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Tumor Necrosis Factor-α (TNF-α) and Interleukin-6 (IL-6) in B-Lymphocyte Function
METHODS: A Companion to Methods in Enzymology 11, 128–132 (1997) Article No. ME960396
Tumor Necrosis Factor-a (TNF-a) and Interleukin-6 (IL-6) in B-Lymphocyte Function P. Rieckmann,* J. M. Tuscano,† and J. H. Kehrl† *Department of Neurology, Julius-Maximilians Universita¨t, Josef Schneider Strasse II, D97080 Wu¨rzburg, Germany; and †Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892
Two cytokines important in the regulation of B-cell function are tumor necrosis factor-a (TNF-a) and interleukin-6 (IL-6). They act at different steps in B-cell differentiation and can be produced by the B cells themselves upon appropriate stimulation. Crosslinking of surface Ig and signaling through CD22 or CD40 lead to increased secretion of both cytokines. Neutralization of TNF-a or IL-6 biologic activity in B-cell cultures results in a significant reduction in B-cell proliferation and Ig secretion. Increased production of these cytokines is found in several diseases associated with aberrant B-cell function. This review will focus on the role of TNF-a and IL-6 in normal and pathophysiological conditions of B-cell function. q 1997 Academic Press Inc.
B-lymphocyte activation is initiated by the interaction of an antigen with the antigen receptor and facilitated by several B-lymphocyte cell surface molecules including CD19 and CD22. Progression of the activated cells through the cell cycle and their subsequent differentiation to immunoglobulin (Ig) secreting cells or memory B cells largely depend upon CD40/CD40L interactions and cytokines. Cognate interactions between antigen-specific T cells and antigen-activated B cells provide several of these signals. T-cell gp39 engages CD40 on B cells, an interaction required for the generation of normal thymus-dependent immune responses (1). Crosslinking of CD40 on the B-cell membrane results in rapid translocation of NF-kB to the nucleus and increased transcription of several cytokines including IL-6 and TNF-a (2, 3). Crosslinking of CD22 on the B-cell membrane amplifies signals provided by the antigen receptor complex (4–6, and J. Tuscano et al., manuscript submitted for publication) and also results in increased B-cell IL-6 and TNF-a secretion (see below). In vitro studies have suggested several roles for TNFa in the regulation of B-cell proliferation and differentiation. Most studies have shown that TNF-a augments
rather than impairs B-cell function. For example, TNFa increases the specific proliferative responses of tonsillar-derived B cells to Ig-crosslinking signals such as Staphylococcus aureus Cowan 1 (SAC) and increases immunoglobulin production by SAC-activated B cells, and spontaneous immunoglobulin secretion by human tonsillar B cells depends upon endogenous TN-a production (7–9). TNF-a augments the effects of other cytokines on B-cell function (7). Furthermore, a 26-kDa transmembrane form of TNF present on activated CD4positive T cells provides costimulatory signals for B-cell activation and immunoglobulin production (10). This mechanism may be important in the polyclonal B-cell expansion observed in HIV-infected individuals (11) and has also been demonstrated with herpesvirus Saimiri transformed human CD4/ T-cell clones (12). TNF-a and the related cytokine, lymphotoxin, both act as autocrine growth factors for Epstein–Barr virus (EBV)-transformed B cells and Burkitt lymphoma cell lines (13). In addition, TNF-a induces NF-kB translocation from the cytoplasm to the nucleus, resulting in the enhanced transcription of a variety of genes important in B-cell function (14). Since TNF-a is a potent inducer of other cytokines, some of its effects on B-cell function may be mediated by these induced cytokines rather than by TNF-a itself. Overall, it appears that TNF-a enhances B-cell function, suggesting that in the setting of an inflammatory focus elevated TNF-a production likely augments the local humoral immune response. IL-6 is known as a late-acting B-cell differentiation factor for mitogen-activated human B cells. It increases immunoglobulin secretion by SAC-activated B cells, even when added as late as Day 4 of the culture period after maximal proliferation has occurred (15, 16). The addition of IL-6 neutralizing antibodies to cultures suggests that immunoglobulin production by pokeweed mitogen-activated B cells depends upon IL-6 (17). In contrast, antigen-specific antibody responses (i.e., against influenza virus) by human B cells apparently do not require IL-6 (18). In addition to its role as a differentia-
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tion factor, IL-6 stimulates proliferation of B-cell hybridomas and myelomas (19). However, there is little evidence to suggest that IL-6 is required for normal Bcell proliferation. In addition to the in vitro studies that suggest an important role for IL-6 in B-cell Ig secretion, overproduction of IL-6 in mice using an IL-6 transgene results in dramatically elevated serum levels of polyclonal IgG1 (20). Various effects of both cytokines on chronic B-cell malignancies have been described (21, 22), which is beyond the scope of this review.
