IFN-α consists of a family of highly homologous proteins, which exert pleiotropic effects on a wide variety of cell types. The biologic activities of IFN-α are mediated by its binding to a multicomponent receptor complex resulting in the activation of the Janus kinase-STAT signaling pathway. In most cell types, activation of Stat1 and Stat2 by IFN-α leads to the formation of either STAT homo-/heterodimers or of the IFN-stimulated gene factor 3 complex composed of Stat1, Stat2, and p48, a non-STAT protein. These distinct transcriptional complexes then target two different sets of cis-elements, γ-activated sites and IFN-stimulated response elements. Here, we report that IFN-α can activate complexes containing Stat6, which, until now, has been primarily associated with signaling by two cytokines with biologic overlap, IL-4 and IL-13. Induction of Stat6 complexes by IFN-α appears to be cell type specific, given that tyrosine phosphorylation of Stat6 in response to IFN-α is predominantly detected in B cells. Activation of Stat6 by IFN-α in B cells is accompanied by the formation of novel Stat2:Stat6 complexes, including an IFN-stimulated gene factor 3-like complex containing Stat2, Stat6, and p48. B cell lines resistant to the antiproliferative effects of IFN-α display a decrease in the IFN-α-mediated activation of Stat6. Activation of Stat6 as well as of Stat2:Stat6 complexes by IFN-α in B cells may allow modulation of target genes in a cell type-specific manner.

Interferon-α consists of a family of highly homologous proteins that exert pleiotropic actions, including immunomodulatory, antiviral, and antiproliferative effects (1, 2, 3). These biologic activities are mediated by the ability of the IFN-α species to bind to a multicomponent receptor complex. Two distinct human receptor chains have been cloned so far: the IFNαR1 and the IFNαR2 chains (4). However, additional receptor components may exist, given that transfection of both human receptor chains in murine cells does not fully reconstitute biologic responses to all human IFN-α subspecies (5). Cell type-specific differences in receptor composition may also occur, as suggested by the finding that IFN-α interacts differently with lymphoid vs epithelial cells (6).

The early intracellular events triggered by IFN-α have recently been elucidated and have served as a paradigm for cytokine signaling (7, 8). Binding of IFN-α to its receptor leads to the activation, via two receptor-associated tyrosine kinases, Tyk2 and Jak1, of latent cytoplasmic factors belonging to the STAT (signal transducers and activators of transcription) family of proteins. Tyrosine phosphorylation of the STATs allows them to homodimerize or heterodimerize and to translocate into the nucleus, where they modulate gene transcription. So far, seven distinct members of the STAT family have been identified in eukaryotes. Some STATs display a restricted pattern of activation. For instance, Stat6 complexes are induced primarily in response to two cytokines with overlapping biologic functions, IL-4 and IL-13. In contrast, other STATs can be activated in response to a variety of cytokines (9). IFN-α has been shown to lead to the tyrosine phosphorylation of Stat1, and of Stat2 in most cell types (9, 10, 11), although activation of Stat3, Stat4, and Stat5 has also been reported (12, 13, 14).

STAT homo-/heterodimers activated in response to IFN-α can drive the expression of a subset of IFN-α-inducible genes, e.g., the gene encoding the transcription factor IFN regulatory factor 1 (IRF-1), by binding to specific promoter elements termed γ-activated sites (GAS).3 Unlike other cytokines, IFN-α activation of STATs also leads to the formation of an additional transcriptional complex termed IFN-stimulated gene factor 3 (ISGF3), which is composed of a Stat1:Stat2 heterodimer and a non-STAT protein, p48. The ISGF3 complex can then induce the expression of a distinct subset of IFN-responsive genes by binding to a different subset of cis-acting elements, termed IFN-stimulated response elements (ISREs) (15).

IFN-α has been widely used as an antiviral as well as an antitumor agent because of its growth-inhibitory effects (16, 17, 18). Up-regulation of IRF-1 is believed to mediate some of the antiproliferative effects of the IFNs (19). The effectiveness of the growth-inhibitory actions of IFNs is, however, limited by the emergence of IFN resistance. In B cells, IFN resistance characterizes EBV transformation of B cells as well as certain subclones of Daudi, a Burkitt’s lymphoma cell line, which is normally exquisitely sensitive to growth inhibition by IFN-α. IFN resistance in EBV-transformed B cells has been attributed to the presence of the EBV nuclear Ag 2 (EBNA2) (20). However, a distinct mechanism is believed to be responsible for the emergence of IFN resistance in Daudi variants, because the EBV genome present in Daudi cells is defective and carries a deletion of the entire coding region of EBNA2 (21). In both of these systems, IFN resistance leads to the selective extinction of the antiproliferative pathways triggered by IFN-α, whereas the induction of other IFN-α responses is maintained at normal levels (20, 22). Studies of the molecular mechanisms underlying IFN resistance in both Daudi as well as EBV-transformed B cells have failed to demonstrate changes in either the affinity or number of IFN-α receptors. A variety of defects in STAT activation has been reported in Daudi resistant clones, including lack of Stat3 activation (12), premature loss of Stat1 phosphorylation (23), and an inability to activate an ISRE binding complex (24). A similar defect in ISGF3 activation has not been detected in EBV-transformed B cells (21), while the ability of these cells to activate other STAT complexes has not been thoroughly investigated.

