1 Instituto Gulbenkian de Ciência, Rua da Quinta Grande #6, 2781 Oeiras, Portugal 2 Present address: Laboratoire dImmunorégulation, Centre de Recherche du CHUM, Hôpital Notre-Dame, 1560 Sherbrooke Est., Montréal H2L4M1 Canada
Correspondence to: J. Demengeot; E-mail: jocelyne{at}igc.gulbenkian.pt
Transmitting editor: T. Hünig
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Abstract |
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Keywords: B lymphocytes, cellular activation, cytokines, immuno-modulation
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Introduction |
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Although best known for activation of macrophages and NK cells (9,10), IFN-/ß has clear pleiotropic effects on the immune system. In addition to its induction during infection, IFN-I is constitutively produced in primary lymphoid organs (bone marrow and thymus) where it interferes with lymphocyte differentiation (11,12). At early stages of lymphocyte development, it impairs IL-7-dependent proliferation of pre-B and pre-T cells (13). Later during B cell differentiation, it is responsible for the low threshold of sensitivity to IgM ligation characteristic of both mature and immature B cells in the bone marrow (14). As a direct consequence of this regulation, it determines the stringency of B cell repertoire selection and the number of IgM-secreting B cells in the bone marrow (BM) (15). During an immune response, IFN-
/ß also appears to prolong the survival of activated T cells and may promote the generation of memory T cells (1618). However, studies on resistance to viral infection of mice deficient for either the IFN-IR (IFN-IR/) or the IFN-
receptor provided a rough generalization that B cell responses are predominantly controlled by IFN-I, while IFN-
regulates T cell activities (19). The authors demonstrated that IFN-I is necessary for the resolution of viral infections controlled by antibodies, but has little or no impact on viral infections that are largely controlled by non-antibody-dependent mechanisms. Little is known, however, on the direct effect of IFN-I on B cell function in the course of an immune response and B cell responses to IFN-I have been sparsely studied.
In order to evaluate the role of IFN-/ß in B lymphocyte responses, we have investigated the direct effect of IFN-I on mature B cells. The responses of freshly isolated B lymphocytes, treated or not with IFN-
/ß, to apoptotic, proliferative and activating stimuli have been compared. The results indicate that IFN-
/ß induces B cells to a state of partial activation associated with a higher sensitivity to further stimulation through the BCR. The finding that IFN-IR acts as an amplifier of the BCR signal suggests that IFN-I enhances B cell function during an inflammatory response, and thus serves as a bridge between the innate and acquired immune responses.
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Methods |
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Cell purification and culture
Comparisons between BM cells originating from wild-type or IFN-IR/ mice were performed using the 129Sv animals, while analyses of splenocytes treated with IFN-/ß was achieved with cells prepared from C57BL/6 mice, unless otherwise mentioned. Cell suspensions were prepared by grinding spleens with nylon mesh tissue or by flushing whole BM from femurs and tibias with a 23-gauge needle in HBSS (Gibco/BRL, Gaithersburg, MD) containing 2% FCS (Gibco/BRL). When required, following T cell depletion by mAb anti-Thy1 (from the J1J hybridoma) and complement (Low-Tox rabbit complement; Hornby, Cedarlane, Ontario, Canada), splenocytes were separated on five layers of Percoll gradient (Pharmacia, Uppsala, Sweden). The 6070% interface contained resting (small, high-density) cells while activated (large, low-density) cells were recovered at the 5060% interface. FACS analyses for FSC and staining with anti-B220 indicated a purity of at least 95 and 80% for resting and activated B220+ cells respectively. Cell cultures were performed in RPMI 1640 (Gibco/BRL) supplemented with 10% FCS (Gibco/BRL), streptomycin (50 µg/ml), penicillin (50 U/ml), 2-mercaptoethanol (50 µM), HEPES buffer (10 mM, pH 7.3) and sodium pyruvate (1 mM) (all from Gibco/BRL). Pre-incubation with IFN-
/ß (Sigma-Aldrich, Madrid, Spain) lasted for 16 or 24 h prior to initiation of each assay unless otherwise indicated and the concentration of IFN-
/ß was maintained at 400 U/ml during the course of the experiment. Control cultures were maintained for the same time in media alone. Actinomycin D (Sigma) was used at 4 µg/ml. Bulk cultures were set at 2 x 106 cells/ml, high-density cultures at 107 cells/ml and proliferation assay at 2.5 or 5 x 105 cells/ml.
