Immunomodulatory activity of mefenamic acid in mice models of cell-mediated and humoral immunity.

OBJECTIVES: Previously, different nonsteroidal anti-inflammatory drugs (NSAIDs) have been evaluated for their potential immunomodulatory activities. Mefenamic acid is a well-known NSAID and is used in the treatment of musculoskeletal disorders, inflammation, fever, and pain. To the best of our knowledge, promising data regarding the immunomodulatory activity of mefenamic acid is scarce. Current study investigates the immunomodulatory activity of mefenamic acid in different models of cell-mediated and humoral immunity.
MATERIALS AND METHODS: Immunomodulatory effects on cell-mediated immunity were evaluated using dinitrochlorobenzene-induced delayed type hypersensitivity (DTH) and cyclophosphamide-induce myelosuppression assays. While effects on humoral immunity were evaluated using hemagglutination assay and mice lethality test.
RESULTS: Hematological analysis showed that mefenamic acid significantly reduced white blood cell count, red blood cell (RBC) count, hemoglobin content, lymphocytes levels, and neutrophils levels in healthy mice as compared with control, suggesting the immunosuppressive activity of mefenamic acid. Treatment with mefenamic acid also significantly reduced all the hematological parameters in cyclophosphamide-induced neutropenic mice, as compared with positive control group. We found that treatment with mefenamic acid significantly suppressed DTH after 24 h, 48 h, and 72 h, as compared with positive control group. Mefenamic acid treated groups showed a significant reduction in antibody titer against sheep RBCs as compared to control group, similar to the effect of cyclophosphamide. We also found increased mice lethality rate in mefenamic acid treated groups, as compared with positive control group.
CONCLUSIONS: The results provided basic information of immunosuppression of mefenamic acid on both cell-mediated and humoral immunity.

Introduction

The structural and functional changes of the immune system caused by drugs and plants are responsible for the immunosuppression or immunostimulation, which, in turn, may cause adaptation of the host defense mechanism against cancer and infection. These changes may also stimulate the abnormal immune response causing autoimmunity and allergy.[12] Mefenamic acid belongs to nonsteroidal anti-inflammatory class of drugs having analgesic, anti-inflammatory, and antipyretic activities. It is used to relieve moderate to severe pain such as muscular aches, menstrual cramps, headache, and dental pain.[3] Previously, different nonsteroidal anti-inflammatory drugs (NSAIDs) have been evaluated for their immunomodulatory activities. Indomethacin showed immunomodulatory activity by suppressing the mitogen-activated T-cell proliferation. Both acetylsalicylic acid and Indomethacin have been reported to attenuate the production of antibodies against sheep red blood cells (SRBC).[45] Piroxicam showed immunomodulatory activity in a rat model of rheumatoid arthritis by reducing the expression levels of tumor necrosis factor-α and nuclear factor-kappa B, and by suppressing concanavalin A-stimulated splenocyte proliferation.[6] Diclofenac sodium salt is known to reduce the number of rosette-forming cells in SRBC vaccinated mice, selectively inhibit Th2 type immune response, and downregulate interleukin-5 (IL-5) synthesis.[78] To the best of our knowledge, mefenamic has not been extensively analyzed for its immunomodulatory activity. Current study evaluates the immunomodulatory activity of mefenamic acid using different models of cell-mediated and humoral immunities.

Materials And Methods

Experimental Animals

Current study utilized 36 BALB/c mice aging between 6 and 8 weeks. Mice were housed in the animal house of Faculty of Pharmacy, The University of Lahore and standard environmental conditions (ambient temperature of 25 ± 2°C and 50 ± 15% relative humidity, along with 12 h light/12 h dark cycles) were provided. Mice were maintained at standard pellet diet and water ad libitum. All the experiments were approved by the Institutional Animal Ethics Committee, Faculty of Pharmacy, The University of Lahore.

