Cockroach allergen extract stimulates protease-activated receptor-2 (PAR-2) expressed in mouse lung fibroblast

Objective: To investigate whether cockroach allergen extract can stimulate Protease-activated receptor 2 (PAR-2) expressed in mouse lung fibroblast.Materials: We established an immortalized lung fibroblast cell line, DM5, from PAR-2 deficient mice. By stable transfection with either an empty vector (DM5/EV) or an expression vector encoding mouse PAR-2 cDNA (DM5/Par2), a pair of lung fibroblast cell lines with or without functional PAR-2 expression were prepared.Treatment: The cells were exposed to cockroach allergen extract {up to 800 protein nitrogen unit (PNU)/ml}, trypsin (up to 100 nM), SLIGRL agonist peptide (up to 500 M), and trans-cinnamoyl-LIGRO agonist peptide (up to 400 M).Methods: The cells were loaded with Fluo-3 calcium indicator and mobilization of intracellular calcium with the stimuli was monitored by a fluorometric plate reader. Extracellular signal-regulated kinase (ERK) phosphorylation was examined by Western blot analysis using an anti-phospho ERK antibody.Results: The cockroach extract induced intracellular calcium transients in a concentration dependent manner in DM5/Par2 but not in DM5/EV. The activity was abolished when the cockroach extract was heat denatured or pre-incubated with PMSF (phenylmethanesulfonyl fluoride) prior to the assay. Concomitantly, ERK phosphorylation was seen in DM5/Par2 with the cockroach extract but not with a heat-denatured extract. The responses were sensitive to an inositol-1,4,5 triphosphate antagonist (2-APB) indicating that calcium was mobilized from intracellular store.Conclusions: Cockroach allergen extract can activate PAR-2 and thereby stimulate mouse lung fibroblasts likely through protease(s). The present study proposes a potential mechanism for cockroach antigens, similar to house dust mite antigens, in the etiology of respiratory diseases.Received 29 February 2004; returned for revision 12 April 2004; accepted by M. Katori 22 April 2004     

A hypo-osmotically induced increase in intracellular Ca2+ in lactating mouse mammary epithelial cells involving Ca2+ influx

 Using a fluorescent Ca²⁺-sensitive dye, we studied the effect of hypo-osmotic stress on the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) in acini freshly isolated from lactating mouse mammary gland. The basal [Ca²⁺]_i of mammary acini was unaffected by a 50% (v/v) dilution of suspensions with isotonic or hypertonic buffer, or after ionic (iso-osmotic) dilution (external Ca²⁺ was 3 mM). Hypo-osmotic dilution (50%) elicited a rapid increase in [Ca²⁺]_i comprising a large, transient elevation, followed by a maintained plateau phase. No hypo-osmotically induced rise in [Ca²⁺]_i was observed in the absence of extracellular Ca²⁺. Neither microtubule disassembly using nocodazole nor actin disruption with cytochalasin D prevented hypo-osmotically evoked stimulation of [Ca²⁺]_i. Pre-incubation of acini with nifedipine did not prevent hypo-osmotically induced stimulation of [Ca²⁺]_i, whereas a non-specific cation channel blocker, gadolinium, partially inhibited the increases in [Ca²⁺]_i induced by hypo-osmotic stress. Furthermore, the transient component was still apparent, and not diminished in magnitude, after [Ca²⁺]_i had been elevated by mobilisation of Ca²⁺ from intracellular stores using thapsigargin. The results demonstrate that hypo-osmotic stress generates an increase in [Ca²⁺]_i in lactating mammary epithelial cells, the major, transient component of which appears to be due to influx of extracellular Ca²⁺. Received: 15 October 1996 / Received after revision and accepted: 1 November 1996     

Caffeine-induced oscillations of cytosolic Ca2+ in GH3 pituitary cells are not due to Ca2+ release from intracellular stores but to enhanced Ca2+ influx through voltage-gated Ca2+ channels

