Tryptase from human mast cells does not activate purified human Hageman Factor

The effect of tryptase, a neural protease released from human lung mast cell secretory granules, on purified human Hageman Factor(Factor XII) was examined. No increase in Hageman Factor enzymatic activity was detected after incubation with tryptase at 37°C; activation of Hageman Factor by bovine trypsin served as a positive control. Furthermore, pre-incubation of Hageman Factor with tryptase did not diminish the subsequent activation of Hageman Factor by trypsin. Polyacrylamide gel electrophoresis was also performed to show that incubation with tryptase does not alter the molecular weight of Hageman Factor. Therefore, tryptase neither activates nor destroys human Hageman Factor.     

Nitric oxide does not affect histamine release from rat peritoneal mast cells

Inflammation Research -     

Platelet activating factor does not release histamine from human dispersed cutaneous mast cells

Histamine H1-antagonists inhibit the weal-and-flare responses to the intradermal injection of platelet activating factor (PAF) in humans, and PAF response is reduced in histamine-depleted skin sites. This indicates that mast cell histamine release is likely to be the mechanism of this response. We have therefore studied the interaction of PAF with cutaneous mast cells by observing whether it releases histamine directly from human dispersed foreskin mast cells, potentiates the activity of known mast cell stimulants or liberates histamine releasing factors (HRFs) from human platelets and leucocytes to release mast cell histamine by an indirect mechanism. At a concentration of 100 microM both PAF C18 and PAF C16 caused near maximal release (83.5 +/- 4.3% and 88.2 +/- 4.5% respectively) of the total histamine content of the cell. This release was not inhibited in the absence of extracellular Ca2+, by the lack of metabolic energy or in the presence of the PAF antagonists WEB 2086 (100 nM-3 microM) or BN 52021 (100 nM-10 microM). These results indicate a cytotoxic mechanism of histamine release by PAF 100 microM. PAF (10 nM-1 microM) failed to potentiate the mast cell-stimulating activity of anti-IgE, calcium ionophore A23187 or substance P and it did not induce the release of HRFs for skin mast cells when incubated with platelets and leucocytes in concentrations up to 1 microM.     

Tryptase and kinin generation: tryptase from human mast cells does not activate human urinary prokallikrein

The effect of tryptase, a neutral protease released from human lung mast cell secretory granules, on the tissue prokallikrein present in human urine was examined. Tryptase has been shown previously to lack activity against plasma prokallikrein. Purified tryptase was incubated with a concentrated preparation of urinary prokallikrein. No increase in kallikrein-like enzymatic activity or immunoreactive tissue kallikrein was detected. Activation of urinary prokallikrein with trypsin served as a positive control. Furthermore, preincubation of urinary prokallikrein with tryptase did not diminish the subsequent activation of urinary prokallikrein by trypsin. Therefore, tryptase neither activates nor destroys human tissue or plasma prokallikreins.     

TH1-dominant granulomatous pathology does not inhibit fibrosis or cause lethality during murine schistosomiasis

Schistosoma mansoni egg-induced inflammation is accompanied by TH2 cell polarization and development of fibrotic granulomas in host tissue. The interleukin (IL)-4 receptor alpha (IL-4Ralpha), which mediates IL-4 and IL-13 signaling, is essential for granulomatous pathology through a putative CD4+ T-cell-dependent mechanism. In this study, we asked whether CD4+ T-cell-specific IL-4Ralpha-deficient mice (Lck(Cre)IL-4Ralpha(-/lox)) developed granulomas and egg-driven collagen production. Although eosinophilia and goblet cell hyperplasia were impaired in Lck(Cre)IL-4Ralpha(-/lox) mice, there was no reduction in size or collagen content of lung and liver granulomas. The lack of CD4+ T-cell IL-4Ralpha expression caused significant increases in interferon-gamma-producing cells, inducible nitric-oxide synthetase production, and hepatic damage, compared with similarly infected wild-type mice. Interestingly, this TH1-associated liver injury did not lead to premature mortality in this strain. Instead, lower levels of serum endotoxin in Lck(Cre)IL-4Ralpha(-/lox) mice suggest that intestinal barrier function may be the dominant factor for survival during natural infection.     

Cardiotrophin-1 is not associated with carotid or coronary disease and is inversely associated with obesity in patients undergoing coronary angiography

Introduction

Cardiotrophin-1 (CT-1) is a member of the interleukin-6 superfamily with known hypertrophic and protective actions upon cardiac myocytes. Although its effects on myocardial tissue and its role in hypertensive heart disease are well documented, there are no studies on CT-1 blood levels in patients with coronary artery disease. In this study we aimed to verify the relationships of serum CT-1 with vascular disease and metabolic parameters in a population of patients undergoing coronary angiography due to clinical indications.

