Modulation of the cannabinoid CB2 receptor in microglial cells in response to inflammatory stimuli

The cannabinoid system is known to be important in neuronal regulation, but is also capable of modulating immune function. Although the CNS resident microglial cells have been shown to express the CB2 subtype of cannabinoid receptor during non-immune-mediated pathological conditions, little is known about the expression of the cannabinoid system during immune-mediated CNS pathology. To examine this question, we measured CB2 receptor mRNA expression in the CNS of mice with experimental autoimmune encephalomyelitis (EAE) and, by real-time PCR, found a 100-fold increase in CB2 receptor mRNA expression during EAE onset. We next determined whether microglial cells specifically express the CB2 receptor during EAE, and found that activated microglial cells expressed 10-fold more CB2 receptor than microglia in the resting state. To determine the signals required for the up-regulation of the CB2 receptor, we cultured microglial cells with combinations of gamma-interferon (IFN-gamma) and granulocyte) macrophage-colony stimulating factor (GM-CSF), which both promote microglial cell activation and are expressed in the CNS during EAE, and found that they synergized, resulting in an eight to 10-fold increase in the CB2 receptor. We found no difference in the amount of the CB2 receptor ligand, 2-arachidonylglycerol (2-AG), in the spinal cord during EAE. These data demonstrate that microglial cell activation is accompanied by CB2 receptor up-regulation, suggesting that this receptor plays an important role in microglial cell function in the CNS during autoimmune-induced inflammation.     

The CB(2) cannabinoid receptor controls myeloid progenitor trafficking: involvement in the pathogenesis of an animal model of multiple sclerosis

Cannabinoids are potential agents for the development of therapeutic strategies against multiple sclerosis. Here we analyzed the role of the peripheral CB(2) cannabinoid receptor in the control of myeloid progenitor cell trafficking toward the inflamed spinal cord and their contribution to microglial activation in an animal model of multiple sclerosis (experimental autoimmune encephalomyelitis, EAE). CB(2) receptor knock-out mice showed an exacerbated clinical score of the disease when compared with their wild-type littermates, and this occurred in concert with extended axonal loss, T-lymphocyte (CD4(+)) infiltration, and microglial (CD11b(+)) activation. Immature bone marrow-derived CD34(+) myeloid progenitor cells, which play a role in neuroinflammatory pathologies, were shown to express CB(2) receptors and to be abundantly recruited toward the spinal cords of CB(2) knock-out EAE mice. Bone marrow-derived cell transfer experiments further evidenced the increased contribution of these cells to microglial replenishment in the spinal cords of CB(2)-deficient animals. In line with these observations, selective pharmacological CB(2) activation markedly reduced EAE symptoms, axonal loss, and microglial activation. CB(2) receptor manipulation altered the expression pattern of different chemokines (CCL2, CCL3, CCL5) and their receptors (CCR1, CCR2), thus providing a mechanistic explanation for its role in myeloid progenitor recruitment during neuroinflammation. These findings demonstrate the protective role of CB(2) receptors in EAE pathology; provide evidence for a new site of CB(2) receptor action, namely the targeting of myeloid progenitor trafficking and its contribution to microglial activation; and support the potential use of non-psychoactive CB(2) agonists in therapeutic strategies for multiple sclerosis and other neuroinflammatory disorders.     

Pathogenic and protective functions of TNF in neuroinflammation are defined by its expression in T lymphocytes and myeloid cells

TNF displays pathogenic activities in many autoimmune disorders. However, anti-TNF therapy in multiple sclerosis patients failed because of poorly understood reasons. We used a panel of gene-targeted mice that allowed cell-type specific ablation of TNF to uncover pathogenic and protective contributions of this cytokine during autoimmune disease of the CNS. T cells and myeloid cells were found to be critical cellular sources of TNF during experimental autoimmune encephalomyelitis (EAE). TNF produced by myeloid cells accelerated the onset of disease by regulation of chemokine expression in the CNS, driving the recruitment of inflammatory cells into the target organ. TNF produced by T cells exacerbated the damage to the CNS during EAE by regulating infiltration of inflammatory myeloid cells into the CNS. In secondary lymphoid organs, TNF expressed by myeloid cells and T cells acted in synergy to dampen IL-12p40 and IL-6 production by APCs, subsequently inhibiting the development of encephalitogenic T cell responses of Th1 and Th17 types. This dual role of TNF during EAE (protective in lymphoid organs and pathogenic in CNS) suggests that global TNF blockade might be inefficient in multiple sclerosis patients because augmented autoreactive T cell development in lymphoid tissues might overwhelm the beneficial effects resulting from TNF inhibition in the CNS.     

