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PTX decreases LPS-induced NF-kappaB activation
Since phosphorylation leads to ubiquitination and degradation of I-kappaBalpha and subsequent nuclear translocation of NF-kappaB, we first examined the effects of PTX on I-kappaBalpha. Cytoplasmic I-kappaBalpha phosphorylation was markedly increased following LPS stimulation when compared to control (100 +/- 0 vs. 20 +/- 18; P < 0.05). The addition of PTX significantly downregulated LPS-induced I-kappaBalpha phosphorylation (P = 0.02; Figure 2)
In a similar fashion, nuclear NF-kappaB p65 phosphorylation was increased following LPS stimulation (100 +/- 0 vs. 38 +/- 20; P < 0.01). PTX similarly decreased LPS-induced NF-kappaB p65 nuclear phosphorylation, a marker of NF-kappaB activation and nuclear translocation (100 +/- 0 vs. 40 +/- 6; P =0.03; Figure 3A).
EMSAs were then performed to verify the PTX-induced alterations in nuclear phosphorylation resulted in similar effects on the DNA binding activity of NF-kappaB. The addition of PTX to LPS-stimulated mononuclear cells resulted in comparable downregulation of DNA binding activity, similar to the observed downregulation of NF-kappaB phosphorylation (Figure 3B).

downregulated

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PTX upregulates CREB phosphorylation and activation after LPS stimulation
Phosphorylation of nuclear CREB was used as a marker for CREB activation. LPS stimulation caused a negligible increase in CREB phosphorylation when compared to control (Figure 4A). PTX alone caused a marked increase in CREB phosphorylation (P < 0.01 vs. HBSS). When LPS and PTX exposure occurred simultaneously, CREB phosphorylation was significantly higher than with LPS stimulation alone (543 +/- 92 vs. 100 +/- 0; P < 0.01). In addition, the amount of CREB phosphorylation seen with concomitant LPS and PTX treatment was less than that seen with PTX alone, although this difference was not statistically significant (P = 0.08).
To determine if CREB-DNA binding was affected by PTX in a manner similar to CREB phosphorylation, an EMSA was performed. Exposure of LPS-stimulated cells to PTX similarly increased CREB DNA-binding compared to LPS alone (Figure 4B).

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Effect of PTX on TNF-alpha production in the presence of PKA inhibition
The role of PKA in PTX-induced downregulation of TNF-alpha production in LPS-stimulated mononuclear cells was assessed by treating mononuclear cells with H89, a PKA inhibitor, prior to LPS and PTX exposure. The addition PTX to LPS-stimulated cells resulted in a reduction in TNF-alpha, similar to that observed in our previous experiments (529 pg/mL +/- 112 vs. 37 pg/mL +/- 10; P < 0.01). Pre-incubation of cells with H89 prior to LPS and PTX exposure did not result in any significant modulation of TNF-alpha production when compared to that demonstrated with PTX alone (P = 0.2; Figure 5). These results suggest that downregulation of TNF-alpha production by PTX in LPS-stimulated mononuclear cells is, at least in part, PKA-independent.

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The exposure of inflammatory cells to a variety of extracellular stimuli results in the initiation of transcriptional and posttranscriptional events, which culminate in the production of pro-inflammatory mediators.11-14 TNF-alpha is released very early following shock and is considered an important mediator of the inflammatory cascade, since it regulates the synthesis of several other critical cytokines and chemokines.15 A clear relationship between increased TNF-alpha synthesis and the development of shock and multiple organ dysfunction has been observed in animal models of sepsis.15-17
Because of its importance in orchestrating multiple steps of the inflammatory response, strategies aimed at decreasing TNF-alpha levels or attenuating its production seem attractive. PDE inhibitors have been shown to downregulate TNF-alpha production in distinct human cell populations. We have previously shown that, in general, TNF-alpha levels continue to increase after LPS exposure and that concomitant infusion of phosphodiesterase inhibitors can markedly decrease TNF-alpha levels.18-19 In this in vitro study, we verified the sustained attenuation of LPS-induced TNF-alpha expression over time in an isolated human mononuclear cell population.
The mechanisms by which PTX, a non-specific phosphodiesterase inhibitor, significantly and consistently decreases TNF-alpha production have not been completely elucidated. It has been postulated that drugs that increase intracellular cAMP exert their anti-inflammatory effects through the activation of PKA. This classic pathway has been challenged as the sole mechanism involved in the modulation of inflammation by cAMP-enhancing drugs, and alternative mechanisms involving PKA-independent pathways have been proposed.6-8 In this series of experiments, we observed a significant attenuation of TNF-alpha production in LPS-stimulated human mononuclear cells following PTX administration, independent of PKA activation.
We used a nonspecific phosphodiesterase inhibitor, PTX, in the present study because it has been used clinically in the treatment of a variety of conditions in which inflammation is an important component of the pathophysiology of the disease process.19 Additionally, the use of PTX in sepsis as an adjunct to other treatments to maintain adequate organ function has been explored based upon its effects on TNF-alpha synthesis. However, the reported beneficial effects of PTX are not only related to the downregulation of TNF-alphabut also to PTX's hemorrheologic properties, its ability to reduce neutrophil activation, and its beneficial effects on microcirculation, cardiac performance, and organ injury.20-25 Herein, we demonstrated that the downregulatory effects of PTX in human mononuclear cells involves modulation of pathways that involve NF-kappaB and CREB, two major transcription factors involved in the inflammatory cascade.
NF-kappaB/Rel constitutes a family of transcriptional factors involved in the regulation of numerous cytokine genes and immune responses in different cell populations. The most abundant form of NF-kappaB is the p50-p65 heterodimer.26 The inactive form of NF-kappaB exists in the cytoplasm bound to an inhibitory complex containing I-kappaBalpha. Following cellular stimulation with LPS and chemotactic factors, NF-kappaB inducible kinase phosphorylates and activates the I-kappaB kinase (IKK) complex consisting of IKK-1 and IKK-2, which in turn phosphorylates the I-kappaBalpha subunit. Phosphorylation, ubiquitination, and subsequent degradation of I-kappaBalpha results in NF-kappaB nuclear translocation. This allows the transcription factor to bind to promoter regions of specific pro-inflammatory genes and influence transcription.27,28 Because a number of potential kappaB sites are present in the nucleotide sequences of the TNF-alpha gene, NF-kappaB is considered to be a critical transcriptional factor involved in TNF-alpha gene expression and protein synthesis.29,30
In this set of experiments, we demonstrated that PTX downregulates cytoplasmic I-kappaBalpha phosphorylation, nuclear NF-kappaB p65 phosphorylation/translocation, as well as NF-kappaB DNA-binding after LPS stimulation, suggesting that PTX exerts its function proximal to or at the level of I-kappaBalpha phosphorylation.
Previous studies that have evaluated the effects of cAMP-elevating drugs on I-kappaBalpha and NF-kappaB have reported conflicting results. Haddad et al. reported results similar to ours with respect to the effects of PTX on I-kappaBalpha and NF-kappaB in pulmonary epithelial cells.31 Conversely, Neumann et al. proposed that increased intracellular cAMP stabilizes the interaction between I-kappaBalpha and NF-kappaB, therefore reducing NF-kappaB activation.32 In contrast, Takahashi et al. showed no difference in I-kappaBalpha degradation in Jurkat T-lymphocytes in the presence and absence of forskolin, an alternative cAMP inducing compound. This group also demonstrated that forskolin did not inhibit the DNA-binding activity of NF-kappaB and that CREB was not involved in forskolin-induced decrease in p65 transcriptional activity.33 Similar results were reported by Ollivier et al. using THP-1 cells and endothelial cells treated with forskolin or dibutyryl cAMP, two agents known to function through cAMP elevation and subsequent PKA activation.9 The differences between our results and those mentioned above may be explained by the use of different cell types and exogenous stimulants. It is also possible that PTX exerts its anti-inflammatory effects on the NF-kappaB pathway independent of PKA, as suggested by the continued attenuation of LPS-induced TNF-alpha production by PTX in the presence or absence of PKA inhibition observed in this study. This hypothesis is currently under investigation in our laboratory. Furthermore, we utilized a broad PKA inhibitor in this study in addition to H89, but did not examine the effect of specific inhibition of various PKA isoforms. This information may yield more insight into the mechanism by which PTX exerts its down-regulatory effects.
PDE inhibitors may also downregulate NF-kappaB transcriptional activity by altering the competitive binding of CREB and NF-kappaB to the promoter regions of pro-inflammatory genes. Herein, we showed that the activation and DNA binding activity of CREB is upregulated in a dose-dependent manner by PTX. Of note, the increase in CREB activity was slightly alleviated by the addition of LPS, suggesting competition between PTX-induced CREB-associated gene transcription and those factors, such as NF-kappaB, which may be upregulated by LPS exposure. This competition has been postulated to involve the recruitment of the co-activator CBP and its homologue p30034, suggesting an alternative mechanism that may be involved in the anti-inflammatory actions of PTX. Future studies are planned to delineate the validity of this potential mechanism.
The effects of PTX on other transcription factors, such as AP-1, c-fos, and c-jun, cannot be ruled out by our experiments. Activation of AP-1 and its components (c-fos and c-jun) by agents that enhance mononuclear cell activity has been implicated in TNF-alpha expression. Moreover, AP-1 and CREB recognize a similar DNA binding sequence in the promoter region of the TNF-alpha gene35. Given these relationships, it is conceivable that PTX may exert some of its anti-inflammatory actions by affecting AP-1 activation and favoring CREB binding, thus inhibiting TNF-alpha gene transcription. Additional studies are necessary to elucidate the complex interactions between CREB, NF-kappaB, and AP-1 following LPS exposure and PDE inhibition. Furthermore, the effect of PTX on the cGMP pathway after LPS stimulation has not been explored, although we plan to do so in the future.
In summary, we have demonstrated that PDE inhibition of human mononuclear cells downregulates TNF-alpha production, at least in part, through a PKA-independent mechanism. In addition, PTX attenuates the activity of NF-B while upregulating CREB activation after LPS stimulation, which may result in modulation of pro-inflammatory mediator synthesis. Therefore, PTX may serve as a potential adjunct therapeutic for the treatment of conditions in which TNF-alpha production plays a significant role.

