Chiu-Yun Chang

Signal transduction pathways of nitric oxide release in primary microglial culture challenged with gram-positive bacterial constituent, lipoteichoic acid

Between one-third and one-half of all cases of sepsis are known to be caused by gram-positive microorganisms through the cell wall component, e.g. lipoteichoic acid (LTA). Gram-positive bacteria are also known to induce encephalomyelitis and meningeal inflammation, and enhance the production of nitric oxide (NO) via expression of inducible nitric oxide synthase (iNOS) in murine tissue macrophages. It remains to be explored if LTA could activate microglia considered to be resident brain macrophages. We report here that LTA derived from gram-positive bacteria (Staphylococcus aureus) significantly induces NO release and iNOS expression in primary microglia. LTA-induced NO accumulation was detected at 2 h in microglial culture and was significantly attenuated by pretreatment with anti-CD14, complement receptor type 3 (CR3) or scavenger receptor (SR) antibodies. LTA activated mitogen-activated protein kinases (MAPKs) such as extracellular signal-regulated kinase, p38 MAPK or c-Jun N-terminal kinase in cultured microglia. LTA-elicited microglial NO production was also drastically suppressed by SB203580 (p38 MAPK inhibitor) or pyrrolidine dithiocarbamate (an inhibitor of nuclear factor kappaB), indicating that p38 MAPK and nuclear factor kappaB were involved in microglial NO release after LTA challenge. These results suggest that gram-positive bacterial product such as LTA can activate microglia to release NO via the signal transduction pathway involving multiple LTA receptors (e.g. CD14, CR3 or SR), p38 MAPK and nuclear factor kappaB. The in vivo study further confirmed that administered intracerebrally LTA induced considerable noticeable iNOS, phospho-IkappaB and phospho-p38 MAPK expression in microglia/macrophages.

HETEROGENEITY OF ANTIGEN EXPRESSION AND LECTIN LABELING ON MICROGLIAL CELLS IN THE OLFACTORY BULB OF ADULT RATS

Microglia in different layers of the rat olfactory bulb expressed a variety of membrane antigens except for CD4 (OX-35). Bulb microglial cells bearing complement receptor type 3 (OX-42) were ubiquitous and their immunoreactivity varied considerably in different bulb layers. Although very few in number, labeled microglia in all layers also expressed major histocompatibility complex class I antigen (OX-18), leukocyte common antigen (OX-1) and unknown macrophage antigen (ED-2). The latter was localized in cells distributed almost exclusively in the perivascular spaces. The immunoreactivity of ED-1, an unknown cytoplasmic or lysosomal membrane antigen in macrophages, was localized in labeled microglia which were concentrated mainly in the granule cell layer and periglomerular zone of the bulb. A variable number of microglial cells were stained with OX-6 (major histocompatibility complex class II antigen) and they were located predominantly in the periglomerular zone and at the junction between the granule cell layer and the subependymal layer. Ultrastructural study using GSA I-B4 lectin labeling showed that microglia in different layers of the bulb exhibited similar labeling patterns in their subcellular structures. A remarkable feature was the occurrence of some microglia in the olfactory nerve layer, subependymal layer and granule cell layer adjacent to the subependymal layer in which the cells showed intense lectin labeling at their Golgi apparatus, a feature which was absent in microglia of other bulb layers. Present results showed the regional differences in microglial antigen expressions and lectin labeling within the olfactory bulb. It is therefore suggested that the cells subserve very different specific functions depending on their ambient microenvironments. The heterogeneity of microglial functions in the olfactory bulb may be related to the progressive regeneration and degeneration of its containing neurons.

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.

CC-CHEMOKINE LIGAND 18/PULMONARY ACTIVATION-REGULATED CHEMOKINE EXPRESSION IN THE CNS WITH SPECIAL REFERENCE TO TRAUMATIC BRAIN INJURIES AND NEOPLASTIC DISORDERS

Pulmonary activation-regulated chemokine (PARC) now designated CC-chemokine ligand 18 (CCL18) has been shown to play a significant role in the pathogenesis of various tissue injuries and diseases in a proinflammatory or immune suppressive way to limit or support the inflammation or disease. While much is known about the roles of CCL18/PARC in non-neural tissues, its expression in the CNS has remained largely unexplored and controversial. Using reverse transcription polymerase chain reaction (RT-PCR) and double immunohistochemical staining, we analyzed the expression of CCL18/PARC in the human brain with special reference to traumatic brain injuries and tumors. The RT-PCR analysis revealed the expression of CCL18/PARC mRNA both in the traumatic brain and glioma tissues examined. Immunoexpression of CCL18/PARC protein was consistently detected in all cases of traumatic brain injuries examined by immunohistochemical staining. Double immunofluorescence labeling has extended the study that CCL18/PARC positive cells were macrophages/microglia, astrocytes or neurons. The CCL18/PARC expression was localized in macrophage-like cells in two of eight glioblastoma tissues whose cancer cells were CCL18/PARC negative. Unexpectedly, CCL18/PARC mRNA weakly and constitutively expressed by glioblastoma cell line was upregulated after endotoxin stimulation. The present results indicated a significant production of CCL18/PARC in different CNS traumatic and neoplasm tissues by specific cellular elements expressing the chemokine. An anti-inflammatory mechanism jointly exerted by these cells via CCL18/PARC may be involved in the CNS immunity after traumatic injury and tumorigenesis.

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.

