Functional KCa3.1 K+ channels are required for human lung mast cell migration.

BACKGROUND: Mast cell recruitment and activation are critical for the initiation and progression of inflammation and fibrosis. Mast cells infiltrate specific structures in many diseased tissues such as the airway smooth muscle (ASM) in asthma. This microlocalisation of mast cells is likely to be key to disease pathogenesis. Human lung mast cells (HLMC) express the Ca2+ activated K+ channel K(Ca)3.1 which modulates mediator release, and is proposed to facilitate the retraction of the cell body during migration of several cell types. A study was undertaken to test the hypothesis that blockade of K(Ca)3.1 would attenuate HLMC proliferation and migration. METHODS: HLMC were isolated and purified from lung material resected for bronchial carcinoma. HLMC proliferation was assessed by cell counts at various time points following drug exposure. HLMC chemotaxis was assayed using standard Transwell chambers (8 microm pore size). Ion currents were measured using the single cell patch clamp technique. RESULTS: K(Ca)3.1 blockade with triarylmethane-34 (TRAM-34) did not inhibit HLMC proliferation and clotrimazole had cytotoxic effects. In contrast, HLMC migration towards the chemokine CXCL10, the chemoattractant stem cell factor, and the supernatants from tumour necrosis factor alpha stimulated asthmatic ASM was markedly inhibited with both the non-selective K(Ca)3.1 blocker charybdotoxin and the highly specific K(Ca)3.1 blocker TRAM-34 in a dose dependent manner. Although K(Ca)3.1 blockade inhibits HLMC migration, K(Ca)3.1 is not opened by the chemotactic stimulus, suggesting that it must be involved downstream of the initial receptor-ligand interactions. CONCLUSIONS: Since modulation of K(Ca)3.1 can inhibit HLMC chemotaxis to diverse chemoattractants, the use of K(Ca)3.1 blockers such as TRAM-34 could provide new therapeutic strategies for mast cell mediated diseases such as asthma.

Activation of human lung mast cells by monomeric immunoglobulin E.

The mechanism of chronic mast cell activation in asthma is unclear. Monomeric immunoglobulin (Ig)E in the absence of allergen induces mediator release from rodent mast cells, indicating a possible role for IgE in the continued activation of mast cells within the asthmatic bronchial mucosa. In this study it was investigated whether monomeric IgE induces Ca2+ influx and mediator release from human lung mast cells (HLMC). Purified HLMC were cultured for 4 weeks and then exposed to monomeric human myeloma IgE. Ratiometric Ca2+ imaging was performed on single fura-2-loaded cells. Histamine release was measured by radioenzymatic assay; leukotriene C4 (LTC4) and interleukin (IL)-8 were measured by ELISA. At concentrations experienced in vivo, monomeric IgE induced dose-dependent histamine release, LTC4 production and IL-8 synthesis. This was associated with a rise in cytosolic free Ca2+. Enhanced histamine release was still evident 1 week after initial exposure to IgE suggesting that continued exposure maintains enhanced secretion. Monomeric immunoglobulin E alone activates cultured human lung mast cells initiating Ca2+ influx, degranulation, arachidonic acid metabolism and cytokine synthesis. These findings support the hypothesis that immunoglobulin E loading of mast cells within the asthmatic airway contributes to the disordered airway physiology of this disease.

Co-cultivation of mast cells and Fc epsilon RI alpha+ dendritic-like cells from human hip bone marrow.

BACKGROUND: Mast cells contribute to the pathogenesis of asthma and allergy through the release of a plethora of pro-inflammatory mediators and cytokines. Their study is hampered by the difficult access to human tissue samples. Human mast cells have been cultured from CD34+ progenitors in the bone marrow of normal volunteers following iliac crest bone marrow biopsy but this is invasive. Hip bone marrow could provide a more convenient less invasive source of mast cell progenitors. OBJECTIVE: To characterize mast cells cultured from human bone marrow obtained at routine hip surgery. METHODS: Mononuclear cells were isolated from the bone marrow reamings of patients undergoing routine hip replacement surgery and were cultured with recombinant stem cell factor (SCF), IL-6 and IL-10. Cell surface markers were examined using flow cytometry, protease expression monitored using immunohistochemistry, histamine measured by radioenzymic assay, Ca2+ responses analysed using ratiometric Ca2+ imaging, and ion currents recorded via the patch-clamp technique. RESULTS: Mast cells were absent at baseline, but accounted for 65 +/- 7% of cells after 8-12 weeks of culture, equating to a mean 0.6 +/- 0.14 x 10(6) mast cells per culture. Fifty-three percent of tryptase+ cells also contained chymase. The remaining cells comprised a population of large CD1a+ HLA-DR+ and Fc epsilon RI alpha+ cells, most likely dendritic cells. All mast cells expressed CD117 and the high-affinity IgE receptor alpha-chain (Fc epsilon RI alpha) constitutively, and developed a Ca2+ response following IgE-dependent activation. These cells exhibited 7.8 +/- 2.9% net IgE-dependent histamine release, and demonstrated a similar ion channel profile to human lung mast cells. In particular, the intermediate conductance Ca(2+)-activated K+ channel opened following IgE-dependent activation. CONCLUSIONS: Mast cells grown from human hip marrow provide a rich non-invasive source of functionally mature mast cells. In addition, this culture system may be useful for the generation of Fc epsilon RI alpha+ dendritic cells.

