Yersinia enterocolitica leads to transient induction of TNF-alpha and activates NF-kappaB in synovial fibroblasts.

OBJECTIVE: The importance of the presence of bacterial antigen or even living bacteria for the pathogenesis of reactive arthritis has been discussed increasingly ever since bacterial antigen was found in inflamed joints. Bacteria may persist in the body and drive the local immune response, maintaining arthritis. Cytokines, in particular tumor necrosis factor-alpha (TNF-alpha) are essential for bacterial elimination. In reactive arthritis, the course of the disease is influenced by several cytokines, including TNF-alpha. TNF-alpha expression can be mediated by transcription factor nuclear factor-kappa B (NF-kappaB). Moreover, TNF-alpha is also one of the strongest activators of NF-kappaB. METHODS: In vitro expression of TNF-alpha and activation of NF-kappaB in synovial fibroblasts after infection with Yersinia enterocolitica or Salmonella enteritidis was analysed by electrophoretic mobility shift assay, Western blot assay and real-time PCR. RESULTS: We found that infection of synovial fibroblasts with yersinia and salmonellae lead to the transient expression of TNF-alpha mRNA and induction of NF-kappaB. CONCLUSION: Induction of TNF-alpha in synovial fibroblasts after infection with yersiniae or salmonellae might be insufficient to eliminate bacteria, and this could allow the intracellular persistence of these bacteria. Our results therefore support the hypothesis that a permissive cytokine pattern might contribute to the pathogenesis of reactive arthritis.

IL-9 and IL-13 production by activated mast cells is strongly enhanced in the presence of lipopolysaccharide: NF-kappa B is decisively involved in the expression of IL-9.

Mast cells, due to their ability to produce a large panel of mediators and cytokines, participate in a variety of processes in adaptive and innate immunity. Herein we report that in primary murine bone marrow-derived mast cells activated with ionomycin or IgE-Ag the bacterial endotoxin LPS strongly enhances the expression of IL-9 and IL-13, but not IL-4. This costimulatory effect of LPS is absent in activated mast cells derived from the LPS-hyporesponsive mouse strain BALB/c-LPS(d), although in these cells the proinflammatory cytokine IL-1 can still substitute for LPS. The enhanced production of mast cell-derived IL-13 in the presence of IL-1 is a novel observation. Coactivation of mast cells with LPS leads to a synergistic activation of NF-kappa B, which is shown by an NF-kappa B-driven reporter gene construct. In the presence of an inhibitor of NF-kappa B activation, the production of IL-9 is strongly decreased, whereas the expression of IL-13 is hardly reduced, and that of IL-4 is not affected at all. NF-kappa B drives the expression of IL-9 via three NF-kappa B binding sites within the IL-9 promoter, which we characterize using gel shift analyses and reporter gene assays. In the light of recent reports that strongly support critical roles for IL-9 and IL-13 in allergic lung inflammation, our results emphasize the potential clinical importance of LPS as an enhancer of mast cell-derived IL-9 and IL-13 production in the course of inflammatory reactions and allergic diseases.

Signal-specific and phosphorylation-dependent RelB degradation: a potential mechanism of NF-kappaB control.

RelB is an unusual member of the Rel/NF-kappaB family of transcription factors which are involved in oncogenic processes. Due to a relaxed control by the IkappaBs, the cytosolic NF-kappaB inhibitors, RelB is constitutively expressed in the nuclei of lymphoid cells. We show here that RelB is inducibly degraded upon activation of T cells in a fashion similar to the IkappaBs. However, RelB degradation differs from that of IkappaBs since it is not induced by TNFalpha but only by T cell receptor or TPA/ionomycin stimulation. Moreover, RelB degradation occurs in three steps: (i) after stimulation RelB is rapidly phosphorylated at amino acids Thr84 and Ser552 followed by (ii) an N-terminal cut and, finally, (iii) the complete degradation in the proteasomes. Since mutation of the two phosphoacceptor sites to non-acceptor sites abolished RelB phosphorylation in vivo and led to the stabilization of the mutated RelB(DM), site-specific phosphorylation appears to be a necessary prerequisite for RelB degradation. RelB is a crucial regulator of NF-kappaB-dependent gene expression. Thus, the signal-induced degradation of RelB should be an important control mechanism of NF-kappaB activity.

