大鼠肺纤维化形成中肺内肥大细胞结缔组织生长因子的表达

目的：观察博莱霉素（BLM）诱导肺纤维化形成中肺肥大细胞（MCs）是否表达结缔组织生长因子（CTGF）。方法：32只雄性SD大鼠,随机分为博莱霉素（BLM）组和对照（Control）组（n=16）。BLM组为气管内一次性滴注BLM（5mg／ks）;Control组为气管内滴注与BLM等容量的生理盐水（NS）。各组分别在气管滴注后第14天和第28天处死大鼠,取肺组织样本。用氯胺-T法检测肺组织羟脯氨酸含量以判断肺纤维化程度;用甲苯胺蓝染色显示肺组织切片中的MCs;免疫组化染色显示肺CTGF的表达和分布。结果：①与对照大鼠比,气管内滴注BLM后第28天大鼠的肺羟脯氨酸含量明显增高（P〈0．01）。②与对照大鼠比,气管内滴注BLM后第14天和第28天大鼠肺内MCs数明显增多（均P〈0．01）,肺内CTGF表达上调（均P〈0．01）。③对照大鼠肺内未见CTGF免疫阳性的MCs;而气管内滴注BLM后第14天和第28天大鼠肺内病灶区中有CTGF免疫阳性的MCs。结论：肺纤维化形成中肺MCs表达CTGF,这可能是MCs促进肺纤维化的作用机制之一。     

Activated MCTC mast cells infiltrate diseased lung areas in cystic fibrosis and idiopathic pulmonary fibrosis

Background

Although mast cells are regarded as important regulators of inflammation and tissue remodelling, their role in cystic fibrosis (CF) and idiopathic pulmonary fibrosis (IPF) has remained less studied. This study investigates the densities and phenotypes of mast cell populations in multiple lung compartments from patients with CF, IPF and never smoking controls.

Methods

Small airways, pulmonary vessels, and lung parenchyma were subjected to detailed immunohistochemical analyses using lungs from patients with CF (20 lung regions; 5 patients), IPF (21 regions; 7 patients) and controls (16 regions; 8 subjects). In each compartment the densities and distribution of MC_T and MC_TC mast cell populations were studied as well as the mast cell expression of IL-6 and TGF-β.

Results

In the alveolar parenchyma in lungs from patients with CF, MC_TC numbers increased in areas showing cellular inflammation or fibrosis compared to controls. Apart from an altered balance between MC_TC and MC_T cells, mast cell in CF lungs showed elevated expression of IL-6. In CF, a decrease in total mast cell numbers was observed in small airways and pulmonary vessels. In patients with IPF, a significantly elevated MC_TC density was present in fibrotic areas of the alveolar parenchyma with increased mast cell expression of TGF-β. The total mast cell density was unchanged in small airways and decreased in pulmonary vessels in IPF. Both the density, as well as the percentage, of MC_TC correlated positively with the degree of fibrosis. The increased density of MC_TC, as well as MC_TC expression of TGF-β, correlated negatively with patient lung function.

Conclusions

The present study reveals that altered mast cell populations, with increased numbers of MC_TC in diseased alveolar parenchyma, represents a significant component of the histopathology in CF and IPF. The mast cell alterations correlated to the degree of tissue remodelling and to lung function parameters. Further investigations of mast cells in these diseases may open for new therapeutic strategies.     

The effect of polymyxin B and some mast-cell constituents on mucosal mast cells in the duodenum of the rat

Summary Mucosal mast cells in the rat duodenum show no morphological signs of exocytosis of granules and do not release histamine after treatment with polymyxin B in doses large enough to cause almost complete degranulation of connective-tissue mast cells of tongue, skin, and mesentery with concomitant release of 60% of the tissue histamine. Administration of polymyxin B in gradually increasing doses over a period of 5ds resulted in a statistically significant increase in mucosal mast cells and a comparable increase in duodenal histamine content, whereas the connective-tissue mast cells in the other tissues examined became fewer in number, the remaining cells showing profound morphological changes, and tissue histamine levels, were reduced to 40% of the controls. A similar increase in mucosal mast cells has been observed after treatment with another mast-cell secretagogue, compound 48/80. This suggests that the increase in mucosal mast cells may be an indirect effect of these compounds, related to their activation of other mast cells and mediated by material(s) secreted by the connective-tissue mast cells. Possible mediators such as heparin, histamine, and 5-hydroxytryptamine injected for 5 ds in doses large enough to account for the amount released from the degranulated mast cells had no effect on the morphology or numbers of mast cells in any of the tissues examined.Supported by grants from the Swedish Medical Research Council, Project no 2235     

IgE-induced degranulation of mucosal mast cells is negatively regulated via nicotinic acetylcholine receptors

The autonomic nervous system is known to mediate mast cell activation. We investigated expression of nicotinic acetylcholine receptors (nAChRs) in mucosal-type mast cells and their contribution to the regulation of mast cell activation. Expression of mRNA of nAChR α4, α7, and β2 subunits were detected in specially differentiated mucosal-type murine bone marrow-derived mast cells (mBMMCs). Pretreatment with non-specific nAChRs agonists, acetylcholine, nicotine and epibatidine and a specific α7 subunit agonist GTS-21 significantly inhibited antigen-induced degranulation of mBMMCs in a dose-dependent manner and GTS-21-induced inhibition was significantly blocked by α7 subunit antagonist, α-bungarotoxin. Furthermore, confocal microscopy also demonstrated surface binding of α-bungarotoxin on mBMMCs. Our findings indicate that mucosal mast cell activation may be negatively regulated mainly through nAChR α7 subunit, suggesting that nAChRs are involved in neuronal-mucosal mast cell interactions.     

