Anne Vroon

GRKs and arrestins: regulators of migration and inflammation

In the immune system, signaling by G protein‐coupled receptors (GPCRs) is crucial for the activity of multiple mediators, including chemokines, leukotrienes, and neurotransmitters. GPCR kinases (GRKs) and arrestins control GPCR signaling by mediating desensitization and thus, regulating further signal propagation through G proteins. Recent evidence suggests that the GRK‐arrestin desensitization machinery fulfills a vital role in regulating inflammatory processes. First, GRK/arrestin levels in immune cells are dynamically regulated in response to inflammation. Second, in animals with targeted deletion of GRKs or arrestins, the progression of various acute and chronic inflammatory disorders, including autoimmunity and allergy, is profoundly affected. Third, chemokine receptor signaling in vitro is known to be tightly regulated by the GRK/arrestin machinery, and even small changes in GRK/arrestin expression can have a marked effect on cellular responses to chemokines. This review integrates data about the role of GRKs and arrestins in inflammation, with results on the molecular mechanism of action of GRKs/arrestins, and describes the pivotal role of GRKs/arrestins in inflammatory processes, with a special emphasis on regulation of chemokine responsiveness.

G protein-coupled receptor kinase 2 in multiple sclerosis and experimental autoimmune encephalomyelitis

Many modulators of inflammation, including chemokines, neuropeptides, and neurotransmitters signal via G protein-coupled receptors (GPCR). GPCR kinases (GRK) can phosphorylate agonist-activated GPCR thereby promoting receptor desensitization. Here we describe that in leukocytes from patients with active relapsing-remitting multiple sclerosis (MS) or with secondary progressive MS, GRK2 levels are significantly reduced. Unexpectedly, cells from patients during remission express even lower levels of GRK2. The level of GRK2 in leukocytes of patients after stroke, a neurological disorder with paralysis but without an autoimmune component, was similar to GRK2 levels in cells from healthy individuals. In addition, we demonstrate that the course of recombinant myelin oligodendrocyte glycoprotein (1–125)-induced experimental autoimmune encephalomyelitis (EAE), an animal model for MS, is markedly different in GRK2+/− mice that express 50% of the GRK2 protein in comparison with wild-type mice. Onset of EAE was significantly advanced by 5 days in GRK2+/− mice. The earlier onset of EAE was associated with increased early infiltration of the CNS by T cells and macrophages. Although disease scores in the first phase of EAE were similar in both groups, GRK2+/− animals did not develop relapses, whereas wild-type animals did. The absence of relapses in GRK2+/− mice was associated with a marked reduction in inflammatory infiltrates in the CNS. Recombinant myelin oligodendrocyte glycoprotein-induced T cell proliferation and cytokine production were normal in GRK2+/− animals. We conclude that down-regulation of GRK2 expression may have important consequences for the onset and progression of MS.

Reduced GRK2 level in T cells potentiates chemotaxis and signaling in response to CCL4

Chemokine receptors belong to the family of G‐protein‐coupled receptors (GPCR). Phosphorylation of GPCR by GPCR kinases (GRKs) is considered to play an important role in desensitization of these receptors. We have recently shown in patients with rheumatoid arthritis that the level of GRK2 in lymphocytes is reduced by ∼50%. However, the physiological relevance of reduced GRK2 levels in lymphocytes is not known. Here, we investigated whether reduced GRK2 expression changes the chemotactic response of T cells to the chemokines CCL3, CCL4, and CCL5. Activated T cells from GRK2+/− mice, which have a 50% reduction in GRK2 protein levels, showed a significant 40% increase in chemotaxis toward the CCR5 ligand CCL4. In addition, chemotaxis toward the CCR1 and CCR5 ligands CCL3 and CCL5 was also increased. Binding of CCL4 to activated T cells from GRK2+/− and wild‐type (WT) mice was similar, but agonist‐induced CCR5 phosphorylation was attenuated in GRK2+/− cells. Moreover, the calcium response and phosphorylation of protein kinase B and extracellular‐regulated kinase in response to CCL4 were significantly increased in GRK2+/− T cells, showing that signaling is increased when the level of GRK2 is reduced. GRK2+/− and WT cells do become refractory to restimulation with CCL4. In conclusion, a 50% decrease in T cell GRK2 expression results in increased responsiveness to CCL3, CCL4, and CCL5, suggesting that the 50% reduction in lymphocyte GRK2 level as observed during inflammation can have functional consequences for the response of these cells to chemokines.

GRK6 deficiency is associated with enhanced CXCR4-mediated neutrophil chemotaxis in vitro and impaired responsiveness to G-CSF in vivo.

