CD200 in growing rat lungs: developmental expression and control by dexamethasone.

CD200 belongs to cell adhesion molecules of the immunoglobulin superfamily. It lacks intracellular signaling motifs and exerts immunosuppressive effect in various tissues. We have reported previously that CD200 is predominantly associated with the capillary network in the alveolar septum of adult rats. The alveolar endothelial cells express CD200, which is confined to their luminal cell membrane facing the blood-air barrier. Our present results show that lung CD200 protein increases gradually with advancing age, being maximally expressed in the early postnatal (P) period. CD200 protein expression, however, declines at P5 but increases again after P7, reaching the adult level at P21. In developing lungs in fetal and neonatal stages, double-immunofluorescence staining has confirmed intense CD200 immunoreactivity delineating the vascular profiles in the double layers of the alveolar capillaries; this staining becomes diffuse and patchy with time. Unlike in adult lungs, immunoelectron microscopy has revealed that CD200 expression in fetal and early postnatal lungs is localized over the entire luminal cell membrane and in the cytoplasm of the endothelia. CD200 expression is progressively redistributed to a specific luminal domain of alveolar endothelia during pulmonary microvascular maturation. In neonatal rats treated with dexamethasone, the amount of lung CD200 significantly increases and is also elevated with time. Upregulation of endothelial CD200 has further been confirmed in isolated pulmonary microvascular endothelial cells treated with dexamethasone. Thus, lung CD200 is developmentally regulated, possibly under hormonal influence.

Extract of Antrodia camphorata exerts neuroprotection against embolic stroke in rats without causing the risk of hemorrhagic incidence.

In this study, the neuroprotective effect of an extract of Antrodia camphorata (A. camphorata), a fungus commonly used in Chinese folk medicine for treatment of viral hepatitis and cancer, alone or in combination with aspirin was investigated in a rat embolic stroke model. An ischemic stroke was induced in rats by a selective occlusion of the middle cerebral artery (MCA) with whole blood clots and then orally treated with A. camphorata (0.25 and 0.75 g/kg/day) alone and combined with aspirin (5 mg/kg/day). Sixty days later, the brains were removed, sectioned, and stained with triphenyltetrazolium chloride and analysed by a commercial image processing software program. Brain infarct volume, neurobehavioral score, cerebral blood perfusion, and subarachnoid and intracerebral hemorrhage incidence were perceived. In addition, potential bleeding side effect of the combinative therapy was assessed by measuring hemoglobin (Hb) content during intracerebral hemorrhage and gastric bleeding, prothrombin time (PT), and occlusion time (OT) after oral administration. Posttreatment with high dose A. camphorata significantly reduced infarct volume and improved neurobehavioral score (P < 0.05). Since A. camphorata alone or with aspirin did not alter the Hb level, this treatment is safe and does not cause hemorrhagic incident. Remarkably, the combination of A. camphorata and aspirin did not show a significant effect on the bleeding time, PT and OT increase suggesting that A. camphorata may have the neuroprotective effect without the prolongation of bleeding time or coagulation time. From these observations, we suggest that combinative therapy of A. camphorata and aspirin might offer enhanced neuroprotective efficacies without increasing side effects.

Curcumin attenuates the expression and secretion of RANTES after spinal cord injury in vivo and lipopolysaccharide-induced astrocyte reactivation in vitro.

Curcumin has been proposed for treatment of various neuroinflammatory and neurodegenerative conditions, including post-traumatic inflammation during acute spinal cord injury (SCI). In this study, we examined whether curcumin anti-inflammation involves regulation of astrocyte reactivation, with special focus on the injury-induced RANTES (regulated on expression normal T-cell expressed and secreted) from astrocytes in acute SCI. Male Sprague-Dawley (SD) rats were subjected to impact injury of the spinal cord followed by treatment with curcumin (40 mg/kg i.p.). RANTES and inducible nitric oxide synthase expression as well as RANTES-positive astrocytes were all induced by injury accompanied by the elevation of lipid peroxidation, and attenuated by the curcumin treatment. In primary cultured rat astrocytes challenged with lipopolysaccharide (LPS) to mimic astrocyte reactivation following SCI, LPS induces robust increase of RANTES expression and the effect was also reduced by 1 µM curcumin treatment. Furthermore, cortical neurons cultured with astrocyte conditioned medium (ACM) conditioned with both LPS and curcumin (LPS-curcumin/ACM), which characteristically exhibited decreased RANTES expression when compared with ACM from astrocytes treated with LPS alone (LPS/ACM), showed higher level of cell viability and lower level of cell death as assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction activity assay and lactate dehydrogenase release assay, respectively. Knockdown of RANTES expression by siRNA (siRANTES) shows reduced RANTES expression and release from LPS-reactivated astrocytes, and ACM obtained from this condition (LPS-siRANTES/ACM) becomes less cytotoxic as compared with the LPS-ACM. Therefore, curcumin reduction of robust RANTES production in reactivated astrocytes both in vitro and in vivo may contribute to its neuroprotection and potential application in SCI.

Glucocorticoid protection of oligodendrocytes against excitotoxin involving hypoxia-inducible factor-1alpha in a cell-type-specific manner.