Caffeine, a well known facilitator of Ca²⁺-induced Ca²⁺ release, induced oscillations of cytosolic free Ca²⁺ ([Ca²⁺]_i) in GH₃ pituitary cells. These oscillations were dependent on the presence of extracellular Ca²⁺ and blocked by dihydropyridines, suggesting that they are due to Ca²⁺ entry through L-type Ca²⁺ channels, rather than to Ca²⁺ release from the intracellular Ca²⁺ stores. Emptying the stores by treatment with ionomycin or thapsigargin did not prevent the caffeine-induced [Ca²⁺]_i oscillations. Treatment with caffeine occluded phase 2 ([Ca²⁺]_i oscillations) of the action of thyrotropin-releasing hormone (TRH) without modifying phase 1 (Ca²⁺ release from the intracellular stores). Caffeine also inhibited the [Ca²⁺]_i increase induced by depolarization with high-K⁺ solutions (56% at 20 mM), suggesting direct inhibition of the Ca²⁺ entry through voltage-gated Ca²⁺ channels. We propose that the [Ca²⁺]_i increase induced by caffeine in GH₃ cells takes place by a mechanism similar to that of TRH, i.e. membrane depolarization that increases the firing frequency of action potentials. The increase of the electrical activity overcomes the direct inhibitory effect on voltage-gated Ca²⁺ channels with the result of increased Ca²⁺ entry and a rise in [Ca²⁺]_i. Consideration of this action cautions interpretation of previous experiments in which caffeine was assumed to increase [Ca²⁺]_i only by facilitating the release of Ca²⁺ from intracellular Ca²⁺ stores.     

Changes in element concentrations induced by agonist in pig pancreatic acinar cells

 Changes in electrolytes of pig pancreatic acinar cells following application of gastrin-cholecystokinin (CCK) were investigated using the technique of X-ray microanalysis of hydrated and dehydrated sections of freshly frozen pancreas. After stimulation by CCK (10^–9 M), Na and Cl increased significantly in the cytoplasm [Na, from 10 mmol/kg wet wt. (48 mmol/kg dry wt.) to 19 mmol/kg (95 mmol/kg); Cl, from 22 mmol/kg (105 mmol/kg) to 49 mmol/kg (245 mmol/kg)] as well as in the luminal interspace [Na, from 53 mmol/kg (189 mmol/kg) to 65 mmol/kg (283 mmol/kg); Cl, from 65 mmol/kg (232 mmol/kg) to 102 mmol/kg (443 mmol/kg)]. In the secretory granules Cl increased significantly from 30 mmol/kg (86 mmol/kg) to 67 mmol/kg (203 mmol/kg). K decreased significantly from 120 mmol/kg (571 mmol/kg) to 81 mmol/kg (405 mmol/kg) in the cytoplasm, while both increased from 38 mmol/kg (109 mmol/kg) to 58 mmol/kg (176 mmol/kg) in the granules and from 46 mmol/kg (164 mmol/kg) to 48 mmol/kg (209 mmol/kg) in the luminal interspace. Ca increased significantly in the cytoplasm as well as in the luminal interspace, and decreased significantly in the secretory granules. CCK evoked Ca release from secretory granules in the secretory pole of acinar cells. The values were measured from dehydrated sections, and agreed well with those from hydrated sections. The effect of furosemide, an inhibitor of the Na⁺-K⁺-2Cl^–co-transporter, on the ion transport of acinar cell was studied. When furosemide (10^–5 M) was added to the external solution, the cytoplasmic Cl and Ca concentrations decreased significantly, while there was a little decrease in Na and K concentrations under the secretory condition. These results indicate that Na⁺-K⁺-2Cl^–co-transport, and Na⁺, Cl^–and K⁺ exits into the lumen are involved in the mechanism of ion secretion in pig pancreatic acinar cells. Received: 17 January 1996 / Accepted: 12 March 1996     

Muscle energetics in short-term training during hypoxia in elite combination skiers

