Differential regulation of MMP-1/9 and TIMP-1 secretion in human monocytic cells in response to Mycobacterium tuberculosis.

In tuberculosis, matrix metalloproteinase (MMP) secretion is involved in leukocyte migration to sites of infection but in excess may contribute to tissue destruction. We demonstrate that human monocytic THP-1 cells and primary monocytes secrete MMP-1 (52 kD collagenase) when phagocytosing live, virulent M. tuberculosis but not inert latex. The magnitude of MMP-1 secretion was approximately 10-fold less when compared to MMP-9 (92 kD gelatinase) secretion. MMP-1 secretion was also relatively delayed (detected at 24 h vs. 4 h). M. tuberculosis, zymosan or latex stimulate similar TIMP-1 secretion within 8 h and increasing over 24 h. MMP-1/9 secretion was decreased by inhibitors of protein kinase (PK) C, PKA or tyrosine kinases (PTK) in a concentration-dependent manner. In contrast, TIMP-1 secretion was not affected by PKC or PTK blockade and only somewhat reduced by high level PKA inhibition. In summary, M. tuberculosis-infected monocytes secrete MMP-1 at lower concentrations than MMP-9 and such MMP secretion is regulated by multiple upstream signalling pathways which do not control TIMP-1 secretion. Divergent effects of i on MMP and TIMP secretion from monocytes may be important in influencing matrix degradation in vivo.     

Mycobacterium tuberculosis H37Rv induces monocytic release of interleukin-6 via activation of mitogen-activated protein kinases: inhibition by N-acetyl-L-cysteine

The release of proinflammatory cytokines after mycobacterial infection is a host immune response that may be propitious or deleterious to the host. Elevated levels of interleukin (IL)-6 are present in plasma of patients with active tuberculosis infection. The aim of this study was to investigate the role of mitogen-activated protein kinases in the secretion of interleukin-6 in THP-1 cells and human primary monocytes that were infected with Mycobacterium tuberculosis H37Rv, and its regulation by N-acetyl-L-cysteine, a potential antimycobacterial agent. Exposure of THP-1 human monocytes to M. tuberculosis H37Rv induced rapidly, in a time-dependent manner, the phosphorylation of mitogen-activated protein kinase kinase 3/6 and p38 mitogen-activated protein kinase, accompanied by an upregulation of interleukin-6. Using highly specific inhibitors of mitogen-activated protein kinase kinase-1, p38 mitogen-activated protein kinase and nuclear factor-kappaB, we found that extracellular-signal regulated kinase 1/2, p38 mitogen-activated protein kinase and nuclear factor-kappaB were essential for M. tuberculosis H37Rv-induced interleukin-6 production in human primary monocytes. Pretreatment with N-acetyl-L-cysteine reduced, in a dose-dependent manner, M. tuberculosis H37Rv-induced activation of mitogen-activated protein kinase kinase 3/6 and interleukin-6 production in THP-1 cells.     

Macrophage-colony stimulating factor and interleukin-34 induce chemokines in human whole blood

The aim of this study is to investigate if macrophage-colony stimulating factor (M-CSF) or interleukin-34 (IL-34) induces cytokines or chemokines using human whole blood (HWB) and if an M-CSF- or IL-34-induced cytokine or chemokine production from HWB is inhibited by soluble M-CSF receptor or c-FMS kinase inhibitors. Among eight cytokines or growth factors tested, only IL-6 level was increased by up to 6-fold by M-CSF or IL-34 in HWB. In contrast, chemokine levels (IP-10/CXCL10, IL-8/CXCL8, and MCP-1/CCL2) were dramatically increased by M-CSF or IL-34 in HWB while exhibiting a large variation among donors. Variability of the MCP-1 signal induced by M-CSF or IL-34 was relatively less among donors compared to the IP-10 and IL-8 signals. The elevation of these chemokine levels was significantly decreased by soluble M-CSF receptor, indicating the elevation of these chemokines was mediated by M-CSF or IL-34. Furthermore, GW2580, a c-FMS kinase inhibitor, inhibited the induction of MCP-1 by M-CSF or IL-34 in a concentration dependent manner. These indicate MCP-1 is the most appropriate chemokine target for a chemokine release assay to evaluate the potency of c-FMS kinase inhibitors and MCP-1 release assay using HWB would be useful, relevant tool for translational pharmacology of c-FMS kinase inhibitors.     

