Role of IL-6 in dendritic cell functions.

Dendritic cells (DCs) are efficient antigen-presenting cells that serve as a link between the innate and adaptive immune systems. These cells are broadly involved in cellular and humoral immune responses by presenting antigens to initiate T cell reactions, cytokine and chemokine secretion, T cell differentiation and expansion, B cell activation and regulation, and the mediation of immune tolerance. The functions of DCs depend on their activation status, which is defined by the stages of maturation, phenotype differentiation, and migration ability, among other factors. IL-6 is a soluble mediator mainly produced by a variety of immune cells, including DCs, that exerts pleiotropic effects on immune and inflammatory responses through interaction with specific receptors expressed on the surface of target cells. Here, we review the role of IL-6, when generated in an inflammatory context or as derived from DCs, in modulating the biologic function and activation status of DCs and emphasize the importance of searching for novel strategies to target the IL-6/IL-6 signaling pathway as a means to diminish the inflammatory activity of DCs in immune response or to prime the immunogenic activity of DCs in immunosuppressive conditions.

G protein-coupled estrogen receptor, a potential therapeutic target of asthma probed by Chinese herb.

Discussion on the identification of GPER as a potential therapeutic target for asthma through Chinese herb-driven drug discovery strategy.

Metallothionein-2 is associated with the amelioration of asthmatic pulmonary function by acupuncture through protein phosphorylation.

BACKGROUND: Acupuncture has long been used for asthma treatment but the underlying mechanism remains unclear. Previous study showed that metallothionein-2 (MT-2) was significantly decreased in asthmatic lung tissue. However, the relationship between acupuncture treatment and MT-2 expression during asthma is still unknown, and the detailed effect analysis of MT-2 on phosphorylation in airway smooth muscle cells (ASMCs) is also unclear. METHODS: The acupuncture effect on pulmonary resistance (RL) was investigated in a rat model of asthma, and the mRNA and protein levels of MT-2 in lung tissue were detected. Primary ASMCs were isolated and treated with MT-2 recombinant protein to study the MT-2 effects on ASMC relaxation. A Phospho Explorer antibody microarray was applied to detect protein phosphorylation changes associated with MT-2-induced ASMC relaxation. Bioinformatic analysis were performed with PANTHER database, DAVID and STRING. Phosphorylation changes in key proteins were confirmed by Western blot. RESULTS: Acupuncture significantly reduced RL at 2-5 min (P < 0.05 vs asthma) in asthmatic rats. Acupuncture continued to increase MT-2 mRNA expression in lung tissue for up to 14 days (P < 0.05 vs asthma). The MT-2 protein expression was significantly decreased in the asthmatic rats (P < 0.05 vs control), while MT-2 protein expression was significantly increased in the asthmatic model group treated with acupuncture (P < 0.05 vs asthma). Primary ASMCs were successfully isolated and recombinant MT-2 protein (100, 200, 400 ng/ml) significantly relaxed ASMCs (P < 0.05 vs control). MT-2 induced phosphorylation changes in 51 proteins. Phosphorylation of 14 proteins were upregulated while 37 proteins were downregulated. PANTHER classification revealed eleven functional groups, and the phosphorylated proteins were identified as transferases (27.8 %), calcium-binding proteins (11.1 %), etc. DAVID functional classification showed that the phosphorylated proteins could be attributed to eight functions, including protein phosphorylation and regulation of GTPase activity. STRING protein-protein interaction network analysis showed that Akt1 was one of the most important hubs for the phosphorylated proteins. The phosphorylation changes of Akt1 and CaMK2ß were consistent in both the Phospho Explorer antibody microarray and Western blot. CONCLUSION: Acupuncture can significantly ameliorate RL, and the MT-2 mRNA and protein levels in lung tissue are increased during treatment. MT-2 significantly relaxes ASMCs and induces a series of protein phosphorylation. These phosphorylation changes, including Akt1 and CaMK2ß, may play important roles in the therapeutic effects of acupuncture on asthma.