In this study, we have found that exposure of B cells to IFN-α leads to activation of Stat6-containing complexes, which are indistinguishable from those induced by IL-4 (25, 26, 27, 28). Stat6 activation in response to IFN-α is not observed in non-B cell lines. The ability of IFN-α to activate Stat6 in addition to previously characterized STATs allows for the formation of novel Stat2:Stat6 complexes in B cells, including an ISGF3-like complex containing Stat2, Stat6, and p48. IFN-α-mediated activation of Stat6 is markedly reduced in both EBV-transformed B cells and D-R cells. A decrease in the IFN-α inducibility of Stat6 binding to the IRF-1 GAS is associated with a diminished transactivation of the IRF-1 gene in response to IFN-α. The ability of IFN-α to activate distinct Stat6-containing transcriptional complexes in B cells may allow it to modulate the expression of target genes in a cell type-specific manner.

The human cell lines Ramos (a kind gift of Dr. Seth Lederman, Columbia University, New York, NY), BL-2, and BJAB (a generous gift of Dr. Riccardo Dalla Favera, Columbia University) are EBV-negative Burkitt’s lymphomas. Namalwa (a generous gift of Dr. Riccardo Dalla Favera, Columbia University) is an EBV-positive Burkitt’s lymphoma cell line. Subclones of the EBV-positive Burkitt’s lymphoma cell line Daudi, which are either IFN sensitive (D-S) or IFN resistant (D-R), were kind gifts of Dr. Sharon Evans (Roswell Park Cancer Institute, Buffalo, NY) and Dr. A. Hovanessian (Institut Pasteur, Paris, France) (29, 30). WIL-2-729HF2 (American Type Culture Collection (ATCC), Manassas, VA) and JY cells (a generous gift of Dr. Riccardo Dalla Favera, Columbia University) are EBV-transformed lymphoblastoid B cell lines. THP-1 (a kind gift of Dr. Kathryne Calame, Columbia University) is a monocytic cell line. Jurkat (a kind gift of Dr. Kathryne Calame) is a human T cell leukemic line, and H9 (ATCC) is derived from a human T cell lymphoma. All cells were grown in IMDM supplemented with 10% FCS (Atlanta Biologicals, Norcross, GA). WI38 VA (a human embryonic lung fibroblast) and WISH (a human epithelial-like amnion tissue-derived cell line) were a kind gift of Dr. Chris Schindler (Columbia University) and were grown in DMEM supplemented with 10% FCS.

Cells (20–50 × 106) were incubated at 37°C for varying periods of times in a final volume of 10 ml. Cells were stimulated utilizing the following cytokine concentrations: human IL-4 (100 U/ml; a generous gift of Dr. Paul Rothman, Columbia University), human IFN-α2a (1000 U/ml; a generous gift of Dr. Chris Schindler), and human IFN-γ (10 ng/ml; PeproTech, Rocky Hill, NJ).

The preparation and use of DNA oligonucleotide probes for mobility shift assays have been described previously (31). The probes used in these studies were as follows: IRF-1 GAS, 5′-gatcGATTTCCCGAAAT-3′; CD23b GAS, 5′-gatcGGGTGAATTTCTAAGAAAGGGAC-3′; and ISG-15 ISRE, 5′-gatcCTCGGGAAAGGGAAACCGAAACTGAAGCC-3′. Double-stranded oligonucleotides used as cold competitors were prepared from single-stranded oligonucleotides (Life Technologies) with the following sequences: βCAS-GAS, 5′-gatcGACTTCTTGGAATT3-′; Iε-GAS (−119 to −104), 5′-gatcAACTTCCCAAGAACAG-3′; Ly6E-GAS, 5′-gatcATATTCCTGTAAGT-3′ (31). STAT antisera were added (final dilution, 1:20) for 30–60 min at 4°C, before a standard 20-min (25°C) incubation of extracts with shift probe. Oligonucleotide competitions were performed by adding a 100-fold excess of cold oligonucleotides for 15 min (25°C) before a standard 20-min (25°C) incubation of extracts with shift probe.

Whole-cell extracts (WCE) were prepared as described previously (31).

Rabbit polyclonal antisera against Stat1, Stat2, and p48 were a generous gift of Dr. Chris Schindler. Rabbit polyclonal Abs against human Stat3, Stat5, and Stat6 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The antiserum against IFN-αRI was a generous gift of Dr. Oscar Colamonici (University of Tennessee).

Extracts were immunoprecipitated with anti-STAT antisera as described previously (31). The immunoprecipitates were fractionated by 7% SDS-PAGE before immunoblotting with either an anti-phosphotyrosine Ab (4G10, Upstate Biotechnology, Lake Placid, NY) or anti-STAT antisera. Bands were detected by enhanced chemiluminescence (Amersham, Arlington Heights, IL).

Total RNA was extracted by utilizing the ULTRA-SPEC II kit (Bioteck Laboratories, Houston, TX). Northern blot analysis was performed with 10 μg of total RNA according to standard protocols. The blot was probed with either a human IRF-1 cDNA (a generous gift of Dr. Richard Pine, Public Health Research Institute, NY) or a GAPDH cDNA radiolabeled by Pharmacia DNA labeling bead (−dCTP) kit.

