations that are a 1000-fold lower than those required of the unconjugated mAb. Furthermore, stimulation of purified B cells with 
-dex required the addition of cytokines to induce Ig secretion, as was previously shown with the TI-2 antigen TNP-Ficoll (1320). These findings guided us in defining an hypothesis that suggests that production of B cell stimulatory cytokines by non-T cells during TI-2 responses plays an important role in regulating the magnitude of antibody induction (21).
IL-4 induces B cell activation and modifies B cell responses to both TD and TI stimuli (22). Originally described as a stimulant necessary for inducing proliferative responses of resting B cells to low concentrations of unconjugated anti-IgM antibody (23), IL-4 has since been shown to exert multiple effects on B cells (2442), many of which can be reversed by the addition of IFN-
(4347). Despite the effects IL-4 has on B cells, it has not been shown to have an effect, either stimulatory or inhibitory, on responses to TI-2 antigens. Since IL-4 is produced by many cell types other than MHC class II-restricted T cells, it is likely that organisms which contain TI epitopes, e.g. encapsulated bacteria, may stimulate IL-4 secretion. Previous examination of the effects of IL-4 on our TI-2 model system demonstrated that this cytokine had no significant effect on 
-dex-induced B cell proliferation nor on the absolute amount of Ig secreted (48), but did direct isotype switching to IgG1, without inducing IgE (49).
Because of recent reports describing the rapid kinetics of IL-4 secretion after infection (5057), with some of these studies describing non-T cells like eosinophils (51) and basophils (52), and NK T cells (53) as the source of this cytokine, we wished to further explore whether there was any role for IL-4 in the response to TI-2 antigen in the presence of other cytokines. Since infectious organisms often display both TD and TI epitopes, the TI-specific B cells may often reside in a milieu of Th1 cytokines that were induced by the TD-specific components of the organism.
We show that pretreatment of sort-purified B cells with IL-4 enhanced the subsequent Ig secretory response induced by 
-dex in combination with IL-1 and IL-2. However, the continuous presence of IL-4 simultaneous with membrane Ig cross-linking inhibited the effect of the cytokines that were tested and only enhanced the response in the presence of IL-5. An analysis of potential antagonism of IFN-
to the effects of IL-4 showed that IFN-
reversed only the stimulatory effects of IL-4 pretreatment, while leaving the inhibitory effects unaffected. This study demonstrates a potentially new and different level of cytokine regulation of TI-2 responses, and suggests that short exposure to IL-4, in the absence of IFN-
, will enhance a TI-2 response when Th1 cytokines are present for the remainder of the response, but will suppress these same responses when present on a persistent basis.
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Methods
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Mice
C57BL/6 mice were obtained from Jackson Laboratories (Bar Harbor, ME) and were used at 810 weeks of age. The experiments were conducted according to the principles set forth in the Guide for the Care and Use of Laboratory Animals, Institute of Animal Resources, National Research Council, Department of Health, Education and Welfare publication (National Institutes of Health) 78-23.
Culture medium
RPMI 1640 (Biowhittaker, Walkersville, MD) supplemented with 10% heat-inactivated FCS (Gibco, Grand Island, NY), L-glutamine (2 mM), HEPES (25 mM), 2-mercaptoethanol (50 mM), non-essential amino acids (100 µM), penicillin (100 U/ml), pyruvate (1 mM) and streptomycin (10 µg/ml) was used for cell culture.
Reagents
A conjugate 
-dex of anti-IgD b allotype specificity was prepared by conjugation of the AF3.33 mAb (58) to a high mol.wt dextran (2x106 Da) as previously described (12). Approximately six mAb were conjugated to each dextran molecule. The concentration of 
-dex reflects the amount of mAb and not the entire dextran conjugate. Murine recombinant IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, IL-13, granulocyte macrophage colony stimulating factor (GM-CSF), IFN-
and tumor necrosis factor (TNF)-
were purchased from R & D Systems (Minneapolis, MN). Anti-B220-FITC, anti-NK1.1phycoerythrin, anti-IgD-biotin and CyChromestreptavidin were from PharMingen (San Diego, CA). Goat anti-mouse
light chain and an alkaline phosphatase-conjugated goat anti-mouse IgM antibodies were purchased from Southern Biotechnology Associates (Birmingham, AL).
Preparation and culture of highly purified splenic B cells
Enriched populations of B cells were obtained from C57BL/6 spleen cells from which T cells were eliminated by treatment with rat anti-Thy-1.2 (59), followed by complement. Cells were then fractionated by centrifugation over a discontinuous Percoll gradient (Pharmacia, Piscataway, NJ) consisting of 70, 60, and 40% Percoll solutions (with densities of 1.086, 1.074 and 1.050 g/ml respectively). The cells were collected from the 7060% interface, washed in cold clear HBSS staining buffer containing 3% heat-inactivated FCS, penicillin (100 U/ml) and streptomycin (100 µg /ml), and incubated at a density of 107 cells/ml in a 1 µg/ml solution of anti-B220FITC and anti-NK1.1phycoerythrin in staining buffer for 30 min on ice. After one wash the cells were resuspended at a density of 107 cells/ml in staining buffer and electronically sorted on an Epics Elite cytometer (Coulter, Hialeah, FL) for a B220bright NK1.1 phenotype. Reanalysis of sorted cells, that were subsequently stained with anti-IgDbiotin and cychromestreptavidin, consistently revealed the presence of >99.5% IgD+. B220bright B cells. These highly purified B cells were incubated in flat-bottom 96-well plates (Costar, Cambridge, MA) at 105 cells/ml in a total volume of 200 µl culture media containing 
-dex at 10 ng/ml and the indicated cytokines for 5 days at 37°C in a humidified atmosphere containing 5% CO2. For preincubation the highly purified B cells were cultured for 16 h at a density of 105 cells/ml in culture flasks (Costar). Unless indicated otherwise, cytokines were used at the following concentrations: IL-1 (100 U/ml), IL-2 (50 U/ml), IL-3 (100 U/ml), IL-4 (100 U/ml), IL-5 (300 U/ml), IL-6 (400 U/ml), IL-10 (100 U/ml), IL-13 (100 U/ml), IFN-
(100 U/ml), GM-CSF (100 U/ml) and TNF-
(500 U/ml).
