Serum leptin levels in rheumatoid arthritis and relationship with disease activity.

Objectives: This study was performed to evaluate serum leptin levels in rheumatoid arthritis (RA) patients and investigate the correlation with serum tumor necrosis factor alpha (TNF-[alpha]) levels and clinical and laboratory parameters of disease activity.

Methods: Fifty patients with RA and 34 control subjects were included. Disease activity score 28 (DAS28) was calculated for each patient. Laboratory activity was assessed by examining erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP). Immunora-diometric assay was used for measuring serum leptin levels (ng/mL). Serum TNF-[alpha] levels (pg/mL) were measured by sandwich enzyme-linked immunosorbent assay method in 41 of 50 RA patients and in 24 control subjects.

Results: Age, sex and body mass index (BMI) did not show a statistically significant difference between RA and control subjects (P > 0.05). Serum leptin levels were higher in RA (P = 0.000). In RA patients, there were no correlations between serum leptin levels and disease duration, swollen and tender joint counts, DAS28, CRP, ESR, serum TNF-[alpha] levels, oral glucocorticoid and methotrexate usage (P > 0.05). There was no statistically significant serum leptin level difference between patients with high disease activity and mild and low disease activity (P = 0.892). Serum leptin levels positively correlated with BMI in both patient and control groups (P < 0.05). In both groups, mean serum leptin levels were higher in women than men.

Conclusions: Even though serum leptin levels were found to be significantly higher in RA patients than in control subjects in this study, there was no correlation between serum leptin levels and TNF-[alpha] levels, clinical and laboratory parameters of disease activity. However serum leptin levels positively correlated with BMI in both patient and control groups. In RA, circulating leptin levels do not seem to reflect disease activity.

Key Words: rheumatoid arthritis, leptin. disease activity

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Leptin is an obese (ob) gene product cytokine-like 16 kDa peptide produced mostly by white adipose tissue cells, but also endothelial cells, T-lymphocytes, bone marrow cells, spleen cells, and platelets. Initially thought to be a satiety factor that regulates body weight by inhibiting food intake and stimulating energy expenditure, leptin is a pleiotropic hormone whose multiple effects include regulation of endocrine function, reproduction, and immunity. (1-5)

Leptin can be considered a proinflammatory cytokine that belongs to the family of long-chain helical cytokines and has structural similarity with interleukin-6 (IL-6), prolactin, growth hormone, IL-12, IL-15, granulocyte colony-stimulating factor and oncostatin M. The role of leptin in the modulation of immune response and inflammation has recently become increasingly evident. The increase in leptin production that occurs during infection and inflammation strongly suggests that leptin is a part of the cytokine network which governs the inflammatory-immune response and the host defense mechanisms. Leptin plays an important role in inflammatory processes involving T-cells and has been reported to modulate T-helper cell activity in the cellular immune response. (6) It is well known that leptin has a dual role in inflammation. Leptin activates monocyte/macrophage cells and potentiates production of the proinflammatory cytokines, tumor necrosis factor alpha (TNF-[alpha]), IL-6 and directs T-cell differentiation to Th1 phenotype, expressing interferon [gamma] and IL-2 (7) but also expresses certain anti-inflammatory substances by releasing IL-1 receptor antagonist. (8) Several studies have implicated leptin in the pathogenesis of autoimmune inflammatory conditions, such as experimental autoimmune encephalomyelitis, type 1 diabetes, rheumatoid arthritis (RA), and intestinal inflammation. (2,9-11)

The role of leptin in the modulation of immune response and inflammation has recently become increasingly evident. (6) It has been suggested that leptin may influence the outcome of RA. (2) In this study, serum leptin levels and correlation with TNF-[alpha] levels and disease activity parameters in RA were studied.

