Activation of E-prostanoid4 and E-prostanoid2 receptors inhibits TNF-alpha release from human alveolar macrophages.

Prostaglandin (PG)E(2) has been shown to inhibit mediator release from human alveolar macrophages (AMs), but the prostanoid receptor(s) mediating this response have not yet been documented. To investigate this, the present authors conducted a range of pharmacological and expression-based studies in monocyte-derived macrophages (MDMs) and AMs. MDMs were obtained by in vitro differentiation of monocytes from the peripheral blood of healthy human volunteers. Human AMs were obtained by perfusion of lung tissue from carcinoma resection patients. In MDMs, PGE(2) potently inhibited lipopolysaccharide-induced tumour necrosis factor (TNF)-alpha release (p[A](50) 8.51+/-0.11, maximum inhibition 95.9+/-4.8%). In human AMs, PGE(2) also inhibited TNF-alpha release but the observed concentration-effect curve was very flat and inhibition was incomplete. The shape of the PGE(2) curve in AMs suggested that its effects were mediated by activation of a heterogeneous receptor population. Expression studies combined with the use of various E-prostanoid (EP) receptor agonists and a selective EP(4)-receptor antagonist (Ono-AE2-227) confirmed that the inhibitory effects of PGE(2) in both AMs and MDMs were mediated by activation of EP(4) and EP(2) receptors. These data indicate that both E-prostanoid(4) and E-prostanoid(2) selective agonists may have anti-inflammatory properties in lung diseases where macrophages play a role.

A positive role for the PP2A catalytic subunit in Wnt signal transduction.

Protein phosphatase-2A (PP2A) is a multisubunit serine/threonine phosphatase involved in intracellular signaling, gene regulation, and cell cycle progression. Different subunits of PP2A bind to Axin and Adenomatous Polyposis Coli, components of the Wnt signal transduction pathway. Using early Xenopus embryos, we studied how PP2A functions in Wnt signal transduction. The catalytic subunit of PP2A (PP2A-C) potentiated secondary axis induction and Siamois reporter gene activation by Dishevelled, a component of the Wnt pathway, indicating a positive regulatory role of this enzyme in Wnt signaling. In contrast, small t antigen, an antagonist of PP2A-C, inhibited Dishevelled-mediated signal transduction, as did the regulatory PP2A-B'epsilon subunit, consistent with the requirement of PP2A function in this pathway. Although Wnt signaling is thought to occur via regulation of beta-catenin degradation, PP2A-C did not significantly affect beta-catenin stability. Moreover, the pathway activated by a stabilized form of beta-catenin was sensitive to PP2A-C and its inhibitors, suggesting that PP2A-C acts downstream of beta-catenin. Because previous work has suggested that PP2A can act upstream of beta-catenin, we propose that PP2A regulates the Wnt pathway at multiple levels.

The chicken B-cell receptor complex and its role in avian B-cell development.

The bursa of Fabricius is critical to normal B-lymphocyte development in birds. During embryonic life, B-cell precursors migrate to the bursal rudiment and those which have undergone productive V(D)J recombination colonize lymphoid follicles and undergo immunoglobulin V gene diversification by gene conversion. The chicken surface IgM complex appears structurally and functionally equivalent to its mammalian counterpart, with homologs to CD79a and CD79b. Expression of a truncated Igmu chain is sufficient to drive the early stages of B-cell development in the embryo bursa. Bursal cells expressing the truncated mu receptor complex proliferate in bursal follicles, and those which contain V gene rearrangements undergo V gene diversification by gene conversion. The bursa is a gut-associated organ and antigen is focused to bursal lymphoid follicles after hatch. While expression of the truncated mu chain is sufficient to support B-cell development in the embryo, B cells expressing this receptor are rapidly eliminated after hatch. We suggest the possibility that B-cell development in the bursa after hatch is driven by encounter with antigen leading to redistribution of B cells within the lymphoid follicle, B-cell proliferation and V gene repertoire development by gene conversion.

Perinatal deletion of B cells expressing surface Ig molecules that lack V(D)J-encoded determinants in the bursa of Fabricius is not due to intrafollicular competition.

