Chantal Moratz

RGS13 Regulates Germinal Center B Lymphocytes Responsiveness to CXC Chemokine Ligand (CXCL)12 and CXCL13

Normal lymphoid tissue development and function depend upon directed cell migration. Providing guideposts for cell movement and positioning within lymphoid tissues, chemokines signal through cell surface receptors that couple to heterotrimeric G proteins, which are in turn subject to regulation by regulator of G protein signaling (RGS) proteins. In this study, we report that germinal center B lymphocytes and thymic epithelial cells strongly express one of the RGS family members, RGS13. Located between Rgs1 and Rgs2, Rgs13 spans 42 kb on mouse chromosome 1. Rgs13 encodes a 157-aa protein that shares 82% amino acid identity with its 159-aa human counterpart. In situ hybridization with sense and antisense probes localized Rgs13 expression to the germinal center regions of mouse spleens and Peyer’s patches and to the thymus medulla. Affinity-purified RGS13 Abs detected RGS13-expressing cells in the light zone of the germinal center. RGS13 interacted with both Giα and Gqα and strongly impaired signaling through Gi-linked signaling pathways, including signaling through the chemokine receptors CXCR4 and CXCR5. Prolonged CD40 signaling up-regulated RGS13 expression in human tonsil B lymphocytes. These results plus previous studies of RGS1 indicate the germinal center B cells use two RGS proteins, RGS1 and RGS13, to regulate their responsiveness to chemokines.

Toll-Like Receptor Signaling Alters the Expression of Regulator of G Protein Signaling Proteins in Dendritic Cells: Implications for G Protein-Coupled Receptor Signaling

Conserved structural motifs on pathogens trigger pattern recognition receptors present on APCs such as dendritic cells (DCs). An important class of such receptors is the Toll-like receptors (TLRs). TLR signaling triggers a cascade of events in DCs that includes modified chemokine and cytokine production, altered chemokine receptor expression, and changes in signaling through G protein-coupled receptors (GPCRs). One mechanism by which TLR signaling could modify GPCR signaling is by altering the expression of regulator of G protein signaling (RGS) proteins. In this study, we show that human monocyte-derived DCs constitutively express significant amounts of RGS2, RGS10, RGS14, RGS18, and RGS19, and much lower levels of RGS3 and RGS13. Engagement of TLR3 or TLR4 on monocyte-derived DCs induces RGS16 and RGS20, markedly increases RGS1 expression, and potently down-regulates RGS18 and RGS14 without modifying other RGS proteins. A similar pattern of Rgs protein expression occurred in immature bone marrow-derived mouse DCs stimulated to mature via TLR4 signaling. The changes in RGS18 and RGS1 expression are likely important for DC function, because both proteins inhibit Gαi- and Gαq-mediated signaling and can reduce CXC chemokine ligand (CXCL)12-, CC chemokine ligand (CCL)19-, or CCL21-induced cell migration. Providing additional evidence, bone marrow-derived DCs from Rgs1−/− mice have a heightened migratory response to both CXCL12 and CCL19 when compared with similar DCs prepared from wild-type mice. These results indicate that the level and functional status of RGS proteins in DCs significantly impact their response to GPCR ligands such as chemokines.

Regulator of G Protein Signaling 1 (RGS1) Markedly Impairs Giα Signaling Responses of B Lymphocytes

Regulator of G protein signaling (RGS) proteins modulate signaling through pathways that use heterotrimeric G proteins as transducing elements. RGS1 is expressed at high levels in certain B cell lines and can be induced in normal B cells by treatment with TNF-α. To determine the signaling pathways that RGS1 may regulate, we examined the specificity of RGS1 for various Gα subunits and assessed its effect on chemokine signaling. G protein binding and GTPase assays revealed that RGS1 is a Giα and Gqα GTPase-activating protein and a potential G12α effector antagonist. Functional studies demonstrated that RGS1 impairs platelet activating factor-mediated increases in intracellular Ca+2, stromal-derived factor-1-induced cell migration, and the induction of downstream signaling by a constitutively active form of G12α. Furthermore, germinal center B lymphocytes, which are refractory to stromal-derived factor-1-triggered migration, express high levels of RGS1. These results indicate that RGS proteins can profoundly effect the directed migration of lymphoid cells.

