Yongduk Lee

Expression of constitutive endothelial and inducible nitric oxide synthase in the sciatic nerve of Lewis rats with experimental autoimmune neuritis

This study examined the expression of constitutive endothelial nitric oxide synthase (eNOS) and inducible NOS (iNOS) in the sciatic nerve of Lewis rats with experimental autoimmune neuritis (EAN). Western blot analysis showed that both eNOS and iNOS expressions in the sciatic nerves of rats increased significantly during the peak stage of EAN, but declined thereafter. Only minimal amounts of these enzymes were identified in normal rat sciatic nerves. Immunohistochemical studies showed that eNOS was increased in vascular endothelial cells and Schwann cells, but not in inflammatory cells, during the peak stage of EAN. However, iNOS was found mainly in inflammatory macrophages in sciatic nerve EAN lesions.These findings suggest that, depending on the stage of peripheral nervous system autoimmune disease, the increased expressions of both eNOS and iNOS might be involved in either the production of detrimental effects during the induction stage of EAN or in the recovery from EAN paralysis.

Pertussis toxin-induced hyperacute autoimmune encephalomyelitis in Lewis rats is correlated with increased expression of inducible nitric oxide synthase and tumor necrosis factor alpha

The involvement of inducible nitric oxide synthase (iNOS) and tumor necrosis factor alpha (TNF-alpha), which have diverse roles in the progression of autoimmune disease models, was studied in pertussis toxin (PT)-induced hyperacute experimental autoimmune encephalomyelitis (EAE) in Lewis rats. The expression of TNF-alpha mRNA (increased 5-fold, P<0.01) and iNOS protein (3-fold, P<0.01) was much greater in the spinal cords with PT(+) EAE at the peak stage of EAE than in those with PT(-) EAE, as shown by competitive PCR and Western blot analysis, respectively. Immunohistochemistry showed that the majority of ED1-positive macrophages in EAE lesions contained iNOS, and that there were many more iNOS-positive cells in the CNS lesions of PT(+) rats than in those of PT(-) rats. These findings suggest that PT-induced hyperacute EAE is partly mediated by the enhanced expression of iNOS and TNF-alpha in the early stages of rat EAE.

Upregulation of osteopontin in Schwann cells of the sciatic nerves of Lewis rats with experimental autoimmune neuritis

We examined the expression of osteopontin (OPN) in the sciatic nerves of rats with experimental autoimmune neuritis (EAN), using immunohistochemistry and immunoblotting, to study its involvement in the pathogenesis of autoimmune peripheral nervous system diseases. Constitutive OPN expression was detected in some Schwann cells; expression was increased after immunization with adjuvant alone. At day 14 after induction of EAN, many Schwann cells had a granular pattern of immunoreactivity, whereas very few inflammatory cells were OPN-positive. Even after recovery from hindlimb paralysis, at 24 days post-immunization, OPN expression remained elevated in the Schwann cells. The results suggest that OPN expression in Schwann cells is easily induced by immunostimulation, and further enhanced by the inflammatory reaction in EAN. Continued elevation of OPN after recovery may represent a functional recovery after a transient inflammatory insult.

Activation of extracellular signal-regulated kinases in the sciatic nerves of rats with experimental autoimmune neuritis

To investigate whether the phosphorylation of extracellular signal-regulated kinase (ERK) is involved in autoimmune injury of the peripheral nervous system (PNS), the expression of phosphorylated ERK (p-ERK) was analyzed in experimental autoimmune neuritis (EAN) in rats. Western blot analysis showed that the level of p-ERK was increased significantly in the sciatic nerves of rats on days 14 (p<0.05) and 24 (p<0.01) post-immunization, compared with controls, and its reaction declined at day 30 post-immunization. Immunohistochemistry showed that p-ERK protein was weakly expressed in Schwann cells and vascular endothelial cells in the sciatic nerves of CFA-immunized control rats. In EAN-affected sciatic nerves, p-ERK immunoreactivity was found mainly in ED1-positive macrophages on days 14 and 24 post-immunization. Moreover, on days 24 and 30 post-immunization, p-ERK immunoreactivity increased gradually in the Schwann cells of rat sciatic nerves with EAN. Based on these results, we postulated that the phosphorylation of ERK has an important role in the differentiation and survival of cells, including inflammatory cells and Schwann cells, in the rat sciatic nerve in EAN. Specifically, the activation of ERK in the recovery phase of EAN paralysis seems to be related in the survival of Schwann cells.

