Signal Transduction Pathways In Macrophages Research Articles

Direct Binding Analysis Between C-Type Lectins and Glycans Using Immunoglobulin Receptor Fusion Proteins.

C-type lectins bind to carbohydrate structures in a Ca2+-dependent manner. Some transmembrane forms of lectins act as innate immune receptors and induce signal transduction pathways in macrophages and dendritic cells (DCs). Expressing these receptors in cells bearing a reporter gene is a useful tool to investigate ligand binding and recognition. However, it cannot be used to quantify the precise affinity of the interaction, and the involvement of other proteins remains a possibility. Direct binding between a receptor and its ligand can be investigated using an immunoglobulin receptor (Ig)-fused soluble protein. This binding can be assessed using enzyme-linked immunosorbent assays and flow cytometry, and the fusion protein may also be used in a glycan array. In this chapter, we explain the generation of Ig fusion proteins and subsequent binding assays using these proteins.

Suppression of inflammation by the rhizome of Anemarrhena asphodeloides via regulation of nuclear factor-κB and p38 signal transduction pathways in macrophages.

The rhizome of Anemarrhena asphodeloides Bunge (A. asphodeloides) has been used as a traditional East Asian medicine for the treatment of various types of inflammatory disease. However, to the best of our knowledge, there have been no systemic studies regarding the molecular mechanisms of action of the A. asphodeloides rhizome anti-inflammatory effects. The aim of the present study was to elucidate the anti-inflammatory effects and underlying mechanism of action of ethanol extracts of the rhizome of A. asphodeloides (EAA) in murine macrophages. Non-cytotoxic concentrations of EAA (10-100 µg/ml) significantly decreased the production of NO and interleukin (IL)-6 in lipopolysaccharide (LPS)-stimulated macrophages, while the production of tumor necrosis factor-α was not regulated by EAA. EAA-mediated reduction of nitric oxide (NO) was due to reduced expression levels of inducible NO synthase (iNOS). Furthermore, protein expression levels of LPS-induced cyclooxygenase-2, another inflammatory enzyme, were alleviated in the presence of EAA. EAA-mediated reduction of those proinflammatory mediators was due to inhibition of nuclear factor-κB (NF-κB) and activator protein 1 transcriptional activities followed by the stabilization of inhibitor of κ Bα and inhibition of p38, respectively. These results indicate that EAA suppresses LPS-induced inflammatory responses by negatively regulating p38 and NF-κB, indicating that EAA is a candidate treatment for alleviating inflammation.

Leishmania donovani phosphoproteins pp41 and pp29 re-establishes host protective immune response in visceral leishmaniasis

As phospho proteins are reported to be involved in virulence and survival, the ability of Leishmania to inhibit macrophage effector functions may result from a direct interference of leishmanial molecules with macrophage signal transduction pathways. Several such proteins such as pp63, pp41 and pp29 have also been identified as a Th1 stimulatory protein in the Leishmania donovani. In the present study, the immunogenicity of a cocktail of pp63+pp41+pp29 was assessed by estimation of serum antibody titre, nitric oxide(NO) production, estimation of Th1 cytokine(IFN-γ) as well as Th2 cytokines(IL-4), and determination of parasite load in L. donovani infected mice. In the group immunized with antigenic cocktail there was a sharp rise in antibody titer up to Day 20 which reduced considerably by Day 50. Groups of mice vaccinated with pp63, pp41, pp29 and the antigenic cocktail expressed 10-fold, 16-fold, 22-fold and 25-fold increase respectively in NO production by splenocytes. The animal groups immunized with pp63, pp41, pp29 and the antigenic cocktail showed reduced parasite load in the liver and spleen, as well as increased IFN-gamma production in the spleen. Furthermore immunized animals remained with a normal hematological profile, whereas L. donovani in unimmunized mice lead to significant anemia.