IFN-α exerts pleiotropic effects on the immune system (1, 2, 3). To investigate whether the diverse biologic activities exerted by IFN-α on distinct cell types are reflected in a differential usage of signaling molecules, we exposed a panel of human cell lines to IFN-α2a. Cells were then harvested, and WCE were obtained and assayed by EMSA utilizing as a probe the GAS element of the IRF-1 promoter. This GAS element is able to mediate binding of a variety of distinct STAT complexes (32). As shown in Fig. 1,A, IFN-α is able to activate multiple IRF-1 GAS binding complexes. The mobility and pattern of the faster-migrating complexes was consistent with the previously described IFN-α-activated Stat1:Stat3 homo-/heterodimers (12). These complexes were detected in all of the cell lines tested. Exposure of human B cell lines to IFN-α, however, led to the induction of an additional slow-mobility complex (complex X). Both EBV-positive (Namalwa and Daudi) and -negative (Ramos, BL-2, and BJAB) Burkitt’s lymphoma cell lines were found to activate this slow-mobility complex, although its intensity varied among the individual B cell lines (Fig. 1,A, and data not shown). This complex was, however, almost undetectable or even absent in two EBV-transformed B cell lines (WIL-2 and JY) (Figs. 1,A and 6A and data not shown). Kinetic experiments revealed that the induction of this B cell-specific complex in response to IFN-α was short lived (Fig. 1,B), because it had disappeared after 2 h of culture with IFN-α. These kinetics differed sharply from those displayed by the Stat1-containing complexes or by the previously characterized ISGF3 complex binding to an ISRE element (Fig. 1,B and data not shown) (11, 33). Kinetic experiments in a non-B cell line (THP-1) failed to reveal the presence of complex X at any of the time points examined, suggesting that differences in the kinetics of activation are not responsible for lack of induction of complex X in non-B cells (Fig. 1 B).

FIGURE 1.

A, IFN-α induces distinct IRF-1 GAS binding complexes in human B cell vs non-B cell lines. Extracts were prepared from a panel of B cell lines (Ramos, Namalwa, and WIL-2) or non-B cell lines (Jurkat, H9, and THP-1) with or without a 20-min stimulation at 37°C with IFN-α2a (1000 U/ml). The WCE were examined by EMSA using an IRF-1 GAS probe. B, Kinetics of IFN-α activation of the B cell-specific IRF-1 GAS binding complex. Extracts were prepared from a human B cell line, Ramos, or a human monocytic cell line, THP-1, before or after stimulation with IFN-α2a (1000 U/ml) for 20 min, 45 min, or 2 h. The WCE were examined by EMSA using an IRF-1 GAS probe.

FIGURE 1.

A, IFN-α induces distinct IRF-1 GAS binding complexes in human B cell vs non-B cell lines. Extracts were prepared from a panel of B cell lines (Ramos, Namalwa, and WIL-2) or non-B cell lines (Jurkat, H9, and THP-1) with or without a 20-min stimulation at 37°C with IFN-α2a (1000 U/ml). The WCE were examined by EMSA using an IRF-1 GAS probe. B, Kinetics of IFN-α activation of the B cell-specific IRF-1 GAS binding complex. Extracts were prepared from a human B cell line, Ramos, or a human monocytic cell line, THP-1, before or after stimulation with IFN-α2a (1000 U/ml) for 20 min, 45 min, or 2 h. The WCE were examined by EMSA using an IRF-1 GAS probe.

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Induction of the slow-mobility IRF-1 GAS binding complex by IFN-α was also detected in extracts from fresh peripheral blood mononuclear cells obtained from a patient with chronic lymphocytic leukemia (which contain a large number of circulating mature B cells) (data not shown). These results suggest that activation of complex X is not due to long-term culture of the Burkitt’s lymphoma cell lines.

These data thus indicate that IFN-α can induce cell type-specific IRF-1 GAS binding complexes.

To determine the identity of the DNA binding activity induced by IFN-α in B cells, we subjected these complexes to oligonucleotide competitions with a panel of GAS elements known to discriminate among distinct STAT complexes (Fig. 2 A). The pattern of competition displayed by the slow-mobility complex found in B cells (complex X) was identical with the one previously described for Stat6 (31) and contrasted to the one exhibited by the Stat1:Stat3-containing complexes. Specifically, competition was observed with the Iε GAS (which binds Stat6 but not Stat1) but only minimally with the LY6E GAS (which binds Stat1 but not Stat6). The β-CAS GAS competed all complexes in a manner identical with that of the IRF-1 GAS, whereas an irrelevant oligonucleotide containing an NF-κB binding site failed to compete any of the complexes.

FIGURE 2.

A, GAS oligonucleotide competition pattern exhibited by the IFN-α-induced IRF-1 GAS binding complexes. Ramos cells were stimulated with human IFN-α2a (1000 U/ml) for 20 min. Extracts were then prepared and examined as described in Fig. 1. Oligonucleotide competition assays were performed either in the absence or in the presence of a 100-fold molar excess of cold GAS oligonucleotides added to the shift reaction as indicated. Competitors included Iε-GAS, Ly6E-GAS, βCAS-GAS, an irrelevant NF-κB binding site, and the IRF-1 GAS. B, The CD23b GAS selectively binds the slow-mobility complex activated by IFN-α in Ramos. Cells either were unstimulated or were stimulated with human IFN-α2a (1000 U/ml) or human IL-4 (100 U/ml) for 20 min. Extracts were prepared as described above and examined by EMSA utilizing either a CD23b GAS probe (left panel) or an IRF-1 GAS probe (right panel).

FIGURE 2.

