Role of histamine in the regulation of intestinal immunity in fish.

In mammals, during the acute inflammatory response, the complex interrelationship and cross-talk among histamine and the immune system has been fairly well characterized. There is a substantial body of information on its structure, metabolism, receptors, signal transduction, physiologic and pathologic effects. However, for early vertebrates, there is little such knowledge. In the case of teleost fish, this lack of knowledge has been due to the widely held belief that histamine is not present in this phylogenetic group. However, it has been recently demonstrated, that granules of mast cells in perciforms contain biologically active histamine. More importantly, the inflammatory response was clearly demonstrated to be regulated by the direct action of histamine on professional phagocytes. Nevertheless, the molecular basis and exact role of this biogenic amine in perciforms is still a matter of speculation. Therefore, this review intends to summarize recent experimental evidence regarding fish mast cells and correlate the same with their mammalian counterparts to establish the possible role of histamine in the fish intestinal inflammatory response.

Acute and subchronic effects on immune responses of carp (Cyprinus carpio L.) after exposure to deoxynivalenol (DON) in feed.

The mycotoxin deoxynivalenol (DON) has been shown to regularly occur at relevant concentrations in feed designed for aquaculture use, but little is known about the consequences of its presence on the organisms that consume the DON-contaminated feed. Previous studies indicated a down-regulation of pro-inflammatory responses in carp (Cyprinus carpio L.) after 4 weeks of feeding DON. The present study examined the time course of innate immune responses of carp to orally administered DON. Changes in mRNA levels of immune genes in different organs (head kidney, trunk kidney, spleen, liver, and intestine) were observed indicating immune-modulating properties of DON. The immune-modulatory effects during the acute phase of DON exposure were characterized by the activation of both pro- and anti-inflammatory cytokines and enzymes in carp. The subchronic responses to DON were characterized by activation of arginases culminating in increased arginase activity in head kidney leukocytes after 26 days of DON treatment. These results suggest profound effects of this mycotoxin on fish in aquaculture.

Isolation of mast cells from the peritoneal exudate of the teleost fish gilthead sea bream (Sparus aurata L.).

Inflammation is the first response of animals to infection or tissue damage. Sparus aurata (Perciformes) was the first fish species shown to possess histamine-containing mast cells at mucosal tissues. We report a separation protocol for obtaining highly enriched (over 95% purity) preparations of fish mast cells in high numbers (5-20 million mast cells per fish). The peritoneal exudate of S. aurata is composed of lymphocytes, acidophilic granulocytes, macrophages and mast cells. We separated the lymphocyte fraction through discontinuous density gradient centrifugation. The remaining cells were cultivated overnight in RPMI-1640 culture medium containing 5% fetal calf serum, which allowed macrophages to adhere to the cell culture flasks. Finally, acidophilic granulocytes were separated from the mast cells though a Magnetic-Activated Cell Separation (MACS) protocol, using a monoclonal antibody against these cells. The purity of mast cells-enriched fractions was analyzed by flow cytometry and by transmission electron microscopy. The functionality of purified mast cells was confirmed by the detection of histamine release by ELISA after stimulation with compound 48/80 and the induction of the pro-inflammatory cytokines IL-1ß and IL-8 following stimulation with bacterial DNA. This fish mast cells separation protocol is a stepping stone for further studies addressing the evolution of vertebrate inflammatory mechanisms.

Histamine regulates the inflammatory response of the tunicate Styela plicata.

Histamine is stored inside hemocytes of the tunicate Styela plicata (Chordata, Tunicata, Ascidiacea), but no evidence on its role in the regulation of the immune response of this species has been reported. We examined whether histamine participated in the regulation of inflammation and host defense in S. plicata. The presence of histamine inside S. plicata hemocytes was confirmed by flow cytometry, and histamine release was detected by ELISA, after in vitro hemocyte stimulation with different PAMPs. In vitro hemocyte treatment with histamine, or specific histamine-receptor agonists, reduced their phagocytic ability. Injection of histamine into the tunic recruited hemocytes to the site of injection. Systemic injection of histamine, or the histamine-releasing agent compound 48/80, decreased the phagocytic ability of hemocytes. Histamine promoted the constriction of tunic hemolymph vessels in vivo, having a direct effect on vasoconstriction in tunic explants. These results provide for the first time clear evidence for the involvement of histamine in the regulation of inflammation and host defense in tunicates.

