MonoMac6 Cells Research Articles

Enhanced Cellular Uptake of a New, in Silico Identified Antitubercular Candidate by Peptide Conjugation

Mycobacterium tuberculosis is a successful pathogen, and it can survive in infected macrophages in dormant phase for years and decades. The therapy of tuberculosis takes at least six months, and the slow-growing bacterium is resistant to many antibiotics. The development of novel antimicrobials to counter the emergence of bacteria resistant to current therapies is urgently needed. In silico docking methods and structure-based drug design are useful bioinformatics tools for identifying new agents. A docking experiment to M. tuberculosis dUTPase enzyme, which plays a key role in the bacterial metabolism, has resulted in 10 new antitubercular drug candidates. The uptake of antituberculars by infected macrophages is limited by extracellular diffusion. The optimization of the cellular uptake by drug delivery systems can decrease the used dosages and the length of the therapy, and it can also enhance the bioavailability of the drug molecule. In this study, improved in vitro efficacy was achieved by attaching the TB5 antitubercular drug candidate to peptide carriers. As drug delivery components, (i) an antimicrobial granulysin peptide and (ii) a receptor specific tuftsin peptide were used. An efficient synthetic approach was developed to conjugate the in silico identified TB5 coumarone derivative to the carrier peptides. The compounds were effective on M. tuberculosis H37Rv culture in vitro; the chemical linkage did not affect the antimycobacterial activity. Here, we show that the OT20 tuftsin and GranF2 granulysin peptide conjugates have dramatically enhanced uptake into human MonoMac6 cells. The TB5-OT20 tuftsin conjugate exhibited significant antimycobacterial activity on M. tuberculosis H37Rv infected MonoMac6 cells and inhibited intracellular bacteria.

Identification and Characterization of a Novel Class of c-Jun N-terminal Kinase Inhibitors

In efforts to identify novel small molecules with anti-inflammatory properties, we discovered a unique series of tetracyclic indenoquinoxaline derivatives that inhibited lipopolysaccharide (LPS)-induced nuclear factor-κB/activating protein 1 activation. Compound IQ-1 (11H-indeno[1,2-b]quinoxalin-11-one oxime) was found to be a potent, noncytotoxic inhibitor of pro-inflammatory cytokine [interleukin (IL)-1α, IL-1β, IL-6, IL-10, tumor necrosis factor (TNF)-α, interferon-γ, and granulocyte-macrophage colony-stimulating factor] and nitric oxide production by human and murine monocyte/macrophages. Three additional potent inhibitors of cytokine production were identified through further screening of IQ-1 analogs. The sodium salt of IQ-1 inhibited LPS-induced TNF-α and IL-6 production in MonoMac-6 cells with IC(50) values of 0.25 and 0.61 μM, respectively. Screening of 131 protein kinases revealed that derivative IQ-3 [11H-indeno[1,2-b]quinoxalin-11-one-O-(2-furoyl)oxime]was a specific inhibitor of the c-Jun N-terminal kinase (JNK) family, with preference for JNK3. This compound, as well as IQ-1 and three additional oxime indenoquinoxalines, were found to be high-affinity JNK inhibitors with nanomolar binding affinity and ability to inhibit c-Jun phosphorylation. Furthermore, docking studies showed that hydrogen bonding interactions of the active indenoquinoxalines with Asn152, Gln155, and Met149 of JNK3 played an important role in enzyme binding activity. Finally, we showed that the sodium salt of IQ-1 had favorable pharmacokinetics and inhibited the ovalbumin-induced CD4(+) T-cell immune response in a murine delayed-type hypersensitivity model in vivo. We conclude that compounds with an indenoquinoxaline nucleus can serve as specific small-molecule modulators for mechanistic studies of JNKs as well as a potential leads for the development of anti-inflammatory drugs.

