Clinical and healthcare burden of disease associated with cytomegalovirus in allogeneic hematopoietic stem cell transplantation - A retrospective single-center study.

BACKGROUND: CMV infection is a common complication in allogeneic hematopoietic stem cell transplantation (HSCT). We investigated the association of clinically significant CMV (CS-CMV) infection with clinical outcomes and healthcare resource utilization in allogeneic HSCT patients in Finland. METHODS: This retrospective study included adult patients who received their first allogeneic HSCT between January 1, 2013, and December 31, 2018, at the Turku University Hospital. Data were collected from the hospital data lake. Clinical and healthcare outcomes were investigated at one year and mortality up to three years. RESULTS: The study included 251 patients. CMV seroprevalence was 69.7%. CS-CMV infection occurred in 59.0% of the patients, and of those, 14.2% had ≥2 infections. The median time to CS-CMV infection was 34.5 days (Q1 -Q3 , 27.0-45.0). Recipient and donor seropositivity, and lymphoproliferative diseases were associated with higher, and HLA identical sibling donors with lower CS-CMV infection risk. CS-CMV infection was not associated with mortality in three years of follow-up. One hundred thirty-three (89.8%) and 75 (72.8%) patients with and without CS-CMV infection, respectively, were readmitted to the hospital. Patients with CS-CMV infection had more hospital readmissions (incidence rate ratio [IRR] 1.38, 95% confidence interval [CI] 1.10-1.73, p = .005) and patients with one CS-CMV infection (IRR 1.48, 95% CI 1.12-1.94, p = .005) or ≥2 infections had longer length of hospital stay (IRR 2.71, 95% CI 1.76-4.35, p < .001). CONCLUSION: CMV seroprevalence is relatively high among Finnish allogeneic HSCT patients. CS-CMV infection was common and associated with a higher readmission rate and longer length of hospital stay.

MKP-1 promotes anti-inflammatory M(IL-4/IL-13) macrophage phenotype and mediates the anti-inflammatory effects of glucocorticoids.

Macrophage polarization refers to the ability of these cells to adopt different functional phenotypes according to their environment. Mitogen-activated protein kinase phosphatase-1 (MKP-1) is known to regulate the classical lipopolysaccharide (LPS)-induced pro-inflammatory macrophage activation and the inflammatory response. Here, we investigated the effects of MKP-1 on the anti-inflammatory and healing-promoting macrophage phenotype induced by cytokines IL-4 and IL-13 and examined the potential mediator role of MKP-1 in glucocorticoid effects on the two macrophage phenotypes. In MKP-1-deficient macrophages treated with IL-4 and IL-13 to induce the anti-inflammatory phenotype, the expression of phenotypic markers arginase 1, Ym-1 and FGF2 was reduced as compared to wild-type cells. In contrast, LPS-induced expression of the pro-inflammatory factors IL-6 and iNOS was significantly higher in MKP-1-deficient macrophages. Dexamethasone suppressed the pro-inflammatory phenotype and enhanced the anti-inflammatory phenotype. Interestingly, both of these glucocorticoid effects were attenuated in macrophages from MKP-1-deficient mice. Accordingly, dexamethasone increased MKP-1 expression in both LPS- and IL4+13-treated wild-type cells. In conclusion, the findings support MKP-1 as an endogenous mechanism able to shift macrophage activation from the classical pro-inflammatory state towards the anti-inflammatory and healing-promoting phenotype. In addition, MKP-1 was found to mediate the anti-inflammatory effects of dexamethasone in a dualistic manner: by suppressing the pro-inflammatory macrophage activation and by enhancing the healing-promoting macrophage phenotype.

Suppression of cytokine production by glucocorticoids is mediated by MKP-1 in human lung epithelial cells.

