TLR9 ligands induce S100A8 in macrophages via a STAT3-dependent pathway which requires IL-10 and PGE2.

S100A8 and S100A9 are highly-expressed calcium-binding proteins in neutrophils and monocytes, and in subsets of macrophages in inflammatory lesions. Unmethylated CpG motifs found in bacterial and viral DNA are potent activators of innate immunity via Toll-like receptor 9 (TLR9). S100A8, but not S100A9, mRNA and protein was directly induced by CpG-DNA in murine and human macrophages. Induction in murine macrophages peaked at 16 h. CpG-DNA-induced S100A8 required de novo protein synthesis; IL-10 and Prostaglandin E2 (PGE2) synergistically enhanced expression and promoted earlier gene induction. Inhibitors of endogenous IL-10, PGE2, and the E prostanoid (EP) 4 receptor strongly suppressed S100A8 expression, particularly when combined. Thus, S100A8 induction by E. coli DNA required both IL-10 and PGE2/EP4 signaling. The MAPKs, PI3K and JAK pathways were essential, whereas ERK1/2 appeared to play a direct role. S100A8 induction by CpG-DNA was controlled at the transcriptional level. The promoter region responsible for activation, either directly, or indirectly via IL-10 and PGE2, was located within a -178 to -34-bp region and required STAT3 binding. Because of the robust links connecting IL-10 and PGE2 with an anti-inflammatory macrophage phenotype, the induction profile of S100A8 strongly indicates a role for this protein in resolution of inflammation.

Relationship between left ventricular dyssynchrony and systolic dysfunction is independent of impaired left ventricular myocardial perfusion in heart failure: assessment with 99mTc-sestamibi gated myocardial scintigraphy.

BACKGROUNDS: Left ventricular (LV) dyssynchrony reduces LV systolic function in patients with heart failure (HF). However, it remains unknown whether this relationship is independent of impaired LV myocardial perfusion. METHODS AND RESULTS: A total of 105 patients with chronic HF (age 71 ± 13 years; 71 men) were enrolled in the present study. (99m)Tc-sestamibi (MIBI) gated myocardial scintigraphy was performed at rest to assess LV myocardial perfusion as evaluated by the total defect score of perfusion Single Photon Emission Computed Tomography images (TDS-MIBI), LV systolic function as evaluated by LV ejection fraction (LVEF), and LV systolic dyssynchrony as evaluated by the maximal difference of time to end systole (MD-TES), which is the time lag between the earliest and the latest end systole among 17 LV segments analyzed with a novel program, "cardioGRAF". The mean ± SD (minimum and maximum range) of the MD-TES was 147.8 ± 117.5 (14.0-458.3)ms. The MD-TES was significantly higher in patients with LVEF<45% (199.4 ± 117.6 ms) than in those with LVEF ≥ 45% (60.5 ± 41.2 ms, p<0.001). In a multiple logistic regression analysis, the MD-TES showed an increased odds ratio for LVEF<45% (2.46 [95% CI; 1.51-4.01] per increment in decile of MD-TES rank, p<0.001), after adjusting for the TDS-MIBI, history of myocardial infarction, and other potential confounders. CONCLUSIONS: LV dyssynchrony is a significant determinant of LV systolic dysfunction in patients with HF, and this relationship is independent of impaired LV myocardial perfusion and history of myocardial infarction.

Effects of statin therapy on the production of monocyte pro-inflammatory cytokines, cardiac function, and long-term prognosis in chronic heart failure patients with dyslipidemia.

BACKGROUND: The effects of statin therapy on the production of monocyte pro-inflammatory cytokines, cardiac function and the long-term prognosis in chronic heart failure (CHF) patients with dyslipidemia remain unclear. METHODS AND RESULTS: A total of 146 CHF patients with a mean left ventricular ejection fraction (LVEF) of 26.9 ± 6.6% were divided into 2 groups based on whether or not statins were included in their treatment: a statin group (n=63) and a no statin group (n=83). Only patients with dyslipidemia were treated with statins. Peripheral blood mononuclear cells (PBMCs) were isolated, and the production of monocyte tumor necrosis factor (TNF)-α and interleukin (IL)-6 were measured at baseline and after 6 months of treatment, and the data expressed as mean ± SD (pg·ml(-1)·10(-6) PBMCs). The LVEF in the statin group improved, and the monocyte TNF-α and IL-6 production decreased (respectively, P<0.001), but the LVEF and cytokine production remained unchanged in the no statin group. Multivariate Cox hazard analysis showed that statin therapy (hazard ratio, 0.14; 95% confidence interval: 0.02-0.97, P=0.046) was an independent predictor of cardiac events. CONCLUSIONS: Statin therapy attenuates the production of monocyte pro-inflammatory cytokines, and ameliorates the cardiac function and may improve long-term prognosis in CHF patients with dyslipidemia.

