Highly expressed cytoplasmic FcεRIß in human mast cells functions as a negative regulator of the FcRγ-mediated cell activation signal.

BACKGROUND: We recently reported that the interaction between Lyn and FcεRIß is indispensable for FcεRI-mediated human mast cell (MC) activation and that FcεRIß functions as an amplifier of FcεRI-mediated activation signal. Some of FcεRIß in cytoplasm appeared not to be co-localized with FcεRIα. The function of FcεRIß in the cytoplasm remains unknown. METHODS: The localization of FcεRIß and FcεRIα in giant papillae specimens from patients with allergic keratoconjunctivitis and of FcεRIß, FcεRIα, and Lyn in cultured human MCs was examined using confocal microscopy. FcεRIß was overexpressed using an adenovirus vector system. Mediators were measured by enzyme immunoassays or enzyme-linked immunosorbent assays. RESULTS: In the subepithelial region, FcεRIß was mainly localized in the cell membrane of MCs. In the perivascular region, FcεRIß expression was scattered throughout the cytoplasm and in the cell membrane of MCs. Overexpression of FcεRIß in MCs mainly increased its cytoplasmic expression and slightly up-regulated cell surface FcεRI expression. However, overexpression of FcεRIß in MCs resulted in down-regulation of the tyrosine phosphorylation levels of FcεRIß and Syk and down-regulation of the Ca(2+) influx soon after FcεRI aggregation and then resulted in down-regulation of degranulation, PGD2 synthesis, and production of a set of cytokines. This negative regulatory effect may be due to inhibition of the redistribution of Lyn to small patches within the plasma membrane. CONCLUSION: Cytoplasmic FcεRIß, which is not co-localized with FcεRIα, may function as a negative regulator, as it can capture important signalling molecules such as Lyn.

The interaction between Lyn and FcεRIß is indispensable for FcεRI-mediated human mast cell activation.

BACKGROUND: FcεRIß reportedly functions as an amplifier of the FcεRIγ-mediated activation signal using a reconstitution system. However, the amplification mechanisms in human mast cells (MCs) are poorly understood. We previously reported the hyperexpression of FcεRIß of MCs in giant papillae from vernal keratoconjunctivitis patients, compared with that in conjunctivae from nonallergic conjunctivitis patients. Elucidation of the molecular mechanisms of the amplification induced by FcεRIß should provide new targets for novel therapeutic interventions. The aim is to understand in greater details the function of FcεRIß in human MC FcεRI expression and signaling. METHODS: FcεRIß and Lyn expression was reduced using a lentiviral shRNA silencing technique. Localization of Lyn and FcεRIß in cultured MCs was examined by confocal microscopic analysis. Mediators were measured by ELISAs. RESULTS: The diminution of FcεRIß significantly downregulated cell surface FcεRI expression and FcεRI-mediated mediator release/production. The downregulation of FcεRI-mediated degranulation was not only due to the decrease in FcεRI expression. The diminution of FcεRIß inhibited the redistribution of Lyn within the cell membrane following IgE sensitization. The diminution of Lyn in MCs significantly downregulated FcεRI-mediated degranulation. The recombinant cell-penetrating forms of phosphorylated FcεRIß immunoreceptor tyrosine-based activation motif (ITAM) for intracellular delivery disturbed the interaction between Lyn and phosphorylated endogenous FcεRIß ITAM, resulted in inhibiting IgE-dependent histamine release from MCs in vitro and from giant papillae specimens ex vivo. CONCLUSION: The interaction between Lyn and FcεRIß is indispensable for FcεRI-mediated human MC activation, and specific inhibition of the interaction may represent a new therapeutic strategy for the treatment of human allergic diseases.

Interleukin 4 is localized to and released by human mast cells.