Rho-kinase mediates lysophosphatidic acid-induced IL-8 and MCP-1 production via p38 and JNK pathways in human endothelial cells

Lysophosphatidic acid (LPA), an inflammatory mediator that is elevated in multiple inflammatory diseases, is a potent activator of Rho kinase (ROCK) signaling and of chemokine production in endothelial cells. In this study, LPA activated ROCK, p38, JNK and NF-κB pathways and induced interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1) mRNA and protein expression in human endothelial cells. We mapped signaling events downstream of ROCK, driving chemokine production. In summary, MCP-1 production was partly regulated by ROCK acting upstream of p38 and JNK and mediated downstream by NF-κB. IL-8 production was largely driven by ROCK through p38 and JNK activation, but with no involvement of NF-κB.     

IL-17 stimulates inflammatory responses via NF-kappaB and MAP kinase pathways in human colonic myofibroblasts

Colonic subepithelial myofibroblasts (SEMFs) may play a role in the modulation of mucosal inflammatory responses. We investigated the effects of interleukin (IL)-17 on IL-6 and chemokine [IL-8 and monocyte chemoattractant protein (MCP)-1] secretion in colonic SEMFs. Cytokine expression was determined by ELISA and Northern blotting. Nuclear factor kappa B (NF-kappaB) DNA-binding activity was evaluated by electrophortetic gel mobility shift assay (EMSA). The activation of mitogen-activated protein kinase (MAPK) was assessed by immunoblotting. IL-6, IL-8, and MCP-1 secretions were rapidly induced by IL-17. IL-17 induced NF-kappaB activation within 45 min after stimulation. A blockade of NF-kappaB activation markedly reduced these responses. MAPK inhibitors (SB-203580, PD-98059, and U-0126) significantly reduced the IL-17-induced IL-6 and chemokine secretion. The combination of either IL-17 + IL-1beta or IL-17 + tumor necrosis factor (TNF)-alpha enhanced cytokine secretion; in particular, the effects of IL-17 + TNF-alpha on IL-6 secretion were much stronger than the other responses. This was dependent on the enhancement of IL-6 mRNA stability. In conclusion, human SEMFs secreted IL-6, IL-8, and MCP-1 in response to IL-17. These responses might play an important role in the pathogenesis of gut inflammation.     

Secretion of IL-1: role of protein kinase C.

In an attempt to define the mechanism by which endotoxin induces its biologic activity, LPS was incorporated into phospholipid vesicles (liposomes) and compared with free LPS for ability to stimulate human monocytes. Activation of human monocytes by free LPS caused the translocation of protein kinase C (PKC) from the cytosol to the plasma membranes, the production of both IL-1, alpha and beta, and IL-1 secretion. Activation by LPS presented in multilamellar vesicles (MLV)-LPS caused IL-1 production but not IL-1 secretion. Moreover, MLV-LPS did not induce PKC translocation. MLV themselves did not inhibit monocyte stimulation by LPS, since LPS presented at the surface of lyophilized liposomes behaved like free LPS in cell activation. In contrast, MLV-LPS primed monocytes for subsequent LPS stimulation. When monocytes were activated by LPS in the presence of PKC inhibitors, no plasma membrane-associated PKC or IL-1 secretion was detected, whereas IL-1 production was observed. PKC inhibitors did not affect IL-1 alpha and IL-1 beta production, showing that PKC is not involved in the production of either IL-1. It can be concluded that IL-1 production and secretion are induced independently, and that IL-1 secretion involves PKC.     

Direct and monocyte-induced innate immune response of human lung epithelial cells to Brucella abortus infection

Although Brucella frequently infects humans through inhalation, its interaction with pulmonary cells has been overlooked. We examined whether human lung epithelial cells produce proinflammatory mediators in response to Brucella infection. Infection with smooth or rough strains of Brucella abortus induced the secretion of IL-8 and GM-CSF by the bronchial epithelial cell lines Calu-6 and 16HBE14o-, but not by the alveolar epithelial cell line A549. Infected Calu-6 cells also produced low levels of MCP-1. Since monocyte-derived cytokines may induce chemokine secretion in epithelial cells, cocultures of human monocytes (THP-1 cell line) and respiratory epithelial cells were used to study such interaction. IL-8 and MCP-1 levels in B. abortus-infected THP-1:A549 and THP-1:Calu-6 cocultures, and MCP-1 levels in THP-1:16HBE14o- cocultures, were higher than those detected in infected epithelial monocultures. Conditioned medium from infected monocytes induced the secretion of IL-8 and/or MCP-1 by A549 and Calu-6 cells, and these effects were mainly mediated by IL-1 (in A549 cells) or TNF-α (in Calu-6 cells). Conversely, culture supernatants from Brucella-infected bronchial epithelial cells induced MCP-1 production by monocytes, an effect largely mediated by GM-CSF. This study shows that human lung epithelial cells mount a proinflammatory response to Brucella, either directly or after interaction with Brucella-infected monocytes.     

