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1.  IL-17RA is Essential for Optimal Localization of Follicular T Helper Cells in the Germinal Center Light Zone to Promote Autoantibody-Producing B cells1 
Journal of immunology (Baltimore, Md. : 1950)  2013;191(4):10.4049/jimmunol.1300479.
Germinal centers provide a microenvironment that promotes and regulates the interactions of B-cells with follicular T-helper cells (TFH). Here we show that there are significantly higher frequencies of CXCR5+ICOS+TFH cells in autoimmune BXD2 mice, and these cells express both interleukin (IL)-21R and IL-17RA. Although IL-17 and IL-21 are both important for the formation of spontaneous GCs and development of pathogenic autoantibodies, IL-21, but not IL-17, is required for the proper development of TFH cells in BXD2 mice. The total numbers of TFH cells and their ability to induce B cell responses in vitro were not affected by a deficiency of IL-17RA in BXD2-Il17ra−/− mice, the majority of CXCR5+ TFH cells from BXD2-Il17ra−/− mice were, however, not localized in the GC light zone (LZ). Interruption of IL-17 signaling, either acutely by AdIL-17R:Fc or chronically by Il17ra−/−, disrupted TFH–B interactions and abrogated the generation of autoantibody-forming B cells in BXD2 mice. IL-17 upregulated the expression of regulator of G-protein signaling (RGS)16 to promote the ability of TFH to form conjugates with B cells which was abolished in TFH cells from BXD2-Rgs16−/− mice. The results suggests that IL-17 is an extrinsic stop signal that it acts on post-differentiated IL-17RA+ TFH to enable its interaction with responder B cells in the LZ niche. These data suggest a novel concept that TFH differentiation and its stabilization in the LZ are two separate checkpoints and that IL-21 and IL-17 act at each checkpoint to enable pathogenic GC development.
doi:10.4049/jimmunol.1300479
PMCID: PMC3819396  PMID: 23858031
2.  Genome-wide SNP analysis of the Systemic Capillary Leak Syndrome (Clarkson disease) 
Rare diseases (Austin, Tex.)  2013;1(1):e27445.
The Systemic Capillary Leak Syndrome (SCLS) is an extremely rare, orphan disease that resembles, and is frequently erroneously diagnosed as, systemic anaphylaxis. The disorder is characterized by repeated, transient, and seemingly unprovoked episodes of hypotensive shock and peripheral edema due to transient endothelial hyperpermeability. SCLS is often accompanied by a monoclonal gammopathy of unknown significance (MGUS). Using Affymetrix Single Nucleotide Polymorphism (SNP) microarrays, we performed the first genome-wide SNP analysis of SCLS in a cohort of 12 disease subjects and 18 controls. Exome capture sequencing was performed on genomic DNA from nine of these patients as validation for the SNP-chip discoveries and de novo data generation. We identified candidate susceptibility loci for SCLS, which included a region flanking CAV3 (3p25.3) as well as SNP clusters in PON1 (7q21.3), PSORS1C1 (6p21.3), and CHCHD3 (7q33). Among the most highly ranked discoveries were gene-associated SNPs in the uncharacterized LOC100130480 gene (rs6417039, rs2004296). Top case-associated SNPs were observed in BTRC (rs12355803, 3rs4436485), ARHGEF18 (rs11668246), CDH13 (rs4782779), and EDG2 (rs12552348), which encode proteins with known or suspected roles in B cell function and/or vascular integrity. 61 SNPs that were significantly associated with SCLS by microarray analysis were also detected and validated by exome deep sequencing. Functional annotation of highly ranked SNPs revealed enrichment of cell projections, cell junctions and adhesion, and molecules containing pleckstrin homology, Ras/Rho regulatory, and immunoglobulin Ig-like C2/fibronectin type III domains, all of which involve mechanistic functions that correlate with the SCLS phenotype. These results highlight SNPs with potential relevance to SCLS.
doi:10.4161/rdis.27445
PMCID: PMC4009617  PMID: 24808988
systemic capillary leak syndrome; genetics, genome-wide SNP study; cell junction; cell adhesion; cytoskeleton; vascular permeability
3.  Genome-wide SNP analysis of the Systemic Capillary Leak Syndrome (Clarkson disease) 
Rare Diseases  2013;1:e27445.