Mitogen-activated protein kinase phosphatase 1 (MKP-1) expression is induced by inflammatory factors and serves as an endogenous p38 MAPK suppressor to limit inflammatory response. Glucocorticoids are very effective anti-inflammatory drugs and they are used for the treatment of many inflammatory diseases, such as asthma and COPD. We investigated the role of MKP-1 in the inhibition of cytokine production by dexamethasone in human A549 bronchial epithelial cells. We found that dexamethasone increased MKP-1 expression, inhibited p38 MAPK phosphorylation, and suppressed TNF and MIP-3α production in A549 cells. Interestingly, the suppression of p38 MAPK phosphorylation and the inhibition of TNF expression by dexamethasone were attenuated in cells, where MKP-1 expression was silenced by siRNA. In conclusion, these data suggest that dexamethasone increases MKP-1 expression and this results in the suppression of p38 MAPK signaling leading to the inhibition of cytokine production in human bronchial epithelial cells. These results point to the role of MKP-1 as an important factor in the therapeutic effects of glucocorticoids in the treatment of inflammatory lung diseases.

ß2-receptor agonists salbutamol and terbutaline attenuated cytokine production by suppressing ERK pathway through cAMP in macrophages.

ß2-receptor agonists are used in the treatment of inflammatory obstructive lung diseases asthma and COPD as a symptomatic remedy, but they have been suggested to possess anti-inflammatory properties, also. ß2-receptor activation is considered to lead to the activation of ERK pathway through G-protein- and cAMP-independent mechanisms. In this study, we investigated the effects of ß2-receptor agonists salbutamol and terbutaline on the production of inflammatory factors in macrophages. We found that ß2-receptor agonists inhibited LPS-induced ERK phosphorylation and the production of MCP-1. A chemical cAMP analog 8-Br-cAMP also inhibited ERK phosphorylation and TNF and MCP-1 release. As expected, MAPK/ERK kinase (MEK)1/2 inhibitor PD0325901 inhibited ERK phosphorylation and suppressed both TNF and MCP-1 production. In conclusion, we suggest that ß2-receptor agonists salbutamol and terbutaline inhibit inflammatory gene expression partly by a mechanism dependent on cAMP leading to the inhibition of ERK signaling in macrophages. Observed anti-inflammatory effects of ß2-receptor agonists may contribute to the clinical effects of these drugs.

Catabolic and proinflammatory effects of leptin in chondrocytes are regulated by suppressor of cytokine signaling-3.

BACKGROUND: Previous studies provide evidence that adipokine leptin increases production of catabolic and proinflammatory factors in chondrocytes and serves as a link between obesity and osteoarthritis (OA). However, the magnitude of the response to leptin treatment varies greatly between chondrocytes from different donor patients. In the present study, we investigated the regulatory role of suppressor of cytokine signaling-3 (SOCS-3) in the leptin-induced responses in OA cartilage. METHODS: Cartilage and synovial fluid samples from 97 patients with OA undergoing knee replacement surgery were collected. Cartilage samples were cultured with leptin (10 µg/ml), and the levels of proinflammatory and catabolic factors in synovial fluid and in the cartilage culture media, and SOCS-3 expression in the cartilage were measured. The role of SOCS-3 in leptin signaling was further studied in H4 murine chondrocytes by downregulating SOCS-3 with siRNA. RESULTS: Leptin-induced expression of matrix metalloproteinases MMP-1, MMP-3, MMP-13, interleukin-6 (IL-6), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) were higher in the cartilage samples with low SOCS-3 expression. Accordingly, downregulation of SOCS-3 by siRNA in H4 chondrocytes led to enhanced leptin-induced expression of MMP-3, MMP-13, IL-6 and iNOS. Synovial fluid leptin was associated positively, and cartilage SOCS-3 negatively with synovial fluid levels of MMPs in a multivariate model in obese (body mass index (BMI) >30 kg/m2) but not in non-obese (BMI <30 kg/m2) patients. CONCLUSIONS: Our results show, for the first time, that SOCS-3 regulates leptin-induced responses in cartilage, and could thus be a future drug target in the treatment or prevention of OA, especially in obese patients.

The Inflammatory Phenotype in Failed Metal-On-Metal Hip Arthroplasty Correlates with Blood Metal Concentrations.

