Early Secreted Antigenic Target of 6 kDa of Mycobacterium tuberculosis Stimulates Macrophage Chemoattractant Protein‐1 Production by Macrophages and Its Regulation by p38 Mitogen‐Activated Protein Kinases and Interleukin‐4

Early secreted antigenic target of 6 kDa (ESAT‐6), the major virulence factor of Mycobacterium tuberculosis, affects host immunity and the formation of granulomas likely through inflammatory cytokines. To understand its role in this regard further, we investigated the effect of ESAT‐6 on macrophages by determining the production of macrophage chemoattractant protein (MCP)‐1, a major chemokine associated with tuberculosis pathogenesis, by murine bone marrow‐derived macrophages (BMDMs) and its regulation by protein kinases and cytokines. The results revealed that ESAT‐6, but not Ag85A and culture filtrate protein 10 kDa (CFP10), induced MCP‐1 production by BMDMs dose and time dependently. Inhibition of p38 but not other mitogen‐activated protein kinases (MAPK) and PI3K further enhanced ESAT‐6‐induced MCP‐1 production by BMDMs. Inhibition of p38 MAPK enhanced ESAT‐6‐induced MCP‐1 mRNA accumulation without affecting mRNA stability. ESAT‐6 also induced TNF‐α from BMDMs and MCP‐1 from mouse lung epithelial cells, and these were suppressed by p38 MAPK inhibition, implying cytokine‐ and cell‐specific effect of p38 MAPK inhibition on ESAT‐6‐induced MCP‐1 by macrophages. Pretreatment of BMDMs with IL‐4, but not other cytokines (IL‐2, IL‐10, TNF‐α, IFN‐γ and IL‐1α) further elevated ESAT‐6‐stimulated MCP‐1 production although IL‐4 did not induce MCP‐1 without ESAT‐6. Both p38 MAPK inhibitor and IL‐4 did not show additive effect on ESAT‐6‐induced MCP‐1 protein level despite such effect on MCP‐1 mRNA level was evident. In conclusion, these results indicate a specific role for both p38 MAPK and IL‐4 in ESAT‐6‐induced MCP‐1 production by macrophages and suggest a pathway with significance in tuberculosis pathogenesis.     

The volatile anaesthetic sevoflurane attenuates lipopolysaccharide‐induced injury in alveolar macrophages

Acute lung injury (ALI) is a well‐defined inflammation whereby alveolar macrophages play a crucial role as effector cells. As shown previously in numerous experimental approaches, volatile anaesthetics might reduce the degree of injury in pre‐ or post‐conditioning set‐ups. Therefore, we were interested to evaluate the effect of the application of the volatile anaesthetic sevoflurane on alveolar macrophages regarding the expression of inflammatory mediators upon lipopolysaccharide (LPS) stimulation in vitro. Alveolar macrophages were stimulated with LPS. Two hours later, cells were exposed additionally to air (control) or to sevoflurane‐containing air for 4, 6, 8, 12 or 24 h. Tumour necrosis factor (TNF)‐α, cytokine‐induced neutrophil chemoattractant‐1 (CINC‐1), macrophage‐inflammatory protein‐2 (MIP‐2) and monocyte chemoattractant protein‐1 (MCP‐1) proteins were determined and chemotaxis assays were performed. To evaluate possible cellular signalling pathways phosphorylation of the kinases extracellular‐regulated kinase (ERK) and Akt was assessed. In the early phase of sevoflurane post‐conditioning expression of TNF‐α, CINC‐1, MIP‐2 and MCP‐1 was attenuated, leading to a diminished chemotaxis reaction for neutrophils. Phosphorylation of ERK seems to be a possible cellular mechanism in the sevoflurane‐induced protection in vitro. Pharmacological post‐conditioning of alveolar macrophages with sevoflurane immunmodulates the inflammatory response upon stimulation with endotoxin. This might be a possible option for a therapeutical approach in ALI.     