LILRA2 selectively modulates LPS-mediated cytokine production and inhibits phagocytosis by monocytes.

The activating immunoglobulin-like receptor, subfamily A, member 2 (LILRA2) is primarily expressed on the surface of cells of the innate immunity including monocytes, macrophages, neutrophils, basophils and eosinophils but not on lymphocytes and NK cells. LILRA2 cross-linking on monocytes induces pro-inflammatory cytokines while inhibiting dendritic cell differentiation and antigen presentation. A similar activating receptor, LILRA4, has been shown to modulate functions of TLR7/9 in dendritic cells. These suggest a selective immune regulatory role for LILRAs during innate immune responses. However, whether LILRA2 has functions distinct from other receptors of the innate immunity including Toll-like receptor (TLR) 4 and FcγRI remains unknown. Moreover, the effects of LILRA2 on TLR4 and FcγRI-mediated monocyte functions are not elucidated. Here, we show activation of monocytes via LILRA2 cross-linking selectively increased GM-CSF production but failed to induce IL-12 and MCP-1 production that were strongly up-regulated by LPS, suggesting functions distinct from TLR4. Interestingly, LILRA2 cross-linking on monocytes induced similar amounts of IL-6, IL-8, G-CSF and MIP-1α but lower levels of TNFα, IL-1ß, IL-10 and IFNγ compared to those stimulated with LPS. Furthermore, cross-linking of LILRA2 on monocytes significantly decreased phagocytosis of IgG-coated micro-beads and serum opsonized Escherichia coli but had limited effect on phagocytosis of non-opsonized bacteria. Simultaneous co-stimulation of monocytes through LILRA2 and LPS or sequential activation of monocytes through LILRA2 followed by LPS led lower levels of TNFα, IL-1ß and IL-12 production compared to LPS alone, but had additive effect on levels of IL-10 and IFNγ but not on IL-6. Interestingly, LILRA2 cross-linking on monocytes caused significant inhibition of TLR4 mRNA and protein, suggesting LILRA2-mediated suppression of LPS responses might be partly via regulation of this receptor. Taken together, we provide evidence that LILRA2-mediated activation of monocytes is significantly different to LPS and that LILRA2 selectively modulates LPS-mediated monocyte activation and FcγRI-dependent phagocytosis.

Upregulation of monocyte tissue factor activity is significantly associated with carotid intima-media thickness in patients with metabolic syndrome.

AIMS: Metabolic syndrome (MS) represents a cluster of cardiovascular risk factors and an increased risk of cardiovascular events. The carotid intima-media thickness (CIMT) is correlated with coronary and carotid atherosclerosis, and is a significant predictor of cardiovascular events. Tissue factor (TF) is an initiator of the extrinsic coagulation cascade and is expressed on peripheral blood monocytes and macrophages in atherosclerotic plaques. TF plays important roles in both thrombosis and atherosclerosis. No study has investigated the relationship between monocyte TF activity and CIMT in MS patients. METHODS: Peripheral blood mononuclear cells (PBMCs) were collected from 39 normal subjects and 110 patients with MS. The procoagulant activity (PCA) in monocytes was measured using a one-stage clotting assay and is expressed as the mean±SD (mU TF/10(6) PBMCs). RESULTS: The PCA in monocytes in MS patients was significantly higher than in normal subjects (86.2 ±69.5 vs. 52.4±9.9 mU TF/10(6) PBMCs, p < 0.001). In multivariate analysis, patient age (ß coefficient= 0.373, p < 0.001), high-density lipoprotein cholesterol (ß coefficient=-0.307, p = 0.001) and PCA (ß coefficient= 0.422, p =0.002) were each significantly and independently associated with CIMT. CONCLUSIONS: These data indicate that the upregulation of monocyte TF activity is significantly associated with CIMT in MS patients.

S100A8 modulates mast cell function and suppresses eosinophil migration in acute asthma.

