Cyclooxygenase-2 (Cox-2) inhibitors do not significantly influence human B-cell viability or proliferation. Viability of human B cells was assessed by 7-aminoactinomycin D (7-AAD) exclusion. (a) Per cent viable cells analysed on days 2, 4 and 6 are represented as bar graphs for SC-58125 treatments. B cells were stained with carboxyfluorescein succinimidyl ester before agonist stimulation and SC-58125 treatment to analyse possible changes in proliferation by flow cytometry after 7 days of culture in two individual donors. Per cent divided cell gates were drawn based on undivided mitomycin-C-treated B cells. Vehicle-treated and SC-58125-treated B-cell CFSE profiles and per cent cells divided are shown for one representative donor (b) and per cent cells divided for multiple donors (n= 3) (c). Data are represented as mean ± SD.

B-cell differentiation to plasma cells and expression of the essential transcription factor, Blimp-1, is cyclooxygenase-2 (Cox-2) dependent. Purified CD19⁺ B cells from spleens of wild-type and Cox-2 deficient mice were cultured with 10 μg/ml lipopolysaccharide (LPS) for 72 hr. Cells were analysed by flow cytometry for CD138 surface expression. (a) Dot plots show events based on forward scatter and CD138 expression. (b) Representative bar graph showing the percentage of CD138⁺ cells after LPS stimulation. (c) Western blot probed for Blimp-1 and GAPDH control following 72 hr of culture with 10 μg/ml LPS. Data are represented as mean ± SD, *P < 0·05.

Cyclooxygenase-2 (Cox-2) inhibitors attenuate immunoglobulin M (IgM) and IgG production. Purified human B cells were left untreated or stimulated with oligodeoxynucleotide 2395 plus anti-IgM and cultured for 7 days in the presence of vehicle control (black), 5 μm, 10 μm or 20 μm SC-58125 (white) or NS-398 (grey). (a) IgM and (b) total IgG were measured in media supernatants by enzyme-linked immunosorbent assay. IgG1 (c), IgG2 (d), IgG3 (e) and IgG4 (f) were assessed using a Luminex multiplex assay. Peripheral blood mononuclear cells were cultured for 24 hr in the presence of SC-58125 or NS-398 following stimulation with CpG plus anti-IgM. (g) Prostaglandin (PG) E₂ was measured in supernatants, demonstrating that concentrations of selective inhibitors used abrogated Cox-2 activity. Data are represented as mean ± SD compared with vehicle alone, *P < 0·05.

Inhibition of cyclooxygenase-2 (Cox-2) prevents the differentiation of B cells to antibody-secreting cells. CD19⁺ B cells cultured for 7 days in the presence of oligodeoxynucleotide 2395 plus anti-immunoglobulin M (IgM) and treated with dimethylsulphoxide vehicle, 5, 10 or 20 μm SC-58125 were stained for surface expression of CD38-phycoerythrin and intracellular IgM- or IgG-fluorescein isothiocyanate. Gates for intracellular staining were drawn based on unpermeabilized cells. Bar graphs depict changes in CD38⁺ (a) CD38⁻ (b) antibody-secreting cell populations in freshly isolated B cells or following activation and treatment with SC-58125. (c) Flow cytometry dot plots show changes in CD38⁺ IgM⁺ and CD38⁺ IgG⁺ in the presence of SC-58125 in another donor. Data shown are representative of two donors; however, multiple donors showed similar decreases in CD38⁺ Ig⁺ cells. Data are represented as mean ± SD, *P < 0·05.

Inhibition of cyclooxygenase-2 (Cox-2) attenuates the expression of transcription factors essential for plasma cell differentiation. RNA was isolated from freshly purified human peripheral blood B cells and cells stimulated with oligodeoxynucleotide (ODN) 2395 plus anti-immunoglobulin M (IgM) and treated with dimethylsulphoxide vehicle or the Cox-2 selective inhibitor SC-58125. Blimp-1, Xbp-1 and Pax5 messenger RNA (mRNA) steady-state levels were first normalized to 7S mRNA steady-state levels. (a) Blimp-1, Xbp-1 and Pax5 mRNA steady-state level fold change compared with freshly isolated B cells from one representative donor. Fold decrease of Blimp-1, Xbp-1 and Pax5 levels were determined by comparing vehicle-treated B cells with those treated with SC-58125. The mRNA fold decrease was averaged for four donors. (b) The mRNA fold decrease in the presence of 10 μm SC-58125 shown over a time–course of 24, 48, 72 and 96 hr; (c) mRNA fold decrease shown at 72 hr with 5, 10 or 20 μm doses of SC-58125; (d) ODN 2395 plus anti-IgM stimulated B-cell protein lysates (10 μg) collected at 72 hr from two separate donors were run using sodium dodecyl sulphate–polyacrylamide gel electrophoresis. The Western blot was probed for Blimp-1, Xbp-1, Pax5 and GAPDH control. (e) Densitometry results were normalized to GAPDH expression. Data are represented as mean ± SEM.