RGS4 controls airway hyperresponsiveness through GAP-independent mechanisms.

Regulators of G protein signaling (RGS) proteins constrain G protein-coupled receptor (GPCR)-mediated and other responses throughout the body primarily, but not exclusively, through their GTPase-activating protein activity. Asthma is a highly prevalent condition characterized by airway hyper-responsiveness (AHR) to environmental stimuli resulting in part from amplified GPCR-mediated airway smooth muscle contraction. Rgs2 or Rgs5 gene deletion in mice enhances AHR and airway smooth muscle contraction, whereas RGS4 KO mice unexpectedly have decreased AHR because of increased production of the bronchodilator prostaglandin E2 (PGE2) by lung epithelial cells. Here, we found that knockin mice harboring Rgs4 alleles encoding a point mutation (N128A) that sharply curtails RGS4 GTPase-activating protein activity had increased AHR, reduced airway PGE2 levels, and augmented GPCR-induced bronchoconstriction compared with either RGS4 KO mice or WT controls. RGS4 interacted with the p85α subunit of PI3K and inhibited PI3K-dependent PGE2 secretion elicited by transforming growth factor beta in airway epithelial cells. Together, these findings suggest that RGS4 affects asthma severity in part by regulating the airway inflammatory milieu in a G protein-independent manner.

PARP15 is a Susceptibility Locus for Clarkson Disease (Monoclonal Gammopathy-Associated Systemic Capillary Leak Syndrome).

BACKGROUND: Vascular leakage is a deadly complication of severe infections, ranging from bacterial sepsis to malaria. Worldwide, septicemia is among the top 10 causes of lethality because of the shock and multiorgan dysfunction that arise from the host vascular response. In the monoclonal gammopathy-associated capillary leak syndrome (MG-CLS), even otherwise mundane infections induce recurrent septic-like episodes of profound microvascular hyperpermeability and shock. There are no defined genetic risk factors for MG-CLS or effective treatments for acute crises. METHODS: We characterized predicted loss-of-function mutations in PARP15 (poly[ADP-ribose] polymerase 15), a protein of unknown function that is absent in mice, in patients with MG-CLS. We analyzed barrier function in PARP15-deficient vascular endothelial cells and vascular leakage in mice engineered to express WT or loss-of-function variant human PARP15. RESULTS: We discovered several loss-of-function PARP15 variants associated with MG-CLS. These mutations severely reduced PARP15 enzymatic function. The presence of the most frequently detected variant (G628R) correlated with clinical markers of severe vascular leakage. In human microvascular endothelial cells, PARP15 suppressed cytokine-induced barrier disruption by ADP-ribosylating the scaffold protein JIP3 (c-Jun N-terminal kinase-interacting protein 3) and inhibiting p38 MAP kinase activation. Mice expressing enzymatically inactive human PARP15(G628R) were significantly more prone to inflammation-associated vascular leakage than mice expressing WT PARP15 in a p38-dependent fashion. CONCLUSIONS: PARP15 represents a previously unrecognized genetic susceptibility factor for MG-CLS. PARP15-mediated ADP ribosylation is an essential and genetically determined mechanism of the human vascular response to inflammation.

Markers of endothelial glycocalyx dysfunction in Clarkson disease.

BACKGROUND: Clarkson disease (monoclonal gammopathy-associated idiopathic systemic capillary leak syndrome, ISCLS) is a rare idiopathic condition marked by transient, relapsing-remitting episodes of systemic microvascular hyper-permeability, which liberates plasma fluid and macromolecules into the peripheral tissues. This pathology manifests clinically as the abrupt onset of hypotensive shock, hemoconcentration, and hypoalbuminemia. METHODS: We analysed endothelial glycocalyx (eGCX)-related markers in plasma from patients with ISCLS during acute disease flares and convalescence by ELISA and comprehensive proteomic profiling. We evaluated eGCX-related components and gene expression in cultured endothelial cells using RNA-sequencing, real-time PCR, and fluorescence staining. RESULTS: Serum levels of eGCX-related core components including hyaluronic acid (HA) and the core proteoglycan soluble syndecan-1 (sCD138) were elevated at baseline and during acute ISCLS flares. Serial measurements demonstrated that sCD138 levels peaked during the recovery (post-leak) phase of the illness. Proteomic analysis of matched acute and convalescent ISCLS plasma revealed increased abundance of eGCX-related proteins, including glypicans, thrombospondin-1 (TSP-1), and eGCX-degrading enzymes in acute compared to remission plasma. Abundance of endothelial cell damage markers did not differ in acute and baseline plasma. Expression of several eGCX-related genes and surface carbohydrate content in endothelial cells from patients with ISCLS did not differ significantly from that observed in healthy control cells. CONCLUSIONS: eGCX dysfunction, but not endothelial injury, may contribute to clinical symptoms of acute ISCLS. Serum levels of of eGCX components including sCD138 may be measured during acute episodes of ISCLS to monitor clinical status and therapeutic responses.

