Platelet-endothelial cell adhesion molecule (PECAM)-1 has been implicated in leukocyte migration through the perivascular basement membrane (PBM) though the mechanisms involved are unclear. The present results demonstrate that the ability of α6 integrins to mediate neutrophil migration through the PBM is PECAM-1 dependent, a response associated with PECAM-1–mediated increased expression of α6β1 on transmigrating neutrophils in vivo. An anti-α6 integrins mAb (GoH3) inhibited (78%, P < 0.001) neutrophil migration through interleukin (IL)-1β–stimulated cremasteric venules, primarily at the level of the PBM, as analyzed by intravital and electron microscopy. In PECAM-1–deficient mice (KO), a reduced level of neutrophil transmigration elicited by IL-1β (4-h reaction) was observed in both the cremaster muscle (55% inhibition, P < 0.05) and in the peritoneum (57% inhibition, P < 0.01) but GoH3 had no additional inhibitory effect on these responses. FACS® analysis of neutrophils demonstrated increased expression of α6β1 on transmigrated peritoneal neutrophils, as compared with blood neutrophils, in wild-type but not KO mice even though neutrophils from both strains of mice exhibited comparable levels of intracellular expression of α6 as observed by immunofluorescent staining and confocal microscopy. Furthermore, mice deficient in either leukocyte or endothelial cell PECAM-1, as developed by bone marrow transplantation, demonstrated a similar level of reduced neutrophil transmigration and expression of α6β1 on transmigrated neutrophils as that detected in KO mice.The results demonstrate a role for PECAM-1 homophilic interaction in neutrophil transmigration and increased expression of α6β1 on the cell surface of transmigrated neutrophils in vivo, a response that could contribute to the mechanism of PECAM-1–mediated neutrophil migration through the PBM.

Parkinson's disease (PD) is a progressive disease related to degeneration of nigrostriatal dopaminergic neurons and can be caused by genetic and environmental factors. N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) can produce Parkinsonian syndrome (Langston et al. 1983) and is an Received February 28, 2010; revised manuscript received April 26, 2010; accepted April 26, 2010. Address correspondence and reprint requests to Kai-Yuan Tzen, MD, Department of Nuclear Medicine, National Taiwan University Hospital, No.7 Chung Shan South Road, Taipei 100, Taiwan. E-mail: tzenky@ntuh.gov.tw or Chun-Jung Lin, School of Pharmacy, College of Medicine, National Taiwan University,1 Jen-Ai Road, Section 1, Taipei, Taiwan 100. E-mail: clementumich@ntu.edu.tw Abbreviations used: 3-MT, 3-methoxytyramine; ARBECs, adult rat brain endothelial cells; AUC, area under the time-concentration curve; BB, blood-brain barrier; BMECs, brain microvessel endothelial cells; BSA, bovine serum albumin; DC, detergent compatible; DOPAC, 3,4-dihydroxyphenylacetic acid; ECF, extracellular fluid; FDOPA, 6-[ AbstractThe cellular localization of organic cation transporter (OCT) 1 and OCT2 in isolated brain microvessel endothelial cells from humans, rats, and mice and in cultured adult rat brain endothelial cells was examined by confocal microscopy and in isolated luminal and abluminal membrane fractions by Western blot analysis. Cellular uptake of N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was measured with or without OCT1/OCT2 silencing. The interaction between MPTP and amantadine was studied by in vitro kinetic analysis and in vivo brain microdialysis. MPTP-induced dopaminergic toxicity was examined by measuring dopamine levels in the brain striatum and by positron emission tomography scanning. The results showed that both OCT1 and OCT2 were mainly expressed on the luminal side of brain microvessel endothelial cells and adult rat brain endothelial cells. Cellular uptake of MPTP was significantly (p < 0.05) decreased by about 53%, 60%, or 91% following silencing of OCT1, OCT2, or both, respectively. Amantadine competitively inhibited MPTP uptake in vitro and significantly (p < 0.05) reduced the area under the time-concentration curve for MPTP and MPP + in the brain extracellular fluid in rats and mice by 65-70% and 35-85%, respectively. MPTP-induced dopaminergic toxicity in mice was ameliorated by amantadine without stimulating dopamine turnover. In conclusion, OCT1 and OCT2 are important for MPTP transfer across the blood-brain barrier and amantadine reduces the blood-brain barrier transfer of MPTP and MPTP-induced dopaminergic toxicity in rodents.

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BackgroundNotch and TLR pathways were found to act cooperatively to activate Notch target genes and to increase the production of TLR-induced cytokines in macrophages. However, the mechanism of LPS-induced Notch activation and its role in sepsis still remains unclear.MethodsWe analyzed the expression patterns of Notch components in a LPS-stimulated murine macrophage cell line using real-time PCR and western blotting. The role of DAPT, a gamma-secretase inhibitor that is known to be a potent Notch inhibitor, in LPS-induced cytokine release and experimental sepsis in mice was also explored. Student's t-test was used to analyze the difference between the two groups.ResultsWe found that Notch signaling was activated after LPS stimulation. The expression of Jagged 1, a Notch ligand, induced by LPS occurred in a JNK-dependent manner. In addition, Notch target genes were upregulated by early Notch-independent activation followed by delayed Notch-dependent activation after LPS stimulation. Disruption of Notch signaling by DAPT attenuated the LPS-induced inflammatory responses, including vascular endothelial growth factor (VEGF) and high-mobility group box chromosomal protein 1 (HMGB1), both in vitro and in vivo and partially improved experimental sepsis survival.ConclusionsThese findings support the existence of a synergistic effect of Notch signaling and the LPS pathway both in vitro and in vivo. Therefore, in the future Notch inhibitors may be utilized as adjunctive agents for the treatment of sepsis syndrome.