INDUCTION AND REGULATION OF IL-6 AND TNF-a PRODUCTION IN B CELLS Activation of several different signal transduction pathways has been described to lead to increased IL-6 or TNF-a production in B cells. Polyclonal activation of human tonsillar B cells with SAC results in sequential induction of TNF-a and IL-6 mRNA as early as 8 to 12 (TNF-a) and 12 to 36 h (IL-6), respectively. A reverse transcriptase–polymerase chain reaction (RT-PCR) study on highly purified peripheral blood B lymphocytes found an early peak of TNF-a and IL-6 mRNA (Days 1–3), whereas IL-10 and transforming growth factor-b1 (TGF-b1) remained relatively constant throughout a 3-week culture period (23). After activation with anti-Ig, splenic B cells rapidly expressed IL6 mRNA with peak expression occurring already after 4 h and declining rapidly thereafter (24). Protein secretion peaked at 20 (TNF-a) and 100 h (IL-6) (8). The early peak of TNF-a production was associated with a proliferative response, whereas IL-6 production was detected at the time when Ig production started. Endogenous IL-6 production is partly dependent on the availability of TNF-a, as TNF-a neutralizing antibodies significantly reduced IL-6 and Ig production in SAC/ IL-2-activated B cells (8). Another study demonstrated the importance of endogenous IL-6 for Ig production in human B cells (25). Interestingly, the inhibitory effects of anti-cytokine antibodies on B-cell differentiation were strictly time dependent. The anti-TNF-a antibody was only effective if present from the beginning of cell culture, whereas the addition of the anti-IL-6 antibody could be delayed until Day 3 without losing blocking activity (8). Established human B-cell lines differentially produce IL-6. Although some produce high levels of this cytokine constitutively, other cell lines need to be induced with mitogen. Normal human peripheral blood B cells can be triggered to secrete large amounts of IL-6 by the combination of IL-4 and PMA (26). Constitutive expression of IL-6 in EBV-transformed B cells leads to an altered growth pattern and malignant phenotype (27). Production of TNF-a in human B cells was first
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described by Sung et al., who investigated different Bcell lines and polyclonally activated tonsillar B cells (28). In addition to polyclonal activation using SAC/IL2, direct anti-Ig treatment rapidly induced transcription of TNF-a in human B cells (29). Another way to trigger cytokine production is via stimulation of B cells through the CD40 receptor–gp39 interactions. Both pathways can be blocked by the immunosuppressants cyclosporin A and FK506 (29, 30). CD40 is a 50-kDa glycoprotein mainly expressed on B cells and belongs to the TNF receptor family. Crosslinking of CD40 results in cytokine production (31), results in enhanced adhesion and proliferation of B cells, and is accompanied by an increased tyrosine phosphorylation of various substrates (32). In addition, in the presence of IL-2, IL-4, or IL-10, CD40 ligand is a potent inducer of IgM, IgE, IgG1, and IgA secretion. This process is inhibited by TGF-b and, to a lesser extent, by IFN-g. Recently, it was demonstrated that activation of the CD40 pathway rapidly induced NF-kB binding activity in the nucleus. Transient transfection studies revealed a marked effect on NF-kB-dependent gene expression (33). Binding of NF-kB to the promoter regions of TNF-a and IL-6 is important for their efficient transcription (34), and functional NF-kB activity is essential for B-cell proliferation (35). CD22 is a B lineage cell membrane protein that functions as an adhesion molecule (4–6). While the nature of the ligand(s) for CD22 remains controversial, several monoclonal antibodies have been developed that block the interaction of CD22 with its ligands (36). Crosslinking of CD22 with such a CD22 mAb induced significant levels of IL-6 (Table 1) and small amounts of TNF-a (data not shown). In contrast, a CD22 mAb that does not block the interaction of CD22 with its ligands failed to induce significant levels of IL-6 production. Consistent with this difference in their abilities to induce IL6 production, the nonblocking antibody induces far less TABLE 1 IL-6 Production after CD22 Crosslinking with Blocking or Nonblocking mAbs Experiment 1 treatment
IL-6 (pg/ml)
Experiment 2 treatment
IL-6 (pg/ml)
Media HB22.5 CD22.5 Anti-Ig HB22.5 / Anti-Ig CD22.5 / Anti-Ig
210 580 180 440 600 450
Media HB22.5 CD22.5 IL-2 HB22.5 / IL-2 CD22.5 / IL-2
220 570 190 220 570 220
Note. Tonsillar B lymphocytes were treated as indicated. HB22.5 is a CD22 blocking mAB while CD22.5 is a nonblocking mAB. Culture supernatants were collected after 24 h and assayed for IL-6 production via ELISA. Similar results were found at 48 h.