Delta-like ligand 4 regulates central nervous system T cell accumulation during experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is a CD4(+) T cell-mediated inflammatory demyelinating disease of the CNS that serves as a model for multiple sclerosis. Notch receptor signaling in T lymphocytes has been shown to regulate thymic selection and peripheral differentiation. In the current study, we hypothesized that Notch ligand-receptor interaction affects EAE development by regulating encephalitogenic T cell trafficking. We demonstrate that CNS-infiltrating myeloid dendritic cells, macrophages, and resident microglia expressed Delta-like ligand 4 (DLL4) after EAE induction. Treatment of mice with a DLL4-specific blocking Ab significantly inhibited the development of clinical disease induced by active priming. Furthermore, the treatment resulted in decreased CNS accumulation of mononuclear cells in the CNS. Anti-DLL4 treatment did not significantly alter development of effector cytokine expression by Ag-specific T cells. In contrast, anti-DLL4 treatment reduced T cell mRNA and functional cell surface expression of the chemokine receptors CCR2 and CCR6. Adoptive transfer of Ag-specific T cells to mice treated with anti-DLL4 resulted in decreased clinical severity and diminished Ag-specific CD4(+) T cell accumulation in the CNS. These results suggest a role for DLL4 regulation of EAE pathogenesis through modulation of T cell chemokine receptor expression and migration to the CNS.     

Gamma delta T cells regulate the extent and duration of inflammation in the central nervous system by a Fas ligand-dependent mechanism

Gamma delta T cells have been shown to regulate immune responses associated with inflammation, but the mechanism of this regulation is largely unknown. Using the experimental autoimmune encephalomyelitis (EAE) model of the human CNS autoimmune disease multiple sclerosis, we demonstrate that gamma delta T cells are important regulators of CNS inflammation. This was shown using gamma delta T cell-deficient mice that were unable to recover from EAE. The chronic disease was accompanied by a prolonged presence of both macrophages and lymphocytes in the CNS. This extended inflammatory response was due to alterations in both cell proliferation and death. In mice lacking gamma delta T cells, proliferation of encephalitogenic T cells was 3-fold higher, and caspase activity, indicating apoptosis, was 2-fold lower compared with those in control mice recovering from EAE. gamma delta T cell-deficient mice reconstituted with wild-type gamma delta T cells recovered from EAE and resolved inflammation in the CNS, whereas mice reconstituted with Fas ligand-dysfunctional gamma delta T cells did not. Thus, gamma delta T cells regulate both inflammation in the CNS and disease recovery via Fas/Fas ligand-induced apoptosis of encephalitogenic T cells, and a quick resolution of inflammation in the CNS is essential to prevent permanent damage to the CNS resulting in chronic disease.     

A2A adenosine receptor signaling in lymphocytes and the central nervous system regulates inflammation during experimental autoimmune encephalomyelitis

Extracellular adenosine has an important role in regulating the severity of inflammation during an immune response. Although there are four adenosine receptor (AR) subtypes, the A2AAR is both highly expressed on lymphocytes and known as a prime mediator of adenosine's anti-inflammatory effects. To define the importance of A2AAR signaling during neuroinflammatory disease progression, we used the experimental autoimmune encephalomyelitis (EAE) animal model for multiple sclerosis. In EAE induction experiments, A2AAR antagonist treatment protected mice from disease development and its associated CNS lymphocyte infiltration. However, A2AAR(-/-) mice developed a more severe acute EAE phenotype characterized by more proinflammatory lymphocytes and activated microglia/macrophages. Interestingly, very high levels of A2AAR were expressed on the choroid plexus, a well-established CNS lymphocyte entry point. To determine the contribution of A2AAR signaling in lymphocytes and the CNS during EAE, we used bone marrow chimeric mice. Remarkably, A2AAR(-/-) donor hematopoietic cells potentiated severe EAE, whereas lack of A2AAR expression on nonhematopoietic cells protected against disease development. Although no defect in the suppressive ability of A2AAR(-/-) regulatory T cells was observed, A2AAR(-/-) lymphocytes were shown to proliferate more and produced more IFN-γ following stimulation. Despite this more proinflammatory phenotype, A2AAR antagonist treatment still protected against EAE when A2AAR(-/-) lymphocytes were adoptively transferred to T cell-deficient A2AAR(+/+) mice. These results indicate that A2AAR expression on nonimmune cells (likely in the CNS) is required for efficient EAE development, while A2AAR lymphocyte expression is essential for limiting the severity of the inflammatory response.     

