Stefano Angiari

A role for leukocyte-endothelial adhesion mechanisms in epilepsy

The mechanisms involved in the pathogenesis of epilepsy, a chronic neurological disorder that affects approximately one percent of the world population, are not well understood. Using a mouse model of epilepsy, we show that seizures induce elevated expression of vascular cell adhesion molecules and enhanced leukocyte rolling and arrest in brain vessels mediated by the leukocyte mucin P-selectin glycoprotein ligand-1 (PSGL-1, encoded by Selplg) and leukocyte integrins α4β1 and αLβ2. Inhibition of leukocyte-vascular interactions, either with blocking antibodies or by genetically interfering with PSGL-1 function in mice, markedly reduced seizures. Treatment with blocking antibodies after acute seizures prevented the development of epilepsy. Neutrophil depletion also inhibited acute seizure induction and chronic spontaneous recurrent seizures. Blood-brain barrier (BBB) leakage, which is known to enhance neuronal excitability, was induced by acute seizure activity but was prevented by blockade of leukocyte-vascular adhesion, suggesting a pathogenetic link between leukocyte-vascular interactions, BBB damage and seizure generation. Consistent with the potential leukocyte involvement in epilepsy in humans, leukocytes were more abundant in brains of individuals with epilepsy than in controls. Our results suggest leukocyte-endothelial interaction as a potential target for the prevention and treatment of epilepsy.

ADIPOSE-DERIVED MESENCHYMAL STEM CELLS AMELIORATE CHRONIC EXPERIMENTAL AUTOIMMUNE ENCEPHALOMYELITIS

Mesenchymal stem cells (MSCs) represent a promising therapeutic approach for neurological autoimmune diseases; previous studies have shown that treatment with bone marrow‐derived MSCs induces immune modulation and reduces disease severity in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. Here we show that intravenous administration of adipose‐derived MSCs (ASCs) before disease onset significantly reduces the severity of EAE by immune modulation and decreases spinal cord inflammation and demyelination. ASCs preferentially home into lymphoid organs but also migrates inside the central nervous system (CNS). Most importantly, administration of ASCs in chronic established EAE significantly ameliorates the disease course and reduces both demyelination and axonal loss, and induces a Th2‐type cytokine shift in T cells. Interestingly, a relevant subset of ASCs expresses activated α4 integrins and adheres to inflamed brain venules in intravital microscopy experiments. Bioluminescence imaging shows that α4 integrins control ASC accumulation in inflamed CNS. Importantly, we found that ASC cultures produce basic fibroblast growth factor, brain‐derived growth factor, and platelet‐derived growth factor‐AB. Moreover, ASC infiltration within demyelinated areas is accompanied by increased number of endogenous oligodendrocyte progenitors. In conclusion, we show that ASCs have clear therapeutic potential by a bimodal mechanism, by suppressing the autoimmune response in early phases of disease as well as by inducing local neuroregeneration by endogenous progenitors in animals with established disease. Overall, our data suggest that ASCs represent a valuable tool for stem cell–based therapy in chronic inflammatory diseases of the CNS. STEM CELLS 2009;27:2624–2635

Neutrophils promote Alzheimer's disease-like pathology and cognitive decline via LFA-1 integrin

Inflammation is a pathological hallmark of Alzheimers disease, and innate immune cells have been shown to contribute to disease pathogenesis. In two transgenic models of Alzheimers disease (5xFAD and 3xTg-AD mice), neutrophils extravasated and were present in areas with amyloid-β (Aβ) deposits, where they released neutrophil extracellular traps (NETs) and IL-17. Aβ42 peptide triggered the LFA-1 integrin high-affinity state and rapid neutrophil adhesion to integrin ligands. In vivo, LFA-1 integrin controlled neutrophil extravasation into the CNS and intraparenchymal motility. In transgenic Alzheimers disease models, neutrophil depletion or inhibition of neutrophil trafficking via LFA-1 blockade reduced Alzheimers disease–like neuropathology and improved memory in mice already showing cognitive dysfunction. Temporary depletion of neutrophils for 1 month at early stages of disease led to sustained improvements in memory. Transgenic Alzheimers disease model mice lacking LFA-1 were protected from cognitive decline and had reduced gliosis. In humans with Alzheimers disease, neutrophils adhered to and spread inside brain venules and were present in the parenchyma, along with NETs. Our results demonstrate that neutrophils contribute to Alzheimers disease pathogenesis and cognitive impairment and suggest that the inhibition of neutrophil trafficking may be beneficial in Alzheimers disease.

