Inflammasome-dependent IL-1ß release depends upon membrane permeabilisation.

Interleukin-1ß (IL-1ß) is a critical regulator of the inflammatory response. IL-1ß is not secreted through the conventional ER-Golgi route of protein secretion, and to date its mechanism of release has been unknown. Crucially, its secretion depends upon the processing of a precursor form following the activation of the multimolecular inflammasome complex. Using a novel and reversible pharmacological inhibitor of the IL-1ß release process, in combination with biochemical, biophysical, and real-time single-cell confocal microscopy with macrophage cells expressing Venus-labelled IL-1ß, we have discovered that the secretion of IL-1ß after inflammasome activation requires membrane permeabilisation, and occurs in parallel with the death of the secreting cell. Thus, in macrophages the release of IL-1ß in response to inflammasome activation appears to be a secretory process independent of nonspecific leakage of proteins during cell death. The mechanism of membrane permeabilisation leading to IL-1ß release is distinct from the unconventional secretory mechanism employed by its structural homologues fibroblast growth factor 2 (FGF2) or IL-1α, a process that involves the formation of membrane pores but does not result in cell death. These discoveries reveal key processes at the initiation of an inflammatory response and deliver new insights into the mechanisms of protein release.

P2X(7) receptor activation enhances SK3 channels- and cystein cathepsin-dependent cancer cells invasiveness.

ATP-gated P2X(7) receptors (P2X(7)R) are unusual plasma membrane ion channels that have been extensively studied in immune cells. More recently, P2X(7)R have been described as potential cancer cell biomarkers. However, mechanistic links between P2X(7)R and cancer cell processes are unknown. Here, we show, in the highly aggressive human breast cancer cell line MDA-MB-435s, that P2X(7) receptor is highly expressed and fully functional. Its activation is responsible for the extension of neurite-like cellular prolongations, of the increase in cell migration by 35% and in cell invasion through extracellular matrix by 150%. The change in cancer cell morphology and the increased migration appeared to be due to the activation of Ca(2+)-activated SK3 potassium channels. The enhanced invasion through the extracellular matrix was related to the increase of mature forms of cysteine cathepsins in the extracellular medium, which was independent of SK3 channel activity and not associated with cell death. Pharmacological targeting of P2X(7)R in vivo was crucial for cell invasiveness in a zebrafish model of metastases. Our results demonstrate a novel mechanistic link between P2X(7)R functionality in cancer cells and invasiveness, a key parameter in tumour growth and in the development of metastases. They also suggest a potential therapeutic role for the newly developed P2X(7)R antagonists.

Intestinal secretory and absorptive function in Trichinella spiralis mouse model of postinfective gut dysfunction: role of bile acids.

OBJECTIVE: Observations showing that bile acid malabsorption is frequent in irritable bowel syndrome (IBS) suggest that alterations in bile acid-induced secretion and absorption could contribute to IBS-associated diarrhoea. The secretory response to bile acids, fluid transport and bile absorption was examined in intestinal tissues from a Trichinella spiralis mouse model of postinfectious gut dysfunction in vitro. Changes in the protein expression of apical sodium-dependent bile acid transporter (ASBT) were also measured. DESIGN: T. spiralis-infected mice were killed at 18 and 25 days postinfection. Jejunal, ileal, proximal and distal colon segments were exposed to taurodeoxycholic acid (TDCA) or cholic acid. Short circuit current (SCC) increases were determined. Tritiated taurocholic acid (3H-TCA) absorption was determined in everted jejunal and ileal sacs. ASBT protein expression was determined by Western blot analysis and immunohistochemistry. RESULTS: Basal SCC increased in ileum and distal colon at 18 and 25 days postinfection, respectively. Ileal SCC responses to TDCA and cholic acid were enhanced at 18 days postinfection. Distal colon SCC response to TDCA was raised at 18 days postinfection but was significantly reduced by 25 days. Ileal 3H-TCA uptake was significantly reduced at 18 and 25 days postinfection. Surprisingly, increased ASBT expression was observed in infected animals. CONCLUSIONS: In a T. spiralis model of postinfectious gut dysfunction, decreased bile absorption and enhanced secretion in response to bile acids was observed. Decreased absorption was not, however, caused by decreased ASBT as increased expression was observed. If similar events occur postinfection, the combined effects of these disturbances may contribute to some symptoms observed in postinfectious IBS patients.

[Blood screening of glucose and lipid disorders as coronary atherosclerosis risks factors in French adolescents 16 to 19-20 years old]. / Dépistage des anomalies sanguines glucidolipidiques à risque coronarien, de 16 à 19-20 ans. A propos d'une enquête menée chez 599 jeunes volontaires.