Role of the Phosphatidylinositol 3-Kinase and Mitogen-Activated Protein Kinase Pathways in the Secretion of Tumor Necrosis Factor-α and Interleukin-10 by the PPD Antigen of Mycobacterium tuberculosis

Here we investigated the role of the phosphatidylinositol 3-kinase (PI 3-K) and mitogen-activated protein kinase (MAPK) pathways in the secretion of tumor necrosis factor (TNF)-α and interleukin (IL)-10 in human primary monocytes after stimulation with the PPD antigen of Mycobacterium tuberculosis. MAPK [extracellular signal-regulated kinase (ERK) 1/2 and p38] and Akt are rapidly phosphorylated in human monocytes stimulated with PPD. We found that the PI 3-K-Akt pathway stimulated by PPD is essential for both IL-10 and TNF-α production, although the inhibition of IL-10 production was more pronounced. The analysis of cytokine production using specific inhibitors of the MAPK pathway revealed that both p38 and ERK activation are essential for PPD-induced TNF-α production, whereas p38, but not ERK, activation is essential for IL-10 secretion. The inhibition of PI 3-K did not significantly activate p38 MAPK or ERK 1/2 in PPD-stimulated human monocytes. Further, the Src inhibitor PP2 inhibited the release of TNF-α but enhanced IL-10 release, suggesting the differential regulation of Src kinase in upstream signaling. Collectively, these data suggest that the PI 3-K and MAPK pathways play a central role in the regulation of both pro- and anti-inflammatory cytokines by the PPD antigen of M. tuberculosis.     

Role of mitogen-activated protein kinase pathways in the production of tumor necrosis factor-alpha,interleukin-10, and monocyte chemotactic protein-1 by Mycobacterium tuberculosis H37Rv-infected human monocytes

The role of mitogen-activated protein kinase (MAPK) pathways in the secretion of tumor necrosis factor (TNF)-, interleukin (IL)-10, IL-8, and monocyte chemotactic protein-1 (MCP-1) was investigated in human monocytes that were infected with Mycobacterium tuberculosis H37Rv. Analysis of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and p38 kinase showed rapid phosphorylation of both subfamilies in response to M. tuberculosis H37Rv. Using highly specific inhibitors of p38 (SB203580) and of MAPK kinase-1 (U0126 and PD98059), we found that both p38 and ERK were essential for M. tuberculosis H37Rv-induced TNF- production, whereas activation of the p38 pathway, but not that of ERK, was essential for M. tuberculosis H37Rv-induced IL-10 production. Interestingly, the ERK pathway, but not that of p38, was critical for MCP-1 secretion from human monocytes that were infected with M. tuberculosis H37Rv. However, IL-8 secretion was not regulated by ERK1/2 or p38 MAPK. Collectively, these results suggest that induction of the MAPK pathway is required for the expression of TNF-, IL-10, and MCP-1 by human monocytes during M. tuberculosis H37Rv infection.     

SOCS3 expression induced by PIM2 requires PKC and PI3K signaling

Initiation of proinflammatory host immunity in response to infection represents as a key event in effective control and containment of the pathogen at the site of infection as well as in elicitation of robust immune memory responses. In the current investigation, we demonstrate that an integral cell wall antigen of the mycobacterial envelope, Phosphatidyl-myo-inositol dimannosides (PIM2) triggers Suppressor of cytokine signaling (SOCS) 3 expression in macrophages in a Toll-like receptor 2 (TLR2)-MyD88 dependent manner. Data derived from signaling perturbations suggest the involvement of phosphoinositide-3 kinase (PI3K) and protein kinase C (PKC) signaling pathways during PIM2 induced SOCS3 expression. Further, pharmacological inhibition of ERK1/2, but not of p38 MAP kinase or JNK abrogated the induced expression of SOCS3. The PIM2 induced activation of ERK1/2 was dependent on the activation of PI3K or PKC signaling which in turn regulated p65 nuclear factor -κB (NF-κB) nuclear translocation. Overall, current study delineates the role for PI3K-PKC axis and ERK1/2 signaling as key signaling events during PIM2 induced SOCS3 expression in macrophages.     

Impaired activation of Stat1 and c‐Jun as a possible defect in macrophages of patients with active tuberculosis