Ganodermycin, a novel inhibitor of CXCL10 expression from Ganoderma applanatum

CXCL10 (inducible protein-10) is a highly inducible chemoattractant, which contributes to the recruitment of inflammatory cells, such as macrophages and T-lymphocytes, and thereby has important roles in chronic inflammatory conditions. In a search for new inhibitors of CXCL10 expression in MonoMac6 cells, a novel compound, designated as Ganodermycin, was isolated from fermentations of the basidiomycete Ganoderma applanatum. The structure was determined by a combination of spectroscopic techniques. Ganodermycin inhibited the lipopolysaccharide (LPS)/interferon (IFN)-γ-induced CXCL10 promoter activity in transiently transfected MonoMac6 cells in a dose-dependent manner with IC(50) values of 15-20 μg ml(-1) (53-71 μM). Ganodermycin also reduced LPS/IFN-γ-induced CXCL10 protein synthesis and excretion.

Loss of Heat Shock Protein gp96 Does Not Impair Toll Like Receptor Signaling In Vitro

Background: The heat shock protein gp96 is an endoplasmic reticulum chaperone for multiple protein substrates. We showed that a lack of gp96 in intestinal macrophages of Crohn's Disease (CD) patients is correlated with loss of tolerance against the host gut flora, leading to chronic inflammation. A recent manuscript suggested gp96 to be the major chaperone for TLRs in murine macrophages. Therefore, we studied the impact of gp96 knockdown on TLR function in the monocytic cell line Mono Mac 6 (MM6). Methods: For stable gp96 knockdown a lentiviral system was used. Stimulations were performed with lipopolysaccharide (LPS), a ligand of TLR 4. Internalization was shown using FITC-labeled LPS. TLR folding and functionality was investigated by Western blotting and flow cytometric analysis. IL-8 secretion upon stimulation with LPS was determined by ELISA. Results: Flow cytometric analysis of TLR 2 and 4 showed similar patterns on the cell surface of gp96knockdown cells as well as on control MM6 cells. Internalization of LPS by gp96-knockdown cells was shown using a FITC-labeled derivative. No reduction by gp96 knockdown was observed. Western blot analysis of phospho-IκB/IκB and phospho-NFκB/NFκB ratios did not reveal a significant difference in TLR mediated NFκB signaling between gp96 knockdown and control cells. Induction of IL-8 secretion upon stimulation with LPS was comparable between knockdown and control cells. Conclusions: In contrast to recent reports loss of gp96 does not have pivotal effects on functionality of TLRs in human MM6 cells. These findings indicate that the loss of tolerance against commensal gut flora is caused by different mechanisms yet to be identified.

Syk Regulates Multiple Signaling Pathways Leading to CX3CL1 Chemotaxis in Macrophages

Several studies have clearly established the importance of the interaction between macrophages and CX3CL1 in the progression of disease. A previous study demonstrated that Syk was required for CX3CL1-mediated actin polymerization and chemotaxis. Here, we delineated the signaling cascade of Syk-mediated cell migration in response to CX3CL1. Inhibition of Syk in bone marrow-derived macrophages or reduction of Syk expression using siRNA in RAW/LR5 cells indicated that Syk was required for the activation of PI3K, Cdc42, and Rac1. Also, reduction in WASP or WAVE2 levels, common downstream effectors of Cdc42 or Rac1, resulted in impaired cell migration to CX3CL1. Syk indirectly regulated WASP tyrosine phosphorylation through Cdc42 activation. Altogether, our data identify that Syk mediated chemotaxis toward CX3CL1 by regulating both Rac1/WAVE2 and Cdc42/WASP pathways, whereas Src family kinases were required for proper WASP tyrosine phosphorylation.