S100A8 inhibits PDGF-induced proliferation of airway smooth muscle cells dependent on the receptor for advanced glycation end-products.

BACKGROUND: Airway remodeling is a key feature of asthma, characterized by increased proliferation of airway smooth muscle cells (ASMCs). S100A8 is a calcium-binding protein with a potential to regulate cell proliferation. Here, the effect of exogenous S100A8 protein on the proliferation of ASMCs induced by platelet-derived growth factor (PDGF) and the underlying molecular mechanism was investigated. METHODS: Rat ASMCs were cultured with or without a neutralizing antibody to the receptor for advanced glycation end-products (RAGE), a potential receptor for S100A8 protein. Purified recombinant rat S100A8 protein was then added into the cultured cells, and the proliferation of ASMCs induced by PDGF was detected by colorimetric-based WST-8 assay and ampedance-based xCELLigence proliferation assay. The expression levels of RAGE in ASMCs were analyzed using western blotting assay. RESULTS: Results showed that exogenous S100A8 inhibited the PDGF-induced proliferation of rat ASMCs in a dose-dependent manner with the maximal effect at 1 µg/ml in vitro. Furthermore, when ASMCs was pre-treated with anti-RAGE neutralizing antibody, the inhibitory effect of S100A8 on PDGF-induced proliferation was significantly suppressed. In addition, neither the treatment with S100A8 or PDGF alone nor the pre-treatment with rS100A8 followed by PDGF stimulation affected the expression levels of RAGE. CONCLUSIONS: Our study demonstrated that S100A8 inhibits PDGF-induced ASMCs proliferation in a manner dependent on membrane receptor RAGE.

S100A8 protein attenuates airway hyperresponsiveness by suppressing the contraction of airway smooth muscle.

Airway hyperresponsiveness (AHR) is a major clinical problem in allergic asthma mainly caused by the hypercontractility of airway smooth muscles (ASM). S100A8 is an important member of the S100 calcium-binding protein family with a potential to regulate cell contractility. Here, we analyze the potential of S100A8 to regulate allergen-induced AHR and ASM contraction. Treatment with recombinant S100A8 (rS100A8) diminished airway hyperresponsiveness in OVA-sensitized rats. ASM contraction assays showed that rS100A8 reduced hypercontractility in both isolated tracheal rings and primary ASM cells treated by acetylcholine. rS100A8 markedly rescued the phosphorylation level of myosin light chain induced by acetylcholine in ASM cells. These results show that rS100A8 plays a protective role in regulating AHR in asthma by inhibiting ASM contraction. These results support S100A8 as a novel therapeutic target to control ASM contraction in asthma.

S100A8 inhibits PDGF-induced proliferation of airway smooth muscle cells dependent on the receptor for advanced glycation end-products

BACKGROUND: Airway remodeling is a key feature of asthma, characterized by increased proliferation of airway smooth muscle cells (ASMCs). S100A8 is a calcium-binding protein with a potential to regulate cell proliferation. Here, the effect of exogenous S100A8 protein on the proliferation of ASMCs induced by platelet-derived growth factor (PDGF) and the underlying molecular mechanism was investigated. METHODS: Rat ASMCs were cultured with or without a neutralizing antibody to the receptor for advanced glycation end-products (RAGE), a potential receptor for S100A8 protein. Purified recombinant rat S100A8 protein was then added into the cultured cells, and the proliferation of ASMCs induced by PDGF was detected by colorimetric-based WST-8 assay and ampedance-based xCELLigence proliferation assay. The expression levels of RAGE in ASMCs were analyzed using western blotting assay. RESULTS: Results showed that exogenous S100A8 inhibited the PDGF-induced proliferation of rat ASMCs in a dose- dependent manner with the maximal effect at 1 µg/ml in vitro. Furthermore, when ASMCs was pre-treated with anti-RAGE neutralizing antibody, the inhibitory effect of S100A8 on PDGF-induced proliferation was significantly suppressed. In addition, neither the treatment with S100A8 or PDGF alone nor the pre-treatment with rS100A8 followed by PDGF stimulation affected the expression levels of RAGE. CONCLUSIONS: Our study demonstrated that S100A8 inhibits PDGF-induced ASMCs proliferation in a manner dependent on membrane receptor RAGE.