Studies of patients with active tuberculosis (TB) and infected healthy individuals have shown that interferon (IFN)‐γ is present in sites of Mycobacterium tuberculosis infection in comparable levels. This suggests that there is a deficiency in the macrophage response to IFN‐γ in TB patients. We used recombinant human IFN‐γ to stimulate adherent monocyte‐derived macrophages from three groups of people: patients with active tuberculosis (TBP), their healthy household contacts (HHC) and healthy uninfected controls from the community (CC). We then evaluated the ability of the macrophages to inhibit the growth of M. tuberculosis H37Rv as well as their cytokine profile at early in infection (48 h). After IFN‐γ treatment, macrophages of healthy individuals (HHC and CC) controlled M. tuberculosis growth and produced mainly nitric oxide (NO) and interleukin (IL)‐12p70, whereas TBP macrophages did not kill M. tuberculosis. Additionally, TBP macrophages produced low levels of NO and IL‐12p70 and high levels of tumour necrosis factor (TNF)‐α and IL‐10. Transforming growth factor (TGF)‐β levels were similar among all three groups. M. tuberculosis infection had little effect on the cytokine response after IFN‐γ stimulus, but infection alone induced more IL‐10 and TGF‐β in TBP macrophages. There were no differences in Stat1 nuclear translocation and DNA binding between the groups. However, the phosphorylated Stat1 and c‐Jun (AP‐1) in nuclear protein extracts was diminished in TBP macrophages compared to macrophages of healthy individuals. These results indicate an impairment of Stat1‐dependent and Stat1‐independent IFN‐γ signalling in macrophages of people with active tuberculosis, suggesting a different molecular regulation that could impact macrophage functionality and disease outcome.     

RIP2/RICK‐Dependent Cytokine Production Upon Yersinia enterocolitica Infection in Macrophages with TLR4 Deficiency

Receptor‐interacting protein 2 (RIP2) is a caspase recruitment domain (CARD)‐containing serine/threonine kinase that is activated by NOD1 or NOD2 recognition of their ligands and essential for the activation of NF‐κB and mitogen‐activated protein kinase (MAPK). RIP2 has been known to play an important role in innate immune responses against certain bacterial infection. However, the role and interplay of RIP2 with TLR signalling on cytokine production in macrophages against Yersinia enterocolitica infection remains poorly understood. In the present study, we examined whether RIP2 is essential for Yersinia‐induced production of cytokines in macrophages. Our results showed that naïve RIP2‐deficient macrophages produced similar level of IL‐6, TNF‐α and IL‐10 upon Y. enterocolitica infection compared with wild‐type macrophages. However, the production of IL‐6, TNF‐α and IL‐10 by Y. enterocolitica was impaired in RIP2‐deficient macrophages after lipopolysaccharide (LPS) pretreatment, a TLR4‐tolerant condition. In addition, RIP2 inhibitors, SB203580, PP2, and gefitinib, reduced IL‐6 production in TLR4‐deficient macrophages in response to Y. enterocolitica, whereas they did not affect the cytokines production in WT cells. These results demonstrate that RIP2 may play an important role in proinflammatory cytokine production in macrophages at the absence of TLR signalling.     

mTOR signaling pathway regulates the IL-12/IL-10 axis in <Emphasis Type="Italic">Leishmania donovani</Emphasis> infection

Leishmania-induced interleukin-12 (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 pathways downstream from PI3K in L. donovani-induced reciprocal regulation of IL-12/IL-10 axis in THP-1-derived macrophages. We show for the first time that in THP-1-derived macrophages and human monocytes, mTOR inhibition by rapamycin reversed L. donovani-induced IL-12 and IL-10 modulation. L. donovani-induced phosphorylation of P70S6K, a correlate of mTOR activity, in TLR-stimulated THP-1 derived macrophages. This increase in P70S6K phosphorylation was completely blocked by rapamycin (mTOR inhibitor) and partially by wortmannin (PI3K inhibitor). These observations suggest that a PI3K independent pathway is operative in the modulation of IL-12 and IL-10. Blocking of TLR2 significantly attenuated IL-10 induced by the parasite, but did not affect IL-12 production. Thus, our data suggests that intracellular network of PI3K and mTOR pathway control IL-12/IL-10 modulation by L. donovani. mTOR inhibitors may be attractive molecules to reverse this modulation and may result in control of disease.     