Discovery of Mycobacterium tuberculosis Protein Tyrosine Phosphatase B (PtpB) Inhibitors from Natural Products

Protein tyrosine phosphatase B (PtpB) is one of the virulence factors secreted into the host cell by Mycobacterium tuberculosis. PtpB attenuates host immune defenses by interfering with signal transduction pathways in macrophages and, therefore, it is considered a promising target for the development of novel anti-tuberculosis drugs. Here we report the discovery of natural compound inhibitors of PtpB among an in house library of more than 800 natural substances by means of a multidisciplinary approach, mixing in silico screening with enzymatic and kinetics studies and MS assays. Six natural compounds proved to inhibit PtpB at low micromolar concentrations (< 30 µM) with Kuwanol E being the most potent with K i = 1.6 ± 0.1 µM. To the best of our knowledge, Kuwanol E is the most potent natural compound PtpB inhibitor reported so far, as well as it is the first non-peptidic PtpB inhibitor discovered from natural sources. Compounds herein identified may inspire the design of novel specific PtpB inhibitors.

Cyclin‐dependent kinase inhibitor p21, via its C‐terminal domain, is essential for resolution of murine inflammatory arthritis

The mechanism responsible for persistent synovial inflammation in rheumatoid arthritis (RA) is unknown. Previously, we demonstrated that expression of the cyclin-dependent kinase inhibitor p21 is reduced in synovial tissue from RA patients compared to osteoarthritis patients and that p21 is a novel suppressor of the inflammatory response in macrophages. The present study was undertaken to investigate the role and mechanism of p21-mediated suppression of experimental inflammatory arthritis. Experimental arthritis was induced in wild-type or p21-/- (C57BL/6) mice, using the K/BxN serum-transfer model. Mice were administered p21 peptide mimetics as a prophylactic for arthritis development. Lipopolysaccharide-induced cytokine and signal transduction pathways in macrophages that were treated with p21 peptide mimetics were examined by Luminex-based assay, flow cytometry, or enzyme-linked immunosorbent assay. Enhanced and sustained development of experimental inflammatory arthritis, associated with markedly increased numbers of macrophages and severe articular destruction, was observed in p21-/- mice. Administration of a p21 peptide mimetic suppressed activation of macrophages and reduced the severity of experimental arthritis in p21-intact mice only. Mechanistically, treatment with the p21 peptide mimetic led to activation of the serine/threonine kinase Akt and subsequent reduction of the activated isoform of p38 MAPK in macrophages. These are the first reported data to reveal that p21 has a key role in limiting the activation response of macrophages in an inflammatory disease such as RA. Thus, targeting p21 in macrophages may be crucial for suppressing the development and persistence of RA.

The function of soluble form of CD163 in severe infection and sepsis

CD163 is one of transmembrane glycoproteins expressed only in activated monocyte-macrophage cell membrane, which acts as a marker of activated macrophages. In sepsis, CD163 activates the intracellular signal transduction pathways in macrophages,regulates TNF-α, IL-10 and IL-6 expression, have antiinflammatory and antioxidant effects in the inflammatory process. Soluble CD163 (sCD163) is a soluble form of CD163 ,which could play anti-inflammatory and immunoregulatory effects by inhibiting T cells proliferation and activation. Serum concentration of sCD163 may be highly increased when severe infection. Detection of serum sCD163 is helpful to diagnose sepsis,and to evaluate the prognosis of severe infection and sepsis. Key words: Severe infection; Sepsis; Soluble CD163

Involvement of SPI-2-encoded SpiC in flagellum synthesis in Salmonella enterica serovar Typhimurium

BackgroundSpiC encoded within Salmonella pathogenicity island 2 on the Salmonella enterica serovar Typhimurium chromosome is required for survival within macrophages and systemic infection in mice. Additionally, SpiC contributes to Salmonella-induced activation of the signal transduction pathways in macrophages by affecting the expression of FliC, a component of flagella filaments. Here, we show the contribution of SpiC in flagellum synthesis.ResultsQuantitative RT-PCR shows that the expression levels of the class 3 fliD and motA genes that encode for the flagella cap and motor torque proteins, respectively, were lower for a spiC mutant strain than for the wild-type Salmonella. Further, this mutant had lower expression levels of the class 2 genes including the fliA gene encoding the flagellar-specific alternative sigma factor. We also found differences in flagella assembly between the wild-type strain and the spiC mutant. Many flagella filaments were observed on the bacterial surface of the wild-type strain, whereas the spiC mutant had only few flagella. The absence of spiC led to reduced expression of the FlhD protein, which functions as the master regulator in flagella gene expression, although no significant difference at the transcription level of the flhDC operon was observed between the wild-type strain and the spiC mutant.ConclusionThe data show that SpiC is involved in flagella assembly by affecting the post-transcription expression of flhDC.

