Impaired production of IL-12 in systemic lupus erythematosus. III: deficient IL-12 p40 gene expression and cross-regulation of IL-12, IL-10 and IFN-gamma gene expression.

Interleukin 12 (IL-12) is a heterodimer comprising p35 and p40 subunits which are encoded and regulated separately. The authors previously demonstrated deficient IL-12 production in SLE which correlates negatively with disease activity. The present study was designed to determine whether deficiency of IL-12 and excess production of IL-10 and IL-6 in systemic lupus erythematosus (SLE) are due to aberrant regulation at the gene level. Using semiquantitative RT-PCR assay, it was shown that constitutive expression of IL-12 p35 gene is somewhat impaired in SLE compared with controls and that IL-12 p40 mRNA, which was present at low levels in controls, was undetectable in unstimulated SLE peripheral blood mononuclear cells (PBMC). Gene expression of IL-12 p35 and p40 was significantly increased in response to SAC, with significantly lower SAC-induced expression of p40 in SLE patients than controls. SAC-stimulated IL-12 p35 and p40 mRNAs were significantly augmented by interferon gamma (IFN-gamma). Exogenous IL-12 or IFN-gamma significantly inhibited IL-10 gene expression, without affecting IL-6 mRNA or other proinflammatory cytokine mRNA levels. These observations were further confirmed by studies of protein production at the single cell level using ELISPOT assay. Downregulation of IL-12 p40 expression appears to be the cause of IL12 p70 deficiency in SLE. If this defect could be repaired, normalization of IL-12 and IFN-gamma production should reduce excessive IL-10 and prevent pathology.     

c-Jun N-terminal kinase negatively regulates lipopolysaccharide-induced IL-12 production in human macrophages: role of mitogen-activated protein kinase in glutathione redox regulation of IL-12 production

Although c-Jun N-terminal kinase (JNK) plays an important role in cytokine expression, its function in IL-12 production is obscure. The present study uses human macrophages to examine whether the JNK pathway is required for LPS-induced IL-12 production and defines how JNK is involved in the regulation of IL-12 production by glutathione redox, which is the balance between intracellular reduced (GSH) and oxidized glutathione (GSSG). We found that LPS induced IL-12 p40 protein and mRNA in a time- and concentration-dependent manner in PMA-treated THP-1 macrophages, and that LPS activated JNK and p38 mitogen-activated protein (MAP) kinase, but not extracellular signal-regulated kinase, in PMA-treated THP-1 cells. Inhibition of p38 MAP kinase activation using SB203580 dose dependently repressed LPS-induced IL-12 p40 production, as described. Conversely, inhibition of JNK activation using SP600125 dose dependently enhanced both LPS-induced IL-12 p40 production from THP-1 cells and p70 production from human monocytes. Furthermore, JNK antisense oligonucleotides attenuated cellular levels of JNK protein and LPS-induced JNK activation, but augmented IL-12 p40 protein production and mRNA expression. Finally, the increase in the ratio of GSH/GSSG induced by glutathione reduced form ethyl ester (GSH-OEt) dose dependently enhanced LPS-induced IL-12 p40 production in PMA-treated THP-1 cells. GSH-OEt augmented p38 MAP kinase activation, but suppressed the JNK activation induced by LPS. Our findings indicate that JNK negatively affects LPS-induced IL-12 production from human macrophages, and that glutathione redox regulates LPS-induced IL-12 production through the opposite control of JNK and p38 MAP kinase activation.     

C5a suppresses the production of IL-12 by IFN-gamma-primed and lipopolysaccharide-challenged human monocytes.

IL-12 is a key mediator of the immune response, skewing T lymphocytes toward a type 1 cytokine pattern. Priming with IFN-gamma or GM-CSF is required for expression of IL-12p70 by cells in which IL-12 is inducible by bacterial products such as LPS. We here show for the first time that the production of bioactive IL-12 by human monocytes can be significantly suppressed by C5a if applied to IFN-gamma-primed monocytes before LPS stimulation. There was a dose-dependent suppression by IL-12 (p70) on the levels of intracellular cytokine production and cytokine secretion. mRNA studies consistently showed a reduction of IL-12p40 and IL-12p35 expression by stimulation in the presence of C5a. The results of several different experimental approaches suggest that IL-12 down-regulation was not due to endogenous IL-10, IL-4, or PGE2 production induced by C5a. Moreover, stimulation of IFN-gamma-primed monocytes with C5a did not lead to a down-regulation of the CD14 Ag, which is an LPS receptor. These findings show that the anaphylatoxin C5a has the capacity to directly interact with the complex regulation of IL-12.     