Impact of ozonation on naphthenic acids speciation and toxicity of oil sands process-affected water to Vibrio fischeri and mammalian immune system.

Oil sands process-affected water (OSPW) is the water contained in tailings impoundment structures in oil sands operations. There are concerns about the environmental impacts of the release of OSPW because of its toxicity. In this study, ozonation followed by biodegradation was used to remediate OSPW. The impacts of the ozone process evolution on the naphthenic acids (NAs) speciation and acute toxicity were evaluated. Ion-mobility spectrometry (IMS) was used to preliminarily separate isomeric and homologous species. The results showed limited effects of the ozone reactor size on the treatment performance in terms of contaminant removal. In terms of NAs speciation, high reactivity of NAs with higher number of carbons and rings was only observed in a region of high reactivity (i.e., utilized ozone dose lower than 50 mg/L). It was also found that nearly 0.5 mg/L total NAs was oxidized per mg/L of utilized ozone dose, at utilized ozone doses lower than 50 mg/L. IMS showed that ozonation was able to degrade NAs, oxidized NAs, and sulfur/nitrogenated NAs. Complete removal of toxicity toward Vibrio fischeri was achieved after ozonation followed by 28-day biodegradation period. In vitro and in vivo assays indicated that ozonation reduced the OSPW toxicity to mice.

A simple and efficient method to detect nuclear factor activation in human neutrophils by flow cytometry.

Neutrophils are the most abundant leukocytes in peripheral blood. These cells are the first to appear at sites of inflammation and infection, thus becoming the first line of defense against invading microorganisms. Neutrophils possess important antimicrobial functions such as phagocytosis, release of lytic enzymes, and production of reactive oxygen species. In addition to these important defense functions, neutrophils perform other tasks in response to infection such as production of proinflammatory cytokines and inhibition of apoptosis. Cytokines recruit other leukocytes that help clear the infection, and inhibition of apoptosis allows the neutrophil to live longer at the site of infection. These functions are regulated at the level of transcription. However, because neutrophils are short-lived cells, the study of transcriptionally regulated responses in these cells cannot be performed with conventional reporter gene methods since there are no efficient techniques for neutrophil transfection. Here, we present a simple and efficient method that allows detection and quantification of nuclear factors in isolated and immunolabeled nuclei by flow cytometry. We describe techniques to isolate pure neutrophils from human peripheral blood, stimulate these cells with anti-receptor antibodies, isolate and immunolabel nuclei, and analyze nuclei by flow cytometry. The method has been successfully used to detect NF-κB and Elk-1 nuclear factors in nuclei from neutrophils and other cell types. Thus, this method represents an option for analyzing activation of transcription factors in isolated nuclei from a variety of cell types.

Mucosal immunity in the gut: the non-vertebrate perspective.

Much is now known about the vertebrate mechanisms involved in mucosal immunity, and the requirement of commensal microbiota at mucosal surfaces for the proper functioning of the immune system. In comparison, very little is known about the mechanisms of immunity at the barrier epithelia of non-vertebrate organisms. The purpose of this review is to summarize key experimental evidence illustrating how non-vertebrate immune mechanisms at barrier epithelia compare to those of higher vertebrates, using the gut as a model organ. Not only effector mechanisms of gut immunity are similar between vertebrates and non-vertebrates, but it also seems that the proper functioning of non-vertebrate gut defense mechanisms requires the presence of a resident microbiota. As more information becomes available, it will be possible to obtain a more accurate picture of how mucosal immunity has evolved, and how it adapts to the organisms' life styles.