A, GAS oligonucleotide competition pattern exhibited by the IFN-α-induced IRF-1 GAS binding complexes. Ramos cells were stimulated with human IFN-α2a (1000 U/ml) for 20 min. Extracts were then prepared and examined as described in Fig. 1. Oligonucleotide competition assays were performed either in the absence or in the presence of a 100-fold molar excess of cold GAS oligonucleotides added to the shift reaction as indicated. Competitors included Iε-GAS, Ly6E-GAS, βCAS-GAS, an irrelevant NF-κB binding site, and the IRF-1 GAS. B, The CD23b GAS selectively binds the slow-mobility complex activated by IFN-α in Ramos. Cells either were unstimulated or were stimulated with human IFN-α2a (1000 U/ml) or human IL-4 (100 U/ml) for 20 min. Extracts were prepared as described above and examined by EMSA utilizing either a CD23b GAS probe (left panel) or an IRF-1 GAS probe (right panel).

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The ability of the slow-mobility complex activated in response to IFN-α to target Stat6-selective GAS elements was further confirmed by simultaneously testing IFN-α-stimulated extracts from a B cell line, Ramos, with two distinct probes, the IRF-1 GAS and the CD23b GAS (Fig. 2 B) (34). In contrast to the IRF-1 GAS, the CD23b GAS only detected the slow-mobility complex activated by IFN-α. The mobility of this complex was identical with that of the IL-4-inducible Stat6 complex, suggesting that complex X might contain Stat6.

To identify further the components of complex X, we subjected the IFN-α-inducible complexes found in Ramos to Ab competitions with a panel of STAT antisera (Fig. 3,A). Consistent with the results obtained above, the B cell-specific IRF-1 GAS binding complex activated by IFN-α was found to migrate with a mobility identical with that of the IL-4-inducible complex and to contain Stat6 or a protein antigenically related to it. As previously reported in other cell types (12), the faster mobility complexes contained Stat1 homodimers, Stat3 homodimers, and Stat1:Stat3 heterodimers. Neither the Stat5 nor the Stat2 antiserum significantly affected any of the IRF-1 GAS binding complexes induced by IFN-α in Ramos cells. Interestingly, the inability of non-B cells to activate Stat6 in response to IFN-α was not due to a global defect in Stat6 activation, because, except for Jurkat cells, these cells displayed normal induction of Stat6 complexes in response to IL-4 (Fig. 3 B and data not shown). These results thus demonstrate that IFN-α is able to activate a B cell-specific DNA binding complex that is indistinguishable from the IL-4-inducible Stat6 complex by gel-shift mobility, as well as by oligonucleotide and antiserum competition patterns.

FIGURE 3.

A, The IRF-1 GAS binding complexes induced by IFN-α in B cells contain Stat6. Ramos cells either were unstimulated or were stimulated with human IFN-α2a (1000 U/ml), human IL-4 (100 U/ml), or human IFN-γ (10 ng/ml) for 20 min. Extracts were then prepared and examined as described in Fig. 1. Ab interference mobility shift assays were conducted by addition of antisera against Stat1, Stat2, Stat3, Stat5, or Stat6 or preimmune serum (control). All antisera were added at a final dilution of 1:20 for 30 min at 4°C before incubation with the IRF-1 GAS probe for 20 min at 25°C. B, Non-B cell lines activate Stat6 in response to IL-4 but not to IFN-α; Ramos, H9, and THP-1 cells either were unstimulated or were stimulated with human IFN-α2a (1000 U/ml) or human IL-4 (100 U/ml) for 20 min. Extracts were prepared and examined by EMSA utilizing an IRF-1 GAS probe as described in Fig. 1.

FIGURE 3.

A, The IRF-1 GAS binding complexes induced by IFN-α in B cells contain Stat6. Ramos cells either were unstimulated or were stimulated with human IFN-α2a (1000 U/ml), human IL-4 (100 U/ml), or human IFN-γ (10 ng/ml) for 20 min. Extracts were then prepared and examined as described in Fig. 1. Ab interference mobility shift assays were conducted by addition of antisera against Stat1, Stat2, Stat3, Stat5, or Stat6 or preimmune serum (control). All antisera were added at a final dilution of 1:20 for 30 min at 4°C before incubation with the IRF-1 GAS probe for 20 min at 25°C. B, Non-B cell lines activate Stat6 in response to IL-4 but not to IFN-α; Ramos, H9, and THP-1 cells either were unstimulated or were stimulated with human IFN-α2a (1000 U/ml) or human IL-4 (100 U/ml) for 20 min. Extracts were prepared and examined by EMSA utilizing an IRF-1 GAS probe as described in Fig. 1.

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To confirm that Stat6 was indeed activated in response to IFN-α in B cells, we then performed immunoprecipitations with a Stat6 antiserum on extracts from a panel of cell lines cultured with IFN-α (Fig. 4). Consistent with our previous observations, tyrosine phosphorylation of a 100-kDa protein was observed in response to both IFN-α and IL-4 in Ramos cells (Fig. 4, top panel). Despite comparable levels of inactive Stat6 in the immunoprecipitates from non-B cell lines (WI38 VA and WISH) (Fig. 4, middle panel), IFN-α treatment led to the tyrosine phosphorylation of Stat6 only in B cell lines (Ramos and Daudi) (Fig. 4, top panel). The inability of IFN-α to activate Stat6 in non-B cells was not due to unresponsiveness of these cells to IFN-α, because IFN-α induction of ISGF3 was detected in both WISH and WI38 VA cells.