Quantitation of Ig-secreting cells in the cultures of stimulated B cells
Ig-secreting cells were detected by spot-ELISA as previously described (60). Briefly, cellulose ester membrane 96-well plates (Millipore Multiscreen; Millipore, Molsheim, France) were coated with 50 µl of a 5 µg/ml solution of goat anti-mouse
light chain antiserum in PBS by incubating overnight at 4°C. The plates were post-coated using culture medium, and cell suspensions from the stimulated B cell cultures were added and incubated for 6 h in a humidified atmosphere containing 5% CO2 at 37°C. A conjugate of isotype-specific goat anti-mouse IgM and alkaline phosphatase was added and incubated overnight at 4°C. Next, the plates were developed using BCIP/NBT substrate solution (Kirkegaard & Perry, Gaithersburg, MD) and spots representing individual Ig-secreting cells were enumerated using an inverted microscope.
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Results
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IL-4 differentially modulates Ig secretory responses in an in vitro TI-2 model system
In previous studies we have demonstrated that in vitro stimulation of purified B cells with the TI-2 model antigen 
-dex requires the addition of various cytokines for the induction of Ig secretion (1320). While the addition of IL-4 to cultures of B cells stimulated with a combination of 
-dex and IL-5 resulted in increased IgM secretion and in isotype switching to IgG1 (14), IL-4 itself did not have a significant effect on 
-dex-induced B cell proliferation (48), in contrast to bivalent anti-Ig stimulation, where IL-4 mediated a significantly enhanced proliferative response. Based on reports describing the rapid in vivo induction of IL-4 in non-T cells by antigen that could be classified as TI (5153), and studies demonstrating the augmentation of bivalent anti-Ig-induced B cell proliferation by pretreatment of the B cells with IL-4 (47), we studied the effects of IL-4 pretreatment on Ig secretion induced by multivalent membrane Ig cross-linking. Sort-purified C57BL/6 B cells were cultured with or without the addition of IL-4 at 100 U/ml for 16 h, washed 3 times, stimulated with 
-dex plus a panel of cytokines with or without IL-4 at 100 U/ml and harvested after 5 days of culture. The data of a spot-ELISA for IgM-secreting cells (IgMSC) shown in Fig. 1
(A) indicate that preincubation with IL-4 resulted in a 6- to 8-fold increase in Ig secretory responses to 
-dex plus IL-1, IL-2 and a combination of these cytokines. In contrast, IL-4 preincubation did not alter the response to 
-dex plus IL-5, by itself or in combination with IL-1 and IL-2 (Fig. 1B
). Addition of IL-4 for the duration of the B cell stimulation culture resulted in the opposite effect. Whereas responses to 
-dex plus IL-5 or plus IL-5 in combination with IL-1 and IL-2 increased 2- to 5-fold (Fig. 1B
), continuous culture with IL-4 inhibited the induction of IgMSC in cultures of stimulated with IL-1, IL-2 or a combination of these cytokines (Fig. 1A
). The level of inhibition varied from 4-fold for cultures stimulated with 
-dex + IL-1 to 12-fold for 
-dex + IL-1 + IL-2. Finally, the results in Fig. 1
show that IL-4 pretreatment did not alter the effects of IL-4 addition for the duration of the B cell stimulation culture and suppression was seen rather than enhancement. Comparison of cultures of B cells that were pretreated in media only, with those of cells that received no preincubation revealed no significant differences (data not shown). Analysis of the B cell cultures stimulated with 
-dex and the members of the cytokine panel revealed no significant effects of either IL-4 pretreatment, IL-4 addition or a combination of the both on the number of viable cells that were recovered (data not shown). While the results in Fig. 1
are limited to a panel consisting of the Th1 cytokines IL-1 and IL-2, and the Th2 cytokine IL-5, these data are representative for results obtained with a larger panel including IL-3, IL-6, IL-10, IL-13, IFN-
, GM-CSF and TNF-
(data not shown).

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Fig. 1. IL-4 differentially modulates Ig secretory responses in an in vitro TI-2 model system. Sort-purified C57BL/6 B cells were cultured for 16 h in culture media without, `' represented by black bars, or with the addition of IL-4 (100 U/ml), `IL-4 pre.', represented by white bars. The cells were washed 3 times in media, and cultured for 5 days in the presence of  -dex (10 ng/ml) and various combina- tions of IL-1 (100 U/ml), IL-2 (50 U/ml) and IL-5 (300 U/ml). Cultures that also received IL-4 (100 U/ml) for the duration of the B cell stimulation culture, `IL-4 cont.', are represented by grey bars, while cultures of B cells that were preincubated with IL-4 and were stimulated in the presence of this cytokine for the duration of the culture, `IL-4 comb.', are represented by hatched bars. (A) Cultures stimulated with  -dex. (B) cultures stimulated with  -dex + IL-5. The induction of IgMSC was analyzed using a spot-ELISA. Results are representative of five individual experiments.
