The Effects of Pain Relievers on Human Cytokine Interleukin-6 and Secretory Immunoglobulin A Production

Transcription

1 The Effects of Pain Relievers on Human Cytokine Interleukin-6 and Secretory Immunoglobulin A Production Angelica A. Co Mentor: Dr. Randy Day BIO 483

2 Abstract Recent studies have shown that the use of NSAIDs and Acetaminophen affect the human immune response but none have focused on the changes of siga and IL-6. This study examined how over the counter (OTC) doses of Ibuprofen, Naproxen, and Acetaminophen affected siga and IL-6 levels after seven days of consumption in fifteen healthy volunteers. Using ELISA, no changes were observed in siga and IL-6 levels after NSAID and Acetaminophen administration. These results suggest that when consumed at clinically available doses, NSAID and Acetaminophen do not affect siga and IL-6 production. Introduction Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used for the treatment of inflammatory disorders by targeting cyclooxygenases 1 and 2 (Cox-1, Cox-2), which are responsible for prostaglandin (PG) production (Ryan et al. 2005). More than 60 million Americans regularly use NSAIDs. A recent study (Bancos et al. 2009) demonstrated that the use of widely available NSAIDs after infection or vaccination may lower host defense. Acetaminophen, another class of pain reliever, which inhibits pain impulse generation, lacks anti-inflammatory properties (Woo 2008). Acetaminophen is among the most commonly used and misused nonprescription medications in the United States (Dlugosz et al. 2006). Prymula et al. (2009) showed that prophylactic administration of Acetaminophen at the time of vaccinations reduces antibody responses to several vaccine antigens. The immune system is a complex network of cells and molecules with specialized roles in host defense. The cell-mediated immunity, activated by Th1 cells and cytokines, involves the destruction of infected cells by NK cells, cytotoxic T-cells and activated macrophages. Humoral immunity depends on the production of antibodies, such as 1

3 secretory immunoglobulin A (siga), by B cells activated by Th2 cells producing interleukin-4, 5, and 6 (IL-4, IL-5, IL-6) (Tanaka et al. 1998). Yamaki et al. (2003) found that administration of indomethacin, a nonselective Cox-1/Cox-2 inhibitor, to mice was followed by a decrease in Th1 responses and by a moderate decrease in Th2 responses. These findings suggest an important role for Cox-1 and Cox-2 in promoting humoral immune responses. Prostaglandins have diverse effects on the regulation and activity of T cells and B cells. Although a few reports suggest PGs inhibit Th2 responses, the majority of reports found that PGs have a Th2 inducing activity on T cells (Harris et al. 2002). IL-6 is an important Th2 cytokine for B cell activation to exert humoral immunity (Tanaka et al. 1998). Several studies have shown how NSAIDs affect IL-6, but none have investigated the effects of Acetaminophen. Acetaminophen and Cox-1 and Cox-2 nonselective NSAIDs, such as ibuprofen, aspirin and naproxen have been found to blunt IgM and IgG synthesis in stimulated human peripheral blood mononuclear cells (Bancos et al. 2009). Ibuprofen reduced IgM by 97% and IgG by 70% in vitro. Martinez and Coleman (1989) concluded that NSAIDs act at an early stage of B cell differentiation. Acting on B cells, PGs also stimulates isotype-class switching to induce the production of IgG1 and IgE. Though siga plays an important role in mucosal immune defense, there are no studies that have investigated the effects of pain relievers on siga production. The purpose of this study was to investigate NSAIDs and Acetaminophen on the human immune response by comparing the production of cytokine IL-6 and siga in pre and post administration of NSAIDs and Acetaminophen. 2

4 Methods Human subjects. After obtaining approval from the Institutional Research Board of Brigham Young University Hawaii, health questionnaires were administered to potential volunteers inquiring about their health history and medication usage. Fifteen healthy volunteers, ages 19 to 28, who showed no visible symptoms of illness, were selected to participate in the study. In order to establish baseline cytokine and immunoglobulin levels, the subjects were instructed to avoid all NSAIDs and Acetaminophen for 30 days prior to sample collection. Administration of Medication. Volunteers were assigned to a test group. They were instructed to take Acetaminophen, Ibuprofen, or Naproxen at the maximum dosage as directed on the package label (Table 1) for seven consecutive days. Subjects were to take drugs at least six hours apart and with a meal. Table 1. The drug dosage and frequency of intake of the pain relievers. Strength # of Tablets Ibuprofen 200mg 1 2 Naproxen 220mg 1 1 Acetaminophen 500mg 1 2 # of Times a Day Data Collection. Saliva samples were collected by salivette on day zero, four, and seven, with the assigned NSAID and Acetaminophen administered on days one through seven. Salivettes were labeled with unique identifiers and analyzed for IL-6 and siga production by ELISA (Salimetrics, LLC). Statistical Analysis and Interpretation of Data. Two sample paired t-test was employed to determine the significance of the observed difference between the means of pre and post administration IL-6 and siga levels. 3

