Specific tools for targeting and expression in Müller glial cells.

Despite their physiological roles, Müller glial cells are involved directly or indirectly in retinal disease pathogenesis and are an interesting target for therapeutic approaches for retinal diseases and regeneration such as CRB1 inherited retinal dystrophies. In this study, we characterized the efficiency of adeno-associated virus (AAV) capsid variants and different promoters to drive protein expression in Müller glial cells. ShH10Y and AAV9 were the most powerful capsids to infect mouse Müller glial cells. Retinaldehyde-binding protein 1 (RLBP1) promoter was the most powerful promoter to transduce Müller glial cells. ShH10Y capsids and RLBP1 promoter targeted human Müller glial cells in vitro. We also developed and tested smaller promoters to express the large CRB1 gene via AAV vectors. Minimal cytomegalovirus (CMV) promoter allowed expression of full-length CRB1 protein in Müller glial cells. In summary, ShH10Y and AAV9 capsids, and RLBP1 or minimal CMV promoters are of interest as specific tools to target and express in mouse or human Müller glial cells.

Shear stress-dependent downregulation of the adhesion-G protein-coupled receptor CD97 on circulating leukocytes upon contact with its ligand CD55.

Adhesion G protein-coupled receptors (aGPCRs) are two-subunit molecules, consisting of an adhesive extracellular α subunit that couples noncovalently to a seven-transmembrane ß subunit. The cooperation between the two subunits and the effect of endogenous ligands on the functioning of aGPCRs is poorly understood. In this study, we investigated the interaction between the pan-leukocyte aGPCR CD97 and its ligand CD55. We found that leukocytes from CD55-deficient mice express significantly increased levels of cell surface CD97 that normalized after transfer into wild-type mice because of contact with CD55 on both leukocytes and stromal cells. Downregulation of both CD97 subunits occurred within minutes after first contact with CD55 in vivo, which correlated with an increase in plasma levels of soluble CD97. In vitro, downregulation of CD97 on CD55-deficient leukocytes cocultured with wild-type blood cells was strictly dependent on shear stress. In vivo, CD55-mediated downregulation of CD97 required an intact circulation and was not observed on cells that lack contact with the blood stream, such as microglia. Notably, de novo ligation of CD97 did not activate signaling molecules constitutively engaged by CD97 in cancer cells, such as ERK and protein kinase B/Akt. We conclude that CD55 downregulates CD97 surface expression on circulating leukocytes by a process that requires physical forces, but based on current evidence does not induce receptor signaling. This regulation can restrict CD97-CD55-mediated cell adhesion to tissue sites.

Phenotyping primary human microglia: tight regulation of LPS responsiveness.

Much is still unknown about mechanisms underlying the phenotypical and functional versatility of human microglia. Therefore, we developed a rapid procedure to isolate pure microglia from postmortem human brain tissue and studied their immediate ex vivo phenotype and responses to key inflammatory mediators. Microglia were isolated, along with macrophages from the choroid plexus by tissue dissociation, density gradient separation, and selection with magnetic microbeads. By flow cytometry, microglia were identified by a CD11b(+) CD45(dim) phenotype and a smaller size compared with CD11b(+) CD45(high) macrophages. Interestingly, white matter microglia from donors with peripheral inflammation displayed elevated CD45 levels and increased size and granularity, but were still distinct from macrophages. The phenotype of isolated microglia was further specified by absent surface expression of CD14, CD200 receptor, and mannose receptor (MR, CD206), all of which were markedly expressed by macrophages. Microglia stimulated immediately after isolation with LPS and IFNγ failed to upregulate TNFα or CCR7. Notably, responsiveness to LPS and IFNγ was clearly instigated in microglia after overnight preculture, which coincided with a strong upregulation of CD14. Culture of microglia with IL-4 resulted in the induction of HLA-DR and CCL18 but not MR, whereas culture with dexamethasone did induce MR, in addition to CD163 and CCL18. In conclusion, this study demonstrates phenotypic changes of microglia associated with peripheral inflammation, and reveals tight regulation of responses to LPS and IFNγ as well as distinct microglial responses to IL-4 and glucocorticoids. These findings are of high relevance to studies on human microglia functioning in health and disease.

A novel role for CD55 in granulocyte homeostasis and anti-bacterial host defense.