Induction and Secretion of the Chemokines Interleukin-8 and Monocyte Chemotactic Protein-1 in Human Immature Leukemia Cell Lines

We investigated expression and secretion of the chemokines interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1) in human myeloid cell lines. Quantitative determination by ELISA revealed a significant constitutive production of both chemokines in the cell lines HL-60 and NB-4 (>1000 pg/ml IL-8 and >400 pg/ml MCP-1 per million cells), while in the cell lines EOL-1, KASUMI-1 and KG-1 only 10–100 pg/ml IL-8 and MCP-1 were detected. Tetradecanoyl phorbol acetate (TPA) strongly increased the IL-8 and MCP-1 amounts in the culture supernatants of all five cell lines. The TPA-induced NB-4 produced the largest amounts of both chemokines (>40,000 pg/ml). The strongest induction was seen in EOL-1 (>100-fold increase). Besides TPA, tumor necrosis factor-α (TNFα) also distinctively enhanced IL-8 and MCP-1 production. The calcium ionophore A-23187 and thapsigargin, an inhibitor of the Ca²⁺-ATPase, differentially induced IL-8 and MCP-1 secretion in the cell lines investigated, suggesting that, at least in some cell lines, intracellular free Ca²⁺ might be important for chemokine secretion. Dexamethasone significantly prevented the IL-8 and MCP-1 production of stimulated cells, emphasizing the potent anti-inflammatory property of glucocorticoids. Similarly, the protein kinase inhibitor staurosporine clearly decreased the TPA-induced chemokine secretion in NB-4 cells, indicating the involvement of protein kinases in the signal transduction pathway which leads to enhanced chemokine secretion.     

Cry1Ac toxin induces macrophage activation via ERK1/2, JNK and p38 mitogen-activated protein kinases

The Cry1Ac toxin from Bacillus thuringiensis is used commercially as a bio-insecticide and is expressed in transgenic plants that are used for human and animal consumption. Although it was originally considered innocuous for mammals, the Cry1Ac toxin is not inert and has the ability to induce mucosal and systemic immunogenicity. Herein, we examined whether the Cry1Ac toxin promotes macrophage activation and explored the signalling pathways that may mediate this effect. Treatment of primary and RAW264.7 macrophages with the Cry1Ac toxin resulted in upregulation of the costimulatory molecules CD80, CD86 and ICOS-L and enhanced production of nitric oxide, the chemokine MCP-1 and the proinflammatory cytokines TNF-α and IL-6. Remarkably, the Cry1Ac toxin induced phosphorylation of the mitogen-activated protein kinases (MAPKs) ERK1/2, JNK and p38 and promoted nuclear translocation of nuclear factor-kappa B (NF-κB) p50 and p65. p38 and ERK1/2 MAPKs were involved in this effect, as indicated by the Cry1Ac-induced upregulation of CD80 and IL-6 and TNF-α abrogation by the p38 MAPK inhibitor SB203580. Furthermore, treatment the MEK1/2 kinase inhibitor PD98059 blocked increases in MCP-1 secretion and augmented Cry1Ac-induced ICOS-L upregulation. These data demonstrate the capacity of the Cry1Ac toxin to induce macrophage activation via the MAPK and NF-κB pathways.     

Key role of regulated upon activation normal T-cell expressed and secreted, nonstructural protein1 and myeloperoxidase in cytokine storm induced by influenza virus PR-8 (A/H1N1) infection in A549 bronchial epithelial cells