Evidence for the presence of functional lipid rafts in immune cells of ectothermic organisms.

The role of lipid rafts in non-mammalian leukocytes has been scarcely investigated. We performed biochemical and functional analysis of lipid rafts in fish leukocytes. Fish Flotillin-1 and a fish GM1-like molecule (fGM1-L) were found in low density detergent-resistant membranes (LD-DRM) in goldfish macrophages and catfish B lymphocytes, similarly to mammals. The presence of flotillin-1 and fGM1-L in LD-DRM was sensitive to increased detergent concentrations, and cholesterol extraction. Confocal microscopy analysis of flotillin-1 and fGM1-L in fish leukocytes showed a distinctive punctuated staining pattern, suggestive of pre-existing rafts. Confocal microscopy analysis of macrophages showed that the membrane of phagosomes containing serum-opsonized zymosan was enriched in fGM1-L, and zymosan phagocytosis was reduced after cholesterol extraction. The presence of flotillin-1 and fGM1-L in LD-DRM, the microscopic evidence of flotillin-1 and fGM1-L on fish macrophages and B-cells, and the sensitivity of phagocytosis to cholesterol extraction, indicate that lipid rafts are biochemically and functionally similar in leukocytes from fish and mammals.

The acute and sub-chronic exposures of goldfish to naphthenic acids induce different host defense responses.

Naphthenic acids (NAs) are believed to be the major toxic component in oil sands process-affected water (OSPW) produced by the oil sands mining industry in Northern Alberta, Canada. We recently reported that oral exposure to NAs alters mammalian immune responses, but the effect of OSPW or NAs on the immune mechanisms of aquatic organisms has not been fully elucidated. We analyzed the effects of acute and sub-chronic NAs exposures on goldfish immune responses by measuring the expression of three pro-inflammatory cytokine genes, antimicrobial functions of macrophages, and host defense after challenge with a protozoan pathogen (Trypanosoma carassii). One week after NAs exposure, fish exhibited increased expression of pro-inflammatory cytokines (IFNγ, IL-1ß1, TNF-α2) in the gills, kidney and spleen. Primary macrophages from fish exposed to NAs for one week, exhibited increased production of nitric oxide and reactive oxygen intermediates. Goldfish exposed for one week to 20 mg/L NAs were more resistant to infection by T. carassii. In contrast, sub-chronic exposure of goldfish (12 weeks) to NAs resulted in decreased expression of pro-inflammatory cytokines in the gills, kidney and spleen. The sub-chronic exposure to NAs reduced the ability of goldfish to control the T. carassii infection, exemplified by a drastic increase in fish mortality and increased blood parasite loads. This is the first report analyzing the effects of OSPW contaminants on the immune system of aquatic vertebrates. We believe that the bioassays depicted in this work will be valuable tools for analyzing the efficacy of OSPW remediation techniques and assessment of diverse environmental pollutants.

Commercial naphthenic acids and the organic fraction of oil sands process water induce different effects on pro-inflammatory gene expression and macrophage phagocytosis in mice.

Naphthenic acids (NAs) are believed to be the major toxic component of oil sands process water (OSPW). Different OSPW preparations have distinct NA compositions, and additional organics, that differ from the commercial NAs (C-NAs) often used for toxicology studies. To evaluate whether C-NAs are an adequate model to study OSPW toxicity in complex organisms, we compared the effects of C-NAs and the extractable organic fraction of OSPW (OSPW-OF) on mice immune mechanisms. Mice were orally exposed to different C-NA doses, or OSPW-OF at the same NA dose, for up to 8 weeks, and the expression of pro-inflammatory genes in different organs was determined using quantitative PCR. C-NAs and OSPW-OF altered the expression of pro-inflammatory genes, inducing either expression down-regulation or up-regulation, depending on the organ examined and time after exposure. The time at which gene expression alterations occurred, and the specific sets of genes whose expression was altered, were very different between animals exposed to C-NAs or to OSPW-OF. We evaluated the ability of mouse peritoneal macrophages to phagocytose yeast cell wall, as a measure of the ability of mice to mount a central function of the innate immune response. Phagocytosis was significantly reduced in animals exposed to C-NAs, but enhanced in mice exposed to OSPW-OF. Our results indicate that studies using C-NAs may not necessarily reflect the possible effects induced in animals by process water from tailing ponds.