S100A8 is implicated in the pathogenesis of inflammatory diseases. S100A8 is upregulated in macrophages by Toll-like receptors (TLR)-3, 4, and 9 agonists in an IL-10-dependent manner, and by corticosteroids in vitro and in vivo, and scavenges oxidants generated by activated phagocytes. Because if its elevated expression in various lung disorders, we asked whether S100A8 was protective in allergic inflammation. S100A8, but not Cys(41)-Ala S100A8, in which the single reactive Cys residue was replaced by Ala, reduced mast cell (MC) degranulation and production of particular cytokines (IL-6, IL-4, and granulocyte macrophage colony-stimulating factor) in response to IgE-crosslinking in vitro, likely by inhibiting intracellular reactive oxygen species production, thereby reducing downstream linker for activation of T cells and extracellular signal regulated kinase/mitogen-activated protein kinase phosphorylation. In lungs of mice with acute asthma, S100A8, but not Cys(41)-Ala S100A8, reduced MC degranulation, production of eosinophil chemoattractants (IL-5, eotaxin, and monocyte chemoattractant protein-1), and eosinophil infiltration. Suppression of IL-6 and IL-13 could have contributed to reduced mucus production seen in lungs of S100A8-treated mice. IgE production was unaffected. In asthma, there is an imbalance of anti-oxidant systems that are generally protective. Our results strongly support a protective role for S100A8 in allergic inflammation by modulating MC activation and eosinophil recruitment, and by scavenging oxidants generated by activated leukocytes, in processes reliant on its thiol-scavenging capacity.

Upregulation of monocyte proinflammatory cytokine production by C-reactive protein is significantly related to ongoing myocardial damage and future cardiac events in patients with chronic heart failure.

BACKGROUND: An elevation of the cardiac troponin T (TnT) level identifies patients with ongoing myocardial damage (OMD) and at increased risk for future cardiac events in chronic heart failure (CHF). C-reactive protein (CRP) upregulates monocyte proinflammatory cytokine production and this upregulation appears to play an important role on OMD. METHODS AND RESULTS: Peripheral blood mononuclear cells (PBMCs) were stimulated by 25 microg/mL CRP in 72 patients with CHF. Tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 production by monocytes was measured by a specific enzyme-linked immunosorbent assay and expressed as the mean +/- SD (pg x mL x 10(6) PBMCs). The patients were divided into 2 groups according to the TnT levels: 27 patients with OMD (TnT >/= 0.01 ng/mL) and 45 patients without OMD. CRP-induced cytokine production was upregulated significantly more in patients with OMD than in those without OMD (TNF-alpha: 200.6 +/- 100.4 vs. 102.1 +/- 73.6 pg/mL, P < .001, IL-6: 4611.7 +/- 2600.0 vs. 1451.6 +/- 1193.5 pg/mL, P < .001). Multivariate Cox regression analyses revealed that CRP-stimulated monocyte production of TNF-alpha >/= 120 pg/mL and TnT >/= 0.03 ng/mL were independent predictors of cardiac events. CONCLUSIONS: The upregulation of monocyte proinflammatory cytokine production by CRP could be significantly related to OMD and future cardiac events in CHF.

IL-10-dependent S100A8 gene induction in monocytes/macrophages by double-stranded RNA.

The S100 calcium-binding proteins S100A8 and S100A9 are elevated systemically in patients with viral infections. The S100A8-S100A9 complex facilitated viral replication in human CD4(+) T lymphocytes latently infected with HIV-1- and S100A8-induced HIV-1 transcriptional activity. Mechanisms inducing the S100 genes and the potential source of these proteins following viral activation are unknown. In this study, we show that S100A8 was induced in murine macrophages, and S100A8 and S100A9 in human monocytes and macrophages, by polyinosinic:polycytidylic acid, a dsRNA mimetic. Induction was at the transcriptional level and was IL-10 dependent. Similar to LPS-induced S100A8, induction by dsRNA was dependent on p38 and ERK MAPK. Protein kinase R (PKR) mediates antiviral defense and participates in MyD88-dependent/independent signaling triggered by TLR4 or TLR3. Like IL-10, S100 induction by polyinosinic:polycytidylic acid and by LPS was inhibited by the specific PKR inhibitor 2-aminopurine, indicating a novel IL-10, PKR-dependent pathway. Other mediators such as IFN-beta, which synergized with dsRNA, may also be involved. C/EBPbeta bound the defined promoter region in response to dsRNA. S100A8 was expressed in lungs of mice infected with influenza virus and was maximal at day 8 with strong immunoreactivity in epithelial cells lining the airways and in mononuclear cells and declined early in the recovery phase, implying down-regulation by mediator(s) up-regulated during resolution of the infection. IL-10 is implicated in viral persistence. Since S100A8/S100A9 levels are likely to be maintained in conditions where IL-10 is raised, these proteins may contribute to viral persistence in patients infected by some RNA viruses.

