A20 (Tnfaip3) expressed in CD4+ T cells suppresses Th2 cell-mediated allergic airway inflammation in mice.

A20 (Tnfaip3), a ubiquitin-editing enzyme, inhibits NF-κB signaling pathways in response to pro-inflammatory cytokines. Previous studies have proved the anti-inflammatory roles of A20 in various cell types, including T cells, B cells, dendritic cells, and intestinal epithelial cells. Moreover, recent studies have shown that A20 expressed in lung epithelial cells is required for LPS-induced protection from asthma. In humans, a single-nucleotide polymorphism in TNFAIP3 is associated with asthma risk. However, the role of A20 expressed in T cells in asthmatic responses has not been elucidated. We addressed this point by generating mice lacking A20 expression in T cells (CD4-CreA20 fl/fl mice). We found that house dust mite (HDM)-induced allergic airway inflammation, mucus production, airway hyperresponsiveness, and Th2 cytokine production were significantly exacerbated in CD4-CreA20 fl/fl mice compared with those in control A20 fl/fl mice. In vitro differentiation of Th2 cells but not of Th1 cells or Th17 cells was enhanced in CD4+ T cells by the absence of A20. Consistently, enforced expression of A20 inhibited the differentiation of Th2 cells but not of Th1 cells or Th17 cells. Notably, the expression of GATA3 was significantly enhanced in A20-deficient CD4+ T cells, and the enhanced GATA3 expression was partly canceled by IL-2 neutralization. These results suggest that A20 functions as a stabilizing factor maintaining GATA3 levels during the induction of Th2 cells to prevent excessive Th2 cell differentiation.

Semaphorin 3G exacerbates joint inflammation through the accumulation and proliferation of macrophages in the synovium.

OBJECTIVES: Methotrexate (MTX) is an anchor drug for the treatment of rheumatoid arthritis (RA). However, the precise mechanisms by which MTX stalls RA progression and alleviates the ensuing disease effects remain unknown. The aim of the present study was to identify novel therapeutic target molecules, the expression patterns of which are affected by MTX in patients with RA. METHODS: CD4+ T cells from 28 treatment-naïve patients with RA before and 3 months after the initiation of MTX treatment were subjected to DNA microarray analyses. The expression levels of semaphorin 3G, a differentially expressed gene, and its receptor, neuropilin-2, were evaluated in the RA synovium and collagen-induced arthritis synovium. Collagen-induced arthritis and collagen antibody-induced arthritis were induced in semaphorin3G-deficient mice and control mice, and the clinical score, histological score, and serum cytokines were assessed. The migration and proliferation of semaphorin 3G-stimulated bone marrow-derived macrophages were analyzed in vitro. The effect of local semaphorin 3G administration on the clinical score and number of infiltrating macrophages during collagen antibody-induced arthritis was evaluated. RESULTS: Semaphorin 3G expression in CD4+ T cells was downregulated by MTX treatment in RA patients. It was determined that semaphorin 3G is expressed in RA but not in the osteoarthritis synovium; its receptor neuropilin-2 is primarily expressed on activated macrophages. Semaphorin3G deficiency ameliorated collagen-induced arthritis and collagen antibody-induced arthritis. Semaphorin 3G stimulation enhanced the migration and proliferation of bone marrow-derived macrophages. Local administration of semaphorin 3G deteriorated collagen antibody-induced arthritis and increased the number of infiltrating macrophages. CONCLUSIONS: Upregulation of semaphorin 3G in the RA synovium is a novel mechanism that exacerbates joint inflammation, leading to further deterioration, through macrophage accumulation.

SOCS3 Expressed in M2 Macrophages Attenuates Contact Hypersensitivity by Suppressing MMP-12 Production.

Numerous studies have clarified the immunological mechanisms of contact hypersensitivity (CHS). In addition, we have recently shown that M2 macrophages play key roles in the development of CHS by producing matrix metalloproteinase-12 (MMP-12). However, regulatory mechanisms of the elicitation phase in CHS remain largely unknown. To determine the roles of suppressor of cytokine signaling (SOCS) family members in M2 macrophages in the regulation of CHS, we investigated the expression of SOCS family members in M2 macrophages at the inflammatory sites of CHS. Transcriptome analysis revealed that among SOCS family members, SOCS3 was highly expressed in M2 macrophages at the site of CHS, and SOCS3 induction was reduced by IFN-? neutralization. 2,4-Dinitrofluorobenzene-induced CHS was significantly enhanced and prolonged in mice lacking SOCS3 expression in monocytes/macrophages (SOCS3(?/?) mice) compared with that in control mice. Importantly, expression of MMP-12 in M2 macrophages was significantly increased in SOCS3(?/?) mice at the site of CHS, and deletion of the MMP-12 gene reduced the exacerbated CHS in SOCS3(?/?) mice. Finally, IFN-? inhibited IL-4-induced MMP-12 expression in a SOCS3-dependent manner. Taken together, these results suggest that SOCS3 expressed in M2 macrophages is involved in the attenuation and/or resolution of CHS, presumably by suppressing MMP-12 production.

