Human mast cells express multiple EP receptors for prostaglandin E2 that differentially modulate activation responses.

Prostaglandin E2 (PGE2) blocks mast-cell (MC)-dependent allergic responses in humans but activates MCs in vitro. We assessed the functions of the EP receptors for PGE2 on cultured human MCs (hMCs). hMCs expressed the EP3, EP2, and EP4 receptors. PGE2 stimulated the accumulation of cyclic adenosine monophosphate (cAMP), and suppressed both Fc epsilonRI-mediated eicosanoid production and tumor necrosis factor-alpha (TNF-alpha) generation. PGE2 also caused phosphorylation of extracellular signal-regulated kinase (ERK), exocytosis, and production of prostaglandin D2 (PGD2), as well as leukotriene C4 (LTC4) when protein kinase A (PKA) was inhibited. An EP3 receptor-selective agonist, AE-248, mimicked PGE2-mediated ERK phosphorylation, exocytosis, and eicosanoid formation. Selective agonists of both EP2 and EP4 receptors (AE1-259-01 and AE-329, respectively) stimulated cAMP accumulation. No selective agonist, alone or in combination, was as effective as PGE2. AE-248, AE1-259-01, and AE-329 all inhibited Fc epsilonRI-mediated TNF-alpha generation, while AE1-259-01 blocked eicosanoid production. PGE2 caused the expression of inducible cAMP early repressor (ICER) by a pathway involving PKA and ERK. Thus, while PGE2 activates MCs through EP3 receptors, it also counteracts Fc epsilonRI-mediated eicosanoid production through EP2 receptors and PKA, and blocks cytokine transcription. These functions explain the potency of PGE2 as a suppressor of early- and late-phase allergic responses.

Lysophosphatidic acid accelerates the development of human mast cells.

Mast cells (MCs) initiate immune responses from mucosal surfaces and perivascular spaces. Stem cell factor (SCF) regulates MC development and viability, but the role of innate serum factors in MC development is unexplored. Cultured cord blood-derived human MCs (hMCs) express mRNA transcripts for all 4 known receptors for lysophosphatidic acid (LPA), an abundant serum-associated lipid growth factor. In an SCF-dependent serum-free culture system, LPA (2.5-10 microM) increased the total number of hMCs by approximately 10-fold compared with cultures maintained in the absence of LPA under otherwise identical conditions. LPA was comitogenic with SCF but did not prolong MC survival. LPA-mediated proliferation was blocked by VPC-32179, a competitive antagonist of LPA(1) and LPA(3) receptors, and by pertussis toxin, and it was also attenuated by GW9662, a selective antagonist of peroxisome proliferator-activated receptor (PPAR)-gamma. LPA accelerated the acquisition of hMC granules and increased Kit expression. hMCs derived in the presence of LPA were functional, as evidenced by their immunoglobulin E (IgE)-dependent histamine release and by their characteristic proliferative responses to interleukin-3 (IL-3), IL-4, and IL-9 in combination with SCF. Thus, LPA acts through LPA receptor and PPAR-gamma-dependent pathways to accelerate hMC proliferation and differentiation, and it modulates their phenotype without providing cytoprotection. LPA could facilitate MC hyperplasia in inflammation associated with either innate or adaptive immunity.

Reconstructing the diversification of subtilisins in the pathogenic fungus Metarhizium anisopliae.

Fungi secrete subtilisin proteinases to acquire nutrients and breach host barriers. Here we sought a global characterization of the diversity of subtilisins in the insect pathogen Metarhizium anisopliae. Expressed sequence tag (EST) analyses showed that a broad host range strain of M. anisopliae sf. anisopliae (strain 2575) expressed 11 subtilisins during growth on insect cuticle, the largest number of subtilisins reported from any fungus. Polymerase chain reaction amplified 10 of their orthologs from a second strain with multiple hosts (strain 820) and seven from the locust specialist M. anisopliae sf. acridum (strain 324). Analyses based on sequence similarities and exon-intron structure grouped M. anisopliae subtilisins into four clusters-a class I ("bacterial") subtilisin (Pr1C), and three clusters of proteinase K-like class II subtilisins: extracellular subfamily 1 (Pr1A, Pr1B, Pr1G, Pr1I and Pr1K), extracellular subfamily 2 (Pr1D, Pr1E, Pr1F and Pr1J) and an endocellular subtilisin (Pr1H). Phylogenetic analysis of homologous sequences from other genera revealed that this subdivision of proteinase K-like subtilisins into three subfamilies preceded speciation of major fungal lineages. However, diversification has continued during the evolution of Metarhizium subtilisins with evidence of gene duplication events after divergence of M. anisopliae sf. anisopliae and M. anisopliae sf. acridum. Comparing alignments and nonsynonymous/synonymous rates for Pr1 isoenzymes within a lineage and between lineages showed that while overall divergence of subtilisins followed neutral expectations, amino acids involved in catalysis were under strong selective constraint. This suggests that each Pr1 paralog contributes to the pathogens fitness. Furthermore, homology modeling predicted differences between the Pr1's in their secondary substrate specificities, adsorption properties to cuticle and alkaline stability, indicative of functional differences.