Fatal Exacerbations of Systemic Capillary Leak Syndrome Complicating Coronavirus Disease.

We report 2 fatal exacerbations of systemic capillary leak syndrome (SCLS), also known as Clarkson disease, associated with coronavirus disease (COVID-19) in the United States. One patient carried an established diagnosis of SCLS and the other sought treatment for new-onset hypotensive shock, hemoconcentration, and anasarca, classic symptoms indicative of an SCLS flare. Both patients had only mild-to-moderate symptoms of COVID-19. This clinical picture suggests that these patients succumbed to complications of SCLS induced by infection with severe acute respiratory syndrome coronavirus 2. Persons with known or suspected SCLS may be at increased risk for developing a disease flare in the setting of mild-to-moderate COVID-19 infection.

RGS4 promotes allergen- and aspirin-associated airway hyperresponsiveness by inhibiting PGE2 biosynthesis.

BACKGROUND: Allergens elicit host production of mediators acting on G-protein-coupled receptors to regulate airway tone. Among these is prostaglandin E2 (PGE2), which, in addition to its role as a bronchodilator, has anti-inflammatory actions. Some patients with asthma develop bronchospasm after the ingestion of aspirin and other nonsteroidal anti-inflammatory drugs, a disorder termed aspirin-exacerbated respiratory disease. This condition may result in part from abnormal dependence on the bronchoprotective actions of PGE2. OBJECTIVE: We sought to understand the functions of regulator of G protein signaling 4 (RGS4), a cytoplasmic protein expressed in airway smooth muscle and bronchial epithelium that regulates the activity of G-protein-coupled receptors, in asthma. METHODS: We examined RGS4 expression in human lung biopsies by immunohistochemistry. We assessed airways hyperresponsiveness (AHR) and lung inflammation in germline and airway smooth muscle-specific Rgs4-/- mice and in mice treated with an RGS4 antagonist after challenge with Aspergillus fumigatus. We examined the role of RGS4 in nonsteroidal anti-inflammatory drug-associated bronchoconstriction by challenging aspirin-exacerbated respiratory disease-like (ptges1-/-) mice with aspirin. RESULTS: RGS4 expression in respiratory epithelium is increased in subjects with severe asthma. Allergen-induced AHR was unexpectedly diminished in Rgs4-/- mice, a finding associated with increased airway PGE2 levels. RGS4 modulated allergen-induced PGE2 secretion in human bronchial epithelial cells and prostanoid-dependent bronchodilation. The RGS4 antagonist CCG203769 attenuated AHR induced by allergen or aspirin challenge of wild-type or ptges1-/- mice, respectively, in association with increased airway PGE2 levels. CONCLUSIONS: RGS4 may contribute to the development of AHR by reducing airway PGE2 biosynthesis in allergen- and aspirin-induced asthma.

Suppression of immunoglobulin E-mediated allergic responses by regulator of G protein signaling 13.

Mast cells elicit allergic responses through degranulation and release of proinflammatory mediators after antigen crosslinking of the immunoglobulin E receptor FcepsilonRI. Proteins of the 'regulator of G protein signaling' (RGS) family negatively control signaling mediated by G protein-coupled receptors through GTPase-accelerating protein activity. Here we show that RGS13 inhibited allergic responses by physically interacting with the regulatory p85alpha subunit of phosphatidylinositol-3-OH kinase in mast cells and disrupting its association with an FcepsilonRI-activated scaffolding complex. Rgs13-/- mice had enhanced immunoglobulin E-mediated mast cell degranulation and anaphylaxis. Thus, RGS13 inhibits the assembly of immune receptor-induced signalosomes in mast cells. Abnormal RGS13 expression or function may contribute to disorders of amplified mast cell activity, such as idiopathic anaphylaxis.

Neutrophil activation in systemic capillary leak syndrome (Clarkson disease).