Molecular imaging of apolipoprotein B-100 in human coronary plaques by color fluorescent angioscopy and microscopy.

Apolipoprotein B-100 (ApoB-100) is an important risk factor for coronary artery disease. However, its localization in human coronary plaques is not well understood. The present study was performed to visualize ApoB-100 in human coronary artery wall. Deposition of native ApoB-100 in excised human coronary plaques and normal segments classified by conventional angioscopy was investigated by color fluorescent angioscopy (CFA) and microscopy (CFM) using Nile blue dye (NB) which elicits a golden fluorescence characteristic of ApoB-100 as a biomarker. By CFA, the % incidence of ApoB-100 was 20 in 40 normal segments, 38 in 42 white, and 11 in 35 yellow plaques (P < 0.05 versus white plaques). There was no significant difference in detection sensitivity between CFA and luminal surface scan by CFM. By CFM transected surface scan, ApoB-100 deposited in superficial, deep, and/or in both layers. Deposition in both layers was frequently observed in white plaques and yellow plaques without necrotic core (NC), less frequently in normal segments, and rarely in yellow plaques with NC. (1) Taking into consideration the well known process of plaque growth, the results suggest that ApoB-100 begins to deposit before plaque formation, increasingly deposits with plaque growth, and disappears after necrotic core formation. (2) CFA is feasible for imaging of ApoB-100 in human coronary artery wall.

Molecular imaging of low-density lipoprotein in human coronary plaques by color fluorescent angioscopy and microscopy.

OBJECTIVES: Low-density lipoprotein (LDL) is an important risk factor for coronary artery disease. However, its localization in human coronary plaques is not well understood. The present study was performed to visualize LDL in human coronary artery wall. METHODS: (1) The fluorescence characteristic of LDL was investigated by color fluorescent microscopy (CFM) with excitation at 470-nm and emission at 515-nm using Nile blue dye (NB) as a biomarker. (2) Native LDL in 40 normal segments, 42 white plaques and 35 yellow plaques (20 with necrotic core) of human coronary arteries was investigated by color fluorescent angioscopy (CFA) and CFM. RESULTS: (1) NB elicited a brown, golden and red fluorescence characteristic of LDL, apolipoprotein B-100, and lysophosphatidylcholine/triglyceride, respectively. (2) The % incidence of LDL in normal segments, white, and yellow plaques was 25, 38 and 14 by CFA and 42, 42 and 14 by CFM scan of their luminal surface, respectively, indicating lower incidence (p<0.05) of LDL in yellow plaques than white plaques, and no significant differences in detection sensitivity between CFA and CFM. By CFM transected surface scan, LDL deposited more frequently and more diffusely in white plaques and yellow plaques without necrotic core (NC) than normal segments and yellow plaques with NC. LDL was localized to fibrous cap in yellow plaques with NC. Co-deposition of LDL with other lipid components was observed frequently in white plaques and yellow plaques without NC. CONCLUSIONS: (1) Taken into consideration of the well-known process of coronary plaque growth, the results of the present study suggest that LDL begins to deposit before plaque formation; increasingly deposits with plaque growth, often co-depositing with other lipid components; and disappears after necrotic core formation. (2) CFA is feasible for visualization of LDL in human coronary artery wall.

ß-actin-positive mononuclear cells participate in coronary microvascular medial hyperplasia by migrating through adventitia into media, with special reference to microvessel angina.