FcepsilonRI-dependent gene expression in human mast cells is differentially controlled by T helper type 2 cytokines.

BACKGROUND: Mast cells (MCs) proliferate in response to T(H)2 cytokines and express genes de novo after activation. Limited information is available concerning the interplay between these events. OBJECTIVE: We explored the potential for T(H)2 cytokines to alter activation-dependent gene expression by MCs. METHODS: Cord blood-derived human (h)MCs maintained in stem cell factor (SCF) alone were compared with replicates treated with IL-4, IL-5, or IL-9, respectively, for their patterns of FcepsilonRI-dependent gene induction using microarray technology. RESULTS: Activation of SCF-treated hMCs upregulated their expression of roughly 140 transcripts at 2 hours, including genes involved in cell cycle progression and arrest. Each cytokine substantially modified this profile; approximately 800 inducible genes apiece were controlled by IL-5 or IL-9, whereas 169 inducible genes were controlled by IL-4. IL-4 favored the induction of cytokines and of genes associated with cell growth arrest (GADD34, GAS-1, CIDE-A, INK4D, and BAX) and completely abolished the enhanced proliferation observed in the other 3 groups after activation. Conversely, IL-5 priming induced preferential upregulation of genes involved in cell proliferation and did not abolish thymidine incorporation. CONCLUSIONS: T(H)2 cytokines differentially modulate gene induction in hMCs after FcepsilonRI cross-linkage. IL-4 uniquely controls cytokine gene expression by hMCs and might also limit their activation-driven proliferation.

Expressed sequence tag (EST) analysis of two subspecies of Metarhizium anisopliae reveals a plethora of secreted proteins with potential activity in insect hosts.

Expressed sequence tag (EST) libraries for Metarhizium anisopliae, the causative agent of green muscardine disease, were developed from the broad host-range pathogen Metarhizium anisopliae sf. anisopliae and the specific grasshopper pathogen, M. anisopliae sf. acridum. Approximately 1,700 5' end sequences from each subspecies were generated from cDNA libraries representing fungi grown under conditions that maximize secretion of cuticle-degrading enzymes. Both subspecies had ESTs for virtually all pathogenicity-related genes cloned to date from M. anisopliae, but many novel genes encoding potential virulence factors were also tagged. Enzymes with potential targets in the insect host included proteases, chitinases, phospholipases, lipases, esterases, phosphatases and enzymes producing toxic secondary metabolites. A diverse array of proteases composed 36 % of all M. anisopliae sf. anisopliae ESTs. Eighty percent of the ESTs that could be clustered into functional groups had significant matches (E<10(-5)) in other ascomycete fungi. These included genes reported to have specific roles in pathogens with plant or vertebrate hosts. Many of the remaining ESTs had their best BLAST match among animal, plant and bacterial sequences. These include genes with plant and microbial counterparts that produce potent antimicrobials. The abundance of transcripts discovered for different functional groups varied between the two subspecies of M. anisopliae in a manner consistent with ecological adaptations of the two pathogens. By hastening gene discovery this project has enhanced development of improved mycoinsecticides. In addition, the M. anisopliae ESTs represent a significant contribution to the extensive database of sequences from ascomycetes that are saprophytes or plant and vertebrate pathogens. Comparative analyses of these sequences is providing important information about the biology and evolutionary history of this clade.

Host recognition by pathogenic fungi through plant flavonoids.

A common characteristic among fungal pathogens of plants is that each specializes on a narrow range of specific plants as hosts. One adaptation to a specific host plant is the recognition of the host's chemicals which can be used to trigger genes or developmental pathways needed for pathogenesis. The production of characteristic flavonoids by plants, particularly those exuded from roots by legumes, appear to be used as signals for various microbes, including symbionts as well as pathogens. Nectria haematococca MPVI (anamorph: Fusarium solani) is a soil-borne pathogen of garden pea (Pisum sativum) which serves as a useful model in studying host flavonoid recognition. This fungus displays flavonoid induction of specific pathogenicity genes as well as stimulation of development needed for pathogenesis. Here, we summarize the study of flavonoid-inducible signal pathways which regulate these trait, through identification of transcription factors and regulatory components which control these responses. The characterization of the components a pathogen uses to specifically recognize its host provides insights into the host adaptation process at the molecular level.