Salmonella virulence factor SpiC is involved in expression of flagellin protein and mediates activation of the signal transduction pathways in macrophages

SpiC is a virulence factor encoded within Salmonella pathogenicity island 2 (SPI-2). We have previously reported that infection of macrophages with Salmonella enterica serovar Typhimurium results in the SPI-2-dependent activation of the mitogen-activated protein kinase (MAPK) signalling pathways, leading to the expression of suppressor of cytokine signalling (SOCS)-3, which is involved in the inhibition of cytokine signalling. Here, we investigated the mechanism by which SpiC mediates the activation of signal transduction pathways in macrophages. Proteomic analysis showed that the level of FliC protein, a component of the flagellar filaments, was lower in the culture supernatant of a spiC mutant than in the supernatant from wild-type Salmonella. Furthermore, quantitative real-time RT-PCR showed that this mutant had a much lower level of fliC mRNA, indicating that SpiC regulates the transcription of FliC. We also found that the level of SOCS-3 in J774 macrophages was lower when they were infected with the fliC mutant than with wild-type Salmonella. FliC participated in the activation of MAPK pathways in Salmonella-infected macrophages, leading to the upregulation of SOCS-3 expression. Collectively, these results indicate that SpiC is involved in the expression of FliC, which plays a significant role in the SPI-2-dependent activation of MAPK pathways in Salmonella-infected macrophages.

Analysis of Signal Transduction Pathways in Macrophages Using Expression Vectors with CMV Promoters: A Cautionary Tale

The cytomegalovirus (CMV) major immediate-early promoter is a strong promoter used for both in vitro and in vivo expression of proteins in signal transduction and gene therapy studies. CMV activity is induced by external stimuli such as endotoxin from Gram-negative bacteria (LPS), TNF-alpha and phorbol esters. This inducibility poses problems when this promoter is used to drive the expression of either wild type or dominant negative mutated proteins as tools in signal transduction studies. This report draws attention to the problem associated with this widely used approach. The role of NF-kappaB and Hypoxia Inducible Factor-1alpha (HIF-1alpha) in the transcriptional regulation of Vascular Endothelial Growth Factor (VEGF) in macrophages was investigated using CMV-promoter-driven expression of either wild type or dominant negative proteins involved in these pathways. Difficulties encountered while interpreting the data due to the inducibility of the CMV promoter by LPS are highlighted in this report and provide a cautionary note for the evaluation of data acquired using this approach.

Homeostatic Role of Interferons Conferred by Inhibition of IL-1-Mediated Inflammation and Tissue Destruction

In addition to their well known immune and proinflammatory activities, IFNs possess homeostatic functions that limit inflammation and tissue destruction in a variety of conditions such as arthritis, osteolysis, and multiple sclerosis. The mechanisms underlying the homeostatic actions of IFNs are not well understood. We report here that both type I and type II IFNs (IFN-alpha, IFN-beta, and IFN-gamma, respectively) suppressed a broad range of proinflammatory and tissue-destructive activities of IL-1, including induction of inflammatory mediators, production of matrix metalloproteinases, macrophage tissue invasion, and cartilage degradation. IFN-alpha attenuated IL-1-mediated cell recruitment in vivo. IFNs completely suppressed the activation of IL-1 signal transduction pathways in macrophages. The mechanism of IFN-mediated inhibition of IL-1 action and signaling was modulation of IL-1R expression, which was also observed in vivo. IFN-gamma-mediated down-regulation of IL-1R type I expression was dependent on Stat1, a transcription factor typically considered to be a key mediator of macrophage activation by IFNs. These results identify cellular and molecular mechanisms that contribute to the homeostatic role of IFNs in limiting inflammation and associated tissue destruction.

Involvement of MAP kinases in apoptosis of macrophage treated with Trichomonas vaginalis.