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tyrosine phosphorylation of CD22 than does the blocking antibody (J. Tuscano et al., manuscript submitted for publication). Besides triggering IL-6 production, the blocking mAb induces B-cell proliferation and costimulates B-cell proliferation in conjunction with anti-Ig, CD40, and IL-2. In addition, in the presence of IL-2 the CD22 mAb induces B-cell Ig production (J. Tuscano et al., manuscript submitted for publication). Various second messenger pathways that can induce TNF-a in B cells have been described. Direct stimulation of protein kinase C (PKC) with PMA results in a rapid induction in TNF-a mRNA transcription. Similar effects were obtained with okadaic acid, a specific inhibitor of the serine/threonine phosphatases 1 and 2A (PP1 and PP2A) (37). When added to intact cells this substance allows the unopposed activity of constitutively active protein kinases, which leads to enhanced phosphorylation of many protein kinase substrates. Significant increases of nuclear factor binding activity (e.g., AP-1 and NF-kB) were detected in normal human tonsillar B cells after the addition of okadaic acid. A strong induction of TNF-a mRNA in the same cells was also observed. These immediate early effects were independent of new protein synthesis (37). Another calcium- and calmodulin-dependent phosphatase, calcineurin, which plays an important role in T-lymphocyte activation (38), mediates protein synthesisindependent TNF-a transcription in human B cells stimulated through their surface immunoglobulin receptors (39). This effect is mediated by modification of the transcription factor NFATp, a 120-kDa phosphoprotein that translocates to the nucleus and binds as a complex to AP-1 binding sites present in various promoters (40). Recently, platelet activating factor (PAF) was shown to directly increase TNF-a mRNA levels in the B-cell line Ramos and costimulate PMA-induced TNF-a production in peripheral blood B cells. Interestingly, PAF alone did not increase protein levels, possibly due to a posttranscriptional block. It can therefore be regarded as a modulator of cytokine production in B cells (41). The immunosuppressant cyclosporin A (CsA) blocks PAF and anti-Ig induced TNF-a mRNA and protein synthesis, but had no effect on PMA-induced cytokine production and CD40-mediated proliferation in freshly isolated and Ramos B cells (30, 41). From these data, it was hypothesized that different B-cell populations originate from a single precursor population depending on the activation pathway. Treatment of small resting B cells with anti-IgM induced proliferation and enhanced surface CD5 expression. In contrast, B cells induced to proliferate in response to CD40 ligand displayed elevated levels of CD23 but no surface CD5 (42). Functional differences in terms of cytokine production were not detected, as both CD5/ and CD50 negative human tonsillar B cells were shown to produce equal
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amounts of TNF-a and IL-6 (43). Similar results were obtained with murine B-cell lines (44). Recently, it was demonstrated that CD27, a 50-kDa transmembrane protein expressed late during B-cell differentiation, can be used to distinguish populations in terms of Ig production, but no such differences were observed for cytokine production. Only CD27/ B cells produced IgM and IgG upon appropriate stimulation, whereas the two subsets secreted similar amounts of IL-6 and TNF-a (45). From the available data it can be concluded that IL-6 and TNF-a production is inducible in various B-cell populations, but apparently is not restricted to a distinct B-cell phenotype.