Examination of the stimulatory signaling potential of a channel catfish leukocyte immune-type receptor and associated adaptor.

Expressed by various subsets of myeloid and lymphoid immune cells, channel catfish (Ictalurus punctatus) leukocyte immune-type receptors (IpLITRs) are predicted to play a key role in the initiation and termination of teleost cellular effector responses. These type I transmembrane proteins belong to the immunoglobulin superfamily and display features of immunoregulatory receptors with inhibitory and/or stimulatory signaling potential. Expanding on our previous work, which demonstrated that putative stimulatory IpLITR-types associated with the catfish adaptor proteins IpFcRγ and FcRγ-L, this study focuses on the functional significance of this immune receptor-adaptor signaling complex. Specifically, we generated an epitope-tagged chimeric receptor construct by fusing the extracellular domain of IpLITR 2.6b with the transmembrane region and cytoplasmic tail of IpFcRγ-L. This chimera was stably expressed in a rat basophilic leukemia (RBL) cell line, RBL-2H3, and following cross-linking of the surface receptor with an anti-hemagglutinin monoclonal antibody or opsonized microspheres, the chimeric teleost receptor induced cellular degranulation and phagocytic responses, respectively. Site-directed mutagenesis of the immunoreceptor tyrosine-based activation motif encoded within the cytoplasmic tail of the chimera confirmed that these functional responses were dependent on the phosphorylated tyrosines within this motif. Using a combination of phospho-specific antibodies and pharmacological inhibitors, we also demonstrate that the IpLITR/IpFcRγ-L-induced degranulation response requires the activity of Src homology 2 domain containing protein tyrosine phosphatases, phosphatidylinositol 3-kinase, protein kinase C, and mitogen-activated protein kinases but appears independent of the c-Jun N-terminal kinase and p38 MAP kinase pathways. In addition to this first look at stimulatory IpLITR-mediated signaling and its influence on cellular effector responses, the advantage of generating RBL-2H3 cells stably expressing a functional IpLITR-adaptor chimera will be discussed.

Ozone treatment ameliorates oil sands process water toxicity to the mammalian immune system.

We evaluated whether ozonation ameliorated the effects of the organic fraction of oil sands process water (OSPW) on immune functions of mice. Ozonation of OSPW eliminated the capacity of its organic fraction to affect various mouse bone marrow-derived macrophage (BMDM) functions in vitro. These included the production of nitric oxide and the expression of inducible nitric oxide synthase, the production of reactive oxygen intermediates and the expression of NADPH oxidase subunits, phagocytosis, and the expression of pro-inflammatory cytokine genes. Ozone treatment also eliminated the ability of OSPW organic fraction to down-regulate the expression of various pro-inflammatory cytokine and chemokine genes in the liver of mice, one week after oral exposure. We conclude that ozone treatment may be a valuable process for the remediation of large volumes of OSPW.

Commercial naphthenic acids and the organic fraction of oil sands process water downregulate pro-inflammatory gene expression and macrophage antimicrobial responses.