Interleukin-12 p40 mRNA Expression in Bovine Leukemia Virus-Infected Animals: Increase in Alymphocytosis but Decrease in Persistent Lymphocytosis

Interleukin-12 (IL-12), a key cytokine in immune regulation, has an important role in activating the cell-mediated immune response in infectious diseases. Recently, a dichotomy between IL-12 and IL-10 regarding progression of a variety diseases has emerged. IL-12 activates type 1 cytokine production and has an antagonistic effect on type 2 cytokines. Here, by using quantitative competitive PCR, we show that peripheral blood mononuclear cells from bovine leukemia virus-infected animals in the alymphocytotic stage of disease express an increased amount of IL-12 p40 mRNA. In contrast, IL-12 p40 mRNA expression by cells from animals with late-stage disease, termed persistent lymphocytosis, was significantly decreased compared to that by normal and alymphocytotic animals. Interestingly, IL-12 p40 mRNA was also detected in tumor-bearing animals. IL-12 p40 expression occurred only in monocytes/macrophages, not B or T lymphocytes. The present study combined with previous findings suggest that IL-12 in bovine leukemia virus-infected animals may regulate production of other cytokines such as gamma interferon and IL-10 and the progression of bovine leukosis in animals that develop more advanced disease such as a persistent lymphocytosis of B cells or B-cell lymphosarcoma.     

Interferon gamma priming is not critical for IL-12 production of murine spleen cells

Interferon gamma (IFN-gamma) priming is considered to be critical for interleukin 12 (IL-12) production of murine macrophages and human monocytes by lipopolysaccharide (LPS) stimulation. In our present experiments, freshly prepared spleen cells (f-spleen cells) were confirmed not to produce detectable level of IL-12 by LPS stimulation, although they produced significant amount of IL-12 by the stimulation with LPS plus IFN-gamma. However, the stimulation only with LPS induced IL-12 production of spleen cells preincubated in the absence of IFN-gamma. Findings on IL-12 p40 mRNA accumulation were consistent with their IL-12 production. Essentially the same results were obtained using spleen cells from IFN-gamma deficient mice. In the presence of anti-IL-10, f-spleen cells produced IL-12 upon LPS stimulation, indicating that the failure of f-spleen cells in IL-12 production is caused by IL-10 produced by themselves upon LPS stimulation. In addition, f-spleen cells produced IL-12 upon CD40 ligand stimulation, and the production was hardly affected by the presence of IFN-gamma or preincubation. These results indicate that IFN-gamma priming is not critical for IL-12 production of spleen cells stimulated with LPS or CD40 ligand, although IFN-gamma enhances the production, especially, in response to LPS stimulation.     

Selective suppression of IL-12 production by chemoattractants

We investigated the ability of chemoattractants to affect IL-12 production by human monocytes and dendritic cells. We found that pretreatment of monocytes with macrophage chemoattractant proteins (MCP-1 to -4), or C5a, but not stromal-derived factor-1, macrophage inflammatory protein-1alpha, RANTES, or eotaxin, inhibited IL-12 p70 production in response to stimulation with Staphylococcus aureus, Cowan strain 1 (SAC), and IFN-gamma. The production of TNF-alpha and IL-10, however, was minimally affected by any of the chemoattractants. The degree of inhibition of IL-12 p70 production by MCP-1 to -4 was donor dependent and was affected by the autocrine inhibitory effects of IL-10. In contrast, C5a profoundly suppressed IL-12 production in an IL-10-independent fashion. Neither TGF-beta1 nor PGE2 was important for the suppression of IL-12 by any of the chemoattractants tested. The accumulation of mRNA for both IL-12 p35 and p40 genes was inhibited by chemokine pretreatment. Interestingly, MCP-1 to -4 and C5a did not suppress IL-12 production by monocyte-derived dendritic cells (DC) stimulated with CD40 ligand and IFN-gamma or by SAC and IFN-gamma, suggesting that these factors may act at the site of inflammation to suppress IL-12 and IFN-gamma production rather than in the lymph node to affect T cell priming. Despite the inability of C5a to inhibit IL-12 production by DCs, the receptor for C5a (CD88) was expressed by these cells, and recombinant C5a induced a Ca2+ flux. Taken together, these results define a range of chemoattractant molecules with the ability to suppress IL-12 production by human monocytes and have broad implications for the regulation of immune responses in vivo.     