FIGURE 4.

Stat6 is tyrosine phosphorylated in response to IFN-α in B cell but not in non-B cell lines. Ramos, Daudi, WISH, and WI38 VA cells either were unstimulated or were stimulated with IFN-α2a (1000 U/ml) for 20 min. Extracts were then immunoprecipitated (IP) with a Stat6 antiserum, resolved by 7% SDS-PAGE, and analyzed by Western blotting (WB) using an antiphosphotyrosine Ab (toppanel). The blot was later stripped and reprobed with a Stat6 antiserum to ensure equal loading of immunoprecipitates (middlepanel). After restripping, the blot was reprobed with a Stat2 antiserum to confirm the presence of Stat2 in the Stat6 immunoprecipitates from IFN-α-treated extracts (bottompanel). Extracts from IL-4-stimulated Ramos cells were included as a control for Stat6 activation.

FIGURE 4.

Stat6 is tyrosine phosphorylated in response to IFN-α in B cell but not in non-B cell lines. Ramos, Daudi, WISH, and WI38 VA cells either were unstimulated or were stimulated with IFN-α2a (1000 U/ml) for 20 min. Extracts were then immunoprecipitated (IP) with a Stat6 antiserum, resolved by 7% SDS-PAGE, and analyzed by Western blotting (WB) using an antiphosphotyrosine Ab (toppanel). The blot was later stripped and reprobed with a Stat6 antiserum to ensure equal loading of immunoprecipitates (middlepanel). After restripping, the blot was reprobed with a Stat2 antiserum to confirm the presence of Stat2 in the Stat6 immunoprecipitates from IFN-α-treated extracts (bottompanel). Extracts from IL-4-stimulated Ramos cells were included as a control for Stat6 activation.

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These experiments had revealed that an additional tyrosine phosphorylated ∼110-kDa protein was coimmunoprecipitated with activated Stat6 in IFN-α-treated extracts (Fig. 4, top panel). Because Stat2 is known to become phosphorylated in response to IFN-α and to migrate on SDS-PAGE gel to a ∼110-kDa position (35, 36), we entertained the notion that Stat2 might complex with Stat6 in B cells. Reprobing of this blot with a Stat2 antiserum indeed confirmed that Stat2 can be detected in Stat6 immunoprecipitates from IFN-α-treated B cells (Fig. 4, bottom panel). Low levels of Stat2 also coimmunoprecipitated with Stat6 in IL-4-treated extracts (Fig. 4, bottom panel), although activation of Stat2 was not detected in response to IL-4 (Fig. 4, top panel).

The ability of IFN-α to activate a variety of STATs in B cells (Fig. 3,A) raised the possibility that Stat2 might interact with other STATs in addition to Stat6 (Fig. 4) or, as previously described, to Stat1 (10). We thus subjected Ramos extracts cultured with or without IFN-α to immunoprecipitations with a panel of antisera against distinct STATs. As shown in Fig. 5,A, tyrosine phosphorylation of all of the STATs assayed was observed in response to IFN-α. Interestingly, in this experiment, we observed that culturing Ramos cells with IFN-α led to the activation of Stat5, which, possibly because of the strong induction of the Stat1:Stat3-containing complexes, had not been detected by EMSA. Multiple tyrosine-phosphorylated proteins were detected only in the Stat2 and Stat6 immunoprecipitates (Fig. 5,A, top panel). Stripping and reprobing of this blot with antisera against Stat2 revealed that Stat2 coimmunoprecipitated only with Stat1 or Stat6 but not with Stat3 or Stat5 (Fig. 5,A, middle panel). As previously described, complexing of Stat2 with Stat1 occurred in the absence of ligand (37), while formation of the Stat2:Stat6 heterodimer was only detected on exposure of cells to IFN-α. Reprobing with Stat6 antiserum demonstrated complexing of Stat6 only with Stat2 but not with other STATs (Fig. 5 A, bottompanel). Reprobing of this Western filter with a Stat1 antiserum confirmed the presence of Stat1 in the Stat2 but not in the Stat6 immunoprecipitation (data not shown). Moreover, reprobing with either Stat3 or Stat5 antisera ensured the adequacy of these immunoprecipitations and established the lack of interaction of these proteins with either Stat2 or Stat6 (data not shown).

FIGURE 5.

A, Stat2 complexes with Stat6 in an IFN-α-stimulated B cell line. Ramos cells were either unstimulated or were stimulated with IFN-α2a (1000 U/ml). Extracts were then immunoprecipitated (IP) with a panel of anti-STAT antiserum, resolved by 7% SDS-PAGE, and analyzed by Western blotting (WB) using an antiphosphotyrosine Ab (toppanel). The blot was later stripped and reprobed with a Stat2 (middlepanel) or a Stat6 (bottompanel) antiserum. B, IFN-α activates a Stat2:Stat6:p48 complex in B cell but not in non-B cell lines. Extracts were prepared from a human B cell line, Ramos, or a non-B cell line, WI38 VA, before or after stimulation with IFN-α2a (1000 U/ml) for 20 min. The WCE were examined by EMSA using an ISG15 ISRE probe. Ab interference mobility shift assays were conducted by addition of antisera against Stat2, Stat6, p48, or preimmune serum (control) as described in Fig. 3 A. C, Stat6 preassociates with the IFNαRI chain. Ramos cells either were unstimulated or were stimulated with IFN-α2a (1000 U/ml) for 20 min. Extracts were then immunoprecipitated (IP) with either an IFNαRI or a Stat6 antiserum, resolved by 7% SDS-PAGE, and analyzed by Western blotting (WB) using an an tiphosphotyrosine Ab (toppanel). The blot was later stripped and reprobed with an IFNαRI antiserum (middlepanel). After restripping, the blot was reprobed with a Stat6 antiserum (bottompanel).