Regulation of cathepsin X overexpression in H. pylori‐infected gastric epithelial cells and macrophages

Cathepsin X (CTSX) is strongly up‐regulated in Helicobacter pylori‐infected gastric mucosa and intestinal‐type gastric cancer. The overexpression of CTSX is mediated predominantly by associated macrophages; depends on a functional type IV‐secretion system; and leads to increased migration of gastric epithelial cells. In the present study, we analysed the role of CagA in CTSX overexpression and identified H. pylori‐induced inflammatory factors and signalling pathways required for stimulating CTSX expression by H. pylori. Gastric epithelial cells were co‐cultured with macrophages in Transwell chambers of 0.4 µm pore size, enabling exchange of fluids but retracting H. pylori. N87 gastric epithelial cells were infected with H. pylori P1 wild‐type strain in the presence of inhibitors for p38, JNK, and ERK1/2 signal transduction pathways. Furthermore, cytokines and growth factors were tested for their regulatory function using inhibitory antibodies, and their gene expression was studied by quantitative RT‐PCRs and western blots. CTSX is strongly up‐regulated at both the mRNA and the protein levels by TNF‐α, IL‐1β, IL‐6, and IL‐8, depending on cell type. All these cytokines were found to be increased by five‐ to ten‐fold in macrophages by H. pylori infection of co‐cultured N87 gastric epithelial cells. In macrophages, H. pylori up‐regulated CTSX via ERK1/2 signalling pathways, and in N87 cells via JNK irrespective of p38 signalling. Our results suggest that H. pylori induced overexpression of CTSX in macrophages and epithelium through specific cytokines that are initiated by CagA‐dependent pathways in a cell type‐dependent manner. Copyright © 2008 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Interleukin‐21 Induces Proliferation and Proinflammatory Cytokine Profile of Fibroblast‐like Synoviocytes of Patients with Rheumatoid Arthritis

Fibroblast‐like synoviocytes (FLS) play a pivotal role in the pathogenesis of rheumatoid arthritis (RA) through aggressive proliferation and invasion, and certain proinflammatory cytokines may affect synoviocyte proliferation. To evaluate whether interleukin‐21 (IL‐21) could promote proliferation and proinflammatory cytokine production by RA‐FLS, immunohistochemistry and immunoblotting were performed to observe the expression of IL‐21 receptor (IL‐21R) in synovial tissues and FLS from RA and osteoarthritis (OA) patients. The MTS assay was used to analyse RA‐FLS proliferation. The concentrations of IL‐6 and tumour necrosis factor‐α (TNF‐α) in culture supernatants were determined by enzyme‐linked immunosorbent assay (ELISA). The signalling pathways triggered by IL‐21 were characterized by immunoblotting. IL‐21R was upregulated in the synovial tissues and FLS of RA patients as compared with OA patients. IL‐21 stimulated RA‐FLS proliferation and promoted the production of TNF‐α and IL‐6 and blockade of IL‐21/IL‐21R pathway with IL‐21R.Fc attenuated IL‐21‐induced proliferation and secretion of TNF‐α and IL‐6. Moreover, IL‐21 induced activation of the ERK1/2, PI3K/AKT and STAT3 pathways, and blockade of these pathways attenuated IL‐21‐induced proliferation and secretion of TNF‐α and IL‐6. These results suggest that IL‐21 could promote RA‐FLS proliferation and production of proinflammatory cytokines. Therefore, therapeutic strategies targeting IL‐21 might be effective for the treatment of RA.     

Lipopolysaccharide‐primed heterotolerant dendritic cells suppress experimental autoimmune uveoretinitis by multiple mechanisms

Exposure of bone‐marrow‐derived dendritic cells (BMDC) to high‐dose ultrapure lipopolysaccharide for 24 hr (LPS‐primed BMDC) enhances their potency in preventing inter‐photoreceptor retinoid binding protein: complete Freund's adjuvant‐induced experimental autoimmune uveoretinitis (EAU). LPS‐primed BMDC are refractory to further exposure to LPS (= endotoxin tolerance), evidenced here by decreased phosphorylation of TANK‐binding kinase 1, interferon regulatory factor 3 (IRF3), c‐Jun N‐terminal kinase and p38 mitogen‐activated protein kinase as well as impaired nuclear translocation of nuclear factor κB (NF‐κB) and IRF3, resulting in reduced tumour necrosis factor‐α (TNF‐α), interleukin‐6 (IL‐6), IL‐12 and interferon‐β secretion. LPS‐primed BMDC also show reduced surface expression of Toll‐like receptor‐4 and up‐regulation of CD14, followed by increased apoptosis, mediated via nuclear factor of activated T cells (NFATc)‐2 signalling. LPS‐primed BMDC are not only homotolerant to LPS but are heterotolerant to alternative pathogen‐associated molecular pattern ligands, such as mycobacterial protein extract (Mycobacterium tuberculosis). Specifically, while M. tuberculosis protein extract induces secretion of IL‐1β, TNF‐α and IL‐6 in unprimed BMDC, LPS‐primed BMDC fail to secrete these cytokines in response to M. tuberculosis. We propose that LPS priming of BMDC, by exposure to high doses of LPS for 24 hr, stabilizes their tolerogenicity rather than promoting immunogenicity, and does so by multiple mechanisms, namely (i) generation of tolerogenic apoptotic BMDC through CD14:NFATc signalling; (ii) reduction of NF‐κB and IRF3 signalling and downstream pro‐inflammatory cytokine production; and (iii) blockade of inflammasome activation.     