Recent attention has focused on the T helper type 2 (Th2) lymphocyte as a source of interleukin 4 (IL-4) in allergic disease. However, Th2 cells themselves require a pulse of IL-4 to initiate this synthesis. Here we provide immunohistochemical evidence of IL-4 localization to human mast cells of the skin and respiratory tract, and demonstrate that immunoglobulin E-dependent stimulation of purified human lung mast cells leads to the rapid release of IL-4 into the extracellular environment. We propose that mast cell activation in an allergic response provides a rapid and local pulse of IL-4 into the local environment essential for the triggering of T lymphocytes into sustained IL-4 production and to initiate inflammatory cell accumulation and activation.

FcepsilonRI-mediated thymic stromal lymphopoietin production by interleukin-4-primed human mast cells.

A significant increase of mRNA expression of thymic stromal lymphopoietin (TSLP) has been reported in the bronchial mast cells (MCs) of asthmatic subjects; however, the mechanism underlying the upregulation of TSLP mRNA and protein remains unknown. FcepsilonRI-mediated activation of human MCs upregulated TSLP mRNA expression by 5.2+/-2.9-fold, while activation of the MCs using lipopolysaccharide and polyriboinosinic:polyribocytidylic acid failed to upregulate TSLP. Stimulation of MCs with interleukin (IL)-4 alone did not affect the TSLP mRNA expression, while pre-incubation of MCs with IL-4 for 48 h significantly enhanced the FcepsilonRI-mediated TSLP mRNA expression (by 53.7+/-15.9-fold; p<0.05) and the amount of TSLP in the cell pellets increased significantly from 23.4+/-4.3 pg mL(-1) to 121.5+/-3.7 pg mL(-1) (p<0.0001). However, the released TSLP was rapidly degraded by proteases that were released by MCs. We identified the population of cells expressing TSLP in the lungs of 16 asthmatic and 11 control subjects by immunohistochemistry. The percentage of TSLP-positive MCs in the total population of MCs was significantly increased in asthmatic airways (p<0.0001). Thus, MCs are able to store TSLP intracellularly and to produce TSLP following aggregation of FcepsilonRI in the presence of IL-4.

The airway smooth muscle CCR3/CCL11 axis is inhibited by mast cells.

BACKGROUND: Airway smooth muscle hyperplasia is a feature of asthma, and increases with disease severity. CCR3-mediated recruitment of airway smooth muscle progenitors towards the airway smooth muscle bundle has been proposed as one possible mechanism involved in airway smooth muscle hyperplasia. Mast cells are microlocalized to the airway smooth muscle bundle and whether mast cells influence CCR3-mediated migration is uncertain. METHODS: We examined the expression of CCR3 by primary cultures of airway smooth muscle cells from asthmatics and nonasthmatics. CCR3 function was examined using intracellular calcium measurements, chemotaxis, wound healing, cell proliferation and survival assays. We investigated the recovery and function of both recombinant and airway smooth muscle-derived CCL11 (eotaxin) after co-culture with beta-tryptase and human lung mast cells. RESULTS: Airway smooth muscle expressed CCR3. Airway smooth muscle CCR3 activation by CCL11 mediated intracellular calcium elevation, concentration-dependent migration and wound healing, but had no effect on proliferation or survival. Co-culture with beta-tryptase or mast cells degraded recombinant and airway smooth muscle-derived CCL11, and beta-tryptase inhibited CCL11-mediated airway smooth muscle migration. CONCLUSIONS: CCL11 mediates airway smooth muscle migration. However co-culture with beta-tryptase or mast cells degraded recombinant and airway smooth muscle-derived CCL11 and inhibited CCL11-mediated airway smooth muscle migration. Therefore these findings cast doubt on the importance of the CCL11/CCR3 axis in the development of airway smooth muscle hyperplasia in asthma.

Assessment of the anti-c-kit monoclonal antibody YB5.B8 in affinity magnetic enrichment of human lung mast cells.