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Interleukin-10 Production by Th1 Cells Requires Interleukin-12-Induced STAT4 Transcription Factor and ERK MAP Kinase Activation by High Antigen Dose
Summary
CD4+ T cells producing interleukin-10 (IL-10) and interferon-gamma (IFN-gamma) are reported in chronic infections. However, the signals that direct the development of IL-10-producing T helper 1 (Th1) cells are undefined. We showed that development of IL-10-producing Th1 cells required high T cell receptor (TCR) ligation, sustained ERK1 and ERK2 MAP kinases phosphorylation, and IL-12-induced STAT4 transcription factor activation. Repeated TCR triggering led to enhanced IL-10 production by Th1 cells, and continued IL-12 action and high-dose TCR signaling were required for the development and maintenance of IL-10-producing Th1 cells. Although Th1, Th2, and Th17 cells require the activation of distinct STATs for their differentiation, activation of ERK1 and ERK2 was a common requirement for production of IL-10 by all Th cell subsets. IL-10 expression also correlated with c-maf expression. Despite having distinct functions in protection against pathogens, all Th cells share the important task of controlling overexuberant immune responses by means of IL-10 production.

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Interleukin-10 (IL-10) is a cytokine with broad anti-inflammatory properties that inhibits macrophage and dendritic cell (DC) function (Moore et al., 2001). IL-10 limits the immune and inflammatory responses to pathogens and gut flora and prevents damage to the host (Moore et al., 2001; O'Garra and Vieira, 2004), but when dysregulated may result in chronic infection (Brooks et al., 2006; Ejrnaes et al., 2006; Moore et al., 2001). IL-10 is expressed by T helper 2 (Th2) cells, B cells, DCs, and macrophages (Moore et al., 2001), and also by Th1 cells (Anderson et al., 2007; Assenmacher et al., 1994; Del Prete et al., 1993; Gerosa et al., 1996; Jankovic et al., 2007; Pohl-Koppe et al., 1998) and (reviewed in O'Garra and Vieira, 2007; Trinchieri, 2007), certain regulatory (Treg) T cells (Moore et al., 2001; O'Garra and Vieira, 2004; Roncarolo et al., 2006), and Th17 cells (Awasthi et al., 2007; Fitzgerald et al., 2007; McGeachy et al., 2007; Stumhofer et al., 2007).
In vitro human CD4+ and CD8+ T cell clones, or mouse CD4+ T cells that produce both interferon-gamma (IFN-gamma) and IL-10, can be differentiated by T cell receptor (TCR)-stimulation in the presence of IL-12 (Chang et al., 2007; Gerosa et al., 1996; Jeannin et al., 1996; Meyaard et al., 1996; Windhagen et al., 1996). Furthermore, Th1 cell clones coproducing IFN-gamma and IL-10 have been isolated from bronchoalveolar lavage (BAL) of active pulmonary tuberculosis (TB) patients (Gerosa et al., 1999). IL-10 production by Th1 cells was also reported in animals infected with Toxoplasma gondii (Jankovic et al., 2002; Shaw et al., 2006) or with Leishmania major (Anderson et al., 2007) and shown to be required for regulation of the immune response in these infections (Anderson et al., 2007; Jankovic et al., 2007). The relative amounts of IL-10 and IFN-gamma produced by Th1 cells may influence the balance between clearance and persistent infection with certain pathogens (Moore et al., 2001; Trinchieri, 2007), thus determining whether chronic infection or immunopathology ensues.
Th1, Th2, and Th17 cell responses differentiate along distinct signaling pathways (Glimcher and Murphy, 2000; Ivanov et al., 2007; Stockinger and Veldhoen, 2007). Th1 cell development requires signal transducer and activator of transcription (STAT)1 activation, induced by type I IFN or IFN-gamma, the transcription factor T-box 21 (T-bet), and IL-12-induced STAT4 signaling, which can couple with IL-18-induced IRAK and NF-kappaB transcription factors to drive the high amounts of IFN-gamma required to eradicate intracellular pathogens (Glimcher and Murphy, 2000). Th2 cell development, with expression of IL-4, IL-5, and IL-13, requires IL-4, STAT6, and the transcription factor GATA binding protein (GATA)-3 (Glimcher and Murphy, 2000). The development of Th17 cells requires IL-6, TGF-beta, and the STAT3-dependent expression of the transcription factor RORgammat (Ivanov et al., 2007; Stockinger and Veldhoen, 2007).
Th1 and Th2 cell responses can also be induced by varying the dose of antigen presented to the naive T cell by the antigen-presenting cell (APC). Whereas high doses of antigen, with sustained TCR signaling and extracellular-signal regulated (ERK) mitogen-activated protein kinase (MAPK) phosphorylation, result in Th1 cells producing IFN-gamma via an IL-12-independent mechanism, low doses of antigen, with transient ERK1 and ERK2 activation, favor Th2 responses and IL-4 secretion (Constant et al., 1995; Hosken et al., 1995; Jorritsma et al., 2003; Yamane et al., 2005).
Because Th1 and Th2 cells cross regulate each other's development and function and can suppress Th17 cell responses, and all differentiate along distinct signaling pathways (Glimcher and Murphy, 2000; Stockinger and Veldhoen, 2007), IL-10 produced by all these Th cells may thus act as a feedback regulator to control the pathology associated with an overexuberant, albeit efficacious, inflammatory response. Whether IL-10 production by these different Th cell subsets is induced by independent and/or common mechanisms is unknown.
Here, we showed that in vitro differentiation of IL-10-producing Th1 cells from naive CD4+ T cells required IL-12-induced STAT4 signaling, strong TCR activation (high antigen dose), and sustained ERK1 and ERK2 phosphorylation. Furthermore, we showed that activation of ERK1 and ERK2 is a requirement for production of IL-10 by Th1, Th2, and Th17 cell subsets. This common but highly regulated pathway for IL-10 induction and maintenance ensures its function as a feedback loop to control damage to the host and also allows a protective response to ensue as opposed to chronic infection.

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IL-12 and High Doses of Antigen Induce the Development of Th1 Cells Producing IL-10
To study the differentiation of Th1 cells coproducing IFN-gamma and IL-10, we cultured purified TCR-transgenic DO11.10 naive CD4+ T cells with purified DCs as APCs and increasing doses of ovalbumin peptide 323-339 (OVA). Culture with high doses of antigen for 7 days gave rise to Th1 cells expressing IFN-gamma upon restimulation (Constant et al., 1995; Hosken et al., 1995), but not IL-10 (Figure S1A available online). Culture with low antigen doses under the same conditions led to the differentiation of Th2 cells, which expressed both IL-4 and IL-10 upon restimulation (Figure S1A). Culture of naive CD4+ T cells in an APC-free system by stimulation with anti-CD3 and anti-CD28 antibodies in the presence of IL-12 resulted in IFN-gamma-producing Th1 cells, a proportion of which coproduced IL-10, as did Th2 cells resulting from culture in IL-4 (Figure S1B).
To investigate whether the lack of IL-10 produced by Th1 cells resulted from inhibition of IL-10 production by DCs and high antigen dose or alternatively required IL-12, we cultured naive CD4+ T cells with increasing doses of antigen presented by DC in the presence of IL-12. At low doses of antigen, IL-12 abrogated the development of Th2 cells and induced IFN-gamma expression but only low levels of IL-10 expression, suggesting that IL-12 per se was not sufficient to induce significant IL-10 production in Th1 cells (Figure 1A). Strikingly, as the antigen dose was increased, Th1 populations driven with IL-12 now contained higher numbers of IL-10-producing cells (Figure 1A) and produced more IL-10 protein upon restimulation (Figures 1A and 1B). Thus, the development of Th1 cells producing IL-10 required both IL-12 and high doses of antigen. Th1 cells differentiated to produce large amounts of IL-10 and IFN-gamma and lost their capacity to produce IL-2 (Figure 1B). Because the presence of IL-12 reduced the proliferation of CD4+ T cells at both high and low antigen doses (Table S1), and only the former showed IL-10 production, the development of high IL-10-producing cells is most likely not related to limited IL-2. Naive CD4+ T cells from DO11.10/recombination-activating gene 1 (Rag1)-deficient animals, cultured with high doses of antigen in the presence of IL-12, also resulted in IL-10 expression by Th1 cells, showing that this expression was not dependent on the presence of effector or memory T cells or Treg cells (Figure S2). Although it has been suggested that TGF-beta can induce IL-10 in CD4+ T cells (Kitani et al., 2003; Schiott et al., 2000), we found that in developing Th1 and Th2 cells this was not the case (data not shown). In fact, neutralization of TGF-beta led to increased IL-10 production by both T cell subsets (Figure S3). Thus, the development of IL-10-producing Th1 cells only depended on the presence of IL-12 together with high antigen dose and not on other soluble factors such as IL-2 or TGF-beta or on the presence of other T cell types.

