http://informahealthcare.com/ipi ISSN: 0892-3973 (print), 1532-2513 (electronic) Immunopharmacol Immunotoxicol, 2015; 37(2): 207–213 ! 2015 Informa Healthcare USA, Inc. DOI: 10.3109/08923973.2015.1017645
RESEARCH ARTICLE
Immunopharmacology and Immunotoxicology Downloaded from informahealthcare.com by UMEA University Library on 04/01/15 For personal use only.
MIF and TNFa serum levels in rheumatoid arthritis patients treated with disease-modifying antirheumatic drugs: a cross-sectional study Lorena Michele Brennan-Bourdon1,2, Ulises De la Cruz-Mosso2, Zyanya Reyes-Castillo2, Gloria Esther Martı´nez-Bonilla3, Marı´a Guadalupe Ramı´rez-Duen˜as1, Marı´a Cristina Islas-Carbajal1, Ana Rosa Rinco´n-Sa´nchez1, Mario Salazar-Pa´ramo1, and Jose´ Francisco Mun˜oz-Valle2 1
Departamento de Fisiologı´a, Centro Universitario de Ciencias de la Salud, Universidad de Guadalajara, Guadalajara, Jalisco, Me´xico, 2Instituto de Investigacio´n en Ciencias Biome´dicas, Centro Universitario de Ciencias de la Salud, Universidad de Guadalajara, Guadalajara, Jalisco, Me´xico, and 3 Servicio de Reumatologı´a, O.P.D. Hospital Civil de Guadalajara ‘‘Fray Antonio Alcalde’’, Guadalajara, Jalisco, Me´xico Abstract
Keywords
Macrophage migration inhibitory factor (MIF) and tumor necrosis factor alpha (TNFa) play a pivotal role in rheumatoid arthritis (RA). MIF is considered a relevant cytokine because it appears before TNFa in the inflammatory cascade thus stimulating TNFa production and MIF’s relationship with traditional synthetic disease modifying antirheumatic drugs (sDMARDs) is unknown. In this cross-sectional study, we investigated the association of MIF and TNFa serum levels with methotrexate (MTX) and in combination with chloroquine (CLQ) and sulfasalazine (SSZ) in RA patients classified according to the ACR/EULAR 2010 criteria. Patients were divided into three groups: MTX-monotherapy group (n ¼ 40), MTX combination therapy groups: MTX + CLQ (n ¼ 41), and MTX + CLQ + SSZ (n ¼ 42). MIF and TNFa serum levels were determined by ELISA. We found high levels of ESR, CRP, RF, and anti-CCP in all therapy groups. Furthermore, we subclassified 97 patients with established RA (2 years of disease duration) and found that TNFa serum levels were lower in the combination therapy group (MTX + CLQ + SSZ) in comparison with the monotherapy MTX group (16.7 pg/mL versus 13.6 pg/mL, p ¼ 0.02). However, we did not find differences between sDMARD therapies in MIF serum levels. We did find a significant reduction in MIF serum levels in patients treated with oral steroids compared with patients without oral steroids (1.7 ng/mL versus 4.3 ng/mL, p50.001). In conclusion, this study supports the role of sDMARDs in modifying TNFa serum levels and oral steroids MIF serum levels. Nevertheless, we found that MIF serum levels are not modified by sDMARD treatment.
Association, macrophage migration inhibitory factor, rheumatoid arthritis, synthetic disease-modifying antirheumatic drugs, tumor necrosis factor alpha
Introduction Rheumatoid arthritis (RA) is an autoimmune disease of unknown etiology that affects primarily the synovial joints. The evolution of the disease is characterized by pain as well as by swollen peripheral joints which affect movement and can cause deformity1. Among the risk factors for RA are female gender, advanced age, and genetic factors such as a positive family history2. Furthermore, RA is an immunemediated disease distinguished by a sustained inflammation process in the diarthrodial joints where cytokines play a key role3. Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine that has been recently linked to the pathogenesis of RA4. MIF is considered a unique pleiotropic cytokine since it is the only cytokine that counter-regulates the inhibitory effects of glucocorticoids5. It also stimulates the
Address for correspondence: Jose´ Francisco Mun˜oz Valle, Insurgentes 244-1, Colonia Lomas de Atemajac, Zapopan, Jalisco CP 45178, Me´xico. Tel: +52 3310585309. E-mail: [email protected]
History Received 2 September 2014 Revised 7 January 2015 Accepted 4 February 2015 Published online 27 February 2015
expression of adhesion molecules and chemokines favoring leukocyte trafficking to the inflamed tissue6, regulates the expression of matrix metalloproteinases associated with cartilage damage7 as well as suppresses the expression of the tumor immunosuppressant protein p53 in fibroblast-like synoviocytes (FLS)8. These characteristics support MIF’s role in the innate and adaptive immune responses that are present in RA. On the contrary, TNFa, an important pro-inflammatory cytokine known to have a primary role in RA, has been proposed as a possible biomarker that could be used to predict DMARD response at the individual level with conventional or synthetic DMARD (sDMARD) therapy and is to date a therapeutic target which has proven successful in the treatment of RA9. Both MIF and TNFa are important elements of the inflammatory cascade that develops in RA. Several studies have reported elevated synovial and serum levels of MIF10,11 as well as TNFa serum levels in RA patients12–15. However, MIF has been reported as an upstream regulator of TNFa since it stimulates TNFa production by macrophages and
208
L. M. Brennan-Bourdon et al.
conversely TNFa is able to augment MIF production in a reciprocal augmenting pro-inflammatory responses loop16. Thus, MIF is implicated in a vast range of regulatory actions in the immune response in RA and has been proposed as a therapeutic target17, but its role with current treatment strategies with traditional sDMARD therapy has not been thoroughly investigated. Therefore, the aim of this crosssectional study was to investigate MIF and TNFa’s relationship with sDMARDs in RA patients.
Immunopharmacology and Immunotoxicology Downloaded from informahealthcare.com by UMEA University Library on 04/01/15 For personal use only.
Methods Patients This was a cross-sectional study, 123 RA patients diagnosed according to the ACR-EULAR 2010 classification criteria18 were randomly enrolled from the Rheumatology Departments: Hospital Civil of Guadalajara ‘‘Fray Antonio Alcalde’’ and Regional Hospital ‘‘Dr. Valentin Go´mez Farias’’ ISSSTE (Instituto de Seguridad y Servicios Sociales de los Trabajadores del Estado). The RA inclusion criteria were as follows: age 418 years and patients were selected independently of disease duration. According to the appointed rheumatologist’s prescription, all patients received sDMARD treatment with MTX or in combination with CLQ and/or SSZ for at least 3 months as well as the concomitant use of low-dose prednisone and non-steroidal anti-inflammatory drugs for several patients. RA patients were subclassified according to the following treatment subgroups: the first group was composed of 40 patients with MTX monotherapy who received doses of 5.0– 20 mg/week, the second combination therapy group (n ¼ 41) with MTX + CLQ received (5.0–15 mg/week of MTX and 150 mg/d of CLQ) and the third combination therapy group (n ¼ 42) with MTX + CLQ + SSZ (5.0–20 mg/week of MTX, 150–300 mg/d of CLQ, and 0.5–3.0 g/d of SSZ). Nonsteroidal anti-inflammatory drugs (NSAIDs) included diclofenac, piroxicam, meloxicam, paracetamol, indomethacin, celecoxib, sulindac, and ketoprofen. Low-dose prednisone was the steroidal anti-inflammatory drug (SAID) of choice by the appointed rheumatologists, with doses of 510 mg of prednisone daily. Ethical guidelines In accordance to the 2008 Declaration of Helsinki ethical guidelines, all patients gave their written informed consent prior enrollment in the study. Approval for this study was obtained from the Institutional ethics review committee of the Centro Universitario de Ciencias de la Salud de la Universidad de Guadalajara (registration no. C-11908). Clinical assessments Clinical assessments and disease activity (DAS28) evaluations were performed by the appointed rheumatologists and blood samples were taken at the time of inclusion to determine with standardized methods the erythrocyte sedimentation rate (ESR) (the Wintrobe method), C-reactive protein (CRP), and rheumatoid factor (RF); both CRP and RF assays were performed by nephelometry with BiosystemsÕ reagents (Beckman Coulter, Inc., Fullerton, CA) and serum
Immunopharmacol Immunotoxicol, 2015; 37(2): 207–213
anti-Cyclic Citrullinated Peptide antibodies (anti-CCP) by a commercial enzyme-linked immunoassay (DIASTATÔ, Diastat, England, UK), all according to manufacturer’s instructions. Quantification of MIF and TNFa serum levels MIF and TNFa serum levels from all RA patients were determined with commercial quantitative sandwich-enzyme immunoassays (human MIF, RayBiotechÕ , Norcross, GA; human TNFa InvitrogenÔ, Waltham, MA) according to manufacturer’s instructions. The MIF assay sensitivity was 6 pg/mL, while for TNFa it was 1.7 pg/mL. Statistical analysis Statistical analysis for this study was performed with GraphPad Prism v 5.0 (GraphPad Software Inc., San Diego, CA) and SPSS v17.0 statistical programs (SPSS Inc., Chicago, IL). For the descriptive analysis, nominal variables were expressed as frequencies and continuous variables with non-parametric distribution were expressed as medians and percentiles 5–95. To determine non-parametric comparisons within therapy groups, we used the Kruskal–Wallis and Mann–Whitney U tests. For the correlation analysis of continuous variables with non-parametric distribution, we used the Spearman correlation test and for those with parametric distribution, Pearson correlation test. A stepwise forward multivariable linear regression analysis was used to identify possible predictors of change in MIF and TNFa levels. Variables included in the MIF analysis were oral steroids, disease activity, and RF and for the TNFa analysis, MTX and CLQ treatment. Probability values with a p50.05 were considered statistically significant in a two-tailed test.