Patients and Methods

Patients and Control Subjects

Fifty patients (female/male = 41/9) fulfilling American College of Rheumatology (ACR) criteria for a diagnosis of RA (12) with a mean age of 56.34 [+ or -] 12.43 years were studied. Thirty-four control subjects (28 female) with no inflammatory disease were chosen from our outpatient clinic. The mean age of the control subjects was 53.22 [+ or -] 9.19 years. Age, sex, body mass index (BMI) (kg/[m.sup.2]), disease duration, concomitant medications, swollen and tender joint counts of the RA patients were recorded. C-reactive protein (CRP), erythrocyte sedimentation rate (ESR) and DAS28 (13) were used to assess disease activity. DAS28 is a validated index of RA disease activity. It consists of four measures: 28 tender (TJC28) and swollen joint counts (SJC28), ESR, and the patient's general health (GH) measured on a 100 mm visual analog scale. It is calculated using the formula: 0.56x[square root of (TJC28)] + 0.28[square root of (SJC28)] + 0.70xIn(ESR) + 0.014x(GH). Patients were grouped according to DAS28 scores as having high (DAS28 > 5.1) or mild to moderate (DAS28 < 5.1) disease activity. No patients met remission criteria. (14)

Forty-two patients were receiving disease modifying antirheumatic drugs (DMARDs) at the time of blood sampling. Thirty-two patients were receiving methotrexate (MTX) alone or in combination with other DMARDs (1 chloroquine, 6 sulfasalazine, 1 both chloroquine and sulfasalazine and 3 TNF inhibitors). Eight patients were not receiving any DMARDs. All patients were on nonsteroidal anti-inflammatory drugs. Twenty-one patients were receiving oral glucocorticoids (2.5-10 mg/d), of whom two were not receiving any DMARDs. Approval was obtained from the ethical committee of Izmir Training and Research Hospital.

Laboratory Parameters

Serum levels of CRP were measured by standard nephelometry, with an established normal range of 0 to 0.8 mg/dL. The ESR was measured by the Westergren method, with a normal range of 0 to 20 mm/h. Serum TNF-[alpha] levels were measured in 41 RA patients and in 24 control subjects. Sandwich enzyme-linked immunosorbent assay (ELISA) method was used for serum TNF-[alpha] level (pg/mL) measurement. The presence of rheumatoid factor in any of the immunoglobulin isotypes was considered positive.

Leptin Levels

Blood sampling. Specimens were collected using standard venipuncture technique after 12 to 16 hours fasting between 9:00 to 10:00 AM and allowed to clot for 30 minutes. Then, all samples were separated by centrifugation for 15 minutes at 1500 g and stored at -20[degrees]C until analysis for leptin determinations.

Immunoradiometric assay (IRMA) for quantitative measurement of leptin. The procedure (Diagnostic Systems Laboratories, Inc., Webster, TX, USA) employs a two-site IRMA principle described by Miles et al. (15) The IRMA is a noncompetitive assay in which the analyte to be measured is 'sandwiched' between two antibodies. The first antibody is immobilized inside the walls of the tubes. The other antibody is radiolabeled for detection. The analyte present in the samples, standards and controls is bound by both antibodies to form a 'sandwich' complex. Unbound reagents are removed by decanting and washing the tubes. An overnight assay procedure was used. All tubes were counted in a gamma counter for one minute.

Intra-assay coefficients of variations are 4.9% for 13.50 [+ or -] 0.67 ng/mL and 2.6% for 73.60 [+ or -] 1.94 ng/mL. Interassay coefficients of variations are 5.3% for 14.35 [+ or -] 0.76 ng/mL and 3.7% for 73.87 [+ or -] 2.71 ng/mL. Leptin concentrations of control serum were also studied concomitantly with unknowns and [+ or -] 2SD was found as 2.90 ng/mL and 16.15 ng/mL at the level of 2.5 [+ or -] 0.8 ng/mL and 15.1 [+ or -] 4.0 ng/mL respectively.

TNF-[alpha] Levels

Principle of the assay. Samples and standards are incubated in the microtiter plate coated with the first monoclonal antibody anti-TNF-[alpha], in the presence of the second anti-TNF-[alpha] monoclonal antibody linked to alkaline phosphatase. After incubation, the wells are washed and the bound enzymatic activity is detected by addition of a chromogenic substrate. Absorbance readings were carried out on a microtiter plate reader (450 nm filter) (TKA-I HD Technolabo A.S.S.I, Milano, Italy).