Anti-inflammatory effects of sodium butyrate on human monocytes: potent inhibition of IL-12 and up-regulation of IL-10 production.

Cytokines are critical in regulating unresponsiveness versus immunity towards enteric antigens derived from the intestinal flora and ingested food. There is increasing evidence that butyrate, a major metabolite of intestinal bacteria and crucial energy source for gut epithelial cells, also possesses anti-inflammatory properties. Its influence on cytokine production, however, is not established. Here, we report that butyrate strongly inhibits interleukin-12 (IL-12) production by suppression of both IL-12p35 and IL-12p40 mRNA accumulation, but massively enhances IL-10 secretion in Staphylococcus aureus cell-stimulated human monocytes. The effect of butyrate on IL-12 production was irreversible upon the addition of neutralizing antibodies to IL-10 or transforming growth factor b1 and of indomethacin. In anti-CD3-stimulated peripheral blood mononuclear cells, butyrate enhanced IL-10 and IL-4 secretion but reduced the release of IL-2 and interferon-g. The latter effect was in part a result of suppressed IL-12 production but also a result of inhibition of IL-12 receptor expression on T cells. These data demonstrate a novel anti-inflammatory property of butyrate that may have broad implications for the regulation of immune responses in vivo and could be exploited as new therapeutic approach in inflammatory conditions.     

IFNgamma enhances IL-23 production during Francisella infection of human monocytes

We previously demonstrated that monocytes produce IL-23 during Francisella infection, and that IL-23 induces IFNgamma from NK cells. Here, we demonstrate that IFNgamma-priming of monocytes enhances IL-23 production during Francisella infection. This effect was seen on the IL12/23 p40 subunit. Induction of IL-12/23 p40 is reported to be enhanced by IRF-1 and IRF-8. Consistently, microarray analysis of IFNgamma-treated monocytes revealed a significant induction of the IRFs. Interestingly, IFNgamma-primed monocytes produced IL-12 p70, a more potent inducer of IFNgamma than IL-23. We propose that there exists an amplification loop between monocyte IL-23 and NK/T cell IFNgamma that leads to IL-12 p70 production.     

Tumor cells deactivate human monocytes by up-regulating IL-1 receptor associated kinase-M expression via CD44 and TLR4

Although blood monocytes possess significant cytotoxic activity against tumor cells, tumor-infiltrating monocytes are commonly deactivated in cancer patients. Monocytes pre-exposed to tumor cells show significantly decreased expression levels of TNF-alpha, IL-12p40, and IL-1R-associated kinase (IRAK)-1. Activation of the Ser/Thr kinase IRAK-1 is an important event in several inflammatory processes. By contrast, another IRAK family member, IRAK-M, negatively regulates this pathway, and is up-regulated in cultures of endotoxin-tolerant monocytes and in monocytes from septic patients within the timeframe of tolerance. In this study, we show that IRAK-M expression is enhanced at the mRNA and protein level in human monocytes cultured in the presence of tumor cells. IRAK-M was induced in monocytes upon coculturing with different tumor cells, as well as by fixed tumor cells and medium supplemented with the supernatant from tumor cell cultures. Moreover, blood monocytes from patients with chronic myeloid leukemia and patients with metastasis also overexpressed IRAK-M. Low concentrations of hyaluronan, a cell surface glycosaminoglycan released by tumor cells, also up-regulated IRAK-M. The induction of IRAK-M by hyaluronan and tumor cells was abolished by incubation with anti-CD44 or anti-TLR4 blocking Abs. Furthermore, down-regulation of IRAK-M expression by small interfering RNAs specific for IRAK-M reinstates both TNF-alpha mRNA expression and protein production in human monocytes re-exposed to a tumor cell line. Altogether, our findings indicate that deactivation of human monocytes in the presence of tumor cells involves IRAK-M up-regulation, and this effect appears to be mediated by hyaluronan through the engagement of CD44 and TLR4.     