This is the first report showing that the organic fraction of oil sands process water (OSPW-OF), and commercial naphthenic acids (C-NAs), cause immunotoxicity. The exposure of mouse bone marrow-derived macrophages (BMDM) to different amounts of C-NAs or OSPW-OF, did not affect cell viability in vitro. We examined whether exposure of BMDM to C-NAs or OSPW-OF affected various antimicrobial responses of these cells. A dose-dependent decrease in nitric oxide response was observed after treatment of BMDM with OSPW-OF, but not with C-NAs. Although OSPW-OF and C-NAs both down-regulated the respiratory burst response of BMDM, the suppression of the production of reactive oxygen intermediates was more pronounced in cells treated with OSPW-OF. Treatment with OSPW-OF or C-NAs reduced BMDM phagocytosis of zymosan and latex beads. The decrease of BMDM antimicrobial response after exposure to OSPW-OF or C-NAs, was accompanied by decreased pro-inflammatory cytokine gene expression. Oral exposure of mice to OSPW-OF caused down-regulation in the expression of genes encoding pro-inflammatory cytokines IFNγ, IL-1ß and CSF-1. Our findings indicated that OSPW causes immunotoxic effects that may impair the ability of an exposed host to defend against infectious disease. Furthermore, given the differences between the effects of OSPW-OF and C-NAs, C-NAs should not be assumed to be a direct surrogate for the immunotoxic chemical species in OSPW.

Fcgamma receptors exhibit different phagocytosis potential in human neutrophils.

In neutrophils, two receptors for IgG antibodies, namely FcgammaRIIA and FcgammaRIIIB are constitutively expressed, and a third one, FcgammaRI, can be upregulated by interferon-gamma. Whether FcgammaRIIIB is capable of triggering phagocytosis by itself is still controversial. The main role of FcgammaRI has not been clearly established in these cells. To address this problem, neutrophils were treated with interferon-gamma, and then phagocytosis mediated by each type of Fcgamma receptor was evaluated by flow cytometry. FcgammaRIIA was the most efficient receptor for phagocytosis. FcgammaRIIIB could mediate phagocytosis but much less efficiently than FcgammaRIIA. Both FcgammaRIIA- and FcgammaRIIIB-mediated phagocytosis were blocked by inhibitors of Src family kinases, Syk, PI 3-K, and ERK. In contrast, interferon-gamma-induced FcgammaRI was not able to mediate phagocytosis. Also, FcgammaRI did not activate ERK in the nucleus, but was however able to stimulate an efficient calcium rise. These data show that different neutrophil Fcgamma receptors possess different phagocytosis capabilities: FcgammaRIIA and FcgammaRIIIB, but not FcgammaRI, promote phagocytosis.

FcgammaRIIA and FcgammaRIIIB mediate nuclear factor activation through separate signaling pathways in human neutrophils.

Receptors for IgG Abs (Fcgamma receptors) are capable of triggering diverse cell responses in leukocytes. In neutrophils, two Fcgamma receptors, namely FcgammaRIIA and FcgammaRIIIB, are constitutively expressed. The signaling pathways that regulate FcgammaRIIA-mediated phagocytosis have been relatively well described. However, the different signaling pathways that lead to NF activation after engagement of each Fcgamma receptor have only been partially described. To address this problem, neutrophils were stimulated by cross-linking selectively each type of Fcgamma receptor with specific mAbs, and NF activation was then analyzed. FcgammaRIIIB, but not FcgammaRIIA, promoted a robust increase in phosphorylated ERK in the nucleus, and also efficient phosphorylation of the NF Elk-1. Complete mAb 3G8 (anti-FcgammaRIIIB) induced a higher response than did F(ab')(2) fragments of mAb 3G8, suggesting a possible synergistic effect of both FcgammaR receptors. However, mAb IV.3 (anti-FcgammaRIIA) alone did not cause an increase of phosphorylated ERK in the nucleus. FcgammaRIIIB-induced nuclear phosphorylation of ERK, and of Elk-1, was not affected by Syk, PI3K, or MEK inhibitors. In contrast, FcgammaRIIA- or FcgammaRIIIB-mediated phosphorylation of cytoplasmic ERK depended on Syk, PI3K, and MEK. Also, ERK, but not MEK, was constitutively present in the nucleus, and FcgammaRIIIB cross-linking did not increase the levels of nuclear ERK or MEK. These data clearly show that different neutrophil Fcgamma receptors possess different signaling capabilities. FcgammaRIIIB, but not FcgammaRIIA, activates a unique signaling pathway leading to the nuclear-restricted phosphorylation of ERK and Elk-1, independently of Syk, PI3K, or MEK.