The stromal cell‐derived factor‐1 (SDF‐1)/CXC chemokine receptor 4 (CXCR4) signaling pathway is thought to play an important role in the induction of neutrophil mobilization from the bone marrow in response to granulocyte‐colony stimulating factor (G‐CSF) treatment. CXCR4 belongs to the family of G protein‐coupled receptors. Multiple members of this receptor family are desensitized by agonist‐induced G protein‐coupled receptor kinase (GRK)‐mediated phosphorylation. Here, we demonstrate that in vitro SDF‐1‐induced chemotaxis of bone marrow‐derived neutrophils from GRK6‐deficient mice is significantly enhanced and that desensitization of the calcium response to SDF‐1 is impaired in GRK6−/− neutrophils. CXCR4 activation by SDF‐1 provides a key retention signal for hematopoietic cells in the bone marrow. It is interesting that we observed that in the absence of GRK6, the G‐CSF‐induced increase in circulating neutrophils is profoundly impaired. Three days after injection of pegylated‐G‐CSF, significantly lower numbers of circulating neutrophils were observed in GRK6−/− as compared with wild‐type (WT) mice. In addition, early/acute neutrophil mobilization in response to G‐CSF (3 h after treatment) was also impaired in GRK6−/− mice. However, blood neutrophil levels in untreated GRK6−/− and WT mice were not different. Moreover, the percentage of neutrophils in the bone marrow after G‐CSF treatment was increased to the same extent in WT and GRK6−/− mice, indicating that neutrophil production is normal in the absence of GRK6. However, the increased chemotactic sensitivity of GRK6−/− neutrophils to SDF‐1 was retained after G‐CSF treatment. In view of these data, we suggest that the impaired G‐CSF‐induced neutrophil mobilization in the absence of GRK6 may be a result of enhanced CXCR4‐mediated retention of PMN in the bone marrow.

Increased Acute Inflammation, Leukotriene B4-Induced Chemotaxis, and Signaling in Mice Deficient for G Protein-Coupled Receptor Kinase 6

Directed migration of polymorphonuclear neutrophils (PMN) is required for adequate host defense against invading organisms and leukotriene B4 (LTB4) is one of the most potent PMN chemoattractants. LTB4 exerts its action via binding to BLT1, a G protein-coupled receptor. G protein-coupled receptors are phosphorylated by G protein-coupled receptor kinases (GRK) in an agonist-dependent manner, resulting in receptor desensitization. Recently, it has been shown that the human BLT1 is a substrate for GRK6. To investigate the physiological importance of GRK6 for inflammation and LTB4 signaling in PMN, we used GRK6-deficient mice. The acute inflammatory response (ear swelling and influx of PMN into the ear) after topical application of arachidonic acid was significantly increased in GRK6−/− mice. In vitro, GRK6−/− PMN showed increased chemokinetic and chemotactic responses to LTB4. GRK6−/− PMN respond to LTB4 with a prolonged increase in intracellular calcium and prolonged actin polymerization, suggesting impaired LTB4 receptor desensitization in the absence of GRK6. However, pre-exposure to LTB4 renders both GRK6−/− as well as wild-type PMN refractory to restimulation with LTB4, indicating that the presence of GRK6 is not required for this process to occur. In conclusion, GRK6 deficiency leads to prolonged BLT1 signaling and increased neutrophil migration.

Changes in the G-protein-coupled receptor desensitization machinery during relapsing–progressive experimental allergic encephalomyelitis

G-protein-coupled receptors (GPCR) play an important role in inflammation. Their responsiveness is regulated by G-protein-coupled receptor kinases (GRKs) and beta-arrestins. We show here that induction of experimental autoimmune encephalomyelitis (EAE) by myelin oligodendrocyte glycoprotein (MOG) resulted in a profound decrease in GRK2 and GRK6 protein in splenocytes during all phases of disease. GRK2 mRNA was also lower during EAE, although the decrease in mRNA was less pronounced than the decrease in GRK2 protein. Interestingly, beta-arrestin protein expression was significantly increased. Downregulation of GRK2 was restricted to the spleen and mesenteric lymph nodes and was not observed in peritoneal macrophages. Furthermore, EAE did not induce alterations in GRK2 expression in heart, liver and pituitary.

The β2-Adrenergic Agonist Salbutamol Potentiates Oral Induction of Tolerance, Suppressing Adjuvant Arthritis and Antigen-Specific Immunity

Therapeutic protocols for treating autoimmune diseases by feeding autoantigens during the disease process have not been very successful to date. In vitro it has been shown that β-adrenergic agonists inhibit pro-inflammatory cytokine production and up-regulate anti-inflammatory cytokine production. We hypothesized that the protective effect of oral administration of Ag would be enhanced by oral coadministration of the β2-adrenergic agonist salbutamol. Here we demonstrate that oral administration of salbutamol in combination with the Ag mycobacterial 65-kDa heat shock protein increased the efficacy of disease-suppressive tolerance induction in rat adjuvant arthritis. To study the mechanism of salbutamol in more detail, we also tested oral administration of salbutamol in an OVA tolerance model in BALB/c mice. Oral coadministration of OVA/salbutamol after immunization with OVA efficiently suppressed both cellular and humoral responses to OVA. Coadministration of salbutamol was associated with an immediate increase in IL-10, TGF-β, and IL-1R antagonist in the intestine. The tolerizing effect of salbutamol/OVA was maintained for at least 12 wk. At this time point IFN-γ production in Ag-stimulated splenocytes was increased in the OVA/salbutamol-treated animals. In conclusion, salbutamol can be of great clinical benefit for the treatment of autoimmune diseases by promoting oral tolerance induction.

