Ibuprofen and other widely used non-steroidal anti-inflammatory drugs inhibit antibody production in human cells

Abstract

The widely used non-steroidal anti-inflammatory drugs (NSAIDs) function mainly through inhibition of cyclooxygenases 1 and 2 (Cox-1 and Cox-2). Unlike Cox-1, Cox-2 is considered an inducible and pro-inflammatory enzyme. We previously reported that Cox-2 is upregulated in activated human B lymphocytes and using Cox-2 selective inhibitors that Cox-2 is required for optimal antibody synthesis. It is not known whether commonly used non-prescription and non-Cox-2 selective drugs also influence antibody synthesis. Herein, we tested a variety of Cox-1/Cox-2 non-selective NSAIDs, namely ibuprofen, tylenol, aspirin and naproxen and report that they blunt IgM and IgG synthesis in stimulated human peripheral blood mononuclear cells (PBMC). Ibuprofen had its most profound effects in inhibiting human PBMCs and purified B lymphocyte IgM and IgG synthesis when administered in the first few days after activation. As shown by viability assays, ibuprofen did not kill B cells. The implications of this research are that the use of widely available NSAIDs after infection or vaccination may lower host defense. This may be especially true for the elderly who respond poorly to vaccines and heavily use NSAIDs.

Figure 1. Non-steroidal anti-inflammatory drugs (NSAIDs) blunt antibody synthesis in human PBMC in vitro

Human PBMCs were stimulated with anti-IgM (2 µg/ml) plus CpG 2395 (1 µg/ml) for 7 days. Cells were exposed to NSAIDs or to a selective Cox-2 inhibitor, as follows: SC-58125 (Cox-2 selective inhibitor; 20 µM), indomethacin (20 µM), ibuprofen (50 µM or 100 µM), aspirin (100 µM or 500 µM), tylenol (20 µM or 80 µM) or naproxen (50 µM or 100 µM). Drugs were added everyday, except for SC-58125, which was added every other day. The control samples (no drug) received only the vehicle (DMSO). At the end of culture period, supernatants were harvested and used for IgM (panel A) or IgG (panel B) detection by ELISA. Each bar represents the means ± SD (n=3). The statistical significance in reduction of antibody synthesis was determined by one-way ANOVA test, followed by Tukey’s post-hoc test. *, p< 0.05, **, p< 0.001 as compared to control (no drug) sample.

Figure 2. Ibuprofen inhibits PGE₂ synthesis in human PBMC in a concentration-dependent manner

Human PBMCs were stimulated with anti-IgM plus CpG 2395 for 2 days. Cells were exposed to increasing concentrations of ibuprofen from 15 µM to 100 µM. Ibuprofen was added at the onset of culture. Control cells received only the vehicle (DMSO). There is a concentration-dependent decrease in PGE₂ synthesis. PGE₂ production was significantly inhibited by 50 µM ibuprofen (~ 300 pg/ml) and further decreased by 100 µM ( ~100 pg/ml). Data are shown as mean ± SD (n=3). *, p< 0.05, **, p< 0.001 were calculated using one-way ANOVA test followed by Tukey’s post-hoc test.

Figure 3. Ibuprofen fails to decrease IgG production in Cox-2 deficient mice

Splenocytes from Cox-2 knock-out mice and their wild-type controls were stimulated with LPS (10 µg/ml) for 6 days and were exposed to ibuprofen (50 µM; added everyday). Control cells (no drug) received only the vehicle (DMSO). Supernatants were harvested and used for determining IgM and IgG synthesis. There is an increase in IgM synthesis in Cox-2 deficient mice (80,000 ng/ml) compared to wild-type controls (~40,000 ng/ml) (Panel A; black bars). Ibuprofen significantly inhibited IgM synthesis in both Cox-2 deficient and wild-type mice (by ~ 60%) (Panel A; striped bars). Cox-2 knock-out mice produce less IgG (~200 ng/ml) compared to wild-type controls (~450 ng/ml) (Panel B; black bars). Ibuprofen significantly inhibited IgG synthesis in wild-type mice (by ~ 70%) but not in Cox-2 deficient mice (Panel B; striped bars). Data are shown as mean ± SD (n=3). *, p< 0.05, **, p < 0.001 were calculated using one-way ANOVA test followed by Tukey’s post-hoc test.