Results Clinical characteristics Clinical characteristics of the 123 RA patients included in this study are shown in Table 1. The average age was 47 years, the predominant gender was female 94% and 6% male, the average disease duration was 6 years and patients had moderate disease activity scores as measured by the DAS28 index (average score 4.5), 28% of RA patients included low-dose oral steroid therapy (510 mg/d of prednisone), 85% included NSAIDs, and 71.5% received folic acid. Furthermore, 91% of patients included in this study were not active smokers at the time of the study. Considering the serum acute phase reactants (ESR and CRP) and autoantibodies (RF and anti-CCP), these were elevated in all three RA therapy groups. We did not find differences within therapy groups for these values. MIF and TNFa serum levels In the case of MIF serum levels, no significant differences were observed in all RA patients within therapy groups (MTX: 2.5 ng/mL; MTX + CLQ: 3.0 ng/mL; MTX + CLQ + SSZ: 4.1 ng/mL). In regard to TNFa therapy groups, all RA patients had increased serum levels and these were not found to be statistically different (MTX: 16.9 pg/mL;
MIF and TNF serum levels in RA patients
DOI: 10.3109/08923973.2015.1017645
209
Table 1. Demographic and clinical characteristics of rheumatoid arthritis patients.
Immunopharmacology and Immunotoxicology Downloaded from informahealthcare.com by UMEA University Library on 04/01/15 For personal use only.
Variables Demographics Age (years)a Genderb Female Male Clinical assessment Disease durationc DAS28 scorec Active smokersb No Yes ESR (mm/h)c C reactive protein (mg/dL)c Rheumatoid factor UI/mLc Anti-CCP (U/mL)c Treatmentb Prednisoneb No Yes NSAIDsb No Yes
All patients, n ¼ 123
MTX, n ¼ 40
MTX + CLQ, n ¼ 41
MTX + CLQ + SSZ, n ¼ 42
47 ± 14
53 ± 13
50 ± 14
39 ± 12
116 (94%) 7 (6%)
38 (95%) 2 (5%)
36 (88%) 5 (12%)
42 (100%) 0 (0)
6 (0.50–23.30) 4.5 (2.4–6.7)
8 (0.56–22.60) 4.2 (2.4–6.80)
8 (0.50–30.80) 4.3 (2.4–6.6)
4.5 (0.34–19.85) 4.5 (2.4–7.2)
112 11 37 8.6 194 109.5
(91%) (9%) (11.0–55.0) (1.0–144.0) (12.2–704.6) (1.6–496.6)
35 5 32 7.6 88 108.4
(88%) (12%) (15.7–55.6) (1.4–152.0) (7.2–711.5) (1.4–602.9)
37 4 37 5.0 208 79.3
(90%) (10%) (12.2–53.2) (0.3–212.0) (13.6–589.8) (1.6–509.4)
40 2 41 11.0 204.5 132.6
(95%) (5%) (7.00–60.9) (0.8–118.9) (13.9–714.6) (1.6–463.6)
88 (72%) 35 (28%)
29 (73%) 11 (27%)
28 (68%) 13 (32%)
31 (74%) 11 (26%)
18 (15%) 105 (85%)
3 (7%) 37 (93%)
5 (12%) 36 (88%)
10 (24%) 32 (76%)
DMARDs, disease modifying anti-rheumatic drugs; NSAIDs, non-steroidal anti-inflammatory drugs; ESR, erythrocyte sedimentation rate; DAS28, disease activity score. RA subgroups: MTX, methotrexate; MTX + CLQ, chloroquine; MTX + CLQ + SSZ, sulphasalazine. a Data are expressed as means ± SD. b n and percentages. c Medians and percentiles p5–p95.
Figure 1. MIF and TNFa serum levels in sDMARD treatment groups in established RA (2 years of disease duration). (A) MIF serum levels, p ¼ 0.13. **Kruskal–Wallis test. (B) TNFa serum levels. No significant differences were found within sDMARD therapies in the established RA group for MIF serum levels, whereas for TNFa serum levels for the established RA group, a significant decrease was found in the MTX + CLQ + SSZ group compared with the MTX group, p ¼ 0.02. *Mann–Whitney U test.
MTX + CLQ: 15.3 pg/mL; MTX + CLQ + SSZ: 14.6 pg/mL). In addition, when RA patients were classified in early RA patients (52 years of disease duration), we did not find significant differences within therapy groups for MIF and TNFa serum levels in individuals with early RA (data not shown). Similarly, in established RA patients (2 years of disease duration), regarding MIF serum levels, we did not find significant differences between therapy groups (MTX: 2.8 ng/mL, MTX + CLQ: 2.8 ng/mL; MTX + CLQ + SSZ: 7.9 ng/mL; Figure 1A). However, in the case of TNFa in established RA patients, we did find a significant difference
between the MTX and the MTX + CLQ + SSZ therapy groups (16.7 pg/mL versus 13.6 pg/mL, p ¼ 0.02; Figure 1B). Relationship between MIF and TNFa serum levels with clinical findings To investigate the relationship of MIF serum levels with clinical markers of the disease, we found a negative correlation of MIF with CRP (r ¼ 0.326, p ¼ 0.001; Figure 2A) and a positive correlation of TNFa with CRP (r ¼ 0.376, p ¼ 0.001; Figure 2B) in the Spearman correlation test.
Immunopharmacology and Immunotoxicology Downloaded from informahealthcare.com by UMEA University Library on 04/01/15 For personal use only.
210
L. M. Brennan-Bourdon et al.
Immunopharmacol Immunotoxicol, 2015; 37(2): 207–213
Figure 2. TNFa and MIF serum levels correlation with CRP. (A) A negative correlation was found in MIF and CRP levels of RA patients. (B) A positive correlation between TNFa and CRP in RA patients was identified; p values were obtained by Spearman’s correlation test.
Figure 3. TNFa and MIF serum levels with and without oral steroid treatment. (A) Significantly diminished serum levels were found in MIF serum levels (p50.001). (B) TNFa serum levels were higher in those patients receiving oral steroids (p ¼ 0.01). *Mann–Whitney U test.
However, MIF and TNFa serum levels did not associate with the DAS28 score, CRP, ESR, or anti-CCP (data not shown). Oral steroid treatment in MIF and TNFa serum levels RA patients with oral steroids were compared with patients without oral steroids. On one hand, MIF serum levels were significantly lower in the oral steroid group compared with those patients who were not receiving steroids (1.7 ng/mL versus 4.3 ng/mL; p50.001; Figure 3A). On the other hand, TNFa soluble levels in this RA group were significantly higher in the oral steroid group compared with those patients who were not receiving steroids (17.7 pg/mL versus 14.5 pg/mL; p ¼ 0.01; Figure 3B). Oral steroids predict changes in MIF and sDMARDs in TNFa serum levels With a stepwise forward multivariable linear regression analysis, we identified significant changes in MIF and TNFa
serum levels according to oral steroids and sDMARDs. Oral steroid treatment contributed to a significant decrease of MIF serum levels ( ¼ 0.362; CI ¼ 5.624 to 1.894; p ¼ 0.001) whereas sDMARD treatment did not contribute to a significant decrease of MIF (data not shown). However, in the case of TNFa serum levels, sDMARDs methotrexate ( ¼ 0.202; CI ¼ 2.266 to 0.126; p ¼ 0.02) and chloroquine ( ¼ 0.190; CI ¼ 0.128 to 0.003; p ¼ 0.03) contributed to a significant decrease of TNFa serum levels.