Systemic capillary leak syndrome (SCLS; Clarkson disease) is a rare orphan disorder characterized by transient yet recurrent episodes of hypotension and peripheral oedema due to diffuse vascular leakage of fluids and proteins into soft tissues. Humoral mediators, cellular responses and genetic features accounting for the clinical phenotype of SCLS are virtually unknown. Here, we searched for factors altered in acute SCLS plasma relative to matched convalescent samples using multiplexed aptamer-based proteomic screening. Relative amounts of 612 proteins were changed greater than twofold and 81 proteins were changed at least threefold. Among the most enriched proteins in acute SCLS plasma were neutrophil granule components including bactericidal permeability inducing protein, myeloperoxidase and matrix metalloproteinase 8. Neutrophils isolated from blood of subjects with SCLS or healthy controls responded similarly to routine pro-inflammatory mediators. However, acute SCLS sera activated neutrophils relative to remission sera. Activated neutrophil supernatants increased permeability of endothelial cells from both controls and SCLS subjects equivalently. Our results suggest systemic neutrophil degranulation during SCLS acute flares, which may contribute to the clinical manifestations of acute vascular leak.

Regulator of G protein signaling 5 restricts neutrophil chemotaxis and trafficking.

Neutrophils are white blood cells that are mobilized to damaged tissues and to sites of pathogen invasion, providing the first line of host defense. Chemokines displayed on the surface of blood vessels promote migration of neutrophils to these sites, and tissue- and pathogen-derived chemoattractant signals, including N-formylmethionylleucylphenylalanine (fMLP), elicit further migration to sites of infection. Although nearly all chemoattractant receptors use heterotrimeric G proteins to transmit signals, many of the mechanisms lying downstream of chemoattractant receptors that either promote or limit neutrophil motility are incompletely defined. Here, we show that regulator of G protein signaling 5 (RGS5), a protein that modulates G protein activity, is expressed in both human and murine neutrophils. We detected significantly more neutrophils in the airways of Rgs5-/- mice than WT counterparts following acute respiratory virus infection and in the peritoneum in response to injection of thioglycollate, a biochemical proinflammatory stimulus. RGS5-deficient neutrophils responded with increased chemotaxis elicited by the chemokines CXC motif chemokine ligand 1 (CXCL1), CXCL2, and CXCL12 but not fMLP. Moreover, adhesion of these cells was increased in the presence of both CXCL2 and fMLP. In summary, our results indicate that RGS5 deficiency increases chemotaxis and adhesion, leading to more efficient neutrophil mobilization to inflamed tissues in mice. These findings suggest that RGS5 expression and activity in neutrophils determine their migrational patterns in the complex microenvironments characteristic of inflamed tissues.

RGS4 Overexpression in Lung Attenuates Airway Hyperresponsiveness in Mice.

A cardinal feature of asthma is airway hyperresponsiveness (AHR) to spasmogens, many of which activate G protein-coupled receptors (GPCRs) on airway smooth muscle (ASM) cells. Asthma subtypes associated with allergy are characterized by eosinophilic inflammation in the lung due to the type 2 immune response to allergens and proinflammatory mediators that promote AHR. The degree to which intrinsic abnormalities of ASM contribute to this phenotype remains unknown. The regulators of G protein signaling (RGS) proteins are a large group of intracellular proteins that inhibit GPCR signaling pathways. RGS2- and RGS5-deficient mice develop AHR spontaneously. Although RGS4 is upregulated in ASM from patients with severe asthma, the effects of increased RGS4 expression on AHR in vivo are unknown. Here, we examined the impact of forced RGS4 overexpression in lung on AHR using transgenic (Tg) mice. Tg RGS4 was expressed in bronchial epithelium and ASM in vivo, and protein expression in lung was increased at least 4-fold in Tg mice compared with wild-type (WT) mice. Lung slices from Tg mice contracted less in response to the m3 muscarinic receptor agonist methacholine compared with the WT, although airway resistance in live, unchallenged mice of both strains was similar. Tg mice were partially protected against AHR induced by fungal allergen challenge due to weakened contraction signaling in ASM and reduced type 2 cytokine (IL-5 and IL-13) levels in Tg mice compared with the WT. These results provide support for the hypothesis that increasing RGS4 expression and/or function could be a viable therapeutic strategy for asthma.

Idiopathic systemic capillary leak syndrome (Clarkson disease).

