Functional characterization of histamine H4 receptor on human mast cells.

Among the four different types of histamine receptors (H1-H4), H4R is predominantly expressed in immune cells and involved in immunomodulatory response. Here, in this study we determined the expression of H4R in human mast cells (HMC-1, LAD-2 and primary cord blood derived CD34+ human mast cells) and characterized its functional properties. Interestingly, we found that human mast cells responded to both histamine (natural ligand) and 4-methylhistamine (selective H4R agonist) for sustained intracellular calcium mobilization, degranulation and cytokine production. However, only histamine induced the release of cAMP, but 4-methylhistamine down regulates cAMP indicating that H4R mediates its effect through Gαi/o protein and H1R via Gαq protein. Furthermore, both histamine and 4-methylhistamine induced the production of cysteinyl leukotrienes and LTB4. Using human inflammation antibody array membrane, we found that H4R induced the expression of various inflammatory proteins, involving pro-inflammatory cytokines and chemokines and these are TGF-ß1, TNF-α, TNF-ß, PDGF-BB, TIMP-2, M-CSF, IP-10, IL-16, IL-6, IL-3, IL-10, MIP-1α, IL-1α, ICAM-1, Eotaxin-2, RANTES, IL-8, MCP-1, and IL-6sR. We also quantified the level of various inflammatory cytokines produced by human mast cells through H4R. It was observed that, the production level of Th2 cytokines IL-4(401.34 pg/ml), IL-5 (64.21 pg/ml) and IL-13 (1044 pg/ml) and classical proinflammatory cytokines IL-6 (221.27 pg/ml) and IL-1ß (34.24 pg/ml) and chemokines MCP-1(106 pg/ml) and IL-8 (818.32 pg/ml). Furthermore, activation of H4R caused the phosphorylation of ERK and PI3K in a time dependent manner. Taken together these data demonstrate that, the activation of H4R in human mast cells produced not only inflammatory mediators that are associated with allergic reactions but also other inflammatory conditions.

Mechanisms of anticancer activity of sulforaphane from Brassica oleracea in HEp-2 human epithelial carcinoma cell line.

Sulforaphane (SFN) an isothiocyanate formed by hydrolysis of glucosinolates found in Brassica oleraceae is reported to possess anticancer and antioxidant activities. In this study, we isolated SFN from red cabbage (Brassica oleraceae var rubra) and evaluated the comparative antiproliferative activity of various fractions (standard SFN, extract and purified SFN) by MTT assay in human epithelial carcinoma HEp -2 and and Vero cells. Probable apoptotic mechanisms mediated through p53, bax and bcl-2 were also examined. The SFN fraction was collected by HPLC, enriched for its SFN content and confirmed. Expression of apoptosis-related proteins was detected by western blotting and RT PCR. Results showed that Std SFN and purified SFN concentration found to have closer IC50 which is equal to 58.96 microgram/ml (HEp-2 cells), 61.2 microgram/ml (Vero cells) and less than the extract which is found to be 113 microgram/ml (HEp-2 cells) and 125 microgram/ml (Vero cells). Further studies on apoptotic mechanisms showed that purified SFN down-regulated the expression of bcl-2 (antiapoptotic), while up-regulating p53 and Bax (proapoptotic) proteins, as well as caspase-3. This study indicates that purified SFN possesses antiproliferative effects the same as Std SFN and its apoptotic mechanism in HEp-2 cells could be mediated through p53 induction, bax and bcl-2 signaling pathways.

Airway smooth muscle cells enhance C3a-induced mast cell degranulation following cell-cell contact.