During embryonic development, the avian bursa of Fabricius selects B cell precursors that have undergone productive V(D)J recombination for expansion in oligoclonal follicles. During this expansion, Ig diversity is generated by gene conversion. We have used retroviral gene transfer in vivo to introduce surface Ig molecules that lack V(D)J-encoded determinants into B cell precursors. This truncated mu heavy chain supports both B cell expansion within embryo bursal lymphoid follicles and gene conversion. We show that individual follicles can be colonized exclusively by cells expressing the truncated mu chain and lacking endogenous surface IgM, ruling out a requirement for V(D)J-encoded determinants in the establishment of bursal lymphoid follicles. In striking contrast to their normal development in the embryo, bursal cells expressing the truncated mu-chain exhibit reduced rates of cell division and increased levels of apoptosis after hatching. The level of apoptosis in individual follicles reflects the proportion of cells within the follicle that express the truncated mu-chain. In particular, high levels of apoptosis are associated with follicles containing exclusively cells expressing the truncated micro receptor. Thus, apoptotic elimination of such cells is not due to competition within the follicle by cells expressing endogenous surface IgM receptors. This provides the first direct demonstration that the regulation of B cell development in the avian bursa after hatching differs fundamentally from that seen in the embryo. The requirement for intact IgM expression when the bursa is exposed to exogenous Ag implicates a role for Ag in avian B cell development after hatching.

Interaction of dishevelled and Xenopus axin-related protein is required for wnt signal transduction.

Signaling by the Wnt family of secreted proteins plays an important role in animal development and is often misregulated in carcinogenesis. Wnt signal transduction is controlled by the rate of degradation of beta-catenin by a complex of proteins including glycogen synthase kinase 3 (GSK3), adenomatous polyposis coli, and Axin. Dishevelled is required for Wnt signal transduction, and its activation results in stabilization of beta-catenin. However, the biochemical events underlying this process remain largely unclear. Here we show that Xenopus Dishevelled (Xdsh) interacts with a Xenopus Axin-related protein (XARP). This interaction depends on the presence of the Dishevelled-Axin (DIX) domains in both XARP and Xdsh. Moreover, the same domains are essential for signal transduction through Xdsh. Finally, our data point to a possible mechanism for signal transduction, in which Xdsh prevents beta-catenin degradation by displacing GSK3 from its complex with XARP.

Avian B cell development: lessons from transgenic models.

The bursa of Fabricius is critical for the development of B lymphocytes in avian species. Despite considerable advances in our understanding of the molecular mechanisms by which avian antibody diversity is generated, many stages of B-cell development in the bursa and the means by which they are regulated remain unclear. Here we discuss the use of productive chicken retroviral vectors which allow gene transfer in vitro or in vivo as tools to probe the requirements for bursal B-cell development. Expression of a truncated form of bursal cell surface IgM, lacking variable region encoded determinants, is sufficient to promote the initial colonization and clonal expansion of B-cells within the bursa. Expression of this truncated IgM does not, however, protect developing bursal cells against the apoptosis that occurs within the bursa after hatch. Conversely, over-expression of the proto-oncogene bcl-2, following retroviral gene transfer, protects cells against apoptotic cell death but is not sufficient to allow B lineage progression in the absence of sIgM expression. Finally we discuss the use of regulated promoters within the retroviral gene transfer system to show that while bursal cells are susceptible to transformation by the v-rel oncogene in vitro, this oncogene preferentially targets mature peripheral cells in vivo.

Efficient antibody diversification by gene conversion in vivo in the absence of selection for V(D)J-encoded determinants.

Antibody diversification in the bursa of Fabricius occurs by gene conversion: pseudogene-derived sequences replace homologous sequences in rearranged immunoglobulin genes. Bursal cells expressing a truncated immunoglobulin mu heavy chain, introduced by retroviral gene transfer, bypass normal requirements for endogenous surface immunoglobulin expression. Immunoglobulin light chain rearrangements in such cells undergo gene conversion under conditions where the products are not selected based on their ability to encode a functional protein. The efficiency with which gene conversion maintains a productive reading frame exceeds 97% under such non-selective conditions. By analysis of donor pseudogene usage we demonstrate that bursal cell development is not driven by a restricted set of antigenic specificities. We further demonstrate that gene conversion can restore a productive reading frame to out-of-frame VJ(L) junctions, providing a rationale for the elimination of cells containing non-productive VJ(L) rearrangements prior to the onset of gene conversion in normal bursal cell development.

