Annemieke Kavelaars

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)

Natural Selection of Human Embryos: Impaired Decidualization of Endometrium Disables Embryo-Maternal Interactions and Causes Recurrent Pregnancy Loss

Background Recurrent pregnancy loss (RPL), defined as 3 or more consecutive miscarriages, is widely attributed either to repeated chromosomal instability in the conceptus or to uterine factors that are poorly defined. We tested the hypothesis that abnormal cyclic differentiation of endometrial stromal cells (ESCs) into specialized decidual cells predisposes to RPL, based on the observation that this process may not only be indispensable for placenta formation in pregnancy but also for embryo recognition and selection at time of implantation. Methodology/Principal Findings Analysis of mid-secretory endometrial biopsies demonstrated that RPL is associated with decreased expression of the decidual marker prolactin (PRL) but increased levels of prokineticin-1 (PROK1), a cytokine that promotes implantation. These in vivo findings were entirely recapitulated when ESCs were purified from patients with and without a history of RPL and decidualized in culture. In addition to attenuated PRL production and prolonged and enhanced PROK1 expression, RPL was further associated with a complete dysregulation of both markers upon treatment of ESC cultures with human chorionic gonadotropin, a glycoprotein hormone abundantly expressed by the implanting embryo. We postulated that impaired embryo recognition and selection would clinically be associated with increased fecundity, defined by short time-to-pregnancy (TTP) intervals. Woman-based analysis of the mean and mode TTP in a cohort of 560 RPL patients showed that 40% can be considered “superfertile”, defined by a mean TTP of 3 months or less. Conclusions Impaired cyclic decidualization of the endometrium facilitates implantation yet predisposes to subsequent pregnancy failure by disabling natural embryo selection and by disrupting the maternal responses to embryonic signals. These findings suggest a novel pathological pathway that unifies maternal and embryonic causes of RPL.

Mesenchymal stem cell treatment after neonatal hypoxic-ischemic brain injury improves behavioral outcome and induces neuronal and oligodendrocyte regeneration.

Birth asphyxia is a frequent cause of perinatal morbidity and mortality and treatment options are very limited. Our aim was to determine the effects of treatment with bone marrow-derived mesenchymal stem cells (MSC) after neonatal hypoxic-ischemic brain injury (HI). Nine-day old mice were exposed to cerebral HI and endogenous cell proliferation was determined by BrdU-incorporation. Maximal endogenous cell proliferation, indicative for a trophic milieu, was observed at 3 days after HI. MSC transplantation at this time point decreased neuronal and oligodendrocyte loss when determined 21 days after HI by 42% and 31%, respectively. MSC treatment enhanced BrdU-incorporation in the ischemic hemisphere mainly in cells of recipient origin. The percentage of recently divided neurons and oligodendrocytes in hippocampus and cortex was increased after MSC transplantation. MSC treatment reduced the percentage of cortical and increased the percentage of hippocampal BrdU+-astrocytes. The percentage of BrdU+-microglia decreased after MSC treatment. Motoric behavior in the cylinder rearing test at 10 and 21 days after HI was significantly improved by MSC treatment 3 days after the insult. Moreover, even when treatment was started at 10 days after HI, there was a significant reduction in lesion size and improvement of behavioral outcome. Our data show that MSC treatment after neonatal HI brain damage improved functional outcome, reduced lesion volume, increased differentiation of recently divided cells towards neurons and oligodendrocytes and decreased proliferating inflammatory cells. We propose that MSC transplantation is a powerful treatment to improve behavioral outcome and cerebral lesion volume after neonatal brain damage via stimulation of endogenous repair processes.