Hepatic mast cells in rats infected with Taenia taeniaeformis

Mast cells appeared in the liver around metacestodes of Taenia taeniaeformis by 13 days after infection (DAI) of rats. The cells often occurred in clusters. The population increased until 28 DAI, then gradually declined. These hepatic mast cells (HMC) were compared to intestinal mucosal mast cells (MMC) and connective tissue mast cells (CTMC) histochemically, morphologically and in their response in vivo to Compound (built|48/80) and dexamethasone. Hepatic mast cells were similar to MMC in that they stained strongly blue with Astra blue at pH 1·0, could not be demonstrated with 0·005% toluidine blue, disappeared after treatment with dexamethasone, and were unaffected by 48/80. Immunoglobulin-containing cells in the liver were characterized by immunofluorescence. Immunoglobulin E-positive, and to a lesser extent IgG_2a-, and IgG_2c-positive cells surround the parasites in increasing numbers until 28 DAI, then declined. Many IgE-positive cells were HMC, and the IgE was frequently located intracytoplasmically. These cells were clearly distinguishable from eosinophils which stained characteristically with Giemsa and did not react with the anti-IgE probe. The results suggest that mast cell progenitors may be induced to localize and proliferate at the host-parasite interface in the Taenia-infected livers, giving rise to a cell population comparable to the MMC often seen at parasitized mucosal surfaces.     

Effects of cigarette smoke on degranulation and NO production by mast cells and epithelial cells

Exhaled nitric oxide (eNO) is decreased by cigarette smoking. The hypothesis that oxides of nitrogen (NO_X) in cigarette smoke solution (CSS) may exert a negative feedback mechanism upon NO release from epithelial (AEC, A549, and NHTBE) and basophilic cells (RBL-2H3) was tested in vitro. CSS inhibited both NO production and degranulation (measured as release of beta-hexosaminidase) in a dose-dependent manner from RBL-2H3 cells. Inhibition of NO production by CSS in AEC, A549, and NHTBE cells was also dose-dependent. In addition, CSS decreased expression of NOS mRNA and protein expression. The addition of NO inhibitors and scavengers did not, however, reverse the effects of CSS, nor did a NO donor (SNP) or nicotine mimic CSS. N-acetyl-cysteine, partially reversed the inhibition of beta-hexosaminidase release suggesting CSS may act via oxidative free radicals. Thus, some of the inhibitory effects of CSS appear to be via oxidative free radicals rather than a NO_X -related negative feedback.     

Different potency of bacterial antigens TLR2 and TLR4 ligands in stimulating mature mast cells to cysteinyl leukotriene synthesis

The aim of study was to compare the potency of different bacterial antigens to induce rat mature mast cell to cysteinyl leukotriene (cysLT) generation. We examined Toll-like receptor (TLR)2 agonists, i.e. lipoteichoic acid (LTA) Staphylococcus faecalis, Streptococcus pyogenes, Bacillus subtilis and Staphylococcus aureus, lipoarabinomannan (LAM) Mycobacterium smegmatis, peptydoglican (PGN) Staphylococcus aureus, as well as TLR4 agonists, i.e. lipopolysaccharide (LPS) Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella enteritidis, Pophyromonas gingivalis and Escherichia coli. We also estimated the effect of tumor necrosis factor (TNF)-, interleukin (IL)-6-, CCL5-, and IL-10-priming on mast cell cysLT synthesis following bacterial antigen activation. We found that all bacterial antigens activated mast cells to cysLT generation; however, the extent of cysLT release in response to stimulation varied. Out of the examined antigens LPS P. gingivalis exhibited the highest potency, as it induced cysLT generation acting at a very low concentration (10(-4) ng/mL). Other LPSs affected mast cells at higher (up to 10(5) -fold) concentrations. LTAs were the most effective at concentrations of 5 × 10(2) ng/mL, while LAM and PGN stimulated mast cells to maximal cysLT generation at concentrations as high as 10(5) ng/mL. Anti-TLR2 and anti-TLR4 antibodies, as well as nuclear factor κB (NF-κB) inhibitor significantly diminished cysLT generation in response to bacterial antigen stimulation. Priming with TNF, IL-6 and CCL5 did not affect bacterial antigen-induced cysLT generation, while IL-10-pretreatment caused significant decrease in cysLT synthesis by mast cells. These observations might have a great pathophysiological importance; inasmuch cysLTs strongly influence the development and intensity of inflammation during bacterial infection.     

A new technique for staining mast cells using ferroin

We describe here a new method for specific staining of mast cells using ferroin. Different hamster tissues were fixed in 4% formalin and processed for paraffin embedding. Sections were stained with hematoxylin followed by ferroin acidified with 2.5 N sulfuric acid to pH 4.0. Mast cells stained an intense orange color that contrasted markedly with bluish violet nuclei. High contrast was also observed when ferroin colored sections were counterstained with light green instead of hematoxylin. To evaluate the specificity of the stain, hamster cheek pouch sections were stained with toluidine blue, alcian blue-safranin O, and ferroin. Quantitative evaluation of mast cells stained with the three techniques showed no statistical difference. The simplicity and selectivity of this method is sufficient for image analysis of mast cells.