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IL-10 Production by Th1 Cells Is Dependent on STAT4 but Not on STAT6, IFN-gamma, or IL-4 Signaling
To further elucidate the mechanisms required for the development of Th1 cells producing IL-10, we investigated the role of STAT4, one of the signaling pathways activated by IL-12 (Murphy et al., 2000). Naive CD4+ D011.10 T cells deficient in STAT4 (Ouyang et al., 1998) were cultured in the presence of IL-12 and OVA. Again, IL-10-producing Th1 cells were differentiated at the high antigen dose in the presence of IL-12 in DO11.10 T cells (Figure 2A). In contrast, in the absence of STAT4, the percentage of cells expressing IFN-gamma was dramatically diminished as expected and resulted in an increase in the percentage of cells expressing IL-4, but not IL-10 (Figure 2A), suggesting that STAT4 contributes to IL-10 expression by Th1 cells.
Because IL-10 expression is associated with an IL-4-induced Th2 cell phenotype, we investigated whether the differentiation of the IL-10-producing Th1 cells depended on signaling through the IL-4 receptor via STAT6 activation (Glimcher and Murphy, 2000; Murphy et al., 2000). The absence of STAT6 did not impair the differentiation of IL-10-producing Th1 cells in the presence of IL-12 and OVA (Figure 2A). In fact, a higher percentage of STAT6-deficient cells compared with WT cells produced both IL-10 and IFN-gamma (Figure 2A), which may be the result of the loss of Th2 cell control over a Th1 cell response. As expected, lack of STAT6 abrogated both IL-4 and IL-10 production by T cells developed with IL-4 or with low antigen dose (Figures 2B and 2C). However, in the absence of STAT4 signaling, IL-10 and IL-4 production by Th2 cells was if anything increased (Figures 2B and 2C). Thus, in contrast to what was observed under Th1 conditions, IL-10 expression by Th2 cells depended on STAT6, but not on STAT4, signaling (Figures 2B and 2C).
To investigate whether the inability of STAT4-deficient T cells to produce IL-10 might be due to the absence of IFN-gamma, as suggested before (Shaw et al., 2006), we differentiated DO11.10 or DO11.10 IFN-gamma-deficient naive CD4+ T cells in the presence of IL-12 and increasing doses of OVA. The secretion of IL-10 as induced by high antigen dose, and IL-12 was not affected by an absence of IFN-gamma (Figure 2D), showing that the expression of IL-10 by Th1 cells is independent of IFN-gamma. In the absence of IFN-gamma, we observed an increase in the secreted IL-4 as expected (data not shown).
We also tested for any potential role of IL-4 in the development of Th1 cells producing IL-10 by culturing DO11.10 or DO11.10 IL-4-deficient naive CD4+ T cells with IL-12 and increasing doses of antigen. As observed in the absence of STAT6 (Figure 2A), IL-4 deficiency had no effect on the development of Th1 cells producing IL-10 (Figure 2E), but compromised the development of Th2 cells producing IL-10 (Figure 2F). Thus, our data suggested that IL-10 production by Th1 or Th2 cells was dependent on the specific signaling pathways required for their differentiation, given that STAT4 is required for the induction of IL-10 production by Th1 cells and STAT6 for Th2 cells.

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High Antigen Doses and STAT4 Are Required for the In Vivo Generation of IL-10-Producing Th1 Cells
To address the mechanisms regulating IL-10 production by Th1 cells in vivo, we transferred DO11.10 cells into BALB/c recipient mice and immunized the recipients with very high doses of OVA-protein with or without added lipopolysacharide (LPS). T cells were recovered from the inguinal lymph nodes 3 days after priming and restimulated in vitro with OVA peptide for 48 hr. This in vivo immunization induced IL-10 and IFN-gamma production, and the amount of IL-10 production was enhanced by addition of LPS in the immunization (Figure 3A) and with higher doses of OVA (3 muM versus 1 muM, data not shown). To test the role of STAT4 and STAT6 signaling in the in vivo development of IL-10-producing Th1 cells, we transferred STAT4- or STAT6-deficient or WT DO11.10 cells into recipient BALB/c mice and immunized with OVA-protein plus LPS as before. In vivo expression of both IL-10 and IFN-gamma was markedly reduced but not completely abrogated in the absence of STAT4 signaling (Figures 3B and 3C), suggesting the existence of compensatory mechanisms that were absent in the in vitro system. Signaling through STAT6 had no effect on IL-10 production by Th1 cells as shown by intracellular cytokine staining (ICS) and by immunoassay in STAT6-deficient T cells (Figures 3B and 3C).

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IL-10 Production Is Maintained by High TCR Signal Strength and IL-12
We next investigated whether repeated strong TCR activation is a compensatory signal for IL-12-induced STAT4 signaling in the induction of IL-10 in Th1 cells. For this, CD4+ T cells were differentiated for 2 consecutive weeks with high antigen doses in the presence or absence of IL-12 throughout (Figures 4A-4D). High antigen dose and IL-12 cooperated to induce maximal IL-10 production (Figures 4A and 4B), given that this combination resulted in the highest numbers of IL-10-producing Th1 cells. Repeated high antigen dose stimulation in the absence of exogenously added IL-12 resulted in the production of IL-10 by Th1 cells, suggesting that repeated strong TCR triggering may overcome the need for IL-12 for IL-10 induction (Figures 4C and 4D). However, IL-10 induction under these conditions was abrogated when IL-12p40-deficient DCs were used as APCs (Figure 4E). Thus, IL-12 is essential during both primary and secondary antigenic stimulation for production of IL-10 by Th1 cells.
To determine the requirements for stability of the IL-10-producing Th1 cells, we differentiated CD4+ T cells for 1 week with high antigen doses with or without IL-12 (Figures 4A and 4C), washed them, and then restimulated them for an additional week with a low antigen dose, in the absence or presence of IL-12 (Figure 4F). Th1 cells induced in the first week to produce IL-10 by culture with high antigen doses and IL-12 lost their ability to express IL-10 when recultured with low doses of OVA, which could be compensated for, to some extent, by addition of IL-12 to the secondary cultures (Figure 4F), again suggesting that antigen dose and IL-12 signals cooperate for the induction of IL-10. Finally, DO11.10 CD4+ cells that were exposed to low doses of antigen and IL-12 during the primary differentiation phase produced high amounts of IFN-gamma but little IL-10, but they could be induced to produce IL-10 when both high antigen dose and IL-12 were present during the recall phase (Figure S4). Thus, high antigen dose and IL-12 are required for sustaining the induction of IL-10 production by Th1 cells.

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Input:
IL-10 Production by Th1 Cells Requires ERK1 and ERK2 Activation
Our data showed that the maintenance of IL-10 induction in Th1 cells required stimulation with high antigen dose, which to some extent could be compensated for by the addition of IL-12. Signaling through the TCR with high doses of antigen induced stronger ERK1 and ERK2 activation than that induced by low antigen dose, not only in naive CD4+ T cells (data not shown) as previously demonstrated (Jorritsma et al., 2003) but also in CD4+ T cells restimulated with the same high and low antigen doses (Figure 5A). Although the apparent peak and amount of ERK1 and ERK2 activation varied slightly between experiments, a consistent finding was that high antigen dose differentiated Th1 cells always showed enhanced and prolonged ERK1 and ERK2 activation in the presence of IL-12, regardless of whether they were restimulated with high or low antigen dose (Figure 5B).
We then investigated whether ERK1 and ERK2 activation was required for the induction of IL-10 in Th1 cells by using U0126 (Figure 5C), a compound that blocks downstream ERK activation. To ensure that only T cell signaling was being affected by U0126, we used an APC-free system in which the T cells were differentiated in the presence of increasing doses of anti-CD3 and a constant amount of IL-12. As in the APC-driven cultures, stronger TCR stimulation together with IL-12 led to higher percentages of cells producing both IL-10 and IFN-gamma after 1 week of culture (Figure 5C). Addition of U0126 to the cultures abrogated the production of IL-10 at all doses of anti-CD3 (Figure 5C). Because U0126 inhibits the MEK5-catalyzed activation of ERK5, as well as the MEK1- and MEK2-catalyzed activation of ERK1 and ERK2 (Bain et al., 2007; Mody et al., 2001), we also used the more specific, structurally unrelated MEK1 and MEK2 inhibitor PD184352 at concentrations in which it inhibits MEK1 and MEK2 but not MEK5 (Bain et al., 2007; Mody et al., 2001). PD184352 caused a similar inhibition of IL-10 production by Th1 cells in a dose-dependent fashion (Figure 5D and Figure S5A). Upon addition of inhibitors to other signaling pathways, including a p38 MAPK inhibitor, SB203580, or the GSK3beta inhibitor, CT99021 (Bain et al., 2007), no effect on IL-10 production was observed (Figure S5B). Our data thus suggested that IL-10 production by Th1 cells in response to high antigen dose and IL-12 requires ERK1 and ERK2 signaling, but not the activation of the p38 or the GSK3beta pathways.

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Input:
IL-10 Production by Th2 and Th17 Cells Also Requires ERK1 and ERK2 Activation
To address whether IL-10 production by Th2 and Th17 cells was also dependent on ERK1 and ERK2 activation, we differentiated these cells with anti-CD3 and anti-CD28 in the absence of APCs (Shoemaker et al., 2006; Veldhoen et al., 2009; Veldhoen et al., 2006), in the presence or absence of the MEK inhibitor (PD184352). We showed that ERK1 and ERK2 activation is a common pathway required for induction of IL-10 in different Th cell subsets because IL-10 production by both Th2 and Th17 cells was markedly inhibited in the presence of the MEK inhibitor (PD184352) (Figure 5D and Figure S5B). In contrast, inhibitors of p38 MAPK or of GSK-3beta activation did not affect the expression of IL-10 by these subsets (Figure S5B). Activation of the ERK1 and ERK2 signaling pathway is therefore a common requirement for the induction of IL-10 production by Th1, Th2, and Th17 cells.