The Systemic Capillary Leak Syndrome (SCLS) is an extremely rare, orphan disease that resembles, and is frequently erroneously diagnosed as, systemic anaphylaxis. The disorder is characterized by repeated, transient, and seemingly unprovoked episodes of hypotensive shock and peripheral edema due to transient endothelial hyperpermeability. SCLS is often accompanied by a monoclonal gammopathy of unknown significance (MGUS). Using Affymetrix Single Nucleotide Polymorphism (SNP) microarrays, we performed the first genome-wide SNP analysis of SCLS in a cohort of 12 disease subjects and 18 controls. Exome capture sequencing was performed on genomic DNA from nine of these patients as validation for the SNP-chip discoveries and de novo data generation. We identified candidate susceptibility loci for SCLS, which included a region flanking CAV3 (3p25.3) as well as SNP clusters in PON1 (7q21.3), PSORS1C1 (6p21.3), and CHCHD3 (7q33). Among the most highly ranked discoveries were gene-associated SNPs in the uncharacterized LOC100130480 gene (rs6417039, rs2004296). Top case-associated SNPs were observed in BTRC (rs12355803, 3rs4436485), ARHGEF18 (rs11668246), CDH13 (rs4782779), and EDG2 (rs12552348), which encode proteins with known or suspected roles in B cell function and/or vascular integrity. 61 SNPs that were significantly associated with SCLS by microarray analysis were also detected and validated by exome deep sequencing. Functional annotation of highly ranked SNPs revealed enrichment of cell projections, cell junctions and adhesion, and molecules containing pleckstrin homology, Ras/Rho regulatory, and immunoglobulin Ig-like C2/fibronectin type III domains, all of which involve mechanistic functions that correlate with the SCLS phenotype. These results highlight SNPs with potential relevance to SCLS.
doi:10.4161/rdis.27445
PMCID: PMC4009617  PMID: 24808988
systemic capillary leak syndrome; genetics, genome-wide SNP study; cell junction; cell adhesion; cytoskeleton; vascular permeability
4.  Inflammatory Markers of the Systemic Capillary Leak Syndrome (Clarkson Disease) 
Objectives
The Systemic Capillary Leak Syndrome (SCLS) is a rare and potentially fatal disorder resembling systemic anaphylaxis that is characterized by transient episodes of hypotensive shock and peripheral edema. The pathogenesis of SCLS is unknown, and triggers for attacks are apparent only in a minority of patients. We introduce a clinical algorithm for the diagnosis of SCLS, and we investigated potential serum biomarkers of acute SCLS episodes.
Methods
We analyzed serum cytokines in a cohort of 35 patients with an established diagnosis of SCLS and characterized the effects of SCLS sera on endothelial cell function. We investigated the cellular source(s) of CXCL10, a chemokine that was significantly elevated in both basal and acute SCLS sera, by flow cytometry.
Results
Several cytokines were elevated in acute SCLS sera compared to baseline or sera from healthy controls, including CXCL10, CCL2, IL-1β, IL-6, IL-8, IL-12 and TNFα. The majority of acute sera failed to activate endothelial cells as assessed by surface adhesion marker expression. Monocytes appear to be the major source of serum CXCL10, and the percentage of CXLC10+ monocytes in response to IFNγ stimulation was increased in SCLS subjects compared to controls.
Conclusions
The presence of proinflammatory cytokines in acute SCLS sera suggests that inflammation or infection may have a role in triggering episodes. The enhanced capacity of monocytes from SCLS patients to produce CXCL10 suggests a new therapeutic avenue for SCLS.
doi:10.4172/2155-9899.1000213
PMCID: PMC4232957  PMID: 25405070
Systemic capillary leak syndrome; Inflammation; Cytokines; CXCL10; Monocytes
5.  Extension of the Germinal Center Stage of B-cell Development Promotes Autoantibodies in BXD2 Mice 
Arthritis and rheumatism  2013;65(10):2703-2712.
Objective
Regulator of G-protein Signaling (RGS) proteins inhibit chemokine signaling by desensitizing G-protein coupled receptor signals. The mechanisms by which RGS13 promotes the generation of pathogenic autoantibodies in germinal centers (GC) were determined using BXD2-Rgs13−/− mice.