5 Results A total of three samples were collected for each individual. There were five test subjects in each drug group. The data for siga levels collected from each individual were compiled and averaged (Table 2). The mean siga level for day zero was μg/ml (SD ), while day seven was μg/ml (SD 91.87). Table 2. The Secretory Immunoglobin A level progression from day zero to day seven (Ibuprofen (I), Naproxen (N), Acetaminophen (A)). siga Levels (μg/ml) Subject Drug Day 0 Day 4 Day 7 1 I I I I I N N N N N A A A A A Mean Secretory Immunoglobulin A levels from day zero and day seven were not statistically different (P>0.05) between pre and post administration of any of the drugs tested. A paired t-test comparing day zero and day seven resulting from ibuprofen, naproxen, and acetaminophen administration indicated that there was also no statistical 4

6 difference in each drug for pre and post administration for siga levels (Figure 1 and Table 3). 400 Boxplot of IgA Day 0, IgA Day 4, IgA Day siga levels (μg/ml) Data IgA Da y 0 IgA Day 4 IgA Da y 7 Figure 1. The median and ranges of siga from days zero, four, and seven. Table 3. The mean and standard deviation for siga from day zero to day seven. Day 0 (μg/ml) Day 7 (μg/ml) P-value Ibuprofen ± ± Naproxen ± ± Acetaminophen ± ± Overall ± ± The data for IL-6 levels collected from the same fifteen individuals were compiled and averaged (Table 4). Day zero had a mean of 1.28 pg/ml (SD 1.48) IL-6 level, and day seven had a mean of 1.77 pg/ml (SD 1.77). 5

7 Table 4. The progression of IL-6 levels from day zero to day seven (Ibuprofen (I), Naproxen (N), Acetaminophen (A)). IL-6 Levels (pg/ml) Subject Drug Day 0 Day 4 Day 7 1 I I I I I N N N N N A A A A A Mean A paired t-test comparing overall IL-6 levels from day zero and day seven indicated that there was no statistical difference (P>0.05) between pre and post administration. A paired t-test comparing day zero and day seven of ibuprofen, naproxen, and acetaminophen found no statistical difference in each drug for pre and post administration for IL-6 levels (Figure 2 and Table 5). 6

8 Boxplot of IL6 Day 0, IL6 Day 4, IL6 Day IL 6 levels (pg/ml) Data IL6 Day 0 IL6 Day 4 IL6 Day 7 Figure 2. The median and range of each IL-6 from day zero, four, and seven. Table 5. The average IL-6 levels of each drug and standard deviation were calculated along with the P-value. Day 0 (pg/ml) Day 7 (pg/ml) P-value Ibuprofen 0.91 ± ± Naproxen 1.57 ± ± Acetaminophen 1.37 ± ± Overall 1.28 ± ± Discussion NSAIDs and Acetaminophen are commonly used pain relievers. Research has shown that they may interfere with cytokine and antibody production. There were no changes in siga production resulting from any of the drugs administered in this study. Reports that suggest that human B cell-derived PG products signal to enhance B cell proliferation and antibody production (Ryan et al. 2005) are difficult to reconcile with the 7