Inhibition of TGF-β signaling, vasculogenic mimicry and proinflammatory gene expression by isoxanthohumol

TGF-β is a multifunctional cytokine that regulates cell proliferation, differentiation, apoptosis and extracellular matrix production. Deregulation of TGF-β production or signaling has been associated with a variety of pathological processes such as cancer, metastasis, angiogenesis and fibrosis. Therefore, TGF-β signaling has emerged as an attractive target for the development of new cancer therapeutics. In a screening program of natural compounds from fungi inhibiting the TGF-β dependent expression of a reporter gene in HepG2 cells, we found that the flavone isoxanthohumol inhibited the binding of the activated Smad2/3 transcription factors to the DNA and antagonized the cellular effects of TGF-β including reporter gene activation and expression of TGF-β induced genes in HepG2 and MDA-MB-231 cells. In an in vitro angiogenesis assay, isoxanthohumol (56μM) strongly decreased the formation of capillary-like tubules of MDA-MB-231 cells on Matrigel. In addition, we found that isoxanthohumol blocked IFN-γ, IL-4 and IL-6 dependent Jak/Stat signaling and strongly inhibited the induction of pro-inflammatory genes in MonoMac6 cells at the transcriptional level after LPS/TPA treatment.

Plasma from preeclamptic women activates endothelial cells via monocyte activation in vitro

In this study we tested whether plasma from preeclamptic women contains factors that can activate endothelial cells in the presence of monocytes in vitro. Plasma from preeclamptic women (n=6), healthy pregnant women (n=6) and nonpregnant women (n=6) was incubated with mono-cultures and co-cultures of human umbilical vein endothelial cells (HUVEC) and monomac-6 monocytes. Reactive oxygen species (ROS) production and ICAM-1 expression were measured using flow cytometry. Whether scavenging of ROS by superoxide dismutase and catalase inhibited HUVEC ICAM-1 expression was also investigated. We found that in HUVEC co-cultured with monomac-6 cells but not in HUVEC cultured alone, ICAM-1 was upregulated after incubation with plasma from preeclamptic women but not plasma from non-pregnant women. Also in co-cultures, monomac-6 ICAM-1 was upregulated by plasma from preeclamptic women, while in both mono- and co-cultures monomac-6 ROS production was upregulated by plasma from pregnant and preeclamptic women, compared with plasma from non-pregnant women. Scavenging of ROS by superoxide dismutase and catalase resulted in a further upregulation of HUVEC ICAM-1 after incubation with plasma from preeclamptic women, compared with incubation without superoxide dismutase and catalase. These results show that endothelial cells in vitro are activated by plasma of preeclamptic women only if they are co-cultured with monocytes. This upregulation appeared not to be due to extracellular ROS production by monocytes or HUVEC, pointing to involvement of other mechanisms. Our data suggest that plasma of preeclamptic women activates monocytes, and that these monocytes subsequently activate endothelial cells.

Influence of apple polyphenols on inflammatory gene expression

Apples (Malus spp., Rosaceae) and products thereof contain high amounts of polyphenols which show diverse biological activities and may contribute to beneficial health effects, like protecting the intestine against inflammation initiated by chronic inflammatory bowel diseases (IBD). IBD are characterized by an excessive release of several proinflammatory cytokines and chemokines by different cell types which results consequently in an increased inflammatory response. In the present study we investigated the preventive effectiveness of polyphenolic juice extracts and single major constituents on inflammatory gene expression in immunorelevant human cell lines (DLD-1, T84, MonoMac6, Jurkat) induced with specific stimuli. Besides the influence on proinflammatory gene expression, the effect on NF-kappaB-, IP-10-, IL-8-promoter-, STAT1-dependent signal transduction, and the relative protein levels of multiple released cytokines and chemokines were studied. DNA microarray analysis of several genes known to be strongly regulated during gastrointestinal inflammation, combined with quantitative real-time PCR (qRT-PCR) revealed that the apple juice extract AE04 (100-200 microg/mL) significantly inhibited the expression of NF-kappaB regulated proinflammatory genes (TNF-alpha, IL-1beta, CXCL9, CXCL10), inflammatory relevant enzymes (COX-2, CYP3A4), and transcription factors (STAT1, IRF1) in LPS/IFN-gamma stimulated MonoMac6 cells without significant effects on the expression of house-keeping genes. A screening of some major compounds of AE04 revealed that the flavan-3-ol dimer procyanidin B(2 )is mainly responsible for the anti-inflammatory activity of AE04. Furthermore, the dihydrochalcone aglycone phloretin and the dimeric flavan-3-ol procyanidin B(1 )significantly inhibited proinflammatory gene expression and repressed NF-kappaB-, IP-10-, IL-8-promoter-, and STAT1-dependent signal transduction in a dose-dependent manner. The influence on proinflammatory gene expression by the applied polyphenols thereby strongly correlated with the increased protein levels investigated by human cytokine array studies. In summary, we evaluated selected compounds responsible for the anti-inflammatory activity of AE04. In particular, procyanidin B(1), procyanidin B(2), and phloretin revealed anti-inflammatory activities in vitro and therefore may serve as transcription-based inhibitors of proinflammatory gene expression.