LILRA5 is expressed by synovial tissue macrophages in rheumatoid arthritis, selectively induces pro-inflammatory cytokines and IL-10 and is regulated by TNF-alpha, IL-10 and IFN-gamma.

Leukocyte immunoglobulin-like receptor A5 (LILRA5) belongs to a family of receptors known to regulate leukocyte activation. There are two membrane-bound and two soluble forms of LILRA5. The transmembrane LILRA5 contain a short cytoplasmic domain and a charged arginine residue within the transmembrane region. Cross-linking of LILRA5 on monocytes induced production of pro-inflammatory cytokines, suggesting that LILRA5 plays a role in inflammation. However, expression of LILRA5 in diseases with extensive inflammatory component is unknown. Rheumatoid arthritis (RA) is a chronic inflammatory synovitis characterized by unregulated activation of leukocytes leading to joint destruction. Here we demonstrate extensive LILRA5 expression on synovial tissue macrophages and in synovial fluid of patients with active RA but not in patients with osteoarthritis. We also show that LILRA5 associated with the common gamma chain of the FcR and LILRA5 cross-linking induced phosphorylation of Src tyrosine kinases and Spleen tyrosine kinase (Syk). Furthermore, LILRA5 induced selective production of pro-inflammatory cytokines as well as IL-10. LILRA5 mRNA and protein expression was tightly regulated by TNF-alpha, IL-10 and IFN-gamma. Increased expression of LILRA5 in rheumatoid tissue, together with its ability to induce key cytokines involved in RA, suggests that this novel receptor may contribute to disease pathogenesis.

FGF-2, IL-1beta and TGF-beta regulate fibroblast expression of S100A8.

Growth factors, including fibroblast growth factor-2 (FGF-2) and transforming growth factor-beta (TGF-beta) regulate fibroblast function, differentiation and proliferation. S100A8 and S100A9 are members of the S100 family of Ca2+-binding proteins and are now accepted as markers of inflammation. They are expressed by keratinocytes and inflammatory cells in human/murine wounds and by appropriately activated macrophages, endothelial cells, epithelial cells and keratinocytes in vitro. In this study, regulation and expression of S100A8 and S100A9 were examined in fibroblasts. Endotoxin (LPS), interferon gamma (IFNgamma), tumour-necrosis factor (TNF) and TGF-beta did not induce the S100A8 gene in murine fibroblasts whereas FGF-2 induced mRNA maximally after 12 h. The FGF-2 response was strongly enhanced and prolonged by heparin. Interleukin-1beta (IL-1beta) alone, or in synergy with FGF-2/heparin strongly induced the gene in 3T3 fibroblasts. S100A9 mRNA was not induced under any condition. Induction of S100A8 in the absence of S100A9 was confirmed in primary fibroblasts. S100A8 mRNA induction by FGF-2 and IL-1beta was partially dependent on the mitogen-activated-protein-kinase pathway and dependent on new protein synthesis. FGF-2-responsive elements were distinct from the IL-1beta-responsive elements in the S100A8 gene promoter. FGF-2-/heparin-induced, but not IL-1beta-induced responses were significantly suppressed by TGF-beta, possibly mediated by decreased mRNA stability. S100A8 in activated fibroblasts was mainly intracytoplasmic. Rat dermal wounds contained numerous S100A8-positive fibroblast-like cells 2 and 4 days post injury; numbers declined by 7 days. Up-regulation of S100A8 by FGF-2/IL-1beta, down-regulation by TGF-beta, and its time-dependent expression in wound fibroblasts suggest a role in fibroblast differentiation at sites of inflammation and repair.

Regulation of S100A8 by glucocorticoids.