Vascular inflammation in central nervous system diseases: adhesion receptors controlling leukocyte–endothelial interactions

Leukocyte trafficking from the blood into the tissues represents a key process during inflammation and requires multiple steps mediated by adhesion molecules and chemoattractants. Inflammation has a detrimental role in several diseases, and in such cases, the molecular mechanisms controlling leukocyte migration are potential therapeutic targets. Over the past 20 years, leukocyte migration in the CNS has been investigated almost exclusively in the context of stroke and MS. Experimental models of ischemic stroke have led to the characterization of adhesion molecules controlling leukocyte migration during acute inflammation, whereas EAE, the animal model of MS, has provided similar data for chronic inflammation. Such experiments have led to clinical trials of antileukocyte adhesion therapy, with consistently positive outcomes in human subjects with MS, showing that interference with leukocyte adhesion can ameliorate chronic inflammatory CNS diseases. This review summarizes our current understanding of the roles of adhesion molecules controlling leukocyte–endothelial interactions in stroke and MS, focusing on recently discovered, novel migration mechanisms. We also discuss the growing evidence suggesting a role for vascular inflammation and leukocyte trafficking in neurodegenerative diseases such as AD. Moreover, we highlight recent findings suggesting a role for leukocyte–endothelial interactions in the pathogenesis of seizures and epilepsy, thus linking endothelial activation and leukocyte trafficking to neuronal electrical hyperactivity. These emerging roles for leukocytes and leukocyte adhesion mechanisms in CNS diseases provide insight into the mechanisms of brain damage and may contribute to the development of novel therapeutic strategies.

TIM-1 Glycoprotein Binds the Adhesion Receptor P-Selectin and Mediates T Cell Trafficking during Inflammation and Autoimmunity

Selectins play a central role in leukocyte trafficking by mediating tethering and rolling on vascular surfaces. Here we have reported that T cell immunoglobulin and mucin domain 1 (TIM-1) is a P-selectin ligand. We have shown that human and murine TIM-1 binds to P-selectin, and that TIM-1 mediates tethering and rolling of T helper 1 (Th1) and Th17, but not Th2 and regulatory T cells on P-selectin. Th1 and Th17 cells lacking the TIM-1 mucin domain showed reduced rolling in thrombin-activated mesenteric venules and inflamed brain microcirculation. Inhibition of TIM-1 had no effect on naive T cell homing, but it reduced T cell recruitment in a skin hypersensitivity model and blocked experimental autoimmune encephalomyelitis. Uniquely, the TIM-1 immunoglobulin variable domain was also required for P-selectin binding. Our data demonstrate that TIM-1 is a major P-selectin ligand with a specialized role in T cell trafficking during inflammatory responses and the induction of autoimmune disease.

Histamine regulates autoreactive T cell activation and adhesiveness in inflamed brain microcirculation

Histamine may contribute to the pathology of MS and its animal model EAE. We explored the effects of histamine and specific HR agonists on activation and migratory capacity of myelin‐autoreactive T cells. We show that histamine in vitro inhibits proliferation and IFN‐γ production of mouse T cells activated against PLP139–151. These effects were mimicked by the H1R agonist HTMT and the H2R agonist dimaprit and were associated with reduced activation of ERK½ kinase and with increased levels of cell cycle inhibitor p27Kip‐1, both involved in T cell proliferation and anergy. H1R and H2R agonists reduced spontaneous and chemokine‐induced adhesion of autoreactive T cells to ICAM‐1 in vitro and blocked firm adhesion of these cells in inflamed brain microcirculation in vivo. Thus histamine, through H1R and H2R, inhibits activation of myelin‐autoreactive T cells and their ability to traffic through the inflamed BBB. Strategies aimed at interfering with the histamine axis might have relevance in the therapy of autoimmune disease of the CNS.

Inverse agonism of cannabinoid CB1 receptor blocks the adhesion of encephalitogenic T cells in inflamed brain venules by a protein kinase A-dependent mechanism

It is well known that the cannabinoid system has a significant role in the regulation of the immune responses. Cannabinoid receptors CB1 and CB2 are expressed on T lymphocytes and mediate the immunomodulatory effects of cannabinoids on T cell functions. Here we show that the treatment of proteolipid protein (PLP)139-151-specific T cells with SR141716A, a CB1 inverse agonist and prototype of the diarylpyrazoles series, induced a strong inhibition of firm adhesion in inflamed brain venules in intravital microscopy experiments. In contrast, SR144528, a potent CB2 inverse agonist, had no significant effect on both rolling and arrest of activated T cells. In addition, two analogs of SR141716A and CB1 inverse agonists, AM251 and AM281 inhibited encephalitogenic T cell adhesion suggesting that selective CB1 inverse agonism interfere with lymphocyte trafficking in the CNS. Flow cytometry experiments showed that CB1 inverse agonists have no effect on adhesion molecule expression suggesting that CB1 blockade interferes with signal transduction pathways controlling T cell adhesion in inflamed brain venules. In addition, integrin clustering was not altered after treatment with CB1 inverse agonists suggesting that adhesion blockade is not due to the modulation of integrin valency. Notably, the inhibitory effect exerted by AM251 and AM281 on the adhesive interactions was completely reverted in the presence of protein kinase A (PKA) inhibitor H89, suggesting that cAMP and PKA activation play a key role in the adhesion blockade mediated by CB1 inverse agonists. To further strengthen these results and unveil a previously unknown inhibitory role of cAMP on activated T cell adhesion in vivo in the context of CNS inflammation, we showed that intracellular increase of cAMP induced by treatment with Bt2cAMP, a permeable analog of cAMP, and phosphodiesterase (PDE) inhibitor theophylline efficiently blocked the arrest of encephalitogenic T cells in inflamed brain venules. Our data show that modulation of CB1 function has anti-inflammatory effects and suggests that inverse agonism of CB1 block signal transduction mechanisms controlling encephalitogenic T cells adhesion in inflamed brain venules by a PKA-dependent mechanism.