Material and methods

Serum levels of CT-1 were investigated in a cohort of 81 consecutive patients (median age 68 years (95% CI: 64–71), 59 males) undergoing coronary angiography and carotid Doppler ultrasound. Exclusion criteria were: acute coronary syndrome, already-established ischemic cardiopathy, chronic inflammatory diseases and presence or past history of cancer.

Results

Levels of CT-1 were inversely correlated with body mass index (BMI) and waist circumference (WC) (ρ = –0.261, p = 0.02; ρ = –0.224, p = 0.05, respectively). Moreover, obese patients showed significantly lower CT-1 concentrations than non-obese ones (1.18 (0.64–1.64) ng/ml vs. 1.56 (1.37–2.04) ng/ml, p = 0.013), and serum CT-1 was significantly reduced in patients with elevated compared to those with normal WC (1.43 (0.94–1.60) ng/ml vs. 1.64 (1.39–2.49) ng/ml, p = 0.047). Concentrations of CT-1 did not correlate either with the other parameters of metabolic syndrome or with markers of cardiovascular disease (carotid intima-media thickness, presence of carotid or coronary artery plaques).

Conclusions

Our results failed to demonstrate any association between CT-1 and carotid or coronary disease. The inverse association with BMI and WC fits with the latest experimental data on the role of CT-1 in dysmetabolic conditions and could help to further clarify the role of CT-1 in obesity and diabetes.     

The effects of endothelin-1 on degranulation, cytokine, and growth factor production by skin-derived mast cells

Endothelin-1 (ET-1), originally described as a vasoconstrictor, is now known to be involved in pathogenesis of various disorders including vascular, inflammatory, and fibrotic diseases. Recent studies suggest that mast cells are also involved in the same pathological conditions. In this study, we tested a hypothesis that ET-1 would affect mast cell functions and contribute to such disease conditions, using fetal skin-derived cultured mast cells (FSMC) and bone marrow-derived cultured mast cells (BMMC). FSMC expressed ET receptors (ET(A) and ET(B)) at mRNA and protein levels, whereas BMMC expressed lower levels of ET(A), and little, if any, ET(B). ET-1 induced degranulation by FSMC, but not by BMMC through ET(A)-mediated pathways. ET-1 at different concentrations exerted the reciprocal effects on degranulation by IgE-bound FSMC. Furthermore, ET-1 induced TNF-alpha and IL-6 production by FSMC, but not by BMMC, and significantly enhanced VEGF production and TGF-beta1 mRNA expression by FSMC. Finally, ET-1 was produced by FSMC, but not by BMMC in response to Toll-like receptor ligands. These results indicate contrasting impacts of ET-1 on distinct mast cell populations. We propose that ET-1 may participate in pathological conditions of various disorders via its multi-functional effects on mast cells under certain conditions.     

The Listeria monocytogenes lemA gene product is not required for intracellular infection or to activate fMIGWII-specific T cells

Clearance of the intracellular bacterial pathogen Listeria monocytogenes requires antigen-specific CD8(+) T cells. Recently it was shown that activation of class Ib major histocompatibility complex (MHC)-restricted CD8(+) T cells alone is sufficient for immune protection against listeriae. A major component of the class Ib MHC-restricted T-cell response is T cells that recognize formylated peptide antigens presented by M3 molecules. Although three N-formylated peptides derived from L. monocytogenes are known to bind to M3 molecules, fMIGWII is the immunodominant epitope presented by M3 during infection of mice. The source of fMIGWII peptide is the L. monocytogenes lemA gene, which encodes a 30-kDa protein of unknown function. In this report, we describe the generation of two L. monocytogenes lemA deletion mutants. We show that lemA is not required for growth of listeriae in tissue culture cells or for virulence during infection of mice. Surprisingly, we found that fMIGWII-specific T cells were still primed following infection with lemA mutant listeriae, suggesting that L. monocytogenes contains at least one additional antigen that is cross-reactive with the fMIGWII epitope. This cross-reactive antigen appears to be a small protease-resistant molecule that is secreted by L. monocytogenes.     