INTRODUCTION: Hip arthroplasty is the standard treatment of a painful hip destruction. The use of modern metal-on-metal (MOM) bearing surfaces gained popularity in total hip arthroplasties during the last decade. Recently, worrisome failures due to adverse reaction to metal debris (ARMD), including pseudotumor response, have been widely reported. However, the pathogenesis of this reaction remains poorly understood. The aim of the present study was to investigate the ARMD response by flow cytometry approach. METHODS: Sixteen patients with a failed Articular Surface Replacement (ASR) hip prosthesis were included in the study. Samples of pseudotumor tissues collected during revision surgery were degraded by enzyme digestion and cells were typed by flow cytometry. Whole blood chromium and cobalt concentrations were analyzed with mass spectrometry before revision surgery. RESULTS: Flow cytometry analysis showed that the peri-implant pseudotumor tissue expressed two principal phenotypes, namely macrophage-dominated and T-lymphocyte-dominated response; the average portions being 54% (macrophages) and 25% (T-lymphocytes) in macrophage-dominated inflammation and 20% (macrophages) and 54% (T-lymphocytes) in T-lymphocyte-dominated response. The percentages of B-lymphocytes and granulocytes were lower in both phenotypes. Interestingly, the levels of blood chromium and cobalt were significantly higher in patients with macrophage-dominated response. CONCLUSIONS: The results suggest that the adverse tissue reactions induced by MOM wear particles contain heterogeneous pathogeneses and that the metal levels are an important factor in the determination of the inflammatory phenotype. The present results support the hypothesis that higher metal levels cause cytotoxicity and tissue injury and macrophages are recruited to clear the necrotic debris. On the other hand, the adverse response developed in association with lower metal levels is T-lymphocyte-dominated and is likely to reflect hypersensitivity reaction.

Anti-Inflammatory Effects of ß2-Receptor Agonists Salbutamol and Terbutaline Are Mediated by MKP-1.

Mitogen-activated protein kinase phosphatase 1 (MKP-1) expression is induced by inflammatory factors, and it is an endogenous suppressor of inflammatory response. MKP-1 expression is increased by PDE4 inhibitor rolipram suggesting that it is regulated by cAMP-enhancing compounds. Therefore, we investigated the effect of ß2-receptor agonists on MKP-1 expression and inflammatory response. We found that ß2-receptor agonists salbutamol and terbutaline, as well as 8-Br-cAMP, increased MKP-1 expression. Salbutamol and terbutaline also inhibited p38 MAPK phosphorylation and TNF production in J774 mouse macrophages. Interestingly, salbutamol suppressed carrageenan-induced paw inflammation in wild-type mice, but the effect was attenuated in MKP-1(-/-) mice. In conclusion, these data show that ß2-receptor agonists increase MKP-1 expression, which seems to mediate, at least partly, the observed anti-inflammatory effects of ß2-receptor agonists.

Regulation of Inflammatory Cytokine Production by MKP-5 in Macrophages.

Mitogen-activated protein kinases (MAPKs) include p38 MAPKs, c-Jun N-terminal kinases (JNKs) and Extracellular signal-regulated kinases (ERKs), and they regulate many cell processes, such as cell division, differentiation and release of inflammatory mediators. MAPK activity is controlled by mitogen-activated protein kinase phosphatases (MKPs), a phosphatase family with 11 members. MKP-1 is the most studied member of MKP family, and it is one of the anti-inflammatory factors induced by glucocorticoids. Less is known about the other MAPK phosphatases although they hold a promise as anti-inflammatory drug targets. In this study, we investigated the effect of MKP-5 on MAPK phosphorylation and cytokine production in J774 mouse macrophages. We used MKP-5 siRNA and an MKP-5 inhibitor (AS077234-4) to modulate MKP-5 function. We found that MKP-5 controlled p38 MAPK phosphorylation, but not that of JNK or ERK. In addition, the production of IL-6 and TNF was suppressed by MKP-5 in macrophages. Our results introduce a novel concept that compounds able to enhance MKP-5 expression and/or activity hold anti-inflammatory potential, because MKP-5 down-regulates the release of inflammatory mediators by controlling p38 MAPK activity.