Exogenous S100A8 protein inhibits PDGF-induced migration of airway smooth muscle cells in a RAGE-dependent manner.

S100A8 is an important member of the S100 protein family, which is involved in intracellular and extracellular regulatory activities. We previously reported that the S100A8 protein was differentially expressed in the asthmatic respiratory tracts. To understand the potential role of S100A8 in asthma, we investigated the effect of recombinant S100A8 protein on the platelet-derived growth factor (PDGF)-induced migration of airway smooth muscle cells (ASMCs) and the underlying molecular mechanism by using multiple methods, such as impedance-based xCELLigence migration assay, transwell migration assays and wound-healing assays. We found that exogenous S100A8 protein significantly inhibited PDGF-induced ASMC migration. Furthermore, the migration inhibition effect of S100A8 was blocked by neutralizing antibody against the receptor for advanced glycation end-products (RAGE), a potential receptor for the S100A8 protein. These findings provide direct evidence that exogenous S100A8 protein inhibits the PDGF-induced migration of ASMCs through the membrane receptor RAGE. Our study highlights a novel role of S100A8 as a potential means of counteracting airway remodeling in chronic airway diseases.

Simultaneous application of BrdU and WST-1 measurements for detection of the proliferation and viability of airway smooth muscle cells

BACKGROUND: BrdU is a commonly used reagent in cell proliferation assays, and WST-1 measurement is widely used to detect cell viability. However, no previous study has formally reported the combination of the two assays, which may be used to detect the proliferation and viability simultaneously. In this study, we examined the effect of adding BrdU 2 h prior to the WST-1 assay and tried to test the possibility of the combined detection using rat airway smooth muscle cells. RESULTS: The WST-1 measurements obtained from the combined detection were consistent with those obtained from the separate detection, which suggested that the addition of BrdU 2 h prior to the WST-1 analysis did not affect the WST-1 results. The BrdU measurements obtained from the combined detection also demonstrated the same trend as that obtained from the separate detection, and dosages of 200, 400 and 800 ng/ml testing reagent significantly inhibited the proliferation of rat airway smooth muscle cells. CONCLUSIONS: Our study suggests that the BrdU and WST-1 measurements can be applied simultaneously without mutual interference, which may increase the efficacy and consistency of these measurements to a certain extent.

Recombinant rat CC10 protein inhibits PDGF-induced airway smooth muscle cells proliferation and migration.

Abnormal migration and proliferation of airway smooth muscle cells (ASMCs) in the airway cause airway wall thickening, which is strongly related with the development of airway remodeling in asthma. Clara cell 10 kDa protein (CC10), which is secreted by the epithelial clara cells of the pulmonary airways, plays an important role in the regulation of immunological and inflammatory processes. Previous studies suggested that CC10 protein had great protective effects against inflammation in asthma. However, the effects of CC10 protein on ASMCs migration and proliferation in airway remodeling were poorly understood. In this study, we constructed the pET-22b-CC10 recombinant plasmid, induced expression and purified the recombinant rat CC10 protein from E. coli by Ni(2+) affinity chromatography and ion exchange chromatography purification. We investigated the effect of recombinant rat CC10 protein on platelet-derived growth factor (PDGF)-BB-induced ASMCs proliferation and migration. Our results demonstrated that the recombinant CC10 protein could inhibit PDGF-BB-induced cell viability, proliferation and migration. Western blot analysis showed that PDGF-BB-induced activation of cyclin D1 was inhibited by CC10. These findings implicated that CC10 could inhibit increased ASMCs proliferation, and migration induced by PDGF-BB, and this suppression effect might be associated with inhibition of cyclin D1 expression, which might offer hope for the future treatment of airway remodeling.