Exacerbation of experimental autoimmune encephalomyelitis in P2X7R-/- mice: evidence for loss of apoptotic activity in lymphocytes

The purinergic receptor P2X7R is a nucleotide-gated ion channel that has been proposed to function as a major regulator of inflammation. In this study we examined the role of this receptor in regulating inflammation in the CNS by determining the effects of the loss of this receptor (P2X7R-/-) on experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. We show here that P2X7R-/- mice developed more severe clinical and pathological expression of EAE than wild type (WT) controls and that spleen and lymph node cells from P2X7R-/- mice proliferated more vigorously to Ag in vitro. Bone marrow (BM) radiation chimeras revealed that enhanced susceptibility to EAE was detected in chimeric mice of WT host engrafted with P2X7R-/- BM cells, indicating that the genotype of the BM cells regulated disease susceptibility. Coculture of P2X7R-/- macrophages with WT lymphocytes and vice versa showed that enhanced proliferative activity resided within the P2X7R-/- lymphocyte population and correlated with reduced levels of IFN-gamma and NO and apoptosis of lymphocytes. mRNA and protein for IFN-gamma were also significantly reduced in the CNS of P2X7R-/- mice with EAE. FACS analysis of cells isolated from the CNS showed significantly fewer annexin V/propidium iodide-positive lymphocytes in the CNS of P2X7R-/- mice early in the disease, and TUNEL staining of inflamed CNS tissues supported this result. From these data we conclude that enhanced susceptibility of P2X7R-/- mice to EAE reflects a loss of apoptotic activity in lymphocytes, supporting an important role for this receptor in lymphocyte homeostasis.     

In vivo blockade of macrophage migration inhibitory factor ameliorates acute experimental autoimmune encephalomyelitis by impairing the homing of encephalitogenic T cells to the central nervous system

Macrophage migration inhibitory factor (MIF) is a cytokine that plays a critical role in the regulation of macrophage effector functions and T cell activation. However, its role in the pathogenesis of T cell-mediated autoimmune diseases, such as experimental autoimmune encephalomyelitis (EAE), has remained unresolved. In this study, we report that anti-MIF Ab treatment of SJL mice with acute EAE improved the disease severity and accelerated the recovery. Furthermore, the anti-MIF treatment impaired the homing of neuroantigen-reactive pathogenic T cells to the CNS in a VCAM-1-dependent fashion. Interestingly, MIF blockade also decreased the clonal size of the neuroantigen-specific Th1 cells and increased their activation threshold. Taken together, the results demonstrate an important role for MIF in the pathogenesis of EAE/multiple sclerosis and suggest that MIF blockade may be a promising new strategy for the treatment of multiple sclerosis.     

Cytokine Control of Inflammation and Repair in the Pathology of Multiple Sclerosis

Cytokines are secreted signaling proteins that play an essential role in propagating and regulating immune responses during experimental autoimmune encephalomyelitis (EAE), the mouse model of the neurodegenerative, autoimmune disease multiple sclerosis (MS). EAE pathology is driven by a myelin-specific T cell response that is activated in the periphery and mediates the destruction of myelin upon T cell infiltration into the central nervous system (CNS). Cytokines provide cell signals both in the immune and CNS compartment, but interestingly, some have detrimental effects in the immune compartment while having beneficial effects in the CNS compartment. The complex nature of these signals will be reviewed.     