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The stimulatory effects of IL-4 pretreatment and the inhibitory effects of the continuous presence of IL-4 show a similar doseresponse relationship
As a first step in analyzing the differential effects of IL-4 treatment on our TI-2 model system we compared the doseresponse relationship of the stimulatory effect of IL-4 pretreatment with that of the inhibitory effect of the continuous presence of IL-4 during B cell stimulation. The data in Fig. 2
(A) illustrate the effect of a 16 h preincubation of sort-purified B cells with various concentrations of IL-4 on the subsequent responses to 
-dex + IL-1 + IL-2 and to 
-dex + IL-5, while Fig. 2
(B) shows the effect of the addition of titrated amounts of IL-4 added to both stimuli for the duration of the 5-day B cell culture. The data indicate similarity in the doseresponse profile of the enhancement of the 
-dex + IL-1 + IL-2 response by IL-4 pretreatment (Fig. 2A
) and its inhibition by the addition of IL-4 during stimulation (Fig. 2B
). The dose of IL-4 that provided maximum enhancement when given as a 16 h pulse was the same dose that induced maximum suppression when present continuously. Figure 2
(A), moreover, illustrates that even preincubation with doses of IL-4 that are an order of magnitude beyond the ones required for optimal enhancement of the 
-dex + IL-5 response, shown in Fig. 2
(B), still did not alter the response to this stimulus. The results obtained by stimulation with 
-dex + IL-1 + IL-2 were representative for those obtained with a broader cytokine panel with the exception of IL-5 and its various combinations. Combining IL-4 pretreatment with continous IL-4 addition the duration of the culture, each over the described concentration range, resulted in data that were not significantly different from the ones shown in Fig. 2
(B) for this stimulus (data not show). Thus, the induction of either enhancement or suppression did not depend on different dose requirements of these effects of IL-4.
IL-4 pretreatment does not enhance the response to 
-dex plus suboptimal doses of IL-5
While the data presented in Figs 1 and 2
indicated a stimulatory effect of IL-4 pretreatment on the response of B cells activated with 
-dex plus various cytokines, responses in the presence of IL-5 remained unaltered. This may, on the one hand, reflect a fundamental difference between the Th2 cytokine IL-5 and the Th1 members of our cytokine panel, or may, on the other hand, reflect that activation of B cells with 
-dex in combination with an optimal dose of IL-5 (300 U/ml) results in a stimulus, that IL-4 pretreatment cannot further enhance. To distinguish between these two hypotheses, sort-purified B cells were pretreated for 16 h with a range of IL-4 concentrations, washed and subsequently activated with 
-dex in combination with titrated concentrations of IL-5 or in combination with IL-1 + IL-2. A comparison of the results of this experiment, shown in Fig. 3
, demonstrates that while the responses to both 
-dex plus IL-5, at the suboptimal concentration of 30 U/ml, and to 
-dex + IL-1 + IL-2 were not significantly different when the B cells were preincubated in the absence of IL-4, only the latter response could be enhanced 8-fold by IL-4 pretreatment, while the former remained unaltered. Similarly, the response to 
-dex plus IL-5 at the suboptimal concentration of 100 U/ml could not be enhanced by IL-4 pretreatment of the B cells. Addition of other cytokines to cultures stimulated with 
-dex plus IL-5 at 30 U/ml did not result in rendering this stimulus sensitive to the effects of IL-4 preincubation of the B cells (data not shown). This suggests that the IL-4-mediated enhancement is operative on B cells that are stimulated only in the presence of multivalent membrane Ig cross-linking and Th1 cytokines.
Kinetics of the effects of IL-4 preincubation and addition to cultures of B cells stimulated with 
-dex in combination with cytokines
In view of the fact that IL-4 production by NK T cells and non-T lymphocytes is brief, we wished to determine the minimum amount of preincubation time with IL-4 that was needed to enhance responsiveness. Thus, sort-purified B cells were preincubated with IL-4 at 100 U/ml for various times, washed and stimulated with either 
-dex + IL-1 + IL-2 or with 
-dex + IL-5 (Fig. 4A
) or cultured directly with these stimuli and with IL-4 at 100 U/ml, added at different times after the start of the B cell stimulation (Fig. 4B
). The results in Fig. 4
(A) show that a minimum of 4 h of preincubation in the presence of IL-4 were required to significantly enhance the subsequent stimulation with 
-dex + IL-1 + IL-2, with an plateau of 10-fold enhancement occurring after 12 h of IL-4 pretreatment. Again IL-4 pretreatment did not alter the response to 
-dex + IL-5. Figure 4
(B) shows the effect of the addition of IL-4 at different time points during B cell stimulation with 
-dex + IL-1 + IL-2 or with 
-dex + IL-5. Comparison of the data of these time courses with the stimulation obtained in the absence of IL-4 indicates that the IL-4-mediated inhibition of the Ig secretory response to 
-dex + IL-1 + IL-2 required addition during the first 2 days of culture, while the addition of IL-4 during the initial 4 days of 
-dex + IL-5 stimulation significantly enhanced this response.