The role of NF-AT transcription factors in T cell activation and differentiation.

The family of genuine NF-AT transcription factors consists of four members (NF-AT1 [or NF-ATp], NF-AT2 [or NF-ATc], NF-AT3 and NF-AT4 [or NF-ATx]) which are characterized by a highly conserved DNA binding domain (is designated as Rel similarity domain) and a calcineurin binding domain. The binding of the Ca(2+)-dependent phosphatase calcineurin to this region controls the nuclear import and exit of NF-ATs. This review deals (1) with the structure of NF-AT proteins, (2) the DNA binding of NF-AT factors and their interaction with AP-1, (3) NF-AT target genes, (4) signalling pathways leading to NF-AT activation: the role of protein kinases and calcineurin, (5) the nuclear entry and exit of NF-AT factors, (6) transcriptional transactivation by NF-AT factors, (7) the structure and expression of the chromosomal NF-AT2 gene, and (8) NF-AT factors in Th cell differentiation. The experimental data presented and discussed in the review show that NF-AT factors are major players in the control of T cell activation and differentiation and, in all likelihood, also of the cell cycle and apoptosis of T lymphocytes.

C/EBPbeta enhances IL-4 but impairs IL-2 and IFN-gamma induction in T cells.

C/EBP transcription factors have been described to control the activity of the human IL-4 promoter. The C/EBP binding sites within the IL-4 promoter overlap with composite NF-AT and AP-1 binding motifs. We show here that similar binding sites are part of the murine IL-4 promoter. Retroviral overexpression of C/EBPbeta in murine EL-4 thymoma cells led to a strong induction of endogenous IL-4 and a reduction in IL-2 and IFN-gamma expression. Similarily, in primary murine T cells C/EBPbeta induction resulted in an increase in IL-4 levels, whereas in human Jurkat T cells a decrease in IL-2 RNA was detected. Like AP-1, C/EBP factors belong to the large class of basic leucine zipper proteins. However, unlike AP-1, C/EBPbeta does not act in synergy with NF-AT in the induction of the murine IL-4 promoter. Instead, both factors compete in their binding to the P4/Pu-bD site, one of the most important sequence elements of the IL-4 promoter. Whereas NF-AT factors require high levels of free Ca2+ and calcineurin activity for induction, C/EBP induction in T cells is Ca2+/calcineurin independent. These observations suggest that various induction conditions lead to the activation of transcription factors, inducing IL-4 promoter activity at specific developmental stages of T cells.

The C-class chemokine, lymphotactin, impairs the induction of Th1-type lymphokines in human CD4(+) T cells.

Chemokines are involved in the regulation of leukocyte migration and for some of them, T-cell costimulation. To date, the only direct property of lymphotactin (Lptn), the unique member of the C class of chemokines, consists of T-cell chemoattraction. This report describes a novel function for Lptn in human T-lymphocyte biology, by demonstrating the direct ability of Lptn to both inhibit and costimulate CD4(+) and CD8(+) T-cell activation, respectively. Lptn but not RANTES inhibited CD4(+) T-cell proliferation, through a decreased production of Th1 (interleukin [IL]-2, interferon [IFN]-gamma) but not Th2 (IL-4, IL-13) lymphokines, and decreased IL-2R alpha expression. Transfections in Jurkat cells showed a Lptn-mediated transcriptional down-regulation of gene-promoter activities specific for Th1-type lymphokines, as well as of nuclear factor of activated T cells (NF-AT) but not AP-1 or NF-KB enhancer activities. This suppressive action of Lptn could be compensated by overexpression of NF-ATc but not NF-ATp. CD4(+) T-cell proliferation was completely restored by exogenous IL-2 or reversed by pertussis toxin, wortmannin, and genistein, suggesting the involvement of multiple partners in Lptn signaling. In contrast to CD4(+) cells, Lptn exerted a potent costimulatory activity on CD8(+) T-cell proliferation and IL-2 secretion. These data provide important insights into the role of Lptn in differential regulation of normal human T-cell activation and its possible implication in immune response disorders. (Blood. 2000;96:420-428)

The environmental pollutant pyrene induces the production of IL-4.