S100A8 (A8) has roles in inflammation, differentiation and development and is associated with oxidative defense. Murine A8 (mA8) is up-regulated in macrophages, fibroblasts, and microvascular endothelial cells by LPS. Glucocorticoids (GCs) amplified LPS-induced mA8 in these cells. Relative to stimulation by LPS, GCs increased mA8 gene transcription and mRNA half-life. Enhancement required new protein synthesis, IL-10 and products of the cyclooxygenase-2 pathway, and both ERK1/2 and p38 MAPK. Protein kinase A positively and protein kinase C negatively regulated this process. Promoter analysis indicated element(s) essential for LPS and dexamethasone enhancement colocated within the region -178 to 0 bp. In the absence of glucocorticoid response elements, NF1 motif at -58 is a candidate for mediation of enhancement. Gel shift analysis detected no differences between LPS- and LPS/dexamethasone-treated complexes within this region. GCs increased constitutive levels of A8 and S100A9 (A9) mRNA in human monocytes. The synovial membrane of rheumatoid patients treated with high dose i.v. methylprednisolone contained higher numbers of A8/A9-positive macrophages than pre- or posttreatment samples. Results support the proposal that A8 has anti-inflammatory properties that may be independent of hetero-complex formation with A9 and may also enable localized defense in the absence of overriding deleterious host responses.

Electrocardiographic and electrophysiological characteristics of atrial fibrillation organized into atrial flutter by oral administration of class I antiarrhythmic agents.

The aim of this study was to evaluate the electrocardiographic (ECG) and electrophysiological characteristics of atrial fibrillation (AF) that organized into atrial flutter during oral administration of class I antiarrhythmic agents. The former clinical study included 72 consecutive patients (58 paroxysmal AF, 14 persistent AF) in whom class I antiarrhythmic agents were orally administered in the outpatient clinic for termination or prophylaxis of AF. The clinical background and ECG variables were compared between the patients with and without atrial flutter during class I antiarrhythmic agents therapy. An electrophysiological study was performed in ten patients with paroxysmal AF (five with [group A] and five without atrial flutter [group B] during oral class I antiarrhythmic agents therapy. Local electrograms from five different atrial sites (high and low right free wall, high and low septum, and distal coronary sinus) were analyzed during induced AF. The activation pattern of the right free wall during AF was also analyzed using a Halo catheter. Atrial flutter was documented during class I antiarrhythmic agents therapy in 14 (24%) patients with paroxysmal AF, whereas in none with persistent AF. The mean cycle length (f-f interval) and amplitude of the fibrillation waves in leads II and V1 from the surface ECG were significantly greater in the patients with than in those without atrial flutter. In the electrophysiological study, the mean cycle lengths for the low and high right free wall were significantly longer in group A than in group B, whereas those for the low septums and distal coronary sinus did not differ between the two groups. During the induced AF, the ratio of time exhibiting a consistent activation pattern (cranio-caudal, caudo-cranial, or undetermined) along the right free wall was significantly greater in group A than in group B. Atrial flutter newly developed during class I antiarrhythmic agents therapy in patients with coarse AF on the surface ECG and a relatively organized activation in the right atrial free wall. The observation of these findings may facilitate the identification of candidates for hybrid pharmacologic and ablative therapies.

Clinical significance of wide QRS complexes at the termination of paroxysmal supraventricular tachycardias.

BACKGROUND: A wide QRS complex is not a rare electrocardiographic phenomenon at the termination of paroxysmal supraventricular tachycardia (PSVT), but no plausible underlying mechanism has yet been proposed. The purpose of the present study was to elucidate the frequency and the underlying mechanism of the wide QRS complexes at the termination of PSVT. METHODS: We retrospectively reviewed 305 electrocardiograms (ECGs) from 100 patients, on which PSVT termination was recorded. The frequency of the wide QRS complexes was analyzed in 181 ECGs to avoid duplication, because there were 124 ECGs obtained from the same patients with same methods. The 181 ECGs were divided by morphology into three groups: Type A, termination with wide QRS complex without pause; Type B, wide QRS complex following initial pause after termination; Type C, wide QRS complex following the first narrow QRS after termination. RESULTS: The wide QRS complex was recorded in 81/181 (44.8%) ECGs (Type A; 3/81 (3.7%), Type B; 44/81 (54.3%), Type C; 62/81 (55.6%) ) and its frequency was not dependent on the mechanism of PSVT. It was more frequently observed after a long pause, and was frequently induced by procedures that increase vagal tone, such as intravenous adenosine 5'-triphosphate administration (16/22: 72.7%) and vagal stimulation maneuvers (16/32: 50%). There were a total of 41 wide QRS complexes (44.6%) which had a preceding sinus P wave, out of a total of 92 wide QRS complexes in all three types. These 41 wide QRS complexes included 30/44 (68.2%) Type B wide QRS, and 11 (24.4%) Type C wide QRS complexes. CONCLUSION: The aberrant conduction or escaped ventricular contraction was suggested to be the underlying mechanism of the majority of wide QRS complexes and ventricular premature contraction is less frequent.