Methods
Confocal and light microscopy imaging was used to determine the location of cells that express RGS13 and activation-induced cytidine deaminase (AID) in the spleen and the number of plasmablasts. The levels of GC and plasma cell program transcripts in GC B cells were determined by quantitative real-time PCR. Differential IL-17-mediated expression of Rgs13 in GC versus non-GC B cells was analyzed using A20 versus 70Z/3 B cells.
Results
In spleens of BXD2 mice, RGS13 was mainly expressed by GC B cells and was stimulated by IL-17 but not IL-21. IL-17 upregulated Rgs13 in A20 GC but not 70Z/3 non-GC B cells. BXD2-Rgs13−/− mice exhibited smaller GCs, lower AID levels, suggesting lower somatic hypermutation and affinity maturation. There were, however, increased IgMbright plasmablasts, upregulation of plasma program genes Irf4, Blimp1, Xbp1 and pCREB target genes Fosb and Obf1, with down-regulation of GC program genes Aicda, Pax5 and Bach2 in GC B cells of BXD2-Rgs13−/− mice. BXD2-Rgs13−/− mice showed lower titers of IgG autoantibodies and IgG deposits in the glomeruli, suggesting reduced autoantibody pathogenicity.
Conclusion
RGS13 deficiency is associated with reduction in GC program genes and exit of less pathogenic IgM plasmablasts in BXD2 mice. Prolonged GC program, mediated by upregulation RGS13, enhanced AID expression and enabled generation of pathogenic autoantibodies in autoreactive GCs.
doi:10.1002/art.38059
PMCID: PMC3979745  PMID: 23818250
6.  Regulator of G-Protein Signaling–5 Inhibits Bronchial Smooth Muscle Contraction in Severe Asthma 
Severe asthma is associated with fixed airway obstruction attributable to inflammation, copious luminal mucus, and increased airway smooth muscle (ASM) mass. Paradoxically, studies demonstrated that the hypertrophic and hyperplastic ASM characteristic of severe asthma has reduced contractile capacity. We compared the G-protein–coupled receptor (GPCR)–induced Ca2+ mobilization and expression of GPCRs and signaling proteins related to procontractile signaling in ASM derived postmortem from subjects who died of nonrespiratory causes, with cells from subjects who died of asthma. Despite the increased or comparable expression of contraction-promoting GPCRs (bradykinin B2 or histamine H1 and protease-activated receptor 1, respectively) in asthmatic ASM cells relative to cells from healthy donors, asthmatic ASM cells exhibited reduced histamine-induced Ca2+ mobilization and comparable responses to bradykinin and thrombin, suggesting a postreceptor signaling defect. Accordingly, the expression of regulator of G-protein signaling–5 (RGS5), an inhibitor of ASM contraction, was increased in cultured, asthmatic ASM cells and in bronchial smooth muscle bundles of both human subjects with asthma and allergen-challenged mice, relative to those of healthy human subjects or naive mice. The overexpression of RGS5 impaired the release of Ca2+ to thrombin, histamine, and carbachol, and reduced the contraction of precision-cut lung slices to carbachol. These results suggest that increased RGS5 expression contributes to decreased myocyte shortening in severe and fatal asthma.
doi:10.1165/rcmb.2011-0110OC
PMCID: PMC3380291  PMID: 22281988
asthma; bronchial smooth muscle; signal transduction; G-protein–coupled receptors
7.  Phosphorylation of RGS13 by the Cyclic AMP-dependent Protein Kinase Inhibits RGS13 Degradation 
Regulators of G-protein signaling (RGS) proteins are scaffolds that control diverse signaling pathways by modulating signalosome formation and by accelerating the GTPase activity of heterotrimeric G proteins. Although expression of many RGS proteins is relatively low in quiescent cells, transcriptional and post-translational responses to environmental cues regulate both their abundance and activity. We found previously that RGS13, one of the smallest RGS proteins in the family, inhibited cyclic AMP-dependent protein kinase (PKA)-induced gene expression through interactions with the transcription factor cAMP-response element-binding (CREB) protein. Here, we show that PKA activation also leads to increased steady-state RGS13 expression through RGS13 phosphorylation, which inhibits RGS13 protein degradation. RGS13 turnover was significantly reduced in cells stimulated with cAMP, which was reversed by expression of the PKA-specific inhibitory peptide PKI. RGS13 phosphorylation was diminished by mutation of an N-terminal Thr residue (T41) identified as a phosphorylation site by mass spectrometry. Mutation of Thr41 in RGS13 to Ala (T41A) reduced steady-state RGS13 levels and its ability to inhibit M2 muscarinic receptor-mediated Erk phosphorylation compared with wild-type RGS13 by attenuating the protective effect of cAMP on RGS13 degradation. RGS13 underwent ubiquitylation, indicating that it is a likely target of the proteasome. These studies are the first to demonstrate post-translational mechanisms controlling the expression of RGS13. Stabilization of RGS13 through PKA-mediated phosphorylation could enhance RGS13 functions, providing negative feedback regulation that promotes cellular desensitization.