The effects of food additives and aflatoxin B1 on histamine release from human mast cells

The effects of the food additives tartrazine, biphenyl, sorbic acid and the mycotoxin contaminant aflatoxin B₁ were studied in mechanically isolated human adenoidal mast cells. Tartrazine inhibited the spontaneous histamine release in the concentration range of 10^–9 to 10^–5 M and the concanavalin A (Con A)-induced histamine release dose-dependently at 10^–11–10^–5 M [10.3%–31.6%]. Biphenyl [10^–9–10^–6 M] neither influenced the spontaneous nor the stimulated histamine release. Sorbic acid [10^–7–10^–4 M] slightly inhibited the Con A-induced release at the highest concentration tested. Aflatoxin B₁ [10^–10–10^–7 M] did not influence mediator release after a preincubation time of 5 min. Extension of the preincubation period inhibited the histamine release slightly. In summary, none of the tested substances enhanced histamine release from human adenoidal mast cells. Tartrazine even had an inhibitory effect.     

Trypanosoma brucei DMC1 does not act in DNA recombination, repair or antigenic variation in bloodstream stage cells

Homologous recombination acts in the repair of cellular DNA damage and can generate genetic variation. Some of this variation provides a discrete purpose in the cell, although it can also be genome-wide and contribute to longer-term natural selection. In Trypanosoma brucei, a eukaryotic parasite responsible for sleeping sickness disease in sub-Saharan Africa, homologous recombination acts to catalyse antigenic variation, an immune evasion strategy involving switches in variant surface glycoprotein. In addition, T. brucei can undergo genetic exchange by homologous recombination in the tsetse vector, and some evidence suggests that this occurs by meiosis. Here, we show that T. brucei, Trypanosoma cruzi and Leishmania major each contain a single copy gene whose product is highly related to the eukaryotic meiosis-specific protein Dmc1, which is structurally and functionally related to Rad51. We show that T. brucei DMC1 is transcribed in the bloodstream stage of the parasite, where the gene can be mutated by reverse genetic disruption. DMC1 mutation does not, however, result in detectable alterations in DNA repair, recombination or antigenic variation efficiency in this life cycle stage.     

The histamine H1-receptor antagonist cetirizine does not interact with bradykinin B1 or B2-receptors in vitro

Inflammation Research -     

The effects of adenosine and of some adenosine analogues on the concanavalin A- or acetylcholine-induced histamine release from human adenoidal mast cells

Histamine release induced by concanavalin A (Con A) or acetylcholine is enhanced by adenosine or the adenosine analogues PIA and NECA. The enhancement is not affected by preincubation with adenosine deaminase.The degree of the Con A-induced histamine release decreases with increasing incubation time. Under these conditions, the enhancing effect of adenosine on histamine release is either abolished or even reversed to inhibition.     

The strain difference in the effect of mercuric chloride on antigen-triggered serotonin release from rat mast cells is not mediated via interferon-gamma.

Previous work has shown that in vitro exposure of Brown-Norway (BN) rat peritoneal mast cells to mercuric chloride (HgCl2) causes enhancement of subsequent mediator release induced by cross-linking of surface immunoglobulin E (IgE). This enhancing effect is seen significantly less often with peritoneal cells from Lewis rats. In addition HgCl2 has been shown to suppress interferon (IFN)-gamma production by BN but not Lewis splenocytes. Given that IFN-gamma is known to inhibit mediator release by mast cells, we hypothesized that the strain difference in the effect of HgCl2 on mediator release was mediated via a differential effect on IFN-gamma release from T cells in the mixed peritoneal cell population: IFN-gamma release would be suppressed in the case of the BN rat, releasing the mast cells from inhibition and resulting in the enhancing effect of HgCl2. The aim of the study was to test two predictions of this hypothesis. Exposure of BN rat mast cells to IFN-gamma inhibited subsequent antigen-induced mediator release but did not significantly reduce HgCl2-mediated enhancement of this release. Exposure of Lewis rat mast cells to blocking concentrations of anti-IFN-gamma did not reveal any HgCl2-mediated enhancement of mediator release. These observations provide strong evidence against the hypothesis that the differential effects of HgCl2 on BN and Lewis rat mast cells are mediated via IFN-gamma. In addition the results revealed that BN rat mast cells are significantly more sensitive than Lewis rat mast cells to the inhibitory effects of IFN-gamma on antigen-induced mediator release.     