Pathogenic Natural Antibodies Recognizing Annexin IV Are Required to Develop Intestinal Ischemia-Reperfusion Injury

Intestinal ischemia-reperfusion (IR) injury is initiated when natural IgM Abs recognize neo-epitopes that are revealed on ischemic cells. The target molecules and mechanisms whereby these neo-epitopes become accessible to recognition are not well understood. Proposing that isolated intestinal epithelial cells (IEC) may carry IR-related neo-epitopes, we used in vitro IEC binding assays to screen hybridomas created from B cells of unmanipulated wild-type C57BL/6 mice. We identified a novel IgM mAb (mAb B4) that reacted with the surface of IEC by flow cytometric analysis and was alone capable of causing complement activation, neutrophil recruitment and intestinal injury in otherwise IR-resistant Rag1−/− mice. mAb B4 was found to specifically recognize mouse annexin IV. Preinjection of recombinant annexin IV blocked IR injury in wild-type C57BL/6 mice, demonstrating the requirement for recognition of this protein to develop IR injury in the context of a complex natural Ab repertoire. Humans were also found to exhibit IgM natural Abs that recognize annexin IV. These data in toto identify annexin IV as a key ischemia-related target Ag that is recognized by natural Abs in a pathologic process required in vivo to develop intestinal IR injury.

Abnormal B-Cell Responses to Chemokines, Disturbed Plasma Cell Localization, and Distorted Immune Tissue Architecture in Rgs1 / Mice

ABSTRACT Normal lymphoid tissue development and function depend upon chemokine-directed cell migration. Since chemokines signal through heterotrimeric G-protein-coupled receptors, RGS proteins, which act as GTPase-activating proteins for Gα subunits, likely fine tune the cellular responses to chemokines. Here we show that Rgs1−/− mice possess B cells that respond excessively and desensitize improperly to the chemokines CXCL12 and CXCL13. Many of the B-cell follicles in the spleens of Rgs1−/− mice have germinal centers even in the absence of immune stimulation. Furthermore, immunization of these mice leads to exaggerated germinal center formation; partial disruption of the normal architecture of the spleen and Peyers patches; and abnormal trafficking of immunoglobulin-secreting cells. These results reveal the importance of a regulatory mechanism that limits and desensitizes chemokine receptor signaling.

IL-17 producing CD4+ T cells mediate accelerated ischemia/reperfusion-induced injury in autoimmunity-prone mice.

Elements of the innate and adaptive immune response have been implicated in the development of tissue damage after ischemic reperfusion (I/R). Here we demonstrate that T cells infiltrate the intestine of C57BL/6 mice subjected to intestinal I/R during the first hour of reperfusion. The intensity of the T cell infiltration was higher in B6.MRL/lpr mice subjected to intestinal I/R and reflected more severe tissue damage than that observed in control mice. Depletion of T cells limited I/R damage in B6.MRL/lpr mice, whereas repletion of B6.MRL/lpr lymph node-derived T cells into the I/R-resistant Rag-1(-/-) mouse reconstituted tissue injury. The tissue-infiltrating T cells were found to produce IL-17. Finally, IL-23 deficient mice, which are known not to produce IL-17, displayed significantly less intestinal damage when subjected to I/R. Our data assign T cells a major role in intestinal I/R damage by virtue of producing the pro-inflammatory cytokine IL-17.

CXCR3 Is Induced Early on the Pathway of CD4+ T Cell Differentiation and Bridges Central and Peripheral Functions

Chemokine receptors on T cells are frequently categorized as functioning either in immune system homeostasis within lymphoid organs, or in peripheral inflammation. CXCR3 is in the latter category and is reported to be expressed selectively on Th1 cells. We found that CXCR3 was expressed in vivo on newly activated tonsillar CD4+ T cells. Using CD4+ T cells from cord blood, we found that CXCR3 was induced by cellular activation in vitro independently of the cytokine milieu, although on resting cells, expression was maintained preferentially on those that had been activated in type 1 conditions. In inflamed tonsils, CXCR3+CD4+ T cells were localized around and within germinal centers. The inference that CXCR3 has a role in germinal center reactions was supported by the finding that the CXCR3 ligand CXC chemokine ligand 9 was expressed in a pattern demarcating a subset of germinal centers both in tonsil and in lymph nodes from an HIV-infected individual. We next investigated the role of CXCR3 on peripheral effector/memory CD4+ T cells by comparing its pattern of expression with that of CCR5, another Th1-cell associated chemokine receptor. Analysis of cells directly from peripheral blood and after activation in vitro suggested that CXCR3 expression preceded that of CCR5, supporting a model of sequential induction of chemokine receptors during CD4+ T cell differentiation. Taken together, our data show that CXCR3 can be expressed at all stages of CD4+ T cell activation and differentiation, bridging central function in lymphoid organs and effector function in peripheral tissues.

