Serum Pro-hepcidin Could Reflect Disease Activity in Patients with Rheumatoid Arthritis

Rheumatoid arthritis (RA) is an autoimmune inflammatory disease mainly affected the peripheral joints, but extra-articular systems are often affected. Anemia is one of the common extra-articular manifestations in RA. The prevalence of anemia is 26.9%-77.6% in patients with RA (1). The leading cause of the anemia in RA is chronic inflammation, with the pattern of a normochromic and normocytic anemia. It is associated with decreased serum iron and total iron-binding capacity (TIBC), but iron store is increased or normal. Iron deficiency anemia due to long-term use of non-steroidal anti-inflammatory drugs (NSAIDs) and folate deficiency due to methotrexate therapy also result in the anemia in RA (2). Hemolytic anemia is a rare cause of anemia in RA.

There are possible mechanisms in the anemia of chronic inflammatory diseases in RA. First, inflammatory cytokines, such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β and interferon-γ, induce inadequate erythropoiesis in bone marrow, inhibiting erythroid progenitor differentiation and driving the apoptosis of erythroid progenitors. Second, the inflammatory cytokines mediate the inhibition of peripheral iron utilization. Third, the erythropoietin production in response to anemia is relatively impaired. Forth, red cell survival is slightly reduced in RA patients (2, 3). In general, a degree of anemia correlates with RA disease activity and serum TNF-α concentration (4). The patients with anemia are likely to have more severe joint diseases than those without anemia in RA.

Hepcidin, a 25 amino acid peptide hormone, regulates intestinal iron absorption, recycling, and tissue storage (5). Beyond the iron regulation, it is closely linked with inflammation and infection (5, 6). The inflammatory signal, chiefly IL-6, induces hepcidin synthesis, then the upregulated hepcidin decreases duodenal iron absorption and increases sequestration of iron by macrophage, resulting in hypoferremia (7). This is another possible mechanism of anemia of chronic inflammatory disease such as RA and systemic lupus erythematosus (SLE). This is supported by following two evidences. First, hepcidin mRNA level in adipose tissue correlates with IL-6 and C-reactive protein (CRP) (8). Second, when IL-6 is infused to healthy individuals, the hepcidin level increases, and serum iron and transferrin saturation decrease (7).

The aim of this study is to analyze the relationship between serum pro-hepcidin, a prohormone of hepcidin, concentration and the anemia profiles of RA and to determine whether the pro-hepcidin could reflect RA disease activity.

The clinical characteristics of the 40 RA patients were as follows: age 57.6±13.3 yr, disease duration 52.6±79.6 months, duration of morning stiffness 45.4±66.5 min, and DAS28 4.64±18.4. RF was positive (>18 IU/mL) in 67.5% of the patients. 75% of the patients had positive anti-CCP Ab (>20 Unit/mL). Table 1 shows the additional clinical data of the RA patients included in the study.

Table 1
Clinical characteristics of the 40 patients with rheumatoid arthritis

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The laboratory profiles relative to anemia were as follows; Hb 11.9±1.5 g/dL, serum iron 63.9±35.9 µg/dL, TIBC 291.6±43.9 µg/dL, ferritin 116.4±168.6 ng/mL, and transferrin 235.3±37.9 ng/mL. When the patients were divided into anemic and non-anemic groups, the patients with anemia of chronic disease had lower serum iron (78.9±39.9 vs. 47.4±20.7 µg/dL, P<0.001), higher ESR (32.2±23.9 vs. 44.8±24.3 mm/hr, P=0.02), and higher CRP (1.3±1.8 vs. 3.4±5.3 mg/dL, P=0.03), than the patients without anemia. Other clinical and laboratory parameters (disease duration, morning stiffness, 100 mm VAS, DAS28, tender/swollen joint counts), RF and anti-CCP Ab titers were not different between anemic and non-anemic groups.

In 40 RA patients, the mean concentration of serum prohepcidin was 237.6±67.9 ng/mL. Serum pro-hepcidin concentration negatively correlated with serum iron (r=-0.23, P=0.04), but it did not correlate with TIBC, ferritin and transferrin. There was no difference of pro-hepcidin concentration between the patients with anemic and non-anemic groups.

Next, we determined the relationship between serum prohepcidin concentration and RA disease activity parameters. The pro-hepcidin concentration correlated with DAS28 (r=0.4, P<0.001), tender joint count (r=0.3, P=0.003), RF titer (r=0.51, P<0.001), ESR (r=0.53, P<0.001), and CRP (r=0.22, P=0.05). When the patients were divided into two groups using DAS28 score: patients with active RA (DAS28 >5.1, n=14) and those with inactive to moderate RA (DAS28 ≤5.1, n=26), the serum pro-hepcidin concentration was higher in patients with active disease than in those inactive to moderate disease (271.7±95.7 ng/mL vs. 219.3±37.6 ng/mL; P=0.01, Table 2). There is no difference of pro-hepcidin concentration in the groups with inactive and moderate diseases. However, the pro-hepcidin concentration was not dependent of current medication including steroid, disease modifying anti-rheumatic drugs (DMARDs), and biologics.