Addition of IFN-
during IL-4 preincubation inhibits the enhancement of the subsequent response to 
-dex + IL-1 + IL-2
Previous studies describing the effects of IL-4 (pre)treatment on the B cell (2435) have shown that the IL-4-induced increase in B cell MHC class II (45,46) and CD23 (32,33) expression, and the effects of IL-4 on the volume increase and preparation for DNA synthesis in resting B cells (47) could be reversed by the addition of IFN-
. To assess whether the mechanism underlying the observed stimulatory effect of IL-4 preincubation on subsequent 
-dex-induced Ig secretory responses might bear similarity to its effect on other modes of B cell stimulation, we added titrated amounts of IFN-
to IL-4 during a 16 h preincubation of sort-purified B cells, washed the cells and stimulated them with 
-dex + IL-1 + IL-2. Figure 5
(A) shows that while preincubation with IFN-
only did not alter the effects of the subsequent stimulation, it did inhibit the IL-4-mediated enhancement in a dose-dependent fashion. This effect was similar for all responses shown to be enhanced by IL-4 pretreatment, while the addition of IFN-
during preincubation had no effect on responses to 
-dex in the presence of IL-5 (data not shown). In contrast, the addition of IFN-
in combination with continuous IL-4 (Fig. 5B
) did not reverse the inhibition, but rather increased it. This effect was similarly observed for all responses shown to be inhibited by the continuous presence of IL-4 during B cell stimulation, while the addition of IFN-
did not inhibit responses in the presence of IL-5, including those enhanced by IL-4 (data not shown). To exclude the possibility that the observed inhibitory effects of IFN-
during pretreatment, and of both IL-4 and IFN-
during B cell stimulation, were due to the induction of isotype switching to IgG, thereby reducing the number of IgMSC, we also analyzed the corresponding cultures for cells secreting the individual IgG isotypes. These analyses revealed that none of the inhibitory effects on the induction of IgMSC were accompanied by significant increases in the number of cells secreting the IgG isotypes (data not shown).
Both the IL-4-mediated enhancement of the Ig secretory responses in the TI-2 model system, and its inhibition by IFN-
are cytokine specific
We wished to determine whether the enhanced responses that we observed with short IL-4 pulses were specific for this factor. Sort-purified B cells were cultured for 16 h in the presence of IL-4 or a panel of other cytokines, or in the presence of a combination of each of these cytokines with IL-4. After washing, the B cells were cultured for 5 days in the presence of either 
-dex + IL-1 + IL-2 or 
-dex + IL-5 and analyzed for the induction of IgMSC. Figure 6
(A) shows that IL-4 was the only cytokine in a panel of 11 to enhance the response to 
-dex + IL-1 + IL-2, whereas none in the panel altered the response to 
-dex + IL-5. Figure 6
(B) demonstrates that upon addition to IL-4 during B cell preincubation, IFN-
was the only cytokine to significantly inhibit the effects of IL-4 on the subsequent response to 
-dex + IL-1 + IL-2, while none of the combinations altered the response to 
-dex + IL-5.

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Fig. 6. Both the IL-4-mediated enhancement of the Ig secretory responses in the TI-2 model system and its inhibition by IFN- are cytokine specific. Sort-purified C57BL/6 B cells were cultured for 16 h in culture media containing one of the following cytokines: IL-1 (100 U/ml), IL-2 (50 U/ml), IL-3 (100 U/ml), IL-4 (100 U/ml), IL-5 (300 U/ml), IL-6 (400 U/ml), IL-10 (100 U/ml), IL-13 (100 U/ml), IFN- (100 U/ml), GM-CSF (100 U/ml) or TNF- (500 U/ml) (A) or a combination of IL-4 (100 U/ml) and the individual members of this cytokine panel (B). The cells were washed 3 times in media, and cultured for 5 days in the presence of  -dex (10 ng/ml) in combination with either IL-1 (100 U/ml) and IL-2 (50 U/ml) or with IL-5 (300 U/ml). The induction of IgMSC was analyzed using a spot-ELISA. Results are representative of three individual experiments.
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Discussion
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Previous studies on the effect of IL-4 on B cell stimulation in our in vitro TI-2 model system have demonstrated that addition of this cytokine to B cell cultures activated with 
-dex plus IL-5 resulted in enhanced IgM secretion and induction of IgG1 isotype switching (49). Yet, the IL-4-mediated enhancement of the proliferative B cell response to unconjugated anti-IgD or anti-IgM antibody (23) was not observed after stimulation with 
-dex (48). These findings suggested that IL-4 may modulate responses in TD and TI-2 model systems differentially. The recent description of rapid induction of IL-4 production during infection (5057), with some of these studies describing non-T cells like eosinophils (51) and basophils (52), and NK T cells (53) as the source of this cytokine, raised the question whether a brief burst of IL-4 could influence responses to TI epitopes of infectious organisms. Because of the evidence in TD systems that prior exposure of T cells to cytokines determines the pattern of cytokines that they will secrete and the nature of the response (61), we wished to determine whether prior exposure of B cells to cytokines could alter their response profile as well. Since the humoral immune response to infectious organisms involves the response to both TI and TD epitopes, we decided to re-examine the modulation of B cell responses in our TI-2 model system by IL-4, focusing on the effect of IL-4 preincubation on Ig production induced by 
-dex in combination with various other cytokines.
While IL-4 pretreatment of sort-purified B cells did not change the kinetics of the proliferative response to subsequent stimulation with 
-dex and left its doseresponse relationship unaltered (data not shown), it profoundly affected the Ig secretory response. Interestingly, the effects of this pretreatment were strictly dependent on the cytokine (combination) used with the B cell stimulation: the responses involving the Th2 cytokine IL-5 remained unchanged, while stimulation in the presence of Th1 cytokines resulted in a 6- to 8-fold increased Ig secretory response. Continuous presence of IL-4 for the duration of the 
-dex stimulation culture resulted in almost the opposite effect: responses to 
-dex in combination with IL-5 increased 2- to 5-fold, while other responses were inhibited 4- to 20-fold. Upon combining IL-4 pretreatment, with continuous culture, the inhibitory effects of the latter prevailed. The lack of effects of IL-4 pretreatment on stimulation with suboptimal IL-5 concentrations established a true dichotomy between this and the other members of the cytokine panel.
