Maria Stella Lombardi

Decreased expression and activity of G-protein-coupled receptor kinases in peripheral blood mononuclear cells of patients with rheumatoid arthritis

β2‐Adrenergic and chemokine receptor antagonists delay the onset and reduce the severity of joint injury in rheumatoid arthritis. β2‐Adrenergic and chemokine receptors belong to the G‐protein‐coupled receptor family whose responsiveness is turned off by the G‐protein‐coupled receptor kinase family (GRK‐1 to 6). GRKs phosphorylate receptors in an agonist‐dependent manner resulting in receptor/G‐protein uncoupling via subsequent binding of arrestin proteins. We assessed the activity of GRKs in lymphocytes of rheumatoid arthritis (RA) patients by rhodopsin phosphorylation. We found a significant decrease in GRK activity in RA subjects that is mirrored by a decrease in GRK‐2 protein expression. Moreover, GRK‐6 protein expression is reduced in RA patients whereas GRK‐5 protein levels were unchanged. In search of an underlying mechanism, we demonstrated that proinflammatory cytokines induce a decrease in GRK‐2 protein levels in leukocytes from healthy donors. Since proinflammatory cytokines are abundantly expressed in RA, it may provide an explanation for the decrease in GRK‐2 expression and activity in patients. No changes in β2‐adrenergic receptor number and Kd were detected. However, RA patients showed a significantly increased cAMP production and inhibition of TNF‐α production by β2‐adrenergic stimulation, suggesting that reduced GRK activity is associated with increased sensitivity to β2‐adrenergic activation.—Lombardi, M. S., Kavelaars, A., Schedlowski, M., Bijlsma, J. W. J., Okihara, K. L., Van de Pol, M., Ochsmann, S., Pawlak, C., Schmidt, R. E., Heijnen, C. J. Decreased expression and activity of G‐protein‐coupled receptor kinases in peripheral blood mono‐nuclear cells of patients with rheumatoid arthritis. FASEB J. 13, 715–725 (1999)

Adjuvant arthritis induces down-regulation of G protein-coupled receptor kinases in the immune system

G protein-coupled receptors (GPCR) play a crucial role in the regulation of the immune response by, e.g., chemokines, PGs, and β2-adrenergic agonists. The responsiveness of these GPCRs is turned off by the family of G protein-coupled receptor kinases (GRK1–6). These kinases act by phosphorylating the GPCR in an agonist-dependent manner, resulting in homologous desensitization of the receptor. Although GRKs are widely expressed throughout the body, leukocytes express relatively high levels of GRKs, in particular GRK2, -3, and -6. We investigated whether in vivo the inflammatory disease adjuvant arthritis (AA) induces changes in GRK expression and function in the immune system. In addition, we analyzed whether the systemic effects of AA also involve changes in GRKs in nonimmune organs. At the peak of the inflammatory process, we observed a profound down-regulation of GRK2, -3, and -6 in splenocytes and mesenteric lymph node cells from AA rats. Interestingly, no changes in GRK were observed in thymocytes and in nonimmune organs such as heart and pituitary. During the remission phase of AA, GRK levels in spleen and mesenteric lymph nodes are returning to baseline levels. The decrease in GRK2 at the peak of AA is restricted to CD45RA+ B cells and CD4+ T cells, and was not observed in CD8+ T cells. In conclusion, we demonstrate in this study, for the first time, that an inflammatory process in vivo induces a tissue-specific down-regulation of GRKs in the immune system.

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.

Bilateral molecular changes in a neonatal rat model of unilateral hypoxic-ischemic brain damage.

Perinatal hypoxia ischemia (HI) is a frequent cause of neonatal brain injury. This study aimed at describing molecular changes during the first 48 h after exposure of the neonatal rat brain to HI. Twelve-day-old rats were subjected to unilateral carotid artery occlusion and 90 min of 8% O2, leading to neuronal damage in the ipsilateral hemisphere only. Phosphorylated-Akt levels were decreased from 0.5 to 6 h post-HI, whereas the level of phosphorylated extracellular signal-related kinases (ERK)1/2 increased during this time frame. Hypoxia-inducible factor (HIF)-1α protein increased with a peak at 3 h after HI. mRNA expression for IL-β and tumor necrosis factor-α and -β started to increase at 6 h with a peak at 24 h post-HI. Expression of heat shock protein 70 was increased from 12 h after HI onwards in the ipsilateral hemisphere only. Surprisingly, HI changed the expression of cytokines, HIF1-α ,and P-Akt to the same extent in both the ipsi- as well as the contralateral hemisphere, although neuronal damage was unilateral. Exposure of animals to hypoxia without carotid artery occlusion induced similar changes in cytokines, HIF-1α, and P-Akt. We conclude that during HI, hypoxia is sufficient to regulate multiple molecular mediators that may contribute, but are not sufficient, to induce long-term neuronal damage.

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.