Microglia in the olfactory bulb of rats during postnatal development and olfactory nerve injury with zinc sulfate: A lectin labeling and ultrastrucutural study

Using isolectin (GSA I-B4) as a marker, this study examined the possible alterations of lectin-labeled membranous glycoproteins in microglial cells in the olfactory bulb of normal development and under experimentally induced degeneration. In light microscopy, several morphological types of microglial cells representing different degrees of cell differentiation were distributed in the bulb laminae. A gradient of microglial differentiation extending from the intermediate to superficial and intermediate to deep occurs in the bulb layers. The differentiation gradient and lectin labeling pattern of microglial cells in the developing bulb resembled those in other areas of the brain tissues. Differentiating microglia showed a gradual diminution of lectin staining when the nascent round cells transformed into the mature ramified cells. Microglia in the external plexiform layer of the olfactory bulb were the first to mature and the cells expressed very weak lectin reactivity. In mature or adult rats, some microglial cells showing intense lectin labeling were observed in the olfactory nerve layer, granule cell layer and subependymal layer. Ultrastructurally, lectin labeling was localized at the trans saccules of the Golgi apparatus. Microglial cells in other bulb laminae, however, exhibited a negative reaction for the isolectin at the Golgi apparatus. Following intranasal irrigation of zinc sulfate, some microglial cells in the olfactory nerve layer and glomerular layer were activated to become phagocytic cells with increased lectin labeling at their ramified processes. GSA I-B4 staining was also localized at their trans saccules of the Golgi apparatus. The lectin labeling pattern of these phagocytic cells resembled that of differentiating microglia in postnatal bulbs, suggesting that bulb microglia in the lesioned sites were activated through cell dedifferentiation into macrophages.

Response of amoeboid and differentiating ramified microglia to glucocorticoids in postnatal rats: a lectin histochemical and ultrastructural study.

After glucocorticoid injection(s), the number of amoeboid microglial cells (AMC) in the corpus callosum labelled by lectin was markedly reduced when compared with the corresponding control rats. In rats killed at the age of 7 days, all the labeled cells differentiated to become ramified microglia. Ultrastructurally, the AMC in glucocorticoid-injected rats were extremely vacuolated and showed increased lipid droplets. Furthermore, the cells displayed varied lectin labelling patterns especially at both the trans saccules of the Golgi apparatus and lysosomes. In differentiating ramified microglia, massive cellular debris and lectin-stained vesicles or vacuoles were observed; some of the latter appeared to fuse with the plasma membrane. The most striking feature after glucocorticoid (GCC) treatment was the complete diminution of lectin labelling at the Golgi saccules in some differentiating ramified microglia. The present results have demonstrated different effects of glucocorticoids on AMC and differentiating ramified microglia. The differential response of AMC and differentiating ramified microglia to the immunosuppressive drugs may be attributed to the fact that these cells in the postnatal brains subserve different functions or that they are at different differentiation stages. In other words, the sensitivity of microglial cells to the immunosuppressive drugs is dependent upon the stage of cell maturation/differentiation.

Variant palmaris profundus enclosed by an unusual loop of the median nerve

According to the usual description in most anatomy texts, the median nerve in the forearm passes between the 2 heads of pronator teres. It continues distally between flexor digitorum superficialis and profundus almost to the retinaculum. Muscular branches leave the nerve near the elbow and supply all superficial muscles of the anterior part of the forearm except flexor carpi ulnaris. Many variations of the median nerve in the forearm have been reported (Urban & Krosman, 1992). The palmaris profundus is also a rare anomaly of the forearm (Dyreby & Engber, 1982). It originates from the radial side of the common flexor tendon in the proximal forearm and inserts into the undersurface of the palmar aponeurosis. The origin of palmaris profundus may be close to the median nerve and its branches, and may be involved in compressive neuropathy of the anterior interosseous nerve. Its tendon crossing through the carpal canal has been implicated in the carpal tunnel syndrome (reviewed by Lahey & Aulicino, 1986). In some cases, palmaris profundus was found enclosed in a common fascial sheath with the median nerve (Stark, 1992; Sahinoglu et al. 1994). To indicate its close association with the median nerve, the palmaris profundus was also named ‘musculus comitans nervi mediani’ (Sahinoglu et al. 1994). This article reports an unusual loop of the median nerve encircling an anomalous palmaris profundus in the forearm, which, to the best of our knowledge, has not been previously described.

NOVEL DISTRIBUTION OF CLUSTER OF DIFFERENTIATION 200 ADHESION MOLECULE IN GLIAL CELLS OF THE PERIPHERAL NERVOUS SYSTEM OF RATS AND ITS MODULATION AFTER NERVE INJURY

This study examined CD200 expression in different peripheral nerves and ganglia. Intense CD200 immunoreactivity was consistently localized in unmyelinated nerve fibers as opposed to a faint immunostaining in the myelinated nerve fibers. By light microscopy, structures resembling the node of Ranvier and Schmidt-Lanterman incisures in the myelinated nerve fibers displayed CD200 immunoreactivity. Ultrastructural study revealed CD200 expression on the neurilemma of Schwann cells whose microvilli and paranodal loops at the node of Ranvier were immunoreactive. The CD200 immunoexpression was also localized in the satellite glial cells of sensory and autonomic ganglia and in the enteric glial cells. Double labeling of CD200 with specific antigens of satellite glia or Schwann cells in the primary cultures of dorsal root ganglia had shown a differential expression of CD200 in the peripheral glial cells. The existence of CD200 in glial cells in the peripheral nervous system (PNS) was corroborated by the expression of CD200 mRNA and protein in a rat Schwann cell line RSC96. Using the model of crush or transected sciatic nerve, it was found that CD200 expression was attenuated or diminished at the site of lesion. A remarkable feature, however, was an increase in incidence of CD200-labelled Schmidt-Lanterman incisures proximal to the injured site at 7 days postlesion. Because CD200 has been reported to impart immunosuppressive signal, we suggest that its localization in PNS glial cells may play a novel inhibitory role in immune homeostasis in both normal and pathological conditions.

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.