Grouping of Experimental Animals

For delayed type hypersensitivity (DTH) and cyclophosphamide-induced myelosuppression assays, mice were divided into six groups, containing six mice in each group. Groups 1 and II served as control groups (negative and positive) and were treated with vehicle (40 µl DMSO for each dose) only. Groups III, IV, and V were intraperitoneally treated with low, medium, and high (1.5 mg/kg, 3 mg/kg, and 5 mg/kg, respectively) doses of mefenamic acid dissolved in the vehicle.[3] Group VI received 0.1 ml/kg dose of immunomodulator, which was used as a reference drug. Immunomodulator was prepared according to the method of Colinet al.[9] and contained 1 g sodium selenite, 15 g Vitamin E, 9 g sodium chloride, and distilled water q.s. to 1000 ml.

Mice were divided into five groups for hemagglutination (HA) test. Group I served as control and received the vehicle only, whereas Groups II, III, and IV served as experimental groups receiving low, medium, and high doses of mefenamic acid, respectively. Cyclophosphamide (25 mg/kg) was used as a reference drug.

For mice lethality test, mice were divided into six groups. Groups 1 and II served as negative control and positive control, respectively. Both groups were treated with the vehicle only. Groups III, IV, and V were treated with low, medium, and high doses of mefenamic acid, respectively. Group VI received cyclophosphamide (25 mg/kg), which was used as a reference drug.

Effects of Mefenamic Acid on Cell-mediated Immunity

Delayed type hypersensitivity test

Intraperitoneal treatment with mefenamic acid and immunomodulator was started at day 1 and continued for 8 days. Mice groups were carefully shaved for DTH assay. All the mice were sensitized with 0.1 ml of 2% dinitrochlorobenzene (DNCB) at day 2, except negative control group which was sham-sensitized with 0.1 ml acetone only. Before applying DNCB, skin thickness of mice was measured with the help of vernier caliper. Previously, sensitized mice were again challenged with 0.2 ml of 2% DNCB at day 8, except negative control group which was false-challenged with 0.2 ml acetone. The skin thickness was measured at 24 h, 48 h, and 72 h after DNCB challenge.[10]

Cyclophosphamide-induced myelosuppression

Mice of the experimental groups were treated with mefenamic acid and immunomodulator for consecutive 10 days. About 200 mg/kg cyclophosphamide was administered subcutaneously at the 10th day. Blood samples were collected from rat tail before and 72 h after administration of cyclophosphamide. Total white blood cell (WBC) count, RBC count, hemoglobin (Hb) content, lymphocyte levels, and neutrophil levels were measured using automated hemocytometer.

Effects of Mefenamic Acid on Humoral Immunity

Hemagglutination test

Preparation of sheep red blood cells

Sheep RBCs were separated from freshly collected blood of sheep. For this purpose, 5 ml blood was taken in a centrifuge tube, and volume was made up to 15 ml with phosphate buffer saline. Blood was centrifuged at 5000 rpm for 10 min. The RBC pellet was collected after removal of supernatant fluid and procedure was repeated twice. SRBCs were counted under the microscope using the neubauer chamber and number of cells was adjusted to 0.5 × 109 cells/0.1 ml. This concentration of cells is known to induce immunological response in mice.[11]

Procedure

Mice were weighed and divided into five groups. All the groups were immunized with 0.5 × 109 cells/0.1 ml of SRBCs at day 0. All the groups were intraperitoneally treated for seven consecutive days. On the 7th day, blood sample was collected from the tail of each mouse and centrifuged at 5000 rpm for 15 min to separate the serum. Antibody titer was determined using HA titer method.[12] 96-well plates were used for performing HA test, and 25 µl of PBS was added in all the wells, except the last column which was denoted as control. Serum (25 µl) was then placed in the first row of microtiter plate and was 2-fold serially diluted up to the 8th row of the microtiter plate. Twenty-five microliters of 10 v/v SRBCs were then dispensed in microtiter plate and the plate was incubated at 37°C for 1 h.[13]

Mice lethality test

Preparation of Pasteurella multocida culture

Pasteurella multocida was reconstituted in normal saline. LD50 (105 cells/0.5 ml) dose of P. multocida was injected subcutaneously into one rabbit. After death of the rabbit, blood samples were collected, and postmortem was performed. Specific organs (liver, heart, spleen, and kidney) were separated, cut into small pieces, and preserved. A small piece of any organ was then placed into blood agar media (pyrogen free) using a Petri dish and incubated for 24 h.