PATHOGENETIC RELEVANCE OF CYTOKINE PRODUCTION IN B CELLS B-cell hyperactivity and polyclonal activation with an increased risk of autoantibody formation is a hallmark of certain immunological disorders such as systemic lupus erythematosus (SLE). It also occurs during the course of HIV infection. The role of stimulatory cytokines in the excessive B-cell activation observed in these diseases has been investigated, and several cytokines have been implicated as important mediators including IL-10, IL-6, and TNF-a (46–49). Increased levels of circulating IL-6 and TNF-a were detected early during the course of HIV-1 infection and their role in the regulation of HIV expression is clearly established (50). Although polyclonal B-cell activation and hypergammaglobulinemia as well as an increased number of activated, spontaneously Ig-secreting cells are present in the circulation of HIV-infected individuals (51), certain B-cell functions, such as polyclonal activation and antibody production after antigenic challenge, are impaired in AIDS patients. Peripheral blood B lymphocytes from HIV-infected individuals with hypergammaglobulinemia spontaneously secrete large amounts of IL-6 and TNF-a compared to healthy seronegative donors. Interestingly, lymph node B cells spontaneously secrete higher levels of both cytokines than do blood B cells from the same patients (43). In addition, these cells are capable of inducing HIV-1 expression in cell lines chronically infected with HIV. This effect is mediated by both TNF-a and IL-6, as neutralizing antibodies against IL-6 and TNF-a reduced the stimulatory activity in B-cell supernatants. Similar results were obtained with an autologous coculture system of B and T cells isolated from lymph nodes of three patients with AIDS (47). Interestingly, during the latent phase of infection the germinal centers of lymph nodes harbor large amounts of virus in the follicular dendritic network in close contact with activated B cells (52). It is conceivable that
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cytokine production by activated B cells in close contact with passaging T cells within the germinal centers of the lymph node contributes to the ongoing production of HIV during the latent phase of the disease. We and others have demonstrated that the major envelope protein of HIV gp120 can induce proliferation and cytokine production in B cells (53–55). Interestingly, stimulation of normal B cells with HIV envelope protein is Tcell dependent (54–56), whereas highly purified B cells from HIV-infected individuals are directly inducible to produce IL-6 and TNF-a without any T-cell help (49, 53). A CD4-independent signaling pathway is apparently responsible for cytokine and Ig production in these patients. Recently, it was demonstrated that a subset of B cells, defined by the utilization of a specific Ig heavy-chain family (VH3), can interact directly with HIV envelope protein and, thereby, could be directly activated by HIV (57). In summary, these studies suggest that germinal centers of lymph nodes provide a unique environment for virus expression and accumulation in which gp120 stimulates B cells to secrete HIV inductive cytokines, such as TNF-a and IL-6, thereby enhancing virus expression in infected cells in a paracrine manner. Whether increased IL-6 production plays a role in the induction of B-cell malignancies that frequently occur in HIV-infected individuals remains unresolved. It has been reported that elevated IL-6 levels may precede the development of B-cell lymphoma in HIV-infected individuals under zidovudine treatment (58). The increase in IL-6 in these individuals is unlikely due to the zidovudine treatment since it does not induce B-cell activation (IL-6 production or Ig secretion) either in vivo or in vitro (59). Spontaneous production of polyclonal and antigenspecific antibodies plays an important role in the pathogenesis of SLE. Several studies revealed that IL-6 and TNF-a support the in vivo differentiation process of B cells in patients with SLE (48, 60–62). High levels of serum IL-6 have been reported in patients with SLE, and IL-6 mRNA was detected in freshly isolated peripheral blood mononuclear cells (48). An autocrine loop for IL-6 in B cells has been described in patients with SLE. High-density B cells from these patients produce large amounts of IL-6, which binds to the IL-6 receptors present on low-density B cells. This interaction, as well as IgG and autoantibody production, could be inhibited by an antibody against IL-6 (63). The pathophysiological role of IL-6 was recently described in an animal model of SLE (62). As IL-6 can be efficiently induced in B cells via autocrine TNF-a, it is of interest whether increased TNF-a levels are also present in patients with SLE. Interestingly, anti-TNF-a antibodies decreased the production of IL-6 and IgG in blood B cells from patients with SLE (48). A genetic association was determined between SLE and TNF-a. This polymor-
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phism revealed a strong correlation with the HLA-DR3 and HLA-B8 allele and may play a role in SLE susceptibility (60). Another nonmalignant B-cell-associated disease with increased cytokine production is the rare Castleman’s syndrome. This disease is characterized by giant lymph node hyperplasia with plasma cell infiltration, fever, anemia, hypergammaglobulinemia, and increased levels of acute phase proteins. Patients with Castelman’s syndrome were found to have increased serum levels of IL6, and it was found that germinal center B cells produce large amounts of this cytokine (64). Overexpression of IL6 in transgenic mice produces a syndrome resembling Castleman’s disease (20, 65). Immunoglobulin production has long been regarded as the preeminent function of B lymphocytes. This review summarizes the evidence that the cytokines IL-6 and TNF-a participate in the regulation of B-cell physiology and the evidence that B cells themselves are an important source and target for these two cytokines. Cytokine production by B cells may serve as an immunoregulatory component in certain diseases associated with dysregulated cytokine production.
