Peter Ludewig

CD38 Exacerbates Focal Cytokine Production, Postischemic Inflammation and Brain Injury after Focal Cerebral Ischemia

Background Converging evidence suggests that inflammatory processes significantly influence brain injury and clinical impairment in ischemic stroke. Although early studies suggested a key role of lymphocytes, recent data has emphasized the orchestrating function of innate immunity, i.e., macrophages and microglia. The bifunctional receptor and ectoenzyme CD38 synthesizes calcium-mobilizing second messengers (e.g., cyclic ADP-ribose), which have been shown to be necessary for activation and migration of myeloid immune cells. Therefore, we investigated the dynamics of CD38 in stroke and the impact of CD38-deficiency on cytokine production, inflammation and cerebral damage in a mouse model of cerebral ischemia-reperfusion. Methodology/Principal Findings We show that the local expression of the chemokine MCP-1 was attenuated in CD38-deficient mice compared with wildtype mice after focal cerebral ischemia and reperfusion. In contrast, no significant induction of MCP-1 expression was observed in peripheral blood after 6 hours. Flow cytometry analysis revealed less infiltrating macrophages and lymphocytes in the ischemic hemisphere of CD38-deficient mice, whereas the amount of resident microglia was unaltered. An up-regulation of CD38 expression was observed in macrophages and CD8+ cells after focal cerebral ischemia in wildtype mice, whereas CD38 expression was unchanged in microglia. Finally, we demonstrate that CD38-deficiency decreases the cerebral ischemic injury and the persistent neurological deficit after three days of reperfusion in this murine temporary middle cerebral artery occlusion (tMCAO) model. Conclusion/Significance CD38 is differentially regulated following stroke and its deficiency attenuates the postischemic chemokine production, the immune cell infiltration and the cerebral injury after temporary ischemia and reperfusion. Therefore CD38 might prove a therapeutic target in ischemic stroke.

The flavonoid fisetin attenuates postischemic immune cell infiltration, activation and infarct size after transient cerebral middle artery occlusion in mice.

The development of the brain tissue damage in ischemic stroke is composed of an immediate component followed by an inflammatory response with secondary tissue damage after reperfusion. Fisetin, a flavonoid, has multiple biological effects, including neuroprotective and antiinflammatory properties. We analyzed the effects of fisetin on infarct size and the inflammatory response in a mouse model of stroke, temporary middle cerebral artery occlusion, and on the activation of immune cells, murine primary and N9 microglial and Raw264.7 macrophage cells and human macrophages, in an in vitro model of inflammatory immune cell activation by lipopolysaccharide (LPS). Fisetin not only protected brain tissue against ischemic reperfusion injury when given before ischemia but also when applied 3 hours after ischemia. Fisetin also prominently inhibited the infiltration of macrophages and dendritic cells into the ischemic hemisphere and suppressed the intracerebral immune cell activation as measured by intracellular tumor necrosis factor α (TNFα) production. Fisetin also inhibited LPS-induced TNFα production and neurotoxicity of macrophages and microglia in vitro by suppressing nuclear factor κB activation and JNK/Jun phosphorylation. Our findings strongly suggest that the fisetin-mediated inhibition of the inflammatory response after stroke is part of the mechanism through which fisetin is neuroprotective in cerebral ischemia.

CEACAM1+ myeloid cells control angiogenesis in inflammation

Local inflammation during cutaneous leishmaniasis is accompanied by accumulation of CD11b(+) cells at the site of the infection. A functional role for these monocytic cells in local angiogenesis in leishmaniasis has not been described so far. Here, we show that CD11b(+) cells express high levels of the myeloid differentiation antigen carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1). In experimental cutaneous leishmaniasis in C57BL/6 wild-type (B6.WT) and B6.Ceacam1(-/-) mice, we found that only B6.Ceacam1(-/-) mice develop edemas and exhibit impairment of both hemangiogenesis and lymphangiogenesis. Because CEACAM1 expression correlates with functional angiogenesis, we further analyzed the role of the CD11b(+) population. In B6.Ceacam1(-/-) mice, we found systemic reduction of Ly-6C(high)/CD11b(high) monocyte precursors. To investigate whether CEACAM1(+) myeloid cells are causally related to efficient angiogenesis, we used reverse bone marrow transplants (BMTs) to restore CEACAM1(+) or CEACAM1(-) bone marrow in B6.Ceacam1(-/-) or B6.WT recipients, respectively. We found that angiogenesis was restored by CEACAM1(+) BMT only. In addition, we observed reduced morphogenic potential of inflammatory cells in Matrigel implants in CEACAM1(-) backgrounds or after systemic depletion of CD11b(high) macrophages. Taken together, we show for the first time that CEACAM1(+) myeloid cells are crucial for angiogenesis in inflammation.