Discussion Long-term pharmacological treatment in RA with sDMARDs, considered the cornerstone of RA therapy, is fundamental in order to reduce joint destruction and eventually minimize functional loss since aiming to achieve remission or low disease activity is the desired treatment goal19. In a 2002 study, Aletaha and Smolen report the gold standard methotrexate as the most prescribed sDMARDs in RA, as well as
Immunopharmacology and Immunotoxicology Downloaded from informahealthcare.com by UMEA University Library on 04/01/15 For personal use only.
DOI: 10.3109/08923973.2015.1017645
chloroquine and sulphasalazine, where a high retention rate for MTX has also been shown20–22. Herein, we show the treatment regimens included in this study are in accordance with published reports and are considered a valid treatment strategy in RA. Cytokines are important therapeutic targets for RA treatment. MIF and TNFa are produced by macrophages, T cells, dendritic cells, and fibroblast-like synoviocytes and a feedback loop has been reported between these two cytokines where TNFa induces the production of MIF and inversely MIF induces the production of TNFa16,23. The relevance of these cytokines in the pathogenic mechanisms of RA is what led us to propose MIF’s relationship with traditional sDMARD treatment, considering sDMARDs as the most common disease-modifying drugs used to date. According to MIF’s relationship with sDMARD therapy, to our knowledge a study by Kim et al., determined MIF’s relationship with clinical measures in 72 patients with RA, and found that the numbers, duration, and kind of disease antirheumatic drug therapy do not associate with MIF serum and synovial fluid levels. However, the specific sDMARD therapy was not described24. Nevertheless, this is the first study that reports MIF serum levels are not modified by sDMARD monotherapy (MTX) and combination therapies (MTX + CLQ and MTX + CLQ + SSZ), which also supports this finding. Previous reports have demonstrated a preference in the prescription of combination regimens with sDMARDs compared with monotherapy primarily due to improved efficacy25–27. More recently, another important study demonstrated that triple therapy is equivalent to the combination of MTX with the biologic DMARD etanercept in patients failing initial MTX monotherapy28. Considering the limitation of our cross-sectional study, we were not able to determine the efficacy of therapy regimens. We found that TNFa levels were significantly lower in the established RA combination therapy group with all three sDMARDs (MTX, CLQ, and SSZ) compared with the monotherapy MTX group. However, these results should be interpreted with caution, since TNFa serum levels in our study were found to be modified by the MTX and CLQ doses. These results may partly reflect TNFa’s relationship with sDMARDs because they have been reported to decrease the production of anti-inflammatory and pro-inflammatory cytokines such as IL-10 and TNFa levels in the synovial membrane in response to anti-rheumatic drug treatment29. However, it is also reasonable to predict that the prescribed medications might not have been active enough and the role of sDMARDs on cytokine serum levels should be considered in future longitudinal studies. In the case of early RA patients, we did not find differences in TNFa serum levels within therapy groups possibly because TNFa is upregulated later in the disease in comparison with anti-CCP, which may be present two decades before RA diagnosis30. However, we must point out that we cannot discard that among RA patients treated with methotrexate, 30–50% do not fulfill response criteria31. This distinction could explain the low number of RA patients in remission in our study group.
MIF and TNF serum levels in RA patients
211
Several studies have demonstrated important reductions in disease activity scores (DAS28) as well as in the acute phase reactants with sDMARD treatment in RA patients32,33. Nonetheless, in our study, we did not find significant differences within therapy groups in ESR, CRP, RF, antiCCP, and DAS28 scores. The discrepancy between our findings and those reported could be attributed to the crosssectional design limitation of our study as well as by the complexity of RA pathogenesis and the heterogeneity of disease manifestations such as severity, differences in disease duration, and disease activity scores, as well as by the individual treatment duration of RA patients included in this study. The positive correlation found between TNFa and CRP is due to the inflammatory process characteristic of RA patients because this cytokine and IL-6 stimulate the hepatocytes to produce acute phase reactants such as CRP34,35. A previous study in our population found a positive correlation between MIF and CRP in RA patients not receiving oral steroids36 and in contrast, other studies have not found an association between MIF serum levels and CRP24. These differences could be accredited to MIF’s response in the local tissue because it appears to be more marked than the systemic response and a positive correlation of synovial MIF and CRP has been reported37. However, the negative correlation found between MIF serum levels and CRP in our study group could be attributed to the heterogeneity in the therapy and disease duration in our selected groups. Moreover, considering TNFa serum levels were overexpressed and MIF values were normal as previously reported (2–6 ng/mL)38 these results could provide further insight into TNFa’s association with disease activity and MIF with disease progression39. MIF expression can be induced by low concentrations of glucocorticoids in macrophages, T cells, and synovial fibroblasts40. However, when the characteristic inflammation process is present in RA and oral glucocorticoids are introduced to therapy, the pharmacological dosage should be superior to the endogenous glucocorticoid concentration to counter-regulate the effects of MIF41. Oral glucocorticoids in low doses are considered as disease-modifying antirheumatic drugs due to their genomic effect by inhibiting inflammatory genes, but are used in RA with caution due to their side effects42,43. In this regard, our results confirm MIF’s relationship with glucocorticoids due to the significantly lower MIF serum levels in RA patients prescribed with oral glucocorticoids. The precise mechanism of action by which MIF counter-regulates the anti-inflammatory effect of glucocorticoids is unknown. On the contrary, our results showed increased TNFa serum levels in RA patients with oral steroids in comparison with patients who did not take steroids. This result could be explained considering that the overall goal in RA treatment is aimed at controlling inflammation. However, we cannot discard that RA patients with oral steroid therapy in our study were possibly at initial treatment phases. Hence, the seemingly negative correlation between MIF and TNFa found in this study that may also reflect the moderate disease activity of RA patients is associated with the tendency of
212
L. M. Brennan-Bourdon et al.
physicians to prescribe oral steroids for patients who show more inflammation.
Immunopharmacology and Immunotoxicology Downloaded from informahealthcare.com by UMEA University Library on 04/01/15 For personal use only.
Conclusion In conclusion, sDMARDs continue to be a valid treatment option for RA patients. This study supports the association that sDMARDs have with TNFa and steroids with MIF. Notably, in this study, we found that sDMARDs do not modify serum MIF levels. However, further longitudinal studies are necessary in order to confirm the efficacy of these therapeutic regimens as well as with biological DMARDs and their effect on TNFa and MIF serum levels in order to have a clearer view of MIF’s role in current RA treatment.
Immunopharmacol Immunotoxicol, 2015; 37(2): 207–213
16. 17. 18.
19. 20. 21.
Declaration of interest The authors report that they have no conflicts of interest. This study was supported by Grant no. 180663 (J. F. M. V.) from the Consejo Nacional de Ciencia y Tecnologı´a (CONACYT; Ciencia Ba´sica-Universidad de Guadalajara).
22.
23.