BACKGROUND: In addition to its complement-regulating activity, CD55 is a ligand of the adhesion class G protein-coupled receptor CD97; however, the relevance of this interaction has remained elusive. We previously showed that mice lacking a functional CD97 gene have increased numbers of granulocytes. METHODOLOGY/RESULTS: Here, we demonstrate that CD55-deficient mice display a comparable phenotype with about two-fold more circulating granulocytes in the blood stream, the marginated pool, and the spleen. This granulocytosis was independent of increased complement activity. Augmented numbers of Gr-1-positive cells in cell cycle in the bone marrow indicated a higher granulopoietic activity in mice lacking either CD55 or CD97. Concomitant with the increase in blood granulocyte numbers, Cd55⁻/⁻ mice challenged with the respiratory pathogen Streptococcus pneumoniae developed less bacteremia and died later after infection. CONCLUSIONS: Collectively, these data suggest that complement-independent interaction of CD55 with CD97 is functionally relevant and involved in granulocyte homeostasis and host defense.

GFAP-driven GFP expression in activated mouse Müller glial cells aligning retinal blood vessels following intravitreal injection of AAV2/6 vectors.

BACKGROUND: Müller cell gliosis occurs in various retinal pathologies regardless of the underlying cellular defect. Because activated Müller glial cells span the entire retina and align areas of injury, they are ideal targets for therapeutic strategies, including gene therapy. METHODOLOGY/PRINCIPAL FINDINGS: We used adeno-associated viral AAV2/6 vectors to transduce mouse retinas. The transduction pattern of AAV2/6 was investigated by studying expression of the green fluorescent protein (GFP) transgene using scanning-laser ophthalmoscopy and immuno-histochemistry. AAV2/6 vectors transduced mouse Müller glial cells aligning the retinal blood vessels. However, the transduction capacity was hindered by the inner limiting membrane (ILM) and besides Müller glial cells, several other inner retinal cell types were transduced. To obtain Müller glial cell-specific transgene expression, the cytomegalovirus (CMV) promoter was replaced by the glial fibrillary acidic protein (GFAP) promoter. Specificity and activation of the GFAP promoter was tested in a mouse model for retinal gliosis. Mice deficient for Crumbs homologue 1 (CRB1) develop gliosis after light exposure. Light exposure of Crb1(-/-) retinas transduced with AAV2/6-GFAP-GFP induced GFP expression restricted to activated Müller glial cells aligning retinal blood vessels. CONCLUSIONS/SIGNIFICANCE: Our experiments indicate that AAV2 vectors carrying the GFAP promoter are a promising tool for specific expression of transgenes in activated glial cells.

Expression of the inhibitory CD200 receptor is associated with alternative macrophage activation.

Classical macrophage activation is inhibited by the CD200 receptor (CD200R). Here, we show that CD200R expression was specifically induced on human in vitro polarized macrophages of the alternatively activated M2a subtype, generated by incubation with IL-4 or IL-13. In mice, peritoneal M2 macrophages, elicited during infection with the parasites Taenia crassiceps or Trypanosoma brucei brucei, expressed increased CD200R levels compared to those derived from uninfected mice. However, in vitrostimulation of mouse peritoneal macrophages and T. crassiceps infection in IL-4-/- and IL-4R-/- mice showed that, in contrast to humans, induction of CD200R in mice was not IL-4 or IL-13 dependent. Our data identify CD200R as a suitable marker for alternatively activated macrophages in humans and corroborate observations of distinct species- and/or site-specific mechanisms regulating macrophage polarization in mouse and man.

Deletion of either CD55 or CD97 ameliorates arthritis in mouse models.

