B Lymphocytes and Plasma Cells Express Functional E-Selectin by Constitutive Activation of NF-κB

Andrographolide Attenuates Inflammation by Inhibition of NF-κB Activation through Covalent Modification of Reduced Cysteine 62 of p50

“…After transfection, the cells were lysed at 44 h, and the lysates were assayed for luciferase activities using the Luciferase Assay System (Promega, Madison, WI) (19,20). Notably, the pCMV␤ vector (BD Clontech, Palo Alto, CA) was cotransfected so that the transfection efficiencies could be normalized with the ␤-galactosidase activities determined using a Luminescent ␤-Gal Detection Kit II (BD Clontech).…”

“…The BAL cells were stained with Wright's staining buffer, and cell differentials were enumerated based on morphology and staining profile. Immunohistochemistry analysis of E-selectin, VCAM-1, and von Willebrand factor (vWF) in fixed and embedded mouse lungs was conducted as described previously (19,30). The positive vWF-staining blood vessels were used as the total number of vessels in the tissues for calculating the ratios of the positive E-selectin-and VCAM-1-staining vessels.…”

Severe Disease, Unaltered Leukocyte Migration, and Reduced IFN-γ Production in CXCR3−/− Mice with Experimental Autoimmune Encephalomyelitis

“…After blocking with CD16/CD32 Abs at 4°C for 30 min, cells were stained for surface markers with directly conjugated Abs in FACS buffer at 4°C for 30 min. Cells were washed twice and resuspended in the 200 -400 l of PBS for flow cytometry analysis as described before (53,54). Abs used in our experiments were CD4-PE, CD8-Cy, CD45-Cy.…”

NF-κB Activation for Constitutive Expression of VCAM-1 and ICAM-1 on B Lymphocytes and Plasma Cells

“…Isolations of total cellular RNAs, reverse transcription reactions and PCR amplifications of cDNAs were carried out as previously described (21,26). Primers specific for detection of mRNAs for human E-selectin, VCAM-1 and ICAM-1 were as follows: human E-selectin sense (ϩ214) 5Ј-ATG ATG AGG CCA GTG CTT ATT G-3Ј and antisense (Ϫ623) 5Ј-GAA GCC AGG GTC ACA CTT GC-3Ј; human VCAM-1 sense (ϩ221) 5Ј-AGG GGA CCA CAT CTA CGC TGA C-3Ј and antisense (Ϫ758) 5Ј-GCT GGT AGA CCC TCG CTG GAA C-3Ј; human ICAM-1 sense (ϩ186) 5Ј-CGT GCT GGT GAC ATG CAG CAC C-3Ј and antisense (Ϫ702) 5Ј-TGG CAG GAC AAA GGT CTG GAG C; ␤-actin sense (ϩ1) 5Ј-ATG GAT GAT GAT ATC GCC GC-3Ј and antisense (Ϫ1127) 5Ј-CTA GAA GCA TTT GCG GTG G-3Ј.…”

“…Although it is present in an inactive but inducible form in pre-B lymphocytes (20) and many other cells including endothelial cells (7), NF-B is known to be constitutively active in mature B lymphocytes and plasma cells for synthesis of immunoglobulin molecules. In the previous study, we have shown that B lymphocytes and plasma cells can synthesize E-selectin mRNA and protein prior to cytokine stimulation (21). Given that NF-B activation can up-regulate the expressions of E-selectin, VCAM-1 and ICAM-1 in activated endothelial cells (17) and that NF-B is reportedly active in B lymphocytes and plasma cells (18,19), we proposed that B lymphocytes and plasma cells might constitutively express these cell adhesion molecules via the mechanism of NF-B activation.…”