Development of B cells expressing surface immunoglobulin molecules that lack V(D)J-encoded determinants in the avian embryo bursa of fabricius.

Immunoglobulin gene rearrangement in avian B cell precursors generates surface Ig receptors of limited diversity. It has been proposed that specificities encoded by these receptors play a critical role in B lineage development by recognizing endogenous ligands within the bursa of Fabricius. To address this issue directly we have introduced a truncated surface IgM, lacking variable region domains, into developing B precursors by retroviral gene transfer in vivo. Cells expressing this truncated receptor lack endogenous surface IgM, and the low level of endogenous Ig rearrangements that have occurred within this population of cells has not been selected for having a productive reading frame. Such cells proliferate rapidly within bursal epithelial buds of normal morphology. In addition, despite reduced levels of endogenous light chain rearrangement, those light chain rearrangements that have occurred have undergone variable region diversification by gene conversion. Therefore, although surface expression of an Ig receptor is required for bursal colonization and the induction of gene conversion, the specificity encoded by the prediversified receptor is irrelevant and, consequently, there is no obligate ligand for V(D)J-encoded determinants of prediversified avian cell surface IgM receptor.

Dephosphorylation of the catenins p120 and p100 in endothelial cells in response to inflammatory stimuli.

Inflammatory mediators such as histamine and thrombin increase the tight-junction permeability of endothelial cells. Tight-junction permeability may be independently controlled, but is dependent on the adherens junction, where adhesion is achieved through homotypic interaction of cadherins, which in turn are associated with cytoplasmic proteins, the catenins. p120, also termed p120(cas)/p120(ctn), and its splice variant, p100, are catenins. p120, originally discovered as a substrate of the tyrosine kinase Src, is also a target for a protein kinase C-stimulated pathway in epithelial cells, causing its serine/threonine dephosphorylation. The present study shows that pharmacological activation of protein kinase C stimulated a similar pathway in endothelial cells. Activation of receptors for agents such as histamine (H1), thrombin and lysophosphatidic acid in the endothelial cells also caused serine/threonine dephosphorylation of p120 and p100, suggesting physiological relevance. However, protein kinase C inhibitors, although blocking the effect of pharmacological activation of protein kinase C, did not block the effects due to receptor activation. Calcium mobilization and the myosin-light-chain-kinase pathway do not participate in p120/p100 signalling. In conclusion, endothelial cells possess protein kinase C-dependent and -independent pathways regulating p120/p100 serine/threonine phosphorylation. These data describe a new connection between inflammatory agents, receptor-stimulated signalling and pathways potentially influencing intercellular adhesion in endothelial cells.

Ascorbic acid-based inhibitors of alpha-amylases.

A series of ascorbic acid and isoascorbic acid derivatives has been evaluated as inhibitors of malt, bacterial, fungal, pancreatic and salivary alpha-amylases using a simple and quick assay procedure. The results demonstrate that the enediol moiety of ascorbic acid is essential for alpha-amylase inhibition. Acylation of the primary and secondary alcohols, and the absolute configuration of the secondary alcohol, do not affect the potency of inhibition.

Dephosphorylation of the cadherin-associated p100/p120 proteins in response to activation of protein kinase C in epithelial cells.

Protein kinase C signaling pathways have been implicated in the disruption of intercellular junctions, but mechanisms are not clear. p100 and p120 are members of the Armadillo family of proteins and are localized to cellular adherens junctions. In strain I Madin-Darby canine kidney cells, protein kinase C activation leads to disruption of tight junctions and an increase in permeability of cell monolayers. We show that this permeability increase is accompanied by dephosphorylation of p100/p120 on serine and threonine residues. The dephosphorylation of these proteins can also be induced by the kinase inhibitors staurosporine, KT5926, and Gö 6976. Treatment of cells with phosphatase inhibitors induced hyperphosphorylation of p100 and p120. Thus, p100 and p120 participate in a regulatable cycle of serine/threonine phosphorylation and dephosphorylation. Protein kinase C must act, directly or indirectly, by perturbing this phosphorylation cycle, by inhibition of a p100/p120 kinase and/or activation of a phosphatase. These data clearly show that p100 and p120 are targets of a novel protein kinase C signaling pathway. Dephosphorylation of these proteins precedes the permeability increase across epithelial cell monolayers seen in response to phorbol esters, raising the possibility that this pathway may play a role in the modulation of intercellular junctions.