These findings suggest, on the one hand, that the effect of an IL-4 exposure prior to a stimulus including a Th2 signal, involving IL-5, would be limited, while the very presence of IL-5 would block potential inhibition by IL-4 itself. On the other hand, the short exposure of B cells to IL-4 before B cell receptor activation by multivalent ligands could lead to enhancement of Ig secretion only if IL-4 did not continue to accompany the membrane Ig cross-linking signal. Thus, the source of IL-4 accompanying the multivalent B cell receptor cross-linking and the duration of its presence would be critical parameters in regulating a TI-2 Ig secretory response.
The differential effects of IL-4 on stimulation of highly purified B cells in the presence of Th1 cytokines like IL-1 and IL-2 versus the Th2 cytokine IL-5 could also be explained by the stimulation of distinctive B cell subsets. Based on the selective, constitutive expression of the IL-5R on B-1 cells (62), it is possible that the observed dichotomy of the effects of IL-4 could be due to activation of B-1 versus B-2 cells. Yet, the method of sort-purification that we employ, results in a contamination of CD5+IgDdullIgMbright cells that is beyond the level of detection of our flow cytometer. Moreover, stimulation of IL-5R B cells with 
-dex results in rapid induction of high levels of this receptor (63).
Preincubation of B cells with a panel of 11 cytokines with potential B cell stimulatory activity demonstrated that IL-4 was the only cytokine that enhanced subsequent Ig secretory responses and that IFN-
was the sole cytokine that inhibited this activity. These results establish the specificity of both actions and warrant an analysis of cells that may be involved in TI-2 responses, and that have the ability to rapidly secrete both IL-4 and IFN-
. In particular, when such cells could migrate from a peripheral site of antigen encounter to the spleen or other lymphoid organs to provide B cells with IL-4 before their actual B cell receptor stimulation. While previous studies have shown that non-T cells like eosinophils (51) and basophils (52), and NK T cells (53) each are capable of rapidly secreting IL-4 upon activation in vivo, the NK T cell has features that set it apart from the other two cell types. These features include the ability to rapidly secrete both IL-4 and IFN-
, the differential regulation of the production of both cytokines by different modes of receptor activation (64), and the influence of the microenvironment on the polarized secretion of these two factors (65).
To analyze the potential mechanism behind the stimulation of subsequent TI-2 responses by IL-4 preincubation and its inhibition by IFN-
, and to assess whether some of the inhibitory effects of IL-4 addition on these responses were caused by IL-2 receptor modulation, as reported in a model involving B cell activation with anti-IgM antibody (66), flow cytometry analysis was performed. Analysis of IL-2R
, CD25, expression showed that 16 h preincubation with IL-4, IFN-
or a combination of the two did not induce significant CD25 expression. Subsequent stimulation with 
-dex by itself, or in combination with cytokines induced a uniformly high expression of CD25, that was not modulated by any of the pretreatments, or by addition of IL-4 and/or IFN-
during stimulation (data not show). Similar analysis of IL-2Rß, CD122, revealed that this antigen was not induced under any of the conditions analyzed for CD25 expression, while analysis of cells subjected to similar pretreatments and stimulations revealed no differences in B cell surface IgD and IgM expression (data not shown). These findings suggest that both the IFN-
-mediated inhibition of the stimulatory effect of IL-4 pretreatment and IL-4-mediated suppression of subsequent Ig secretory responses are due to modulation of down-stream elements in the B cell signaling cascade.
While the results presented here are restricted to the effects of IL-4 on our in vitro TI-2 model system, we are currently exploring in vivo models. Among them, the TI-2 immunization of B cell-deficient mice, that received B cells that were either cultured in media or in the presence of IL-4, has yielded preliminary results that suggest that the effects of IL-4 pre- treatment can be observed in vivo as well.
Taken together, our data demonstrate that, depending on the kinetics of secretion and the presence of other cytokines, IL-4 can mediate a new level of cytokine regulation of TI-2 responses.
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Acknowledgments
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The authors thank Dr Andrew Lees for the preparation of 
-dex and Ms Karen Wolcott for expert assistance in electronic cell sorting. This work was supported by National Institutes of Health grant AI36588. Opinions and assertions herein are the private ones of the authors and are not to be construed as official or reflecting the views of the Department of Defense or the Uniformed Services University of the Health Sciences.
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Abbreviations
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 -dex dextran-conjugated anti-IgD mAb |
GM-CSF granulocyte monocyte colony stimulating factor |
IgMSC IgM-secreting cell |
TD T cell-dependent |
TI T cell-independent |
TI-2 T cell independent type 2 |
TNF tumor necrosis factor |
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Notes
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Transmitting editor: Z. Ovary
Received 16 March 2000,
accepted 29 May 2000.
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References
|
---|
-
Parker, D. C. 1993. T cell-dependent B cell activation. Annu. Rev. Immunol. 11:331.[ISI][Medline]
-
Coutinho, A. and Moller, G. 1975. Thymus-independent B-cell induction and paralysis. Adv. Immunol. 21:114.