A primitive protozoan parasite Trichomonas vaginalis selectively activates the signal transduction pathways in macrophages (RAW264.7). This study evaluated the correlation of these signaling pathways and T. vaginalis-induced cell apoptosis. In macrophages infected with T. vaginalis, apoptosis was assessed on the basis of DNA fragmentation on agarose gel electrophoresis. Infection of macrophages with T. vaginalis induced tyrosine phosphorylation of several proteins. Infected cells with T. vaginalis were shown to associate with phosphorylation of the extracellular signal-regulated (ERK)1/2 kinase, p38, c-Jun N-terminal kinase (JNK) mitogen-activated protein (MAP) kinases on Western blot analysis. The present finding also demonstrated a link between the ERK1/2, JNK and p38 apoptotic pathways that was modulated by T. vaginalis infection.

Lipopolysaccharide desensitization of macrophages provides protection against Yersinia enterocolitica-induced apoptosis.

Pathogenic Yersinia spp. uncouple an array of signal transduction pathways in macrophages to disrupt their response to infection. This compels the macrophage to undergo apoptosis. Our study shows that macrophages that had acquired tolerance to Yersinia infection by preexposure to lipopolysaccharide were considerably protected against Y. enterocolitica-induced apoptosis. The desensitization of macrophages by lipopolysaccharide, which is thought to be a self-protective, adaptive response to sustained bacterial stimulation, may represent an immune mechanism that aids in overcoming Yersinia-mediated apoptosis and infection.

Role of MacMARCKS in Integrin-dependent Macrophage Spreading and Tyrosine Phosphorylation of Paxillin

The cellular function of the MARCKS family of protein kinase C substrates is unknown. In this report, we present evidence that indicates a role for MacMARCKS, a member of the MARCKS family, in the integrin-dependent signal transduction pathways in macrophages. Using a dominant negative mutant of MacMARCKS, we showed that MacMARCKS participates in several integrin-dependent macrophage functions, including the phorbol ester-stimulated macrophage spreading, a process involving multiple integrins. The dominant negative mutant also blocks macrophage spreading on immune complex-coated surfaces, a process again requiring beta2 integrin. More direct evidence of the role of MacMARCKS in the integrin-dependent pathway is the ablation of macrophage binding to complement iC3b-coated sheep erythrocytes by MacMARCKS mutant, suggesting an effect of this mutant on the avidity of complement receptor 3, a member of the beta2 integrin family. To further evaluate the possible mechanism of MacMARCKS function, the integrin-dependent tyrosine phosphorylation of paxillin was examined. Concomitant with the inhibition of macrophage spreading and rosette formation, MacMARCKS mutant also inhibits integrin-dependent tyrosine phosphorylation of paxillin. Furthermore, immunofluorescent microscopy data showed that MacMARCKS and paxillin colocalize in the membrane ruffles at the leading edge of the spreading cells, providing a potential site and opportunity for MacMARCKS to participate in the regulation of integrin-dependent tyrosine phosphorylation of paxillin. Together, these data strongly suggest that MacMARCKS plays a role in integrin-dependent signal transduction pathways in macrophages.

A pleiotropic effect of fluoride on signal transduction in macrophages: is it mediated by GPT-binding proteins?

The activation of GTP-binding proteins (G-proteins) by sodium fluoride + aluminum (AlF4-) was shown in several cell free systems. In the intact cell, NaF +/- aluminum was shown to activate various signal transduction pathways and indirect evidence is in line with effector mechanisms involving regulation of G-protein activity. We have explored the effect of NaF on several components of signal transduction pathways in macrophages. NaF was shown to reduce intracellular ATP levels and to suppress agonist-induced protein tyrosine phosphorylation and reactive oxygen species formation. NaF led to in situ activation of nitrogen activated protein kinase, phospholipase A2 and PtdIns-phospholipase C. Addition of AlCl(3) or deferoxamine, a chelator of aluminum, had little or no effect on NaF mediated enzyme activation. The results suggest that at least some of the pleiotropic effects of NaF in intact cells may not be mediated by G-protein activation but rather by depletion of ATP which is essential for protein phosphorylation reactions.