Mitogen-activated protein kinase phosphatase 1 as an inflammatory factor and drug target.

Mitogen-activated protein kinases (MAPKs) are signaling proteins that are activated through phosphorylation, and they regulate many physiological and pathophysiological processes in cells. Mitogen-activated protein kinase phosphatase 1 (MKP-1) is an inducible nuclear phosphatase that dephosphorylates MAPKs, and thus, it is a negative feedback regulator of MAPK activity. MKP-1 has been found as a key endogenous suppressor of innate immune responses, as well as a regulator of the onset and course of adaptive immune responses. Altered MKP-1 signaling is implicated in chronic inflammatory diseases in man. Interestingly, MKP-1 expression and protein function have been found to be regulated by certain anti-inflammatory drugs, namely by glucocorticoids, antirheumatic gold compounds and PDE4 inhibitors, and MKP-1 has been shown to mediate many of their anti-inflammatory effects. In this Mini Review, we summarize the effect of MKP-1 in the regulation of innate and adaptive immune responses and its role as a potential anti-inflammatory drug target and review recent findings concerning the role of MKP-1 in certain anti-inflammatory drug effects.

Attenuation of TNF production and experimentally induced inflammation by PDE4 inhibitor rolipram is mediated by MAPK phosphatase-1.

BACKGROUND AND PURPOSE: 3',5'-Cyclic nucleotide PDE4 is expressed in several inflammatory and immune cells, and PDE4 catalyses the hydrolysis of cAMP to 5'AMP, down-regulating cAMP signalling in cells. MAPK phosphatase-1 (MKP-1) is an endogenous p38 MAPK signalling suppressor and limits inflammatory gene expression and inflammation. In the present study, we investigated the effect of a PDE4 inhibitor rolipram on MKP-1 expression and whether MKP-1 is involved in the anti-inflammatory effects of rolipram. EXPERIMENTAL APPROACH: The effect of rolipram on TNF production was investigated in J774 mouse macrophage cell line and in primary mouse peritoneal macrophages (PM) from wild-type (WT) and MKP-1(-/-) mice. We also investigated the effect of rolipram on carrageenan-induced paw inflammation in WT and MKP-1(-/-) mice. KEY RESULTS: MKP-1 expression was enhanced by rolipram, by a non-selective PDE inhibitor IBMX and by a cAMP analogue 8-Br-cAMP in J774 cells and in PM. Enhanced MKP-1 mRNA expression by rolipram was reversed by a PKA inhibitor. Rolipram, IBMX and 8-Br-cAMP also inhibited TNF production in activated macrophages. Accordingly, rolipram inhibited TNF production in PMs from WT mice but, interestingly, not in PMs from MKP-1(-/-) mice. Furthermore, rolipram attenuated carrageenan-induced paw inflammation in WT but not in MKP-1(-/-) mice. CONCLUSIONS AND IMPLICATIONS: PDE4 inhibitor rolipram was found to enhance the expression of MKP-1, and MKP-1 mediated, at least partly, the anti-inflammatory effects of PDE4 inhibition. The results suggest that compounds that enhance MKP-1 expression and/or MKP-1 activity hold potential as novel anti-inflammatory drugs.

Down-regulation of protein kinase Cδ inhibits inducible nitric oxide synthase expression through IRF1.