Ribosomal Protein S19 Interacts with Macrophage Migration Inhibitory Factor and Attenuates Its Pro-inflammatory Function

Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine that has been implicated in the pathogenesis of inflammatory disorders such as infection, sepsis, and autoimmune disease. MIF exists preformed in cytoplasmic pools and exhibits an intrinsic tautomerase and oxidoreductase activity. MIF levels are elevated in the serum of animals and patients with infection or different inflammatory disorders. To elucidate how MIF actions are controlled, we searched for endogenous MIF-interacting proteins with the potential to interfere with key MIF functions. Using in vivo biotin-tagging and endogenous co-immunoprecipitation, the ribosomal protein S19 (RPS19) was identified as a novel MIF binding partner. Surface plasmon resonance and pulldown experiments with wild type and mutant MIF revealed a direct physical interaction of the two proteins (K(D) = 1.3 x 10(-6) m). As RPS19 is released in inflammatory lesions by apoptotic cells, we explored whether it affects MIF function and inhibits its binding to receptors CD74 and CXCR2. Low doses of RPS19 were found to strongly inhibit MIF-CD74 interaction. Furthermore, RPS19 significantly compromised CXCR2-dependent MIF-triggered adhesion of monocytes to endothelial cells under flow conditions. We, therefore, propose that RPS19 acts as an extracellular negative regulator of MIF.

Hyperthermia induced NFκB mediated apoptosis in normal human monocytes

Conceptual approaches of heat-induced cytotoxic effects against tumor cells must address factors affecting therapeutic index, i.e., the relative toxicity for neoplastic versus normal tissues. Accordingly, we investigated the effect of hyperthermia treatment (HT) on the induction of DNA fragmentation, apoptosis, cell-cycle distribution, NFkappaB mRNA expression, DNA-binding activity, and phosphorylation of IkappaBalpha in the normal human Mono Mac 6 (MM6) cells. For HT, cells were exposed to 43 degrees C. FACS analysis showed a 48.5% increase in apoptosis, increased S-phase fraction, and reduced G2 phase fraction after 43 degrees C treatments. EMSA analysis showed a dose-dependent inhibition of NFkappaB DNA-binding activity after HT. This HT-mediated inhibition of NFkappaB was persistent even after 48 h. Immunoblotting analysis revealed dose-dependent inhibition of IkappaBalpha phosphorylation. Similarly, RPA analysis showed that HT persistently inhibits NFkappaB mRNA. These results demonstrate that apoptosis upon HT exposure of MM6 cells is regulated by IkappaBalpha phosphorylation mediated suppression of NFkappaB.