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Input:
c-maf Expression Correlates with IL-10 Production in Th1, Th2, and Th17 Cells
To investigate further the downstream factors involved in regulating IL-10 production, we differentiated DO11.10 CD4+ T cells with increasing doses of OVA, in the presence or absence of IL-12, and quantified the expression of cytokines and transcription factors by real-time RT-PCR. Low-dose antigen resulted in transcription of Il4 and this was abrogated by both high antigen doses and IL-12 (Figure 6A). A low amount of transcription of Ifngamma was induced by IL-12 when cells were differentiated with low antigen dose, but this effect of IL-12 was markedly upregulated with increasing doses of antigen (Figure 6A). A low amount of Il10 transcription was observed at low doses of antigen accompanying Il4 expression (Th2 cell response), and this was abrogated by increased doses of antigen as was Il4 expression (Figure 6A). At low doses of antigen, IL-12 had little effect to increase IL-10 mRNA expression (Figure 6A) in keeping with the protein data (Figure 1A). However, IL-12 induced a high amount of Il10 transcription as well as Ifngamma expression with increased antigen doses (Figure 6A), again in keeping with the protein data (Figure 1A).
CD4+ T cells differentiated with increasing doses of antigen did not express high amounts of Tbx-21 (T-bet) mRNA, unless they were cocultured with IL-12 (Figure 6B). In contrast, high amounts of GATA-3 mRNA expression were only observed under Th2 cell differentiation conditions (low-dose antigen) (Figure 6B), and this expression was markedly downregulated by both increasing antigen dose and coculture in IL-12 (Figure 6B). Differentiation of T cells under low antigen dose led to expression of c-maf, in keeping with the Th2 cell profile (Ho et al., 1996), which was almost completely abrogated by increasing doses of antigen (Figure 6B). Interestingly, IL-12 sustained the high expression of c-Maf mRNA even at the highest antigen dose (Figure 6B). Moreover, IL-12 maintenance of c-maf expression required STAT4 activation (data not shown).
In Th17 cells that expressed IL-17a as well as IL-10 mRNA (Figure 6C), T-bet and GATA-3 mRNA were undetectable (data not shown), whereas that of ROR-gammat was high (Figure 6C) (Ivanov et al., 2007). Th17 cells also expressed high amounts of c-maf (Figure 6C), confirming a recent report (Bauquet et al., 2009). c-Maf is therefore expressed in all IL-10-expressing T cell populations tested (Figures 6B and 6C) and may not be just a Th2 cell-specific transcription factor as originally thought (Ho et al., 1996). We showed also that like Il10 expression, c-maf expression was inhibited in Th1 and Th17 cells in the presence of the MEK1 and MEK2 inhibitor (PD184352), whereas T-bet and RORgammat expression was hardly affected (Figure 6D).

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Input:
IL-10 expression by cells of the innate and adaptive immune systems reflects the importance of this cytokine in the tight regulation of the immune response, to minimize pathology during infection. IL-10 expression by Th1 cells has been reported to regulate the immune response in leishmaniasis and toxoplasmosis. However, in many situations, IL-10 is not produced by Th1 cells in response to antigenic stimulation. Our goal was to address the molecular signals that determine whether Th1 cells develop to produce IL-10 (Trinchieri, 2007). Here, we showed that Th1 cells required high-antigen-dose-induced ERK1 and ERK2 phosphorylation and IL-12-induced STAT-4 activation to produce IL-10. Our findings that ERK1 and ERK2 activation was a common pathway required for the production of IL-10 by Th1, Th2, and Th17 cell subsets, which differentiate along distinct pathways, such that IL-10 provides a highly regulated feedback loop to avoid the extremes of excessive inflammation or chronic infections and also allow a protective response to diverse pathogens.
In certain viral or parasitic infections (Anderson et al., 2007; Brooks et al., 2006; Ejrnaes et al., 2006), high amounts of stimulation may lead to the chronic nonhealing infection shown to be regulated by IL-10. During the course of infection, after initial triggering with antigen, T cells migrate to the tissue encountering high doses of antigen and factors produced by the innate immune response. Under these conditions, we speculate that Th1 cells will be induced to express high amounts of IL-10, in keeping with reports that IL-10-producing Th1 cells were found in CD4+ clones isolated from BAL but not blood of TB patients (Gerosa et al., 1999). Similarly, the immune response to a clinical isolate of L. major, which produces heavily infected nonhealing lesions, was found to be regulated by IL-10 derived from Foxp3- Th1 cells that coproduce IL-10 and IFN-gamma (Anderson et al., 2007), and the immune response during T. gondii infection was found also to be regulated by Foxp3- Th1 cells (Jankovic et al., 2007). It is likely that IL-10 production by Th1 cells is evoked under conditions of high inflammation and antigenic stimulation, whereas regulatory CD4+ T cells producing IL-10 may operate to regulate the immune response under conditions in which the pathogen is clinically controlled, such as in infection with L. major (Friedlin strain) (Belkaid et al., 2002; Suffia et al., 2006). We now also reported that CD4+ T cells cultured with high antigen dose and IL-12 differentiate into canonical Th1 effector cells, which, in addition to expressing large amounts of IFN-gamma and IL-10, lose their IL-2 expression as described before in certain chronic infection models (Sallusto et al., 2004). Our demonstration that loss of IL-2 is accompanied by production of IL-10 offers potential additional mechanisms whereby effector T cell responses may be dampened during chronic disease.
Using an in vivo transfer model of DO11.10 TCR transgenic cells (Castro et al., 2000), we showed that IL-10-producing Th1 cells were differentiated in the presence of high doses of OVA protein and LPS. We showed here that this induction of IL-10 in Th1 cells in vivo was markedly, but not totally, reduced in STAT4-deficient T cells as observed during T. gondii infection (Jankovic et al., 2002). A high antigenic activation during T. gondii infection or high antigen doses delivered in the presence of LPS, as seen in our system, may compensate for an absolute requirement for IL-12 in the induction of IL-10 by Th1 cells.
In our in vitro system, repeated stimulation of Th1 cells with high antigen doses allowed the development of Th1 cells producing IL-10 in an IL-12-dependent manner. IL-10 production by Th1 cells induced by high antigen dose and IL-12 was independent of IFN-gamma, in keeping with previous findings (Jankovic et al., 2002). However, a role for IFN-gamma in mediating IL-10 reactivation by Th1 cells during secondary infection with T. gondii has been suggested (Shaw et al., 2006). We have found that CD4+ T cells exposed to a high dose of antigen do not express IL-10 upon restimulation, but can be induced to produce IL-10 upon re-exposure to a high dose of antigen in the recall phase in the absence of added IL-12. However, this is dependent on the induction of IL-12 by antigen-presenting DCs. The combination of both high antigen dose and IL-12 resulted in the highest levels of IL-10 production and correlated with the high levels of ERK1 and ERK2 activation. The increased expression of IFN-gamma observed during the secondary phase will induce increased IL-12 production by DCs and suggests that repeated high-level TCR activation feeds back to upregulate IL-12 production by DC. It is thus likely that in T. gondii infection in vivo (Shaw et al., 2006), the requirement for IFN-gamma to induce IL-10, was for feedback upregulation of IL-12 by DCs, which in turn induced IL-10 in the Th1 cells.
Although IL-10 may be differentially regulated in Th1 and Th2 cells as has been reported (Chang et al., 2007; Wang et al., 2005), some studies suggest the existence of common pathways, but the molecular basis for these is as yet unclear. Costimulatory OX-40 signals have been shown to negatively regulate IL-10 production (Ito et al., 2005) both in Th1 and Th2 cells, whereas ICOS signaling has been suggested to induce IL-10 (Ito et al., 2007; Witsch et al., 2002) in both Th1 and Th2 cells. However, in some cases, ICOS signaling also regulates IL-4 production and Th2 responses (Greenwald et al., 2005). We now provide a common mechanism of ERK1 and ERK2 activation for the regulation of IL-10 production in Th1, Th2, and Th17 cells, although each subset differentiates along a distinct and subset-specific transcriptional pathway. This reinforces the fact that IL-10 is not a Th cell-subset-specific cytokine, but instead is produced in a tightly regulated fashion during each differentiation pathway. Of note, a role for ERK1 and ERK2 activation in the induction of IL-10 production has already been described for macrophages and DC (Agrawal et al., 2006; Hacker et al., 1999).
Differential transcriptional regulation of IL-10 in Th1 and Th2 cells has been suggested (Chang et al., 2007; Wang et al., 2005), and extensive histone acetylation of the IL-10 gene is detectable in fully polarized Th2 cells, but not Th1 cells (Chang et al., 2007). We provide evidence that IL-10 is produced in canonical Th1 cells and that its expression correlates with the expression of T-bet and the highest IFN-gamma production, in keeping with our observations that high-dose antigen stimulation and IL-12 signaling are required for IL-10 and IFN-gamma expression. It has also been shown that maintenance of IL-10 expression is conditional on IL-12 or IL-4 unless the IL-10 gene is imprinted by GATA-3 (Chang et al., 2007), which can remodel the IL-10 locus, thus explaining the highest amounts of IL-10 produced by Th2 cells (Chang et al., 2007; Shoemaker et al., 2006). We show here that high antigen dose and IL-12 drastically downregulate Gata-3 expression, suggesting that additional factors are in place to induce IL-10 expression in Th1 cells, albeit transiently. Expression of c-maf was greatly diminished by high antigen doses in T cells and yet was unexpectedly maintained by IL-12 and present in Th17 cells. That c-maf expression is common to IL-10-producing Th1, Th2, and Th17 cells and, like IL-10, is dependent on ERK activation in Th1 and Th17 cells for its expression is of interest because c-Maf has been shown to be an essential transcription factor for IL-10 expression in macrophages (Cao et al., 2005).
In summary, we show that although Th1, Th2, and Th17 CD4+ T cell subsets differentiate along distinct signaling and transcriptional pathways, they can all be induced to make IL-10. ERK1 and ERK2 activation is required for IL-10 production by all these Th cell subsets. With regard to the expression of IL-10 by Th1 cells, our data provide a mechanism for how IL-10 expression is induced and then amplified and regulated by the levels of antigen and IL-12 encountered in the environment. This provides a mechanism whereby a Th1 cell responds to extrinsic signals, reflecting increased inflammation in the tissue, to tightly regulate the production of IL-10 so as to allow a protective response to eradicate a pathogen with minimal damage to the host and also prevent chronic infection. Moreover, our findings have important implications for the regulation of IL-10 production during an inflammatory Th1 response in infection and may be of relevance for the design of vaccines and for strategies in immunotherapy in infectious diseases.

expression

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Input:
Mice, Cytokines, Antibodies, and Other Reagents
BALB/c DO11.10 mice transgenic for OVA-specific TCR WT or crossed back with Rag1-, IL-4-, IFN-gamma-, STAT4-, and STAT6-deficient mice were used as a source of antigen-specific T cells (Murphy et al., 1990; Ouyang et al., 1998; Shoemaker et al., 2006) and were bred and maintained under SPF conditions at the NIMR, London, Home Office, UK, Animals (Scientific Procedures) Act 1986 or at the Washington University School of Medicine. Female mice were used at 8-12 weeks old, and animal protocols were approved according to the Animals (Scientific Procedures) Act 1986, Home Office, UK. Reagents, including antibodies for T cell and DC preparation, purification and culture, media, cytokines, and cytokine mAbs have been described (Hosken et al., 1995; Shoemaker et al., 2006; Veldhoen et al., 2009; Veldhoen et al., 2006). LPS (S. minnesota) was from Alexis, chicken ovalbumin protein (OVA protein) was from from Sigma-Aldrich, and ovalbumin peptide323-339 (OVA) (endotoxin-free) was from Biosynthesis. U0126 was from BioMol International. PD184352 (MEK inhibitors), SB203580 (p38 inhibitor), and CT99021 (GSK3beta inhibitor) were kind gifts from P. Cohen and N. Shpiro, University of Dundee, UK.