References 1. Smith HS, Smith AR, Seidner P. Painful rheumatoid arthritis. Pain Physician 2011;14:E427–E458. 2. Goronzy JJ, Weyand CM. Rheumatoid arthritis. Immunol Rev 2005;204:55–73. 3. Brennan FM, McInnes IB. Evidence that cytokines play a role in rheumatoid arthritis. J Clin Invest 2008;118:3537–3545. 4. Morand EF, Leech M. Macrophage migration inhibitory factor in rheumatoid arthritis. Front Biosci 2005;10:12–22. 5. Calandra T, Bucala R. Macrophage migration inhibitory factor (MIF): a glucocorticoid counter-regulator within the immune system. Crit Rev Immunol 1997;17:77–88. 6. Cheng Q, McKeown SJ, Santos L, et al. Macrophage migration inhibitory factor increases leukocyte–endothelial interactions in human endothelial cells via promotion of expression of adhesion molecules. J Immunol 2010;185:1238–1247. 7. Onodera S, Kaneda K, Mizue Y, et al. Macrophage migration inhibitory factor up-regulates expression of matrix metalloproteinases in synovial fibroblasts of rheumatoid arthritis. J Biol Chem 2000;275:444–450. 8. Leech M, Lacey D, Xue JR, et al. Regulation of p53 by macrophage migration inhibitory factor in inflammatory arthritis. Arthritis Rheum 2003;48:1881–1889. 9. Maillefert JF, Puechal X, Falgarone G, et al. Prediction of response to disease modifying antirheumatic drugs in rheumatoid arthritis. Joint Bone Spine 2010;77:558–563. 10. Radstake TR, Sweep FC, Welsing P, et al. Correlation of rheumatoid arthritis severity with the genetic functional variants and circulating levels of macrophage migration inhibitory factor. Arthritis Rheum 2005;52:3020–3029. 11. Leech M, Metz C, Hall P, et al. Macrophage migration inhibitory factor in rheumatoid arthritis: evidence of proinflammatory function and regulation by glucocorticoids. Arthritis Rheum 1999;42: 1601–1608. 12. Sivalingam SP, Thumboo J, Vasoo S, et al. In vivo pro-and antiinflammatory cytokines in normal and patients with rheumatoid arthritis. Ann Acad Med Singapore 2007;36:96–99. 13. Oregon-Romero E, Vazquez-Del Mercado M, Ruiz-Quezada SL, et al. Tumor necrosis factor alpha-308 and -238 polymorphisms in rheumatoid arthritis. Association with messenger RNA expression and sTNF-alpha. J Investig Med 2008;56:937–943. 14. Manicourt DH, Triki R, Fukuda K, et al. Levels of circulating tumor necrosis factor alpha and interleukin-6 in patients with rheumatoid arthritis. Relationship to serum levels of hyaluronan and antigenic keratan sulfate. Arthritis Rheum 1993;36:490–499. 15. Klimiuk PA, Sierakowski S, Latosiewicz R, et al. Circulating tumour necrosis factor alpha and soluble tumour necrosis factor
24.
25.
26.
27.
28. 29.
30.
31. 32. 33.
34. 35.
receptors in patients with different patterns of rheumatoid synovitis. Ann Rheum Dis 2003;62:472–475. Calandra T, Bernhagen J, Mitchell RA, Bucala R. The macrophage is an important and previously unrecognized source of macrophage migration inhibitory factor. J Exp Med 1994;179:1895–1902. Hoi AY, Iskander MN, Morand EF. Macrophage migration inhibitory factor: a therapeutic target across inflammatory diseases. Inflamm Allergy Drug Targets 2007;6:183–190. Aletaha D, Neogi T, Silman AJ, et al. 2010 rheumatoid arthritis classification criteria: an American College of Rheumatology/ European League Against Rheumatism collaborative initiative. Ann Rheum Dis 2010;69:1580–1588. Upchurch KS, Kay J. Evolution of treatment for rheumatoid arthritis. Rheumatology (Oxford) 2012;51:vi28–vi36. Aletaha D, Smolen JS. The rheumatoid arthritis patient in the clinic: comparing more than 1,300 consecutive DMARD courses. Rheumatology (Oxford) 2002;41:1367–1374. Maetzel A, Wong A, Strand V, et al. Meta-analysis of treatment termination rates among rheumatoid arthritis patients receiving disease-modifying anti-rheumatic drugs. Rheumatology (Oxford) 2000;39:975–981. Kinder AJ, Hassell AB, Brand J, et al. The treatment of inflammatory arthritis with methotrexate in clinical practice: treatment duration and incidence of adverse drug reactions. Rheumatology (Oxford) 2005;44:61–66. Wijbrandts CA, van Leuven SI, Boom HD, et al. Sustained changes in lipid profile and macrophage migration inhibitory factor levels after anti-tumour necrosis factor therapy in rheumatoid arthritis. Ann Rheum Dis 2009;68:1316–1321. Kim HR, Park MK, Cho ML, et al. Macrophage migration inhibitory factor upregulates angiogenic factors and correlates with clinical measures in rheumatoid arthritis. J Rheumatol 2007; 34:927–936. de Jong PH, Hazes JM, Barendregt PJ, et al. Induction therapy with a combination of DMARDs is better than methotrexate monotherapy: first results of the tREACH trial. Ann Rheum Dis 2013;72: 72–78. Gubar EE, Bochkova AG, Bunchuk NV. Comparison of efficacy and tolerability of triple combination therapy (methotrexate + sulfasalazine + hydroxychloroquine) with methotrexate monotherapy in patients with rheumatoid arthritis. Ter Arkh 2008;80: 25–30. O’Dell JR, Haire CE, Erikson N, et al. Treatment of rheumatoid arthritis with methotrexate alone, sulfasalazine and hydroxychloroquine, or a combination of all three medications. N Engl J Med 1996;334:1287–1291. O’Dell JR, Mikuls TR, Taylor TH, et al. Therapies for active rheumatoid arthritis after methotrexate failure. N Engl J Med 2013; 369:307–318. Smith MD, Slavotinek J, Au V, et al. Successful treatment of rheumatoid arthritis is associated with a reduction in synovial membrane cytokines and cell adhesion molecule expression. Rheumatology (Oxford) 2001;40:965–977. Jorgensen KT, Wiik A, Pedersen M, et al. Cytokines, autoantibodies and viral antibodies in premorbid and postdiagnostic sera from patients with rheumatoid arthritis: case–control study nested in a cohort of Norwegian blood donors. Ann Rheum Dis 2008;67: 860–866. Wolf J, Stranzl T, Filipits M, et al. Expression of resistance markers to methotrexate predicts clinical improvement in patients with rheumatoid arthritis. Ann Rheum Dis 2005;64:564–568. Weinblatt ME, Kaplan H, Germain BF, et al. Methotrexate in rheumatoid arthritis: effects on disease activity in a multicenter prospective study. J Rheumatol 1991;18:334–338. Proudman SM, Keen HI, Stamp LK, et al. Response-driven combination therapy with conventional disease-modifying antirheumatic drugs can achieve high response rates in early rheumatoid arthritis with minimal glucocorticoid and nonsteroidal anti-inflammatory drug use. Semin Arthritis Rheum 2007;37: 99–111. Fleck A. Clinical and nutritional aspects of changes in acute-phase proteins during inflammation. Proc Nutr Soc 1989;48:347–354. Swaak AJ, van Rooyen A, Nieuwenhuis E, Aarden LA. Interleukin6 (IL-6) in synovial fluid and serum of patients with rheumatic diseases. Scan J Rheumatol 1988;17:469–474.
MIF and TNF serum levels in RA patients
DOI: 10.3109/08923973.2015.1017645
Immunopharmacology and Immunotoxicology Downloaded from informahealthcare.com by UMEA University Library on 04/01/15 For personal use only.
36. Llamas-Covarrubias MA, Valle Y, Navarro-Hernandez RE, et al. Serum levels of macrophage migration inhibitory factor are associated with rheumatoid arthritis course. Rheumatol Int 2012; 32:2307–2311. 37. Santos LL, Morand EF. The role of macrophage migration inhibitory factor in the inflammatory immune response and rheumatoid arthritis. Wien Med Wochenschr 2006;156:11–18. 38. Donn RP, Ray DW. Macrophage migration inhibitory factor: molecular, cellular and genetic aspects of a key neuroendocrine molecule. J Endocrinol 2004;182:1–9. 39. Llamas-Covarrubias MA, Valle Y, Bucala R, et al. Macrophage migration inhibitory factor (MIF): genetic evidence for
40. 41. 42. 43.
213
participation in early onset and early stage rheumatoid arthritis. Cytokine 2013;61:759–765. Calandra T, Bernhagen J, Metz CN, et al. MIF as a glucocorticoidinduced modulator of cytokine production. Nature 1995;377:68–71. Morand EF, Bucala R, Leech M. Macrophage migration inhibitory factor: an emerging therapeutic target in rheumatoid arthritis. Arthritis Rheum 2003;48:291–299. Barnes PJ. Corticosteroids: the drugs to beat. Eur J Pharmacol 2006;533:2–14. Blanco-Morales EA, Bravo-Ferrer Acosta JM, Rubio Romero E, et al. Glucocorticoids in rheumatoid arthritis: almost always or hardly ever? Reumatol Clin 2011;7:407–411.
