Figure 1

Inhibition of cAMP-dependent signaling by the shared epitope (SE). (a) Epstein-Barr virus-transformed B-cell lines expressing SE-positive or SE-negative human leukocyte antigen-DRB1 (HLA-DRB1) alleles were stimulated with the adenylyl cyclase (AC) activator forskolin (25 μM) for 12 minutes, and protein kinase A (PKA) activity was determined. The values in parentheses represent the number of donors in each group. (b) L-cell transfectants expressing SE-positive or SE-negative HLA-DRB1 alleles were stimulated with the AC activator forskolin (25 μM) over a time course, and cAMP levels were quantified. (c) L-cell transfectants were stimulated with forskolin as in (b), and PKA activity was determined. (d) M1 cells were pre-incubated overnight with 50 μg/ml of different soluble 15 mer synthetic peptides corresponding to the HV3 region (residues 65 to 79) encoded by SE-positive or SE-negative HLA-DRB1 alleles. Cells were then stimulated with 25 μM forskolin, and cell membrane AC activity was determined. (e) Intact M1 cells were pre-incubated overnight with either SE-positive or SE-negative soluble 15 mer peptides, and cAMP levels were determined 12 minutes after stimulation with forskolin. (f) M1 cells were pre-incubated for 1 hour with different 15 mer peptides immobilized on Sepharose beads and then stimulated with 2-chloroadenosine (2CA, 1 μM), and PKA activity was measured over a time course. Black circles indicate SE-positive peptide 65–79*0401, and white circles indicate SE-negative peptide 65–79*0402. (g) L-cell transfectants expressing SE-positive or SE-negative HLA-DRB1 alleles were pre-incubated for 15 minutes in the presence or absence of the non-selective nitric oxide synthase inhibitor N-nitro-L-arginine methyl ester (L-NAME, 4 mM) and then stimulated with forskolin (25 μM), and cAMP levels were measured. NS, not significant.