BACKGROUND: Epidemiologic studies and experiments with mouse models suggest that polyaromatic hydrocarbons contained in, among others, diesel exhaust particles can promote the development of allergy. OBJECTIVE: Because IL-4 organizes allergic responses in vivo, we have investigated whether pyrene, a major compound of diesel exhaust particles, can affect the production of IL-4. METHODS: IL-4 production by primary human T cells was assessed by ELISA and messenger RNA transcription was detected by Northern blotting. Activation of the IL-4 promoter was tested in reporter gene assays with transiently transfected cell lines. RESULTS: Pyrene induced transcription of IL-4 messenger RNA and expression of IL-4 protein in primary human T cells. Pyrene, but not related polyaromatic hydrocarbons, enhanced basal transcription of the human and mouse IL-4 promoter. CONCLUSION: Our results suggest that pyrene may promote allergic diseases by inducing the production of IL-4.

STAT6 and the regulation of CD23 expression in B-chronic lymphocytic leukemia.

High CD23 expression is a hallmark of B-CLL cells. It is lost during in vitro culture and can be reinduced by IL-4, albeit to a lower extent than in normal B cells. To elucidate the events controlling CD23 expression in B-CLL cells, the IL-4 mediated induction of STAT6 was investigated. Western-blot analysis demonstrated that B-CLL cells contain comparable amounts of STAT6. Electrophoretic mobility shift assays (EMSA) showed no constitutive nuclear translocation of STAT6. IL-4 induced the translocation of STAT6 in B-CLL cells from all 22 patients investigated. The increase was transient, dose and time dependent without a distinct difference between B-CLL cells and non-malignant B cells. However, in contrast to normal B lymphocytes no strict correlation between CD23 expression and STAT6 activation was detected in B-CLL. Therefore further signalling pathways and transcription factors in addition to STAT6 have to be activated to explain the high expression of CD23 in B-CLL cells. For example, STAT1 which is induced by IFN-gamma and binds to the classical STAT6 site. It might be involved in the strong induction of CD23 on B-CLL cells after cotreatment with IL-4 and IFN-gamma, while in non-malignant B lymphocytes IFN-gamma leads to a reduction of IL-4 mediated CD23 expression.

17beta-estradiol prevents programmed cell death in cardiac myocytes.

The cardioprotective effects of estrogens are clearly established. However, the underlying mechanisms are poorly understood. Because programmed cell death (apoptosis) probably contributes to the loss of cardiac myocytes in heart failure and because estrogens prevent apoptosis in breast cancer cells, we investigated whether the loss of cardiac myocytes by programmed cell death could be prevented by physiological doses of 17beta-estradiol. Apoptosis of cultured cardiac myocytes was induced by staurosporine. 17beta-estradiol (10 nM) had an antiapoptotic effect as determined by morphological analysis, vital staining using the Hoechst dye 33342 and terminal transferase dUTP nick-end labeling (TUNEL). As a potential mechanism for the antiapoptotic effect of 17beta-estradiol we found a reduced activity of the ICE-like protease caspase-3 in hormone-treated myocytes. Furthermore, inhibition of apoptosis by estradiol was associated with a reduced activity of NF-kappaB transcription factors, particularly p65/RelA and p50. To our knowledge, these data provide the first indication that 17beta-estradiol in physiological concentrations inhibits apoptosis in cardiac myocytes. The antiapoptotic effect of estrogens might contribute to the known cardioprotective effect of estrogens and provides a starting point for the development of future treatment options.

Differential expression of Rel/NF-kappaB and octamer factors is a hallmark of the generation and maturation of dendritic cells.