The monoclonal antibody, YB5.B8 binds to the second domain of the c-kit proto-oncogene product on human mast cells, a receptor associated with tyrosine kinase activity. This molecule is involved with cell proliferation, maturation and viability as well as cell activation and its natural ligand is stem cell factor (SCF). We have used this antibody coupled to Dynabeads to perform positive affinity enrichment of human lung mast cells. This procedure results in enrichment of mast cells from 2.6 +/- 0.3% to 85.0 +/- 1.6% purity (n = 29) with yields of 41.9 +/- 3.7% (n = 29). As YB5.B8 interacts with the same receptor domain as does SCF, it is important to demonstrate that this procedure does not modify mast cell function. Incubation of mast cells with 1-5000 ng/ml YB5.B8 for 30 min neither induced histamine release nor modulated histamine release induced by anti-IgE. Furthermore, incubation with YB5.B8 did not alter prolonged culture with SCF. Examination of cells enriched using YB5.B8 showed that they had a normal histamine content (3.8 +/- 0.3 pg/cell compared with 3.9 +/- 0.7 pg/cell unpurified, n = 20) and had unchanged behaviour in both histamine secretion and cell survival studies. These studies indicate that YB5.B8 does not influence mast cell function and thus its use in magnetic affinity purification procedures offers a simple and effective method for enriching human mast cell preparations.

Interleukin-3 production by mast cells from human lung.

The cytokine interleukin (IL)-3 is important in the proliferation of eosinophils and basophils in the airway. We investigated IL-3 production by human lung mast cells as a possible mechanism of the airway inflammation constituting the late asthmatic response. Mast cells were purified using affinity magnetic selection with the monoclonal antibody YB5.B8 and then stimulated with anti-human IgE antibody. IL-3 release was detectable by enzyme-linked immunosorbent assay 8 h after anti-IgE stimulation. IL-3 release 24 h after anti-IgE stimulation was significantly greater than its controls. By reverse transcription-polymerase chain reaction, IL-3 mRNA was detected weakly 2 h after anti-IgE stimulation, peaking at 4 h and waning at 8 h. Immunocytochemistry to localize IL-3 demonstrated mast cell staining. These results suggest that mast cells release IL-3 in response to high-affinity IgE receptor.

Retroperitoneal malignant fibrous histiocytoma causing variegated colonic lesions.

A 68-year-old man visited our hospital because of heartburn. A firm mass was palpated in the left upper abdominal quadrant. Ultrasonography and computed tomography revealed a large left sided retroperitoneal tumor. A barium enema examination showed shallow irregularly depressed or elevated lesions. Colonoscopy revealed an irregularly shaped ulcer and multiple submucosal masses suggesting invasion by an extrinsic malignant tumor. Although colonoscopic biopsy was negative, a resected tumor was histologically diagnosed as a malignant fibrous histiocytoma (MFH). When such varigated lesions are detected in the colon, MFH should be considered, and an attempt to sample the submucosal layer may be necessary.

[Effect of phorbol myristate acetate (PMA) on diphosphoinositide (DPI) synthesis in rat mast cell granules].

Rat mast cell granules were obtained by homogenization of highly purified rat mast cells and isolated in a Percoll gradient. DPI synthesis in rat mast cell granules was assayed by measuring the incorporation of 32P from [gamma 32P] ATP into DPI in the absence of exogenous phosphatidylinositol (PI). Lipids were isolated with methanol/chloroform/HC1 and were separated by thin-layer chromatography on oxalic acid impregnated silica gel plates. DPI areas were identified by staining with iodine, scraped and measured for 32P radioactivity. The addition of PMA to the granules caused an increase of DPI synthesis, which can be catalysed by PI kinase. Neither an inactive phorbol ester, 4-alpha-phorbol-12, 13-didecanoate, nor dimethyl sulfoxide (DMSO) used as a solvent for PMA had any effect. The effect of PMA in the DPI synthesis was dose-dependent and maximal effects were observed at 10-100 ng/ml. Dose-response curves of the effects of PMA in DPI synthesis in the granules corresponded to those of other biochemical effects of PMA in rat mast cells, such as mediator release mediated through the activation of protein kinase C. These results suggest that PMA may directly affect PI kinase or indirectly regulate its activity in rat mast cell granules.