RESEARCH ARTICLE
Immunopharmacology and Immunotoxicology Downloaded from informahealthcare.com by UMEA University Library on 04/01/15 For personal use only.
MIF and TNFa serum levels in rheumatoid arthritis patients treated with disease-modifying antirheumatic drugs: a cross-sectional study Lorena Michele Brennan-Bourdon1,2, Ulises De la Cruz-Mosso2, Zyanya Reyes-Castillo2, Gloria Esther Martı´nez-Bonilla3, Marı´a Guadalupe Ramı´rez-Duen˜as1, Marı´a Cristina Islas-Carbajal1, Ana Rosa Rinco´n-Sa´nchez1, Mario Salazar-Pa´ramo1, and Jose´ Francisco Mun˜oz-Valle2 1
Departamento de Fisiologı´a, Centro Universitario de Ciencias de la Salud, Universidad de Guadalajara, Guadalajara, Jalisco, Me´xico, 2Instituto de Investigacio´n en Ciencias Biome´dicas, Centro Universitario de Ciencias de la Salud, Universidad de Guadalajara, Guadalajara, Jalisco, Me´xico, and 3 Servicio de Reumatologı´a, O.P.D. Hospital Civil de Guadalajara ‘‘Fray Antonio Alcalde’’, Guadalajara, Jalisco, Me´xico Abstract
Keywords
Macrophage migration inhibitory factor (MIF) and tumor necrosis factor alpha (TNFa) play a pivotal role in rheumatoid arthritis (RA). MIF is considered a relevant cytokine because it appears before TNFa in the inflammatory cascade thus stimulating TNFa production and MIF’s relationship with traditional synthetic disease modifying antirheumatic drugs (sDMARDs) is unknown. In this cross-sectional study, we investigated the association of MIF and TNFa serum levels with methotrexate (MTX) and in combination with chloroquine (CLQ) and sulfasalazine (SSZ) in RA patients classified according to the ACR/EULAR 2010 criteria. Patients were divided into three groups: MTX-monotherapy group (n ¼ 40), MTX combination therapy groups: MTX + CLQ (n ¼ 41), and MTX + CLQ + SSZ (n ¼ 42). MIF and TNFa serum levels were determined by ELISA. We found high levels of ESR, CRP, RF, and anti-CCP in all therapy groups. Furthermore, we subclassified 97 patients with established RA (2 years of disease duration) and found that TNFa serum levels were lower in the combination therapy group (MTX + CLQ + SSZ) in comparison with the monotherapy MTX group (16.7 pg/mL versus 13.6 pg/mL, p ¼ 0.02). However, we did not find differences between sDMARD therapies in MIF serum levels. We did find a significant reduction in MIF serum levels in patients treated with oral steroids compared with patients without oral steroids (1.7 ng/mL versus 4.3 ng/mL, p50.001). In conclusion, this study supports the role of sDMARDs in modifying TNFa serum levels and oral steroids MIF serum levels. Nevertheless, we found that MIF serum levels are not modified by sDMARD treatment.
Association, macrophage migration inhibitory factor, rheumatoid arthritis, synthetic disease-modifying antirheumatic drugs, tumor necrosis factor alpha
Introduction Rheumatoid arthritis (RA) is an autoimmune disease of unknown etiology that affects primarily the synovial joints. The evolution of the disease is characterized by pain as well as by swollen peripheral joints which affect movement and can cause deformity1. Among the risk factors for RA are female gender, advanced age, and genetic factors such as a positive family history2. Furthermore, RA is an immunemediated disease distinguished by a sustained inflammation process in the diarthrodial joints where cytokines play a key role3. Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine that has been recently linked to the pathogenesis of RA4. MIF is considered a unique pleiotropic cytokine since it is the only cytokine that counter-regulates the inhibitory effects of glucocorticoids5. It also stimulates the
Address for correspondence: Jose´ Francisco Mun˜oz Valle, Insurgentes 244-1, Colonia Lomas de Atemajac, Zapopan, Jalisco CP 45178, Me´xico. Tel: +52 3310585309. E-mail: [email protected]
History Received 2 September 2014 Revised 7 January 2015 Accepted 4 February 2015 Published online 27 February 2015
expression of adhesion molecules and chemokines favoring leukocyte trafficking to the inflamed tissue6, regulates the expression of matrix metalloproteinases associated with cartilage damage7 as well as suppresses the expression of the tumor immunosuppressant protein p53 in fibroblast-like synoviocytes (FLS)8. These characteristics support MIF’s role in the innate and adaptive immune responses that are present in RA. On the contrary, TNFa, an important pro-inflammatory cytokine known to have a primary role in RA, has been proposed as a possible biomarker that could be used to predict DMARD response at the individual level with conventional or synthetic DMARD (sDMARD) therapy and is to date a therapeutic target which has proven successful in the treatment of RA9. Both MIF and TNFa are important elements of the inflammatory cascade that develops in RA. Several studies have reported elevated synovial and serum levels of MIF10,11 as well as TNFa serum levels in RA patients12–15. However, MIF has been reported as an upstream regulator of TNFa since it stimulates TNFa production by macrophages and
208
L. M. Brennan-Bourdon et al.
conversely TNFa is able to augment MIF production in a reciprocal augmenting pro-inflammatory responses loop16. Thus, MIF is implicated in a vast range of regulatory actions in the immune response in RA and has been proposed as a therapeutic target17, but its role with current treatment strategies with traditional sDMARD therapy has not been thoroughly investigated. Therefore, the aim of this crosssectional study was to investigate MIF and TNFa’s relationship with sDMARDs in RA patients.
Immunopharmacology and Immunotoxicology Downloaded from informahealthcare.com by UMEA University Library on 04/01/15 For personal use only.
Methods Patients This was a cross-sectional study, 123 RA patients diagnosed according to the ACR-EULAR 2010 classification criteria18 were randomly enrolled from the Rheumatology Departments: Hospital Civil of Guadalajara ‘‘Fray Antonio Alcalde’’ and Regional Hospital ‘‘Dr. Valentin Go´mez Farias’’ ISSSTE (Instituto de Seguridad y Servicios Sociales de los Trabajadores del Estado). The RA inclusion criteria were as follows: age 418 years and patients were selected independently of disease duration. According to the appointed rheumatologist’s prescription, all patients received sDMARD treatment with MTX or in combination with CLQ and/or SSZ for at least 3 months as well as the concomitant use of low-dose prednisone and non-steroidal anti-inflammatory drugs for several patients. RA patients were subclassified according to the following treatment subgroups: the first group was composed of 40 patients with MTX monotherapy who received doses of 5.0– 20 mg/week, the second combination therapy group (n ¼ 41) with MTX + CLQ received (5.0–15 mg/week of MTX and 150 mg/d of CLQ) and the third combination therapy group (n ¼ 42) with MTX + CLQ + SSZ (5.0–20 mg/week of MTX, 150–300 mg/d of CLQ, and 0.5–3.0 g/d of SSZ). Nonsteroidal anti-inflammatory drugs (NSAIDs) included diclofenac, piroxicam, meloxicam, paracetamol, indomethacin, celecoxib, sulindac, and ketoprofen. Low-dose prednisone was the steroidal anti-inflammatory drug (SAID) of choice by the appointed rheumatologists, with doses of 510 mg of prednisone daily. Ethical guidelines In accordance to the 2008 Declaration of Helsinki ethical guidelines, all patients gave their written informed consent prior enrollment in the study. Approval for this study was obtained from the Institutional ethics review committee of the Centro Universitario de Ciencias de la Salud de la Universidad de Guadalajara (registration no. C-11908). Clinical assessments Clinical assessments and disease activity (DAS28) evaluations were performed by the appointed rheumatologists and blood samples were taken at the time of inclusion to determine with standardized methods the erythrocyte sedimentation rate (ESR) (the Wintrobe method), C-reactive protein (CRP), and rheumatoid factor (RF); both CRP and RF assays were performed by nephelometry with BiosystemsÕ reagents (Beckman Coulter, Inc., Fullerton, CA) and serum
Immunopharmacol Immunotoxicol, 2015; 37(2): 207–213
anti-Cyclic Citrullinated Peptide antibodies (anti-CCP) by a commercial enzyme-linked immunoassay (DIASTATÔ, Diastat, England, UK), all according to manufacturer’s instructions. Quantification of MIF and TNFa serum levels MIF and TNFa serum levels from all RA patients were determined with commercial quantitative sandwich-enzyme immunoassays (human MIF, RayBiotechÕ , Norcross, GA; human TNFa InvitrogenÔ, Waltham, MA) according to manufacturer’s instructions. The MIF assay sensitivity was 6 pg/mL, while for TNFa it was 1.7 pg/mL. Statistical analysis Statistical analysis for this study was performed with GraphPad Prism v 5.0 (GraphPad Software Inc., San Diego, CA) and SPSS v17.0 statistical programs (SPSS Inc., Chicago, IL). For the descriptive analysis, nominal variables were expressed as frequencies and continuous variables with non-parametric distribution were expressed as medians and percentiles 5–95. To determine non-parametric comparisons within therapy groups, we used the Kruskal–Wallis and Mann–Whitney U tests. For the correlation analysis of continuous variables with non-parametric distribution, we used the Spearman correlation test and for those with parametric distribution, Pearson correlation test. A stepwise forward multivariable linear regression analysis was used to identify possible predictors of change in MIF and TNFa levels. Variables included in the MIF analysis were oral steroids, disease activity, and RF and for the TNFa analysis, MTX and CLQ treatment. Probability values with a p50.05 were considered statistically significant in a two-tailed test.