Carcinoembryonic Antigen–Related Cell Adhesion Molecule 1 Inhibits MMP-9–Mediated Blood–Brain–Barrier Breakdown in a Mouse Model for Ischemic Stroke

Rationale: Blood–brain–barrier (BBB) breakdown and cerebral edema result from postischemic inflammation and contribute to mortality and morbidity after ischemic stroke. A functional role for the carcinoembryonic antigen–related cell adhesion molecule 1 (CEACAM1) in the regulation of reperfusion injury has not yet been demonstrated. Objective: We sought to identify and characterize the relevance of CEACAM1-expressing inflammatory cells in BBB breakdown and outcome after ischemic stroke in Ceacam1−/− and wild-type mice. Methods and Results: Focal ischemia was induced by temporary occlusion of the middle cerebral artery with a microfilament. Using MRI and Evans blue permeability assays, we observed increased stroke volumes, BBB breakdown and edema formation, reduction of cerebral perfusion, and brain atrophy in Ceacam1−/− mice. This translated into poor performance in neurological scoring and high poststroke-associated mortality. Elevated neutrophil influx, hyperproduction, and release of neutrophil-related matrix metalloproteinase-9 in Ceacam1−/− mice were confirmed by immune fluorescence, flow cytometry, zymography, and stimulation of neutrophils. Importantly, neutralization of matrix metalloproteinase-9 activity in Ceacam1−/− mice was sufficient to alleviate stroke sizes and improve survival to the level of CEACAM1-competent animals. Immune histochemistry of murine and human poststroke autoptic brains congruently identified abundance of CEACAM1+matrix metalloproteinase-9+ neutrophils in the ischemic hemispheres. Conclusions: CEACAM1 controls matrix metalloproteinase-9 secretion by neutrophils in postischemic inflammation at the BBB after stroke. We propose CEACAM1 as an important inhibitory regulator of neutrophil-mediated tissue damage and BBB breakdown in focal cerebral ischemia.Rationale: Blood-brain-barrier (BBB) breakdown and cerebral edema result from postischemic inflammation and contribute to mortality and morbidity after ischemic stroke. A functional role for the carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) in the regulation of reperfusion injury has not yet been demonstrated. Objective: We sought to identify and characterize the relevance of CEACAM1- expressing inflammatory cells in BBB breakdown and outcome after ischemic stroke in Ceacam1-/- and wild type (WT) mice. Methods and Results: Focal ischemia was induced by temporary occlusion of the middle cerebral artery (tMCAO) with a microfilament. Using magnetic resonance imaging (MRI) and Evans blue permeability assays, we observed increased stroke volumes, BBB breakdown and edema formation, reduction of cerebral perfusion and brain atrophy in Ceacam1-/- mice.This translated into poor performance in neurological scoring and high post stroke-associated mortality. Elevated neutrophil influx, hyperproduction and release of neutrophil-related matrix metalloproteinase (MMP)-9 in Ceacam1-/- mice were confirmed by immune fluorescence, flow cytometry, zymography and stimulation of neutrophils. Importantly, neutralization of MMP-9 activity in Ceacam1-/- mice was sufficient to alleviate stroke sizes and improve survival to the level of CEACAM1-competent animals. Immune histochemistry of murine and human post-stroke autoptic brains congruently identified abundance of CEACAM1+MMP9+ neutrophils in the ischemic hemispheres. Conclusions: CEACAM1 controls MMP-9 secretion by neutrophils in postischemic inflammation at the BBB after stroke. We propose CEACAM1 as an important inhibitory regulator of neutrophil-mediated tissue damage and BBB breakdown in focal cerebral ischemia.

CEACAM1 Inhibits MMP-9-Mediated Blood-Brain-Barrier Breakdown in a Mouse Model for Ischemic Stroke