In inflammation, pro-inflammatory cytokines and bacterial products induce the production of high amounts of NO by inducible nitric oxide synthase (iNOS) in inflammatory and tissue cells. NO is an effector molecule in innate immunity, and it also has regulatory and pro-inflammatory/destructive effects in the inflammatory process. Protein kinase Cδ (PKCδ) is an important signaling protein regulating B lymphocyte functions, but less is known about its effects in innate immunity and inflammatory gene expression. In the present study we investigated the role of PKCδ in the regulation of iNOS expression in inflammatory conditions. NO production and iNOS expression were induced by LPS or a combination of cytokines IFNγ, IL-1ß, and TNFα. Down-regulation of PKCδ by siRNA and inhibition of PKCδ by rottlerin suppressed NO production and iNOS expression in activated macrophages and fibroblasts. PKCδ directed siRNA and inhibition of PKCδ by rottlerin suppressed also the expression of transcription factor IRF1, possibly through inhibition of STAT1 activation. Accordingly, down-regulation of IRF1 by siRNA reduced iNOS expression in response to inflammatory stimuli. In addition, inhibition of PKCδ showed anti-inflammatory effects in carrageenan induced paw inflammation in mice as did iNOS inhibitor L-NIL. These results suggest that inhibitors of PKCδ have anti-inflammatory effects in disease states complicated by enhanced NO production through iNOS pathway.

Regulation of tristetraprolin expression by mitogen-activated protein kinase phosphatase-1.

Tristetraprolin (TTP) is an acute phase protein, and its expression is rapidly up-regulated by inflammatory signals, such as lipopolysaccharide (LPS) and cytokines. TTP regulates gene expression by governing the mRNA stability of its target genes, which include cytokines and growth factors. MAP kinase phosphatase-1 (MKP-1) is a nuclear phosphatase that inhibits p38 mitogen-activated protein kinase (MAPK) signaling. This study investigated the role of MKP-1 in TTP expression in A549 human lung epithelial cells, THP-1 human macrophages, J774 mouse macrophages, and primary mouse macrophages. TTP and MKP-1 expression was increased by cytokines or LPS. Silencing of MKP-1 by siRNA enhanced TTP expression in response to LPS, and LPS-induced TTP expression was increased in macrophages from MKP-1 (-/-) mice as compared with that in macrophages from wild-type animals. The inhibition of p38 MAPK by SB202190 reduced TTP expression. In conclusion, MKP-1 suppressed TTP expression by inhibiting p38 MAPK pathway.

Flavonoids eupatorin and sinensetin present in Orthosiphon stamineus leaves inhibit inflammatory gene expression and STAT1 activation.

Cytokines and other inflammatory mediators, such as prostaglandin E2 (PGE2) and nitric oxide (NO) produced by cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS), respectively, activate and drive inflammation and therefore serve as targets for anti-inflammatory drug development. Orthosiphon stamineus is an indigenous medicinal plant of Southeast Asia that has been traditionally used in the treatment of rheumatoid arthritis, gout, and other inflammatory disorders. The present study investigated the anti-inflammatory properties of Orthosiphon stamineus leaf chloroform extract (CE), its flavonoid-containing CE fraction 2 (CF2), and the flavonoids eupatorin, eupatorin-5-methyl ether (TMF), and sinensetin, identified from the CF2. It was found that CE (20 and 50 µg/mL) and CF2 (20 and 50 µg/mL) inhibited iNOS expression and NO production, as well as PGE2 production. Eupatorin and sinensetin inhibited iNOS and COX-2 expression and the production of NO (IC50 5.2 µM and 9.2 µM for eupatorin and sinensetin, respectively) and PGE2 (IC50 5.0 µM and 2.7 µM for eupatorin and sinensetin, respectively) in a dose-dependent manner. The extracts and the compounds also inhibited tumor necrosis factor α (TNF-α) production (IC50 5.0 µM and 2.7 µM for eupatorin and sinensetin, respectively). Eupatorin and sinensetin inhibited lipopolysaccharide (LPS)-induced activation of transcription factor signal transducers and activators of transcription 1α (STAT1α). Furthermore, eupatorin (50 mg/kg i. p.) and sinensetin (50 mg/kg i. p.) inhibited carrageenan-induced paw inflammation in mice. The results suggest that CE and CF2, as well as the known constituents of CF2, i.e., eupatorin and sinensetin, have meaningful anti-inflammatory properties which may be utilized in the development of novel anti-inflammatory treatments.

TRPA1 contributes to the acute inflammatory response and mediates carrageenan-induced paw edema in the mouse.