Procedure

All the mice groups were treated with experimental drugs for 21 days starting from day 1. On 7th and 17th day of experiment, all the groups were immunized through intraperitoneal route with hemorrhagic septicemia vaccine, except negative control. On the 21st day of experiment, all the mice were challenged with lethal dose of P. multocida subcutaneously and were examined for about 72 h.

Statistical Analysis

The data obtained from above mention experiments were statistically analyzed using GraphPad Prism version 6 software. All the data were expressed as mean ± standard error of the mean and analyzed using one-way ANOVA followed by Tukey's test or Student's t-test where applicable. P < 0.05 was considered statistically significant.

Results

Treatment with Mefenamic Acid Significantly Reduced Delayed Type Hypersensitivity Test After 24 h, 48 h, and 72 h

We found that treatment with DNCB caused increase in skin thickness in all groups after 24 h. The data showed a significant (1.5 ± 0.1; P < 0.001) increase in skin thickness in positive control group as compared with negative control group (0.01 ± 0.005). Treatment with low (0.9 ± 0.1; P < 0.01), medium (0.8 ± 0.08; P < 0.001), and high (0.7 ± 0.07; P < 0.001) doses of mefenamic acid significantly inhibited the increase in skin thickness as compared with positive control group. Immunomodulator also showed a significant (1.5 ± 0.1; P < 0.001) increased skin thickness [Figure 1a].

Figure 1

Treatment with mefenamic acid significantly reduced delayed-type hypersensitivity when analyzed after 24 h (a), 48 h (b), and 72 h (c). Mefenamic acid also significantly alleviated anti-sheep red blood cell antibody titer in hemagglutination test (d). Mean ± standard error of the mean is given to represent the data, where n = 6 for delayed type hypersensitivity assay and n = 3 for hemagglutination test. **P < 0.01 and ***P < 0.001 represent comparison of experimental groups with positive control. ###P < 0.001 represents comparison with negative control group

Analysis after 48 h showed a significant increase in skin thickness in positive control group (1.3 ± 0.1; P < 0.001) as compared with negative control group (0.020 ± 0.005). Treatment with immunomodulator (1.8 ± 0.13; P < 0.001) also showed a significant increase as compared with negative control group. Treatment with low dose (0.7 ± 0.07; P < 0.05), medium dose (0.46 ± 0.07; P < 0.01), and high dose (0.36 ± 0.04; P < 0.01) caused significant decrease in skin thickness as compared with positive control group [Figure 1b].

Analysis after 72 h showed that treatment with low dose, medium dose, and high dose showed significantly (0.32 ± 0.06; P < 0.001, 0.16 ± 0.05; P < 0.001, and 0.07 ± 0.01; P < 0.001, respectively) decreased skin thickness when compared with positive control group. We determined a significant elevation in positive control group (0.7 ± 0.06; P < 0.001) as compared with negative control group (0.04 ± 0.01). Treatment with immunomodulator (1.18 ± 0.1; P < 0.001) also showed a significant increase in skin thickness as compared to negative control [Figure 1c].

Mefenamic Acid Significantly Reduced White Blood Cell Counts, Lymphocyte Levels, Neutrophil Levels, Red Blood Cell Counts, and Hemoglobin Content in Healthy Mice

We compared control group (10.7 ± 0.1) with experimental groups and results showed nonsignificant difference of immunomodulator (11.5 ± 0.2) as compared with control group. The data revealed that there were significant reduction in WBC counts in low dose (9.3 ± 0.3; P < 0.01), medium dose (7.1 ± 0.2; P < 0.001), and high dose (5.6 ± 0.2; P < 0.001) treated groups [Figure 2a].