G(i)-protein-dependent inhibition of IL-12 production is mediated by activation of the phosphatidylinositol 3-kinase-protein 3 kinase B/Akt pathway and JNK

Ligands for certain G(i)-protein-coupled receptors (GiPCRs) potently inhibit the production of IL-12 by human monocytes. We addressed the intracellular signaling mechanisms by which this occurs using primary human cells. Stimulation with the GiPCR ligands C5a and 1-deoxy-1-[6-[(3-iodophenyl)methyl]amino]-9H-purine-9-y1]-N-methyl-beta-D-ribofuranuronamide (IB-MECA) blocked the production of IL-12 p70 by human monocytes stimulated with LPS and IFN-gamma. In addition, C5a reduced the expression of mRNA for IL-12 p35, p40, IL-23 p19, and IL-27 p28. This effect was due neither to a down-regulation of TLR4 or IFN-gamma receptor on the cell surface nor to interference with IFN-gamma signaling, because IFN-gamma-induced up-regulation of HLA-DR and CD40 were unaffected. C5a or IB-MECA activated the PI3K/Akt signaling pathway and induced the phosphorylation of the MAPK p38, ERK, and JNK. Inhibition of the PI3K/Akt signaling pathway with wortmannin or an inhibitor of Akt activity, and inhibition of JNK but not ERK prevented IL-12 and IL-23 suppression by C5a. These data extend observations on IL-12 suppression by C5a to IL-23 and IL-27, and are the first to demonstrate the intracellular signaling events leading to IL-12 and IL-23 inhibition after GiPCR activation.     

CD40 ligation induces macrophage IL-10 and TNF-alpha production: differential use of the PI3K and p42/44 MAPK-pathways.

Interleukin 10 (IL-10) is an anti-inflammatory cytokine produced in the rheumatoid arthritis (RA) joint by macrophages/monocytes and infiltrating peripheral blood derived lymphocytes. Recent data suggest a role for physical cell-to-cell interactions in the production of IL-10. In this report, we have investigated the signalling mechanisms involved in IL-10 production by peripheral blood-derived macrophages upon interaction with fixed CD40L transfectants. IL-10 and tumour necrosis factor alpha (TNF-alpha) are produced by macrophage colony-stimulating factor (M-CSF)-primed monocytes/macrophages in response to CD40 ligation. The utilization of the inhibitors, wortmannin and LY294002, demonstrated a role for phosphatidylinositol 3-kinase (PI3K) whereas rapamycin demonstrated p70 S6-kinase (p70S6K) involvement in the production of IL-10 by these monocytes. The production of TNF-alpha was enhanced by wortmannin and LY294002, suggesting negative regulation by PI3K; however, it was dependent on p70S6K suggesting a PI3K-independent mechanism of p70S6K activation. One alternative pathway that activates p70S6K independently of PI3K and also differentiates between IL-10 and TNF-alpha is the p42/44 mitogen-activated protein kinase (MAPK), which regulates TNF-alpha production in a PI3K-independent manner. These observations suggest that CD40 ligation induces macrophage IL-10 and TNF-alpha production, the mechanism of which is p70S6K-dependent yet bifurcates at the level of PI3K and p42/44 MAPK.     

Intracellular network of phosphatidylinositol 3-kinase, mammalian target of the rapamycin/70 kDa ribosomal S6 kinase 1, and mitogen-activated protein kinases pathways for regulating mycobacteria-induced IL-23 expression in human macrophages