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Tumor Necrosis Factor-a (TNF-a) and Interleukin-6 (IL-6) in B-Lymphocyte Function P. Rieckmann,* J. M. Tuscano,† and J. H. Kehrl† *Department of Neurology, Julius-Maximilians Universita¨t, Josef Schneider Strasse II, D97080 Wu¨rzburg, Germany; and †Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892
Two cytokines important in the regulation of B-cell function are tumor necrosis factor-a (TNF-a) and interleukin-6 (IL-6). They act at different steps in B-cell differentiation and can be produced by the B cells themselves upon appropriate stimulation. Crosslinking of surface Ig and signaling through CD22 or CD40 lead to increased secretion of both cytokines. Neutralization of TNF-a or IL-6 biologic activity in B-cell cultures results in a significant reduction in B-cell proliferation and Ig secretion. Increased production of these cytokines is found in several diseases associated with aberrant B-cell function. This review will focus on the role of TNF-a and IL-6 in normal and pathophysiological conditions of B-cell function. q 1997 Academic Press Inc.
B-lymphocyte activation is initiated by the interaction of an antigen with the antigen receptor and facilitated by several B-lymphocyte cell surface molecules including CD19 and CD22. Progression of the activated cells through the cell cycle and their subsequent differentiation to immunoglobulin (Ig) secreting cells or memory B cells largely depend upon CD40/CD40L interactions and cytokines. Cognate interactions between antigen-specific T cells and antigen-activated B cells provide several of these signals. T-cell gp39 engages CD40 on B cells, an interaction required for the generation of normal thymus-dependent immune responses (1). Crosslinking of CD40 on the B-cell membrane results in rapid translocation of NF-kB to the nucleus and increased transcription of several cytokines including IL-6 and TNF-a (2, 3). Crosslinking of CD22 on the B-cell membrane amplifies signals provided by the antigen receptor complex (4–6, and J. Tuscano et al., manuscript submitted for publication) and also results in increased B-cell IL-6 and TNF-a secretion (see below). In vitro studies have suggested several roles for TNFa in the regulation of B-cell proliferation and differentiation. Most studies have shown that TNF-a augments
rather than impairs B-cell function. For example, TNFa increases the specific proliferative responses of tonsillar-derived B cells to Ig-crosslinking signals such as Staphylococcus aureus Cowan 1 (SAC) and increases immunoglobulin production by SAC-activated B cells, and spontaneous immunoglobulin secretion by human tonsillar B cells depends upon endogenous TN-a production (7–9). TNF-a augments the effects of other cytokines on B-cell function (7). Furthermore, a 26-kDa transmembrane form of TNF present on activated CD4positive T cells provides costimulatory signals for B-cell activation and immunoglobulin production (10). This mechanism may be important in the polyclonal B-cell expansion observed in HIV-infected individuals (11) and has also been demonstrated with herpesvirus Saimiri transformed human CD4/ T-cell clones (12). TNF-a and the related cytokine, lymphotoxin, both act as autocrine growth factors for Epstein–Barr virus (EBV)-transformed B cells and Burkitt lymphoma cell lines (13). In addition, TNF-a induces NF-kB translocation from the cytoplasm to the nucleus, resulting in the enhanced transcription of a variety of genes important in B-cell function (14). Since TNF-a is a potent inducer of other cytokines, some of its effects on B-cell function may be mediated by these induced cytokines rather than by TNF-a itself. Overall, it appears that TNF-a enhances B-cell function, suggesting that in the setting of an inflammatory focus elevated TNF-a production likely augments the local humoral immune response. IL-6 is known as a late-acting B-cell differentiation factor for mitogen-activated human B cells. It increases immunoglobulin secretion by SAC-activated B cells, even when added as late as Day 4 of the culture period after maximal proliferation has occurred (15, 16). The addition of IL-6 neutralizing antibodies to cultures suggests that immunoglobulin production by pokeweed mitogen-activated B cells depends upon IL-6 (17). In contrast, antigen-specific antibody responses (i.e., against influenza virus) by human B cells apparently do not require IL-6 (18). In addition to its role as a differentia-
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tion factor, IL-6 stimulates proliferation of B-cell hybridomas and myelomas (19). However, there is little evidence to suggest that IL-6 is required for normal Bcell proliferation. In addition to the in vitro studies that suggest an important role for IL-6 in B-cell Ig secretion, overproduction of IL-6 in mice using an IL-6 transgene results in dramatically elevated serum levels of polyclonal IgG1 (20). Various effects of both cytokines on chronic B-cell malignancies have been described (21, 22), which is beyond the scope of this review.