Topological analysis of the Escherichia coli ferrichrome-iron receptor by using monoclonal antibodies.

Ferrichrome-iron transport in Escherichia coli is initiated by the outer membrane receptor FhuA. Thirty-five anti-FhuA monoclonal antibodies (MAbs) were isolated to examine the surface accessibility of FhuA sequences and their contribution to ligand binding. The determinants of 32 of the MAbs were mapped to eight distinct regions in the primary sequence of FhuA by immunoblotting against (i) five internal deletion FhuA proteins and (ii) four FhuA peptides generated by cyanogen bromide cleavage. Two groups of MAbs bound to FhuA in outer membrane vesicles but not to intact cells, indicating that their determinants, located between residues 1 and 20 and 21 and 59, are exposed to the periplasm. One of the 28 strongly immunoblot-reactive MAbs bound to FhuA on intact cells in flow cytometry, indicating that its determinant, located between amino acids 321 and 381, is cell surface exposed. This MAb and four others which in flow cytometry bound to cells expressing FhuA were tested for the ability to block ligand binding. While no MAb inhibited growth promotion by ferrichrome or cell killing by microcin 25, some prevented killing by colicin M and were partially able to inhibit the inactivation of T5 phage. These data provide evidence for spatially distinct ligand binding sites on FhuA. The lack of surface reactivity of most of the immunoblot-reactive MAbs suggests that the majority of FhuA sequences which lie external to the outer membrane may adopt a tightly ordered organization with little accessible linear sequence.

Identification and analysis of the expression of CD8 alpha beta and CD8 alpha alpha isoforms in chickens reveals a major TCR-gamma delta CD8 alpha beta subset of intestinal intraepithelial lymphocytes.

Expression screening has been used to clone cDNAs encoding the alpha- and beta-chains of chicken CD8. Amino acid sequence similarities with the mammalian sequences were about 30%. Many amino acid residues of structural or functional importance were more highly conserved, as were the overall structures of both chains. Like human CD8 alpha, the chicken alpha-chain lacked sites for N-linked glycosylation, but the beta-chain contained three such sites. In COS cells transfected with CD8 beta cDNA, surface expression of the beta-chain was dependent on co-transfection of the alpha-chain cDNA, indicating that, as in mammals, chicken CD8 can be expressed as a CD8 alpha alpha homodimer or as a CD8 alpha beta heterodimer. Immunofluorescence analysis with mAbs that were shown to identify the CD8 alpha- and CD8 beta-chains revealed that the vast majority of the CD8+ cells in the thymus, spleen, and blood of adult chickens express both CD8 alpha- and CD8 beta-chains. However, a relatively large proportion of the CD8+ TCR-gamma delta cells in the spleens of embryos and young chicks express only the alpha-chain of CD8. Among intestinal epithelial lymphocytes the major CD8+ T cell populations present in mice are conserved, but there is a population of TCR-gamma delta CD8 alpha beta cells that is not found in rodents. This observation is important in interpretation of experiments examining the pathways of development of intestinal intraepithelial lymphocytes in chickens.

Loss of surface immunoglobulin expression precedes B cell death by apoptosis in the bursa of Fabricius.

The vast majority of lymphocytes generated daily in the chicken bursa of Fabricius do not emigrate to the periphery but die in situ. Apoptotic cells in the bursa can be readily detected by the presence of fragmented DNA and by the large numbers of condensed cellular nuclei observed by electron microscopy. Consequently, most newly generated lymphocytes die by programmed cell death. We show that bursal cells divide rapidly and apoptotic cells are derived from rapidly dividing precursors. Analysis of the phenotype of bursal cells undergoing apoptosis demonstrated that cell death does not occur in the most mature bursal cell population and is therefore not random. High levels of surface Ig are expressed on bursal cells entering S phase of the cell cycle. In contrast, bursal cells in the early stages of apoptosis in vivo express very low to undetectable levels of surface Ig but were unequivocally confirmed as being of the B lineage by polymerase chain reaction (PCR) detection of rearranged Ig genes. Bursal cells induced to undergo apoptosis in vitro express high levels of surface Ig demonstrating that induction of apoptosis does not in itself induce a loss of surface Ig expression. Consequently, loss of surface Ig expression precedes bursal cell death by apoptosis in vivo, suggesting that maintenance of a threshold level of surface Ig may be a requirement for the continued progression of chicken B lymphocyte development in the bursa.