Figure 2

Mefenamic acid showed immunosuppressive effect by significantly reducing white blood cell counts (a), lymphocyte levels (b), neutrophil levels (c), red blood cell counts (d), and hemoglobin content (e) in healthy mice. Immunomodulator prevented the suppression in hematological parameters. Mean ± standard error of the mean is given to represent the data, where n = 6. *P < 0.05, **P < 0.01, and ***P < 0.001 represent comparison of experimental groups with positive control

Treatment with low dose (71.5 ± 3; P < 0.05), Medium dose (65 ± 1.4; P < 0.01), and high dose (51.0 ± 2.7; P < 0.001) showed a significant decrease in lymphocytes levels as compared with control group. However, nonsignificant difference was found when the result of immunomodulator (74.8 ± 1.6) group was compared with control group (79.5 ± 1.6) [Figure 2b].

We found nonsignificant difference in neutrophil levels when immunomodulator group (80.8 ± 0.9) was compared with control group. Treatment with low (69.8 ± 2.8; P < 0.05), medium (65.8 ± 1.8; P < 0.001), and high doses (52.2 ± 3.6; P < 0.001) showed a significant alleviation as compared with control group (81.3 ± 1.6) [Figure 2c].

We did not find statistical significant difference when low dose (6.5 ± 0.1) and immunomodulator (7.0 ± 0.2) groups were compared with control group (6.8 ± 0.2). The data showed a significant reduction in RBC counts in medium (6.2 ± 0.1; P < 0.05) and high dose treated groups (6.0 ± 0.1; P < 0.01) as compared with control group [Figure 2d].

Treatment with low dose (10.5 ± 0.4) of mefenamic acid and immunomodulator (11.2 ± 0.37) showed nonsignificant difference in Hb levels as compared to control group (11.2 ± 0.3). Whereas medium (9.0 ± 0.5; P < 0.01) and high dose groups (8.2 ± 0.4; P < 0.001) demonstrated significant decrease in Hb levels when compared with control group [Figure 2e].

Mefenamic Acid Significantly Reduced White Blood Cell Counts, Lymphocyte Levels, Neutrophil Levels, Red Blood Cell Counts, and Hemoglobin Content in Cyclophosphamide-induced Myelosuppressive Mice

We found significant (P < 0.001) alleviation in WBC counts in positive control group (8.4 ± 0.1; P < 0.001), as compared with negative control group (10.7 ± 0.1). Treatment with low dose (6.4 ± 0.1), medium dose (5.2 ± 0.1), and high dose (4.3 ± 0.2) of mefenamic acid significantly (P < 0.001) attenuated WBC counts as compared with positive control. Whereas immunomodulator (10.4 ± 0.15; P < 0.01) showed a significant prevention in WBC reduction caused by cyclophosphamide administration [Figure 3a].

Figure 3

Treatment with mefenamic acid significantly reducing white blood cell counts (a), lymphocyte levels (b), neutrophil levels (c), red blood cell counts (d), and hemoglobin content (e) in cyclophosphamide-induced myelosuppressive mice. Immunomodulator significantly prevented the suppression in hematological parameters. Mean ± standard error of the mean is given to represent the data, where n = 6. *P < 0.05, ***P < 0.001 represent comparison of experimental groups with positive control. ###P < 0.001 represents comparison with negative control group

Lymphocytes levels were found significantly (P < 0.001) alleviated in positive control (79.5 ± 1.6) as compared with negative control group (64.5 ± 1.6). Treatment with low dose (51.7 ± 0.7), medium dose (48.8 ± 1.6), and high dose (40.8 ± 1.4) of mefenamic acid significantly (P < 0.001) attenuated the lymphocyte levels as compared with positive control group. Treatment with immunomodulator (75.6 ± 2; P < 0.001) caused significant elevation as compared to positive control [Figure 3b].