doi:10.1093/jmcb/mjq031
PMCID: PMC3031343  PMID: 20974683
RGS proteins; cAMP; protein kinase; phosphorylation
8.  Enhanced Effector Function of CD8+ T Cells From Healthy Controls and HIV-Infected Patients Occurs Through Thrombin Activation of Protease-Activated Receptor 1 
The Journal of Infectious Diseases  2012;207(4):638-650.
Disruption of vascular integrity by trauma and other tissue insults leads to inflammation and activation of the coagulation cascade. The serine protease thrombin links these 2 processes. The proinflammatory function of thrombin is mediated by activation of protease-activated receptor 1 (PAR-1). We found that peripheral blood effector memory CD4+ and CD8+ T lymphocytes expressed PAR-1 and that expression was increased in CD8+ T cells from human immunodeficiency virus (HIV)–infected patients. Thrombin enhanced cytokine secretion in CD8+ T cells from healthy controls and HIV-infected patients. In addition, thrombin induced chemokinesis, but not chemotaxis, of CD8+ T cells, which led to structural changes, including cell polarization and formation of a structure rich in F-actin and phosphorylated ezrin-radexin-moesin proteins. These findings suggest that thrombin mediates cross-talk between the coagulation system and the adaptive immune system at sites of vascular injury through increased T-cell motility and production of proinflammatory cytokines.
doi:10.1093/infdis/jis730
PMCID: PMC3549602  PMID: 23204166
PAR-1; T cells; thrombin; HIV pathogenesis; coagulation; inflammation
9.  R4 RGS Proteins: Regulation of G Protein Signaling and Beyond 
Pharmacology & therapeutics  2007;116(3):473-495.
The Regulators of G protein Signaling (RGS) proteins were initially characterized as inhibitors of signal transduction cascades initiated by G-protein-coupled receptors (GPCRs) because of their ability to increase the intrinsic GTPase activity of heterotrimeric G proteins. This GTPase accelerating (GAP) activity enhances G protein deactivation and promotes desensitization. However, in addition to this signature trait, emerging data have revealed an expanding network of proteins, lipids, and ions that interact with RGS proteins and confer additional regulatory functions. This review highlights recent advances in our understanding of the physiological functions of one subfamily of RGS proteins with a high degree of homology (B/R4) gleaned from recent studies of knockout mice or cells with reduced RGS expression. We also discuss some of the newly-appreciated interactions of RGS proteins with cellular factors that suggest RGS control of several components of G-protein-mediated pathways as well as a diverse array of non-GPCR-mediated biological responses.
doi:10.1016/j.pharmthera.2007.09.005
PMCID: PMC2156173  PMID: 18006065
10.  RGS10 exerts a neuroprotective role through the PKA/c-AMP response-element (CREB) pathway in dopaminergic neuron-like cells 
Journal of neurochemistry  2012;122(2):333-343.