Growing evidence suggests that anaphylatoxins, C3a and C5a, play important roles in innate immunity and may also participate in the pathogenesis of asthma. Previous studies with animal models and immunohistochemistry analysis of lung tissue indicated that anaphylatoxins may regulate airway hyperresponsiveness (AHR) in asthma via the activation of their cell surface G protein-coupled receptors (C3aR and C5aR) in airway smooth muscle (ASM) cells. Using RT-PCR, flow cytometry, and confocal microscopy, we made the surprising observation that while C3aR and C5aR were expressed in human mast cells, they were not present in cultured primary human or murine ASM cells. Furthermore, we could not detect C3aR in smooth muscle-positive cells of human trachea or bronchus. Interestingly, incubation of human mast cells with ASM cells, but not its culture supernatant, caused a significant enhancement of C3a-induced mast cell degranulation. Although stem cell factor (SCF) and its receptor c-kit are constitutively expressed on ASM cells and mast cells, respectively, neutralizing antibodies to SCF and c-kit failed to inhibit ASM cell-mediated enhancement of mast cell degranulation. However, dexamethasone-treated ASM cells were normal for cell surface SCF expression but were significantly less effective in enhancing C3a-induced mast cell degranulation when compared with untreated cells. These findings suggest that cell-cell interaction between ASM cells and mast cells, via a SCF-c-kit-independent but dexamethasone-sensitive mechanism, enhances C3a-induced mast cell degranulation, which likely regulates ASM function, thus contributing to the pathogenesis of asthma.

C3a enhances nerve growth factor-induced NFAT activation and chemokine production in a human mast cell line, HMC-1.

Activation of cell surface G protein-coupled receptors leads to transphosphorylation and activation of a number of receptor tyrosine kinases. Human mast cells express G protein-coupled receptors for the complement component C3a (C3aR) and high affinity nerve growth factor (NGF) receptor tyrosine kinase, TrkA. To determine whether C3a cross-regulates TrkA signaling and biological responses, we used a human mast cell-line, HMC-1, that natively expresses both receptors. We found that NGF caused tyrosine phosphorylation of TrkA, resulting in a sustained Ca(2+) mobilization, NFAT activation, extracellular-signal regulated kinase (ERK) phosphorylation, and chemokine, macrophage inflammatory protein-1beta (MIP-1beta) production. In contrast, C3a induced a transient Ca(2+) mobilization and ERK phosphorylation but failed to stimulate TrkA phosphorylation, NFAT activation, or MIP-1beta production. Surprisingly, C3a significantly enhanced NGF-induced NFAT activation, ERK phosphorylation, and MIP-1beta production. Pertussis toxin, a G(i/o) inhibitor, selectively blocked priming by C3a but had no effect on NGF-induced responses. Mitogen-activated protein/ERK kinase inhibitor U0126 caused approximately 30% inhibition of NGF-induced MIP-1beta production but had no effect on priming by C3a. However, cyclosporin A, an inhibitor of calcineurin-mediated NFAT activation, caused substantial inhibition of NGF-induced MIP-1beta production both in the absence and presence of C3a. These data demonstrate that NGF caused tyrosine phosphorylation of TrkA to induce chemokine production in HMC-1 cells via a pathway that mainly depends on sustained Ca(2+) mobilization and NFAT activation. Furthermore, C3a enhances NGF-induced transcription factor activation and chemokine production via a G protein-mediated pathway that does not involve TrkA phosphorylation.

Purification and characterization of alkaline protease from Alcaligenes faecalis.

Extracellular alkaline protease from the alkalophilic bacterium Alcaligenes faecalis was purified by a combination of ion-exchange and size-exclusion chromatographic methods, and its properties were examined. The purified enzyme had a specific activity of 563.8 micromol of tyrosine/min per mg of protein and gave a single band on native PAGE and SDS/PAGE with a molecular mass of 67 kDa. Gelatin zymogram also revealed one clear zone of proteolytic activity which corresponded to the band obtained with native PAGE and SDS/PAGE. The enzyme had an optimal pH of 9.0 and exhibited its highest activity at 55 degrees C. The enzyme activity was inhibited by PMSF, suggesting the presence of serine residues at the active site. The enzyme had a K(m) of 1.66 mg/ml and a V(max) of 526 units/min per mg of protein with casein as the substrate.