Low Endogenous G-Protein-Coupled Receptor Kinase 2 Sensitizes the Immature Brain to Hypoxia–Ischemia-Induced Gray and White Matter Damage

Hypoxic–ischemic brain injury is regulated in part by neurotransmitter and chemokine signaling via G-protein-coupled receptors (GPCRs). GPCR-kinase 2 (GRK2) protects these receptors against overstimulation by inducing desensitization. Neonatal hypoxic–ischemic brain damage is preceded by a reduction in cerebral GRK2 expression. We determined the functional importance of GRK2 in hypoxic–ischemic brain damage. Nine-day-old wild-type and GRK2+/− mice with a ∼50% reduction in GRK2 protein were exposed to unilateral carotid artery occlusion and hypoxia. In GRK2+/− animals, gray and white matter damage was aggravated at 3 weeks after hypoxia–ischemia. In addition, cerebral neutrophil infiltration was increased in GRK2+/− animals. Neutrophil depletion reduced brain damage, but neuronal loss was still more pronounced in GRK2+/− animals. Onset of neuronal loss was advanced in GRK2+/− animals regardless of neutrophil depletion. White matter injury was advanced in GRK2+/− animals and was not affected by neutrophil depletion. Activation/infiltration of microglia/macrophages was stronger in GRK2+/− brains but only occurred 24 h after hypoxia–ischemia and is therefore not the primary cause of increased damage. During hypoxia, cerebral blood flow was reduced to the same extent in both genotypes. In vitro, GRK2+/− hippocampal slices and cerebellar granular neurons were more sensitive to glutamate-induced death. We propose the novel concept that the kinase GRK2 regulates onset and magnitude of hypoxic–ischemic brain damage. Increased gray and white matter damage in GRK2+/− animals was not dependent on infiltrating neutrophils and occurred before microglia/macrophage activation was detected. Collectively, our data suggest that cerebral GRK2 has an important endogenous neuroprotective role in ischemic cerebral damage.

Down-regulation of GRK2 after oxygen and glucose deprivation in rat hippocampal slices: role of the PI3-kinase pathway.

G protein‐coupled receptor kinase 2 (GRK2) modulates G protein‐coupled receptor desensitization and signaling. We previously described down‐regulation of GRK2 expression in vivo in rat neonatal brain following hypoxia‐ischemia. In this study, we investigated the molecular mechanisms involved in GRK2 down‐regulation, using organotypic cultures of neonatal rat hippocampal slices exposed to oxygen and glucose deprivation (OGD). We observed a 40% decrease in GRK2 expression 4 h post‐OGD. No changes in GRK2 protein occurred after exposure of hippocampal slices to glucose deprivation only. No significant alterations in GRK2 mRNA expression were detected, suggesting a post‐transcriptional effect of OGD on GRK2 expression. Blockade of the proteasome pathway by MG132 prevented OGD‐induced decrease of GRK2. It has been shown that extracellular signal‐regulated kinase‐dependent phosphorylation of GRK2 at Ser670 triggers its turnover via the proteasome pathway. However, despite a significant increase of pSer670‐GRK2 after OGD, inhibition of the extracellular signal‐regulated kinase pathway by PD98059 did neither prevent the hypoxia‐ischemia‐induced increase in pSer670‐GRK2 nor the down‐regulation of GRK2 protein. Interestingly, inhibition of phosphoinositide‐3‐kinase with wortmannin inhibits both OGD‐induced phosphorylation of GRK2 on Ser670 and the GRK2 decrease. In conclusion, OGD‐induced phosphoinositide‐3‐kinase‐dependent phosphorylation of GRK2 on Ser670 is a novel mechanism leading to down‐regulation of GRK2 protein via a proteasome‐dependent pathway.

In vitro adrenergic modulation of cellular immune functions in experimental autoimmune encephalomyelitis

OBJECTIVE To analyze the effects in vitro of alpha- and beta-adrenoceptor agonists on splenocyte proliferation and on proinflammatory cytokine production in splenocytes and peritoneal macrophages (MF) in different stages of EAE. METHODS Splenocytes and peritoneal macrophages were harvested in the acute phase of EAE and in remission, and from controls. The beta-agonist terbutaline, the alpha(1)-agonist methoxamine, and the alpha(2)-agonist UK-14304 were added with ConA or lipopolysaccharide (LPS). TNF-alpha and IFN-gamma contents in supernatant and splenocyte proliferation were determined. RESULTS Terbutaline and UK-14304 significantly suppressed TNF-alpha production by MF. However, EAE acute phase rats were resistant to the suppressive effect of UK-14304. Terbutaline significantly suppressed IFN-gamma and TNF-alpha production by splenocytes. EAE acute phase and remission animals showed reduced terbutaline-induced inhibition of IFN-gamma production. CONCLUSIONS Disturbed sympathetic-immune communication in EAE is characterized by alterations in adrenergic sensitivity via both alpha- and beta-adrenergic pathways.