In 1960, Dr Bayard Clarkson described a woman experiencing sporadic recurrent episodes of shock and anasarca. Plasma from an acute attack induced a shock-like syndrome when injected into rats. The enigmatic systemic capillary leak syndrome (SCLS) named for Dr Clarkson is characterized by transient and severe but reversible hemoconcentration and hypoalbuminemia caused by leakage of fluids and macromolecules into tissues. Although less than 500 cases of SCLS have been reported in the literature since 1960, the condition is probably underdiagnosed because of a lack of awareness and a high mortality without treatment. Allergists should be vigilant of this diagnosis because its presentation can resemble more common plasma leakage syndromes, including angioedema or systemic anaphylaxis. Although the precise molecular cause of SCLS remains unknown, substantial advances over the last 5 years have increased our understanding of SCLS pathogenesis.

CXCL12 regulates platelet activation via the regulator of G-protein signaling 16.

The regulators of G protein signaling (RGS) protein superfamily negatively controls G protein-coupled receptor signal transduction pathways. One of the members of this family, RGS16, is highly expressed in megakaryocytes and platelets. Studies of its function in platelet and megakaryocyte biology have been limited, in part, due to lack of pharmacological inhibitors. For example, RGS16 overexpression inhibited CXC chemokine receptor 4 (CXCR4)-mediated megakaryocyte migration. More recent studies showed that the chemokine stromal cell-derived factor (SDF1α or CXCL12) regulates platelet function via CXCR4. Based on these considerations, the present study investigated the capacity of RGS16 to regulate CXCL12-dependent platelet function, using the RGS16 knockout mouse model (Rgs16(-/-)). RGS16-deficient platelets had increased protease activated receptor 4 and collagen-induced aggregation, as well as increased CXCL12-dependent agonist-induced aggregation, dense and alpha granule secretion, integrin αIIbß3 activation and phosphatidylserine exposure compared to those from WT littermates. CXCL12 alone did not stimulate aggregation or secretion in either RGS16-deficient or WT platelets. Furthermore, platelets from Rgs16(-/-) mice displayed enhanced phosphorylation of ERK and Akt following CXCL12 stimulation relative to controls. Finally, we also found that PKCδ is involved in regulating CXCL12-dependent activation of ERK and Akt, in the Rgs16-deficient platelets. Collectively, our findings provide the first evidence that RGS16 plays an important role in platelet function by modulating CXCL12-dependent platelet activation.

Eosinophil-associated ribonuclease 11 is a macrophage chemoattractant.

RNase A is the prototype of an extensive family of divergent proteins whose members share a unique disulfide-bonded tertiary structure, conserved catalytic motifs, and the ability to hydrolyze polymeric RNA. Several members of this family maintain independent roles as ribonucleases and modulators of innate immunity. Here we characterize mouse eosinophil-associated RNase (Ear) 11, a divergent member of the eosinophil ribonuclease cluster, and the only known RNase A ribonuclease expressed specifically in response to Th2 cytokine stimulation. Mouse Ear 11 is differentially expressed in somatic tissues at baseline (brain ≪ liver < lung < spleen); systemic stimulation with IL-33 results in 10-5000-fold increased expression in lung and spleen, respectively. Ear 11 is also expressed in response to protective priming of the respiratory mucosa with Lactobacillus plantarum; transcripts are detected both locally in lung as well as systemically in bone marrow and spleen. Mouse Ear 11 is enzymatically active, although substantially less so than mEar 1 and mEar 2; the relative catalytic efficiency (kcat/Km) of mEar 11 is diminished ∼1000-1500-fold. However, in contrast to RNase 2/EDN and mEar 2, which have been characterized as selective chemoattractants for CD11c(+) dendritic cells, mEar 11 has prominent chemoattractant activity for F4/80(+)CD11c(-) tissue macrophages. Chemoattractant activity is not dependent on full enzymatic activity, and requires no interaction with the pattern recognition receptor, Toll-like receptor 2 (TLR2). Taken together, this work characterizes a divergent RNase A ribonuclease with a unique expression pattern and function, and highlights the versatility of this family in promoting innate immunity.

RGS13 acts as a nuclear repressor of CREB.

Cyclic AMP-induced phosphorylation of the transcription factor CREB elicits expression of genes mediating diverse biological functions. In lymphoid organs, the neurotransmitter norepinephrine stimulates beta(2)-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 cofactors 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 Ca(2+) 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 beta(2)-agonist-induced OCA-B expression. Thus, RGS13 inhibits CREB-dependent transcription of target genes through disruption of complexes formed at the promoter.

Increased myocardial extracellular volume in active idiopathic systemic capillary leak syndrome.