We found significant suppression of neutrophil levels in positive control group (59.3 ± 3.6; P < 0.001) as compared with negative control group (81.3 ± 1.6). Treatment with immunomodulator (73.83 ± 2.1; P < 0.05) nearly normalized the neutrophils level. Therapy with low dose (43.3 ± 3.8; P < 0.01), medium dose (29.5 ± 2.8; P < 0.001), and high dose (19.3 ± 2.1; P < 0.001) displayed a significant reduction in neutrophil levels as compared with positive control group [Figure 3c].

Treatment with low dose (4.6 ± 0.2) and medium dose (4.1 ± 0.3) did not show significant difference in RBC counts as compared with positive control group. However, high dose treated group showed (3.4 ± 0.1; P < 0.001) significant reduction as compared with positive control group. We also found significant (P < 0.001) alleviation in RBC counts in positive control group (4.7 ± 0.1; P < 0.001), as compared with negative control group (6.8 ± 0.08). Treatment with immunomodulator (7.3 ± 0.2; P < 0.001) normalized the RBC counts [Figure 3d].

We found significantly decreased levels of Hb in the positive control (8.4 ± 0.17; P < 0.001) group as compared with negative control group (11.3 ± 0.3). Treatment with low dose (6.7 ± 0.09), medium dose (5.46 ± 0.1), and high dose (4.6 ± 0.2) of mefenamic acid showed a significant (P < 0.001) attenuation of Hb content as compared with positive control group. Treatment with immunomodulator significantly (10.4 ± 0.15; P < 0.001) prevented the cyclophosphamide-induce decrease in Hb content [Figure 3e].

Mefenamic Acid Significantly Suppressed Antibody Titer in Hemagglutination Assay

We found that treatment with low (85.8 ± 2.3), medium (79.8 ± 0.4), and high (65.03 ± 2.15) doses of mefenamic acid significantly (P < 0.001) decreased the antibody titer as compared with the control group (136 ± 11). Similarly, treatment with cyclophosphamide (reference drug) also significantly (52.7 ± 1.7; P < 0.001) attenuated the antibody titer [Figure 1d].

Mefenamic Acid Increased the Mortality Rate in Mice Lethality Test

Administration of P. multocida caused 100% mortality in 24 h in the negative control group, whereas 33% mortality was observed in positive control group within 72 h. Treatment with low dose and medium dose increased the mortality rate to 50% and 66.6%, respectively as compared with positive control group. We observed the death of one mouse after 48 h and two mice later after 72 h in low dose treated group. In medium dose treated group, we found one mouse dead with 12–24 h. Subsequently, two mice died after 48 h, and one mouse was found dead after 72 h. Treatment with high dose of mefenamic acid caused 100% mortality within 24 h, whereas cyclophosphamide used as a reference drug caused death of all five mice within 12 h [Table 1].

Table 1

Mice lethality ratio at 12 h, 24 h, 48 h, and 72 h

Discussion

We evaluated the immunosuppressive effect of mefenamic acid through DTH assay. DNCB was used as an allergen to cause hypersensitivity reaction. When DNCB is applied on the skin, it causes accumulation of macrophages on reaction site which release IL-12 and IL-18. These cytokines, in turn, are responsible for differentiation of Th1 cell.[14] These factors cause increase in skin thickness at the site of reaction which is widely considered as a measure of DTH.[15] In our study, inflammatory response toward allergen was greater in positive control group as compared with mefenamic acid treated groups. In mefenamic acid treated groups, we found dose-dependent reduction in skin thickness as compared with positive control group. DTH is a T-cell-mediated reaction, and T-cell-mediated immunity is regulated by different prostaglandins that are product of cyclooxygenase (COX) pathway, for example, PGE2, PGI2, and PGD2, and by T-cell specific cytokines.[16] NSAIDs inhibit COX pathway which eventually leads to T-cell suppression.[17] Ketoprofen is another NSAID, which is known to suppress the T-cell-mediated DTH reaction.[18]