Results Clinical characteristics Clinical characteristics of the 123 RA patients included in this study are shown in Table 1. The average age was 47 years, the predominant gender was female 94% and 6% male, the average disease duration was 6 years and patients had moderate disease activity scores as measured by the DAS28 index (average score 4.5), 28% of RA patients included low-dose oral steroid therapy (510 mg/d of prednisone), 85% included NSAIDs, and 71.5% received folic acid. Furthermore, 91% of patients included in this study were not active smokers at the time of the study. Considering the serum acute phase reactants (ESR and CRP) and autoantibodies (RF and anti-CCP), these were elevated in all three RA therapy groups. We did not find differences within therapy groups for these values. MIF and TNFa serum levels In the case of MIF serum levels, no significant differences were observed in all RA patients within therapy groups (MTX: 2.5 ng/mL; MTX + CLQ: 3.0 ng/mL; MTX + CLQ + SSZ: 4.1 ng/mL). In regard to TNFa therapy groups, all RA patients had increased serum levels and these were not found to be statistically different (MTX: 16.9 pg/mL;
MIF and TNF serum levels in RA patients
DOI: 10.3109/08923973.2015.1017645
209
Table 1. Demographic and clinical characteristics of rheumatoid arthritis patients.
Immunopharmacology and Immunotoxicology Downloaded from informahealthcare.com by UMEA University Library on 04/01/15 For personal use only.
Variables Demographics Age (years)a Genderb Female Male Clinical assessment Disease durationc DAS28 scorec Active smokersb No Yes ESR (mm/h)c C reactive protein (mg/dL)c Rheumatoid factor UI/mLc Anti-CCP (U/mL)c Treatmentb Prednisoneb No Yes NSAIDsb No Yes
All patients, n ¼ 123
MTX, n ¼ 40
MTX + CLQ, n ¼ 41
MTX + CLQ + SSZ, n ¼ 42
47 ± 14
53 ± 13
50 ± 14
39 ± 12
116 (94%) 7 (6%)
38 (95%) 2 (5%)
36 (88%) 5 (12%)
42 (100%) 0 (0)
6 (0.50–23.30) 4.5 (2.4–6.7)
8 (0.56–22.60) 4.2 (2.4–6.80)
8 (0.50–30.80) 4.3 (2.4–6.6)
4.5 (0.34–19.85) 4.5 (2.4–7.2)
112 11 37 8.6 194 109.5
(91%) (9%) (11.0–55.0) (1.0–144.0) (12.2–704.6) (1.6–496.6)
35 5 32 7.6 88 108.4
(88%) (12%) (15.7–55.6) (1.4–152.0) (7.2–711.5) (1.4–602.9)
37 4 37 5.0 208 79.3
(90%) (10%) (12.2–53.2) (0.3–212.0) (13.6–589.8) (1.6–509.4)
40 2 41 11.0 204.5 132.6
(95%) (5%) (7.00–60.9) (0.8–118.9) (13.9–714.6) (1.6–463.6)
88 (72%) 35 (28%)
29 (73%) 11 (27%)
28 (68%) 13 (32%)
31 (74%) 11 (26%)
18 (15%) 105 (85%)
3 (7%) 37 (93%)
5 (12%) 36 (88%)
10 (24%) 32 (76%)
DMARDs, disease modifying anti-rheumatic drugs; NSAIDs, non-steroidal anti-inflammatory drugs; ESR, erythrocyte sedimentation rate; DAS28, disease activity score. RA subgroups: MTX, methotrexate; MTX + CLQ, chloroquine; MTX + CLQ + SSZ, sulphasalazine. a Data are expressed as means ± SD. b n and percentages. c Medians and percentiles p5–p95.
Figure 1. MIF and TNFa serum levels in sDMARD treatment groups in established RA (2 years of disease duration). (A) MIF serum levels, p ¼ 0.13. **Kruskal–Wallis test. (B) TNFa serum levels. No significant differences were found within sDMARD therapies in the established RA group for MIF serum levels, whereas for TNFa serum levels for the established RA group, a significant decrease was found in the MTX + CLQ + SSZ group compared with the MTX group, p ¼ 0.02. *Mann–Whitney U test.
MTX + CLQ: 15.3 pg/mL; MTX + CLQ + SSZ: 14.6 pg/mL). In addition, when RA patients were classified in early RA patients (52 years of disease duration), we did not find significant differences within therapy groups for MIF and TNFa serum levels in individuals with early RA (data not shown). Similarly, in established RA patients (2 years of disease duration), regarding MIF serum levels, we did not find significant differences between therapy groups (MTX: 2.8 ng/mL, MTX + CLQ: 2.8 ng/mL; MTX + CLQ + SSZ: 7.9 ng/mL; Figure 1A). However, in the case of TNFa in established RA patients, we did find a significant difference
between the MTX and the MTX + CLQ + SSZ therapy groups (16.7 pg/mL versus 13.6 pg/mL, p ¼ 0.02; Figure 1B). Relationship between MIF and TNFa serum levels with clinical findings To investigate the relationship of MIF serum levels with clinical markers of the disease, we found a negative correlation of MIF with CRP (r ¼ 0.326, p ¼ 0.001; Figure 2A) and a positive correlation of TNFa with CRP (r ¼ 0.376, p ¼ 0.001; Figure 2B) in the Spearman correlation test.
Immunopharmacology and Immunotoxicology Downloaded from informahealthcare.com by UMEA University Library on 04/01/15 For personal use only.
210
L. M. Brennan-Bourdon et al.
Immunopharmacol Immunotoxicol, 2015; 37(2): 207–213
Figure 2. TNFa and MIF serum levels correlation with CRP. (A) A negative correlation was found in MIF and CRP levels of RA patients. (B) A positive correlation between TNFa and CRP in RA patients was identified; p values were obtained by Spearman’s correlation test.
Figure 3. TNFa and MIF serum levels with and without oral steroid treatment. (A) Significantly diminished serum levels were found in MIF serum levels (p50.001). (B) TNFa serum levels were higher in those patients receiving oral steroids (p ¼ 0.01). *Mann–Whitney U test.
However, MIF and TNFa serum levels did not associate with the DAS28 score, CRP, ESR, or anti-CCP (data not shown). Oral steroid treatment in MIF and TNFa serum levels RA patients with oral steroids were compared with patients without oral steroids. On one hand, MIF serum levels were significantly lower in the oral steroid group compared with those patients who were not receiving steroids (1.7 ng/mL versus 4.3 ng/mL; p50.001; Figure 3A). On the other hand, TNFa soluble levels in this RA group were significantly higher in the oral steroid group compared with those patients who were not receiving steroids (17.7 pg/mL versus 14.5 pg/mL; p ¼ 0.01; Figure 3B). Oral steroids predict changes in MIF and sDMARDs in TNFa serum levels With a stepwise forward multivariable linear regression analysis, we identified significant changes in MIF and TNFa
serum levels according to oral steroids and sDMARDs. Oral steroid treatment contributed to a significant decrease of MIF serum levels ( ¼ 0.362; CI ¼ 5.624 to 1.894; p ¼ 0.001) whereas sDMARD treatment did not contribute to a significant decrease of MIF (data not shown). However, in the case of TNFa serum levels, sDMARDs methotrexate ( ¼ 0.202; CI ¼ 2.266 to 0.126; p ¼ 0.02) and chloroquine ( ¼ 0.190; CI ¼ 0.128 to 0.003; p ¼ 0.03) contributed to a significant decrease of TNFa serum levels.