9 results of this study. Bancos et al. (2009) showed that aspirin, ibuprofen, Tylenol, and naproxen (used at OTC concentrations) blunt antibody production in human PBMC in vitro. The major effects were exhibited when Ibuprofen was administered early (day 2 and 3) while it had little effect on days 3-5 in their culture system and at a concentration of μm. They reported that Ibuprofen s ability to reduce antibody production was concentration and time-dependent. This study showed that at OTC concentrations, NSAIDs and Acetaminophen do not alter siga production in vivo. The difference between these studies may be due to the difference in dosage. Those investigations which found effects in cytokine and Ig production used high drug dosages. The studies were also conducted in vitro where the drugs can have a direct effect on cell function. Like siga, IL-6 levels also demonstrated no changes from days zero to day seven. Harris et al. (2002) stated that PGs can have no effect on or enhance production of Th2 cytokines such as IL-4, IL-5, and IL10. However, reports suggest that indomethacin down-regulates production of Th2 type cytokines IL-4 and IL-6 (Tsuboi et al. 1995). Tanaka et al. (1998) suggested that the down-regulation of IL-6 production by NSAIDs in their study may be partly independent of the inhibition of PG production suggesting that NSAIDs have a direct effect on immune competent cells. Although there are conflicting studies, the majority supports the Th2 inducing effects of PGs. Cho (2007) found that aspirin and phenylbutazone dose-dependently (10 to 100 μg/ml) suppressed the proliferation of T cells. NSAIDs may inhibit Ig production by mediating cytokine production. This study however, found that NSAIDs and Acetaminophen have no effect on IL-6 production at OTC dosages. 8

10 Future studies to determine if vaccinated subjects taking NSAIDS at different time points before or after vaccination might resolve the question of whether or not there was a commensurate decrease/increase in antibody synthesis. Changing the concentration of the different drugs is also suggested for future research. In vivo, and at OTC concentrations, the selected NSAIDs and Acetaminophen did not affect IL-6 production by the Th2 cells and thus did not attenuate siga production by B cells. Acknowledgements I would like to thank Dr. Randy Day for his mentorship, all the BYUH-faculty, and to the volunteers of this research. 9

11 References Bancos, S., M.P. Bernard, D.J. Topham, and R.P. Phipps. Ibuprofen and other widely used non-steroidal anti-inflammatory drugs inhibit antibody production in human cells. Cellular Immunology 258(1): 18-28, Cho, J.Y. Immunomodulatory Effect of Nonsteroidal Anti-inflammatory Drugs at the Clinically Available Doses. Archives of Pharmacal Research 30(1): 64-74, Dlugosz, C.K., R.W. Chater, and J.P. Engle. Appropriate Use of Nonprescription Analgesics in Pediatric Patients. Journal of Pediatric Health care 20(5): , Harris, S.G., J. Padilla, L. Koumas, D. Ray, and R.P. Phipps. Prostaglandins as modulators of immunity. Trends in Immunology 23(3): , Martinez, F. and J.W. Coleman. The effects of selected drugs, including chlorpromazine and non-steroidal anti-inflammatory agents, on polyclonal IgG synthesis and interleukin 1 production by human peripheral blood mononuclear cells in vitro. Clinical and Experimental Immunology 76: , Prymula, R., C.A. Siergrist, R. Chlibek, H. Zemlickova, M. Vackova, J. Smetana, P. Lommel, E. Kaliskova, D. Borys, and L. Schuerman. Effect of prophylactic paracetamol administration at time of vaccination on febrile reactions and antibody responses in children: two open-label, randomized controlled trails. Lancet 374: , Ryan, E.P., S.J. Pollack, T.I. Murrant, S.H. Bernstein, R.E. Felgar, R.P. Phipps. Activated Human B Lymphocytes Express Cyclooxygenase-2 and Cyclooxygenase Inhibitors Attenuate Antibody Production. The Journal of Immunology 174: , Tanaka, K., H. Tanaka, Y. Kanemoto, and I. Tsuboi. The effects of nonsteroidal antiinflammatory drugs on immune functions of human peripheral blood mononuclear cells. Immunopharmacology 40(3): , Tsuboi, I., H. Tanaka, M. Nakao, S. Shichijo, and K. Ito. Nonsteroidal antiinflammatory drugs differentially regulate cytokine production in human lymphocytes: up-regulation of TNF, IFN-γ, and IL-2, in contrast to down-regulation of IL-6 production. Cytokine 7: , Woo, T. Pharmacology of Cough and Cold Medicines. Journal of Pediatric Healthcare 22(2): 73-79, Yamaki, K., H. Uchida, Y. Harada, R. Yanagisawa, H. Takano, H. Hayashi, Y. Mori, and S. Yoshino. Effect of the Nonsteroidal Anti-inflammatory Drug Indomethacin on Th1 and Th2 Immune Responses in Mice. Journal of Pharmaceutical Sciences 92(8): ,