Commercial kit: Cellular Communication Investigations TNF[alpha] ELISA, Immunotech, Beckman Coulter, France. Catalog number: IM1121 -IM11121. Standard concentrations: 1000-250-62.5 to 15.6 to 0 pg/mL, Intra-assay coefficients of variation: (n = 10) 1.6%, Interassay coefficients of variation: (n = 10) 5.4%.

Statistical Analyses

Serum leptin and TNF-[alpha] levels were compared using independent samples t test between RA and control groups. Patients were grouped according to sex, DAS28 scores, methotrexate and glucocorticoid usage. Serum leptin levels were compared between patient groups by using independent samples t test. The correlation between leptin levels and disease duration, BMI. swollen and tender joint counts, DAS28, ESR, CRP and TNF-[alpha] levels were analyzed by Pearson correlation analyses. The comparison of leptin concentrations between the patient and control groups were performed after adjustment for BMI using analyses of covariance. To compare leptin concentrations (adjusted for BMI) between patient groups (with and without glucocorticoid usage) and control subjects, Bonferroni test was used. Leptin concentration comparison was also made between patient groups (DAS28 < 5.1 v >5.1) after adjustment for glucocorticoid usage by using two-way variance analyses. All analyses were performed using the Statistical Package for the Social Sciences for Windows 13.0. For all evaluations, values of P < 0.05 were considered significant.

Results

The clinical and demographic characteristics of patients and control subjects are shown in Table 1. The mean serum leptin level in the patient group was higher than the control group (53.17[+ or -]37.70 ng/mL v 23.03[+ or -]17.40 ng/mL respectively) (P = 0.000) (Fig. 1). This comparison was also made between these groups after adjustment for BMI and a similar result was obtained (P = 0.000). TNF-[alpha] levels were also higher in RA patients (37.16[+ or -]25.76 pg/mL v 25.27[+ or -]11.52 pg/mL, P = 0.013) (Fig. 2). Serum leptin levels did not show correlation with disease duration (r = -0.178, P = 0.217), ESR (r = -0.266, P = 0.062), CRP (r = -0.044, P = 0.760), number of swollen joints (r = -0.046, P = 0.749), number of tender joints (r = 0.072. P = 0.619). DAS28 (r = -0.111, P = 0.442) or serum TNF-[alpha] levels (r = 0.002, P = 0.989). Both in RA patients and controls, there was a correlation between leptin levels and BMI (r = 0.410 P = 0.003, r = 0.486 P = 0.004). In both groups, the mean serum leptin levels were higher in women (62.46[+ or -]35.20, 25.55[+ or -]18.07) than men (10.85[+ or -]7.46, 11.27[+ or -]5.80) (P = 0.000, P = 0.002).

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

Serum leptin levels did not differ between patients with high (DAS28 > 5.1) or mild to moderate (DAS28 < 5.1) disease activity (P = 0.892) (Table 2). Patients were grouped according to medications being used at the time of blood sampling to assess their influence on leptin levels. There was no difference in leptin levels in patients receiving MTX or not (P = 0.172). Patients receiving glucocorticoids seem to have higher leptin levels, but this difference did not reach statistical significance (P = 0.097) (Table 3). The leptin concentrations (adjusting for BMI) between patients (with and without glucocorticoid usage) and control subjects were statistically different (respectively P = 0.000, 0.013). After adjustment for glucocorticoid usage, leptin concentrations between patient groups (DAS28 < 5.1 and DAS28 > 5.1) were compared. There was no statistically significant difference (P = 0.356).