OBJECTIVE: CD55 (decay-accelerating factor) is best known for its role in the negative regulation of the complement system. Indeed, lack of this molecule leads to disease aggravation in many autoimmune disease models. However, CD55 is abundantly present on fibroblast-like synoviocytes and is also a ligand of the adhesion-class heptahelical receptor CD97, which is expressed by infiltrating macrophages. Treatment with antibodies to CD97 ameliorates the collagen-induced model of rheumatoid arthritis (RA) in DBA/1 mice, but the net contribution of CD55 is unknown. This study was undertaken to investigate the role of CD55 in experimental RA. METHODS: Arthritis was induced in wild-type, CD55(-/-), and CD97(-/-) mice using collagen-induced and K/BxN serum-transfer models. Incidence of arthritis was monitored over time, and disease activity was assessed by clinical and immunohistochemical evaluation. RESULTS: In contrast to observations in many inflammatory disease models, lack of CD55 resulted in decreased arthritis in experimental models of RA. Consistent with the previously reported effects of anti-CD97 antibody treatment, CD97(-/-) mice had reduced arthritis activity compared with wild-type controls. CONCLUSION: Our findings indicate that the lack of CD55 or CD97 in 2 different models of arthritis increases resistance to the disease. These findings provide insight into a role for CD55 interaction with CD97 in the pathogenesis of RA and suggest that therapeutic strategies that disrupt CD55/CD97 may be clinically beneficial.

Restoring immune suppression in the multiple sclerosis brain.

Multiple sclerosis is a very disabling inflammatory demyelinating disease of the brain of unknown etiology. Current therapies can reduce new lesion development and partially prevent clinical disease activity, but none can halt the progression, or cure the disease. We will review current therapeutic strategies, which are mostly discussed in literature in terms of their effective inhibition of T cells. However, we argue that many of these treatments also influence the myeloid compartment. Interestingly, recent evidence indicates that myelin phagocytosis by infiltrated macrophages and activated microglia is not just a hallmark of multiple sclerosis, but also a key determinant of lesion development and disease progression. We reason that severe side effects and/or insufficient effectiveness of current treatments necessitates the search for novel therapeutic targets, and postulate that these should aim at manipulation of the activation and phagocytic capacity of macrophages and microglia. We will discuss three candidate targets with high potential, namely the complement receptor 3, CD47-SIRPalpha interaction as well as CD200-CD200R interaction. Blocking the actions of complement receptor 3 could inhibit myelin phagocytosis, as well as migration of myeloid cells into the brain. CD47 and CD200 are known to inhibit macrophage/microglia activation through binding to their receptors SIRPalpha and CD200R, expressed on phagocytes. Triggering these receptors may thus dampen the inflammatory response. Our recent findings indicate that the CD200-CD200R interaction is the most specific and hence probably best-suited target to suppress excessive macrophage and microglia activation, and restore immune suppression in the brain of patients with multiple sclerosis.

Lack of CD200 enhances pathological T cell responses during influenza infection.

Influenza virus infection can be accompanied by life-threatening immune pathology most likely due to excessive antiviral responses. Inhibitory immune receptors may restrain such overactive immune responses. To study the role of the inhibitory immune receptor CD200R and its ligand CD200 during influenza infection, we challenged wild-type and CD200(-/-) mice with influenza virus. We found that CD200(-/-) mice in comparison to wild-type controls when inoculated with influenza virus developed more severe disease, associated with increased lung infiltration and lung endothelium damage. CD200(-/-) mice did develop adequate adaptive immune responses and were able to control viral load, suggesting that the severe disease was caused by a lack of control of the immune response. Interestingly, development of disease was completely prevented by depletion of T cells before infection, despite dramatically increased viral load, indicating that T cells are essential for the development of disease symptoms. Our data show that lack of CD200-CD200R signaling increases immune pathology during influenza infection, which can be reduced by T cell depletion.

GITR triggering induces expansion of both effector and regulatory CD4+ T cells in vivo.

Glucocorticoid-induced TNF receptor family-related protein (GITR) is expressed on activated and regulatory T cells, but its role on these functionally opposing cell types is not fully understood. Here we describe that transgenic expression of GITR's unique ligand (GITRL) induces a prominent increase of both effector and regulatory CD4(+) T cells, but not CD8(+) T cells. Regulatory T cells from GITRL transgenic mice are phenotypically activated and retain their suppressive capacity. The accumulation of effector and regulatory T cells is not due to enhanced differentiation of naive T cells, but is a direct result of increased proliferation. Functional consequences of increased numbers of both regulatory and effector T cells were tested in an autoimmune model and show that GITR stimulation is protective, as it significantly delays disease induction. These data indicate that GITR regulates the balance between regulatory and effector CD4(+) T cells by enhancing proliferation of both populations in parallel.

Distribution of the immune inhibitory molecules CD200 and CD200R in the normal central nervous system and multiple sclerosis lesions suggests neuron-glia and glia-glia interactions.