A key feature of maturation of dendritic cells is the down-regulation of antigen-processing and up-regulation of immunostimulatory capacities. To study the differential expression of transcription factors in this process, we investigated the nuclear translocation and DNA binding of Rel/NF-kappaB and octamer factors during in vitro generation and maturation of dendritic cells compared with macrophage development. RelB was the only factor strongly up-regulated during the generation of both immature dendritic cells and macrophages. Cytokine-induced maturation of dendritic cells resulted in an increase in nuclear RelB, p50, p52, and especially c-Rel, whereas cytokine-treated macrophages responded poorly. This up-regulation of NF-kappaB factors did not correlate with lower levels of cytosolic NF-kappaB inhibitors, the IkappaBs. One IkappaB, Bcl-3, was strongly expressed only in mature dendritic cells. Furthermore, generation and maturation of dendritic cells led to a continuous down-regulation of the octamer factor Oct-2, whereas monocytes and macrophages displayed high Oct-2 levels. A similar pattern of maturation-induced changes in transcription factor levels was found in cultured murine epidermal Langerhans cells, suggesting a general physiological significance of these findings. Finally, this pattern of differential activation of Rel and octamer factors appears to be suitable in determining the maturation stage of dendritic cells generated by treatment with different cytokine combinations in vitro. (Blood. 2000;95:277-285)

CD4-mediated signals induce T cell dysfunction in vivo.

Triggering of CD4 coreceptors on both human and murine T cells can suppress TCR/CD3-induced secretion of IL-2. We show here that pretreatment of murine CD4+ T cells with the CD4-specific mAb YTS177 inhibits the CD3-mediated activation of the IL-2 promoter factors NF-AT and AP-1. Ligation of CD4 molecules on T cells leads to a transient stimulation of extracellular signal-regulated kinase (Erk) 2, but not c-Jun N-terminal kinase (JNK) activity. Pretreatment with anti-CD4 mAb impaired anti-CD3-induced Erk2 activation. Costimulation with anti-CD28 overcame the inhibitory effect of anti-CD4 Abs, by induction of JNK activation. The in vivo relevance of these studies was demonstrated by the observation that CD4+ T cells from BALB/c mice injected with nondepleting anti-CD4 mAb were inhibited in their ability to respond to OVA Ag-induced proliferation and IL-2 secretion. Interestingly, in vivo stimulation with anti-CD28 mAb restored IL-2 secretion. Furthermore, animals pretreated with anti-CD4 elicited enhanced IL-4 secretion induced by OVA and CD28. These observations suggest that CD4-specific Abs can inhibit T cell activation by interfering with signal 1 transduced through the TCR, but potentiate those delivered through the costimulatory molecule CD28. These studies have relevance to understanding the mechanism of tolerance induced by nondepleting anti-CD4 mAb used in animal models for allograft studies, autoimmune pathologies, and for immunosuppressive therapies in humans.

CBP/p300 integrates Raf/Rac-signaling pathways in the transcriptional induction of NF-ATc during T cell activation.

NF-ATc, an inducibly expressed transcription factor, controls gene expression in T lymphocytes and cardiomyocytes. We show here that the transcriptional co-activators CBP/p300 bind to and control the activity of the inducible N-terminal transactivation domain of NF-ATc, TAD-A. Similar to the N terminal transactivation domain of c-Jun, TAD-A is inducibly phosphorylated, but this phosphorylation is dispensable for the interaction with CBP/p300. Constitutive active versions of c-Raf and Rac synergistically enhance the CBP/p300-mediated increase of TAD-A activity, indicating the important role CBP/p300 plays in the integration of T cell activation signals. Since a mutation of CBP abolishing HAT activity is almost as active as wild-type CBP in T cells, functions of CBP/p300 other than histone acetylation appear to control the NF-AT-dependent transcription in T cells.

Multiple NF-ATc isoforms with individual transcriptional properties are synthesized in T lymphocytes.

The transcription factor NF-ATc that controls gene expression in T lymphocytes and embryonic cardiac cells is expressed in three prominent isoforms. This is due to alternative splice/polyadenylation events that lead to the predominant synthesis of two long isoforms in naive T cells and a shorter NF-ATc isoform in effector T cells. Whereas the previously described isoform NF-ATc/A contains a relatively short C terminus, the longer isoforms, B and C, span extra C-terminal peptides of 128 and 246 aa, respectively. We show here that in addition to the strong N-terminal trans-activation domain, TAD-A, which is common to all three NF-ATc isoforms, NF-ATc/C contains a second trans-activation domain, TAD-B, in its C-terminal peptide. Various stimuli of T cells that induce the activity of TAD-A also enhance the activity of TAD-B, but, unlike TAD-A, TAD-B remains unphosphorylated by protein from 12-O-tetradecanoyl 12-phorbol 13-acetate-stimulated T cells. The shorter C-terminal peptide of isoform NF-ATc/B exerts a suppressive transcriptional effect. These properties of NF-ATc/B and -C might be of importance for gene regulation in naive T lymphocytes in which NF-ATc/B and -C are predominantly synthesized.