-
Mosier, D. E., and Subbarao, B. 1982. Thymus-independent antigens: complexity of B-lymphocyte activation revealed. Immunol. Today 3:217.[ISI]
-
Cosgrove, D., Gray, D., Dierich, A., Kaufman, J., Lemeur, M., Benoist, C. and Mathis, D. 1991. Mice lacking MHC class II molecules. Cell 66:1051.[ISI][Medline]
-
Mond, J. J., Vos, Q., Lees, A. and Snapper, C. M. 1995. T cell independent antigens. Curr. Opin. Immunol. 7: 349.[ISI][Medline]
-
Mosier, D. E., Mond, J. J. and Goldings, E. A. 1977. The ontogeny of thymic independent antibody responses in vitro in normal mice and mice with an X-linked B cell defect. J. Immunol. 119:1874.[Abstract]
-
Mond, J. J., Scher, I., Blaese, M., Mosier, D. E. and Paul, W. E. 1978. T independent responses in B cell defective CBA/N mice to B. abortus and TNP-B. abortus. Eur. J. Immunol. 8:459.[ISI][Medline]
-
Mond, J. J., Lees, A. and Snapper, C. M. 1995. T cell-independent antigens type 2. Annu. Rev. Immunol. 13:655.[ISI][Medline]
-
Khan, W. N., Alt, F. W., Gerstein, R. M., Malynn, B. A., Larsson, I., Rathbun, G., Davidson, L., Muller, S., Kantor, A. B., Herzenberg, L. A., Herzenberg, L. A., Rosen, F. S. and Sideras, P. S. 1995. Defective B cell development and function in Btk-deficient mice. Immunity 3:283.[ISI][Medline]
-
Drabek, D., Raguz, S., De Wit, T. P., Dingjan, G. M., Savelkoul, H. F. J., Grosveld, F. and Hendriks, R. W. 1997. Correction of the X-linked immunodeficiency phenotype by transgenic expression of human Bruton tyrosine kinase under control of the class II major histocompatibility complex Ea locus control region. Proc. Natl Acad. Sci. USA 94:610.[Abstract/Free Full Text]
-
Yamada, H., June, C. H., Finkelman, F. D., Brunswick, M., Ring, M. S., Lees, A. and Mond, J. J. 1992. Persistent calcium elevation correlates with the induction of surface immunoglobulin-mediated B cell DNA synthesis. J. Exp. Med. 177:1613.[Abstract]
-
Brunswick, M., Finkelman, F. D., Highet, P. F., Inman, J. K., Dintzis, H. M. and Mond, J. J. 1988. Picogram quantities of anti-Ig antibodies coupled to dextran induce B cell proliferation. J. Immunol. 140:3364.[Abstract/Free Full Text]
-
Peçanha, L. M. T., Snapper, C.M., Finkelman, F. D. and Mond, J. J. 1991. Dextran-conjugated anti-Ig antibodies as a model for T cell-independent type 2 antigen-mediated stimulation of Ig secretion in vitro. I. Lymphokine dependence. J. Immunol. 146:833.[Abstract/Free Full Text]
-
Snapper, C. M., Pecianha, L. M. T., Levine, A. D. and Mond, J. J. 1991. IgE class switching is critically dependent upon the nature of the B cell activator, in addition to the presence of IL-4. J. Immunol. 147:1163.[Abstract/Free Full Text]
-
Snapper, C. M., McIntyre, T. M., Mandler, R., Peçanha, L. M., Finkelman, F. D., Lees, A. and Mond, J. J. 1992. Induction of IgG3 secretion by interferon gamma: a model for T cell independent class switching in response to T cell-independent type 2 antigens. J. Exp. Med. 175:1367.[Abstract]
-
Peçanha, L. M. T., Yamaguchi, H., Lees, A., Noelle, R., Mond, J. J. and Snapper, C. M. 1993. Dextran-conjugated anti-IgD antibodies inhibit T cell-mediated IgE production but augment the synthesis of IgM and IgG. J. Immunol. 150:2160.[Abstract/Free Full Text]
-
Snapper, C. M., Yamada, H., Smoot, D., Sneed, R., Lees, A. and Mond, J. J. 1993. Comparative in vitro analysis of proliferation, Ig secretion, and Ig class switching by murine marginal zone and follicular B cells. J. Immunol. 150:2737.[Abstract/Free Full Text]
-
Peçanha, L. M. T., Snapper, C. M., Lees, A., Yamaguchi, H. and Mond, J. J. 1993. IL-10 inhibits T cell-independent but not T cell-dependent responses in vitro. J. Immunol. 150:3215.[Abstract/Free Full Text]
-
Snapper, C. M., Moorman, M. A., Rosas, F. R., Kehry, H. R., Maliszewski, C. R. and Mond, J. J. 1995. IL-3 and granulocyte-macrophage colony-stimulating factor strongly induce Ig secretion by sort-purified murine B cells activated through the membrane Ig, but not the CD40, signaling pathway. J. Immunol. 154:5842.[Abstract/Free Full Text]
-
Snapper, C. M., Rosas, F., Moorman, M. A., Jin, L., Shanebeck, K., Klinman, D., Kehry, M. R., Mond, J. J. and Maliszewski, C. R. 1995. IFN-
is a potent inducer of Ig secretion by sort-purified B cells activated through the mIg, but not the CD40, signaling pathway. Int. Immunol. 6:877.