Regulator of G-protein signaling-10 (RGS10) is a GTPase activating protein (GAP) for Gαi/q/z subunits that is highly expressed in the immune system and in a broad range of brain regions including the hippocampus, striatum, dorsal raphe, and ventral midbrain. Previously, we reported that RGS10-null mice display increased vulnerability to chronic systemic inflammation-induced degeneration of nigral dopaminergic (DA) neurons. Given that RGS10 is expressed in DA neurons, we investigated the extent to which RGS10 regulates cell survival under conditions of inflammatory stress. Because of the inherent limitations associated with use of primary DA neurons for biochemical analyses, we employed a well-characterized ventral mesencephalon DA neuroblastoma cell line (MN9D) for our studies. We found that stable over-expression of RGS10 rendered them resistant to TNF-induced cytotoxicity; whereas MN9D cells expressing mutant RGS10-S168A (which is resistant to phosphorylation by protein kinase A (PKA) at a serine residue that promotes its nuclear translocation) showed similar sensitivity to TNF as the parental MN9D cells. Using biochemical and pharmacological approaches, we identified protein kinase A (PKA) and the downstream phospho-cAMP response element-binding (CREB) signaling pathway (and ruled out ERK 1/2, JNK, and NFkB) as key mediators of the neuroprotective effect of RGS10 against inflammatory stress.
doi:10.1111/j.1471-4159.2012.07780.x
PMCID: PMC3435458  PMID: 22564151
RGS10; MN9D; dopaminergic; CREB; neuroinflammation; TNF
11.  RGS16 attenuates pulmonary T helper type 2/T helper type 17 inflammatory responses 
The Regulators of G protein signaling (RGS) protein superfamily negatively controls G-protein-coupled receptor (GPCR) signal transduction pathways. RGS16 is enriched in activated/effector T lymphocytes. Here, we show that RGS16 constrains pulmonary inflammation by regulating chemokine-induced T-cell trafficking in response to challenge with Schistosoma mansoni. Naïve Rgs16–/– mice were “primed” for inflammation by accumulation of CCR10+ T cells in the lung. Upon pathogen exposure, these mice developed more robust granulomatous lung fibrosis than wild-type (WT) counterparts. Distinct TH2 or putative TH17 subsets expressing CCR4 or CCR10 accumulated more rapidly in Rgs16–/– lungs following challenge and produced pro-inflammatory cytokines IL-13 and IL-17B. CCR4+ Rgs16–/– TH2 cells migrated excessively to CCL17 and localized aberrantly in challenged lungs. T lymphocytes were partially excluded from lung granulomas in Rgs16–/– mice, instead forming peribronchial/perivascular aggregates. Thus, RGS16-mediated confinement of T cells to Schistosome granulomas mitigates widespread cytokine-mediated pulmonary inflammation.
doi:10.4049/jimmunol.1103781
PMCID: PMC3522182  PMID: 22593615
Chemokines; G proteins; RGS proteins; Schistosoma mansoni; fibrosis; TH2; TH17; cytokines
12.  Naïve T cells sense the cysteine protease allergen papain through protease-activated receptor 2 and propel T helper type 2 immunity 
Background
Sensitization to protease allergens, such as papain, or helminth infection is associated with basophil recruitment to draining lymph nodes. Basophils have the capacity to present antigen to naïve T cells and promote TH2 differentiation directly or indirectly through IL-4 production.
Objective
We studied how papain induces basophil migration to lymph nodes and the contribution of various leukocytes to papain-induced immune responses.
Methods
We immunized mice in the footpad with papain and studied leukocyte recruitment and inflammatory cytokine and chemokine production in the draining popliteal lymph nodes.
Results
Papain directly activated naïve T cells through protease-activated receptor 2 (PAR2) to initiate a chemokine/cytokine program that includes CCL17, CCL22, and IL-4. Papain-triggered innate immune responses were dependent on both CD4 T cells and PAR2 and were strongly reduced in the absence of CCR4, the primary receptor for CCL17/22.
Conclusion
These results elucidate a novel innate allergen recognition pathway mediated by naïve T cells through PAR2, which provide an immediate source of chemokines and IL-4 upstream of basophils and antigen-restricted TH2 differentiation. PAR2 antagonism may thus hold promise for the treatment of allergic disease.
doi:10.1016/j.jaci.2012.02.035
PMCID: PMC3340436  PMID: 22460072
Chemokines; basophils; chemotaxis; TH2; allergens
13.  Extracellular volume fraction mapping in the myocardium, part 2: initial clinical experience 
Background
Diffuse myocardial fibrosis, and to a lesser extent global myocardial edema, are important processes in heart disease which are difficult to assess or quantify with cardiovascular magnetic resonance (CMR) using conventional late gadolinium enhancement (LGE) or T1-mapping. Measurement of the myocardial extracellular volume fraction (ECV) circumvents factors that confound T1-weighted images or T1-maps. We hypothesized that quantitative assessment of myocardial ECV would be clinically useful for detecting both focal and diffuse myocardial abnormalities in a variety of common and uncommon heart diseases.