Survivors of bursal B cell production and emigration.

The bursa of Fabricius is the site of primary B cell generation in the chicken. Analysis of the rate of bursal cell emigration demonstrated that about 1% of the blood B cell pool was replaced per hour by bursal emigrants. Surgical bursectomy distinguished three populations of blood B cells. About 60% of blood B cells in 3-wk-old chickens were short lived, with a lifespan of 2 to 3 d, and included most bursal emigrants. These cells migrate directly from the bursal follicular cortex to the periphery, express the LT2 antigen, and are proposed to represent a diversified repertoire of B cell specificities that have emigrated from the bursa in the absence of interaction with environmentally derived antigens in the follicular medulla. About 35% of blood B cells were much longer lived cells, having a lifespan exceeding 2 wk. These cells represent about 10% of bursal emigrants, which do not express the LT2 antigen and do not divide in the peripheral blood following emigration from the bursa. This population may represent cells that have undergone positive selection in the bursa by interacting with environmentally derived antigens and should, therefore, contain a restricted repertoire of B cell specificities. The third population, about 5% of blood B cells, were short-lived cells that represent the progeny of postbursal B cell production. The frequency of these cells progressively increases with time, taking over from the short-lived bursal emigrants as the bursa involutes, likely as a diversified repertoire of B cell specificities.

Genetic insertion and exposure of a reporter epitope in the ferrichrome-iron receptor of Escherichia coli K-12.

The ferrichrome-iron receptor of Escherichia coli K-12 is FhuA (M(r), 78,992), the first component of an energy-dependent, high-affinity iron uptake pathway. FhuA is also the cognate receptor for bacteriophages T5, T1, phi 80, and UC-1, for colicin M and microcin 25, and for albomycin. To probe the topological organization of FhuA which enables recognition of these different ligands, we generated a library of 16 insertion mutations within the fhuA gene. Each insertion spliced a 13-amino-acid antigenic determinant (the C3 epitope of poliovirus) at a different position within FhuA. Immunoblotting of outer membranes with anti-FhuA and anti-C3 antibodies indicated that 15 of 16 FhuA.C3 proteins were present in the outer membrane in amounts similar to that observed for plasmid-encoded wild-type FhuA. One chimeric protein with the C3 epitope inserted after amino acid 440 of FhuA was present in the outer membrane in greatly reduced amounts. Strains overexpressing FhuA.C3 proteins were subjected to flow cytometric analysis using anti-FhuA monoclonal antibodies. Such analysis showed that (i) the chimeric proteins were properly localized and (ii) the wild-type FhuA protein structure had not been grossly altered by insertion of the C3 epitope. Twelve of sixteen strains expressing FhuA.C3 proteins were proficient in ferrichrome transport and remained sensitive to FhuA-specific phages. Three FhuA.C3 proteins, with insertions after amino acid 321, 405, or 417 of FhuA, were detected at the cell surface by flow cytometry using anti-C3 antibodies. These three chimeric proteins were all biologically active. We conclude that amino acids 321, 405, and 417 are surface accessible in wild-type FhuA.

Rearrangement of immunoglobulin genes in chicken B cell development.

Immunoglobulin gene rearrangement in the chicken has evolved not to generate antibody diversity per se but to generate an immunoglobulin variable region which can be diversified by subsequent somatic gene conversion events. While the molecular mechanism of V(D)J recombination in chickens cannot be distinguished from that seen in other species, the way in which this recombination is regulated during chicken B lymphocyte development does differ from the more widely known models of gene rearrangement in humans and rodents. In this review we focus on these differences, relating V(D)J recombination to the progression of chicken B cell development in the bursa of Fabricius.