Table 2
Laboratory parameters relative to anemia and the serum concentrations of pro-hepcidin in the 14 patients with active RA (DAS>5.1) and the 26 patients with inactive to moderate RA (DAS≤5.1)

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Finally, we analyzed the relationship between pro-hepcidin with proinflammatory cytokines such as TNF-α, IL-1β, and IL-6. Mean concentrations of serum TNF-α, IL-1β, and IL-6 were 197.8±22.6 pg/mL, 252.5±5.8 pg/mL, and 262.3±17.3 pg/mL, respectively. There was significant correlation between the serum concentration of pro-hepcidin with the serum concentrations of TNF-α (r=0.58, P<0.001), IL-1β (r=0.59, P<0.001), and IL-6 (r=0.4, P=0.01).

Hepcidin synthesis is regulated by extrinsic or intrinsic iron loading, anemia, and hypoxemia. Moreover, the hepcidin production is induced by a particular cytokine, IL-6, during inflammation (7). In a previous study, IL-6 is induced within 3 hr after injection of lipopolysaccharide (LPS) and urinary hepcidin peaks within 6 hr, followed by decrease in serum iron (12). IL-6 is one of the proinflammatory cytokines, which has central role in the pathogenesis of RA, a representative inflammatory disease. IL-6 induces acute phase response, and differentiates B and T cells, and promotes joint destruction in RA (13-15). Serum levels of IL-6 are correlated with RA disease activity and clinical symptoms. Because anemia is a common extra-articular manifestation of RA, we hypothesized IL-6-hepcidin could be the possible link in RA to the anemia.

A recent study showed that RA patients have higher serum concentration of pro-hepcidin than patients with SLE and healthy volunteers, but the pro-hepcidin concentration doesn't correlate with RA disease activity scores, TNF-α, or IL-6 (14). However, in our study, serum pro-hepcidin concentration correlated with RA disease activity parameters such as DAS28, tender joint count, ESR and CRP. The serum concentration of pro-hepcidin also correlated with proinflammatory cytokines, such as TNF-α, IL-1β, and IL-6. In the previous study, serum concentrations of TNF-α, IL-1β, and IL-6 are correlated with RA disease activity indexes, such as DAS28, ESR, and CRP (16-18). The roles of these proinflammatory cytokines in the RA pathogenesis are well known and the inhibitors of each cytokines are used universally as the therapy for active RA. Correlation of pro-hepcidin with proinflammatory cytokines suggests that pro-hepcidin was induced by the proinflammatory cytokines, especially IL-6. Moreover, when the patients were divided two groups by disease activities, the RA patients with active disease had higher concentration of serum pro-hepcidin than those with inactive to moderate disease. This result suggested that serum pro-hepcidin concentration could be another useful marker for RA disease activity as acute phase reactants, active joint counts or proinflammatory cytokines. Our results showed different results from the previous study in RA (14), it is plausible that the patients in our study were older Asian with more active RA than the patients who were enrolled in the previous study (mean age: 57.6 vs. 46.4 yr, and mean DAS28: 4.64 vs. 3.59). These differences of characteristics of patients could be the cause of different results. Further study should be warranted to elucidate this contradictory result.

On the other hand, pro-hepcidin did not correlate anemiazzzz profiles, except for negative correlation with serum iron concentration. It was postulated that serum pro-hepcidin could not represent the bioactive hepcidin (12), thus serum pro-hepcidin concentration did not reflect actual anemic parameters, as supported by a previous studies (19, 20). After injection of LPS, serum pro-hepcidin levels don't change significantly, although pro-inflammatory cytokine induction, urinary hepcidin excretion, and serum iron are decreased (12). No difference of serum pro-hepcidin between the RA patients with or without anemia may be explained that serum pro-hepcidin could not reflect the iron state exactly in human disease setting. Another explanation is that hepcidin is overproduced by the inflammatory response in RA, and the over-expressed hepcidin might disturb the normal physiologic response in the iron metabolism. Many arguments on the correlation between pro-hepidin and anemia indicate that measurement of serum pro-hepcidin could not substitute bioactive hepcidin. Therefore, available method for measurement of hepdicin is needed to reveal accurate relationship of inflammatory diseases and anemia. We postulate that there is different clinical significance between hepcidin and pro-hepcidin, the former might regulate iron metabolism and the latter might regulate inflammatory process. More research is needed to serial measurement of serum pro-hepcidin in same patients as the disease activity changes.

In conclusion, proinflammatory cytokines play a major role in the development of anemia of RA, as they inhibit erythropoiesis, and hepcidin might involve in minor mechanism of anemia in the inflammatory disease, as it regulates iron metabolism inappropriately. Hepcidin or pro-hepcidin might play an important role in RA, as the mediator linking anemia and the inflammatory cytokines. Pro-hepcidin could be another useful marker for RA disease activity.