BACKGROUND: The Systemic Capillary Leak Syndrome (SCLS) is a rare disorder of unknown etiology presenting as recurrent episodes of shock and peripheral edema due to leakage of fluid into soft tissues. Insights into SCLS pathogenesis are few due to the scarcity of cases, and the etiology of vascular barrier disruption in SCLS is unknown. Recent advances in cardiovascular magnetic resonance (CMR) allow for the quantitative assessment of the myocardial extracellular volume (ECV), which can be increased in conditions causing myocardial edema. We hypothesized that measurement of myocardial ECV may detect myocardial vascular leak in patients with SCLS. METHODS: Fifty-six subjects underwent a standard CMR examination at the NIH Clinical Center from 2009 until 2014: 20 patients with acute intermittent SCLS, six subjects with chronic SCLS, and 30 unaffected controls. Standard volumetric measurements; late gadolinium enhancement imaging and pre- and post-contrast T1 mapping were performed. ECV was calculated by calibration of pre- and post-contrast T1 values with blood hematocrit. RESULTS: Demographics and cardiac parameters were similar in both groups. There was no significant valvular disorder in either group. Subjects with chronic SCLS had higher pre-contrast myocardial T1 compared to healthy controls (T1: 1027 ± 44 v. 971 ± 41, respectively; p = 0.03) and higher myocardial ECV than patients with acute intermittent SCLS or controls: 33.8 ± 4.6, 26.9 ± 2.6, 26 ± 2.4, respectively; p = 0.007 v. acute intermittent; P = 0.0005 v. controls). When patients with chronic disease were analyzed together with five patients with acute intermittent disease who had just experienced an acute SCLS flare, ECV values were significantly higher than in subjects with acute intermittent SCLS in remission or age-matched controls and (31.2 ± 4.6 %, 26.5 ± 2.7 %, 26 ± 2.4 %, respectively; p = 0.01 v. remission, p = 0.001 v. controls). By contrast, T1 values did not distinguish these three subgroups (1008 ± 40, 978 ± 40, 971 ± 41, respectively, p = 0.2, active v. remission; p = 0.06 active v. controls). Abundant myocardial edema without evidence of acute inflammation was detected in cardiac tissue postmortem in one patient. CONCLUSIONS: Patients with active SCLS have significantly higher myocardial ECV than age-matched controls or SCLS patients in remission, which correlated with histopathological findings in one patient.

IL-17RA is essential for optimal localization of follicular Th cells in the germinal center light zone to promote autoantibody-producing B cells.

Germinal centers (GCs) provide a microenvironment that promotes and regulates the interactions of B cells with follicular Th (TFH) cells. In this study, we show that there are significantly higher frequencies of CXCR5(+)ICOS(+) TFH cells in autoimmune BXD2 mice, and these cells express both 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 16 (RGS16) 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 postdifferentiated 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.

Loss of regulator of G protein signaling 5 promotes airway hyperresponsiveness in the absence of allergic inflammation.

BACKGROUND: Although eosinophilic inflammation typifies allergic asthma, it is not a prerequisite for airway hyperresponsiveness (AHR), suggesting that underlying abnormalities in structural cells, such as airway smooth muscle (ASM), contribute to the asthmatic diathesis. Dysregulation of procontractile G protein-coupled receptor (GPCR) signaling in ASM could mediate enhanced contractility. OBJECTIVE: We explored the role of a regulator of procontractile GPCR signaling, regulator of G protein signaling 5 (RGS5), in unprovoked and allergen-induced AHR. METHODS: We evaluated GPCR-evoked Ca(2+) signaling, precision-cut lung slice (PCLS) contraction, and lung inflammation in naive and Aspergillus fumigatus-challenged wild-type and Rgs5(-/-) mice. We analyzed lung resistance and dynamic compliance in live anesthetized mice using invasive plethysmography. RESULTS: Loss of RGS5 promoted constitutive AHR because of enhanced GPCR-induced Ca(2+) mobilization in ASM. PCLSs from naive Rgs5(-/-) mice contracted maximally at baseline independently of allergen challenge. RGS5 deficiency had little effect on the parameters of allergic inflammation, including cell counts in bronchoalveolar lavage fluid, mucin production, ASM mass, and subepithelial collagen deposition. Unexpectedly, induced IL-13 and IL-33 levels were much lower in challenged lungs from Rgs5(-/-) mice relative to those seen in wild-type mice. CONCLUSION: Loss of RGS5 confers spontaneous AHR in mice in the absence of allergic inflammation. Because it is selectively expressed in ASM within the lung and does not promote inflammation, RGS5 might be a therapeutic target for asthma.