Inhibition of pain responses by activation of CB(2) cannabinoid receptors

Cannabinoid receptor agonists diminish responses to painful stimuli. Extensive evidence demonstrates that CB(1) cannabinoid receptor activation inhibits pain responses. Recently, the synthesis of CB(2) cannabinoid receptor-selective agonists has allowed testing whether CB(2) receptor activation inhibits pain. CB(2) receptor activation is sufficient to inhibit acute nociception, inflammatory hyperalgesia, and the allodynia and hyperalgesia produced in a neuropathic pain model. Studies using site-specific administration of agonist and antagonist have suggested that CB(2) receptor agonists inhibit pain responses by acting at peripheral sites. CB(2) receptor activation also inhibits edema and plasma extravasation produced by inflammation. CB(2) receptor-selective agonists do not produce central nervous system (CNS) effects typical of cannabinoids retaining agonist activity at the CB(1) receptor. Peripheral antinociception without CNS effects is consistent with the peripheral distribution of CB(2) receptors. CB(2) receptor agonists may have promise for the treatment of pain and inflammation without CNS side effects.     

CXCL10 (IFN-gamma-inducible protein-10) control of encephalitogenic CD4+ T cell accumulation in the central nervous system during experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is a CD4(+) Th1-mediated demyelinating disease of the CNS that serves as a model for multiple sclerosis. A critical event in the pathogenesis of EAE is the entry of both Ag-specific and Ag-nonspecific T lymphocytes into the CNS. In the present report, we investigated the role of the CXC chemokine CXCL10 (IFN-gamma-inducible protein-10) in the pathogenesis of EAE. Production of CXCL10 in the CNS correlated with the development of clinical disease. Administration of anti-CXCL10 decreased clinical and histological disease incidence, severity, as well as infiltration of mononuclear cells into the CNS. Anti-CXCL10 specifically decreased the accumulation of encephalitogenic PLP(139-151) Ag-specific CD4+ T cells in the CNS compared with control-treated animals. Anti-CXCL10 administration did not affect the activation of encephalitogenic T cells as measured by Ag-specific proliferation and the ability to adoptively transfer EAE. These results demonstrate an important role for the CXC chemokine CXCL10 in the recruitment and accumulation of inflammatory mononuclear cells during the pathogenesis of EAE.     

GM-CSF production by autoreactive T cells is required for the activation of microglial cells and the onset of experimental autoimmune encephalomyelitis

Multiple sclerosis (MS) is a CNS autoimmune disease believed to be triggered by T cells secreting Th1-specific proinflammatory cytokines, such as GM-CSF. In the animal model of MS, experimental autoimmune encephalomyelitis (EAE), Th1 but not Th2 cells have been shown to induce disease; however, to date, no single encephalitogenic T cell-derived cytokine has been shown to be required for EAE onset. Because GM-CSF-deficient mice have been shown to be resistant to EAE following immunization with myelin self-Ag, we investigated the cellular source of the required GM-CSF and found that GM-CSF production by encephalitogenic T cells, but not CNS resident or other peripheral cells, was required for EAE induction. Furthermore, we showed that microglial cell activation, but not peripheral macrophage activation, was a GM-CSF-dependent process. Activation of microglial cells by the injection of LPS abrogated the GM-CSF requirement for EAE induction, suggesting that microglial cell activation is required for EAE onset. These data also demonstrate that GM-CSF is a critical Th1 cell-derived cytokine required for the initiation of CNS inflammation associated with EAE, and likely MS.     

Noncanonical autophagy in dendritic cells triggers CNS autoimmunity

Reactivation and expansion of myelin-reactive CD4⁺ T cells within the central nervous system (CNS) are considered to play a key role in the pathogenesis of multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). We demonstrated that accumulation of myelin-specific CD4⁺ T cells within the CNS and subsequent clinical disease development require autophagy related (ATG) protein-dependent phagocytosis in dendritic cells (DCs). Genetic ablation of this pathway impairs presentation of myelin-associated antigen following phagocytosis of injured, phosphatidylserine-exposing oligodendroglial cells. Thus, DCs use ATG-dependent phagocytosis for enhanced presentation of myelin antigen, thereby linking oligodendrocyte injury with antigen processing and T cell-pathogenicity during autoimmune CNS inflammation.     