Resting and activation-dependent ion channels in human mast cells.

The mechanism of mediator secretion from mast cells in disease is likely to include modulation of ion channel activity. Several distinct Ca(2+), K(+), and Cl(-) conductances have been identified in rodent mast cells, but there are no data on human mast cells. We have used the whole-cell variant of the patch clamp technique to characterize for the first time macroscopic ion currents in purified human lung mast cells and human peripheral blood-derived mast cells at rest and following IgE-dependent activation. The majority of both mast cell types were electrically silent at rest with a resting membrane potential of around 0 mV. Following IgE-dependent activation, >90% of human peripheral blood-derived mast cells responded within 2 min with the development of a Ca(2+)-activated K(+) current exhibiting weak inward rectification, which polarized the cells to around -40 mV and a smaller outwardly rectifying Ca(2+)-independent Cl(-) conductance. Human lung mast cells showed more heterogeneity in their response to anti-IgE, with Ca(2+)-activated K(+) currents and Ca(2+)-independent Cl(-) currents developing in approximately 50% of cells. In both cell types, the K(+) current was blocked reversibly by charybdotoxin, which along with its electrophysiological properties suggests it is carried by a channel similar to the intermediate conductance Ca(2+)-activated K(+) channel. Charybdotoxin did not consistently attenuate histamine or leukotriene C(4) release, indicating that the Ca(2+)-activated K(+) current may enhance, but is not essential for, the release of these mediators.

Voltage-dependent and calcium-activated ion channels in the human mast cell line HMC-1.

The mechanisms underlying the recruitment, differentiation, and sustained activation of mast cells in disease are likely to include modulation of ion channels. Specific Ca(2+), K(+), and Cl(-) conductances have been identified in rodent mast cells, but there are no equivalent data on human mast cells. We have used the whole-cell patch-clamp technique to characterize macroscopic ion currents in both the human mast cell line HMC-1 and human skin mast cells (HSMCs) at rest and in HMC-1 after activation with calcium ionophore. HSMCs were electrically silent at rest. In contrast, HMC-1 expressed a strong outwardly rectifying voltage-dependent Cl(-) conductance characteristic of ClC-4 or ClC-5 and a small inwardly rectifying K(+) current not carried by the classical Kir family of K(+) channels. Calcium ionophore induced the appearance of outwardly rectifying Ca(2+)-activated Cl(-) and K(+) currents, while hypotonicity induced another outwardly rectifying conductance typical of ClC-3. Reverse transcription-PCRs confirmed that mRNAs for the voltage-dependent Cl(-) channels ClC-3 and -5 were expressed. This is the first definitive description of a ClC-4/5-like current in a native leukocyte. We suggest that this current may contribute to the malignant phenotype while the Ca(2+)-activated K(+) and Cl(-) currents may be involved in cell activation.

Satisfaction and commitment in homosexual and heterosexual relationships.

Rusbult's (1980, 1983) investment model was utilized to explore the determinants of satisfaction with and commitment to maintain romantic relationships among male and female homosexuals and male and female heterosexuals. The study employed a questionnaire designed to obtain both specific and global measures of rewards, costs, alternatives, and investments, and to obtain global measures of satisfaction and commitment. Women, both lesbians and heterosexuals, reported that they had invested more in their relationships and were more committed to maintaining their relationships than did men. Heterosexuals, male and female, reported greater costs and marginally greater investments in their relationships. In general, the investment model effectively predicted satisfaction and commitment for the sample as a whole and for all four groups of respondents. Greater satisfaction with relationships was associated with higher levels of rewards and lower levels of costs. Greater commitment was associated with greater satisfaction, greater investments, and poorer quality alternatives. Relationship costs were more strongly related to satisfaction and commitment for females than for males. Differences in the average level and the importance of a wide variety of specific predictors were also examined. In general, gender appeared to be a more important predictor of the behaviors explored in this study than was sexual preference.