Transient receptor potential ankyrin 1 (TRPA1) is an ion channel involved in thermosensation and nociception. TRPA1 is activated by exogenous irritants and also by oxidants formed in inflammatory reactions. However, our understanding of its role in inflammation is limited. Here, we tested the hypothesis that TRPA1 is involved in acute inflammatory edema. The TRPA1 agonist allyl isothiocyanate (AITC) induced inflammatory edema when injected intraplantarly to mice, mimicking the classical response to carrageenan. Interestingly, the TRPA1 antagonist HC-030031 and the cyclo-oxygenase (COX) inhibitor ibuprofen inhibited not only AITC but also carrageenan-induced edema. TRPA1-deficient mice displayed attenuated responses to carrageenan and AITC. Furthermore, AITC enhanced COX-2 expression in HEK293 cells transfected with human TRPA1, a response that was reversed by HC-030031. This study demonstrates a hitherto unknown role of TRPA1 in carrageenan-induced inflammatory edema. The results also strongly suggest that TRPA1 contributes, in a COX-dependent manner, to the development of acute inflammation.

The expression of interleukin-12 is increased by MAP kinase phosphatase-1 through a mechanism related to interferon regulatory factor 1.

Mitogen-activated protein kinase phosphatase-1 (MKP-1) is a nuclear tyrosine/threonine phosphatase that inhibits p38 mitogen-activated protein kinase (MAPK) activity. We and others have shown that MKP-1 deficiency leads to excessive activation of innate immunity and inflammatory gene expression. Surprisingly, the present study shows that MKP-1 is a positive regulator of IL-12 expression in macrophages suggesting a stimulatory effect on Th1 type immune response. In the present study, we found that LPS-induced expression of IL-12p40 was lower in primary mouse peritoneal macrophages (PMs) and bone marrow-derived macrophages from MKP-1 deficient mice than in cells from wild-type mice whereas TNF expression was enhanced as expected. Correspondingly, the inhibition of p38 MAPK by pharmacologic inhibitors BIRB 796 and SB 202190 enhanced LPS-induced IL-12p40 production. Silencing of interferon regulatory factor 1 (IRF1) by siRNA inhibited the expression of IL-12p40 in J774 macrophages, showing that IRF1 is an important factor regulating IL-12p40 expression. BIRB 796 enhanced LPS-induced expression of IRF1 in J774 macrophages and in PMs from wild-type mice, and IRF1 expression was reduced in PMs from MKP-1 deficient mice. In conclusions, our results show that MKP-1 increases and p38 MAPK decreases the expression of IL-12 by enhancing the expression of IRF1. MKP-1, through regulation of IRF1 and IL-12, therefore may be an important factor supporting the development of Th1 type of immune response and anti-microbial defense.

Attenuation of the acute inflammatory response by dual specificity phosphatase 1 by inhibition of p38 MAP kinase.

Dual specificity phosphatase 1 (DUSP1) dephosphorylates and, hence, regulates the activity of MAP kinases. The present study investigated the effect of DUSP1 on inflammatory gene expression and on the development of carrageenan-induced inflammation. It was found that DUSP1 expression was increased by LPS, and the down-regulation of DUSP1 by siRNA enhanced the phosphorylation of p38 MAPK, while JNK phosphorylation was not affected in murine macrophages. LPS-induced interleukin (IL)-6, tumor-necrosis factor (TNF) and cyclooxygenase-2 (COX2) expression were enhanced in bone marrow-derived macrophages (BMMs) from DUSP1(-/-) mice as compared to those from wild-type mice. In addition, down-regulation of DUSP1 by siRNA enhanced IL-6, TNF and COX2 expression in J774 macrophages, while p38 MAPK inhibitors SB202190 and BIRB 796 inhibited the expression of those inflammatory factors. In vivo, the intensity of the carrageenan-induced paw edema reaction was increased in DUSP1(-/-) mice as compared to the wild-type animals. In conclusion, DUSP1 is an important negative regulator of the acute inflammatory response by limiting p38 MAPK, and compounds which enhance DUSP1 expression or activity may hold a promise as anti-inflammatory drugs.