Antibodies, cytokines and FACS analyses
In vitro assays of BCR ligation used antibodies directed against the µ chain of mouse Ig, either monoclonal rat IgG (purified in the laboratory from the hybridoma 331.12 (20)), goat polyclonal F(ab')2 (Pierce, Rockford, IL) or affinity-isolated goat polyclonal Ig (Sigma). The rat anti-mouse CD40 antibody was produced in the laboratory using the FGK45 hybridoma (a gift from J. Andersson, Basel Institute of Immunology, Switzerland). Lipopolysaccharide (LPS) from Salmonella typhimurium was purchased from Sigma. Culture supernatant from the transfected 3T3-IL-4 cell line (a gift from A. Cumano, Pasteur Institute, France) diluted 1/100 was used as a source of IL-4. All labeled antibodies used for flow cytometry analyses were purchased from Becton Dickinson (Mountain View, CA). Single-cell suspensions of BM or splenic B cells were stained with various antibodies coupled to FITC, phycoerythrin, biotin or allophycocyanin at previously determined concentrations. Biotinylated antibodies were revealed by streptavidinphycoerythrin (Becton Dickinson). Dead cells were excluded from analysis by gating out propidium iodide (Sigma) stained cells. Data were collected from 5 to 10 x 103 cells on a FACScan or a FACSCalibur flow cytometer (Becton Dickinson) and analyzed using the CellQuest program.
Measure of apoptosis by TUNEL
The measures of apoptosis were performed using a TUNEL kit (Roche Diagnostics, Mannheim, Germany) following the instructions of the manufacturer. The cells were stained with phycoerythrin-B220 antibody prior to fixation. Acquisition was completed on a FACSCalibur flow cytometer (Becton Dickinson). All results were confirmed by propidium iodide staining of ethanol fixed cells.
Measurements of proliferation
[3H]Thymidine (ICN, Aurora, OH; sp. act. 5 Ci/mol) uptake was performed at day 3 or 4 of culture by adding 1 µCi/culture, 6 h before harvest. Pulsed cultures were harvested on glass-fiber filter paper using a 96-well microtiter plate harvester (TOMTEC; Pharmacia/LKB, Bromma, Sweden). After drying, the filters were immersed in scintillation fluid and counted in a Rack-Beta Plate liquid scintillation counter (Pharmacia/LKB). The data represent the mean and SD of three cultures expressed as c.p.m. per culture.
Measurements of Ca2+ influxes
A protocol adapted from Sei and Akora (21) was followed. Briefly, 107 cells/ml were incubated for 20 min at 37°C in HBSS (Gibco/BRL) containing 4 µg/ml Fluo-3 AM (Molecular Probes, Eugene, OR). Cells were then diluted 5-fold in HBSS containing 1% FCS and incubated for 40 min at 37°C. Following surface staining with phycoerythrinanti-B220 antibody, the cells were washed and resuspended at 2 x 106/ml in HEPES buffer (137 mM NaCl, 5 mM KCl, 1 mM Na2HPO4, 5 mM glucose, 1 mM CaCl2, 0.5 mM MgCl2, 1 g/ml BSA and 10 mM HEPES). Before each test, cells were incubated during 10 min at 37°C. The stimulation was performed with mAb anti-µ 331.12 at doses ranging from 0.1 to 10 µg/ml.