Besides, DTH assay, we also evaluated the immunomodulatory effects of mefenamic acid on cell-mediated immunity using cyclophosphamide-induced myelosuppression assay. Cyclophosphamide causes bone marrow suppression by alkylation of DNA.[19] Effects of mefenamic acid were evaluated in cyclophosphamide-induced neutropenic mice, as well as, in healthy mice. Mefenamic acid showed immunosuppressive effect by dose-dependently decreasing the total leukocyte counts (TLCs) and differential leukocyte counts (DLCs). WBCs are fundamental constituent of immune system. Elevation or attenuation in WBCs directly affects the immune system as they are known to identify the pathogen and support the immune response.[20] WBCs fight against infection causing agents by phagocytosis and deliver the pathogens to phagosomes, where pathogens are destroyed.[21] Neutropenia is associated with the treatment of different disease conditions such as cancer chemotherapy, rheumatoid arthritis, and allergic asthma.[6] Lymphocytes are considered as an essential parameter of immune system which plays a key role in modulating immune system through the differentiation and proliferation of T-cells and B-cells.[22] Previous studies showed that other NSAIDs such as ketoprofen demonstrated immunosuppressive effects by significantly decreasing the TLC and DLC, before and after administration of cyclophosphamide. It was suggested that NSAIDs enhance cell apoptosis and reduce granulocyte colony stimulating factor which plays an important role in the development of new neutrophils.[18]

Antigen-specific antibody production is the main component of humoral immunity. In this study, we evaluated the effects of mefenamic acid on humoral immunity through HA assay and mice lethality test. For experimental purpose, the most frequently used method for evaluating humoral immunity is testing of antibody titer against sheep RBCs.[23] Antigens are neutralized either directly by antibody or antibodies convert antigen in such form that is easily phagocytized.[24] Antibody titer is the direct way to evaluate humoral immunity. In the current study, all the doses of mefenamic acid were found to decrease antibody titer in a dose-dependent manner as compared with control group, similar to the effect of cyclophosphamide used as a reference drug. Hamdaniet al.[18] suggested that ketoprofen treated mice decreased the production of IgG and IgM antibody against sheep RBCs. The decreased antibody titer found in our study might also be due to the decrease production of IgG and IgM antibodies in the serum of mefenamic acid-treated mice. However, further studies are required to confirm evaluate the effects of mefenamic acid on IgG and IgM levels.

Mice lethality test is used to evaluate the survival rate after injecting challenging dose of P. multocida in hemorrhagic septicemia vaccine immunized mice.[25] P. multocida is pathogenic to experimental animals, so mice are first immunized with vaccine to produce antibodies and then P. multocida is administered to evaluate the effect of drugs or plant extracts on lethality ratio. If the drug is immunostimulant and increases the antibody production, it would cause survival of mice against P. multocida culture, and if the drug is immunosuppressant, it would cause the lethality in experimental animal. In the current study, mefenamic acid treated group showed increase in mice lethality ratio as compared with positive control group. Immunization with hemorrhagic septicemia vaccine produces IgG and IgM antibodies in mice blood which causes mice to survive against P. multocida antigen.[25] It may be assumed that mefenamic acid treated groups might have decreased antibodies (IgG and IgM) production which led to increase in the mortality rate as compared to positive control.

Conclusion

The data suggest that mefenamic acid possesses immunosuppressive activity which is evident by reduction in TLCs, lymphocytes levels, neutrophil levels, RBC counts, and Hb content in healthy mice. Mefenamic acid showed immunosuppressive effect on cell-mediated immunity by attenuation of DTH and all hematological parameters in a mouse model of cyclophosphamide-induced myelosuppression. Immunosuppressive activity of mefenamic acid on humoral immunity was also evident in our study as different doses significantly reduced antibody titer in HA assay and increased mice lethality ratio.

Financial Support and Sponsorship

Nil.

Conflicts of Interest

There are no conflicts of interest.