Influenza virus infection causes severe respiratory disease such as that due to avian influenza (H5N1). Influenza A viruses proliferate in human epithelial cells, which produce inflammatory cytokines/chemokines as a "cytokine storm" attenuated with the viral nonstructural protein 1 (NS1). Cytokine/chemokine production in A549 epithelial cells infected with influenza A/H1N1 virus (PR-8) or nonstructural protein 1 (NS1) plasmid was examined in vitro. Because tumor necrosis factor-α (TNF-α) and regulated upon activation normal T-cell expressed and secreted (RANTES) are predominantly produced from cells infected with PR-8 virus, the effects of mRNA knockdown of these cytokines were investigated. Small interfering (si)TNF-α down-regulated RANTES expression and secretion of RANTES, interleukin (IL)-8, and monocyte chemotactic protein-1 (MCP-1). In addition, siRANTES suppressed interferon (IFN)-γ expression and secretion of RANTES, IL-8, and MCP-1, suggesting that TNF-α stimulates production of RANTES, IL-8, MCP-1, and IFN-γ, and RANTES also increased IL-8, MCP-1, and IFN-γ. Furthermore, administration of TNF-α promoted increased secretion of RANTES, IL-8, and MCP-1. Administration of RANTES enhanced IL-6, IL-8, and MCP-1 production without PR-8 infection. These results strongly suggest that, as an initial step, TNF-α regulates RANTES production, followed by increase of IL-6, IL-8, and MCP-1 and IFNs concentrations. At a later stage, cells transfected with viral NS1 plasmid showed production of a large amount of IL-8 and MCP-1 in the presence of the H(2)O(2)-myeloperoxidse (MPO) system, suggesting that NS1 of PR-8 may induce a "cytokine storm" from epithelial cells in the presence of an H(2)O(2)-MPO system.     

Simian virus 5 is a poor inducer of chemokine secretion from human lung epithelial cells: identification of viral mutants that activate interleukin-8 secretion by distinct mechanisms

We have compared chemokine secretion from human lung A549 cells infected with simian virus 5 (SV5) with other members of the Rubulavirus genus of paramyxoviruses. High levels of the chemokines interleukin-8 (IL-8) and macrophage chemoattractant protein-1 (MCP-1) were secreted from A549 cells infected with Human parainfluenza virus type 2 (HPIV-2) but not from cells infected with wild-type (WT) SV5. The lack of IL-8 secretion from SV5-infected cells was not due to a global block in all signal transduction pathways leading to IL-8 secretion, since SV5-infected A549 cells secreted IL-8 after stimulation with exogenously added tumor necrosis factor alpha or by coinfection with HPIV-2. A previously described, recombinant SV5 containing substitutions in the shared region of the P/V gene (rSV5-P/V-CPI-) induced IL-8 secretion by a mechanism that was dependent on viral gene expression. By contrast, an SV5 variant isolated from persistently infected cells (Wake Forest strain of Canine parainfluenza virus) induced IL-8 secretion by a mechanism that was largely not affected by inhibitors of viral gene expression. Together, these data demonstrate that SV5 is unusual compared to other closely related paramyxoviruses, since SV5 is a very poor inducer of the cytokines IL-8 and MCP-1. The isolation of two recombinant SV5 mutants that are defective in preventing chemokine induction will allow an identification of mechanisms utilized by WT SV5 to avoid activation of host cell innate immune responses to infection.     

Differential signaling mechanisms of HNP-induced IL-8 production in human lung epithelial cells and monocytes

Human neutrophil peptides (HNP) kill microorganisms but also modulate immune responses through upregulation of the chemokine IL-8 by activation of the nucleotide P2Y(6) receptor. However, the intracellular signaling mechanisms remain yet to be determined. Human lung epithelial cells (A549) and monocytes (U937) were stimulated with HNP in the absence and presence of the specific kinase inhibitors for Src, extracellular signal-regulated kinase-1 and -2 (ERK1/2), p38 mitogen-activated protein kinase (MAPK), c-Jun-N-terminal kinases (JNK), and Akt. HNP induced a rapid phosphorylation of the kinases in both cell types associated with a dose-dependent, selective production of IL-8 among 10 cytokines assayed. The HNP-induced IL-8 production was blocked by the Src tyrosine kinase inhibitor PP2, MEK1/2 inhibitor U0126, and the phosphatidylinositol 3 kinase (PI3K) inhibitor LY294002, but not by the JNK inhibitor SP600125 in both cell types. Treatment with the p38 inhibitor SB203580 attenuated the HNP-induced IL-8 production only in monocytes. Blockade of Src kinase blunted HNP-induced phosphorylation of the ERK1/2 and Akt but not p38 in monocytes. In contrast, Src inhibition had no effect on phosphorylation of the other kinases in the lung epithelial cells. We conclude that the activation of ERK1/2 and PI3K/Akt pathways is required for HNP-induced IL-8 release which occurs in a Src-independent manner in lung epithelial cells, while is Src-dependent in monocytes.