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Results |
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IFN-/ß induces the expression of various B cell activation markers
To further characterize the physiological status of IFN-/ß-treated B splenocytes, we measured the expression level of several surface markers associated with B cell activation. In agreement with what has been reported for various cell types (22,23), wild-type splenic B cells treated with IFN-
/ß for 48 h up-regulate class I molecule expression (Kb, shown in Fig. 2A). The level of IgM expressed at the cell membrane was decreased (Fig. 2B), a feature often associated with BCR-mediated activation. Expression of CD69 was rapidly induced and peaked at
6 h after initiation of the treatment (Fig. 2C). Induction of CD86 (B7-2) was maximal after 8 h (Fig. 2D), whereas expression of CD25 (the
chain of the IL-2 receptor) was detectable on up to 60% of treated B cells after 1012 h of incubation (Fig. 2E). Expression of MHC class II, CD38 or CD62 ligand remained unaffected by IFN-
/ß treatment (data not shown). While the percentages of B cells induced for expression of Kb, CD69, CD86 or CD25 was dependent on the concentration of IFN-
/ß added to the media, the levels of expression per cell remained constant. In addition, the induction was transient and resolved after 2448 h. The same pattern of expression was obtained when purified resting B cells were analyzed (not shown), indicating that IFN-
/ß directly stimulates B cells. In each assay, lymphocytes prepared from mice deficient for the type I IFN receptor (IFN-IR/) served as controls for the specificity of the responses. Wild-type lymphocytes cultured with medium alone and IFN-IR/ splenocytes treated or not with IFN-
/ß showed equivalent levels of expression for each of the markers tested. We conclude that IFN-I directly and specifically induces a transient activation of B cells.
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Our previous work demonstrated that IFN-/ß amplifies the inhibition of LPS-induced terminal differentiation by BCR ligation (14). We tested whether it would also increase the sensitivity of B cells to anti-µ stimulation in the anti-CD40 proliferation assay. Control or IFN-
/ß-treated splenocytes were stimulated with a fixed amount of anti-CD40 mAb together with various doses of F(ab')2 anti-µ fragments and incorporation of [3H]thymidine was monitored on day 3 (Fig. 3C). For each cell population, proliferation followed a doseresponse relationship that reached the same plateau. However, evaluation of the F(ab')2 concentration resulting in 50% of the maximum proliferation (dotted line in Fig. 3C), revealed that IFN-
/ß-treated splenocytes are 100 times more sensitive to anti-µ-mediated proliferation than untreated splenocytes. Since IFN-ß is constitutively produced in the BM (11), the same co-stimulation was performed on freshly isolated BM cells prepared from wild-type and IFN-IR/ mice. For a low concentration of F(ab')2 anti-µ, cells deficient for the IFN-IR proliferated less than wild-type lymphocytes (data not shown), thus confirming that IFN-I enhances B cell proliferative response to F(ab')2 anti-µ. These analyses therefore indicate that IFN-
/ß endows B lymphocytes with a higher sensitivity to proliferative responses induced by their antigen receptor ligation.
IFN-/ß amplifies BCR-mediated induction of surface activation markers
Cross-linking of the BCR at the surface of a resting mature B cell has been shown to induce expression of various molecules such as B7-2 and MHC II (24,25). To further characterize the synergy of BCR and IFN-IR signals, wild-type splenocytes treated or not for 24 h with IFN-/ß as well as BM cells prepared either from IFN-IR/ or wild-type animals were incubated for 16 h with various doses of the non-proliferative anti-µ mAb. In both spleen and BM, measurement of the induction of several surface molecules was restricted to mature B cells (B220high). Since IFN-
/ß alone induces the transient up-regulation of several activation markers on wild-type cells, all measurements of surface marker expression following BCR engagement were normalized to the level of expression before BCR stimulation. The dose-dependent induction of the four surface markers I-Ab (MHC-II), CD23, CD69 and CD86 following treatment of both spleen and BM mature B cells with the anti-µ mAb was dramatically enhanced by IFN-
/ß, either added in the cultures (splenocytes) or locally produced (wild-type BM). Typical profiles of induction for each molecule are shown in Fig. 4. Noteworthy, the same expression profiles were obtained after stimulation of BM mature B cells or IFN-I-treated B splenocytes. This result indicates that a concentration of 400 U/ml in vitro reflects closely the IFN-
/ß amount actually present in the BM. The expression levels of other activation markers (CD25, CD38 and CD44) or differentiation markers associated with membrane (m) IgM triggering (CD19, CD22) remained constant after BCR ligation, independently of the presence or absence of IFN-
/ß (data not shown). These results indicate that IFN-
/ß and BCR ligation act in synergy to stimulate B cells.