Alternative polyadenylation events contribute to the induction of NF-ATc in effector T cells.

The transcription factor NF-ATc is synthesized in three prominent isoforms. These differ in the length of their C terminal peptides and mode of synthesis. Due to a switch from the use of a 3' polyA site to a more proximal polyA site, NF-ATc expression switches from the synthesis of the two longer isoforms in naive T cells to that of short isoform A in T effector cells. The relative low binding affinity of cleavage stimulation factor CstF-64 to the proximal polyA site seems to contribute to its neglect in naive T cells. These alternative polyadenylation events ensure the rapid accumulation of high concentrations of NF-ATc necessary to exceed critical threshold levels of NF-ATc for gene induction in effector T cells.

GA-binding protein factors, in concert with the coactivator CREB binding protein/p300, control the induction of the interleukin 16 promoter in T lymphocytes.

Interleukin 16 (IL-16) is a chemotactic cytokine that binds to the CD4 receptor and affects the activation of T cells and replication of HIV. It is expressed as a large 67-kDa precursor protein (pro-IL-16) in lymphocytes, macrophages, and mast cells, as well as in airway epithelial cells from asthmatics after challenge with allergen. This pro-IL-16 is subsequently processed to the mature cytokine of 13 kDa. To study the expression of IL-16 at the transcriptional level, we cloned the human chromosomal IL-16 gene and analyzed its promoter. The human IL-16 gene consists of seven exons and six introns. The 5' sequences up to nucleotide -120 of the human and murine IL-16 genes share >84% sequence homology and harbor promoter elements for constitutive and inducible transcription in T cells. Although both promoters lack any TATA box, they contain two CAAT box-like motifs and three binding sites of GA-binding protein (GABP) transcription factors. Two of these motifs are part of a highly conserved and inducible dyad symmetry element shown previously to control a remote IL-2 enhancer and the CD18 promoter. In concert with the coactivator CREB binding protein/p300, which interacts with GABPalpha, the binding of GABPalpha and -beta to the dyad symmetry element controls the induction of IL-16 promoter in T cells. Supplementing the data on the processing of pro-IL-16, our results indicate the complexity of IL-16 expression, which is tightly controlled at the transcriptional and posttranslational levels in T lymphocytes.

The interleukin 2 receptor alpha chain/CD25 promoter is a target for nuclear factor of activated T cells.

The expression of the murine interleukin (IL)-2 receptor alpha chain/CD25 is strongly induced at the transcriptional level after T cell activation. We show here that nuclear factor of activated T cell (NF-AT) factors are involved in the control of CD25 promoter induction in T cells. NF-ATp and NF-ATc bind to two sites around positions -585 and -650 located upstream of the proximal CD25 promoter. Immediately 3' from these NF-AT motifs, nonconsensus sites are located for the binding of AP-1-like factors. Mutations of sites that suppress NF-AT binding impair the induction and strong NF-ATp-mediated transactivation of the CD25 promoter in T cells. In T lymphocytes from NF-ATp-deficient mice, the expression of CD25 is severely impaired, leading to a delayed IL-2 receptor expression after T cell receptor (TCR)/CD3 stimulation. Our data indicate an important role for NF-AT in the faithful expression of high affinity IL-2 receptors and a close link between the TCR-mediated induction of IL-2 and IL-2 receptor alpha chain promoters, both of which are regulated by NF-AT factors.

Retarded thymic involution and massive germinal center formation in NF-ATp-deficient mice.