Porphyromonas gingivalis culture supernatants differentially regulate Interleukin-1β and Interleukin-18 in human monocytic cells

Porphyromonas gingivalis is a major bacterial species implicated in chornic periodontitis, a disease characterized by inflammatory destruction of the tooth supporting tissues. Its main virulence factors are lipopolysaccharide (LPS) and gingipains, a group of cysteine proteinases. Interleukin (IL)-18 is a potent pro-inflammatory cytokine with structural similarities to IL-1β. This study aimed to investigate if P .gingivalis regulates IL-1β and IL-18 in monocytic cells. Monomac-6 cells were challenged with P. gingivalis culture supernatants. Quantitative real-time PCR and ELISA were used to investigate IL-1β and IL-18 mRNA expression and protein secretion, respectively. P. gingivalis enhanced IL-1β and IL-18 mRNA expression, the former being induced earlier, but transiently. IL-18 up-regulation was not affected by P. gingivalis heat-inactivation or chemical inhibition of its gingipains, whereas both treatments resulted in 50% reduction of IL-1β expression. Purified P. gingivalis LPS enhanced both IL-1β and IL-18 expression. However, only IL-1β, but not IL-18, secretion was detected, and was up-regulated by P. gingivalis. In conclusion, although IL-1β and IL-18 belong to the same cytokine family, their gene expression and secretion are differentially regulated in human monocytic cells in response to P. gingivalis. Therefore, cytokines of the IL-1 family may participate via different pathways in the complex pathogenesis of periodontitis.

Design, Synthesis, and In Vitro Activity of Novel Drug Delivery Systems Containing Tuftsin Derivatives and Methotrexate

During the past decade, biodegradable polymers or oligopeptides recognized by cell-surface receptors have been shown to increase drug specificity, lowering systemic drug toxicity in contrast to small-size fast-acting drugs. The goal of the present study was to develop anticancer bioconjugates based on chemotactic drug targeting (CDT). These constructs are composed of methotrexate (Mtx) attached to a tuftsin-like peptide carrier through an enzyme-labile pentapeptide spacer (GFLGC) and several copies of a chemotactic targeting moiety (H-TKPR, For-TKPR, H-TKPKG, and Ac-TKPKG). Carriers with targeting moieties in the branches were prepared by solid-phase synthesis using mixed Boc and Fmoc strategies. The drug molecule connected to an enzyme-labile spacer was attached to the branched oligopeptide in solution. In vitro chemotaxis, cellular uptake, and cytotoxicity assays were carried out on the MonoMac6 cell line. The most effective conjugates with H-TKPR or Ac-TKPKG targeting moieties in the branches, which have the most advantageous chemotactic properties, can be internalized rapidly, and these conjugates trigger higher toxic effect than the free drug (Mtx). The results suggest that our tuftsin-based drug delivery systems might be potential candidates for targeting cancer chemotherapy.

Rosiglitazone and 15-Deoxy-Δ12,14-Prostaglandin J2, PPARγ Agonists, Differentially Regulate Cigarette Smoke-Mediated Pro-Inflammatory Cytokine Release in Monocytes/Macrophages

Peroxisome Proliferator-Activated Receptor gamma (PPARgamma) ligands have the potential for use as anti-inflammatory agents in chronic airway diseases. We hypothesized that cigarette smoke (CS)-mediated pro-inflammatory cytokine release would be downregulated in the monocyte-macrophage cell line (MonoMac6) by synthetic and natural PPARgamma ligands. Surprisingly, treatment of MonoMac6 cells with the natural PPARgamma ligand 15-deoxy-Delta12,14-prostaglandin J2 led to increased cytokine (IL-8) release in response to either TNF-alpha or CS extract (CSE). However, exposure to rosiglitazone, a synthetic agonist, led to decreased TNF-alpha, but not CSE, mediated cytokine release. Cytokine release correlated with nuclear PPARgamma localization; CSE reduced the amount of activated PPARgamma located in the nucleus and formed aldehyde adducts as PPARgamma protein carbonyls. Furthermore, it was shown that PPARgamma interacts with the RelA/p65 subunit of NF-kappaB under TNF-alpha exposure conditions, but this interaction was disrupted by CS exposure, suggesting that CS blocks this important anti-inflammatory pathway involving PPARgamma. Thus, these new data show that activation of PPARgamma with natural or synthetic ligands have differential inhibitory effects on CS-mediated pro-inflammatory mediator release. These data have implications in designing therapies for treatment of COPD and pulmonary fibrosis.