INDUCTION AND REGULATION OF IL-6 AND TNF-a PRODUCTION IN B CELLS Activation of several different signal transduction pathways has been described to lead to increased IL-6 or TNF-a production in B cells. Polyclonal activation of human tonsillar B cells with SAC results in sequential induction of TNF-a and IL-6 mRNA as early as 8 to 12 (TNF-a) and 12 to 36 h (IL-6), respectively. A reverse transcriptase–polymerase chain reaction (RT-PCR) study on highly purified peripheral blood B lymphocytes found an early peak of TNF-a and IL-6 mRNA (Days 1–3), whereas IL-10 and transforming growth factor-b1 (TGF-b1) remained relatively constant throughout a 3-week culture period (23). After activation with anti-Ig, splenic B cells rapidly expressed IL6 mRNA with peak expression occurring already after 4 h and declining rapidly thereafter (24). Protein secretion peaked at 20 (TNF-a) and 100 h (IL-6) (8). The early peak of TNF-a production was associated with a proliferative response, whereas IL-6 production was detected at the time when Ig production started. Endogenous IL-6 production is partly dependent on the availability of TNF-a, as TNF-a neutralizing antibodies significantly reduced IL-6 and Ig production in SAC/ IL-2-activated B cells (8). Another study demonstrated the importance of endogenous IL-6 for Ig production in human B cells (25). Interestingly, the inhibitory effects of anti-cytokine antibodies on B-cell differentiation were strictly time dependent. The anti-TNF-a antibody was only effective if present from the beginning of cell culture, whereas the addition of the anti-IL-6 antibody could be delayed until Day 3 without losing blocking activity (8). Established human B-cell lines differentially produce IL-6. Although some produce high levels of this cytokine constitutively, other cell lines need to be induced with mitogen. Normal human peripheral blood B cells can be triggered to secrete large amounts of IL-6 by the combination of IL-4 and PMA (26). Constitutive expression of IL-6 in EBV-transformed B cells leads to an altered growth pattern and malignant phenotype (27). Production of TNF-a in human B cells was first
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described by Sung et al., who investigated different Bcell lines and polyclonally activated tonsillar B cells (28). In addition to polyclonal activation using SAC/IL2, direct anti-Ig treatment rapidly induced transcription of TNF-a in human B cells (29). Another way to trigger cytokine production is via stimulation of B cells through the CD40 receptor–gp39 interactions. Both pathways can be blocked by the immunosuppressants cyclosporin A and FK506 (29, 30). CD40 is a 50-kDa glycoprotein mainly expressed on B cells and belongs to the TNF receptor family. Crosslinking of CD40 results in cytokine production (31), results in enhanced adhesion and proliferation of B cells, and is accompanied by an increased tyrosine phosphorylation of various substrates (32). In addition, in the presence of IL-2, IL-4, or IL-10, CD40 ligand is a potent inducer of IgM, IgE, IgG1, and IgA secretion. This process is inhibited by TGF-b and, to a lesser extent, by IFN-g. Recently, it was demonstrated that activation of the CD40 pathway rapidly induced NF-kB binding activity in the nucleus. Transient transfection studies revealed a marked effect on NF-kB-dependent gene expression (33). Binding of NF-kB to the promoter regions of TNF-a and IL-6 is important for their efficient transcription (34), and functional NF-kB activity is essential for B-cell proliferation (35). CD22 is a B lineage cell membrane protein that functions as an adhesion molecule (4–6). While the nature of the ligand(s) for CD22 remains controversial, several monoclonal antibodies have been developed that block the interaction of CD22 with its ligands (36). Crosslinking of CD22 with such a CD22 mAb induced significant levels of IL-6 (Table 1) and small amounts of TNF-a (data not shown). In contrast, a CD22 mAb that does not block the interaction of CD22 with its ligands failed to induce significant levels of IL-6 production. Consistent with this difference in their abilities to induce IL6 production, the nonblocking antibody induces far less TABLE 1 IL-6 Production after CD22 Crosslinking with Blocking or Nonblocking mAbs Experiment 1 treatment
IL-6 (pg/ml)
Experiment 2 treatment
IL-6 (pg/ml)
Media HB22.5 CD22.5 Anti-Ig HB22.5 / Anti-Ig CD22.5 / Anti-Ig
210 580 180 440 600 450
Media HB22.5 CD22.5 IL-2 HB22.5 / IL-2 CD22.5 / IL-2
220 570 190 220 570 220
Note. Tonsillar B lymphocytes were treated as indicated. HB22.5 is a CD22 blocking mAB while CD22.5 is a nonblocking mAB. Culture supernatants were collected after 24 h and assayed for IL-6 production via ELISA. Similar results were found at 48 h.