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Discussion |
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Our results showing an anti-apoptotic effect of IFN-/ß on resting B cells are in accordance with previous studies conducted on B cell lines. Inhibition of BCR-mediated apoptosis by IFN-
/ß in Ramos cells (which are phenotypically representative of germinal center B cells) has been correlated with increased expression of the anti-apoptotic Bcl-2 and Bcl-XL genes (27). In addition, IFN-
activates the NF-
B transcription factor, protecting human Daudi cells against pro-apoptotic stimuli such as virus infection and BCR cross-linking (28). Several studies indicate that IFN-
/ß is a T cell survival and/or anti-apoptotic factor (7,1618), an effect that appears to be restricted to activated and memory lymphocytes (17). In contrast, our results indicate that IFN-
/ß increases the survival of resting B cells and does not significantly affect naturally activated B cell recovery after culture. However, B cells activated through the CD40 receptor, a mimic of T cell help in germinal centers, show increased resistance to Fas-mediated apoptosis when treated with IFN-
/ß. This apparent discrepancy may reflect the particular nature of naturally activated B cells, which do not seem to result from bona fide inductive TB cell interaction and cycle poorly [e.g. (29,30)]. In turn, these results suggest that IFN-
/ß may affect the outcome of Fas-mediated B cell selection during germinal center reactions. Analyses of the dynamic of Ig affinity maturation and evaluation of in situ cell death in the germinal center during the course of a T-dependent immune response in IFN-IR/ mice will help clarify this point.
We show that IFN-/ß treatment leads to increased B cell survival and up-regulation of several activation markers, although it does not induce B cell proliferation. The increased survival and clonal expansion of CD8+ T cells mediated by IFN-
/ß has been attributed to the effect of IL-15 produced by activated macrophages. Purified B cells respond to IFN-
/ß no differently than whole spleen cell populations. It is therefore unlikely that a much-reduced population of activated macrophages could secrete significant amounts of IL-15, which would account for the effects we monitored following IFN-
/ß treatment. Moreover, splenocytes cultured in medium supplemented with IL-15 instead of IFN-
/ß do not show any increase in CD25 expression. Thus, in our assay, IL-15 does not appear to mediate the effects of IFN-
/ß.
The responses of B cells to antigen begin with a Ca2+ flux, receptor internalization and antigen uptake, then proceed to antigen processing, induction of several activation surface molecules, and finally result in proliferation and differentiation to antibody-secreting cells. The earlier events lead B cells to a state of competence for cognate interaction with primed Th cells, which in turn induces proliferation and differentiation into effector cells. We show that pre-treatment of B lymphocytes with IFN-/ß increases Ca2+ flux, receptor internalization as well as MHC II expression following BCR ligation. Similarly, expression of several activation markers, including B7-2, a molecule necessary for interactions with T cells, is dramatically increased. Finally B cell proliferative response to co-ligation of the BCR and CD40 molecules, an experimental mimicry of TB cell interaction, was also enhanced by IFN-
/ß. It is worth recalling that the level of IgM at the B cell surface is substantially decreased after overnight culture with IFN-
/ß. Therefore the higher sensitivity to BCR ligation after IFN-
/ß treatment does not depend on the number of available receptors at the cell surface and, thus, should result from modifications of the cell responsiveness, most probably related to its partial-activation status. The increase of sensitivity to BCR ligation characterized here is of the same order of magnitude as the one we previously reported measuring the inhibition of IgM secretion after LPS stimulation (14,31). The doses of IFN-
/ß required to monitor such effects (100400 U/ml) are relatively low and therefore close to physiological levels. Moreover, the effect is similar when considering peripheral B cells treated ex vivo with IFN-
/ß and mature BM B cells from wild-type animal, a tissue where IFN-
/ß is constitutively produced (11). Finally, because the various IFN-I share a single specific receptor, the comparison between BM cells prepared from either wild-type mice, where IFN-
/ß is constitutively expressed (11), or IFN-IR/ animals, where cells cannot respond to this cytokine, indicates that the role of IFN-I on B cells responses is of physiological relevance.