Coronary microvascular hyperplasia is a cause of microvessel angina, although the underlying cellular mechanisms remain unclear. We examined how mononuclear cells expressing ß-actin (ß-MNCs), which were identified in coronary vessels, induce coronary microvascular hyperplasia.The presence of ß-MNCs in coronary hyperplastic arterial (HAM) and venous microvessels (HVM) was examined by endomyocardial biopsy in 25 patients with suspected microvessel angina. ß-MNCs were identified in 14 HAMs obtained from 11 patients. Basic fibroblast growth factor and heparin sulfate were injected into the infarcted myocardium to induce HAM and HVM in 28 beagles, and then we examined the role of ß-MNCs in the onset of HAM and HVM. The following changes were observed after infarction induction in beagles: (a) migration of ß-MNCs from the existing microvessels into the interstitial space at 1-2 weeks; (b) those traversing the adventitia into the media, but not intima, of microvessels; (c) their transformation to smooth muscle cells (SMCs) and/or connective tissues (collagen and elastin fibers); (d) and medial hyperplasia without intimal hyperplasia. Medial hyperplasia was classified into SMC-proliferative and both SMC- and connective tissue-proliferative types. ß-MNCs expressed CD(34) but did not express other major vessel-related cell markers.ß-MNCs are a vascular progenitor, and migrate out of the adventitia into media, and participate in the etiology of coronary microvascular medial hyperplasia.

Migration of mononuclear cells expressing ß-actin through the adventitia into media and intima in coronary arteriogenesis and venogenesis in ischemic myocardium.

It was previously thought that arteriogenesis and venogenesis are induced not only by proliferation of vessel-resident smooth muscle cells (SMCs) and endothelial cells (ECs) but also by migration of their precursors. However, it is not well understood through what route(s) the precursors migrate into the existing vessels.We examined through what route or routes circulating mononuclear cells expressing ß-actin (ß-MNCs), which we identified in canine coronary vessels, migrate into coronary vessel walls and cause arteriogenesis and venogenesis at 1, 2, 4 and 8 weeks after induction of myocardial infarction.The following changes were observed: (1) The ß-MNCs migrated via coronary microvessels to the interstitial space at one week; (2) ß-MNCs traversed the adventitia into the media and settled in parallel with pre-existing smooth muscle cells (SMCs) in arterioles and arteries and lost ß-actin and acquired α-smooth muscle actin (α-SMA) to become mature SMCs at 2-4 weeks; (3) at the same time, other ß-MNCs migrated across the adventitia and media into the intima and settled in parallel with pre-existing endothelial cells (ECs) and lost ß-actin, while acquiring CD(31), to become mature ECs, resulting in arteriogenesis; (4) Similarly, ß-MNCs migrated into venular and venous walls and became SMCs or ECs, resulting in venogenesis.ß-MNCs in the interstitial space expressed CD(34) but not other major vascular cell markers.ß-MNCs, possibly a vascular progenitor, migrate not from the lumen but across the adventitia into the media or intima of coronary vessels and transit to SMCs or ECs, and participate in arteriogenesis and venogenesis in ischemic myocardium.

Nitroglycerin-induced heterogeneous subendocardial myocardial blood flow observed by cardioscopy in patients with coronary artery disease.

It is controversial as to whether or not nitroglycerin (NTG) increases subendocardial myocardial blood flow (SMBF), and if it does, whether arterial or venous blood flow is increased in patients with coronary artery disease. This study was performed to examine NTG-induced changes in SMBF.Changes in SMBF induced by NTG (200 µg, i.v.) were examined by cardioscopy in 58 left ventricular wall segments of 58 patients with coronary artery disease. NTG-induced red and purple endocardial colors were defined as increased arterial and venous SMBF, respectively. Endocardial color before NTG administration was classified into brown, light brown, pale and white. Endomyocardial biopsy of the observed portion and (201)Tl scintigraphy were performed in 40 of these patients immediately after cardioscopy and several days after cardioscopy, respectively.Upon administration of NTG, SMBF increased in 48 of 58 wall segments; arterial SMBF in 34 and venous SMBF in 12 wall segments; arterial SMBF in all 24 brown to light brown segments; venous SMBF, arterial SMBF and no change in 12, 10 and 5 of pale segments, respectively; and no change in all 10 white wall segments. (201)Tl-scintigraphy and endomyocardial biopsy revealed that brown, light brown, pale and white endocardial color represented no ischemia, mild ischemia, severe ischemia and fibrosis, respectively.NTG caused an increase in either arterial or venous SMBF depending on control endocardial color, wall motion and severity of coronary stenosis.

Role of calcium-activated potassium channels in the genesis of 3,4-diaminopyridine-induced periodic contractions in isolated canine coronary artery smooth muscles.