Four well-trained combination skiers were studied through pre- and post-training for the effects of short-term intermittent training during hypoxia on muscle energetics during submaximal exercise as measured by Phosphorus-31 nuclear magnetic resonance and maximal aerobic power ( O_2max). The hypoxia and training in the cold was conducted in a hypobaric chamber and comprised 60-min aerobic exercise (at an intensity equivalent to the blood lactate threshold), using a cycle ergometer or a treadmill twice a day for 4, consecutive days at 5°C, in conditions equivalent to an altitude of 2000 m (593 mm Hg). No change in O_2max was observed over the training period, while in the muscle energetics during submaximal exercise, the values of phosphocreatine/(phosphocreatine + inorganic phosphate) and intracellular pH were found to be significantly increased by training during hypoxia. During recovery, the time constant of phosphocreatine was found to have been significantly reduced [pre, 27.9 (SD 6.7) s; post, 22.5 (SD 4.7) s, P < 0.01]. The observed inhibition of phosphocreatine as well as that of intracellular pH changes after training during hypoxia and quicker recovery of phosphocreatine in submaximal exercise tests, may indicate improved oxidative capacity (i.e. a high adenosine 5-triphosphate formation rate) despite the short-term hypoxia training. Present address: Department Life Sciences, Univ. of Tokyo, Komaba 3-8-1, Meguro-ku 153, Japan     

Production of IL-12, IL-23 and IL-27p28 by bone marrow-derived conventional dendritic cells rather than macrophages after LPS/TLR4-dependent induction by Salmonella Enteritidis

Induction of the interleukin-12 (IL-12) cytokine family comprising IL-12, IL-23, IL-27, and IL-12p40 by intracellular pathogens is required for orchestration of cell-mediated immune responses. Macrophages (MΦ) have been shown to be a source of IL-12 following TLR4-dependent activation by Salmonella (S.). In this study another antigen-presenting cell type, the conventional dendritic cell (cDC), was analyzed and its cytokine responses compared with those of MΦ. We generated bone marrow-derived conventional dendritic cells (BMDC) and macrophages (BMMΦ) by incubating murine bone marrow cells with supernatants containing granulocyte/macrophage colony-stimulating factor (GM-CSF) or macrophage colony-stimulating factor (M-CSF), respectively. Stimulation of BMDC and BMMΦ with S. enterica serovar Enteritidis (SE) or LPS resulted in the release of IL-12 and IL-23 by BMDC but not by BMMΦ. Furthermore, BMDC secreted approx. 20-fold more IL-12p40 and IL-27p28 than BMMΦ. However, BMDC and BMMΦ produced similar levels of IL-10. Using BMDC originating from wild-type (wt), TLR2^def and TLR4^def mice, we show that in BMDC the induction of IL-12, IL-23, and IL-27p28 by SE is dependent on TLR4, whereas low-level production of p40 is also mediated by pattern recognition receptors (PRR) other than TLR4. Interestingly, LPS- and SE-provoked responses of BMDC were remarkably similar indicating that LPS is the primary danger molecule of SE. Taken together, our results point to cDC rather than MΦ as the major producers of the IL-12 family members during in vitro infection with SE. The mechanisms of recognition of SE, however, appear to be the same for cDC and MΦ     

Surface immunoglobulins mediate efficient transport of antigen to lysosomal compartments resulting in enhanced specific antigen presentation by B cells