Discussion Long-term pharmacological treatment in RA with sDMARDs, considered the cornerstone of RA therapy, is fundamental in order to reduce joint destruction and eventually minimize functional loss since aiming to achieve remission or low disease activity is the desired treatment goal19. In a 2002 study, Aletaha and Smolen report the gold standard methotrexate as the most prescribed sDMARDs in RA, as well as
Immunopharmacology and Immunotoxicology Downloaded from informahealthcare.com by UMEA University Library on 04/01/15 For personal use only.
DOI: 10.3109/08923973.2015.1017645
chloroquine and sulphasalazine, where a high retention rate for MTX has also been shown20–22. Herein, we show the treatment regimens included in this study are in accordance with published reports and are considered a valid treatment strategy in RA. Cytokines are important therapeutic targets for RA treatment. MIF and TNFa are produced by macrophages, T cells, dendritic cells, and fibroblast-like synoviocytes and a feedback loop has been reported between these two cytokines where TNFa induces the production of MIF and inversely MIF induces the production of TNFa16,23. The relevance of these cytokines in the pathogenic mechanisms of RA is what led us to propose MIF’s relationship with traditional sDMARD treatment, considering sDMARDs as the most common disease-modifying drugs used to date. According to MIF’s relationship with sDMARD therapy, to our knowledge a study by Kim et al., determined MIF’s relationship with clinical measures in 72 patients with RA, and found that the numbers, duration, and kind of disease antirheumatic drug therapy do not associate with MIF serum and synovial fluid levels. However, the specific sDMARD therapy was not described24. Nevertheless, this is the first study that reports MIF serum levels are not modified by sDMARD monotherapy (MTX) and combination therapies (MTX + CLQ and MTX + CLQ + SSZ), which also supports this finding. Previous reports have demonstrated a preference in the prescription of combination regimens with sDMARDs compared with monotherapy primarily due to improved efficacy25–27. More recently, another important study demonstrated that triple therapy is equivalent to the combination of MTX with the biologic DMARD etanercept in patients failing initial MTX monotherapy28. Considering the limitation of our cross-sectional study, we were not able to determine the efficacy of therapy regimens. We found that TNFa levels were significantly lower in the established RA combination therapy group with all three sDMARDs (MTX, CLQ, and SSZ) compared with the monotherapy MTX group. However, these results should be interpreted with caution, since TNFa serum levels in our study were found to be modified by the MTX and CLQ doses. These results may partly reflect TNFa’s relationship with sDMARDs because they have been reported to decrease the production of anti-inflammatory and pro-inflammatory cytokines such as IL-10 and TNFa levels in the synovial membrane in response to anti-rheumatic drug treatment29. However, it is also reasonable to predict that the prescribed medications might not have been active enough and the role of sDMARDs on cytokine serum levels should be considered in future longitudinal studies. In the case of early RA patients, we did not find differences in TNFa serum levels within therapy groups possibly because TNFa is upregulated later in the disease in comparison with anti-CCP, which may be present two decades before RA diagnosis30. However, we must point out that we cannot discard that among RA patients treated with methotrexate, 30–50% do not fulfill response criteria31. This distinction could explain the low number of RA patients in remission in our study group.
MIF and TNF serum levels in RA patients
211
Several studies have demonstrated important reductions in disease activity scores (DAS28) as well as in the acute phase reactants with sDMARD treatment in RA patients32,33. Nonetheless, in our study, we did not find significant differences within therapy groups in ESR, CRP, RF, antiCCP, and DAS28 scores. The discrepancy between our findings and those reported could be attributed to the crosssectional design limitation of our study as well as by the complexity of RA pathogenesis and the heterogeneity of disease manifestations such as severity, differences in disease duration, and disease activity scores, as well as by the individual treatment duration of RA patients included in this study. The positive correlation found between TNFa and CRP is due to the inflammatory process characteristic of RA patients because this cytokine and IL-6 stimulate the hepatocytes to produce acute phase reactants such as CRP34,35. A previous study in our population found a positive correlation between MIF and CRP in RA patients not receiving oral steroids36 and in contrast, other studies have not found an association between MIF serum levels and CRP24. These differences could be accredited to MIF’s response in the local tissue because it appears to be more marked than the systemic response and a positive correlation of synovial MIF and CRP has been reported37. However, the negative correlation found between MIF serum levels and CRP in our study group could be attributed to the heterogeneity in the therapy and disease duration in our selected groups. Moreover, considering TNFa serum levels were overexpressed and MIF values were normal as previously reported (2–6 ng/mL)38 these results could provide further insight into TNFa’s association with disease activity and MIF with disease progression39. MIF expression can be induced by low concentrations of glucocorticoids in macrophages, T cells, and synovial fibroblasts40. However, when the characteristic inflammation process is present in RA and oral glucocorticoids are introduced to therapy, the pharmacological dosage should be superior to the endogenous glucocorticoid concentration to counter-regulate the effects of MIF41. Oral glucocorticoids in low doses are considered as disease-modifying antirheumatic drugs due to their genomic effect by inhibiting inflammatory genes, but are used in RA with caution due to their side effects42,43. In this regard, our results confirm MIF’s relationship with glucocorticoids due to the significantly lower MIF serum levels in RA patients prescribed with oral glucocorticoids. The precise mechanism of action by which MIF counter-regulates the anti-inflammatory effect of glucocorticoids is unknown. On the contrary, our results showed increased TNFa serum levels in RA patients with oral steroids in comparison with patients who did not take steroids. This result could be explained considering that the overall goal in RA treatment is aimed at controlling inflammation. However, we cannot discard that RA patients with oral steroid therapy in our study were possibly at initial treatment phases. Hence, the seemingly negative correlation between MIF and TNFa found in this study that may also reflect the moderate disease activity of RA patients is associated with the tendency of
212
L. M. Brennan-Bourdon et al.
physicians to prescribe oral steroids for patients who show more inflammation.
Immunopharmacology and Immunotoxicology Downloaded from informahealthcare.com by UMEA University Library on 04/01/15 For personal use only.
Conclusion In conclusion, sDMARDs continue to be a valid treatment option for RA patients. This study supports the association that sDMARDs have with TNFa and steroids with MIF. Notably, in this study, we found that sDMARDs do not modify serum MIF levels. However, further longitudinal studies are necessary in order to confirm the efficacy of these therapeutic regimens as well as with biological DMARDs and their effect on TNFa and MIF serum levels in order to have a clearer view of MIF’s role in current RA treatment.
Immunopharmacol Immunotoxicol, 2015; 37(2): 207–213
16. 17. 18.
19. 20. 21.
Declaration of interest The authors report that they have no conflicts of interest. This study was supported by Grant no. 180663 (J. F. M. V.) from the Consejo Nacional de Ciencia y Tecnologı´a (CONACYT; Ciencia Ba´sica-Universidad de Guadalajara).
22.
23.