NATURAL SELECTION OF HUMAN EMBRYOS: DECIDUALIZING ENDOMETRIAL STROMAL CELLS SERVE AS SENSORS OF EMBRYO QUALITY UPON IMPLANTATION

Background Pregnancy is widely viewed as dependent upon an intimate dialogue, mediated by locally secreted factors between a developmentally competent embryo and a receptive endometrium. Reproductive success in humans is however limited, largely because of the high prevalence of chromosomally abnormal preimplantation embryos. Moreover, the transient period of endometrial receptivity in humans uniquely coincides with differentiation of endometrial stromal cells (ESCs) into highly specialized decidual cells, which in the absence of pregnancy invariably triggers menstruation. The role of cyclic decidualization of the endometrium in the implantation process and the nature of the decidual cytokines and growth factors that mediate the crosstalk with the embryo are unknown. Methodology/Principal Findings We employed a human co-culture model, consisting of decidualizing ESCs and single hatched blastocysts, to identify the soluble factors involved in implantation. Over the 3-day co-culture period, approximately 75% of embryos arrested whereas the remainder showed normal development. The levels of 14 implantation factors secreted by the stromal cells were determined by multiplex immunoassay. Surprisingly, the presence of a developing embryo had no significant effect on decidual secretions, apart from a modest reduction in IL-5 levels. In contrast, arresting embryos triggered a strong response, characterized by selective inhibition of IL-1β, -6, -10, -17, -18, eotaxin, and HB-EGF secretion. Co-cultures were repeated with undifferentiated ESCs but none of the secreted cytokines were affected by the presence of a developing or arresting embryo. Conclusions Human ESCs become biosensors of embryo quality upon differentiation into decidual cells. In view of the high incidence of gross chromosomal errors in human preimplantation embryos, cyclic decidualization followed by menstrual shedding may represent a mechanism of natural embryo selection that limits maternal investment in developmentally impaired pregnancies.

β-Endorphin: Cytokine and neuropeptide

A decade of cytokine research has revealed that these mediators play a pivotal role in the functioning of many organ systems. The immune system is not the exclusive producer of cytokines. These mediators are also produced in, e.g., the brain, gastro-inlestinal tract and liver (Breder et al. 1988, Farrar et al. 1987, Dinarello 1984, Oppenheim & Gery 1982). In view of the fact that the immune system can react to cytokines produced by cell types other than leukocytes, immunologists had to accept that the immune system is not functioning autonomously, but that it is constantly communicating with other organ systems in order to maintain its homeostasis.

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.

Endometrial secretion analysis identifies a cytokine profile predictive of pregnancy in IVF

BACKGROUND The study of human endometrial-embryonic interactions is complicated by the disruptive impact of endometrial sample collection on the process of implantation itself. Endometrial secretion analysis is a novel technique, non-disruptive to implantation. The primary aim of this prospective cohort study was to explore whether a cytokine profile predictive of implantation and clinical pregnancy can be identified in endometrial secretions aspirated immediately prior to embryo transfer following IVF. METHODS Endometrial secretions, aspirated immediately prior to embryo transfer from 210 women undergoing IVF, were analyzed using a multiplex immunoassay for 17 soluble regulators of implantation, namely interleukin (IL)-1beta, IL-5, IL-6, IL-10, IL-12, IL-15, IL-17, IL-18, tumor necrosis factor (TNF)-alpha, interferon (IFN)-gamma, macrophage migration inhibitory factor, eotaxin, IFN-gamma-inducible 10 kDa protein (IP-10), monocyte chemo-attractant protein-1 (MCP-1), Dickkopf homolog 1, heparin-binding epidermal growth factor and vascular endothelial growth factor (VEGF). In order to detect implantation, daily urine samples were collected after embryo transfer, and human Chorionic Gonadotropin (hCG) concentrations were analyzed by an immunoassay. RESULTS Multivariable logistic regression analysis revealed significant associations (negative and positive association, respectively) between MCP-1 (P = 0.005) and IP-10 (P = 0.037) levels and implantation, and between IL-1beta (P = 0.047) and TNF-alpha (P = 0.023) levels and clinical pregnancy. The predictive value for pregnancy of IL-1beta and TNF-alpha was observed to be equivalent and additive to that of embryo quality. CONCLUSIONS Endometrial secretion cytokine profiling offers a novel, non-disruptive approach to study the role of the endometrium in human embryo implantation and identifies a profile which appears to be conducive to clinical pregnancy. CLINICAL TRIAL REGISTRATION www.clinicaltrials.gov (nCT00264992).