A BCL₁ immunoglobulin (Ig) transfectant, expressing wild-type surface (s)IgM with the TEPC-15 idiotype (T15-Id) and anti-phosphorylcholine (PC) specificity, was previously shown to present PC-conjugated hen egg-white lysozyme (PC-HEL) to a HEL-specific T cell hybridoma at a lower antigen (Ag) concentration than that required for native HEL. Two variant Ig transfectants, expressing T15-Id sIgM with substitutions either in the entire spacer, transmembrane (TM) domain and cytoplasmic tail (B186 variant) or in the NH₂-terminal third of TM domain only (TM2 variant), failed to display this sIgM-mediated, enhanced presentation of PC-HEL at low concentrations. However, prolonged treatment with anti-T15-Id monoclonal antibody (mAb) led to a reduction of surface expression of the T15-Id sIgM in the wild-type and TM2 variant, but not in the B186 variant sIgM transfectants. Treatment with anti-T15-Id mAb also resulted in an increased intracellular accumulation of T15-Id sIgM in the wild-type transfectant, but not in the B186 variant. Subcellular fractionation analysis revealed that the ligands bound to the T15-Id sIgM are not efficiently transported to the dense lysosomal compartments in both B186 and TM2 transfectants, as compared to the wild-type sIgM transfectant. A significant increase in tyrosine phosphorylation after cross-linking of the T15-Id sIgM was observed only in the wild-type sIgM transfectant. These results suggest that, while the NH₂-terminal third of the TM region is not involved in the process responsible for the ligand-induced reduction of surface expression of sIgM, it appears to be essential for subsequent transport of sIgM/ligand complexes to the lysosomal compartments, as well as efficient activation of tyrosine kinases. These results strongly suggest that sIg-mediated enhancement of specific antigen presentation reflects the ability of sIg to efficiently transport antigen to the lysosomal compartments, and possibly the activation of protein tyrosine kinases.     

细胞间粘附分子—1^125I标记及其纯度、免疫活性的鉴定

目的：建立细胞间粘附分子－1（intracellular adhesion molecule-1 ICAM-1)^125I标记方法及鉴定其纯度和免疫活性。方法：用氯胺－T法标记ICAM－1,用Sephadex G-50柱层析分离,用纸层析法鉴定^125I-ICAM-1的纯度,放免法检测其免疫活性,结果：^125I-ICAM-1比活度为77．84uCi/ug蛋白,标记率为65．54％,^125I-Na的放化纯度为97．27％,^125I-ICAM-1能够与ICAM－1－Ab的最大结合为88．64％,并且随ICAM－1－Ab滴度的降低而增高。结论：成功建立^125I标记ICAM－1的方法,并且^125I-ICAM-1具有一定的免疫活性。     

Intracellular production of IL-2, IL-4 and IFN-γ by peripheral blood CD3+ cells in intermittent allergic rhinitis

Background: Allergic inflammation is mainly driven by type 2 T helper cells. The aim was to assess the changes in production of type 1 and 2 cytokines by CD3⁺ T cells dependent on natural exposure to allergens in subjects with intermittent allergic rhinitis (IAR) and in non-atopic subjects.Material: A total of 13 patients with IAR and 13 healthy non-atopics were recruited into the study. 11 patients with IAR were examined during the grass pollen season and 11 patients outside the season, 9 of them were assessed on both occasions.Methods: A flow cytometric assessment of intracellular expression of IL-2, IL-4 and IFN- by CD3⁺ cells was performed. For statistical analysis non-parametric tests were used.Results: A tendency to decreased production of IL-4 outside the season was observed (6.94% [3.42–13.33] in season vs. 2.06% [0.7–3.6] out of season). The production of IL-4 was higher in the rhinitic group in the season than in the control group (1.93% [1.07–4.97], p = 0.0034) and production of IL-2 was higher both in and outside the season (9.1% [3.94–15.09] and 10.0% [4.79–25.35] vs. 3.64% (2.64–5.03), p = 0.037 and 0.045, respectively). IL-4/IL-2 and IL-4/IFN- ratios were higher in the IAR group in the season than outside the season.Conclusion: A tendency towards a switch from a predominant type 2 response during natural allergen exposure to its suppression outside the season was found, together with a stable type 1 response.Received 22 July 2004; returned for revision 27 August 2004; accepted by M. J. Parnham 2 November 2004     

A convenient method for repeated intracellular recording of action potentials from the same muscle fibre without membrane damage

Summary Repeated intracellular recording of end-plate and action potentials from the same muscle fibre is often impossible because the membrane is damaged by the microelectrode when the muscle twitches. Membrane damage can be avoided by rolling and simultaneously stretching the muscle around a polyethylene rod; the amount of stretch necessary is about 50% of the “in-situ” length. Supported by the Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 38 “Membranforschung”, Bad Godesberg. Established Investigator of the Consejo Nacional de Investigaciones Científicas y Tecnicas, Argentina