NF-ATp and NF-ATc are the most prominent nuclear NF-AT transcription factors in peripheral T lymphocytes. After T cell activation both factors bind to and control the promoters and enhancers of numerous lymphokine and receptor ligand genes. In order to define a specific role for NF-ATp in vivo we have inactivated the NF-ATp gene by gene targeting in mice. We show that NF-ATp deficiency leads to the accumulation of peripheral T cells with a "preactivated" phenotype, enhanced immune responses of T cells after secondary stimulation in vitro and severe defects in the proper termination of antigen responses, as shown by a reduced deletion of superantigen-reactive CD4+ T cells. These alterations in the function of the immune system are correlated with drastic changes in the morphology of lymphoid organs. Approximately 25 % of NF-ATp-deficient mice older than 6 months develop large germinal centers in the spleen and peripheral lymph nodes. In addition, they exhibit a pronounced retardation in the involution of the thymus. The thymus of these NF-ATp-deficient mice exhibits large cortical areas typical for newborn mice and a massive infiltration of IgM+/ IgD+ B lymphocytes. Contrary to the T lymphocytes from IL-2-deficient mice which develop a phenotype similar to the NF-ATp-/- mice, NF-ATp-/- T cells do not show obvious defects in Fas-mediated apoptosis. This might indicate defects in other types of programmed cell death which are controlled by the activity of NF-ATp.

A CD28-associated signaling pathway leading to cytokine gene transcription and T cell proliferation without TCR engagement.

Stimulation of resting human T cells with the CD28-specific mAb BW 828 induces proliferation and cytokine synthesis without further requirement for TCR coengagement. This observation prompted us to postulate that signal 2 (costimulatory signal) alone without signal 1 (TCR signal) can activate T cells. To test whether this putative function of CD28 is mediated via a particular signaling pathway, we compared early signaling events initiated in resting T cells by the stimulatory mAb BW 828 with signals triggered by the nonstimulating CD28 mAb 9.3. Stimulation of T cells with BW 828 induced an increase in intracellular Ca2+, but did not lead to detectable activation of the protein kinases p56(lck) and c-Raf-1. This pathway resulted in the induction of the transcription factors NF-kappa B, NF-AT, and proteins binding to the CD28 response element of the IL-2 promoter. On the other hand, stimulation of T cells with mAb 9.3 increased the level of intracellular Ca2+ and triggered the activation of p56(lck) and c-Raf-1, but was unable to induce the binding of transcription factors to the IL-2 promoter. In contrast to the differential signaling of BW 828 and 9.3 in resting T cells, the two mAbs exhibited a similar pattern of early signaling events in activated T cells and Jurkat cells (p56(lck) activation, association of phosphatidylinositol 3-kinase with CD28), indicating that the signaling capacity of CD28 changes with activation. These data support the view that stimulation through CD28 can induce some effector functions in T cells and suggest that this capacity is associated with a particular pattern of early signaling events.

The GABP-responsive element of the interleukin-2 enhancer is regulated by JNK/SAPK-activating pathways in T lymphocytes.

T cell activation leads via multiple intracellular signaling pathways to rapid induction of interleukin-2 (IL-2) expression, which can be mimicked by costimulation with 12-O-tetradecanoylphorbol-13-acetate (TPA) and ionomycin. We have identified a distal IL-2 enhancer regulated by the Raf-MEK-ERK signaling pathway, which can be induced by TPA/ionomycin treatment. It contains a dyad symmetry element (DSE) controlled by the Ets-like transcription factor GA-binding protein (GABP), a target of activated ERK. TPA/ionomycin treatment of T cells stimulates both mitogen-activated ERK, as well as the stress-activated mitogen-activated protein kinase family members JNK/SAPK and p38. In this study, we investigated the contribution of the stress-activated pathways to the induction of the distal IL-2 enhancer. We show that JNK- but not p38-activating pathways regulate the DSE activity. Furthermore, the JNK/SAPK signaling pathway cooperates with the Raf-MEK-ERK cascade in TPA/ionomycin-induced DSE activity. In T cells, overexpression of SPRK/MLK3, an activator of JNK/SAPK, strongly induces DSE-dependent transcription and dominant negative kinases of SEK and SAPK impair TPA/ionomycin-induced DSE activity. Blocking both ERK and JNK/SAPK pathways abolishes the DSE induction. The inducibility of the DSE is strongly dependent on the Ets-core motifs, which are bound by GABP. Both subunits of GABP are phosphorylated upon JNK activation in vivo and three different isoforms of JNK/SAPK, but not p38, in vitro. Our data suggest that GABP is targeted by signaling events from both ERK and JNK/SAPK pathways. GABP therefore is a candidate for signal integration and regulation of IL-2 transcription in T lymphocytes.