Activation of p38 mitogen-activated protein kinase in the early and peak phases of autoimmune neuritis in rat sciatic nerves

To examine the involvement of p38 mitogen-activated protein kinase (MAPK) in autoimmune disorders of the peripheral nerve system, we analyzed the phosphorylation of p38 MAPK protein in the sciatic nerves of Lewis rats with experimental autoimmune neuritis (EAN). Western blot analysis showed that phosphorylated p38 (p-p38) MAPK protein was significantly increased in the sciatic nerves of rats in the early and peak phases of EAN, and declined gradually thereafter. Immunohistochemistry showed that p-p38 MAPK levels were increased in the infiltrating inflammatory cells, including T cells and macrophages, as well as in blood vessels and some Schwann cells in EAN-affected sciatic nerves, as compared to the sciatic nerves of controls. Some inflammatory cells and a few Schwann cells were also positive for TUNEL reaction at the peak and recovery phases of EAN. In conclusion, we postulate that the phosphorylation of p38 MAPK is involved in the elimination of infiltrating inflammatory cells during the course of EAN and may possibly modulate recovery in autoimmune disorders of the peripheral nervous system.

Heat shock protein 27 upregulation and phosphorylation in rat experimental autoimmune encephalomyelitis

Following stress or inflammation, the 27-kDa heat shock protein (HSP27) is induced in various cell types, where it promotes cell survival and inhibits inflammatory reactions. We examined the expression of HSP27 and phosphorylated HSP27 (p-HSP27) in the spinal cords of Lewis rats with experimental autoimmune encephalomyelitis (EAE). Western blotting analysis revealed low levels of HSP27 and p-HSP27 in the normal spinal cords and significantly higher levels in EAE-affected spinal cords. Immunohistochemistry revealed that HSP27 was expressed constitutively in the neurons and some fibrous astrocytes of the spinal cords of normal rats. However, in EAE-affected spinal cords, HSP27 immunoreactivity was higher and located primarily in the fibrous astrocytes of the white matter, whereas few of the inflammatory cells were immunopositive for HSP27. Immunoreactivity for p-HSP27 was detected predominantly in the fibrous astrocytes of the normal controls and was markedly increased in EAE-affected spinal cords. Therefore, the levels of HSP27 expression and phosphorylation of HSP27 were increased primarily during reactive astrogliosis of spinal white matter affected by EAE. These observations suggest that in rat EAE, the increased expression and elevated activation of HSP27 modulate host cell activity, survival, and inflammation to counter the autoimmune inflammatory injury. Our results also suggest that HSP27 plays a role in spontaneous recovery from EAE-induced paralysis.

Involvement of Cyclooxygenase‐1 and ‐2 in the Sciatic Nerve of Rats with Experimental Autoimmune Neuritis

The expression of both cyclooxygenase (COX)‐1 and COX‐2, which are representative enzymes in prostaglandin synthesis, was evaluated in the sciatic nerve of rats with experimental autoimmune neuritis (EAN). Western blot analysis showed that both COX‐1 and COX‐2 were significantly increased in the sciatic nerve at the peak stage of EAN and declined during the recovery stage. Vascular endothelial cells in normal sciatic nerves immunostained for both COX‐1 and COX‐2. COX‐1 was mainly detected in macrophages, and not in other cell types, while COX‐2 was detected in Schwann cells and axons as well as inflammatory macrophages in EAN lesions. This suggests that COXs are involved in the pathogenesis of peripheral demyelinating disease, including EAN, and the major cellular source of both COXs in EAN lesions is inflammatory macrophages. Furthermore, COX‐2 is enhanced in some Schwann cells and neural elements, possibly mediating peripheral nervous system inflammation.