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Input:
Isolation of CD4+ T Cells and of Splenic DC and Cell Culture for T Cell Phenotype Differentiation
T cells were sorted for CD4+CD62Lhi, CD4+CD62LhiCD25-, or CD4+CD44loCD25- to >98% on a Moflo cytometer (Cytomation) as before (Shoemaker et al., 2006; Veldhoen et al., 2009). In most cases, experiments were reproduced with each type of purified CD4+ T cell population with similar results obtained. Splenic DCs were prepared as described (Hosken et al., 1995), and sort purified CD11c+ cells were added to the T cell culture. Purified DO11.10 CD4+ T cells (1 x 105 cells/ml) were cultured as before (Hosken et al., 1995), in a total volume of 1 ml cRPMI medium in a 48-well plate, with splenic DCs (2 x 104 cells/ml), and varying amounts of OVA and of IL-12. APC-independent differentiation of naive CD4+ T cells into Th1 and Th2 cells used stimulation with anti-CD3 and anti-CD28 and appropriate cytokine conditions, and control Th1 and Th2 cells were cultured as described before (Hosken et al., 1995; Shoemaker et al., 2006). Culture conditions for Th17 cells were as described before (Veldhoen et al., 2006). Importantly, Th1 and Th2 cells could be differentiated in cRPMI or IMDM (Hosken et al., 1995; Shoemaker et al., 2006; Veldhoen et al., 2006), but Th17 cells were only differentiated optimally in IMDM (Veldhoen et al., 2009). When indicated, U0126 or PD184352 (MEK inhibitors), SB203580 (p38 inhibitor), CT99021 (GSK3beta inhibitor), or a similar amount of DMSO were present in the culture. More details of specific culture conditions are provided in Figures S6 and S7.

inhibitors

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Input:
Cytokine Detection by ICS and ELISA
At day 5 or 7, cells were restimulated with immobilized anti-CD3 (2 mug/ml) and anti-CD28 (2 mug/ml) (4 hr with BrefeldinA [10 mug/ml] in the last 2 hr) or with PdBU and Ionomycin (5 hr with BrefeldinA [1 mug/ml]). After ICS FACS, data were collected on a FACSCalibur (Becton Dickinson) and analyzed with FlowJo (Tree Star). For ELISA, cells were similarly restimulated for 48 hr and supernatant was collected and analyzed for IL-4, IL-10, and IFN-gamma as described before (Shoemaker et al., 2006).


Input:
In Vivo Studies
BALB/c mice were injected intravenously (i.v.) with a red blood cell-depleted single-cell spleen suspension (2.5 x 107 cells) (Castro et al., 2000) from DO11.10 WT or STAT-deficient mice. After 48 hr, they were injected subcutaneously with PBS or with OVA protein (5 mg) plus LPS (5 mug). The inguinal lymph nodes were removed 48 hr later. A single-cell suspension (1 x 106 cells) was restimulated for 24 or 48 hr with 1 muM or 3 muM of OVA and with BrefeldinA for the last 6 hr. Half of the supernatant was removed before the addition of BrefeldinA for use in an ELISA assay. The cells were fixed and stained as before. Those positive for KJ1-26-Bio and for CD4-PerCP were gated, and IFN-gamma and IL-10 staining was examined for this population and analyzed as before.

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Input:
Real-Time Quantitative RT-PCR
Cells were harvested and restimulated in the presence of immobilized anti-CD3 (2 mug/ml) plus anti-CD28 (2 mug/ml) for 3 hr or immediately lysed. RNA was extracted and reverse-transcribed and cDNA was analyzed for the expression of cytokines and transcription factors by real-time PCR assay as before (Shoemaker et al., 2006). Target gene mRNA expression was quantified either with SYBR Green (Applied Biosystems) or with Master Mix (Applied Biosystems) and normalized to ubiquitin or HPRT mRNA levels, respectively.

mRNA levels

ubiquitin

mRNA levels

HPRT


Input:
Immunoblotting
Differentiated CD4+ T cells were rested for 5 hr in 1% FCS-containing medium and restimulated as described for specific experiments. Cell lysates were prepared, equal amounts of protein were separated by SDS-PAGE, and phosphorylated or total ERK and actin were detected as described before (Beinke et al., 2004).


Input:
Differential cytokine regulation by NF-kappaB and AP-1 in Jurkat T-cells
Background
Activator protein (AP)-1 and nuclear factor (NF)-kappaB largely control T-cell activation, following binding of foreign antigens to the T-cell receptor leading to cytokine secretion. Elevated levels of pro-inflammatory cytokines and chemokines such as TNF, IL-6 and CXCL8 are associated with several human diseases including cystic fibrosis, pulmonary fibrosis and AIDS. The aim of this study was to investigate the role of the transcription factors, AP-1 and NF-kappaB, in IL-6 and CXCL8 regulation in Jurkat T-cells.
Results
Phorbol myristate acetate (PMA) exposure resulted in an up-regulation of AP-1 and down-regulation of NF-kappaB activity, however, exposure to heat killed (HK) Escherichia. coli MG1655 resulted in a dose-dependent increase in NF-kappaB activity without affecting AP-1. The cytokine profile revealed an up-regulation of the chemokine CXCL8 and the pro-inflammatory cytokines TNF, IL-2 and IL-6 following treatment with both PMA and HK E. coli, while the levels of the anti-inflammatory cytokine IL-10 were not affected by PMA but were significantly down-regulated by HK E. coli. AP-1 activation was significantly increased 2 h after PMA exposure and continued to increase thereafter. In contrast, NF-kappaB responded to PMA exposure by a rapid up-regulation followed by a subsequent down-regulation. Increased intracellular Ca2+ concentrations countered the down-regulation of NF-kappaB by PMA, while similar treatment with calcium ionophore resulted in a reduced NF-kappaB activity following induction with HK E. coli. In order to further study NF-kappaB activation, we considered two up-stream signalling proteins, PKC and Bcl10. Phosphorylated-PKC levels increased in response to PMA and HK E. coli, while Bcl10 levels significantly decreased following PMA treatment. Using an NF-kappaB activation inhibitor, we observed complete inhibition of IL-6 expression while CXCL8 levels only decreased by 40% at the highest concentration. Treatment of Jurkat T-cells with PMA in the presence of JNK-inhibitor suppressed both CXCL8 and IL-6 while PKC-inhibitor primarily decreased CXCL8 expression.
Conclusion
The present study shows that NF-kappaB regulated IL-6 but not CXCL8. This complex regulation of CXCL8 suggests that there is a need to further evaluate the signalling pathways in order to develop new treatment for diseases with elevated CXCL8 levels, such as AIDS and autoimmune diseases.

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Cytokines and chemokines are important in immune cell recruitment and in regulation of inflammatory responses [1]. T-cells produce a broad range of inflammatory mediators, including IL-2, IL-6, TNF and CXCL8, all of which are important in cell proliferation, differentiation, communication and initiation of inflammatory responses [2]. Elevated levels of pro-inflammatory cytokines and chemokines, such as TNF, IL-6 and CXCL8, are associated with several human diseases including cystic fibrosis [3-5], pulmonary fibrosis [6,7] and AIDS [8,9]. Induction of CXCL8 has been suggested to be mediated through NF-kappaB in cooperation with AP-1 [10,11], however the precise mechanism is not fully elucidated, and treatment strategies aimed at inhibiting CXCL8 have failed [12]. Persistent production of IL-6 and CXCL8 leads to chronic inflammation and enhanced survival of lymphocytes increasing serum cytokine/chemokine levels. This forms the basis of several autoimmune disorders including plasmacytosis and hyperplasia [13]. To develop viable CXCL8 based treatment strategies, it is necessary to identify the signalling pathways regulating CXCL8 and determine how this is coupled to NF-kappaB, AP-1 and IL-6.
The signalling pathways leading to NF-kappaB and AP-1 activation are overlapping, where both are involved in the induction and regulation of cytokines/chemokines. NF-kappaB is activated in response to stress, such as oxidative stress, bacterial toxins, viruses and UV light [14], and is essential for differentiation, proliferation and survival of many cell types including T-lymphocytes [15]. AP-1 activation requires Fos (c-Fos, FosB, Fra-1, Fra-2) and Jun (c-Jun, v-Jun, JunB, JunD) through the formation of homo- and hetero-dimers [16,17], and regulates transcription of a broad range of genes involved in immune responses [18-21]. Both AP-1 and NF-kappaB binding sites have been identified in the promoter region of IL-6 and CXCL8 [12,22], however, the mechanism by which these interleukins are regulated in T-cells is still not clear. CXCL8 is a C-X-C chemokine with properties enabling it to recruit T-cells and basophils and to activate neutrophils and monocytes [23]. IL-6 is a cytokine that possesses both pro- and anti-inflammatory characteristics and that plays a key role in haematopoiesis and acute-phase responses [24,25].
The present study suggests that the regulation of CXCL8 and IL-6 is uncoupled. Using Jurkat T-cells exposed to PMA and heat killed (HK) Escherichia coli MG1655 in combination with inhibitors of NF-kappaB, JNK and PKC, we demonstrated that NF-kappaB regulates IL-6 expression while the regulation of CXCL8 more closely correlated to AP-1 activity. These results indicate that inhibition of NF-kappaB is not an effective strategy in countering the high CXCL8 activities in diseases such as cystic fibrosis, AIDS and pulmonary fibrosis.

