Characterization of the necrotic protein that regulates the Toll-mediated immune response in Drosophila

Necrotic (Nec) is an important component of the proteolytic cascade that activates the Toll-mediated immune response in Drosophila. The Nec protein is a member of the serpin (SERine Protease INhibitor) superfamily and is thought to regulate the cascade by inhibiting the serine protease Persephone. Nec was expressed in Escherichia coli, and the purified protein folded to the active native conformation required for protease inhibitory activity. Biochemical analysis showed that Nec had a broad inhibitory specificity and inhibited elastase, thrombin, and chymotrypsin-like proteases. It did not inhibit trypsin or kallikrein. These data show that Necrotic is likely to inhibit a wide range of proteases in Drosophila and that Nec has the specificity requirements to act as the physiological inhibitor of Persephone in vivo.     

Microgravity modifies protein kinase C isoform translocation in the human monocytic cell line U937 and human peripheral blood T-cells

Individual protein kinase C (PKC) isoforms fulfill distinct roles in the regulation of the commitment to differentiation, cell cycle arrest, and apoptosis in both monocytes and T-cells. The human monocyte like cell line U937 and T-cells were exposed to microgravity, during spaceflight and the translocation (a critical step in PKC signaling) of individual isoforms to cell particulate fraction examined. PKC activating phorbol esters induced a rapid translocation of several PKC isoforms to the particulate fraction of U937 monocytes under terrestrial gravity (1 g) conditions in the laboratory. In microgravity, the translocation of PKC beta II, delta, and epsilon in response to phorbol esters was reduced in microgravity compared to 1 g, but was enhanced in weak hypergravity (1.4 g). All isoforms showed a net increase in particulate PKC following phorbol ester stimulation, except PKC delta which showed a net decrease in microgravity. In T-cells, phorbol ester induced translocation of PKC delta was reduced in microgravity, compared to 1 g, while PKC beta II translocation was not significantly different at the two g-levels. These data show that microgravity differentially alters the translocation of individual PKC isoforms in monocytes and T-cells, thus providing a partial explanation for the modifications previously observed in the activation of these cell types under microgravity.     

Erythroid expression of the human alpha-spectrin gene promoter is mediated by GATA-1- and NF-E2-binding proteins

alpha-Spectrin is a highly expressed membrane protein critical for the flexibility and stability of the erythrocyte. Qualitative and quantitative defects of alpha-spectrin are present in the erythrocytes of many patients with abnormalities of red blood cell shape including hereditary spherocytosis and elliptocytosis. We wished to determine the regulatory elements that determine the erythroid-specific expression of the alpha-spectrin gene. We mapped the 5' end of the alpha-spectrin erythroid cDNA and cloned the 5' flanking genomic DNA containing the putative alpha-spectrin gene promoter. Using transfection of promoter/reporter plasmids in human tissue culture cell lines, in vitro DNase I footprinting analyses, and gel mobility shift assays, an alpha-spectrin gene erythroid promoter with binding sites for GATA-1- and NF-E2-related proteins was identified. Both binding sites were required for full promoter activity. In transgenic mice, a reporter gene directed by the alpha-spectrin promoter was expressed in yolk sac, fetal liver, and erythroid cells of bone marrow but not adult reticulocytes. No expression of the reporter gene was detected in nonerythroid tissues. We conclude that this alpha-spectrin gene promoter contains the sequences necessary for low level expression in erythroid progenitor cells.     