CD200 is a membrane glycoprotein that suppresses immune activity via its receptor, CD200R. CD200-CD200R interactions have recently been considered to contribute to the "immune privileged" status of the central nervous system (CNS). The mechanisms by which these interactions take place are not well understood in part because there is limited detailed information on the distribution of CD200 and CD200R in the CNS. Here, we used immunohistochemistry to characterize the distinct anatomical and cellular distribution of these molecules in multiple sclerosis (MS) lesions and controls. CD200 was robustly expressed in gray matter areas including the cerebral cortex, hippocampus, striatum, cerebellum, and spinal cord, where neurons appeared immunopositive. CD200 expression was also detected in oligodendrocytes, but not in astrocytes or microglia. In CNS samples from MS patients, CD200 expression was additionally observed on reactive astrocytes in chronic active plaque centers, despite our previous finding of an overall decrease ofCD200 expression in MS lesions. In contrast to CD200, the immunolocalization pattern of CD200R was very distinct, showing high expression on perivascular macrophages in both gray and white matter. Using flow cytometry, we also found that human primary microglia express low levels of CD200R. These data suggest that CD200-mediated immune suppression may occur not only via neuron-microglia interactions, but also via glia-glia interactions, especially in inflammatory conditions in which an immune-suppressive environment needs to be restored; this may occur as a result of increased CD200 expression on reactive astrocytes.

Analysis of CD97 expression and manipulation: antibody treatment but not gene targeting curtails granulocyte migration.

The heptahelical receptor CD97 is a defining member of the EGF-TM7 family of adhesion class receptors. In both humans and mice, CD97 isoforms are expressed with variable numbers of tandemly arranged N-terminal epidermal growth factor-like domains that facilitate interactions with distinct cellular ligands. Results from treatment of mice with mAbs in various disease models have suggested a role for CD97 in leukocyte trafficking. Here, we aimed to thoroughly characterize the expression profile of CD97, and delineate its biological function. To this end, we applied a novel polyclonal Ab, which is the first antiserum suitable for immunohistochemistry, and combined this analysis with the study of Cd97-lacZ knock-in mice. We show that similar to the situation in humans, hematopoietic, epithelial, endothelial, muscle, and fat cells expressed CD97. Despite this broad expression pattern, the Cd97(-/-) mouse that we created had no overt phenotype, except for a mild granulocytosis. Furthermore, granulocyte accumulation at sites of inflammation was normal in the absence of CD97. Interestingly, application of CD97 mAbs blocked granulocyte trafficking after thioglycollate-induced peritonitis in wild-type but not in knock-out mice. Hence, we conclude that CD97 mAbs actively induce an inhibitory effect that disturbs normal granulocyte trafficking, which is not perturbed by the absence of the molecule.

The inhibitory CD200R is differentially expressed on human and mouse T and B lymphocytes.

To ensure an adequate response against pathogens and prevent unwanted self-reactivity, immune cells need to functionally express both activating and inhibitory receptors. CD200R is an inhibitory receptor mainly expressed on myeloid cells that down-modulates cellular activation both in vivo and in vitro. Although previously mainly studied as a regulator of myeloid function, we now show that CD200R is differentially expressed on human and mouse T-cell subsets. In both species, CD4+ T cells express higher amounts of CD200R than CD8+ T cells, and memory cells express higher amounts of CD200R than naïve or effector cells. CD200R expression is up-regulated on both CD4+ and CD8+ T cells after stimulation in vitro. Furthermore, we show CD200R expression on human and mouse B cells. In human tonsils, CD200R is differentially expressed on B cells, with high expression on memory cells and plasmablasts. Mice lacking the ligand for CD200R, CD200-/- mice, do not show abnormal composition of the lymphocyte compartment and have normal B cell responses to antigenic challenge. Although the functional implications remain to be elucidated, the expression of CD200R on lymphocytes suggests a much broader role for CD200R-mediated immune regulation than previously anticipated.

Downregulation of macrophage inhibitory molecules in multiple sclerosis lesions.