FIGURE 5.

A, Stat2 complexes with Stat6 in an IFN-α-stimulated B cell line. Ramos cells were either unstimulated or were stimulated with IFN-α2a (1000 U/ml). Extracts were then immunoprecipitated (IP) with a panel of anti-STAT antiserum, resolved by 7% SDS-PAGE, and analyzed by Western blotting (WB) using an antiphosphotyrosine Ab (toppanel). The blot was later stripped and reprobed with a Stat2 (middlepanel) or a Stat6 (bottompanel) antiserum. B, IFN-α activates a Stat2:Stat6:p48 complex in B cell but not in non-B cell lines. Extracts were prepared from a human B cell line, Ramos, or a non-B cell line, WI38 VA, before or after stimulation with IFN-α2a (1000 U/ml) for 20 min. The WCE were examined by EMSA using an ISG15 ISRE probe. Ab interference mobility shift assays were conducted by addition of antisera against Stat2, Stat6, p48, or preimmune serum (control) as described in Fig. 3 A. C, Stat6 preassociates with the IFNαRI chain. Ramos cells either were unstimulated or were stimulated with IFN-α2a (1000 U/ml) for 20 min. Extracts were then immunoprecipitated (IP) with either an IFNαRI or a Stat6 antiserum, resolved by 7% SDS-PAGE, and analyzed by Western blotting (WB) using an an tiphosphotyrosine Ab (toppanel). The blot was later stripped and reprobed with an IFNαRI antiserum (middlepanel). After restripping, the blot was reprobed with a Stat6 antiserum (bottompanel).

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We next proceeded to determine whether the interaction of Stat2 and Stat6 in IFN-α-stimulated B cells could lead to the formation of a multimeric complex with p48. To address this possibility, we conducted EMSA experiments with the ISG15 ISRE probe (Fig. 5,B). These assays revealed that stimulation of Ramos cells with IFN-α leads to the induction of two distinct complexes. The faster-mobility IFN-α-inducible complex was activated by IFN-α in both Ramos and WI38 VA and corresponds to the previously described ISGF3 complex. In contrast, the slower-mobility IFN-α-inducible complex was activated only in Ramos and was found, by supershifting experiments, to contain Stat2, Stat6, and p48 (Fig. 5 B). Thus, stimulation of B cells with IFN-α2a leads to the interaction of Stat2 and Stat6 and to the formation of ISGF3-like complexes containing Stat2, Stat6, and p48.

To assess whether, in B cells, Stat6 can associate with the IFN-α receptor, we then conducted immunoprecipitation experiments with an antiserum directed against the IFNαRI chain (38). These studies revealed that Stat6 interacts with the IFNαRI chain in the absence of IFN-α stimulation (Fig. 5,C, bottompanel). Exposure of Ramos cells to IFN-α led to a decrease in the association of Stat6 with IFNαRI as well as to the concomitant tyrosine phosphorylation of the IFNαRI chain (Fig. 5 C, toppanel). Interestingly, preassociation of Stat2 with the IFNαRII chain has been previously reported and is believed to be a critical step in Stat2 activation (39).

Our survey of different B cell lines displayed heterogeneity in the intensity of the IFN-α-inducible Stat6 complex. In particular, we noticed that EBV-transformed B cell lines (i.e., JY or WIL-2), despite a robust activation of Stat6 in response to IL-4, displayed minimal Stat6 induction on exposure to IFN-α (Figs. 1,A and 6A and data not shown). EBV-transformed B cell lines have been shown to be resistant to the antiproliferative effects of IFN-α on B cells (20). We thus became interested in determining whether IFN-α resistance in B cells was accompanied by a decrease in Stat6 activation. To investigate this issue, we utilized two distinct subclones of the B cell line Daudi. This cell line displays exquisite sensitivity toward growth inhibition by IFN-α. However, subclones that are resistant to these effects have been derived (29, 40, 41, 42). We thus utilized an IRF-1 GAS probe to assess the IFN-α inducibility of STAT complexes in IFN-sensitive (D-S) and -resistant (D-R) cell lines. As shown in Fig. 6 A, the most striking difference between the two Daudi subclones consisted of a markedly reduced activation of Stat6 complexes in the IFN-resistant cells. Interestingly, both the D-S and D-R subclones were unable to activate Stat6 in response to IL-4 (data not shown).

FIGURE 6.