Hypoxia/Ischemia Modulates G Protein–Coupled Receptor Kinase 2 and β-Arrestin-1 Levels in the Neonatal Rat Brain

Background and Purpose— Neurotransmitters, neuropeptides, chemokines, and many other molecules signal through G protein–coupled receptors (GPCRs). GPCR kinases (GRKs) and &bgr;-arrestins play a crucial role in regulating the responsiveness of multiple GPCRs. Reduced expression of GRK and &bgr;-arrestins leads to supersensitization of GPCRs and will thereby increase the response to neuropeptides and neurotransmitters. We analyzed GRK and &bgr;-arrestin expression after cerebral hypoxia/ischemia (HI). Materials and Methods— Twelve-day-old rat pups were exposed to 90 minutes of hypoxia (fraction of inspired oxygen [FiO2] 0.08) after ligation of the right carotid artery, a procedure that induces unilateral damage in the right hemisphere. At 6, 12, 24, and 48 hours after HI, the left (hypoxic) and right (hypoxic/ischemic) hemispheres were analyzed for GRK and &bgr;-arrestin protein and mRNA expression by Western blotting and real-time polymerase chain reaction, respectively. In addition, we analyzed GRK2 expression in the hippocampus by immunohistochemistry. Results— HI downregulated GRK2 protein expression in both hemispheres at 24 to 48 hours after HI, and the effect was more pronounced in the ipsilateral hemisphere. HI induced no global change in GRK6 protein expression. However, GRK2 was markedly decreased in the hippocampal region of the ipsilateral hemisphere that will be severely damaged after HI. No changes in global mRNA levels for GRK2 were detected. In contrast, HI increased &bgr;-arrestin-1 protein expression as well as mRNA levels at 6 to 12 hours after HI. Conclusions— Neonatal HI-induced brain damage is associated with specific changes in the GPCR desensitization machinery. We hypothesize that these changes result in supersensitization of multiple GPCRs and might therefore contribute to HI-induced brain damage.

Selective Inhibition of Nuclear Factor-κB Activation After Hypoxia/Ischemia in Neonatal Rats Is Not Neuroprotective

Activated nuclear factor-κB (NFκB) has been shown to increase transcription of several genes that could potentially contribute to neuronal damage, such as proinflammatory cytokines, chemokines, and inducible nitric oxide synthase. The aim of our study was to investigate whether inhibition of NFκB activation could prevent hypoxia/ischemia (HI)-induced cerebral damage in neonatal rats. We used a cell permeable peptide (NEMO binding domain [NBD] peptide) that is known to prevent the association of the regulatory protein NEMO with IKK, the kinase that activates NFκB. Via this mechanism, the NBD peptide can specifically block the activation of NFκB, without inhibiting basal NFκB activity. Cerebral HI was induced in neonatal rats by occlusion of the right carotid artery followed by 90 min of hypoxia (Fio2 = 0.08). Immediately upon reoxygenation, as well as 6 and 12 h later, rats were treated with vehicle or NBD peptide (20 mg/kg i.p.). Histologic analysis of brain damage was performed at 6 wk after HI. To assess NFκB activation, electromobility shift assays (EMSAs) were performed on brain nuclear extracts obtained 6 h after reoxygenation. Increased NFκB activity could be shown at 6 h after HI in both hemispheres. Peripheral administration of NBD peptide prevented this HI-induced increase in NFκB activity in both hemispheres. Histologic analysis of long-term cerebral damage revealed that inhibition of NFκB activation by administration of NBD peptide at 0, 6, and 12 h after HI resulted in an increment of neuronal damage. In conclusion, our data suggest that inhibition of NFκB activation using NBD peptide early after HI increases brain damage in neonatal rats.

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.

#55 Inhibition of nuclear factor κB activation by NBD peptide following hypoxia–ischemia in neonatal rats aggravates damage

blood was cultured and ex vivo cytokine production in the presence of 5 lg/ml PHA and lipopolysaccharide (LPS) was measured. Leukocytes were prepared and stained for flow cytometry, with CD3, CD4, CD8, CD19, and CD56 measured. The results indicated that rape subjects had evidence of increased inflammatory responses and immune activation. They had higher levels of CD3, CD4, and CD56 cells, an increased CD4/CD8 ratio, lower mean cortisol and ACTH and higher levels of IL-6, IFN-c, neopterin, and CRP than matched controls. Rape subjects lymphocytes proliferated at a lower rate than controls when stimulated with T cell mitogens. Women reporting rape often display polar behavior, either very controlled and quiet or uncontrolled and agitated. There was a correlation between uncontrolled behavior and higher levels of CRP, and extreme controlled behavior and cortisol at the time of examination. The long term effects of rape include psychological distress (post-traumatic stress disorder, depression), and physical illnesses (irritable bowel syndrome, fibromyalgia, cancer, cardiovascular disease, and arthritis). Women reporting rape often are hypocortisolemic, a factor predictive of later pathology. By studying the early behavior and physiology in the post-rape period, the biobehavioral mechanisms for these sequelae may be elucidated.