A screening of fasting blood glucose and lipids disorders, presumely linked to premature atherosclerosis namely affecting Coronary arteries, has been performed among 599 adolescents of both sexes with the goal of establishing the actual prevalence of these disorders in French population recruited through different areas of the country. All of them were between ages of 16 and 19-20 years old, and invited to give, in total gratuity, their blood samples to private and accreditable laboratories close to their living habitation. After 262 exclusions due to either previous screening not signaled before or present use of contraceptive pill in girls, only 202 boys and 135 girls remained eligible for such a prevalence study. Using plasma enzymatic dosages of CT, HDL C, (calculated) LDLC, TG, and blood glucose, cut off points for each of these parameters, were analysed as well as calculated international index of CT/HDLC and CT minus HDLC. But the first one index was shown the best tool for the final estimation of the frequency of lipid disorders, which requires primary prevention. Indeed, despite of a rather high frequency of overlaps of CT and LDLC respectively found at 16.3 and 22.5% for boys, and 27.3 and 27.5% for girls, the still higher increase of frequency of HDL C at 31% for boys and 28.1% for girls has shown a very significant compensation of these previous increases. In such a way as the authentic prevalence of atherogenic lipid disorders is found reduced in boys to 8.4% and in girls to 7.4% for CT/HDLC>/=4.5 ratio, and to 5.4% in boys and 5.2% in girls for CT less HDLC. A Familial Dominant Hypercholesterolemia was discovered only two times in two girls 16 years old. Other abnormal lipid profiles were rather those of Mixed H., type IV, chiefly mild Hypercholesterolemia, and some rare cases of HypoHDLemia. The only greater linked cardiovascular risk factor was direct parental C.V. heredity, round 30% among lipid disorders. Obesity remained rare, as well as Metabolic Syndrome in the present recruitment. Contraceptive pill increases significantly all lipid parameters and atherogenic index: chiefly CT minus HDLC which reaches almost the double of frequency (15%) versus that of girls without pill. But 53% of boys with proatherogenic lipid disorders are smokers, while only 10% of these dyslipidemic girls smoke.

Identification of Thr283 as a key determinant of P2X7 receptor function.

BACKGROUND AND PURPOSE: The ATP-gated P2X(7) receptor is an unusual ion channel that couples to multiple downstream signalling cascades. We noted differences in mouse cDNA sequences that may indicate polymorphisms; the aim of this study was to compare function and expression of these mouse P2X(7) receptor mutations. EXPERIMENTAL APPROACH: There are three differences in the sequences of P2X(7) cDNA cloned from mouse NTW8 microglial cells or C57 BL/6 mice: [Phe(11),Ala(221),Met(283)]P2X(7) in the former and [Leu(11),Thr(221),Thr(283)]P2X(7) in the latter. We expressed these receptors and measured membrane currents, ethidium uptake, calcium influx and surface membrane expression. We also carried out these assays on the previously described polymorphism observed between C57 BL/6 and Balb/c mice ([Leu(451)]P2X(7) vs [Pro(451)]P2X(7)). KEY RESULTS: Maximum current densities at [Phe(11),Ala(221),Met(283)]P2X(7) were <12% of those at [Leu(11),Thr(221),Thr(283)]P2X(7) without change in the agonist concentration-response. Replacing methionine with threonine at residue 283 yielded a receptor whose properties were the same as [Leu(11),Thr(221),Thr(283)]P2X(7). Replacing T283 in the rat P2X(7) receptor with methionine yielded currents that were <10% of wildtype and no ethidium uptake was associated with its activation. Maximum current densities and agonist EC(50) values were the same at mouse [Thr(283),Leu(451)]P2X(7) and [Thr(283),Pro(451)]P2X(7) but ethidium uptake and Fluo4 fluorescence were significantly reduced at the [Thr(283),Leu(451)]P2X(7) receptor. There was equivalent surface membrane expression of all P2X(7) receptors. CONCLUSIONS: This study has revealed a residue (Thr(283)) in the ectodomain that is critical for P2X(7) receptor function and suggests that the intracellular residue 451 alters downstream signalling independently of ion channel activity.

The tumor necrosis factor alpha of the bony fish seabream exhibits the in vivo proinflammatory and proliferative activities of its mammalian counterparts, yet it functions in a species-specific manner.

Information on the bioactivities of non-mammalian cytokines is scant due to the lack of the recombinant molecules and specific antibodies. We produced the mature predicted peptide of tumor necrosis factor alpha (TNF alpha) from the bony fish gilthead seabream (Sparus aurata L.) (sbTNF alpha), and its biological role was determined in vitro and in vivo. We first demonstrated by analytical size-exclusion chromatography that sbTNF alpha is an oligomeric protein but the dimer appears to predominate over the trimeric form, in contrast to mammalian TNF alpha. Intraperitoneal injection of native sbTNF alpha resulted in (i) priming of the respiratory burst of the peritoneal exudate and head-kidney (HK) leukocytes, the latter being the bone marrow equivalent in fish; (ii) rapid recruitment of phagocytic granulocytes to the injection site, and (iii) induction of granulopoiesis in the HK. Interestingly, sbTNF alpha was able to induce a strong proliferation of HK cells in vitro, whereas human TNF alpha did not. Conversely, sbTNF alpha was not cytotoxic for murine L929 fibroblasts.

A role for acidophilic granulocytes in the testis of the gilthead seabream (Sparus aurata L., Teleostei).