Increased phosphorylation of cyclic AMP response element-binding protein in the spinal cord of Lewis rats with experimental autoimmune encephalomyelitis

To investigate whether the phosphorylation of cyclic AMP response element-binding protein (CREB) is implicated in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), the change in the level of CREB phosphorylation was analyzed in the spinal cord of Lewis rats with EAE. Western blot analysis showed that the phosphorylation of CREB in the spinal cord of rats increased significantly at the peak stage of EAE compared with the controls (p<0.05) and declined significantly in the recovery stage (p<0.05). Immunohistochemistry showed that the phosphorylated form of CREB (p-CREB) was constitutively immunostained in few astrocytes and dorsal horn neurons in the spinal cord of normal rats. In the EAE-affected spinal cord, p-CREB was mainly found in ED1-positive macrophages at the peak stage of EAE, and the number of p-CREB-immunopositive astrocytes was markedly increased in the spinal cord with EAE compared with the controls. Moreover, p-CREB immunoreactivity of sensory neurons, which are closely associated with neuropathic pain, was significantly increased in the dorsal horns at the peak stage of EAE. Based on these results, we suggest that the increased phosphorylation of CREB in EAE lesions was mainly attributable to the infiltration of inflammatory cells and astrogliosis, possibly activating gene transcription, and that its increase in the sensory neurons in the dorsal horns is involved in the generation of neuropathic pain in the rat EAE model.

The expression and cellular localization of phospholipase D isozymes in the developing mouse testis.

To examine the involvement of phospholipase D (PLD) isozymes in postnatal testis development, the expression of PLD1 and PLD2 was examined in the mouse testis at postnatal weeks 1, 2, 4, and 8 using Western blot analysis and immunohistochemistry. The expression of both PLD1 and PLD2 increased gradually with development from postnatal week 1 to 8. Immunohistochemically, PLD immunoreactivity was detected in some germ cells in the testis and interstitial Leydig cells at postnatal week 1. PLD was mainly detected in the spermatocytes and residual bodies of spermatids in the testis after 8 weeks after birth. The intense immunostaining of PLD in Leydig cells remained unchanged by postnatal week 8. These findings suggest that PLD isozymes are involved in the spermatogenesis of the mouse testis.

Increased expression of phospholipase D in the heart with experimental autoimmune myocarditis in Lewis rats

The expression of phospholipase D (PLD) in the hearts of rats with experimental autoimmune myocarditis (EAM) was studied to elucidate the functional role of PLD in the pathogenesis of EAM. Western blot analysis showed that the level of the PLD1 isoform was significantly increased in the hearts of rats with EAM on days 14, 17 and 21 postimmunization (pi) (P < 0.01; control vs EAM at 14 pi, 17 pi and 21 pi). The phenotypes of cells exhibiting increased PLD1 expression were primarily inflammatory cells, including ED1 positive macrophages, in the inflammatory EAM lesions. Some cardiomyocytes also showed increased PLD1 immunoreaction in and around EAM lesions. Some PLD1‐positive cells were also positive for proliferating cell nuclear antigen in some cardiomyocytes or terminal deoxynucleotidyl transferase (TdT)‐mediated dUTP nick end‐labeling in some macrophages, suggesting that PLD1 positive cells have a capacity for proliferation or apoptosis depending on cell types in the target organ. Thus, it is postulated that increased expression of PLD1 in EAM may support an early inflammatory response in proliferating inflammatory cells, and its expression in cardiomyocytes may help them to survive by activation of survival factors in hearts with EAM.