Methods
A total of 156 subjects were imaged including 62 with normal findings, 33 patients with chronic myocardial infarction (MI), 33 with hypertrophic cardiomyopathy (HCM), 15 with non-ischemic dilated cardiomyopathy (DCM), 7 with acute myocarditis, 4 with cardiac amyloidosis, and 2 with systemic capillary leak syndrome (SCLS). Motion corrected ECV maps were generated automatically from T1-maps acquired pre- and post-contrast calibrated by blood hematocrit. Abnormally-elevated ECV was defined as >2SD from the mean ECV in individuals with normal findings. In HCM the size of regions of LGE was quantified as the region >2 SD from remote.
Results
Mean ECV of 62 normal individuals was 25.4 ± 2.5% (m ± SD), normal range 20.4%-30.4%. Mean ECV within the core of chronic myocardial infarctions (without MVO) (N = 33) measured 68.5 ± 8.6% (p < 0.001 vs normal). In HCM, the extent of abnormally elevated ECV correlated to the extent of LGE (r = 0.72, p < 0.001) but had a systematically greater extent by ECV (mean difference 19 ± 7% of slice). Abnormally elevated ECV was identified in 4 of 16 patients with non-ischemic DCM (38.1 ± 1.9% (p < 0.001 vs normal) and LGE in the same slice appeared “normal” in 2 of these 4 patients. Mean ECV values in other disease entities ranged 32-60% for cardiac amyloidosis (N = 4), 40-41% for systemic capillary leak syndrome (N = 2), and 39-56% within abnormal regions affected by myocarditis (N = 7).
Conclusions
ECV mapping appears promising to complement LGE imaging in cases of more homogenously diffuse disease. The ability to display ECV maps in units that are physiologically intuitive and may be interpreted on an absolute scale offers the potential for detection of diffuse disease and measurement of the extent and severity of abnormal regions.
doi:10.1186/1532-429X-14-64
PMCID: PMC3442966  PMID: 22967246
Fibrosis; Edema; Gadolinium; Myocardial infarction; Hypertrophic cardiomyopathy; Dilated cardiomyopathy; Myocarditis; Systemic capillary leak syndrome
14.  An RGS4-Mediated Phenotypic Switch of Bronchial Smooth Muscle Cells Promotes Fixed Airway Obstruction in Asthma 
PLoS ONE  2012;7(1):e28504.
In severe asthma, bronchodilator- and steroid-insensitive airflow obstruction develops through unknown mechanisms characterized by increased lung airway smooth muscle (ASM) mass and stiffness. We explored the role of a Regulator of G-protein Signaling protein (RGS4) in the ASM hyperplasia and reduced contractile capacity characteristic of advanced asthma. Using immunocytochemical staining, ASM expression of RGS4 was determined in endobronchial biopsies from healthy subjects and those from subjects with mild, moderate and severe asthma. Cell proliferation assays, agonist-induced calcium mobilization and bronchoconstriction were determined in cultured human ASM cells and in human precision cut lung slices. Using gain- and loss-of-function approaches, the precise role of RGS proteins was determined in stimulating human ASM proliferation and inhibiting bronchoconstriction. RGS4 expression was restricted to a subpopulation of ASM and was specifically upregulated by mitogens, which induced a hyperproliferative and hypocontractile ASM phenotype similar to that observed in recalcitrant asthma. RGS4 expression was markedly increased in bronchial smooth muscle of patients with severe asthma, and expression correlated significantly with reduced pulmonary function. Whereas RGS4 inhibited G protein-coupled receptor (GPCR)-mediated bronchoconstriction, unexpectedly RGS4 was required for PDGF-induced proliferation and sustained activation of PI3K, a mitogenic signaling molecule that regulates ASM proliferation. These studies indicate that increased RGS4 expression promotes a phenotypic switch of ASM, evoking irreversible airway obstruction in subjects with severe asthma.
doi:10.1371/journal.pone.0028504
PMCID: PMC3257220  PMID: 22253691
15.  Narrative Review: Clarkson Disease-Systemic Capillary Leak Syndrome 
Annals of internal medicine  2010;153(2):90-98.