formation of homo- and hetero-dimers

c-Fos

c-Jun

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FosB

c-Jun

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c-Jun

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c-Jun

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v-Jun

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JunB

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Input:
Regulation of AP-1 and NF-kappaB activation
The transcription factors NF-kappaB and AP-1 play key roles in the initiation of an inflammatory response by inducing the expression and secretion of chemokines and cytokines that attract and activate immune cells. However, the signal transduction pathways and subsequent inflammatory cytokine induction by these transcription factors is not fully elucidated. The present study is aimed at determining the involvement of AP-1 and NF-kappaB in cytokine induction and regulation. PMA treatment resulted in an up-regulation of AP-1 after 2 h exposure and continued to increase throughout the analysis period (figure 1a). HK E. coli treatment did not affect AP-1 activation in Jurkat T-cells (figure 1b). To determine the involvement of associated pathways, we exposed cells to Ca2+ ionophore with or without PMA and observed a modest involvement of Ca2+ in PMA-dependent AP-1 activation (figure 1c) while Ca2+ alone did not alter AP-1 activity (data not shown). Furthermore, AP-1 activity decreased in a TCR-deficient Jurkat cell line when exposed to PMA compared to the parent cell line indicating that regulation of AP-1 was only partially T-cell receptor dependent (figure 1d).
NF-kappaB levels showed a transient increase at 1 min after exposure to PMA (figure 2a). However, 1 h after exposure the NF-kappaB levels began to drop reaching the lowest levels by 6 h, after which they increased again by 24 h. Exposure of Jurkat T-cells to HK E. coli resulted in a dose-dependent NF-kappaB activation, with the highest activity observed at a relative concentration of 5 x 107 CFU/ml (figure 2b). The time-dependent activation of NF-kappaB by HK E. coli was assessed further using the optimal concentration obtained from figure 2b and showed that the NF-kappaB activity increased after 3 h of exposure (figure 2c). Furthermore, increased intracellular Ca2+ reversed the PMA dependent NF-kappaB inhibition (figure 2d) and reduced the HK E. coli -dependent NF-kappaB activation (figure 2e).


Input:
Induction of inflammatory responses
The ability of PMA and HK E. coli to induce an inflammatory response in Jurkat T-cells was evaluated using a multiplex cytokine assay following 24 h stimulation. The cytokine profile revealed an enhanced induction of the pro-inflammatory cytokines IL-2, IL-6, TNF and the chemokine CXCL8. The levels of the anti-inflammatory cytokine IL-10 were unaffected by PMA but were significantly decreased by HK E. coli (Table 1). These results confirmed that PMA and HK E. coli induced an inflammatory response in the Jurkat T-cells. It is interesting to note that PMA was 120-fold more effective at inducing CXCL8 than HK E. coli. PMA-dependent induction of AP-1 and down-regulation of NF-kappaB suggests an involvement of AP-1 in CXCL8 regulation. Determination of the time course of cytokine induction in response to PMA showed that CXCL8 was already released between 2-6 h, while TNF and IL-6 were released between 6-24 h (figure 3). These results indicated that the cytokines were differentially regulated and that the release was not associated with the early transient induction of NF-kappaB. The temporal induction of AP-1 correlated to the CXCL8 levels and preceded the TNF and IL-6 release. This suggests an association between CXCL8 release and AP-1 signalling.

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Cooperative induction of cytokines by AP-1 and NF-kappaB
To further characterize the involvement of NF-kappaB in cytokine regulation, we treated cells with an NF-kappaB activation inhibitor (NAI). The results showed that NAI selectively down-regulated NF-kappaB activation (figures 4a and 4b) and did not alter AP-1 activity (figures 4c and 4d). Exposure of Jurkat T-cells to NAI resulted in a modest reduction of CXCL8 following PMA exposure, while it did not alter the CXCL8 release following HK E. coli exposure (figure 5a). NAI did not affect TNF expression (figure 5b) indicating that NF-kappaB is not the main regulator of CXCL8 or TNF following either PMA or HK E. coli exposure in Jurkat T-cells. In contrast, NAI resulted in a complete inhibition of IL-6 following PMA exposure and a 45% inhibition following HK E. coli exposure (figure 5c), suggesting an involvement of NF-kappaB in IL-6 regulation.
Ca2+ was observed to increase AP-1 activity (figure 1c) and reduce NF-kappaB activity (figure 2e); therefore, we exposed T-cells to a PKC inhibitor together with PMA to determine its effect on cytokine expression. Inhibition of PKC reduced CXCL8 release from 7 ng/ml to 3 ng/ml while it had a modest effect on IL-6 and TNF (figure 6a-c). This prompted us to test the effect of JNK inhibition on PMA-induced cytokine expression. JNK is involved in the regulation of a multitude of different transcription factors, including the phosphorylation and activation of c-Jun, c-Fos and p53, leading to cellular apoptosis [26]. Inhibition of the JNK pathways resulted in a down-regulation of both CXCL8 and IL-6, while no clear effect was observed on TNF expression (figure 6a-c). Analysis of mRNA levels using RT-qPCR (table 2) showed that PMA induced both il-6 and cxcl8 mRNA (5.1-fold and 111.8 fold respectively). Addition of the NF-kappaB inhibitor NAI and the JNK inhibitor reduced the il-6 expression below basal levels. In contrast, while the cxcl8 levels were suppressed by the same treatments the levels remained elevated above basal level.

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NF-kappaB inhibition due to PKC-dependent Bcl10 degradation
Western blot analysis revealed an up-regulation of phosphorylated-PKC after 24 h treatment of Jurkat T-cells with PMA or HK E. coli (figure 7a), while IkappaBbeta levels remained unaffected (figure 7b). Bcl10 is a signalling protein that acts upstream of NF-kappaB in concert with CARMA1 and MALT1 and has been suggested to directly regulate NF-kappaB activity in T-cells [27]. Therefore, Bcl10 activation was evaluated in both control and PMA stimulated cells after 10 min, 1 h, 6 h, and 24 h using western blot analysis. The Bcl10 levels decreased following treatment with PMA, while in control cells, Bcl10 returned to higher levels by 24 h (figure 7c). This suggests that Bcl10 is involved in the PMA dependent inhibition of NF-kappaB activation.