References 1. Smith HS, Smith AR, Seidner P. Painful rheumatoid arthritis. Pain Physician 2011;14:E427–E458. 2. Goronzy JJ, Weyand CM. Rheumatoid arthritis. Immunol Rev 2005;204:55–73. 3. Brennan FM, McInnes IB. Evidence that cytokines play a role in rheumatoid arthritis. J Clin Invest 2008;118:3537–3545. 4. Morand EF, Leech M. Macrophage migration inhibitory factor in rheumatoid arthritis. Front Biosci 2005;10:12–22. 5. Calandra T, Bucala R. Macrophage migration inhibitory factor (MIF): a glucocorticoid counter-regulator within the immune system. Crit Rev Immunol 1997;17:77–88. 6. Cheng Q, McKeown SJ, Santos L, et al. Macrophage migration inhibitory factor increases leukocyte–endothelial interactions in human endothelial cells via promotion of expression of adhesion molecules. J Immunol 2010;185:1238–1247. 7. Onodera S, Kaneda K, Mizue Y, et al. Macrophage migration inhibitory factor up-regulates expression of matrix metalloproteinases in synovial fibroblasts of rheumatoid arthritis. J Biol Chem 2000;275:444–450. 8. Leech M, Lacey D, Xue JR, et al. Regulation of p53 by macrophage migration inhibitory factor in inflammatory arthritis. Arthritis Rheum 2003;48:1881–1889. 9. Maillefert JF, Puechal X, Falgarone G, et al. Prediction of response to disease modifying antirheumatic drugs in rheumatoid arthritis. Joint Bone Spine 2010;77:558–563. 10. Radstake TR, Sweep FC, Welsing P, et al. Correlation of rheumatoid arthritis severity with the genetic functional variants and circulating levels of macrophage migration inhibitory factor. Arthritis Rheum 2005;52:3020–3029. 11. Leech M, Metz C, Hall P, et al. Macrophage migration inhibitory factor in rheumatoid arthritis: evidence of proinflammatory function and regulation by glucocorticoids. Arthritis Rheum 1999;42: 1601–1608. 12. Sivalingam SP, Thumboo J, Vasoo S, et al. In vivo pro-and antiinflammatory cytokines in normal and patients with rheumatoid arthritis. Ann Acad Med Singapore 2007;36:96–99. 13. Oregon-Romero E, Vazquez-Del Mercado M, Ruiz-Quezada SL, et al. Tumor necrosis factor alpha-308 and -238 polymorphisms in rheumatoid arthritis. Association with messenger RNA expression and sTNF-alpha. J Investig Med 2008;56:937–943. 14. Manicourt DH, Triki R, Fukuda K, et al. Levels of circulating tumor necrosis factor alpha and interleukin-6 in patients with rheumatoid arthritis. Relationship to serum levels of hyaluronan and antigenic keratan sulfate. Arthritis Rheum 1993;36:490–499. 15. Klimiuk PA, Sierakowski S, Latosiewicz R, et al. Circulating tumour necrosis factor alpha and soluble tumour necrosis factor
24.
25.
26.
27.
28. 29.
30.
31. 32. 33.
34. 35.
receptors in patients with different patterns of rheumatoid synovitis. Ann Rheum Dis 2003;62:472–475. Calandra T, Bernhagen J, Mitchell RA, Bucala R. The macrophage is an important and previously unrecognized source of macrophage migration inhibitory factor. J Exp Med 1994;179:1895–1902. Hoi AY, Iskander MN, Morand EF. Macrophage migration inhibitory factor: a therapeutic target across inflammatory diseases. Inflamm Allergy Drug Targets 2007;6:183–190. Aletaha D, Neogi T, Silman AJ, et al. 2010 rheumatoid arthritis classification criteria: an American College of Rheumatology/ European League Against Rheumatism collaborative initiative. Ann Rheum Dis 2010;69:1580–1588. Upchurch KS, Kay J. Evolution of treatment for rheumatoid arthritis. Rheumatology (Oxford) 2012;51:vi28–vi36. Aletaha D, Smolen JS. The rheumatoid arthritis patient in the clinic: comparing more than 1,300 consecutive DMARD courses. Rheumatology (Oxford) 2002;41:1367–1374. Maetzel A, Wong A, Strand V, et al. Meta-analysis of treatment termination rates among rheumatoid arthritis patients receiving disease-modifying anti-rheumatic drugs. Rheumatology (Oxford) 2000;39:975–981. Kinder AJ, Hassell AB, Brand J, et al. The treatment of inflammatory arthritis with methotrexate in clinical practice: treatment duration and incidence of adverse drug reactions. Rheumatology (Oxford) 2005;44:61–66. Wijbrandts CA, van Leuven SI, Boom HD, et al. Sustained changes in lipid profile and macrophage migration inhibitory factor levels after anti-tumour necrosis factor therapy in rheumatoid arthritis. Ann Rheum Dis 2009;68:1316–1321. Kim HR, Park MK, Cho ML, et al. Macrophage migration inhibitory factor upregulates angiogenic factors and correlates with clinical measures in rheumatoid arthritis. J Rheumatol 2007; 34:927–936. de Jong PH, Hazes JM, Barendregt PJ, et al. Induction therapy with a combination of DMARDs is better than methotrexate monotherapy: first results of the tREACH trial. Ann Rheum Dis 2013;72: 72–78. Gubar EE, Bochkova AG, Bunchuk NV. Comparison of efficacy and tolerability of triple combination therapy (methotrexate + sulfasalazine + hydroxychloroquine) with methotrexate monotherapy in patients with rheumatoid arthritis. Ter Arkh 2008;80: 25–30. O’Dell JR, Haire CE, Erikson N, et al. Treatment of rheumatoid arthritis with methotrexate alone, sulfasalazine and hydroxychloroquine, or a combination of all three medications. N Engl J Med 1996;334:1287–1291. O’Dell JR, Mikuls TR, Taylor TH, et al. Therapies for active rheumatoid arthritis after methotrexate failure. N Engl J Med 2013; 369:307–318. Smith MD, Slavotinek J, Au V, et al. Successful treatment of rheumatoid arthritis is associated with a reduction in synovial membrane cytokines and cell adhesion molecule expression. Rheumatology (Oxford) 2001;40:965–977. Jorgensen KT, Wiik A, Pedersen M, et al. Cytokines, autoantibodies and viral antibodies in premorbid and postdiagnostic sera from patients with rheumatoid arthritis: case–control study nested in a cohort of Norwegian blood donors. Ann Rheum Dis 2008;67: 860–866. Wolf J, Stranzl T, Filipits M, et al. Expression of resistance markers to methotrexate predicts clinical improvement in patients with rheumatoid arthritis. Ann Rheum Dis 2005;64:564–568. Weinblatt ME, Kaplan H, Germain BF, et al. Methotrexate in rheumatoid arthritis: effects on disease activity in a multicenter prospective study. J Rheumatol 1991;18:334–338. Proudman SM, Keen HI, Stamp LK, et al. Response-driven combination therapy with conventional disease-modifying antirheumatic drugs can achieve high response rates in early rheumatoid arthritis with minimal glucocorticoid and nonsteroidal anti-inflammatory drug use. Semin Arthritis Rheum 2007;37: 99–111. Fleck A. Clinical and nutritional aspects of changes in acute-phase proteins during inflammation. Proc Nutr Soc 1989;48:347–354. Swaak AJ, van Rooyen A, Nieuwenhuis E, Aarden LA. Interleukin6 (IL-6) in synovial fluid and serum of patients with rheumatic diseases. Scan J Rheumatol 1988;17:469–474.
MIF and TNF serum levels in RA patients
DOI: 10.3109/08923973.2015.1017645
Immunopharmacology and Immunotoxicology Downloaded from informahealthcare.com by UMEA University Library on 04/01/15 For personal use only.
36. Llamas-Covarrubias MA, Valle Y, Navarro-Hernandez RE, et al. Serum levels of macrophage migration inhibitory factor are associated with rheumatoid arthritis course. Rheumatol Int 2012; 32:2307–2311. 37. Santos LL, Morand EF. The role of macrophage migration inhibitory factor in the inflammatory immune response and rheumatoid arthritis. Wien Med Wochenschr 2006;156:11–18. 38. Donn RP, Ray DW. Macrophage migration inhibitory factor: molecular, cellular and genetic aspects of a key neuroendocrine molecule. J Endocrinol 2004;182:1–9. 39. Llamas-Covarrubias MA, Valle Y, Bucala R, et al. Macrophage migration inhibitory factor (MIF): genetic evidence for
40. 41. 42. 43.
213
participation in early onset and early stage rheumatoid arthritis. Cytokine 2013;61:759–765. Calandra T, Bernhagen J, Metz CN, et al. MIF as a glucocorticoidinduced modulator of cytokine production. Nature 1995;377:68–71. Morand EF, Bucala R, Leech M. Macrophage migration inhibitory factor: an emerging therapeutic target in rheumatoid arthritis. Arthritis Rheum 2003;48:291–299. Barnes PJ. Corticosteroids: the drugs to beat. Eur J Pharmacol 2006;533:2–14. Blanco-Morales EA, Bravo-Ferrer Acosta JM, Rubio Romero E, et al. Glucocorticoids in rheumatoid arthritis: almost always or hardly ever? Reumatol Clin 2011;7:407–411.