Rationale: Blood–brain–barrier (BBB) breakdown and cerebral edema result from postischemic inflammation and contribute to mortality and morbidity after ischemic stroke. A functional role for the carcinoembryonic antigen–related cell adhesion molecule 1 (CEACAM1) in the regulation of reperfusion injury has not yet been demonstrated. Objective: We sought to identify and characterize the relevance of CEACAM1-expressing inflammatory cells in BBB breakdown and outcome after ischemic stroke in Ceacam1−/− and wild-type mice. Methods and Results: Focal ischemia was induced by temporary occlusion of the middle cerebral artery with a microfilament. Using MRI and Evans blue permeability assays, we observed increased stroke volumes, BBB breakdown and edema formation, reduction of cerebral perfusion, and brain atrophy in Ceacam1−/− mice. This translated into poor performance in neurological scoring and high poststroke-associated mortality. Elevated neutrophil influx, hyperproduction, and release of neutrophil-related matrix metalloproteinase-9 in Ceacam1−/− mice were confirmed by immune fluorescence, flow cytometry, zymography, and stimulation of neutrophils. Importantly, neutralization of matrix metalloproteinase-9 activity in Ceacam1−/− mice was sufficient to alleviate stroke sizes and improve survival to the level of CEACAM1-competent animals. Immune histochemistry of murine and human poststroke autoptic brains congruently identified abundance of CEACAM1+matrix metalloproteinase-9+ neutrophils in the ischemic hemispheres. Conclusions: CEACAM1 controls matrix metalloproteinase-9 secretion by neutrophils in postischemic inflammation at the BBB after stroke. We propose CEACAM1 as an important inhibitory regulator of neutrophil-mediated tissue damage and BBB breakdown in focal cerebral ischemia.Rationale: Blood-brain-barrier (BBB) breakdown and cerebral edema result from postischemic inflammation and contribute to mortality and morbidity after ischemic stroke. A functional role for the carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) in the regulation of reperfusion injury has not yet been demonstrated. Objective: We sought to identify and characterize the relevance of CEACAM1- expressing inflammatory cells in BBB breakdown and outcome after ischemic stroke in Ceacam1-/- and wild type (WT) mice. Methods and Results: Focal ischemia was induced by temporary occlusion of the middle cerebral artery (tMCAO) with a microfilament. Using magnetic resonance imaging (MRI) and Evans blue permeability assays, we observed increased stroke volumes, BBB breakdown and edema formation, reduction of cerebral perfusion and brain atrophy in Ceacam1-/- mice.This translated into poor performance in neurological scoring and high post stroke-associated mortality. Elevated neutrophil influx, hyperproduction and release of neutrophil-related matrix metalloproteinase (MMP)-9 in Ceacam1-/- mice were confirmed by immune fluorescence, flow cytometry, zymography and stimulation of neutrophils. Importantly, neutralization of MMP-9 activity in Ceacam1-/- mice was sufficient to alleviate stroke sizes and improve survival to the level of CEACAM1-competent animals. Immune histochemistry of murine and human post-stroke autoptic brains congruently identified abundance of CEACAM1+MMP9+ neutrophils in the ischemic hemispheres. Conclusions: CEACAM1 controls MMP-9 secretion by neutrophils in postischemic inflammation at the BBB after stroke. We propose CEACAM1 as an important inhibitory regulator of neutrophil-mediated tissue damage and BBB breakdown in focal cerebral ischemia.

Tumor-associated Neu5Ac-Tn and Neu5Gc-Tn antigens bind to C-type lectin CLEC10A (CD301, MGL)

In human tumors, glycoproteins often exhibit abnormal glycosylation patterns, e.g. certain Lewis structures, TF antigen, Tn antigen and/or their sialylated forms, creating additional binding sites for glycoreceptors. In the present study, we have analyzed the carbohydrate specificity of the C-type lectin CLEC10A using glycan profiling by enzyme-linked immunosorbent assay (ELISA). In addition to the known ligands, we show binding to two tumor-associated antigens, namely Neu5Acα2,6-Tn and Neu5Gcα2,6-Tn, with an affinity of CLEC10A in the micromolar range. Detailed analyses of the glycan-lectin interactions were carried out by surface plasmon resonance (SPR) and saturation transfer difference (STD) NMR. CLEC10A binds Neu5Acα2,6-Tn and Neu5Gcα2,6-Tn with dissociation constants of 297 and 80 µM, respectively, as determined by SPR. Comparison of the STD nuclear magnetic resonance (NMR) binding epitopes of Tn and Neu5Acα2,6-Tn revealed a constant binding mode of the N-acetylgalactosamine moiety. This finding is in good agreement with binding studies of CLEC10A transfectomas, which show a well-defined interaction of transmembrane CLEC10A with 6-sialylated-Tn structures. Since both Neu5Acα2,6-Tn and Neu5Gcα2,6-Tn together with the previously known Tn antigen are expressed in human tumors such as mammary carcinoma, the interaction with CLEC10A expressed by macrophages and dendritic cells could be of major functional significance in tumor progression.

Deficiency in Serine Protease Inhibitor Neuroserpin Exacerbates Ischemic Brain Injury by Increased Postischemic Inflammation

The only approved pharmacological treatment for ischemic stroke is intravenous administration of plasminogen activator (tPA) to re-canalize the occluded cerebral vessel. Not only reperfusion but also tPA itself can induce an inflammatory response. Microglia are the innate immune cells of the central nervous system and the first immune cells to become activated in stroke. Neuroserpin, an endogenous inhibitor of tPA, is up-regulated following cerebral ischemia. To examine neuroserpin-dependent mechanisms of neuroprotection in stroke, we studied neuroserpin deficient (Ns−/−) mice in an animal model of temporal focal ischemic stroke. Infarct size and neurological outcome were worse in neuroserpin deficient mice even though the fibrinolytic activity in the ischemic brain was increased. The increased infarct size was paralleled by a selective increase in proinflammatory microglia activation in Ns−/− mice. Our results show excessive microglial activation in Ns−/− mice mediated by an increased activity of tPA. This activation results in a worse outcome further underscoring the potential detrimental proinflammatory effects of tPA.