Discussion

In this study, serum leptin and TNF-[alpha] levels were statistically higher than age-, sex-, and BMI-matched control subjects but showed no correlation with disease activity parameters in RA patients. After adjustment for BMI, serum leptin levels remained higher in the patient group. Leptin levels were correlated with BMI and were found to be higher in women than men in both RA patients and controls. The role of leptin in human rheumatic diseases became the subject of various studies. In RA, serum leptin levels were reported to be similar, (16,17) lower, (18) and higher (2,19,20) compared with healthy subjects in different studies. In these studies, leptin levels were not found to correlate with disease activity parameters. Serum leptin levels were higher, but did not correlate with disease activity parameters in systemic lupus erythematosus (SLE) in a study performed by Garcia-Gonzalez et al (21) Based on these findings in a review article, Palmer and Gabay (3) stated that leptin can not be used to evaluate disease activity in RA and SLE patients.

Leptin likely plays a major role in the pathogenesis of rheumatoid arthritis. In patients with rheumatoid arthritis, it was reported that fasting led to an improvement of different clinical and biologic measures of disease activity, which was associated with a marked decrease in serum leptin and a shift toward Th2 cytokine production. (22) These features, resembling those previously depicted in ob/ob mice, suggest that leptin may also influence the inflammatory mechanisms of arthritis in humans through the induction of Th1 responses. (6) Bokarewa et al (2) reported that in RA patients, synovial fluid leptin levels were lower than serum levels. Because synovial fluid leptin levels were found to be lower in nonerosive disease, they suggested that consumption of intra-articular leptin may be protective against the destructive process in RA.

To date, no study has investigated the relationship between serum leptin and TNF-[alpha] levels in RA patients. In several studies it was demonstrated that proinflammatory cytokines such as TNF-[alpha] and IL 1[beta] stimulate leptin release from adipose tissue. (23,24) Some authors (25,26) demonstrated that TNF-[alpha] exerts dual effects on leptin synthesis and release by adipose tissue. While TNF-[alpha] 4 to 8 hour treatment increased leptin release fourfold, this effect was lost at 24 hours and leptin accumulation in culture medium decreased after 24 to 48 hours. Thus, leptin may be an acute phase protein of fat tissue which supports the immune system during a short-term infectious disease. In our study, we did not observe a correlation between serum leptin and TNF-[alpha] levels in RA patients. This result is probably due to the fact that some patients experience an acute flare during serum sampling. In a recent study performed in RA patients, even though leptin concentrations were similar to healthy controls, there was an inverse correlation between the severity of inflammation evaluated by CRP and IL-6. These findings suggest that chronic active inflammation decreases plasma leptin concentrations, contrary to acute inflammation, as seen in sepsis and surgery. (27)

In our study, serum leptin levels did not differ between patients receiving MTX versus those not on MTX. Patients on glucocorticoids had higher plasma leptin levels but this did not reach statistical significance. After adjustment for BMI, it was observed that serum leptin levels were higher in patients treated with glucocorticoids; however, after correction for glucocorticoid usage, there was no serum leptin level difference between the two patient groups having a DAS28 below or above 5.1. Glucocorticoids also have a regulatory effect on serum leptin levels. Glucocorticoids have been shown to increase leptin production in vitro and in humans. A sustained rise in circulating leptin levels with the use of exogenously administered glucocorticoids was reported. (28-31) Janssen et al (30) showed that after an overnight dexamethasone suppression test, leptin levels increased significantly at the 9th hour. Posttreatment leptin levels were positively related to dexamethasone levels. In vitro studies have shown that the effect of dexamethasone on leptin levels may be due to a direct effect on leptin production in adipose cells by increasing leptin mRNA expression or to the presence of a glucocorticoid responsive element in the promoter of the ob gene. (32) Dagogo-Jack et al (33) demonstrated that plasma leptin response to dexamethasone treatment was similar in both sexes at all ages. Bokarewa et al (2) studied the effect of different medications on leptin levels. There was no difference in patients receiving glucocorticoids or not. This was attributed to low-dose and long-term steroid use. Leptin levels were higher in patients receiving MTX compared with other DMARDs. This difference could not be explained by known folic acid's (34) increasing effect on leptin levels because there was no significant difference in folic acid levels between groups. Besides, there are studies performed in RA and juvenile idiopathic arthritis with no evidence of DMARD or MTX effect on leptin levels. (20,35)