Composition and characteristics of distinct macrophage subpopulations in the mouse thymus.

The aim of the present study was to investigate the composition, morphology, characteristics, distribution and function of distinct macrophage subpopulations in the mouse thymus. Apoptosis of mouse thymocytes was induced by glucocorticoids and three monoclonal antibodies against Mac-2, F4/80 and ED1 were used for immunofluorescence staining and immunohistochemical analysis. The morphology of thymic macrophages was examined by transmission electron microscopy. Four subpopulations of mouse thymic macrophages were identified. Dendritic macrophages were identified using anti-Mac-2 and anti-F4/80 antibodies, and were demonstrated to be distributed in the entire thymus. Phagocytes were also observed. In addition, plate-shaped macrophages, identified using the anti-F4/80 antibody, were distributed under the thymic cortex capsule. Small oval macrophages, identified using the anti-Mac-2 antibody, were distributed in the thymic medulla and corticomedullary region (CMR), while phagocytes were not observed in these types of cell. ED1+ thymic macrophages with irregular forms were distributed in the CMR. All of the four subpopulations of mouse thymic macrophages described above exhibited acid phosphate activity. This study indicated the existence of macrophage subpopulations with different shapes, distribution and functions in the mouse thymus.

[S100A8 protein in inflammation].

S100A8, an important member of the S100 protein family, is a low-molecular-weight (10.8 kDa) calcium-binding protein containing conserved EF-hand structural motifs. Previous studies have shown that the biological function of S100A8 protein is associated with a variety of inflammatory diseases, for example asthma. S100A8 protein plays important roles in the regulation of inflammation. It can activate inflammatory cells and cytokines via chemotactic activity for neutrophils, and bind to the receptor for advanced glycation end products (RAGE) and Toll-like receptor 4 (TLR4), thus mediating intracellular inflammatory signaling transduction. Additionally, recent studies have reported the anti-inflammation activity of S100A8 protein, which indicates that S100A8 may have a more complex function of biological regulation in the different pathophysiological conditions. In this review, we summarized the studies on the functions and molecular mechanisms of S100A8 protein in inflammation, which would propose a novel strategy for the prophylaxis and treatment of asthma and other inflammatory diseases.

The early asthmatic response is associated with glycolysis, calcium binding and mitochondria activity as revealed by proteomic analysis in rats.

BACKGROUND: The inhalation of allergens by allergic asthmatics results in the early asthmatic response (EAR), which is characterized by acute airway obstruction beginning within a few minutes. The EAR is the earliest indicator of the pathological progression of allergic asthma. Because the molecular mechanism underlying the EAR is not fully defined, this study will contribute to a better understanding of asthma. METHODS: In order to gain insight into the molecular basis of the EAR, we examined changes in protein expression patterns in the lung tissue of asthmatic rats during the EAR using 2-DE/MS-based proteomic techniques. Bioinformatic analysis of the proteomic data was then performed using PPI Spider and KEGG Spider to investigate the underlying molecular mechanism. RESULTS: In total, 44 differentially expressed protein spots were detected in the 2-DE gels. Of these 44 protein spots, 42 corresponded to 36 unique proteins successfully identified using mass spectrometry. During subsequent bioinformatic analysis, the gene ontology classification, the protein-protein interaction networking and the biological pathway exploration demonstrated that the identified proteins were mainly involved in glycolysis, calcium binding and mitochondrial activity. Using western blot and semi-quantitative RT-PCR, we confirmed the changes in expression of five selected proteins, which further supports our proteomic and bioinformatic analyses. CONCLUSIONS: Our results reveal that the allergen-induced EAR in asthmatic rats is associated with glycolysis, calcium binding and mitochondrial activity, which could establish a functional network in which calcium binding may play a central role in promoting the progression of asthma.