Dual specificity phosphatase 1 regulates human inducible nitric oxide synthase expression by p38 MAP kinase.

The role of dual specificity phosphatase 1 (DUSP1) in inducible nitric oxide synthase (iNOS) expression in A549 human pulmonary epithelial cells, J774 mouse macrophages and primary mouse bone marrow-derived macrophages (BMMs) was investigated. iNOS expression was induced by a cytokine mixture (TNF, IFNγ and IL-1ß) in A549 cells and by LPS in J774 cells, and it was inhibited by p38 MAPK inhibitors SB202190 and BIRB 796. Stimulation with cytokine mixture or LPS enhanced also DUSP1 expression. Down-regulation of DUSP1 by siRNA increased p38 MAPK phosphorylation and iNOS expression in A549 and J774 cells. In addition, LPS-induced iNOS expression was enhanced in BMMs from DUSP1((-/-)) mice as compared to that in BMMs from wild-type mice. The results indicate that DUSP1 suppresses iNOS expression by limiting p38 MAPK activity in human and mouse cells. Compounds that enhance DUSP1 expression or modulate its function may be beneficial in diseases complicated with increased iNOS-mediated NO production.

Aurothiomalate inhibits cyclooxygenase 2, matrix metalloproteinase 3, and interleukin-6 expression in chondrocytes by increasing MAPK phosphatase 1 expression and decreasing p38 phosphorylation: MAPK phosphatase 1 as a novel target for antirheumatic drugs.

OBJECTIVE: Aurothiomalate is a disease-modifying antirheumatic drug that suppresses inflammation and retards cartilage degradation and bone erosion in arthritis. The molecular mechanisms of action of aurothiomalate are not known in detail. MAPK pathways are major signaling pathways in inflammation that regulate the production of many inflammatory and destructive factors in arthritis. The purpose of the present study was to investigate the effects of aurothiomalate on the activity of p38 MAPK and on the expression of MAPK phosphatase 1 (MKP-1), cyclooxygenase 2 (COX-2), matrix metalloproteinase 3 (MMP-3), and interleukin-6 (IL-6) in immortalized murine H4 chondrocytes and in intact human and murine cartilage. METHODS: Protein expression was examined by Western blotting or by enzyme-linked immunosorbent assay, and messenger RNA (mRNA) expression was examined by real-time reverse transcription-polymerase chain reaction analysis. The mediator role of MKP-1 was investigated by using small interfering RNA (siRNA) methods to down-regulated MKP-1 expression in chondrocytes in culture and by comparing the responses in intact cartilage from MKP-1-deficient and wild-type mice. The effects of aurothiomalate were also confirmed in human rheumatoid cartilage by using tissue samples obtained at the time of total knee replacement surgery. RESULTS: Aurothiomalate inhibited IL-1beta-induced COX-2 expression and prostaglandin E(2) production by destabilizing COX-2 mRNA, as did the p38 MAPK inhibitor SB203580. Interestingly, aurothiomalate also increased the expression of MKP-1 and reduced the IL-1beta-induced phosphorylation of p38 MAPK. Knockdown of MKP-1 by siRNA significantly impaired the ability of aurothiomalate to inhibit the phosphorylation of p38 MAPK and the expression of COX-2, MMP-3, and IL-6. Likewise, aurothiomalate reduced COX-2, MMP-3, and IL-6 expression in articular cartilage from patients with rheumatoid arthritis, as well as in articular cartilage from wild-type mice but not from MKP-1(-/-) mice. CONCLUSION: Our findings indicate a novel mechanism for the antiinflammatory and antierosive actions of aurothiomalate, through increased expression of MKP-1, which leads to reduced activation of p38 MAPK and suppressed expression of COX-2, MMP-3, and IL-6. The results suggest that manipulation of MKP-1 levels is a promising new mechanism to be directed in the search and development of novel antiinflammatory and antierosive compounds that have the good efficacy of gold compounds but not their toxicity.

Mitogen-activated protein kinase phosphatase-1 negatively regulates the expression of interleukin-6, interleukin-8, and cyclooxygenase-2 in A549 human lung epithelial cells.