Regulatory T Cells Suppress the Late Phase of the Immune Response in Lymph Nodes through P-Selectin Glycoprotein Ligand-1

Regulatory T cells (Tregs) maintain tolerance toward self-antigens and suppress autoimmune diseases, although the underlying molecular mechanisms are unclear. In this study, we show that mice deficient for P-selectin glycoprotein ligand-1 (PSGL-1) develop a more severe form of experimental autoimmune encephalomyelitis than wild type animals do, suggesting that PSGL-1 has a role in the negative regulation of autoimmunity. We found that Tregs lacking PSGL-1 were unable to suppress experimental autoimmune encephalomyelitis and failed to inhibit T cell proliferation in vivo in the lymph nodes. Using two-photon laser-scanning microscopy in the lymph node, we found that PSGL-1 expression on Tregs had no role in the suppression of early T cell priming after immunization with Ag. Instead, PSGL-1-deficient Tregs lost the ability to modulate T cell movement and failed to inhibit the T cell–dendritic cell contacts and T cell clustering essential for sustained T cell activation during the late phase of the immune response. Notably, PSGL-1 expression on myelin-specific effector T cells had no role in T cell locomotion in the lymph node. Our data show that PSGL-1 represents a previously unknown, phase-specific mechanism for Treg-mediated suppression of the persistence of immune responses and autoimmunity induction.

Mutations of Cystic Fibrosis Transmembrane Conductance Regulator Gene Cause a Monocyte-Selective Adhesion Deficiency

RATIONALE Cystic fibrosis (CF) is a common genetic disease caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Persistent lung inflammation, characterized by increasing polymorphonuclear leukocyte recruitment, is a major cause of the decline in respiratory function in patients with CF and is a leading cause of morbidity and mortality. CFTR is expressed in various cell types, including leukocytes, but its involvement in the regulation of leukocyte recruitment is unknown. OBJECTIVES We evaluated whether CF leukocytes might present with alterations in cell adhesion and migration, a key process governing innate and acquired immune responses. METHODS We used ex vivo adhesion and chemotaxis assays, flow cytometry, immunofluorescence, and GTPase activity assays in this study. MEASUREMENTS AND MAIN RESULTS We found that chemoattractant-induced activation of β1 and β2 integrins and of chemotaxis is defective in mononuclear cells isolated from patients with CF. In contrast, polymorphonuclear leukocyte adhesion and chemotaxis were normal. The functionality of β1 and β2 integrins was restored by treatment of CF monocytes with the CFTR-correcting drugs VRT325 and VX809. Moreover, treatment of healthy monocytes with the CFTR inhibitor CFTR(inh)-172 blocked integrin activation by chemoattractants. In a murine model of lung inflammation, we found that integrin-independent migration of CF monocytes into the lung parenchyma was normal, whereas, in contrast, integrin-dependent transmigration into the alveolar space was impaired. Finally, signal transduction analysis showed that, in CF monocytes, chemoattractant-triggered activation of RhoA and CDC42 Rho small GTPases (controlling integrin activation and chemotaxis, respectively) was strongly deficient. CONCLUSIONS Altogether, these data highlight the critical regulatory role of CFTR in integrin activation by chemoattractants in monocytes and identify CF as a new, cell type-selective leukocyte adhesion deficiency disease, providing new insights into CF pathogenesis.

Use of imaging to study leukocyte trafficking in the central nervous system

The migration of leukocytes from the bloodstream into the central nervous system (CNS) is a key event in the pathogenesis of inflammatory neurological diseases and typically involves the movement of cells through the endothelium of post‐capillary venules, which contains intercellular tight junctions. Leukocyte trafficking has predominantly been studied in animal models of multiple sclerosis, stroke and infection. However, recent evidence suggests that immune cells and inflammation mechanisms play an unexpected role in other neurological diseases, such as epilepsy and Parkinsons disease. Imaging leukocyte trafficking in the CNS can be achieved by epifluorescence intravital microscopy (IVM) and multiphoton microscopy. Epifluorescence IVM is ideal for the investigation of leukocyte–endothelial interactions, particularly tethering and rolling, signal transduction pathways controlling integrin activation, slow rolling, arrest and adhesion strengthening in CNS vessels. Multiphoton microscopy is more suitable for the investigation of intraluminal crawling, transmigration and motility inside CNS parenchyma. The mechanisms of leukocyte trafficking in the CNS are not well understood but the use of in vivo imaging techniques to unravel the underlying regulatory pathways will provide insight into the mechanisms of brain damage and may contribute to the development of novel therapeutic strategies. In this review, we discuss recent work in this field, highlighting the development and use of in vivo imaging to investigate leukocyte recruitment in the CNS.