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tyrosine phosphorylation of CD22 than does the blocking antibody (J. Tuscano et al., manuscript submitted for publication). Besides triggering IL-6 production, the blocking mAb induces B-cell proliferation and costimulates B-cell proliferation in conjunction with anti-Ig, CD40, and IL-2. In addition, in the presence of IL-2 the CD22 mAb induces B-cell Ig production (J. Tuscano et al., manuscript submitted for publication). Various second messenger pathways that can induce TNF-a in B cells have been described. Direct stimulation of protein kinase C (PKC) with PMA results in a rapid induction in TNF-a mRNA transcription. Similar effects were obtained with okadaic acid, a specific inhibitor of the serine/threonine phosphatases 1 and 2A (PP1 and PP2A) (37). When added to intact cells this substance allows the unopposed activity of constitutively active protein kinases, which leads to enhanced phosphorylation of many protein kinase substrates. Significant increases of nuclear factor binding activity (e.g., AP-1 and NF-kB) were detected in normal human tonsillar B cells after the addition of okadaic acid. A strong induction of TNF-a mRNA in the same cells was also observed. These immediate early effects were independent of new protein synthesis (37). Another calcium- and calmodulin-dependent phosphatase, calcineurin, which plays an important role in T-lymphocyte activation (38), mediates protein synthesisindependent TNF-a transcription in human B cells stimulated through their surface immunoglobulin receptors (39). This effect is mediated by modification of the transcription factor NFATp, a 120-kDa phosphoprotein that translocates to the nucleus and binds as a complex to AP-1 binding sites present in various promoters (40). Recently, platelet activating factor (PAF) was shown to directly increase TNF-a mRNA levels in the B-cell line Ramos and costimulate PMA-induced TNF-a production in peripheral blood B cells. Interestingly, PAF alone did not increase protein levels, possibly due to a posttranscriptional block. It can therefore be regarded as a modulator of cytokine production in B cells (41). The immunosuppressant cyclosporin A (CsA) blocks PAF and anti-Ig induced TNF-a mRNA and protein synthesis, but had no effect on PMA-induced cytokine production and CD40-mediated proliferation in freshly isolated and Ramos B cells (30, 41). From these data, it was hypothesized that different B-cell populations originate from a single precursor population depending on the activation pathway. Treatment of small resting B cells with anti-IgM induced proliferation and enhanced surface CD5 expression. In contrast, B cells induced to proliferate in response to CD40 ligand displayed elevated levels of CD23 but no surface CD5 (42). Functional differences in terms of cytokine production were not detected, as both CD5/ and CD50 negative human tonsillar B cells were shown to produce equal
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amounts of TNF-a and IL-6 (43). Similar results were obtained with murine B-cell lines (44). Recently, it was demonstrated that CD27, a 50-kDa transmembrane protein expressed late during B-cell differentiation, can be used to distinguish populations in terms of Ig production, but no such differences were observed for cytokine production. Only CD27/ B cells produced IgM and IgG upon appropriate stimulation, whereas the two subsets secreted similar amounts of IL-6 and TNF-a (45). From the available data it can be concluded that IL-6 and TNF-a production is inducible in various B-cell populations, but apparently is not restricted to a distinct B-cell phenotype.