The role of antigen presenting cells in multiple sclerosis

Multiple sclerosis (MS) is a debilitating T cell mediated autoimmune disease of the central nervous system (CNS). Animal models of MS, such as experimental autoimmune encephalomyelitis (EAE) and Theiler's murine encephalomyelitis virus-induced demyelinating disease (TMEV-IDD) have given light to cellular mechanisms involved in the initiation and progression of this organ-specific autoimmune disease. Within the CNS, antigen presenting cells (APC) such as microglia and astrocytes participate as first line defenders against infections or inflammation. However, during chronic inflammation they can participate in perpetuating the self-destructive environment by secretion of inflammatory factors and/or presentation of myelin epitopes to autoreactive T cells. Dendritic cells (DC) are also participants in the presentation of antigen to T cells, even within the CNS. While the APCs alone are not solely responsible for mediating the destruction to the myelin sheath, they are critical players in perpetuating the inflammatory milieu. This review will highlight relevant studies which have provided insight to the roles played by microglia, DCs and astrocytes in the context of CNS autoimmunity.     

Selective CB2 receptor activation ameliorates EAE by reducing Th17 differentiation and immune cell accumulation in the CNS

CB2, the cannabinoid receptor expressed primarily on hematopoietic cells and activated microglia, mediates the immunoregulatory functions of cannabinoids. The involvement of CB2 in EAE has been demonstrated by using both endogenous and exogenous ligands. We showed previously that CB2 selective agonists inhibit leukocyte rolling and adhesion to CNS microvasculature and ameliorate clinical symptom in both chronic and remitting-relapsing EAE models. Here we showed that Gp1a, a highly selective CB2 agonist, with a four log higher affinity for CB2 than CB1, reduced clinical scores and facilitated recovery in EAE in conjunction with long term reduction in demyelination and axonal loss. We also established that Gp1a affected EAE through at least two different mechanisms, i.e. an early effect on Th1/Th17 differentiation in peripheral immune organs, and a later effect on the accumulation of pathogenic immune cells in the CNS, associated with reductions in the expression of CNS and T cell chemokine receptors, chemokines and adhesion molecules. This is the first report on the in vivo CB2-mediated Gp1a inhibition of Th17/Th1 differentiation. We also confirmed the Gp1a-induced inhibition of Th17/Th1 differentiation in vitro, both in non-polarizing and polarizing conditions. The CB2-induced inhibition of Th17 differentiation is highly relevant in view of recent studies emphasizing the importance of pathogenic self-reactive Th17 cells in EAE/MS. In addition, the combined effect on Th17 differentiation and immune cell accumulation into the CNS, emphasize the relevance of CB2 selective ligands as potential therapeutic agents in neuroinflammation.     

Estrogen receptor alpha signaling in inflammatory leukocytes is dispensable for 17beta-estradiol-mediated inhibition of experimental autoimmune encephalomyelitis

Estrogen treatment has been shown to exert a protective effect on experimental autoimmune encephalomyelitis (EAE), and is under clinical trial for multiple sclerosis. Although it is commonly assumed that estrogens exert their effect by modulating immune functions, we show in this study that 17beta-estradiol (E2) treatment can inhibit mouse EAE without affecting autoantigen-specific T cell responsiveness and type 1 cytokine production. Using mutant mice in which estrogen receptor alpha (ERalpha) has been unambiguously inactivated, we found that ERalpha was responsible for the E2-mediated inhibition of EAE. We next generated irradiation bone marrow chimeras in which ERalpha expression was selectively impaired in inflammatory T lymphocytes or was limited to the radiosensitive hemopoietic compartment. Our data show that the protective effect of E2 on clinical EAE and CNS inflammation was not dependent on ERalpha signaling in inflammatory T cells. Likewise, EAE development was not prevented by E2 treatment in chimeric mice that selectively expressed ERalpha in the systemic immune compartment. In conclusion, our data demonstrate that the beneficial effect of E2 on this autoimmune disease does not involve ERalpha signaling in blood-derived inflammatory cells, and indicate that ERalpha expressed in other tissues, such as CNS-resident microglia or endothelial cells, mediates this effect.