Regulation of chemokine-induced lymphocyte migration by RGS proteins

G-protein-coupled receptors (GPCRs) activate heterotrimeric G proteins by inducing the G-protein alpha (Galpha) subunit to exchange guanosine diphosphate for guanosine triphosphate. Regulators of G-protein signaling (RGS) proteins enhance the deactivation of Galpha subunits, thereby reducing the activation of downstream effectors. Several members of the RGS family are expressed in lymphocytes. Among RGS proteins with the highest levels of expression are RGS1, RGS2, RGS10, RGS13, RGS14, RGS16, and RGS19. Perhaps the most important G-protein-coupled receptors in lymphocytes potentially subject to regulation by RGS proteins are the chemokine receptors. By signaling through these receptors, chemokines help orchestrate immune cell trafficking both during the development of the immune system and during responses to exogenous or infectious agents. Thus, the level and regulation of RGS proteins in lymphocytes likely significantly impact lymphocyte migration and function. This article provides some tools for the analysis of RGS protein expression in lymphocytes and outlines a number of methods for the analysis of the effects of RGS proteins on lymphocyte migration and chemokine receptor signaling.

Anti-ribonucleoprotein antibodies mediate enhanced lung injury following mesenteric ischemia/reperfusion in Rag-1−/− mice

Natural Abs and autoantibodies bind antigens displayed by ischemia-conditioned tissues, followed by complement activation and enhanced tissue injury during reperfusion. Anti-ribonucleoprotein (RNP) Ab is associated with lung disease in patients with autoimmune disease but it is not known whether these abs contribute to lung injury. Mesenteric I/R in mice leads to local and remote lung injury. Accordingly, we used this model to investigate whether anti-RNP Abs would reconstitute I/R damage with prominent lung damage in injury-resistant Rag1− / − animals. Rag1− / − mice injected with anti-RNP Ab containing serum and subjected to mesenteric I/R suffered greater intestinal injury than control-treated and sham-operated animals. The magnitude of the reconstituted damage was anti-RNP Ab titer-dependent. Anti-RNP Ab-treated animals demonstrated a dose-dependent increase in lung histologic injury scores compared to control and sham animals. Anti-RNP mediated injury was shown to be complement dependent. These experiments reveal a novel mechanism whereby anti-RNP Abs contributes to the development of pulmonary pathology in patients with autoimmune diseases following exposure of remote organs to I/R injury.

Decay-Accelerating Factor Mitigates Controlled Hemorrhage-Instigated Intestinal and Lung Tissue Damage and Hyperkalemia in Swine

BACKGROUND Activation of complement system has been associated with tissue injury after hemorrhage and resuscitation in rats and swine. This study investigated whether administration of human recombinant decay-accelerating factor (DAF; a complement regulatory protein that inhibits classical and alternative pathways) reduces tissue damage in a porcine model of hemorrhagic shock. METHODS Male Yorkshire swine assigned to four groups were subjected to controlled, isobaric hemorrhage over 15 minutes to a target mean arterial pressure of 35 mm Hg. Hypotension was maintained for 20 minutes followed by a bolus intravenous injection of DAF or vehicle and then animals were observed for 200 minutes. Blood chemistry and physiologic parameters were recorded. Tissue samples from lung and small intestine were subjected to histopathological evaluation and detection of tissue deposition of complement proteins by immunohistochemistry and Western blot analyses. RESULTS Administration of DAF significantly reduced intestinal and lung tissue damage in a dose-dependent manner (5, 25, and 50 μg/kg). In addition, DAF treatment improved hemorrhage-induced hyperkalemia. The protective effects of DAF appear to be related to its ability to reduce tissue complement activation and deposition on affected tissues. CONCLUSIONS DAF treatment decreased tissue complement activation and deposition in hemorrhaged animals and attenuated tissue damage at 200 minutes after treatment. The observed beneficial effects of DAF treatment on tissue injury after 20 minutes of severe hypotension presents an attractive model of small volume resuscitation, particularly in situations with a restrictive medical logistical footprint such as far-forward access to first responders in the battlefield or in remote rural or mountainous environments.