[Gallstone classification and analysis of their constituents].

Macroscopic classification of gallstones as proposed by the Japanese Society of Gastroenterology is based on the presence of characteristic structures on the cut surface. According to this classification, gallstones can be broadly divided into three types: cholesterol gallstones, pigment gallstones and rare gallstones. This classification is widely accepted in Japan because of its clinical applicability. A more accurate classification requires analysis of the constituents of gallstone. In particular, infrared absorption spectroscopy has been utilized for this purpose because it enables a rapid and easy analysis, requiring only small amounts of sample. In the selection of patients with gallstones for non-surgical treatment, such as ESWL and dissolution therapy, qualitative diagnosis is necessary. With this in mind, we have attempted to produce CT-Profile curves of individual gallstones, depicting changes in CT values assessed along cross-sectional lines passing from the center of the stone to their maximum diameters. This method allowed qualitative diagnosis almost of all gallstones.

Basophils in the giant papillae of chronic allergic keratoconjunctivitis.

AIMS: The essential role of basophils as an initiator of chronic allergic reaction has been elucidated in mouse models. The aim of this present study was to analyse the in situ immunolocalisation of basophils and other relevant inflammatory cells in chronic allergic keratoconjunctivitis. METHODS: Transmission electron microscopic (TEM) analysis was carried out to examine the existence of basophils in the giant papillae obtained from atopic keratoconjunctivitis (AKC) and vernal keratoconjunctivitis (VKC) patients. Cryostat sections of giant papillae were immunostained with basophil-specific antibody BB-1, and with anti-CD4, anti-CD8, anti-CD20, anti-major basic protein (MBP), anti-IgE and anti-FcepsilonRI-beta antibodies. RESULTS: TEM analysis confirmed the existence of basophils in the giant papillae. Small clusters of basophils were observed in the substantia propria of giant papillae, especially at the vicinity of vascular endothelium and subepithelial regions. BB-1-positive basophil clusters were surrounded by T cells, B cells, IgE-positive cells and MBP-positive eosinophils. No BB-1-positive basophils were observed in the control conjunctivae. CONCLUSION: Basophils may infiltrate from either vascular endothelium into the giant papillae. The existence of basophils at the centre of inflammatory cells suggests the role of basophils as an initiator of chronic allergic conjunctivitis.

Impacts of excision repair cross-complementing gene 1 (ERCC1), dihydropyrimidine dehydrogenase, and epidermal growth factor receptor on the outcomes of patients with advanced gastric cancer.

Using laser-captured microdissection and a real-time RT-PCR assay, we quantitatively evaluated mRNA levels of the following biomarkers in paraffin-embedded gastric cancer (GC) specimens obtained by surgical resection or biopsy: excision repair cross-complementing gene 1 (ERCC1), dihydropyrimidine dehydrogenase (DPD), methylenetetrahydrofolate reductase (MTHFR), epidermal growth factor receptor (EGFR), and five other biomarkers related to anticancer drug sensitivity. The study group comprised 140 patients who received first-line chemotherapy for advanced GC. All cancer specimens were obtained before chemotherapy. In patients who received first-line S-1 monotherapy (69 patients), low MTHFR expression correlated with a higher response rate (low: 44.9% vs high: 6.3%; P=0.006). In patients given first-line cisplatin-based regimens (combined with S-1 or irinotecan) (43 patients), low ERCC1 correlated with a higher response rate (low: 55.6% vs high: 18.8%; P=0.008). Multivariate survival analysis of all patients demonstrated that high ERCC1 (hazard ratio (HR): 2.38 (95% CI: 1.55-3.67)), high DPD (HR: 2.04 (1.37-3.02)), low EGFR (HR: 0.34 (0.20-0.56)), and an elevated serum alkaline phosphatase level (HR: 1.00 (1.001-1.002)) were significant predictors of poor survival. Our results suggest that these biomarkers are useful predictors of clinical outcomes in patients with advanced GC.