We previously demonstrated that Mycobacterium tuberculosis (M. tbc)-induced interleukin (IL)-12 expression is negatively regulated by the phosphatidylinositol 3-kinase (PI3K) and extracellular signal-regulated kinase (ERK) 1/2 pathways in human monocyte-derived macrophages (MDMs). To extend these studies, we examined the nature of the involvement of toll-like receptors (TLRs) and intracellular signalling pathways downstream from PI3K in M. tbc-induced IL-23 expression in human MDMs. M. tbc-induced Akt activation and IL-23 expression were essentially dependent on TLR2. Blockade of the mammalian targets of rapamycin (mTOR)/70 kDa ribosomal S6 kinase 1 (S6K1) pathway by the specific inhibitor rapamycin greatly enhanced M. tbc-induced IL-12/IL-23 p40 (p40) and IL-23 p19 (p19) mRNA and IL-23 protein expression. In sharp contrast, p38 mitogen-activated protein kinase (MAPK) inhibition abrogated the p40 and p19 mRNA and IL-23 protein expression induced by M. tbc. Furthermore, the inhibition of PI3K-Akt, but not ERK 1/2 pathway, attenuated M. tbc-induced S6K1 phosphorylation, whereas PI3K inhibition enhanced p38 phosphorylation and apoptosis signal-regulating kinase 1 activity during exposure to M. tbc. Although the negative or positive regulation of IL-23 was not reversed by neutralization of IL-10, it was significantly modulated by blocking TLR2. Collectively, these findings provide new insight into the homeostatic mechanism controlling type 1 immune responses during mycobacterial infection involving the intracellular network of PI3K, S6K1, ERK 1/2 and p38 MAPK pathways in a TLR2-dependent manner.     

Effective stimulation for IL-12 p35 mRNA accumulation and bioactive IL-12 production of antigen-presenting cells interacted with Th cells.

Bioactive IL-12 is composed of two subunits, p35 and p40. In the APC-Th cell interaction, p40 mRNA accumulation in APC was shown to be up-regulated by stimulation with CD40 ligand (CD40L) on Th cells. However, the CD40-CD40L interaction scarcely induced p35 mRNA accumulation in APC. In the present experiments, p35 mRNA accumulation was induced in splenic macrophages/dendritic cells by the interaction with paraformaldehyde-fixed Th1 cells in the presence of Ag, and the p35 mRNA accumulation was abrogated by the inclusion of anti-I-A in cultures to block TCR/MHC class II interaction. The accumulation was also induced by the stimulation with agonistic anti-I-A. These results indicate that the interaction of the MHC class II molecule with TCR evokes an activation signal for p35 mRNA accumulation in APC. Furthermore, the production of bioactive IL-12 in macrophages/dendritic cells stimulated with CD40L was enhanced by the inclusion of agonistic anti-I-A. The p35 mRNA accumulation and IL-12 production of macrophages/dendritic cells induced by stimulation with OVA-specific fixed Th1 clone expressing CD40L were also enhanced by adding OVA in cultures. These results indicate that the p35 mRNA accumulation induced by MHC class II stimulation plays a role in bioactive IL-12 production.     

Suppression of IL-12 production by soluble CD40 ligand: evidence for involvement of the p44/42 mitogen-activated protein kinase pathway

IL-12 is a key cytokine in skewing immune responses toward Th1-like reactions. Human monocytes/macrophages produce high amounts of bioactive IL-12 when a priming signal (IFN-gamma or GM-CSF) precedes a second signal (e.g., LPS). We and others have previously shown that preincubation with LPS before this stimulation procedure can efficiently and selectively suppress the production of IL-12 by human monocytes. In this study, we show that an almost complete suppression of IL-12 production can also be observed after preincubation of monocytes with costimulatory cell surface molecules that bind to members of the TNFR superfamily (CD40 ligand, TNF-related activation-induced cytokine (TRANCE)). The suppression of IL-12 was observable on the mRNA and protein levels and was not due to endogenous production of known IL-12 antagonists (i.e., IL-10, IL-4, and PGE(2)), to an increased number of cells undergoing apoptosis, nor to down-regulation of the IFN-gamma or CD40 receptor. Cell surface expression of the costimulatory molecules CD80 and CD86 was not reduced by the preincubation procedure, and only a moderate reduction of IL-6 production was observed. Several studies have identified signal transduction pathways that are activated by CD40 signaling, including activation of mitogen-activated protein kinases. The presence of the extracellular signal-related kinase-specific mitogen-activated protein kinase kinase 1/2-specific inhibitors PD98059 and U0126 abrogated suppression induced by sCD40 ligand or other second signals. This indicates that activation of extracellular signal-regulated kinase 1/2 contributes to the underlying mechanism of IL-12 suppression. This mechanism may be relevant in other inflammatory responses and may help to develop therapeutic strategies in Th1-mediated diseases.