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Input:
NF-kappaB and AP-1 are critical regulators of inflammatory responses, proliferation and differentiation of T-cells [28-30], however, the signal transduction and subsequent cytokine/chemokine expression is not fully understood. The aim of the present study was to investigate IL-6 and CXCL8 regulation by NF-kappaB and AP-1 in Jurkat T-cells.
Our results demonstrated that PMA induced AP-1 activation, indicating a specific activation of the MAPK pathway. Furthermore, we demonstrated that PMA dependent AP-1 activation in T-cells was delayed (>2 h) and increased following long-term treatment. MAPK is one of the main signalling pathways in T-cells that regulate cell- and transcriptional activation [31,32]. Several studies [20,33,34] have indicated the importance of AP-1 in T-cell activation and the induction of inflammatory responses [35], including pro-inflammatory cytokine release. In contrast to AP-1, NF-kappaB activity rapidly increased (1 min) during PMA exposure followed by a down-regulation to the lowest levels at 6 h. This is in line with Park and colleagues [36] who demonstrated a rapid increase in NF-kappaB activity following short-term stimulation with PMA, but prolonged challenge resulted in a persistent inhibition of NF-kappaB. They showed that the inhibition of NF-kappaB was due to PKC-dependent degradation of IkappaB kinase beta and gamma in response to PMA. Interestingly, the HK E. coli exposure induced NF-kappaB activation without affecting AP-1 activity. Wang and colleagues [37] reported an elevated inflammatory response by obtaining expression of IL-6 following the exposure of T-cells to peptidoglycan. Reduction in NF-kappaB by calcium ionophore following HK E. coli stimulation may be due to the Ca2+ binding protein calmodulin (CaM), which has been shown to negatively regulate c-Rel when activated [38]. The regulation of NF-kappaB and AP-1 observed in the present study was in agreement with earlier studies.
T-cells produce a broad range of pro- and anti-inflammatory cytokines, including IL-2, IL-6, CXCL8, TNF and IL-10, in response to infections or other stress factors [39]. The assessment of Jurkat T-cell inflammatory responses (Table 1) revealed an enhanced IL-2 expression upon exposure to PMA or HK E. coli due to PKC activation [40]. In addition to the transcription factor NF-kappaB, AP-1 binding sites have been identified in the IL-6 promoter region, indicating multiple regulation [22]. AP-1 and NF-kappaB have also been demonstrated to regulate CXCL8 expression during induction of inflammatory responses in T-cells [12]. Furthermore, IL-6 and CXCL8 gene-expression is associated with an early immune response in Jurkat T-cells [41]. In the present study, both PMA and HK E. coli resulted in comparable increases in IL-6 while PMA was more potent at activating CXCL8 release. PMA and HK E. coli treatment also induced TNF expression. TNF is one of the first cytokines induced by T-cells [42] and its expression is regulated by calcineurin, NFAT and ATF-2/Jun [20]. However, PMA-stimulated Jurkat T-cells showed no difference in IL-10 expression indicating an induced inflammatory response. HK E. coli treatment resulted in a significant reduction of IL-10 expression. The anti-inflammatory cytokine IL-10 is known to inhibit T-cell activation, proliferation and the expression of pro-inflammatory cytokines, such as IL-2, IL-5 and INF-gamma [39,43] and regulate inflammatory responses by inducing T-cell anergy [44].
The time course analysis of cytokine expression showed a correlation between AP-1 and the chemokine CXCL8 where CXCL8 expression was significantly elevated already at 2-6 h after PMA exposure. The CXCL8 expression did not correlate with the early NF-kappaB activation (1 min) or with the down-regulation of NF-kappaB at 2 h post exposure. Both IL-6 and TNF expression were up regulated between 6-24 h. During this period, NF-kappaB increased from its minimum level at 6 h. PKC has been shown to be associated with the activation of AP-1, but not with NF-kappaB activation of the IL-2 promoter [45]. Mutation of the NF-kappaB site did not affect IL-2 expression, whereas mutation of the AP-1 site or PKC depletion almost revoked IL-2 release. These observations indicate that the MAPK pathway and the transcription factor AP-1 play an important role in the induction of inflammatory responses in Jurkat T-cells [12,20,22]. The obtained results signify that CXCL8 was primarily regulated through the MAPK pathway.
The NF-kappaB activation inhibitor (NAI) showed specificity against NF-kappaB and resulted in a complete IL-6 inhibition following induction with PMA and significant IL-6 reduction after HK E. coli treatment. CXCL8 was highly up regulated by PMA and the addition of NAI resulted in a minor reduction in CXCL8 expression. Furthermore, CXCL8 expression was not affected by NAI following HK E. coli treatment, indicating a lack of correlation between CXCL8 and NF-kappaB. Further analysis of CXCL8 expression revealed a down-regulation by PKC- and JNK- inhibitors, suggesting an involvement of AP-1 via PKC and JNK, respectively. Analysis of gene expression further confirmed that il-6 and cxcl8 were upregulated by PMA. Expression of il-6 dropped below basal levels following inhibition of NF-kappaB and JNK whereas cxcl8 remained elevated above basal levels (16.5-fold and 7.1-fold respectively) following the same treatment. Furthermore, inhibition of PKC did not result in a significant decrease of il-6 or cxcl8, which is in accordance with protein data (figure 6). These results suggest that NF-kappaB is involved in IL-6 regulation and release while it is not required for the expression of CXCL8 in Jurkat T cells.
The transcription factor NF-kappaB is responsible for a rapid immune response which is followed by an increase in transcription of IkappaB thus inhibiting NF-kappaB [46]. Activation of NF-kappaB in Jurkat T-cells is dependent on Bcl10 activation, which in turn is regulated by PKC. Recent studies have established the importance of a protein complex consisting of CARMA1, Bcl10 and MALT1 (CBM), in the induction of NF-kappaB. Investigating Bcl10, Scharschmidt and colleagues [47] demonstrated that it is a critical regulator of NF-kappaB activity. Down-regulation of Bcl10 from signals transduced via the TCR/CD28 and PKC resulted in a concomitant down-regulation of NF-kappaB. They suggested that Bcl10 is initially activated by TCR/PKC but that continued activation (>1 h) promotes its degradation. Narayan and colleagues [27] suggested that deletion in any of the three CBM complex proteins impairs antigen-receptor dependent activation of NF-kappaB. They showed that NF-kappaB activation via Akt requires CARMA1 and acts in cooperation with PKC following short-term exposure (30 min) of Jurkat T-cells with PMA. Akt phosphorylates and thus activates Bcl10. These studies indicate that PKC is crucial for NF-kappaB activation following short-term treatment through signals via membrane bound receptors such as the TCR and the co-stimulatory receptor CD28. Thus, the CBM complex proteins play a key role in this signalling process. PMA diffuses into the cytosol and directly activates PKC since it is an analogue to diacylglycerol [48]. Several studies have demonstrated that inhibition of PKC blocks NF-kappaB and AP-1 activity, suggesting a direct regulation of these transcription factors by PKC [49,50]. PKC is activated at an early stage following T-cell stimulation and is therefore an important regulator of downstream inflammatory signalling pathways leading to cytokine expression [50]. We have shown that phosphorylated-PKC is up regulated in response to PMA and HK E. coli, indicating an association between PKC and the transcription factors AP-1 and NF-kappaB. Bcl10 levels were down-regulated following extended treatment of Jurkat T-cells with PMA. In the control groups, a loss of Bcl10 occurred after 6 h, followed by an increase after 24 h, which is in accordance with the observed NF-kappaB activity (figure 7). These results are supported by an earlier study [47], demonstrating that prolonged PKC activation by PMA leads to an inhibition of Bcl10 and NF-kappaB.
It has been shown that NF-kappaB is an important transcription factor complex involved in almost every aspect of cell regulation including apoptosis, differentiation, proliferation and initiation of immune responses [51-53]. NF-kappaB is constitutively active in many human malignancies, which makes it an attractive therapeutic target [54]. Elevated CXCL8 levels during chronic inflammation result in an enhanced recruitment of immune cells to the site of infection, which may lead to the development of autoimmune diseases following secretion of pro-inflammatory cytokines. In HIV infected persons, serum CXCL8 levels are elevated and this could recruit more T-cells potentially leading to a more rapid progression of the disease since there will be more T-cells available to infect.

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Input: In the present study, IL-6 release was found to be associated with NF-kappaB activity while CXCL8 release more closely correlated with AP-1 activity. Treatment of Jurkat T-cells with PMA was more potent than HK E. coli at elevating the CXCL8 levels. PMA induced AP-1 activation and down-regulated NF-kappaB while HK E. coli up-regulated NF-kappaB without affecting AP-1 activity. In addition, the temporal induction pattern of AP-1 correlated to the release of CXCL8 while IL-6 followed the NF-kappaB activity. Likewise, blocking NF-kappaB activation resulted in a complete inhibition of IL-6 while the CXCL8 levels remained elevated as shown both at the protein and mRNA level. Furthermore, the CXCL8 release was down-regulated by inhibition of JNK activity. The present study indicates that in Jurkat T-cells, IL-6 is regulated through NF-kappaB while CXCL8 regulation is independent of NF-kappaB and closely associated with AP-1 activation.

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Input:
Chemicals
The following chemicals were used in the present study: PMA (Phorbol 12-myristate 13-acetate, (Sigma #P1585, USA)); NF-kappaB activation inhibitor (NAI), (InSolution(TM) NF-kappaB Activation Inhibitor, Calbiochem #481407, USA); JNK inhibitor, (InSolution(TM) JNK Inhibitor II, Calbiochem #420128, USA); PKC Inhibitor, (InSolution(TM) Bisindolylmaleimide I, Calbiochem #203293, USA); Calcium Ionophore, (Calcium Ionophore A23187 mixed calcium magnesium salt, Sigma #C5149, USA).


Input:
Heat killed (HK) Escherichia coli
E. coli MG1655 were grown on Luria-Bertani (LB) agar and incubated at 37degreesC overnight. One colony was inoculated into 10 ml LB broth and incubated on a shaker (200 rpm) at 37degreesC overnight. The bacteria were centrifuged for 10 min at 3000 x g, washed with 3 ml phosphate buffered saline (PBS; 8 g NaCl, 1.16 g Na2HPO4, 0.2 g KH2PO4, 0.2 g KCl, pH7) and resuspended in 50 mul PBS. The bacteria were killed by heating to 70degreesC for 1 h. To ensure that the bacteria were killed; 10 mul of the heat-killed suspension was spread on a LB plate and incubated overnight at 37degreesC.


Input:
Cell culturing, transfection and stimulation
Jurkat T-cells (wild type and TCR deficient- TCR-/-) were maintained in 90% RPMI 1640 medium (PAA laboratories, Austria) with 1.5 mM L-glutamine (Invitrogen, USA), 10% foetal bovine serum (Invitrogen, USA) and 1% antibiotic-antimycotic (Invitrogen, USA) and incubated in a stable environment of 5% CO2 at 37degreesC.
The cells were centrifuged at 1000 x g for 8 min and resuspended in fresh media to a final cell density of 1.6 x 107 cells/ml in a 24-well plate. Reporter plasmid (pNFkappaB-Luc, pAP1 (PMA)-TA-Luc, pNFkappaB-SEAP), internal control plasmid (pRL) (Promega, USA) and lipofectamine 2000 (Invitrogen, USA) were added to each well at 0.54 mug/well, 0.06 mug/well and 1.5 mul/well, respectively. Initially, the reporter plasmid and pRL were mixed separately with OptiMEM (Gibco, USA). After 5 min of incubation at room temperature, lipofectamine 2000 was added and the mixture was incubated further for 20 min at room temperature. The transfection was allowed to proceed overnight at 37degreesC, after which, the cells were centrifuged, the media removed and fresh pre-warmed media added. The cells were pre-incubated with NF-kappaB, JNK and PKC inhibitors and stimulated in 24-well plates with different concentrations of PMA, HK E. coli MG1655 and Calcium Ionophore A23187.
The cells were lysed and luciferase activity (NF-kappaB and AP-1) was measured using the Dual-Luciferase(R) reporter assay system (Promega, USA) according to the manufacturer's instructions on a TD 20/20 luminometer (Turner Designs, Sunnyvale, CA). Secreted alkaline phosphatase (NFkappaB-SEAP, figure 4a, b) levels were measured using Great EscAPe(TM) SEAP Detection Kit (Clontech, USA).

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Input:
Multiplex cytokine assay
Quantification of the levels of cytokines IL-2, IL-6, IL-10 and TNF and the chemokine CXCL8 was performed on culture supernatants using multiplexed biomarker immunoassay kits according to manufacturer's instructions (Bio-Rad Laboratories, Hercules, CA). A Bio-Plex(TM) 200 readout System was used (Bio-Rad), which utilizes Luminex(R) xMAP(TM) fluorescent bead-based technology (Luminex Corp., Austin). Levels were automatically calculated from standard curves using Bio-Plex Manager software (v.4.1.1, Bio-Rad).