Extent of bilateral neuronal network reorganization and functional recovery in relation to stroke severity.

Remodeling of neuronal structures and networks is believed to significantly contribute to (partial) restoration of functions after stroke. However, it has been unclear to what extent the brain reorganizes and how this correlates with functional recovery in relation to stroke severity. We applied serial resting-state functional MRI and diffusion tensor imaging together with behavioral testing to relate longitudinal modifications in functional and structural connectivity of the sensorimotor neuronal network to changes in sensorimotor function after unilateral stroke in rats. We found that gradual improvement of functions is associated with wide-ranging changes in functional and structural connectivity within bilateral neuronal networks, particularly after large stroke. Both after medium and large stroke, brain reorganization eventually leads to (partial) normalization of neuronal signal synchronization within the affected sensorimotor cortical network (intraregional signal coherence), as well as between the affected and unaffected sensorimotor cortices (interhemispheric functional connectivity). Furthermore, the bilateral network configuration shifts from subacutely increased “small-worldness,” possibly reflective of initial excessive neuronal clustering and wiring, toward a baseline small-world topology, optimal for global information transfer and local processing, at chronic stages. Cortical network remodeling was accompanied by recovery of initially disrupted structural integrity in corticospinal tract regions, which correlated positively with retrieval of sensorimotor functions. Our study demonstrates that the degree of functional recovery after stroke is associated with the extent of preservation or restoration of ipsilesional corticospinal tracts in combination with reinstatement of interhemispheric neuronal signal synchronization and normalization of small-world cortical network organization.

Neuroinflammation and Comorbidity of Pain and Depression

Comorbid depression and chronic pain are highly prevalent in individuals suffering from physical illness. Here, we critically examine the possibility that inflammation is the common mediator of this comorbidity, and we explore the implications of this hypothesis. Inflammation signals the brain to induce sickness responses that include increased pain and negative affect. This is a typical and adaptive response to acute inflammation. However, chronic inflammation induces a transition from these typical sickness behaviors into depression and chronic pain. Several mechanisms can account for the high comorbidity of pain and depression that stem from the precipitating inflammation in physically ill patients. These mechanisms include direct effects of cytokines on the neuronal environment or indirect effects via downregulation of G protein–coupled receptor kinase 2, activation of the tryptophan-degrading enzyme indoleamine 2,3-dioxygenase that generates neurotropic kynurenine metabolites, increased brain extracellular glutamate, and the switch of GABAergic neurotransmission from inhibition to excitation. Despite the existence of many neuroimmune candidate mechanisms for the co-occurrence of depression and chronic pain, little work has been devoted so far to critically assess their mediating role in these comorbid symptoms. Understanding neuroimmune mechanisms that underlie depression and pain comorbidity may yield effective pharmaceutical targets that can treat both conditions simultaneously beyond traditional antidepressants and analgesics.

Functional α1-adrenergic receptors on leukocytes of patients with polyarticular juvenile rheumatoid arthritis

During the last decade it has been shown that the central nervous system can influence the immune system. In healthy individuals, catecholamines can inhibit the production of pro-inflammatory cytokines like interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-alpha) via interaction with beta 2-adrenergic receptors. In contrast, we show here that catecholamines can stimulate the production of the interleukin-6 (IL-6) in children with the chronic inflammatory disease polyarticular juvenile rheumatoid arthritis (JRA). The induction of IL-6 is mediated by triggering of alpha 1-adrenergic receptors on peripheral blood leucocytes of the patients with polyarticular JRA. Functional alpha 1-adrenergic receptors are absent on leukocytes of normal donors and on leukocytes of patients with the oligoarticular form of the disease.