In mammals, a complex interaction between the immune and the reproductive systems has been described, in which testicular immune cells produce cytokines and growth factors which modulate gonad functions, while specific gonad cells influence the immune response in this organ. In this study we describe the presence of acidophilic granulocytes in the testis of the hermaphrodite teleost fish gilthead seabream (Sparus aurata L.) by using a specific monoclonal antibody. During the post-spawning stage of the testis, this cell type appears in the germinal compartment, accumulates interleukin (IL)-1beta and does not seem to be involved in the phagocytosis of degenerating cells. Moreover, in vitro, 11-ketotestosterone and 17beta-oestradiol, the principal fish sexual steroids, regulate the respiratory burst activity of acidophilic granulocytes obtained from the head-kidney (the bone marrow equivalent in fish) and the intracellular accumulation of IL-1beta by these cells. It is likely, therefore, that IL-1beta produced by testicular acidophilic granulocytes regulates important functions of the testis in fish.

Characterisation of gilthead seabream acidophilic granulocytes by a monoclonal antibody unequivocally points to their involvement in fish phagocytic response.

The various cell types involved in fish phagocytic defence have not been properly established because of the morphological heterogeneity of leucocytes and the lack of appropriate cell-surface markers. In this study, we report the production and characterisation of a monoclonal antibody, G7, which specifically recognises gilthead seabream acidophilic granulocytes, as assayed by immunofluorescence and immunoelectron microscopy. The antibody reacted with 40%-50% of head-kidney and peritoneal exudate leucocytes and 10%-20% of spleen and peripheral blood leucocytes. More importantly, G7(+) acidophils constituted 85% of the head-kidney leucocytes showing phagocytic activity towards the fish pathogenic bacterium Vibrio anguillarum. The results are discussed in relation to the role played by this cell type in fish immune responses.

Interleukin-1beta isolated from a marine fish reveals up-regulated expression in macrophages following activation with lipopolysaccharide and lymphokines.

The gilthead seabream IL-1beta gene consists of five exons/four introns. The complete coding sequence contains a 102 bp 5' untranslated region (UTR), a single open reading frame of 762 bp which translates into a 253 amino acid molecule, and a 407 bp 3'UTR with a polyadenylation signal 14 nucleotides upstream of the poly(A)tail. The seabream sequence has the highest degree of nucleotide (61.7%) and amino acid (53%) identity with the trout IL-1beta sequences. The IL-1beta message was detected by RT-PCR in head-kidney, blood, spleen, liver, gill and peritoneal exudate of both non-infected and Vibrio anguillarum-challenged fish. More importantly, IL-1beta was highly expressed by purified macrophage monolayers and was up-regulated by lipopolysaccharide and lymphocyte-derived macrophage-activating factor stimulation.

A fish cell surface receptor defined by a mAb mediates leukocyte aggregation and deactivation.

Cell adhesion molecules play a key role in the inflammatory response. Selectins, integrins and immunoglobulin gene superfamily adhesion receptors mediate the different steps of leukocyte migration from the blood-stream towards inflammatory foci. In addition to their adhesive function, these receptors modulate major cellular processes such as cell activation, growth, differentiation and death. To characterise the fish molecules involved in cell adhesion, a panel of mAbs was raised by immunising mice with macrophages from the marine fish gilthead seabream (Sparus aurata L.). One of these mAbs, which we named anti-Aggregatin, was found to induce a rapid heterotypic aggregation of seabream leukocytes. Anti-Aggregatin defined a 140-kDa cell surface receptor which was highly expressed by macrophages and was up-regulated after co-stimulation with LPS and MAF. Functionally, the cell adhesion which occurred upon exposure to anti-Aggregatin required Ca(2+), an intact cytoskeleton and an active cell metabolism. More importantly, Aggregatin engagement resulted in strong inhibition of the phagocyte respiratory burst, although the cells showed neither loss of viability nor DNA fragmentation. The results are discussed in relation to the potential role of cell adhesion molecules in fish immune responses.

Phylogeny of cytokines: molecular cloning and expression analysis of sea bass Dicentrarchus labrax interleukin-1beta.

In this paper the cloning of interleukin-1beta (IL-1beta) from the fish Dicentrarchus labrax (sea bass) is described. Using degenerate primers designed from known IL-1beta sequences, a cDNA fragment was amplified by PCR and elongated by 3' and 5' RACE to give the full-length coding sequence for sea bass IL-1beta. The cDNA is 1292 bp, lacks a putative ICE cut site, and codes for a deduced peptide of 29.4 kDa with a pI of 5.1. Sequence analysis showed highest amino acid similarity with rainbow trout (62%), Xenopus (46%), and carp (45.5%) IL-1beta sequences. Expression studies show that sea bass IL-1beta can be upregulated by bacterial lipopolysaccharide both in vitro and in vivo in leucocytes from blood, head-kidney, spleen, gills and liver, whereas the IL-1beta transcript was not detectable in thymus and gut-associated lymphoid tissue. Northern blot analysis with head-kidney leucocyte RNA showed a main LPS-upregulated band at 1.3 kb, and two minor bands at 0.9 and 3.0 kb, respectively. Phylogenetic comparisons with IL-1beta from other vertebrates is presented.