Glucocorticoids are commonly used in treating diseases with white matter lesions, including demyelinating diseases and spinal cord injury (SCI). However, glucocorticoids are ineffective in gray matter injuries, such as head injury and stroke. The differential glucocorticoid effects in white and gray matter injuries are unclear. We report here a novel mechanism of methylprednisolone (MP), a synthetic glucocorticoid widely used for treating multiple sclerosis and SCI, in protecting oligodendrocytes (OLGs) against AMPA-induced excitotoxicity, which has been implicated in the white matter injuries and diseases. The cytoprotective action of MP in OLGs is causally related to its upregulation of a neuroprotective cytokine erythropoietin (Epo). MP transactivation of Epo expression involves dual transcription factors: glucocorticoid receptor (GR) and hypoxia-inducible factor-1alpha (HIF-1alpha). Coimmunoprecipitation, chromatin immunoprecipitation analysis, yeast two-hybrid analysis, and structure modeling of three-dimensional protein-protein interactions confirm that MP induces interaction between GR DNA binding domain and HIF-1alpha PAS domain, with subsequent recruitment of HIF-1beta to transactivate Epo expression in OLGs. In contrast, MP activates GR but does not induce GR-HIF-1alpha interaction, HIF-1alpha binding to Epo enhancer/promoter, or Epo expression in cultured cortical neurons. The OLG-specific GR-HIF-1alpha transactivation of Epo provides novel insights into the development of more effective therapies for diseases affecting the white matter.

Distribution and expression of CD200 in the rat respiratory system under normal and endotoxin-induced pathological conditions.

In vivo and in vitro studies have clearly demonstrated that signaling mediated by the interaction of CD200 and its cognate receptor, CD200R, results in an attenuation of inflammatory or autoimmune responses through multiple mechanisms. The present results have shown a differential expression of CD200 in the respiratory tract of intact rats. Along the respiratory passage, CD200 was specifically distributed at the bronchiolar epithelia with intense CD200 immunoreactivity localized at the apical surface of some ciliated epithelial cells; only a limited expression was detected on the Clara cells extending into the alveolar duct. In the alveolar septum, double immunofluorescence showed intense CD200 immunolabeling on the capillary endothelia. A moderate CD200 labeling was observed on the alveolar type II epithelial cells. It was, however, absent in the alveolar type I epithelial cells and the alveolar macrophages. Immunoelectron microscopic study has revealed a specific distribution of CD200 on the luminal front of the thin portion of alveolar endothelia. During endotoxemia, the injured lungs showed a dose- and time-dependent decline of CD200 expression accompanied by a vigorous infiltration of immune cells, some of them expressing ionized calcium binding adapter protein 1 or CD200. Ultrastructural examination further showed that the marked reduction of CD200 expression was mainly attributable to the loss of alveolar endothelial CD200. It is therefore suggested that CD200 expressed by different lung cells may play diverse roles in immune homeostasis of normal lung, in particular, the molecules on alveolar endothelia that may control regular recruitment of immune cells via CD200-CD200R interaction. Additionally, it may contribute to intense infiltration of immune cells following the loss or inefficiency of CD200 under pathological conditions.

Endothelial CD200 is heterogeneously distributed, regulated and involved in immune cell-endothelium interactions.

CD200 is a highly glycosylated cell surface protein containing two immunoglobulin superfamily domains in the extracellular region and performs immunosuppressive activities. It is widely distributed in various tissues including the vascular endothelium. We report here the distribution of CD200 in rat endothelia from different vascular beds. Endothelial CD200 immunoreactivity was weakly expressed in most arteries but was intensely expressed in the arterioles, most veins and venules, as well as continuous and fenestrated capillaries. The distribution of CD200 in the sinusoidal and lymphatic endothelia was variable. Immunoelectron microscopic studies revealed that endothelial CD200 varied considerably not only in different microvasculatures but also in the membrane domains at the subcellular level. Endothelial CD200 expression was differentially regulated by lipopolysaccharide in cell types both in vivo and in vitro. Functional assessments of endothelial CD200 suggested that the physical binding between CD200 and CD200 receptor (CD200R) was involved in T-cell adhesion to the endothelium but not in macrophage-endothelium interaction. In the latter, however, CD200 agonist, a synthetic peptide from complementarity-determining region 3 of mouse CD200, may trigger CD200R signaling in macrophages to suppress their adhesion to the endothelium. Our findings demonstrate that the distribution, subcellular localization, and lipopolysaccharide-regulation of endothelial CD200 are heterogeneous, and provide evidence elucidating the functional roles of endothelial CD200 during tissue inflammation.

Responses of microglia in vitro to the gram-positive bacterial component, lipoteichoic acid.

An increase in incidence and severity of gram-positive infections has emerged in the past decade. In this regard, attention has been focused recently on immune responses of microglial cells in the central nervous system to gram-positive bacteria. The underlying immunological and cellular events in microglial activation induced by specific bacterial toxin of gram-positive bacteria, however, have not yet been clarified fully. This study reports that a simple cell wall product, lipoteichoic acid (LTA), derived from gram-positive bacteria (Staphylococcus aureus) could trigger microglial activation in vitro. Microglia challenged with LTA showed intense ruffling of plasma membrane in the form of lamellipodia or rounded up forming cell aggregates. MTT assay and Western blot analysis with anti-proliferating cell nuclear antigen antibody showed a significant microglial proliferation that may be induced at the later phases of LTA treatment with low doses but at the early period with a high dose. Concentrated LTA also caused apoptotic death of cultured microglia showing fragmented nuclei and increased expression of annexin V or caspase 3. In response to LTA, isolated microglia increased the expression of inducible nitric oxide synthase and major histocompatibility complex class II antigen. Microglial LTA receptors such as CD14 molecule, complement receptor type 3, and macrophage scavenger receptor were upregulated concurrently. In conclusion, staphylococcal LTA can exert an immunomodulatory effect on microglial morphology, cell cycle, and immunomolecules, including its receptors.