Vascular endothelial hyperpermeability induces the clinical symptoms of Clarkson disease (the systemic capillary leak syndrome).

The systemic capillary leak syndrome (SCLS) is a rare disorder characterized by transient episodes of hypotensive shock and anasarca thought to arise from reversible microvascular barrier dysfunction. Although the high prevalence of a monoclonal gammopathy of unknown significance in SCLS suggests a pathogenic contribution of endogenous immunoglobulins, the mechanisms of vascular hyperpermeability remain obscure. Herein, we report clinical and molecular findings on 23 patients, the largest SCLS case series to date. Application of episodic SCLS sera, but neither the purified immunoglobulin fraction nor sera obtained from patients during remission, to human microvascular endothelial cells caused vascular endothelial cadherin internalization, disruption of interendothelial junctions, actin stress fiber formation, and increased permeability in complementary functional assays without inducing endothelial apoptosis. Intravenous immunoglobulin, one promising therapy for SCLS, mitigated the permeability effects of episodic sera. Consistent with the presence of endogenous, nonimmunoglobulin, circulating permeability factor(s) constrained to SCLS episodes, we found that vascular endothelial growth factor (VEGF) and angiopoietin 2 (Ang2), were elevated in episodic SCLS sera but not in remission sera. Ab-based inhibition of Ang2 counteracted permeability induced by episodic SCLS sera. Comparable experiments with anti-VEGF Ab (bevacizumab) yielded less interpretable results, probably because of endothelial toxicity of VEGF withdrawal. Our results support a model of SCLS pathogenesis in which nonimmunoglobulin humoral factors such as VEGF and Ang2 contribute to transient endothelial contraction, suggesting a molecular mechanism for this highly lethal disorder.

Extension of the germinal center stage of B cell development promotes autoantibodies in BXD2 mice.

OBJECTIVE: Regulator of G protein signaling (RGS) proteins inhibit chemokine signaling by desensitizing G protein-coupled receptor signals. This study was undertaken to determine the mechanisms by which RGS13 promotes the generation of pathogenic autoantibodies in germinal centers (GCs), using BXD2-Rgs13-/- mice. METHODS: Confocal and light microscopy imaging techniques were used to determine the location of cells that express RGS13 and activation-induced cytidine deaminase (AID) in the mouse spleen, and the number of plasmablasts. The levels of GC and plasma cell program transcripts in GC B cells were determined by real-time quantitative polymerase chain reaction (qPCR). Differential interleukin-17 (IL-17)-mediated expression of RGS13 in GC versus non-GC B cells was analyzed using A20 and 70Z/3 B cells. RESULTS: In the spleens of BXD2 mice, RGS13 was mainly expressed by GC B cells and was stimulated by IL-17 but not IL-21. IL-17 up-regulated RGS13 in A20 GC cells but not 70Z/3 non-GC B cells. BXD2- Rgs13-/- mice exhibited smaller GCs and lower AID levels, suggesting lower somatic hypermutation and affinity maturation. However, GC B cells from BXD2- Rgs13-/- mice showed increased levels of IgMbright plasmablasts, up-regulation of the genes encoding plasma program, including interferon regulatory factor 4, B lymphocyte-induced maturation protein 1, and X-box binding protein 1 and the p-CREB target genes Fosb and Obf1, and down-regulation of the GC program genes Aid, Pax5, and Bach2 compared to BXD2 mice. BXD2-Rgs13-/- mice had lower titers of IgG autoantibodies and IgG deposits in the glomeruli, suggesting reduced autoantibody pathogenicity. CONCLUSION: RGS13 deficiency is associated with a reduction in GC program genes and the exit of fewer pathogenic IgM plasmablasts in BXD2 mice. Our findings indicate that prolonged GC program, mediated by up-regulation of RGS13, enhances AID expression and enables the generation of pathogenic autoantibodies in autoreactive GCs.

RGS16 attenuates pulmonary Th2/Th17 inflammatory responses.

The regulators of G protein signaling (RGS) protein superfamily negatively controls G protein-coupled receptor signal transduction pathways. RGS16 is enriched in activated/effector T lymphocytes. In this paper, we show that RGS16 constrains pulmonary inflammation by regulating chemokine-induced T cell trafficking in response to challenge with Schistosoma mansoni. Naive 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 counterparts. Distinct Th2 or putative Th17 subsets expressing CCR4 or CCR10 accumulated more rapidly in Rgs16(-/-) lungs following challenge and produced proinflammatory 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.