In 1960, Dr. Bayard Clarkson described a patient experiencing sporadic episodes of hypovolemia, hypotension, and edema. Plasma during the acute attack induced a “shock”-like syndrome when given systemically in rats. The unusual and enigmatic “Systemic Capillary Leak Syndrome” (SCLS) named for Dr. Clarkson is of unknown etiology and is characterized by transient, severe, reversible hemoconcentration and hypoalbuminemia due to leakage of fluids and macromolecules (up to 900 kDa) into tissues (1). Fewer than 150 cases of SCLS have been reported since 1960, but the nonspecific presenting symptoms and signs and high mortality rate may have resulted in under-recognition of this disorder. Given the substantial overlap of SCLS with other “shock” syndromes, including sepsis, anaphylaxis, and angioedema, clinicians should consider this diagnosis in patients with unexplained edema, increased hematocrit, and hypotension.
doi:10.1059/0003-4819-153-2-201007200-00005
PMCID: PMC3017349  PMID: 20643990
16.  RGS13 acts as a nuclear repressor of CREB 
Molecular cell  2008;31(5):660-670.
SUMMARY
Cyclic AMP-induced phosphorylation of the transcription factor CREB elicits expression of genes mediating diverse biological functions. In lymphoid organs, the neurotransmitter norepinephrine stimulates β2-adrenergic receptors on B lymphocytes to promote CREB-dependent expression of genes like the B-cell Oct 2 coactivator (OCA-B). Although CREB phosphorylation recruits co-factors such as CBP/p300 to stimulate transcription, bona fide endogenous inhibitors of CREB-coactivator or CREB-DNA interactions have not emerged. Here, we identified RGS13, a member of the Regulator of G protein Signaling (RGS) protein family, as a nuclear factor that suppresses CREB-mediated gene expression. cAMP or Ca2+ signaling promoted RGS13 accumulation in the nucleus, where it formed a complex with phosphorylated CREB and CBP/p300. RGS13 reduced the apparent affinity of pCREB for both the CRE and CBP. B lymphocytes from Rgs13−/− mice had more β2-agonist-induced OCA-B expression. Thus, RGS13 inhibits CREB-dependent transcription of target genes through disruption of complexes formed at the promoter.
doi:10.1016/j.molcel.2008.06.024
PMCID: PMC2600481  PMID: 18775326
17.  RGS13 controls G-protein-coupled receptor-evoked responses of human mast cells 
IgE-mediated mast cell degranulation and release of vasoactive mediators induced by allergens elicits allergic responses. Although G-protein-coupled receptor (GPCR)-induced signals may amplify IgE-dependent degranulation, how GPCR signaling in mast cells is regulated remains incompletely defined. We investigated the role of Regulator of G protein signaling (RGS) proteins in the modulation of these pathways in human mast cells. Several RGS proteins were expressed in mast cells including RGS13, which we previously showed inhibited IgE-mediated mast cell degranulation and anaphylaxis in mice. To characterize how RGS13 affects GPCR-mediated functions of human mast cells, we analyzed human mast cell lines (HMC-1 or LAD2) depleted of RGS13 by specific siRNA or shRNA and HMC-1 cells overexpressing RGS13. Transient RGS13 knockdown in LAD2 cells led to increased degranulation to sphingosine-1-phosphate, but not to antigen/IgE or C3a. Relative to control cells, HMC-1 cells stably expressing an RGS13-specific shRNA had greater Ca2+ mobilization in response to several GPCR ligands such as adenosine, C5a, sphingosine-1-phosphate (S-1P), and CXCL12 than wild-type cells. Akt phosphorylation, chemotaxis and cytokine (interleukin 8, IL-8) secretion induced by CXCL12 were also greater in shRGS13-HMC-1 cells compared to control. RGS13 overexpression inhibited CXCL12-evoked Ca2+ mobilization, Akt phosphorylation and chemotaxis. These results suggest that RGS13 restricts certain GPCR-mediated biological responses of human mast cells.