A, IFN-α activation of Stat6 is markedly diminished in IFN-resistant human B cell lines. Cells from a Daudi subclone sensitive to IFN-α (D-S), a Daudi subclone resistant to IFN-α (D-R), and an EBV-transformed B cell line (JY) were stimulated with human IFN-α2a (1000 U/ml) for 1 h. Extracts were then prepared and examined utilizing an IRF-1 GAS probe as described in Fig. 1. B, IFN-α activation of Stat2:Stat6:p48 complexes is absent in IFN-resistant human B cell lines. Cells from D-S subclone, D-R subclone, JY cell line, and a non-B cell line, WI38 VA, were stimulated with human IFN-α2a (1000 U/ml) for 20 min. Extracts were then prepared and examined utilizing an ISG15 ISRE probe as described in Fig. 5 B. C, Stat6 is not tyrosine phosphorylated in response to IFN-α in a D-R subclone. Cells from Ramos, D-S, and D-R either were unstimulated or were stimulated with IFN-α2a (1000 U/ml) for 20 min. Extracts were then immunoprecipitated (IP) with a Stat6 antiserum, resolved by 7% SDS-PAGE, and analyzed by Western blotting (WB) using an antiphosphotyrosine Ab (upperpanel). The blot was later stripped and reprobed with a Stat6 antiserum (lowerpanel). D, Induction of IRF-1 in response to IFN-α stimulation is diminished in DR cells. Cells either were unstimulated or were stimulated with human IFN-α2a (1000 U/ml) for 1 h. Total RNA was then extracted, and 10 μg of RNA was assayed by Northern blotting as per standard protocols. The blot was probed with 32P-labeled IRF-1 cDNA (upperpanel), stripped, and then reprobed with 32P-labeled GAPDH cDNA (lowerpanel).

FIGURE 6.

A, IFN-α activation of Stat6 is markedly diminished in IFN-resistant human B cell lines. Cells from a Daudi subclone sensitive to IFN-α (D-S), a Daudi subclone resistant to IFN-α (D-R), and an EBV-transformed B cell line (JY) were stimulated with human IFN-α2a (1000 U/ml) for 1 h. Extracts were then prepared and examined utilizing an IRF-1 GAS probe as described in Fig. 1. B, IFN-α activation of Stat2:Stat6:p48 complexes is absent in IFN-resistant human B cell lines. Cells from D-S subclone, D-R subclone, JY cell line, and a non-B cell line, WI38 VA, were stimulated with human IFN-α2a (1000 U/ml) for 20 min. Extracts were then prepared and examined utilizing an ISG15 ISRE probe as described in Fig. 5 B. C, Stat6 is not tyrosine phosphorylated in response to IFN-α in a D-R subclone. Cells from Ramos, D-S, and D-R either were unstimulated or were stimulated with IFN-α2a (1000 U/ml) for 20 min. Extracts were then immunoprecipitated (IP) with a Stat6 antiserum, resolved by 7% SDS-PAGE, and analyzed by Western blotting (WB) using an antiphosphotyrosine Ab (upperpanel). The blot was later stripped and reprobed with a Stat6 antiserum (lowerpanel). D, Induction of IRF-1 in response to IFN-α stimulation is diminished in DR cells. Cells either were unstimulated or were stimulated with human IFN-α2a (1000 U/ml) for 1 h. Total RNA was then extracted, and 10 μg of RNA was assayed by Northern blotting as per standard protocols. The blot was probed with 32P-labeled IRF-1 cDNA (upperpanel), stripped, and then reprobed with 32P-labeled GAPDH cDNA (lowerpanel).

Close modal

Because the EMSA experiments performed with the ISG-15 ISRE probe had previously identified a Stat2:Stat6:p48 complex (Fig. 5,B), we then proceeded to investigate whether assembly of such a complex was also impaired in IFN-resistant cells. As shown in Fig. 6,B, formation of a Stat2:Stat6:p48 complex on IFN-α stimulation could not be detected in either D-R subclones or EBV-transformed B cells. Consistent with previous reports, ISGF3 complex formation was defective in D-R subclones but occurred normally in EBV-transformed B cells (21, 24). To confirm that the lack of Stat6 DNA binding activity in IFN-resistant cells was due to a lack of appropriate activation of Stat6 in response to IFN-α, we then subjected extracts from D-S as well as D-R subclones to immunoprecipitation with a Stat6 antiserum. As shown in Fig. 6 C, stimulation with IFN-α led to tyrosine phosphorylation of Stat6 only in D-S but not in D-R cells.

Because the IRF-1 GAS has previously been shown to be the critical element regulating IFN-α inducibility of the IRF-1 gene (43), we then tested whether the diminished ability of D-R subclones to activate Stat6 binding to the IRF-1 GAS is reflected in a decreased induction of the IRF-1 gene in response to IFN-α. We therefore assayed by Northern analysis total RNA obtained from D-S and D-R cells that had either been unstimulated or stimulated with IFN-α (Fig. 6 D). Probing the Northern blot with a radiolabeled IRF-1 cDNA probe demonstrated that, when compared with the D-S cells, the D-R subclone displayed a 30% reduction (by densitometry) in the IFN-α-mediated induction of IRF-1. Thus, in B cells, a decrease in the Stat6 activation in response to IFN-α correlates with diminished IFN-α inducibility of the IRF-1 gene and with the acquisition of IFN resistance.

It has long being recognized that IFN-α exerts pleiotropic actions on a variety of target cells (1, 2, 3). The molecular mechanisms leading to such diverse effects, however, have not been fully elucidated. We have now demonstrated that, in B cells, IFN-α leads to the activation of a Stat6-containing complex that is indistinguishable from that activated by IL-4 (25, 26, 27, 28). The ability of IFN-α to activate Stat6 is intriguing, because, unlike other STATs, Stat6 has been primarily linked to the signaling pathways triggered by cytokines that exhibit overlapping biologic activities, i.e., IL-4 and IL-13 (9). In contrast to the IL-4-mediated Stat6 activation, however, appearance of the Stat6 complex in response to IFN-α is short lived (Fig. 1 B and data not shown). This restricted pattern of Stat6 activation may thus allow IFN-α to exert B cell-specific effects without fully mimicking the IL-4-mediated biologic responses. The ability of IFN-α to activate Stat6 and possibly to target some IL-4-responsive genes may also play a role in the inappropriate induction of CD23b exhibited by B cells of CLL patients in response to IFN-α (44).