hINADl/PATJ, a homolog of discs lost, interacts with crumbs and localizes to tight junctions in human epithelial cells

dCrumbs is an apical organizer crucial for the maintenance of epithelial polarity in Drosophila (1). It is known that dCrumbs interacts with Discs lost (Dlt), a protein with four PDZ (PSD95/Discs Large/ZO-1) domains (2), and Stardust (Sdt), a protein of the MAGUK (membrane-associated guanylate kinase) family (3, 4). We have searched for potential homologs of Dlt in human epithelial cells and characterized one of them in intestinal epithelial cells. Human INAD-like (hINADl) contains 8 PDZ domains, is concentrated in tight junctions, and is also found at the apical plasma membrane. Overexpression of hINADl disrupted the tight junctions localization of ZO-1 and 3. We also identified a partial cDNA coding the transmembrane and cytoplasmic domains of a new human crumbs (CRB3) expressed in Caco-2 cells. This CRB3 was able to interact through its C-terminal end with the N-terminal domain of hINADl. Taken together, the data indicate that hINADl is likely to represent a Dlt homolog in mammalian epithelial cells and might be involved in regulating the integrity of tight junctions. We thus propose to rename hINADl PATJ for protein associated to tight junctions.     

Extracellular mRNA induces dendritic cell activation by stimulating tumor necrosis factor-alpha secretion and signaling through a nucleotide receptor

We previously demonstrated that dendritic cell (DC) pulsing with antigen-encoded mRNA resulted in the loading of both major histocompatibility complex class I and II antigen presentation pathways and the delivery of an activation signal. Coculture of mRNA-pulsed DC with T cells led to the induction of a potent primary immune response. DC, in addition to recognizing foreign antigens through pattern recognition receptors, also must respond to altered self, transformed, or intracellularly infected cells. This occurs through cell surface receptors that recognize products of inflammation and cell death. In this report, we characterize two signaling pathways utilized by extracellular mRNA to activate DC. In addition, a novel ligand, poly(A), is identified that mediates signaling through a receptor that can be inhibited by pertussis toxin and suramin and can be desensitized by ATP and ADP, suggesting a P2Y type nucleotide receptor. The role of this signaling activity in vaccine design and the potential effect of mRNA released by damaged cells in the induction of immune responsiveness is discussed.     

TNF-mediated toxicity after massive induction of specific CD8+ T cells following immunization of mice with a tumor-specific peptide

We immunized mice with antigenic peptide P815E, which is presented by H-2K(d) and recognized by tumor-specific CTL raised against P815 tumor cells. This peptide is encoded by the ubiquitously expressed gene MsrA and carries a mutated residue conferring tumor specificity. Unexpectedly, we observed a severe toxicity occurring in the early hours after the third injection, resulting in the death of most mice within 24 h. The toxic syndrome was reminiscent of TNF-induced shock, and the sera of ill mice contained high levels of TNF. Toxicity was prevented by injection of neutralizing anti-TNF Abs, confirming the involvement of TNF. Depletion of CD8+ T cells could also prevent toxicity, and ex vivo experiments confirmed that CD8+ lymphocytes were the major cellular source of TNF in immunized mice. Tetramer analysis of the lymphocytes of immunized mice indicated a massive expansion of P815E-specific T cells, up to >60% of circulating CD8+ lymphocytes. A similar toxicity was observed after massive expansion of specific CD8+ T cells following immunization with another P815 peptide, which is encoded by gene P1A and was injected in a form covalently linked to an immunostimulatory peptide derived from IL-1. We conclude that the toxicity is caused by specific CD8+ lymphocytes, which are extensively amplified by peptide immunization in a QS21-based adjuvant and produce toxic levels of TNF upon further stimulation with the peptide. Our results suggest that immunotherapy trials involving new peptides should be pursued with caution and should include a careful monitoring of the T cell response.     

IFN-gamma-induced apoptosis and microbicidal activity in monocytes harboring the intracellular bacterium Coxiella burnetii require membrane TNF and homotypic cell adherence