OBJECTIVE: Inflammatory and demyelinating activity of activated resident macrophages (microglia) and recruited blood-borne macrophages are considered crucial in multiple sclerosis (MS) lesion development. The membrane glycoproteins CD200 and CD47, highly expressed on neurons, are mediators of macrophage inhibition via their receptors CD200R and signal-regulatory protein alpha, respectively, on myeloid cells. We determined the expression pattern of immune inhibitory molecules in relation to genes involved in macrophage activation and MS lesion pathology. METHODS: Laser dissection microscopy was combined with real-time polymerase chain reaction to quantitatively study these gene expression patterns in specific subareas (rim, center, and normal-appearing white matter) of chronic active and inactive MS lesions. RESULTS: Hallmarks of MS pathology were confirmed by messenger RNA expression patterns of glial fibrillary acidic protein, neurofilament (NF), myelin basic protein, growth factors, chemokines and receptors, and macrophage activation markers, although expression of osteopontin and alphaB-crystallin was decreased. CD200 and CD47 were downregulated in the center of chronic active and inactive MS lesions. CD47 expression was also decreased in the rim of chronic active lesions, where complement expression was increased. This expression profile was also found in normal-appearing white matter surrounding these lesions, but not surrounding inactive lesions. Expression of CD200R and signal-regulatory protein alpha was not altered. INTERPRETATION: These data suggest that diminished immune inhibition via decreased CD200 and CD47 expression contributes to a disturbed equilibrium in macrophage and microglia activation in MS lesions. Furthermore, this may result in a proinflammatory predisposition in the area surrounding chronic active lesions, thereby contributing to axonal injury, demyelination, and possible lesion expansion.

Ligation of CD200R by CD200 is not required for normal murine myelopoiesis.

CD200R is an inhibitory receptor involved in the regulation of myeloid cells. It recruits Dok-1 and Dok-2, which are potent inhibitors of the Ras signalling pathway used by colony-stimulating factor (CSF) receptors. Dok-1/Dok-2 double knockout (DKO) mice develop leukaemia at 10-12 months of age. We investigated whether disturbed CD200R signalling could be responsible for this phenotype. Therefore, we studied whether CD200(-/-) mice have altered myelopoiesis and develop leukaemia. We report that CD200R is expressed on haematopoietic progenitor cells. However, CD200(-/-) mice have normal numbers of myeloid progenitors in the bone marrow and these cells have normal proliferative capacity. These results indicate that the development of leukaemia in Dok-1/Dok-2 DKO mice is not solely due to an absence of CD200R signalling. In addition, we show that the previously reported enhanced numbers of myeloid cells do not occur in all CD200(-/-) mice. We determined whether variations in the numbers of peripheral myeloid cells were due to an enhanced response to granulocyte-CSF (G-CSF) or an inflammatory stimulus. Mobilisation of immature neutrophils via G-CSF and infiltration of mature neutrophils and macrophages upon thioglycolate injection were not altered in CD200(-/-) mice. We conclude that CD200(-/-) mice exhibit normal myelopoiesis and that development of leukaemia in Dok-1/Dok-2 DKO mice is not caused by a lack of CD200-mediated CD200R signalling.

The human EGF-TM7 receptor EMR3 is a marker for mature granulocytes.

EMR3 is a member of the epidermal growth factor-seven-transmembrane (EGF-TM7) family of adhesion class TM7 receptors. This family also comprises CD97, EMR1, EMR2, and EMR4. To characterize human EMR3 at the protein level, we generated Armenian hamster mAb. Using the mAb 3D7, we here demonstrate that EMR3, like other EGF-TM7 receptors, is expressed at the cell surface as a heterodimeric molecule consisting of a long extracellular alpha-chain, which possesses at its N-terminus EGF-like domains and a membrane-spanning beta-chain. Flow cytometric analysis revealed that all types of myeloid cells express EMR3. In peripheral blood, the highest expression of EMR3 was found on granulocytes. More mature CD16(+) monocytes express high levels of EMR3, and CD16(-) monocytes and myeloid dendritic cells (DC) are EMR3(dim/low). Lymphocytes and plasmacytoid DC are EMR3(-). It is interesting that in contrast with CD97 and EMR2, CD34(+)CD33(-)/CD38(-) committed hematopoietic stem cells and CD34(+)CD33(+)/CD38(+) progenitors in bone marrow do not express EMR3. In vitro differentiation of HL-60 cells and CD34(+) progenitor cells revealed that EMR3 is only up-regulated during late granulopoiesis. These results demonstrate that the expression of EGF-TM7 receptors on myeloid cells is differentially regulated. EMR3 is the first family member found mainly on granulocytes.