The rat orthologue to the inhibitory receptor gp49B is expressed by neutrophils and monocytes, but not by NK cells or mast cells

Mouse gp49B is a member of the leukocyte immunoglobulin-like receptor family. It is constitutively expressed by mast cells and certain myeloid cells, and expression can be induced on natural killer (NK) cells and T cells. We have cloned several rat cDNA, 78% identical to mouse gp49B at the amino acid level, that represent the rat orthologue to mouse gp49B. A mouse monoclonal antibody (WEN29) against rat gp49B was generated. By flow cytometry and Northern blot analysis, gp49B was found to be expressed by neutrophils and monocytes, but not NK cells (primary or IL-2-activated), T cells (resting or concanavalin A-stimulated) or peritoneal mast cells. Following pervanadate treatment, the tyrosine phosphatase SHP-1 was co-immunoprecipitated with gp49B in the macrophage cell line R2. In glutathione S-transferase pull-down experiments, the cytoplasmic tail of rat gp49B associated with the SH2 domains of both SHP-1 and SHP-2, dependent on intact and phosphorylated immunoreceptor tyrosine-based inhibition motifs (ITIM). Compared to mouse, the cytoplasmic domain of rat gp49B contains a third ITIM-like sequence (YLYASV) that was phosphorylated by several Src family tyrosine kinases, enhanced the phosphorylation of other ITIM, and bound to the SH2 domains of SHP-2, suggesting a role in the recruitment of downstream phosphatases.     

Gender differences in non-ischemic myocardial remodeling: are they due to estrogen modulation of cardiac mast cells and/or membrane type 1 matrix metalloproteinase

This review is focused on gender differences in cardiac remodeling secondary to sustained increases in cardiac volume (VO) and generated pressure (PO). Estrogen has been shown to favorably alter the course of VO-induced remodeling. That is, the VO-induced increased extracellular matrix proteolytic activity and mast cell degranulation responsible for the adverse cardiac remodeling in males and ovariectomized rodents do not occur in intact premenopausal females. While less is known regarding the mechanisms responsible for female cardioprotection in PO-induced stress, gender differences in remodeling have been reported indicating the ability of premenopausal females to adequately compensate. In view of the fact that, in male mice with PO, mast cells have been shown to play a role in the adverse remodeling suggests favorable estrogen modification of mast cell phenotype may also be responsible for cardioprotection in females with PO. Thus, while evidence is accumulating regarding premenopausal females being cardioprotected, there remains the need for in-depth studies to identify critical downstream molecular targets that are under the regulation of estrogen and relevant to cardiac remodeling. Such studies would result in the development of therapy which provides cardioprotection while avoiding the adverse effects of systemic estrogen delivery.     

The baculovirus anti-apoptotic protein Op-IAP does not inhibit Drosophila caspases or apoptosis in Drosophila S2 cells and instead sensitizes S2 cells to virus-induced apoptosis

The Op-IAP protein from the baculovirus Orgyia pseudotsugata M nucleopolyhedrovirus (OpMNPV) is highly effective at inhibiting apoptosis triggered by a variety of different stimuli in lepidopteran cells as well as in several different mammalian cell types, suggesting that it functions at a highly conserved step in the apoptotic pathway. However, the mechanism by which Op-IAP inhibits apoptosis is unclear. Since some IAP proteins can bind and inhibit caspases, we tested whether Op-IAP could inhibit the activity of caspases from Drosophila melanogaster. We found that recombinant Op-IAP protein was not able to bind or directly inhibit the activity of the Drosophila caspases DRONC, DrICE, or DCP-1 in vitro. In addition, expression of Op-IAP was unable to inhibit apoptosis triggered by either actinomycin D or UV light in D. melanogaster S2 cells. Surprisingly, Op-IAP expression in S2 cells enhanced apoptosis caused by baculovirus infection, but did not cause increased sensitivity to either actinomycin D or UV damage-induced apoptosis. The observation that Op-IAP cannot inhibit these insect caspases suggests that it functions by a mechanism that does not involve direct caspase inhibition.     

The effects of Fructus Xanthii extract on cytokine release from human mast cell line (HMC-1) and peripheral blood mononuclear cells

In the present study, human mast cell line (HMC-1) and peripheral blood mononuclear cells (PBMNC) were pre-incubated with various concentrations of Fructus xanthii extract solution followed by being stimulated with phorbol myristate acetate and further incubated for 24 hours. The cytokines, in cell culture supernatants, of interleukin (IL)-4, IL-6, IL-8, GM-CSF, and TNF-alpha in the supernatant were measured by enzyme-linked immunosorbent assay. The results demonstrated that Fructus xanthii could modulate the mast cell-mediated and PBMNC-mediated inflammatory and immunological reactions modulated by cytokines. And our study supplied the evidence of the mechanisms of Fructus Xanthii in treating inflammatory and allergic diseases.