An fMLP receptor is involved in activation of phagocytosis by hemocytes from specific insect species.

In mammalian phagocytes, the bacterial formylated peptide fMLP functions both as a potent enhancer of phagocytosis and chemoattractant. fMLP has been reported to be chemotactic for hemocytes of two marine invertebrates, and of the insect Manduca sexta (Lepidoptera). Whether fMLP is also able to activate phagocytosis has not been explored in hemocytes of any invertebrate. To determine the effect of fMLP on insect hemocyte phagocytosis, in vitro phagocytosis assays were performed with hemocytes from the insects: Gromphadorhina portentosa (Blattodea), Acheta domesticus (Orthoptera), Zophobas morio (Coleoptera), and Galleria mellonella (Lepidoptera). Phagocytosis of latex, zymosan (yeast), Gram-positive and Gram-negative bacteria was measured by flow cytometry, in the presence of increasing fMLP concentrations. G. portentosa hemocytes showed no enhancement of phagocytosis by fMLP. A. domesticus hemocytes had increased phagocytosis of latex and Gram-negative bacteria in the presence of fMLP. Z. morio hemocytes increased phagocytosis of latex, yeast, and Gram-negative bacteria after fMLP stimulation. Galleria mellonella hemocytes increased phagocytosis of latex after fMLP stimulation. Treating hemocytes with Pertussis toxin, a known inhibitor of the signaling pathway initiated by the mammalian fMLP receptor, returned phagocytosis to basal levels. Also, hemocytes from all insect species tested presented a similar chemotactic response to fMLP. These data suggest that, whereas the ability of hemocytes to chemotactically-respond to fMLP is conserved in insects ranging from Blattodea to Lepidoptera, the ability to respond to fMLP by activating phagocytosis is restricted to specific insect species.

Insulin-like growth factor 1 is expressed in mouse developing testis and regulates somatic cell proliferation.

Testicular development occurs prenatally in mammals. The developmental underlying mechanism is only partially understood. The aim of the present investigation was to study the expression of the gene coding for insulin-like growth factor 1 (Igf-1) and Igf-1 type 1 receptor (Igf-1r) and their respective proteins in mouse Sertoli and Leydig cells at gestation day 12 (E12)-E18. Moreover, we sought to determine the effect of IGF-1 on the proliferation of both cell types and to establish the signal transduction mechanism involved in the IGF-1 pathway. Transcripts for the Igf-1 and Igf-1r genes were found in Sertoli and Leydig cells from E12-E18. Highest IGF-1 and IGF-Ir protein expression levels were found in both cell types at E18. Exogenous IGF-1 administration increased Sertoli and Leydig cell proliferation at E14-E18 in vitro. Inhibition of the pathway mitogen-activated extracellular signal-regulated protein kinase (MEK) 1/2 with UO126 diminished the proliferation of the Sertoli and Leydig cells in vitro. We propose that IGF-1 and IGF-1r regulate Sertoli and Leydig cell proliferation through the MEK/extracellular-signal-regulated kinase (ERK) 1/2 signal transduction pathway, leading to development and growth of the mouse embryonic testis.

Immune responses of mussel hemocyte subpopulations are differentially regulated by enzymes of the PI 3-K, PKC, and ERK kinase families.