IFN-α-mediated Stat6 activation is accompanied by the formation of novel Stat2:Stat6 complexes. In contrast to Stat1:Stat2 heterodimers that, consistent with previous studies, were detected as preexisting latent complexes (37), association of Stat2 and Stat6 appears to be dependent on the presence of ligand. Assembly of the Stat2:Stat6 dimeric complex may thus require a distinct set of interactions from those leading to Stat1:Stat2 complex formation. Also, in contrast to the Stat1:Stat2 heterodimer, the Stat2:Stat6 heterodimer does not appear to target the IRF-1 GAS, because Ab interference assays with a Stat2 antiserum did not affect the Stat6 complex activated by IFN-α. It remains to be determined whether the Stat2:Stat6 heterodimer can target a distinct set of GAS-like elements.

Our studies have also revealed that interaction of Stat6 with Stat2 can lead to the formation of an ISGF3-like complex composed of Stat2, Stat6, and p48. Because previous studies utilizing a yeast two-hybrid system failed to detect a direct interaction between p48 and Stat6 (45), presence of Stat2 and/or specific posttranslational modifications may be required for the formation of this ternary complex. Consistent with studies from other groups, we have found that IFN-α induces ISGF3 normally in EBV-transformed B cells (20) but not in D-R cells (24). However, both cell types are unable to activate the Stat2:Stat6:p48 complex on stimulation with IFN-α. Because transactivation of the ISG15 gene in response to IFN-α has previously been shown to be defective in both cell types (20, 24), these findings suggest that, in B cells, activation of the Stat2:Stat6:p48 complex may be required for the appropriate IFN-α induction of this gene.

IL-4-mediated Stat6 phosphorylation has been linked to activation of Jak1 and Jak3 (46, 47) or of Jak1 alone (48, 49, 50, 51, 52, 53). Preliminary experiments have failed to detect IFN-α-inducible activation of Jak3 in B cells (data not shown). Thus, tyrosine phosphorylation of Stat6 in response to IFN-α does not appear to be mediated by a different set of Janus kinases, and IFN-α activation of Jak1 is likely to be sufficient for Stat6 phosphorylation. The mechanism underlying the selective ability of IFN-α to activate Stat6 in B cell lines is at present unclear. However, we favor the hypothesis that Stat6 activation may be due either to different posttranslational modifications of the IFNαRI receptor chain in B cells or to the recruitment of additional, possibly B cell-specific, components to the IFN-α receptor complex.

The phenomenon of IFN resistance has long been recognized as carrying profound implications for the diverse clinical uses of IFN-α. Recent studies have suggested that multiple mechanisms may be involved in the acquisition of IFN resistance. For example, in T cell lines, changes in the ability of the IFN-α receptor to complex with CD45 (54) as well as Stat1-dependent mechanisms have been described (55). IFN resistance in Daudi cells instead has been associated with either lack of Stat3 phosphorylation (12) or premature loss of Stat1 phosphorylation (23). Our finding that Stat6 activation by IFN-α was markedly diminished in both D-R as well as EBNA2-containing B cell lines suggests that changes in Stat6 phosphorylation are also associated with the acquisition of IFN resistance. The decreased/absent Stat6 binding to the IRF-1 GAS in IFN-resistant cells was associated with a diminished induction of IRF-1, a gene involved in the regulation of cellular proliferation. The residual IRF-1 induction detected in d-R cells is likely to be mediated by the other STAT complexes, which bind to the IRF-1 GAS (Fig. 3,A), because activation of these complexes can still occur normally in D-R cells (Fig. 6 A). However, IRF-1 function is antagonized by the closely related factor IRF-2, the levels of which are normally higher than those of IRF-1 and the growth arrest of which has been shown to be related to changes in the ratio of these two factors (56). Therefore, the submaximal IRF-1 induction displayed by D-R cells may be unable to overcome the antagonistic effects of IRF-2, thus providing a link between defects in Stat6 activation and protection against the antiproliferative actions of IFN-α.

The multiple differences in STAT activation displayed by IFN-resistant cell lines are likely not to be mutually exclusive and may stem from a common mechanism. Differential IFN-γ responsiveness in T cell subsets has previously been ascribed to changes in receptor components (57, 58). It will thus be interesting to determine whether modulation of specific receptor components, e.g., by the employment of alternatively spliced forms of the receptor chains, may allow IFN-α-resistant cell lines to selectively extinguish the antiproliferative effects of IFN-α.

We thank J. Siu for his critical reading of this manuscript.

1

This work was supported by a Grant-in-Aid from the American Heart Association, the Silverberg Award, and the Saydman Trust Fund for Research in Septicemia in honor of Dr. Harold C. Neu at Columbia University.

3

Abbreviations used in this paper: GAS, γ-activated site; IRF, IFN regulatory factor; ISRE, IFN-stimulated response element; ISGF3, IFN-stimulated gene factor 3; EBNA2, EBV nuclear Ag 2; WCE, whole-cell extract.

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