Our finding that IFN-/ß enhances B lymphocyte responses to antigen receptor ligation and acts as a regulator of B cell function is reminiscent of the role that has been attributed to C3 when binding to the CD19/CD21/CD81 co-receptor complex (32). Co-receptors are molecules physically associated with the BCR and their co-ligation modulates the strength of the antigen receptor signal. This regulatory function has never been attributed to a cytokine receptor. The parameters we varied in our analyses were both the dose of IFN-
/ß and the expression of the IFN-IR; therefore we conclude that IFN-IR upon binding IFN-
/ß, acts as an amplifier of BCR-mediated responses.
There are two possible ways that binding of IFN-/ß to the IFN-IR could influence BCR signaling. It may exert a direct effect on intermediates of the BCR signaling pathway or induce changes in the expression of a number of componentsboth intracellular and at the cell surfaceincreasing the likelihood that a BCRligand binding event will produce a functional signal. The former hypothesis suggests that the BCR and IFN-IR signaling pathways share common elements. However, to date, none of the proteins identified in either of the signaling cascades seem to be identical (33,34). Our kinetic analyses indicate that pretreatment with IFN-
/ß has to last for several hours before the cell sensitivity to BCR ligation is increased. Moreover, we show that inhibition of RNA synthesis prevents amplification of the BCR signal by IFN-
/ß, demonstrating that it requires de novo synthesis.
It is striking that IFN-/ß treatment alone leads B cells to a physiological state closely resembling that induced by BCR triggering. Continuous BCR engagement has been suggested as a sine qua non requirement for B cell survival (3537). It is thus possible that the small number (15%) of B cells recovered after 2 days of culture in medium alone survived through their possession of receptors recognizing serum proteins in the medium. Under this hypothesis, and as a direct consequence of a lower threshold of sensitivity to BCR ligation, the higher fraction of surviving cells in cultures containing IFN-
/ß may correspond to a larger number of cells for which BCR engagement was strong enough to provide a survival signal. Experiments conducted in serum-free media supplemented with suboptimal doses of BCR ligand and/or involving monoclonal population of B cells sharing the same BCR specificity (purified from BCR transgenic mice) may help to clarify this issue.
Our present analyses, taken together with the previous works conducted on T cells (7,1618), lead us to propose a new scheme for IFN-/ß function during an immune response. In the early step after infection, induction of IFN-
/ß enhances B cell responses to antigen and to their subsequent interaction with helper T cells. Once the primary phase of the response is performed, IFN-
/ß potentates survival of activated and memory T cells which are later responsible for the maintenance of an efficient immune response. These data thus provide a further functional link between the innate immune system largely associated with IFN-I and the acquired immune system.
The anti-proliferative properties of IFN-I have been exploited through the therapeutic usage of this cytokine in the treatment of viral infections or malignant disease (1). The analyses presented here suggest that these approaches may in addition augment B cell responses to viral or tumor antigens. However, an undesirable complication of the IFN-I therapy is the development of autoreactive antibodies, which frequently necessitates the interruption of the treatment [e.g. (3841)]. The impaired Fas-mediated apoptosis, increased sensitivity to BCR activation as well as the apparent enhanced ability to receive T cell help evidenced here suggest that IFN-/ß direct effects on B cells are responsible for the production of autoreactive antibodies by these patients. Analyses of the role of IFN-I on the onset and development of a lupus-like syndrome in mice will test this hypothesis.
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Acknowledgements |
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Abbreviations |
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IFN-IRIFN type I receptor
LPSlipopolysaccharide
mIgMmembrane IgM
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References |
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