Figure 4. Ibuprofen inhibits antibody synthesis by human PBMC in a concentration-dependent manner

Stimulated (anti-IgM plus CpG 2395) PBMCs were exposed to ibuprofen (15 µM, 30 µM, 50 µM or 100 µM). Ibuprofen was added everyday. Control cells received only the vehicle (DMSO). Supernatants were harvested on day 5, 6, 7 and 8 and IgM (panel A) and IgG (panel B) synthesis was determined. Ibuprofen (50 or 100 µM) caused a statistically significant decrease in IgM and IgG synthesis from day 5 to day 8. Fifteen µM ibuprofen significantly decreased IgM synthesis only on day 7, whereas 30 µM cause a significant decrease in IgM and IgG synthesis on day 6 and day 7 and day 6 to day 8, respectively. Statistical significance, *, p< 0.05, **, p< 0.001 was calculated using one-way ANOVA test followed by Tukey’s post-hoc test.

Figure 5. Time course of ibuprofen addition and inhibition of antibody synthesis

PBMC were stimulated with anti-IgM plus CpG 2395 for 7 days in the presence or absence of ibuprofen (added on different days). Panel A and panel B show that only one dose of ibuprofen added at the beginning (day 1) or end of culture (day 7) does not affect IgM or IgG synthesis. Panel C and Panel D show that daily doses of ibuprofen (50 µM) caused a ~ 4 fold decrease in IgM and completely abrogated IgG synthesis. Two doses of ibuprofen decreased IgM and IgG production to the same extent as three doses. *, p< 0.05, **, p< 0.001 were calculated using one-way ANOVA test followed by Tukey’s post-hoc test.

Figure 6. Ibuprofen decreases the number of antibody producing cells

PBMC were stimulated with anti-IgM plus CpG 2395 for 7 days in the presence or absence of ibuprofen (50 µM; added everyday). Panel A and panel B show that ibuprofen reduced the numbers of antibody secreting cells. In panels C the number of spots was counted and their means +/− SD were plotted on the graph. There are more IgM-producing cells (~ 30 cells / 10⁵ PBMC) compared to IgG (~18 cells/ 10⁵ PBMC). In the presence of ibuprofen there are fewer IgM (~ 15 cells / 10⁵ PBMC) and IgG (~ 3 cells/ 10⁵ PBMC) producing cells. *, p< 0.05 was calculated using one-way ANOVA test followed by Tukey’s post-hoc test.

Figure 7. Ibuprofen inhibits antibody synthesis in purified human B lymphocytes

Human B lymphocytes were stimulated with anti-IgM (2 µg/ml) plus CpG 2395 (1 µg/ml). Cells were exposed to ibuprofen (50 µM; added on various days of culture). Control cells (no drug) received only the vehicle (DMSO). IgG and IgM synthesis was determined on day 7 of culture. There is a decrease in IgM (panel A) and IgG (panel B) synthesis in B lymphocytes exposed to ibuprofen everyday or on the first days of culture (days 1+2+3). Ibuprofen added on the last days of culture (days 4+5+6) did not reach statistical significance. **, p< 0.001 (as determined by one-way ANOVA test followed by Tukey’s post-hoc test).

Figure 8. Ibuprofen only modestly inhibits purified B cell viability

Purified human peripheral blood B cells isolated from 2 different donors were stimulated with anti-IgM (2 µg/ml) plus CpG 2395 (1 µg/ml) and were exposed to ibuprofen (50 µM; added daily) for 7 days. Cell viability was determined by MTT assay (donor 1) and by 7-AAD staining (donor 2). (A) The absorbance (A510 nm) values of ibuprofen-treated samples were similar to control (no drug) samples throughout day 2 to day 7. (B) Cells were surface stained for CD20 followed by 7-AAD staining and were analyzed by flow cytometry. A total of 15,000 events were acquired in each sample. Viable cells do not incorporate 7-AAD compared to apoptotic (7-AAD intermediate) and dead cells (7-AAD bright). The percentage of live, apoptotic and dead cells is similar in ibuprofen- (panel C) and S-Ibuprofen (panel D) treated samples. Cell percentages represent the mean values (n=3) with the corresponding SD. *, p< 0.05 (see panel A) was calculated using one-way ANOVA test followed by Tukey’s post-hoc test.