Input:
Enzyme-linked immunosorbent assay (ELISA)
ELISA was performed on supernatants from challenged Jurkat T-cells to quantify IL-6, CXCL8 and TNF (BD OptEIA Human IL-6 Elisa Set, BD OptEIA Human CXCL8 Elisa Set and BD OptEIA Human TNF Elisa Set, Biosciences, USA) according to the manufacturer's instructions. Briefly, Jurkat T-cells were stimulated with PMA (162 nM) for 1 h, centrifuged (1000 x g, 8 min) and the supernatants were collected and stored at -80degreesC until use. Following centrifugation, the cells were resuspended in 1 h aged media, where cells have been grown in, containing PMA. The same procedure was performed to collect media after 2 h and 6 h. The final collection of media was performed after 24 h.


Input:
Western blot analysis
Following stimulation, Jurkat T-cells were centrifuged at 1000 x g for 8 min and lysed on ice for 2 h using sodium hydroxide with the addition of a protease inhibitor cocktail (Roche, Mannheim). The cells were further centrifuged at 8000 x g, 4degreesC for 10 min and the supernatants were transferred to new tubes. Cytoplasmic proteins (~8 mug) were separated by SDS-PAGE (10%) followed by western blotting using anti-IkappaBbeta, phospho-PKC (pan)(zeta Thr410)(190D10), anti-Bcl10 (Cell Signalling Technology, Boston) and beta-actin (Abcam, Cambridge). Detection was performed following incubation with ECL(TM) Anti-rabbit IgG, horseradish peroxidase linked whole antibodies (Amersham Biosciences, Buckinghamshire) and developed using ECL(TM) Western Blotting Detection Reagents (GE Healthcare, UK).


Input:
RNA extraction
Jurkat T-cells were treated with NF-kappaB, JNK and PKC inhibitors for 2 h in 6-well plates followed by stimulation with 162 nM PMA for 24 h. At sampling the cells were pelleted followed by RNA extraction using 100 mul TRI-reagent (Sigma, USA). This was followed by addition of 100 mul chloroform/isoamylalcohol (24/1). The solutions were mixed by vortexing followed by centrifugation at 12,000 rpm for 15 min at 4degreesC. The upper phase was transferred to a new tube followed by addition of 100 mul iso-propanol and incubated at room temperature for 10 min. RNA was then pelleted by centrifugation at 12,000 rpm for 15 min at 4degreesC and washed with 70% ethanol. The RNA pellet was dissolved in 25 mul RNase free water and the yield and ratio (A260/A280) was determined using NanoVue (GE Healthcare, UK). The samples were stored at -80degreesC until further use.


Input:
Reverse transcription quantitative PCR (RT-qPCR)
RT-qPCR was used to determine gene expression levels of il-6 and cxcl8 in response to PMA following inhibition of NF-kappaB, JNK and PKC. The following primer sequences were used, il-6: forward- TGTGAAAGCAGCAAAGAGGCACTG, reverse- ACAGCTCTGGCTTGTTCCTCACTA; cxcl8: forward- ACCACACTGCGCCAACACAGAAAT, reverse- AAACTTCTCCACAACCCTCTGCAC. Thermocycling conditions for CYBR Green (Quanta, USA) consisted of a denaturation step for 10 min at 95 degreesC followed by 60 cycles of 95degreesC for 1s and 60degreesC for 30s. Gene expression was analysed using Stratagene (Mx3000p(TM)) (AH diagnostics). The obtained Ct values were normalized against 18S. Initially, all measured 18S Ct values were used to calculate a mean Ct value that was used to determine the deltaCt values for each sample. Gene expression patterns for il-6 and cxcl8 were then normalized with regard to the samples 18S deltaCt.

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Statistical analysis
Statistical significant differences were determined using two-tailed Student's t-test (*p < 0.05; **p < 0.01; ***p < 0.001).


Input: AP-1 activation following long-term exposure of Jurkat T-cells to PMA. Jurkat T-cells were transfected with luciferase reporter plasmids containing the AP-1 cis-elements. (A) Time-dependent AP-1 activation in response to PMA. (B) HK E. coli does not activate AP-1. (C) Dose-dependent activation of AP-1 were performed using PMA alone (grey bars) and in combination with calcium ionophore (CaI 955 muM, black bars). (D) TCR-/- deficient cells responded to PMA (162 nM) by up-regulating AP-1 activity. Statistical significance from the control was determined using Student's t-test. (n = 4). Controls were arbitrarily set to 1.


Input: NF-kappaB down-regulation by PMA and up-regulation by heat killed E. coli MG1655 following long-term stimulation. Transfection of Jurkat T-cells was performed using luciferase reporter plasmids containing NF-kappaB cis-elements. (A) Time-dependent stimulation of Jurkat T-cells using PMA. NF-kappaB activation was evaluated using HK E. coli in a (B) dose- and (C) time-dependant manner. Calcium ionophore increased NF-kappaB activity following PMA exposure (E) and resulted in a negative regulation in response to HK E. coli stimulation (D). Statistical significance from the control was determined using Student's t-test. (n = 4). Controls were arbitrarily set to 1.


Input: CXCL8, IL-6 and TNF expression following long-term stimulation with PMA. Jurkat T-cells were either treated with media (white bars) or stimulated with PMA (black bars) and incubated for 1 h, 2 h, 6 h and 24 h. Aged media was added following each centrifugation step representative to the stimulation time (see Materials and methods). Cytokine levels (A) CXCL8, (B) IL-6 and (C) TNF were detected by ELISA. Statistical significance from the control was determined using Student's t-test. (n = 3).

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Input: Inhibition of NF-kappaB activity by NAI. Jurkat T-cells were transfected with luciferase reporter plasmids containing either NF-kappaB or AP-1 cis-acting elements. The cells were incubated with NF-kappaB activation inhibitor (NAI) for 1 h followed by stimulation with PMA (162 nM, A and C) or HK E. coli (5 x 107 CFU/ml, B and D) for 24 h. (A, B) NF-kappaB, but not (C, D) AP-1 was inhibited. Statistical significance from the positive control (PMA/HK E. coli) was determined using Student's t-test. (n = 4). Controls were set to 1.


Input: Involvement of NF-kappaB in cytokine regulation. Cytokine/chemokine levels were determined using ELISA following incubation of Jurkat T-cells with NAI (1 h) and stimulation (24 h). (A) CXCL8 expression was partially inhibited following PMA stimulation (grey bars), whereas the levels were not altered following stimulation with HK E. coli (black bars), this indicates an induction mainly regulated by AP-1. (B) TNF expression was not affected by NAI. (C) IL-6 release was completely inhibited by NAI following PMA exposure, indicating regulation through NF-kappaB since IL-6 expression was significantly increased in response to HK E. coli than PMA. Statistical significance from the positive control (PMA/HK E. coli) was determined using Student's t-test. (n = 3).

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Input: Involvement of PKC and JNK in cytokine expression. Jurkat T-cells were incubated with PKC- and JNK inhibitors for 1 h followed by stimulation with PMA (162 nM) for 24 h. (A) CXCL8 expression was inhibited in a dose-dependent manner by both inhibitors. (B) JNK-I revealed a stronger inhibitory effect on IL-6 expression than the PKC-I. (C) TNF expression was not affected by either inhibitor. Statistical significance from the positive control (PMA) was determined using Student's t-test. (n = 3).

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Input: NF-kappaB inhibition by PMA correlated to PKC-dependent Bcl10 degradation. Levels of intracellular protein were assessed following 24 h stimulation with PMA (162 nM) or HK E. coli (5 x 107 CFU/ml). (A) Phospho-PKC increased in response to PMA and HK E. coli stimulation. (B) IkappaB decreased following stimulation with HK E. coli indicating NF-kappaB activation. (C) Bcl10 activation was inhibited following long-term stimulation with PMA, which explains the inhibitory effect of PMA on NF-kappaB activation. beta-actin was used as a loading control. (n = 3).

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Input: Jurkat T-cells were stimulated with 162 nM PMA and 5 x 107 CFU/ml HK E. coli for 24 h.


Input: Jurkat T-cells were treated with 10 nM NAI, 10 muM JNK-I or10 nM PKC-I for 2 h followed by induction with 162 nM PMA for 24 h.


Input:
HOIL-1L Interacting Protein (HOIP) Is Essential for CD40 Signaling
CD40 is a cell surface receptor important in the activation of antigen-presenting cells during immune responses. In macrophages and dendritic cells, engagement of CD40 by its ligand CD154 provides signals critical for anti-microbial and T cell-mediated immune responses, respectively. In B cells, CD40 signaling has a major role in regulating cell proliferation, antibody production, and memory B cell development. CD40 engagement results in the formation of a receptor-associated complex that mediates activation of NF-kappaB, stress-activated protein kinases, and other signaling molecules. However, the mechanisms that link CD40 to these signaling events have been only partially characterized. Known components of the CD40 signaling complex include members of the TNF receptor-associated factor (TRAF) family of proteins. We previously showed that the TRAF family member TRAF2 mediates recruitment of HOIL-1L-interacting protein (HOIP) to the cytoplasmic domain of CD40, suggesting that HOIP has a role in the CD40 signaling pathway. To determine the role of HOIP in CD40 signaling, we used somatic cell gene targeting to generate mouse B cell lines deficient in HOIP. We found that the CD40-induced upregulation of CD80 and activation of germline immunoglobulin epsilon transcription were defective in HOIP-deficient cells. We also found that the CD40-mediated activation of NF-kappaB and c-Jun kinase was impaired. Recruitment of IkappaB kinase proteins to the CD40 signaling complex was undetectable in HOIP-deficient cells, potentially explaining the defect in NF-kappaB activation. Restoration of HOIP expression reversed the defects in cellular activation and signaling. These results reveal HOIP as a key component of the CD40 signaling pathway.

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