Various hemocyte cell types have been described in invertebrates, but for most species a functional characterization of different hemocyte cell types is still lacking. In order to characterize some immunological properties of mussel (Mytilus galloprovincialis) hemocytes, cells were separated by flow cytometry and their capacity for phagocytosis, production of reactive oxygen species (ROS), and production of nitric oxide (NO), was examined. Phosphatidylinositol 3-kinase (PI 3-K), protein kinase C (PKC), and extracellular signal-regulated kinase (ERK) inhibitors were also used to biochemically characterize these cell responses. Four morphologically distinct subpopulations, designated R1-R4, were detected. R1, R2, and R3 cells presented different levels of phagocytosis towards zymosan, latex beads, and two bacteria species. Similarly, R1 to R3, but not R4, cells produced ROS, while all subpopulations produced NO, in response to zymosan. Internalization of all phagocytic targets was blocked by PI 3-K inhibition. In addition, internalization of latex particles, but not of bacteria, was partially blocked by PKC or ERK inhibition. Interestingly, phagocytosis of zymosan was impaired by PKC, or ERK inhibitors, only in R2 cells. Zymosan-induced ROS production was blocked by PI 3-K inhibition, but not by PKC, or ERK inhibition. In addition, zymosan-stimulated NO production was affected by PI 3-K inhibition in R1 and R2, but not in R3 or R4 cells. NO production in all cell types was unaffected by PKC inhibition, but ERK inhibition blocked it in R2 cells. These data reveal the existence of profound functional and biochemical differences in mussel hemocytes and indicate that M. galloprovincialis hemocytes are specialized cells fulfilling specific tasks in the context of host defense.

Nuclear factor activation by FcgammaR in human peripheral blood neutrophils detected by a novel flow cytometry-based method.

In mammals, neutrophils are the most abundant circulating leukocytes. Neutrophils are short-lived cells presenting at least two important transcriptionally regulated cellular responses, initiated by cell activation: the production of pro-inflammatory cytokines and the inhibition of apoptosis. The study of transcriptionally regulated processes in these cells cannot be approached through conventional reporter gene strategies, as there are currently not available methods for neutrophil transfection. Here we describe a novel flow cytometry-based method that allowed quantification of nuclear factor NF-kappaB activation in neutrophils, in response to FcgammaIIA and FcgammaRIIIB stimulation. The sensitivity of this method allowed the detection of small changes in NF-kappaB activation, due to pharmacological inhibition of receptor-initiated signaling pathways. NF-kappaB activation was also detected by this method in various leukocyte cell lines. In addition, quantification of Fcgamma receptor-initiated nuclear activation of ERK and Elk-1 was successfully achieved through this method. The broad applicability and versatility of this flow cytometry-based method position it as a fast and reliable alternative to traditional methods for analyzing activation of transcription factors in a variety of cell types.

Transmembrane mutations to FcgammaRIIA alter its association with lipid rafts: implications for receptor signaling.

Many immunoreceptors have been reported to associate with lipid rafts upon ligand binding. The way in which this association is regulated is still obscure. We investigated the roles for various domains of the human immunoreceptor FcgammaRIIA in regulating its association with lipid rafts by determining the resistance of unligated, or ligated and cross-linked, receptors to solubilization by the nonionic detergent Triton X-100, when expressed in RBL-2H3 cells. Deletion of the cytoplasmic domain, or destruction of the cytoplasmic palmitoylation site, had no effect on the association of the receptor with lipid rafts. A transmembrane mutant, A224S, lost the ability to associate with lipid rafts upon receptor cross-linking, whereas transmembrane mutants VA231-2MM and VVAL234-7GISF showed constitutive lipid raft association. Wild-type (WT) FcgammaRIIA and all transmembrane mutants activated Syk, regardless of their association with lipid rafts. WT FcgammaRIIA and mutants that associated with lipid rafts efficiently activated NF-kappaB, in an ERK-dependent manner. In contrast, WT FcgammaRIIA and the A224S mutant both presented efficient phagocytosis, while VA231-2MM and VVAL234-7GISF mutants presented lower phagocytosis, suggesting that phagocytosis may proceed independently of lipid raft association. These data identify the transmembrane domain of FcgammaRIIA as responsible for regulating its inducible association with lipid rafts and suggest that FcgammaRIIA-mediated responses, like NF-kappaB activation or phagocytosis, can be modulated by lipid raft association of the ligated receptor.