IKKα Causes Chromatin Modification on Pro-Inflammatory Genes by Cigarette Smoke in Mouse Lung

Cigarette smoke (CS) induces abnormal and sustained lung inflammation; however, the molecular mechanism underlying sustained inflammation is not known. It is well known that activation of I kappaB kinase beta (IKK beta) leads to transient translocation of active NF-kappaB (RelA/p65-p50) in the nucleus and transcription of pro-inflammatory genes, whereas the role of IKK alpha in perpetuation of sustained inflammatory response is not known. We hypothesized that CS activates IKK alpha and causes histone acetylation on the promoters of pro-inflammatory genes, leading to sustained transcription of pro-inflammatory mediators in mouse lung in vivo and in human monocyte/macrophage cell line (MonoMac6) in vitro. CS exposure to C57BL/6J mice resulted in activation of IKK alpha, leading to phosphorylation of ser10 and acetylation of lys9 on histone H3 on the promoters of IL-6 and MIP-2 genes in mouse lung. The increased level of IKK alpha was associated with increased acetylation of lys310 RelA/p65 on pro-inflammatory gene promoters. The role of IKK alpha in CS-induced chromatin modification was confirmed by gain and loss of IKK alpha in MonoMac6 cells. Overexpression of IKK alpha was associated with augmentation of CS-induced pro-inflammatory effects, and phosphorylation of ser10 and acetylation of lys9 on histone H3, whereas transfection of IKK alpha dominant-negative mutants reduced CS-induced chromatin modification and pro-inflammatory cytokine release. Moreover, phosphorylation of ser276 and acetylation of lys310 of RelA/p65 was augmented in response to CS extract in MonoMac6 cells transfected with IKK alpha. Taken together, these data suggest that IKK alpha plays a key role in CS-induced pro-inflammatory gene transcription through phospho-acetylation of both RelA/p65 and histone H3.

SIRT1, an Antiinflammatory and Antiaging Protein, Is Decreased in Lungs of Patients with Chronic Obstructive Pulmonary Disease

Abnormal inflammation and accelerated decline in lung function occur in patients with chronic obstructive pulmonary disease (COPD). Human sirtuin (SIRT1), an antiaging and antiinflammatory protein, is a metabolic NAD(+)-dependent protein/histone deacetylase that regulates proinflammatory mediators by deacetylating histone and nonhistone proteins. To determine the expression of SIRT1 in lungs of smokers and patients with COPD, and to elucidate the regulation of SIRT1 in response to cigarette smoke in macrophages, and its impact on nuclear factor (NF)-kappaB regulation. SIRT1 and NF-kappaB levels were assessed in lung samples of nonsmokers, smokers, and patients with COPD. Human monocyte-macrophage cells (MonoMac6) were treated with cigarette smoke extract (CSE) to determine the mechanism of CSE-mediated regulation of SIRT1 and its involvement in RelA/p65 regulation and IL-8 release. Peripheral lungs of smokers and patients with COPD showed decreased levels of nuclear SIRT1, as compared with nonsmokers, associated with its post-translational modifications (formation of nitrotyrosine and aldehyde carbonyl adducts). Treatment of MonoMac6 cells with CSE showed decreased levels of SIRT1 associated with increased acetylation of RelA/p65 NF-kappaB. Mutation or knockdown of SIRT1 resulted in increased acetylation of nuclear RelA/p65 and IL-8 release, whereas overexpression of SIRT1 decreased IL-8 release in response to CSE treatment in MonoMac6 cells. SIRT1 levels were reduced in macrophages and lungs of smokers and patients with COPD due to its post-translational modifications by cigarette smoke-derived reactive components, leading to increased acetylation of RelA/p65. Thus, SIRT1 plays a pivotal role in regulation of NF-kappaB-dependent proinflammatory mediators in lungs of smokers and patients with COPD.