We found that 3,4-diaminopyridine (3,4-DAP), a voltage-gated potassium channel (K(V)) inhibitor, elicits pH-sensitive periodic contractions (PCs) of coronary smooth muscles. Underlying mechanisms of PCs, however, remained to be elucidated. The present study was performed to examine the roles of ion channels in the genesis of PCs. To determine the electromechanical changes of smooth muscles, isolated coronary arterial rings from beagles were suspended in organ chambers filled with Krebs-Henseleit solution, and 10(-2) M 3,4-DAP was added to elicit PCs. 3,4-DAP caused periodic spike-and-plateau depolarization accompanied by contraction. PCs were not produced when the CaCl(2) concentration in the chamber was ≤ 0.3 × 10(-3) or ≥ 10(-2) M. PCs were eliminated by a CaCl(2) concentration ≥ 5 × 10(-3) M or by lowering pH below 7.20 with HCl and recovered by the addition of iberiotoxin or charybdotoxin, which inhibit large-conductance calcium-activated potassium channels (K(Ca)), or by elevating pH above 7.35 with NaOH. PCs, as well as the spike-and-plateau depolarization, were eliminated by nifedipine, which inhibits L-type voltage-gated calcium channels (Ca(V)). Influx of Ca(2+) through L-type Ca(V), which was opened because closing of K(Ca), secondary to 3,4-DAP-induced closing of K(V), resulted in contraction; the intracellular Ca(2+) increased by this influx opened K(Ca), leading to closure of Ca(V) and consequent cessation of Ca(2+) influx with resultant relaxation. These processes were repeated spontaneously to cause PCs. H(+) and OH(-) were considered to act as the opener and closer of K(Ca), respectively.

IL-23 and Th17 cells enhance Th2-cell-mediated eosinophilic airway inflammation in mice.

RATIONALE: The IL-23-IL-17A-producing CD4(+) T-cell (Th17 cell) axis plays an important role in the development of chronic inflammatory diseases, including autoimmune diseases. However, the role of the IL-23-Th17 cell axis in the regulation of allergic airway inflammation is still largely unknown. OBJECTIVES: To determine the role of IL-23 and Th17 cells in allergic airway inflammation. METHODS: We examined the effect of anti-IL-23 antibody on antigen-induced airway inflammation. We also investigated the effect of enforced expression of IL-23 on allergic airway inflammation by generating lung-specific IL-23 transgenic mice. Moreover, we examined the effect of adoptive transfer of antigen-specific Th17 cells on allergic airway inflammation. MEASUREMENTS AND MAIN RESULTS: IL-23 mRNA was expressed in the lung of sensitized mice upon antigen inhalation, and the neutralization of IL-23 decreased antigen-induced eosinophil recruitment and Th2 cytokine production in the airways. The enforced expression of IL-23 in the airways significantly enhanced antigen-induced eosinophil and neutrophil recruitment into the airways; Th2 cytokine, IL-17A, and tumor necrosis factor (TNF)-alpha production in the airways; goblet cell hyperplasia; and airway hyperresponsiveness. Moreover, IL-23-mediated enhancement of antigen-induced Th2 cytokine production and eosinophil recruitment in the airways was still observed in the mice lacking IL-17A. Furthermore, although adoptive transfer of antigen-specific Th17 cells alone induced neutrophil but not eosinophil recruitment into the airways upon antigen inhalation, cotransfer of Th17 cells with Th2 cells significantly enhanced antigen-induced Th2-cell-mediated eosinophil recruitment into the airways and airway hyperresponsiveness. CONCLUSIONS: IL-23 and Th17 cells not only induce Th17-cell-mediated neutrophilic airway inflammation but also up-regulate Th2-cell-mediated eosinophilic airway inflammation.

IL-4-Stat6 signaling induces tristetraprolin expression and inhibits TNF-alpha production in mast cells.

Increasing evidence has revealed that mast cell-derived tumor necrosis factor alpha (TNF-alpha) plays a critical role in a number of inflammatory responses by recruiting inflammatory leukocytes. In this paper, we investigated the regulatory role of interleukin 4 (IL-4) in TNF-alpha production in mast cells. IL-4 inhibited immunoglobulin E-induced TNF-alpha production and neutrophil recruitment in the peritoneal cavity in wild-type mice but not in signal transducers and activators of transcription 6 (Stat6)-deficient mice. IL-4 also inhibited TNF-alpha production in cultured mast cells by a Stat6-dependent mechanism. IL-4-Stat6 signaling induced TNF-alpha mRNA destabilization in an AU-rich element (ARE)-dependent manner, but did not affect TNF-alpha promoter activity. Furthermore, IL-4 induced the expression of tristetraprolin (TTP), an RNA-binding protein that promotes decay of ARE-containing mRNA, in mast cells by a Stat6-dependent mechanism, and the depletion of TTP expression by RNA interference prevented IL-4-induced down-regulation of TNF-alpha production in mast cells. These results suggest that IL-4-Stat6 signaling induces TTP expression and, thus, destabilizes TNF-alpha mRNA in an ARE-dependent manner.