In our study, serum leptin levels showed correlation with BMI in both RA patients and controls (16,19) and women had higher levels, as seen in previous studies. (35,36) In one study, this sex difference was reported not to be related to sex hormones or fat distribution, but possibly to differences in hypothalamic regulation of leptin production or in adipose tissue biologic characteristics. (35) Besides, there are studies reporting that gonadal steroids have an effect on circulating leptin levels. Testosterone inhibited the expression of this hormone, whereas it was increased by ovarian sex steroids. (37)

In conclusion, even though leptin levels were higher in RA patients, there was no correlation with disease activity parameters. Leptin levels do not seem to reflect disease activity. Whether leptin has a role in outcome, joint damage and the relationship with cytokines in RA and other inflammatory diseases needs further evaluation.

References

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3. Palmer G. Gabay C. A role for leptin in rheumatic diseases? Ann Rheum Dis 2003;62:913-915.

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Do not go gentle into that good night, Old age should burn and rave at
close of day; Rage, rage against the dying of the light.
--Dylan Thomas

Rezzan Gunaydin, MD, Taciser Kaya, MD, Aysenur Atay, MD, Nese Olmez, MD, Aysel Hur, MD, and Mehmet Koseoglu, MD

From the Clinic of Physical Medicine and Rehabilitation, Izmir Training and Research Hospital, Izmir Turkey and the Clinics of Biochemistry and Physical Medicine and Rehabilitation. Ataturk Training and Research Hospital, Izmir, Turkey.

Reprint requests to Rezzan Gunaydin, Associate Professor. MD. 116/16 Sok. No:8/15 35050 Bornova, Izmir. Turkey. Email: [e-mail]

RELATED ARTICLE: Key Points

* Leptin may play a part in the regulation of autoimmune inflammatory conditions.

* Leptin may influence rheumatoid arthritis in opposing ways: enhance the expression of Th1 cytokines or limit the inflammatory responses.

* In rheumatoid arthritis, serum leptin levels were reported to be similar, lower or higher compared to healthy subjects in different studies.

* In this study, serum leptin and TNF-[alpha] levels were statistically higher than in age-, sex-, and BMI-matched control subjects, but showed no correlation with disease activity parameters in rheumatoid arthritis patients.

Table 1. Clinical and demographic characteristics of patients with
rheumatoid arthritis (RA) and of control subjects

                      RA                    Control subjects
                      (n = 50)              (n = 34)             P

Age (years)           56.34 [+ or -] 12.43  53.22 [+ or -] 9.19  0.231
  (mean [+ or -] SD)
Sex (F/M)             41/9                  28/6                 0.967
BMI (weight/          26.77 [+ or -] 5.70   25.77 [+ or -] 3.56  0.368
  [height.sup.2])
  (mean [+ or -] SD)
Disease duration      10 [+ or -] 9.1       NA
  (years)
  (mean [+ or -] SD)
Rheumatoid factor     73.17                 NA
  (%)
Number of swollen      4.5 [+ or -] 4.4     NA
  joints
Number of tender      12.24 [+ or -] 7.3    NA
  joints
DAS28 score            5.4 [+ or -] 1.17    NA
  (mean [+ or -] SD)
Medications
  MTX                 32                    NA
  Other DMARDs        10                    NA
  None                 8                    NA
  Oral                21                    NA
    glucocorticoids

NA, not applicable.

Table 2. Serum leptin levels according to disease activity

                     DAS > 5.1             DAS < 5.1
                     (n = 19)              (n = 31)              P

Serum leptin levels  54.11 [+ or -] 40.78  52.59 [+ or -] 36.37  0.892
  (ng/mL)

Table 3. Serum leptin levels in patients receiving methotrexate or oral
glucocorticoids

                                          Serum leptin
                                          levels (ng/mL)        P

Treated with MTX (n=32)                   58.66 [+ or -] 38.02  0.172
Not treated with MTX (n=18)               43.39 [+ or -] 36.09
Treated with oral glucocorticoids (n=21)  63.59 [+ or -] 41.48  0.097
Not treated with oral glucocorticoids     45.62 [+ or -] 33.44
  (n=29)