A comparison of gene expression profiles between primary human AML cells and AML cell line

In acute myeloid leukemia (AML), hematologic malignancies are characterized by recurring chromosomal abnormalities. Chromosome translocation t(9;11)(p22;q23) is one of the most common genetic aberrations and results in the formation of the MLL-AF9 fusion gene that functions as a facilitator of cell growth directly. In order to study this type of AML, the cell lines with cytogenetically diagnosed t(9;11)(p22;q23), such as Mono Mac 6 (MM6), have been widely used. To examine whether there is any difference in gene expression between the primary human t(9;11) AML cells and MM6 cell line, genome-wide transcriptome analysis was performed on MM6 cell line using SAGE and the results were compared to the profile of primary human t(9;11) AML cells. 884 transcripts which were alternatively expressed between MM6 cells and primary human t(9;11) cells were identified through statistical analysis (P < 0.05) and 4-fold expression change. Of these transcripts, 830 (94%) matched to known genes or EST were classified by functional categories (http://david.abcc.ncifcrf.gov/). The majority of alternatively expressed genes in MM6 were involved in biosynthetic and metabolic processes, but HRAS, a protein that is known to be associated with leukemogenesis, was expressed only in MM6 cells and several other genes involved in Erk1/Erk2 MAPK pathway were also over-expressed in MM6. Therefore, since MM6 cell line has a similar expression profile to primary human t(9;11) AML in general and expresses uniquely a strong Erk1/Erk2 MAPK pathway including HRAS, it can be used as a model for HRAS-positive t(9;11) AML.

A Novel Secondary Acyl Chain in the Lipopolysaccharide of Bordetella pertussis Required for Efficient Infection of Human Macrophages

Lipopolysaccharide is one of the major constituents of the Gram-negative bacterial outer membrane and is a potent stimulator of the host innate immune response. The biosynthesis of the lipid A moiety of lipopolysaccharide is a complex process in which multiple gene products are involved. Two late lipid A acyl transferases, LpxL and LpxM, were first identified in Escherichia coli and shown to be responsible for the addition of secondary acyl chains to the 2' and 3' positions of lipid A, respectively. Here, we describe the identification of two lpxL homologues in the genome of Bordetella pertussis. We show that one of them, LpxL2, is responsible for the addition of the secondary myristate group that is normally present at the 2' position of B. pertussis lipid A, whereas the other one, LpxL1, mediates the addition of a previously unrecognized secondary 2-hydroxy laurate at the 2 position. Increased expression of lpxL1 results in the appearance of a hexa-acylated lipopolysaccharide form with strongly increased endotoxic activity. In addition, we show that an lpxL1-deficient mutant of B. pertussis displays a defect in the infection of human macrophages.

Differential regulation of matrix metalloproteinase 2 and matrix metalloproteinase 9 by activated protein C: Relevance to inflammation in rheumatoid arthritis