Mitogen-activated protein kinase phosphatase (MKP)-1 is a protein phosphatase that regulates the activity of p38 mitogen-activated protein (MAP) kinase and c-Jun NH2-terminal kinase (JNK) and, to lesser extent, p42/44 extracellular signal-regulated kinase. Studies with MKP-1(-/-) mice show that MKP-1 is a regulating factor suppressing excessive cytokine production and inflammatory response. The data on the role of MKP-1 in the regulation of inflammatory gene expression in human cells are much more limited. In the present study, we investigated the effect of MKP-1 on the expression of interleukin (IL)-6, IL-8 and cyclooxygenase (COX)-2 in response to stimulation with cytokines (tumor necrosis factor, IL-1beta, and interferon-gamma; 10 ng/ml each) in A549 human lung epithelial cells. Cytokines enhanced p38 and JNK phosphorylation and MKP-1 expression. p38 MAP kinase inhibitors 4-[4-(4-fluorophenyl)-5-(4-pyridinyl)-1H-imidazol-2-yl] phenol (SB202190) and 1-(5-tert-butyl-2-p-tolyl-2H-pyrazol-3-yl)-3(4-(2-morpholin-4-yl-ethoxy)naphthalen-1-yl)urea (BIRB 796) inhibited cytokine-induced phosphorylation of p38 substrate MAP kinase-activated protein kinase 2 and expression of IL-6, IL-8, and COX-2. An aminopyridine-based JNK inhibitor, N-(4-amino-5-cyano-6-ethoxypyridin-2-yl)-2-(2,5-dimethoxyphenyl)acetamide (JNK inhibitor VIII), inhibited phosphorylation of a JNK substrate c-Jun but did not have any effect on IL-6, IL-8, or COX-2 expression. Down-regulation of MKP-1 with small interfering RNA enhanced p38 and JNK phosphorylation and increased IL-6, IL-8, and COX-2 expression in A549 cells. In conclusion, cytokine-induced MKP-1 expression was found to negatively regulate p38 phosphorylation and the expression of IL-6, IL-8, and COX-2 in human pulmonary epithelial cells. Our results suggest that MKP-1 is an important negative regulator of inflammatory gene expression in human pulmonary epithelial cells, and compounds that enhance MKP-1 may have anti-inflammatory effects and control inflammatory response in the human lung.

Inhibition of protein kinase Cdelta reduces tristetraprolin expression by destabilizing its mRNA in activated macrophages.

Tristetraprolin (TTP) binds to AU-rich elements within the mRNAs of several inflammatory genes and causes destabilization of the target mRNAs. The protein kinase C (PKC) pathway represents a major signalling system in inflammation and PKCdelta is one of the key isoenzymes in the regulation of inflammatory processes. In the present study, we investigated the role of PKCdelta in the regulation of the expression of tristetraprolin in activated macrophages by using the PKCdelta inhibitor, rottlerin, and by downregulating PKCdelta expression by using PKCdelta siRNA. TTP protein and mRNA expression were measured by Western blotting and quantitative RT-PCR, respectively. TTP and TNFalpha mRNA decays were studied by the actinomycin D assay. In addition, we measured nuclear translocation of transcription factors believed to be important for TTP transcription, i.e. NF-kappaB, AP-2, SP1 and EGR1. Downregulation of PKCdelta by siRNA decreased significantly TTP expression in activated macrophages. Rottlerin also decreased TTP expression in wild type cells but not in cells in which PKCdelta had been downregulated by siRNA. Rottlerin decreased TTP mRNA half-life as measured by actinomycin D assay but it did not affect the nuclear translocation of transcription factors NF-kappaB, Sp1, AP-2 or EGR1 (which have been shown to be involved in TTP transcription). In addition, rottlerin reduced the decay of TNFalpha mRNA, which is an important target of TTP. The results suggest that PKCdelta up-regulates the expression of TTP by stabilizing its mRNA which may serve as a feed-back loop to regulate the inflammatory response.