-
Snapper, C. M. and Mond, J. J. 1996. A model for the induction of T cell-independent humoral immunity to polysaccharide antigens. J. Immunol. 157:2229.[Abstract]
-
Brown, M. A. and Hural, J. 1997. Functions of IL-4 and control of its expression. Crit. Rev. Immunol. 17:1.[ISI][Medline]
-
Howard, M., Farrar, J., Hilfiker, M., Johnson, B., Takatsu, K., Hamaoka, T. and Paul, W. E. 1982. Identification of a T cell-derived B cell growth factor distinct from interleukin 2. J. Exp. Med. 155:914.[Abstract]
-
Rabin, E. M., Ohara, J. and Paul, W. E. 1985. B-cell stimulatory factor 1 activates resting B cells. Proc. Natl Acad. Sci. USA 81:6149.[ISI]
-
Thompson, C. B., Schaefer, M. E., Finkelman, F. D., Scher, I., Farrar, J. and Mond, J. J. 1985. T cell-derived growth factor(s) can induce stimulation of both resting and activated B cells. J. Immunol. 134:369.[Abstract/Free Full Text]
-
Ellenstrom, C. and Severinson, E. 1989. Interleukin 4 induces cellular adhesion among B lymphocytes. Growth Factors 2:73.[Medline]
-
Wilkinson, P. C. and Islam, L. N. 1989. Recombinant IL-4 and IFN-gamma activate locomotor capacity in human B lymphocytes. Immunology 67:237.[ISI][Medline]
-
Clinchy, B. C., Elenstrom, C., Severinson, E. and Moller, G. 1991. T and B cell colaboration: induction of motility in small, resting B cells by interleukin 4. Eur. J. Immunol. 21:1445.[ISI][Medline]
-
Davey, E. J., Thyberg, J., Conrad, D. H. and Severinson, E. 1998. Regulation of cell morphology in B lymphocytes by IL-4: evidence for induced cytoskeletal changes. J. Immunol. 160:5366.[Abstract/Free Full Text]
-
Roehm, N. W., Leibson, H. J., Zlotnick, A., Kappler, J., Marrack, P. and Cambier, J. C. 1984. Interleukin-induced increase in Ia expression by normal mouse B cells. J. Exp. Med. 160:679.[Abstract]
-
Noelle, R., Krammer, P. H., Ohara, J., Uhr, J. W. and Vitetta, E. S. 1984. Increased expression of Ia antigens on resting B cells: an additional role for B-cell growth factor. Proc. Natl Acad. Sci. USA 81:6149.[Abstract]
-
Defrance, T., Aubry, J. P., Rousset, F., Vanbervliet, B., Bonnefoy, J. Y., Arai, N., Takebe, Y., Yokota, T, Lee, F., Arai, K., DeVries, J. and Banchereau, J. 1987. Human recombinant interleukin 4 induces Fc epsilon receptors (CD23) on normal human B lymphocytes. J. Exp. Med. 165:1459.[Abstract]
-
Hudak, S. A., Gollnick, S. O., Conrad, D. H. and Kehry, M. R. 1987. Murine B-cell stimulatory factor 1 (interleukin 4) increases expression of the Fc receptor for IgE on mouse B cells. Proc. Natl Acad. Sci. USA 84:4606.[Abstract]
-
Oliver, K., Noelle, R. J., Uhr, J. W., Krammer, P. H. and Vitetta, E. S. 1985. B-cell growth factor (B-cell growth factor 1 or B-cell-stimulating factor, provisional 1) is a differentiation factor for resting B cells and may not induce cell growth. Proc. Natl Acad. Sci. USA 82:2465.[Abstract]
-
Rabin E. M., Mond, J. J., Ohara, J. and Paul, W. E.. 1986. B cell stimulatory factor 1 (BSF-1) prepares resting B cells to enter S phase in response to anti-IgM and lipopolysaccharide. J. Exp. Med. 164:517.[Abstract]
-
Isakson, P. C., Pure, E., Vitetta, E. S. and Krammer, P. H. 1982. T cell-derived B cell differentiation factor(s). Effect on the isotype switch of murine B cells. J. Exp. Med. 155:734.[Abstract]
-
Sideras, P., Bergstedt-Lidqvist, S. and Severinson, E. 1985. Partial biochemical characterization of IgG1-inducing factor. Eur. J. Immunol. 15:593.[ISI][Medline]
-
Noma, Y., Sideras, T., Naito, T., Bergstedt-Lidqvist, S., Azuma, C., Severinson, E., Tanabe, T., Kinashi, T., Matsuda, F., Yaoita, Y. and Honjo, T. 1986. Cloning of cDNA encoding the murine IgG1 induction factor by a novel strategy using SP6 promoter. Nature 319:640.[ISI][Medline]
-
39 Lee, F. T., Yokota, T., Otsuka, T., Meyerson, P., Villaret, D., Coffman, R., Mosmann, T., Rennick, D., Roehm, N., Smith, C., Zlotnick, A. and Arai, K. 1986. Isolation and characterization of a mouse interleukin cDNA clone that expresses B-cell stimulatory factor-1 activities and T-cell and mast-cell-stimulating activities. Proc. Natl Acad. Sci. USA 83:2061.[Abstract]
-
Isakson, P. 1986. Anti-immunoglobulin-treated B cells respond to a B cell differentiation factor for IgG1. J. Exp. Med. 164:303.[Abstract]
-
Coffman, R. L., Ohara, J., Bond, M. W., Carty, J., Zlotnick, A. and Paul, W. E. 1986. B cell stimulatory factor-1 enhances the IgE response of lipopolysaccharide-activated B cells. J. Immunol. 136:4538.[Abstract/Free Full Text]
-
Finkelman, F. D., Katona, I., Urban, J., Snapper, C.M., Ohara, J. and Paul, W. E. 1986. Suppression of in vivo polyclonal IgE responses by monoclonal antibody to the lymphokine BSF-1. Proc. Natl Acad. Sci. USA 83:9675.[Abstract]
-
Snapper, C. M. and Paul, W. E. 1987. Interferon-gamma and B cell stimulatory factor-1 reciprocally regulate Ig isotype production. Science 235:944.