To investigate the in vitro effect of activated protein C (APC), a natural anticoagulant and novel antiinflammatory agent, on the regulation of the gelatinases matrix metalloproteinase 2 (MMP-2) and MMP-9. Synovial fibroblasts and peripheral blood monocytes isolated from patients with rheumatoid arthritis (RA) or osteoarthritis (OA) and Mono Mac6 cells were used in this study. After treatment, cells and culture supernatants were collected for zymography, enzyme-linked immunosorbent assay, reverse transcription-polymerase chain reaction, and Western blot analysis. Fibroblasts and monocytes from RA patients produced substantially more MMP-9 than did those from OA patients; however, there was no difference in MMP-2 production. The addition of recombinant APC markedly reduced MMP-9 at the gene and protein levels. In contrast, APC up-regulated and activated MMP-2. Using a blocking antibody to the endothelial protein C receptor (EPCR), we showed that the inhibition of MMP-9 by APC was EPCR-dependent. Furthermore, APC directly suppressed the production of tumor necrosis factor (TNF) and the activation of NF-kappaB and MAP kinase p38, and inhibitors of NF-kappaB or p38 reduced the production of MMP-9, suggesting that APC inhibits MMP-9 by blocking TNF, NF-kappaB, and p38. Thus, APC acts on MMP-9 by binding to EPCRs on the cell surface and, subsequently, inhibiting the intracellular activation of the proinflammatory signaling molecules NF-kappaB and p38. APC appears to be the first physiologic agent to inhibit the production of proinflammatory MMP-9, yet increase antiinflammatory MMP-2 activity. Our results provide the initial evidence that APC may be beneficial in the prevention of inflammation and joint destruction in RA.

Tiotropium suppresses acetylcholine-induced release of chemotactic mediators in vitro

The driving force in the progression of COPD is the development of exacerbations which are mostly the result of excessive inflammation. Bronchodilatators play an important role in the treatment of COPD. The reported reduction in exacerbation rates in COPD is due to the inhibition of vagal-mediated bronchoconstriction and mucus secretion. However, recent studies have highlighted the existence of muscarinic receptors on inflammatory cells and we have explored the possibility that tiotropium bromide might also inhibit neutrophil migration. We analysed the influence of tiotropium on the release of neutrophil chemotactic activity in response to acetylcholine (ACh) and the expression of muscarinic receptors on human alveolar macrophages (AM), A549 cells, MonoMac6 cells, and human lung fibroblasts. We found significant levels of all muscarinic receptor subtypes on all analysed cells except the fibroblasts. Fibroblasts expressed predominantly M2, receptors and did not release chemotactic activity. AM, A549 cells, and MonoMac6 cells released chemotactic active mediators after incubation with ACh. The secretion could be suppressed by more than 70% after coincubation with tiotropium. Tiotropium alone did not influence the granulocyte migration. Most of the chemotactic activity could be attributed to leukotriene B4 (LTB4). The release of interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1) was not induced by ACh. From this, we suggest that the suppression of the Ach-mediated release of chemotactic substances like LTB4 modulates the inflammatory reaction. This may contribute to the decreased rate of exacerbations in COPD, which was observed in clinical trials.

Porphyromonas gingivalis antagonises Campylobacter rectus induced cytokine production by human monocytes

Porphyromonas gingivalis and Campylobacter rectus are two major bacterial species implicated in the pathogenesis of periodontitis. P. gingivalis can antagonise the inflammatory response to other periodontal pathogens, a property commonly attributed to its lipopolysaccharide (LPS). The aim of this study was to investigate the capacity of P. gingivalis to antagonise C. rectus induced cytokine stimulation from human monocytes, and to investigate the involvement of its LPS. Primary human monocytes and Monomac-6 cells were challenged with culture supernatants from P. gingivalis and C. rectus, and levels of IL-1β, IL-6 and IL-8 produced were measured by ELISA after 6 h incubation. Purified P. gingivalis LPS was also added alone or in combination with C. rectus culture supernatant. Both species significantly stimulated the production of all three cytokines from the two cell lines, but P. gingivalis was considerably weaker inducer. Co-stimulation of the cells with P. gingivalis and C. rectus suppressed the cytokine-stimulatory capacity of the latter. P. gingivalis LPS alone was sufficient to antagonise IL-6 and IL-8, but not IL-1β stimulation by C. rectus. In conclusion, mixed infections may impair host immune responses by reducing pro-inflammatory cytokine levels, which may be of relevance to the pathogenesis of periodontitis.