PMCID: PMC2693264  PMID: 19017978
Signal transduction; Chemokines; Mast cells/basophils
18.  Regulation of G-protein-coupled signaling pathways in allergic inflammation 
Immunologic research  2009;43(1-3):62-76.
Abstract Allergic diseases such as asthma are elicited by maladaptive activation of immune cells such as mast cells and lymphocytes by otherwise innocuous allergens. The numerous mediators secreted by such cells promote both acute inflammation and, in many instances, chronic tissue remodeling. Most of these compounds exert their effects on end-organ targets such as epithelial and endothelial cells and airway smooth muscle by activating G-protein-coupled receptors (GPCRs), which are by far the most abundant type of cell surface receptor. Since GPCRs are also the most common target of allergy therapeutics, a better understanding of their intracellular signaling mechanisms is vital to improve the efficacy of such drugs or to develop new targets. In this review, we focus on some of the new regulatory elements that control the duration and amplitude of GPCR signal transduction pathways in immune effector cells and end-organ structural cells affected by allergic inflammation.
doi:10.1007/s12026-008-8050-0
PMCID: PMC2687145  PMID: 18810336
G proteins; RGS proteins; Signal transduction; Allergy; Asthma; Inflammation; Mast cells; Lymphocytes; Bronchial smooth muscle
19.  Suppression of IgE-mediated allergic responses by Rgs13 
Nature immunology  2007;9(1):73-80.
Mast cells provoke allergic responses through degranulation and release of proinflammatory mediators after antigen crosslinking of the high affinity immunoglobulin E (IgE) receptor (FcεRI). Regulator of G protein Signaling (RGS) proteins negatively control G-protein-coupled receptor-mediated signaling through GTPase accelerating protein (GAP) activity. Here, we show that Rgs13 inhibits allergic responses by physically interacting with the regulatory p85α subunit of PI3K in mast cells and disrupting its association with an FcεRI-activated scaffold complex. Rgs13−/− mice exhibited increased IgE-mediated mast cell degranulation and anaphylaxis. Thus, apart from its regulation of GPCRs, Rgs13 inhibits immune receptor-induced signalosome assembly in MCs. Abnormal Rgs13 expression or function may underlie some cases of idiopathic anaphylaxis or disorders of amplified MC activity.
doi:10.1038/ni1533
PMCID: PMC2387203  PMID: 18026105
20.  RGS4 and RGS2 Bind Coatomer and Inhibit COPI Association with Golgi Membranes and Intracellular Transport 
Molecular Biology of the Cell  2000;11(9):3155-3168.
COPI, a protein complex consisting of coatomer and the small GTPase ARF1, is an integral component of some intracellular transport carriers. The association of COPI with secretory membranes has been implicated in the maintenance of Golgi integrity and the normal functioning of intracellular transport in eukaryotes. The regulator of G protein signaling, RGS4, interacted with the COPI subunit β′-COP in a yeast two-hybrid screen. Both recombinant RGS4 and RGS2 bound purified recombinant β′-COP in vitro. Endogenous cytosolic RGS4 from NG108 cells and RGS2 from HEK293T cells cofractionated with the COPI complex by gel filtration. Binding of β′-COP to RGS4 occurred through two dilysine motifs in RGS4, similar to those contained in some aminoglycoside antibiotics that are known to bind coatomer. RGS4 inhibited COPI binding to Golgi membranes independently of its GTPase-accelerating activity on Giα. In RGS4-transfected LLC-PK1 cells, the amount of COPI in the Golgi region was considerably reduced compared with that in wild-type cells, but there was no detectable difference in the amount of either Golgi-associated ARF1 or the integral Golgi membrane protein giantin, indicating that Golgi integrity was preserved. In addition, RGS4 expression inhibited trafficking of aquaporin 1 to the plasma membrane in LLC-PK1 cells and impaired secretion of placental alkaline phosphatase from HEK293T cells. The inhibitory effect of RGS4 in these assays was independent of GTPase-accelerating activity but correlated with its ability to bind COPI. Thus, these data support the hypothesis that these RGS proteins sequester coatomer in the cytoplasm and inhibit its recruitment onto Golgi membranes, which may in turn modulate Golgi–plasma membrane or intra-Golgi transport.
PMCID: PMC14982  PMID: 10982407

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