-
Coffman, R. L. and Carty, J. 1986. A T cell activity that enhances polyclonal IgE production and its inhibition by interferon-
. J. Immunol. 136:4538.[Abstract/Free Full Text]
-
Mond, J. J., Carman, J., Sarma, C., Ohara, J. and Finkelman, F. D. 1986. Interferon-gamma suppresses B cell stimulation factor (BSF-1) induction of class II MHC determinants on B cells. J. Immunol. 137:3534.[Abstract/Free Full Text]
-
Oliver, K., Krammer, P. H., Tucker, P. W. and Vitetta, E. S. 1987. The effects of cytokines and adherent cells on the interleukin 4-mediated induction of Ia antigens on resting B cells. Cell. Immunol 106:428.[ISI][Medline]
-
Rabin, E. M., Mond, J. J., Ohara, J. and Paul, W. E. 1986. Interferon-gamma inhibits the action of B cell stimualtory factor (BSF)-1 on resting B cells. J. Immunol. 137:1573.[Abstract/Free Full Text]
-
Lindsberg, M. -L., Brunswick, M., Keegan, A. and Mond, J. J. 1990. IL4 is not required for proliferative responses in B cells stimulated by anti-IgDdextran conjugates even under limiting conditions of cell-cell interaction. Cell. Immunol. 130:320.[ISI][Medline]
-
Snapper, C. M., Pecianha, L. M. T., Levine, A. D. and Mond, J. J. 1991. IgE class switching is critically dependent upon the nature of the B cell activator, in addition to the presence of IL-4. J. Immunol. 147:1163.[Abstract/Free Full Text]
-
Sabin, E. A. and Pearce, E. J. 1995. Early IL-4 production by non-CD4+ cells at the site of antigen deposition predicts the development of a T helper 2 cell response to Schistosoma mansoni eggs. J. Immunol. 155:4844.[Abstract]
-
Sabin, E. A., Kopf, M. A. and Pearce, E. J. 1996. Schistosoma mansoni egg-induced early IL-4 production is dependent upon IL-5 and eosinophils. J. Exp. Med. 184:1871.[Abstract]
-
Falcone F. H., Dahinden, C. A., Gibbs, B. F., Noll, T., Amon, U., Hebestreit, H., Abrahamsen, O., Klaucke, J., Schlaak, M. and Haas, H.. 1996. Human basophils release interleukin-4 after stimulation with Schistosoma mansoni egg antigen. Eur. J. Immunol. 26:1147.[ISI][Medline]
-
Bendelac, A., Rivera, M. N, Park, S. H. and Roark, J. H. 1997. Mouse CD1-specific NK1 T cells: development, specificity, and function. Annu. Rev. Immunol. 15:535.[ISI][Medline]
-
Launois, P., Swihart, K. G., Milon, G. and Louis, J. A. 1997. Early production of IL-4 in suceptible mice infected with Leishmania major rapidly induces IL-12 unresponsiveness. J. Immunol. 158:3317.[Abstract]
-
Launois, P, Maillard, I., Pingel, S., Swihart, K. G., Xenarios, I., Acha-Orbea, H., Diggelmann, H., Locksley, R. M., MacDonald, H. R. and Louis, J. A. 1997. IL-4 rapidly produced by Vß4 V
8 CD4+ T cells instructs Th2 development and susceptibility to Leishmania major in BALB/c mice. Immunity 6:541.[ISI][Medline]
-
Himmelrich, H., Parra-Lopez, C., Tacchini-Cottier, F., Louis, J. A. and Launois, P. 1998. The IL-4 rapidly produced in BALB/c mice after infection with Leishmania major down-regulates IL-12 receptor ß2-chain expression on CD4+ T cells resulting in a state of unresponsiveness to IL-12. J. Immunol. 161:6156.[Abstract/Free Full Text]
-
Himmelrich, H., Launois, P., Tacchini-Cottier, F. and Louis, J. A. 1999. Some of the early events underlying Th2 cell maturation and susceptibility to Leishmania major infection in BALB/c mice. Biol. Chem. 380:909.[ISI][Medline]
-
Stall, A. and Loken, M. 1984. Allotypic specificities of murine IgD and IgM recognized by monoclonal antibodies. J. Immunol. 132:787[Abstract/Free Full Text]
-
Ledbetter, J. A. and Herzenberg, L. A. 1979. Xenogeneic monoclonal antibodies to mouse lymphoid differentiation antigens. Immunol. Rev. 47:63.[ISI][Medline]
-
Vos, Q., Snapper, C. M. and Mond, J. J. 1999. Heterogeneity in the ability of cytotoxic NK cell clones to enhance Ig secretion in vitro. Int. Immunol. 11: 159.[Abstract/Free Full Text]
-
O'Garra, A. 1998. Cytokines induce the development of functionally heterogeneous T helper cell subsets. Immunity 8:275.[ISI][Medline]
-
Hitoshi, Y., Yamaguchi, N., Mita, S., Sonoda, E., Takaki, S., Tominaga, A. and Takatsu, K. 1990. Distribution of IL-5 receptor positive B cells: expression of IL-5 receptors on Ly-1(CD5)+ B cells. J. Immunol. 144:4218.[Abstract/Free Full Text]
-
Allison, K. C., Strober, W. and Harriman, G. R. 1991. Induction of IL-5 receptors on normal B cells by crosslinking surface Ig with anti-Igdextran. J. Immunol. 146:4197.[Abstract/Free Full Text]
-
Arase, H., Arase, N. and Saito, T. 1996. Interferon gamma production by natural killer (NK) cells and NK1.1 T cells upon NKR-P1 cross-linking. J. Exp. Med. 183:2391.[Abstract]
-
Leite-De-Moraes, M. C., Moreau, G., Arnould, A., Machavoine, F., Garcia, C., Papiernik, M. and Dy, M.. 1998. IL-4-producing NK T cells are biased toward IFN-
production by IL-12. Influence of the microenvironment on the functional capacities of NK T cells. Eur. J. Immunol. 28:1507.[ISI][Medline]
-
Moreau, J. L., Chastagner, P., Tanaka, T., Miyasaka, M., Kondo, M., Sugamura, K. and Theze, J. 1995. Control of the IL-2 responsiveness of B lymphocytes by IL-2 and IL-4. J. Immunol. 155:3401.[Abstract]