Amomum tsao‐ko fruit extract suppresses lipopolysaccharide‐induced inducible nitric oxide synthase by inducing heme oxygenase‐1 in macrophages and in septic mice

Amomum tsao‐ko Crevost et Lemarié (Zingiberaceae) has traditionally been used to treat inflammatory and infectious diseases, such as throat infections, malaria, abdominal pain and diarrhoea. This study was designed to assess the anti‐inflammatory effects and the molecular mechanisms of the methanol extract of A. tsao‐ko (AOM) in lipopolysaccharide (LPS)‐induced RAW 264.7 macrophages and in a murine model of sepsis. In LPS‐induced RAW 264.7 macrophages, AOM reduced the production of nitric oxide (NO) by inhibiting inducible nitric oxide synthase (iNOS) expression, and increased heme oxygenase‐1 (HO‐1) expression at the protein and mRNA levels. Pretreatment with SnPP (a selective inhibitor of HO‐1) and silencing HO‐1 using siRNA prevented the AOM‐mediated inhibition of NO production and iNOS expression. Furthermore, AOM increased the expression and nuclear accumulation of NF‐E2‐related factor 2 (Nrf2), which enhanced Nrf2 binding to antioxidant response element (ARE). In addition, AOM induced the phosphorylation of extracellular regulated kinase (ERK) and c‐Jun N‐terminal kinase (JNK) and generated reactive oxygen species (ROS). Furthermore, pretreatment with N‐acetyl‐l ‐cysteine (NAC; a ROS scavenger) diminished the AOM‐induced phosphorylation of ERK and JNK and AOM‐induced HO‐1 expression, suggesting that ERK and JNK are downstream mediators of ROS during the AOM‐induced signalling of HO‐1 expression. In LPS‐induced endotoxaemic mice, pretreatment with AOM reduced NO serum levels and liver iNOS expression and increased HO‐1 expression and survival rates. These results indicate that AOM strongly inhibits LPS‐induced NO production by activating the ROS/MAPKs/Nrf2‐mediated HO‐1 signalling pathway, and supports its pharmacological effects on inflammatory diseases.     

Interplay of T-cell receptor and interleukin-2 signalling in V��2V��2 T-cell cytotoxicity

Human peripheral blood Vγ2Vδ2 T cells are important for host defence and tumour immunity. Their unusual T‐cell receptor (TCR) recognizes small molecule phosphoantigens; stimulated cells produce inflammatory cytokines and are potently cytotoxic for a variety of tumours. However, molecular mechanisms linking phosphoantigen stimulation and cytotoxicity are incompletely understood. We know that isopentenyl pyrophosphate (IPP) activates mitogen‐activated protein kinase kinase/extracellular signal‐regulated kinase (MEK/Erk) and phosphoinositide 3‐kinase (PI‐3K)/Akt pathways; specific inhibition of Erk or Akt significantly impairs the functional response to IPP. We now show that interleukin‐2 also activates MEK/Erk and PI‐3K/Akt pathways but on its own, fails to induce cytokine expression or cytotoxicity. Hence, MEK/Erk and PI‐3K/Akt activation are necessary but not sufficient to induce effector responses in Vγ2Vδ2 T cells and a TCR‐dependent signal is still required for tumour cell killing. Cyclosporin A, an inhibitor of calcineurin, blocked calcium‐dependent nuclear translocation of nuclear factor of activated T cell (NFAT) and significantly reduced IPP‐induced cytokine production, degranulation and cytotoxicity. The IPP‐induced calcium mobilization and NFAT translocation were necessary to activate Vγ2Vδ2 effector functions; interleukin‐2, acting on the MEK/Erk pathway, regulated the strength of these responses. The TCR has a specific role in Vγ2Vδ2 T‐cell killing of tumour cells, which is distinct from its role in triggering cellular proliferation in response to phosphoantigens.