Plasma levels of neuron specific enolase quantify the extent of neuronal injury in murine models of ischemic stroke and multiple sclerosis

OBJECTIVE We aimed at validating a plasma biomarker for neuronal damage that can be used in acute and chronic models of neurological diseases. METHODS We investigated two different models, middle cerebral artery occlusion followed by reperfusion and MOG35-55-induced experimental autoimmune encephalomyelitis (EAE). In stroke experiments we measured infarct sizes by magnetic resonance imaging and vital stainings and correlated them with plasma levels of neuron specific enolase (NSE) at different time points after reperfusion. Equally, in EAE experiments, we correlated NSE levels with neurological scores and histopathological damage of axons at different time points. We detected plasma NSE levels by ELISA. RESULTS Plasma NSE levels correlated significantly with stroke size, EAE score and histopathological damage in EAE. Investigations into the dynamics of neuronal loss over time correlated well with the dynamics of NSE levels. NSE even predicted the onset of EAE, before clinical signs were recordable. CONCLUSIONS Plasma NSE is a valid and simple experimental biomarker that allows quantifying the degree of neuronal injury in a non-invasive approach.

Dendritic cells in brain diseases

Abstract Dendritic cells (DCs) are professional antigen presenting cells that constantly survey the environment acting as sentinels of the immune system, including in the CNS. DCs are strategically located near the cerebrospinal fluid, but they can potentially migrate to draining cervical lymph nodes either triggering immunogenic T cell responses or displaying tolerogenic functions. Under physiological conditions, the presence of DCs in the brain parenchyma is minimal but their numbers increase in neuroinflammation. Although DCs belong to a distinct immune cell lineage, they show various phenotypes and share certain common markers with monocytes, macrophages, and microglia. All these cells can express major histocompatibility complex class II, and acquire similar morphologies hampering their precise identification. Neuroinflammation is increasingly recognized in many brain disorders; here we review the literature reporting DCs in the inflamed brain in disease conditions and corresponding animal models of multiple sclerosis, stroke, brain tumors, Alzheimers disease, Parkinsons disease, and epilepsy. This article is part of a Special Issue entitled: Neuro Inflammation edited by Helga E. de Vries and Markus Schwaninger.

CCR6 (CC Chemokine Receptor 6) Is Essential for the Migration of Detrimental Natural Interleukin-17–Producing γδ T Cells in Stroke

Background and Purpose— Immune-mediated tissue damage after stroke evolves within the first days, and lymphocytes contribute to the secondary injury. Our goal was to identify T-cell subpopulations, which trigger the immune response. Methods— In a model of experimental stroke, we analyzed the immune phenotype of interleukin-17 (IL-17)–producing &ggr;&dgr; T cells and explored the therapeutic potential of neutralizing anti-IL-17 antibodies in combination with mild therapeutic hypothermia. Results— We show that brain-infiltrating IL-17–positive &ggr;&dgr; T cells expressed the V&ggr;6 segment of the &ggr;&dgr; T cells receptor and were largely positive for the chemokine receptor CCR6 (CC chemokine receptor 6), which is a characteristic for natural IL-17–producing &ggr;&dgr; T cells. These innate lymphocytes are established as major initial IL-17 producers in acute infections. Genetic deficiency in Ccr6 was associated with diminished infiltration of natural IL-17–producing &ggr;&dgr; T cells and a significantly improved neurological outcome. In the ischemic brain, IL-17 together with tumor necrosis factor-&agr; triggered the expression of CXC chemokines and neutrophil infiltration. Therapeutic targeting of synergistic IL-17 and tumor necrosis factor-&agr; pathways by IL-17 neutralization and therapeutic hypothermia resulted in additional protective effects in comparison to an anti-IL-17 antibody treatment or therapeutic hypothermia alone. Conclusions— Brain-infiltrating IL-17–producing &ggr;&dgr; T cells belong to the subset of natural IL-17–producing &ggr;&dgr; T cells. In stroke, these previously unrecognized innate lymphocytes trigger a highly conserved immune reaction, which is known from host responses toward pathogens. We demonstrate that therapeutic approaches targeting synergistic IL-17 and tumor necrosis factor-&agr; pathways in parallel offer additional neuroprotection in stroke.