IFN-gamma is critical for the protection against intracellular bacteria through activation of the antimicrobial machinery of phagocytes. Coxiella burnetii, the etiological agent of Q fever, is a strictly intracellular bacterium that inhabits monocytes/macrophages. We previously showed that IFN-gamma induced C. burnetii killing by promoting the apoptosis of infected monocytes. We show in this study that IFN-gamma-induced apoptosis of infected monocytes was characterized by a time- and dose-dependent activation of caspase-3. IFN-gamma-mediated caspase-3 activation and C. burnetii killing depend on the expression of membrane TNF. Indeed, TNF was transiently expressed on the cell surface of infected monocytes a few hours after IFN-gamma treatment. In addition, anti-TNF Abs inhibited IFN-gamma-mediated caspase-3 activation whereas soluble TNF had no effect on infected cells. Concomitantly, IFN-gamma induced homotypic adherence of C. burnetii-infected monocytes. The latter required the interaction of beta(2) integrins with CD54. When adherence was disrupted by pipetting, by a combination of Abs specific for CD11b, CD18, and CD54, or by an antisense oligonucleotide targeting CD18 mRNA, both cell apoptosis and bacterial killing induced by IFN-gamma were inhibited. Thus, adherence via CD54/beta(2) integrins together with membrane TNF are required to eliminate C. burnetii-infected cells through cell contact-dependent apoptosis. Our results reveal a new component of the antimicrobial arsenal mobilized by IFN-gamma against infection by intracellular bacteria.     

A model for the enhancement of fitness in cyanobacteria based on resonance of a circadian oscillator with the external light-dark cycle

Fitness enhancement based on resonating circadian clocks has recently been demonstrated in cyanobacteria [Ouyang et al. (1998). Proc. Natl Acad. Sci. U.S.A.95, 8660-8664]. Thus, the competition between two cyanobacterial strains differing by the free-running period (FRP) of their circadian oscillations leads to the dominance of one or the other of the two strains, depending on the period of the external light-dark (LD) cycle. The successful strain is generally that which has an FRP closest to the period of the LD cycle. Of key importance for the resonance phenomenon are observations which indicate that the phase angle between the circadian oscillator and the LD cycle depends both on the latter cycle's length and on the FRP. We account for these experimental observations by means of a theoretical model which takes into account (i) cell growth, (ii) secretion of a putative cell growth inhibitor, and (iii) the existence of a cellular, light-sensitive circadian oscillator controlling growth as well as inhibitor secretion. Building on a previous analysis in which the phase angle was considered as a freely adjustable parameter [Roussel et al. (2000). J. theor. Biol.205, 321-340], we incorporate into the model a light-sensitive version of the van der Pol oscillator to represent explicitly the cellular circadian oscillator. In this way, the model automatically generates a phase angle between the circadian oscillator and the LD cycle which depends on the characteristic FRP of the strain and varies continuously with the period of the LD cycle. The model provides an explanation for the results of competition experiments between strains of different FRPs subjected to entrainment by LD cycles of different periods. The model further shows how the dominance of one strain over another in LD cycles can be reconciled with the observation that two strains characterized by different FRPs nevertheless display the same growth kinetics in continuous light or in LD cycles when present alone in the medium. Theoretical predictions are made as to how the outcome of competition depends on the initial proportions and on the FRPs of the different strains. We also determine the effect of the photoperiod and extend the analysis to the case of a competition between three cyanobacterial strains.     

Morphology and ultrastructure of paired prototergal glands in the adult rove beetle Philonthus varians (Coleoptera, Staphylinidae)

Philonthus and other genera of Philonthina possess a pair of prototergal glands located in the first abdominal tergum and hidden at rest by hind wings and elytra. In Philonthus varians they occupy the whole length of the tergum and form a pouch-like invaginated reservoir with a scaly glandular zone and a smooth outlet. A grille of long setae covers the opening of each gland. The fine structure of these glands is given for the first time. Three types of cells are found in the glandular epithelium. Epidermal cells underlie the cuticular scales, numerous class 1 secretory cells open in the centre of calyces made of finger-like processes of the cuticle, and class 3 cells are connected to pored tubercles. A cytological comparison is made with the diverse class 1 cells described to date in Coleoptera. In these cells different evolutionary trends are shown in the structure of the cuticular apparatus, particularly in the number, size and position of the cuticular apertures as well as in the length and abundance of epicuticular filaments. A possible defensive function of the prototergal glands against pathogens and their interest for the phylogenetic study of Staphylininae are discussed.