PATHOGENETIC RELEVANCE OF CYTOKINE PRODUCTION IN B CELLS B-cell hyperactivity and polyclonal activation with an increased risk of autoantibody formation is a hallmark of certain immunological disorders such as systemic lupus erythematosus (SLE). It also occurs during the course of HIV infection. The role of stimulatory cytokines in the excessive B-cell activation observed in these diseases has been investigated, and several cytokines have been implicated as important mediators including IL-10, IL-6, and TNF-a (46–49). Increased levels of circulating IL-6 and TNF-a were detected early during the course of HIV-1 infection and their role in the regulation of HIV expression is clearly established (50). Although polyclonal B-cell activation and hypergammaglobulinemia as well as an increased number of activated, spontaneously Ig-secreting cells are present in the circulation of HIV-infected individuals (51), certain B-cell functions, such as polyclonal activation and antibody production after antigenic challenge, are impaired in AIDS patients. Peripheral blood B lymphocytes from HIV-infected individuals with hypergammaglobulinemia spontaneously secrete large amounts of IL-6 and TNF-a compared to healthy seronegative donors. Interestingly, lymph node B cells spontaneously secrete higher levels of both cytokines than do blood B cells from the same patients (43). In addition, these cells are capable of inducing HIV-1 expression in cell lines chronically infected with HIV. This effect is mediated by both TNF-a and IL-6, as neutralizing antibodies against IL-6 and TNF-a reduced the stimulatory activity in B-cell supernatants. Similar results were obtained with an autologous coculture system of B and T cells isolated from lymph nodes of three patients with AIDS (47). Interestingly, during the latent phase of infection the germinal centers of lymph nodes harbor large amounts of virus in the follicular dendritic network in close contact with activated B cells (52). It is conceivable that
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cytokine production by activated B cells in close contact with passaging T cells within the germinal centers of the lymph node contributes to the ongoing production of HIV during the latent phase of the disease. We and others have demonstrated that the major envelope protein of HIV gp120 can induce proliferation and cytokine production in B cells (53–55). Interestingly, stimulation of normal B cells with HIV envelope protein is Tcell dependent (54–56), whereas highly purified B cells from HIV-infected individuals are directly inducible to produce IL-6 and TNF-a without any T-cell help (49, 53). A CD4-independent signaling pathway is apparently responsible for cytokine and Ig production in these patients. Recently, it was demonstrated that a subset of B cells, defined by the utilization of a specific Ig heavy-chain family (VH3), can interact directly with HIV envelope protein and, thereby, could be directly activated by HIV (57). In summary, these studies suggest that germinal centers of lymph nodes provide a unique environment for virus expression and accumulation in which gp120 stimulates B cells to secrete HIV inductive cytokines, such as TNF-a and IL-6, thereby enhancing virus expression in infected cells in a paracrine manner. Whether increased IL-6 production plays a role in the induction of B-cell malignancies that frequently occur in HIV-infected individuals remains unresolved. It has been reported that elevated IL-6 levels may precede the development of B-cell lymphoma in HIV-infected individuals under zidovudine treatment (58). The increase in IL-6 in these individuals is unlikely due to the zidovudine treatment since it does not induce B-cell activation (IL-6 production or Ig secretion) either in vivo or in vitro (59). Spontaneous production of polyclonal and antigenspecific antibodies plays an important role in the pathogenesis of SLE. Several studies revealed that IL-6 and TNF-a support the in vivo differentiation process of B cells in patients with SLE (48, 60–62). High levels of serum IL-6 have been reported in patients with SLE, and IL-6 mRNA was detected in freshly isolated peripheral blood mononuclear cells (48). An autocrine loop for IL-6 in B cells has been described in patients with SLE. High-density B cells from these patients produce large amounts of IL-6, which binds to the IL-6 receptors present on low-density B cells. This interaction, as well as IgG and autoantibody production, could be inhibited by an antibody against IL-6 (63). The pathophysiological role of IL-6 was recently described in an animal model of SLE (62). As IL-6 can be efficiently induced in B cells via autocrine TNF-a, it is of interest whether increased TNF-a levels are also present in patients with SLE. Interestingly, anti-TNF-a antibodies decreased the production of IL-6 and IgG in blood B cells from patients with SLE (48). A genetic association was determined between SLE and TNF-a. This polymor-
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phism revealed a strong correlation with the HLA-DR3 and HLA-B8 allele and may play a role in SLE susceptibility (60). Another nonmalignant B-cell-associated disease with increased cytokine production is the rare Castleman’s syndrome. This disease is characterized by giant lymph node hyperplasia with plasma cell infiltration, fever, anemia, hypergammaglobulinemia, and increased levels of acute phase proteins. Patients with Castelman’s syndrome were found to have increased serum levels of IL6, and it was found that germinal center B cells produce large amounts of this cytokine (64). Overexpression of IL6 in transgenic mice produces a syndrome resembling Castleman’s disease (20, 65). Immunoglobulin production has long been regarded as the preeminent function of B lymphocytes. This review summarizes the evidence that the cytokines IL-6 and TNF-a participate in the regulation of B-cell physiology and the evidence that B cells themselves are an important source and target for these two cytokines. Cytokine production by B cells may serve as an immunoregulatory component in certain diseases associated with dysregulated cytokine production.
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