An unusual mode of concerted evolution of the EGF-TM7 receptor chimera EMR2.

The epidermal growth factor (EGF)-TM7 receptors CD97, EMR1, EMR2, EMR3, and EMR4 form a group of adhesion class heptahelical molecules predominantly expressed by cells of the immune system. These receptors bind cellular ligands through EGF-like domains, localized N-terminal to a large extracellular region. Remarkably, EMR2 possesses a chimeric structure with a seven-span transmembrane (TM7) region most related to EMR3 and an EGF domain region nearly identical to CD97. By comparing EGF-TM7 receptors in primates and dogs, we identified an intriguing pattern of concerted evolution, apparently mediated by gene conversion, among EMR2 and the oppositely orientated and physically adjacent genes CD97 and EMR3. This concerted evolution has continuously maintained the chimeric structure of EMR2 since early mammal radiation. Most highly conserved between EMR2 and CD97 is the fourth EGF domain, which mediates binding to chondroitin sulfate, a ligand specificity shared by both receptors. Another ligand, CD55, is bound effectively only by CD97. We show that different molecular mechanisms (mutations vs. alternative splicing) prevent CD55 binding by EMR2 in hominoids. Our findings illustrate how various and partially opposing evolutionary events have shaped the structure and ligand specificity of a modern mammalian gene family.

Interactions between hemopoietically derived TNF and central nervous system-resident glial chemokines underlie initiation of autoimmune inflammation in the brain.

Tumor necrosis factor is a proinflammatory cytokine that induces directly many of the components required for inflammation to proceed rapidly. We show in this study that the interplay between TNF and chemokines, now recognized to be essential for normal secondary lymphoid tissue development, is also a feature of CNS inflammation, and that the two apparently dissimilar biological processes share many properties. Thus, induction of seven chemokines, including T cell activation gene 3 (TCA3), monocyte chemoattractant protein-1, and IFN-gamma-inducible protein-10 within the CNS during experimental autoimmune encephalomyelitis fails to occur early in the inflammatory process in TNF-deficient mice, despite local expression of monokines and IFN-gamma. The critical source of TNF in CNS inflammation is the infiltrating hemopoietic cell, and, in its absence, chemokine expression by irradiation-resistant CNS-resident cells fails. The CCR8 ligand, TCA3, is shown to be produced predominantly by resident microglia of the CNS in response to TNF. Using CCR8(-/-) mice, evidence is provided that TCA3-CCR8 interactions contribute to rapid-onset CNS inflammation. Thus, through TNF production, the hemopoietic compartment initiates the signals for its own movement into tissues, although the tissue ultimately defines the nature of that movement. Chemokines are a major, although not exclusive, mechanism by which tissues regulate leukocyte movement in response to TNF.

Constitutive retinal CD200 expression regulates resident microglia and activation state of inflammatory cells during experimental autoimmune uveoretinitis.

Recent evidence supports the notion that tissue OX2 (CD200) constitutively provides down-regulatory signals to myeloid-lineage cells via CD200-receptor (CD200R). Thus, mice lacking CD200 (CD200(-/-)) show increased susceptibility to and accelerated onset of tissue-specific autoimmunity. In the retina there is extensive expression of CD200 on neurons and retinal vascular endothelium. We show here that retinal microglia in CD200(-/-) mice display normal morphology, but unlike microglia from wild-type CD200(+/+) mice are present in increased numbers and most significantly, express inducible nitric oxide synthase (NOS2), a macrophage activation marker. Onset and severity of uveitogenic peptide (1-20) of interphotoreceptor retinoid-binding protein-induced experimental autoimmune uveoretinitis is accelerated in CD200(-/-) mice and although tissue destruction appears no greater than seen in CD200(+/+) mice, there is continued increased ganglion and photoreceptor cell apoptosis. Myeloid cell infiltrate was increased in CD200(-/-) mice during experimental autoimmune uveoretinitis, although NOS2 expression was not heightened. The results indicate that the CD200:CD200R axis regulates retinal microglial activation. In CD200(-/-) mice the release of suppression of tonic macrophage activation, supported by increased NOS2 expression in the CD200(-/-) steady state accelerates disease onset but without any demonstration of increased target organ/tissue destruction.