We have read with interest the article by Knevel et al, entitled “Genetic variants in IL15 associate with progression of joint destruction in rheumatoid arthritis: a multicohort study” published on-line on March 22, 2012.1 The authors describe an elegant candidate gene study using four datasets of European rheumatoid arthritis (RA) patients with longitudinal radiological data, which let them conclude that genetic variants in IL15 are associated with the intensity of radiological progression in RA. With a different approach, we have recently reported data suggesting that serum-IL15 (sIL15) could be a potential biomarker of severe disease for patients with early arthritis,2 and then we demonstrated that those with increased levels of the cytokine may require earlier and more intensive treatment, based on two major findings: first, early arthritis patients with increased sIL15 levels at baseline (30% of the study population) presented higher disease activity throughout follow-up; second, rheumatologists—blinded to sIL15 levels— prescribed treatment with DMARDs and glucocorticoids more intensively to these patients than to those with normal or low IL15 levels.3 Hence, the findings from the genetic study by Knevel et al. add further evidence for IL15 as a marker of severity in RA.
In addition, we would like to share new data that may contribute to the better understanding of the relation between IL15 and radiological progression. It has been described that IL15 can mediate the development of osteoclasts 4 through the induction of IL17 5 6 that, in turns, promotes RANKL production 7 8. Based on these findings, we have analyzed whether the amounts of RANKL and osteoprotegerin (OPG) in serum correlated with sIL15 in 102 early arthritis patients from our cohort in which blood samples from 376 visits have been analysed for either marker. We measured OPG concentration by using a DuoSet kit from R&D Systems (Abingdon, United Kingdom), and total RANKL serum levels through a specific kit from Apotech (Epalinges, Switzerland), following the manufacturer recommendations. We fitted multivariable models adjusted for repeated measures (xtgee command of Stata 12) to find correlates of either RANKL or OPG.
RANKL serum levels were 3795 pg/ml (interquartile range[IQR]: 1464 – 10095) in patients with positive rheumatoid factor (RF+) and 1125 pg/ml (IQR: 569 – 2867) in those with negative RF (RF-), being the differences statistically significant at the multivariable analysis (β coefficient 5913 ± 1835 [RF+ vs RF-], p=0.001). In addition, RANKL was 2532 pg/ml (IQR: 912 – 7242) in patients with increased serum IL15 compared to 1441 pg/ml (IQR: 632 – 3920) in those with low levels of IL15, being significant the correlation between serum levels of these molecules (β coefficient 22.7 ± 11.3 [by pg/ml IL15], p=0.044). On the other hand, we did not observe any significant association between RANKL serum levels and age, gender, disease activity assessed by DAS28, ACPA, or OPG levels. Conversely, OPG serum levels were significantly associated with disease activity (β coefficient 337 ± 121, p=0.006) and age (β coefficient 70 ± 16.6, p <0.001. We did not observe any significant association between OPG serum levels and either gender, rheumatoid factor, ACPA, or IL15 levels.
In conclusion, our findings, and those reported by Knevel et al. appear to harmonize. It would be interesting to know whether these genetic variants could conceivably explain the high baseline sIL15 values that we found in our early arthritis subpopulation, in association with severe disease and intensive therapy requirement.

References

1. Knevel R, Krabben A, Brouwer E, et al. Genetic variants in IL15 associate with progression of joint destruction in rheumatoid arthritis: a multicohort study. Ann Rheum Dis 2012. doi:10.1136/annrheumdis-2011-200724

2. Lamana A, Ortiz AM, Alvaro-Gracia JM, et al. Characterization of serum interleukin-15 in healthy volunteers and patients with early arthritis to assess its potential use as a biomarker. Eur Cytokine Netw 2010;21(3):186-94.

3. Gonzalez-Alvaro I, Ortiz AM, Alvaro-Gracia JM, et al. Interleukin 15 levels in serum may predict a severe disease course in patients with early arthritis. PLoS One 2011;6(12):e29492.

4. Ogata Y, Kukita A, Kukita T, et al. A novel role of IL-15 in the development of osteoclasts: inability to replace its activity with IL-2. J Immunol 1999;162(5):2754-60.

5. Gonzalez-Alvaro I, Ortiz Garcia AM, Dominguez-Jimenez C, et al. Inhibition of TNF and IL-17 production by leflunomide involves the JAK/STAT pathway. Ann Rheum Dis 2009;68(10):1644-50.

6. Ziolkowska M, Koc A, Luszczykiewicz G, et al. High levels of IL-17 in rheumatoid arthritis patients: IL-15 triggers in vitro IL-17 production via cyclosporin A-sensitive mechanism. J Immunol 2000;164(5):2832-8.

7. Neumann E, Gay S, Muller-Ladner U. The RANK/RANKL/osteoprotegerin system in rheumatoid arthritis: new insights from animal models. Arthritis Rheum 2005;52(10):2960-7.

8. Schett G, Hayer S, Zwerina J, et al. Mechanisms of Disease: the link between RANKL and arthritic bone disease. Nature clinical practice. Rheumatology 2005;1(1):47-54.

Conflict of Interest:

None declared

We read with interest the paper of M Sebastianni et al about the predictive role of capillaroscopic skin ulcer risk index in systemic sclerosis(1). The authors suggest that the CSURI score "can represent a feasible and reliable clinical tool in the routine evaluation of patients". However, we feel this report raises serious issues concerning the reproducibility of the methodology. When calculating the score, the number of capillaries ("N") was the most important predictive index. The definition of the "N" is clearly detailed and the clinicians have to count "all the capillaries detectable in the entire distal front line, including capillaries placed at different levels". The measurement was illustrated in figure 1 and we noticed that that the author were counting 13 capillaries on the panel C as specified in the legend.

However, the same picture was used 3 years ago, when the same authors described the score(2), excepted that the picture was a little larger and included one more capillary on the right of the picture, and the authors counted only 11 capillaries. We were surprised about such a huge difference in capillary counting, especially in didactic pictures which have the aim to illustrate the methodology of the score. This difference may be induced by the difficulty of defining the "entire distal front line". It also reflects the difficulty of capillary counting using native videocapilaroscopy that could be overcome by using fluorescent dyes. It is difficult to estimate the extrapolation of this error on the score MxD/N2 but, it seems clear that the reproducibility of the CSURI remains questionable. Although the aim to build-up an informative and easy to use capillaroscopic score is a highly valuable purpose, the reproducibility of such measurements remains a stumbling stone difficult to get round.

References

1. Sebastianni M, Manfredi A, Vukatana G, et al. Predictive role of capillaroscopic skin ulcer risk index in systemic sclerosis: a multicentre validation study. Ann Rheum Dis 2012;71:67-70.

2. Sebastiani M, Manfredi A, Colaci M, et al. Capillaroscopic skin ulcer risk index: a new prognostic tool for digital skin ulcer development in systemic sclerosis patients. Arthritis Rheum 2009;61:688-94.

Conflict of Interest:

none

Dear Editor,

The maintenance of serum calcium within normal limits is important to sustain life. In vitamin D deficiency calcium cannot adequately be absorbed by the intestinal mucosa. The lack of both of these important players in bone health and can bring about secondary hyperparathyroidism[1].

The excess PTH, among other things, stimulates osteoclastogenesis[2] and liberates calcium from the skeleton to maintain an adequate serum calcium level[3]. The recent study by Boumans, et al., (2012)[4] finding that rituximab inhibits osteoclastogenesis raises a significant question: what impact does this have on maintaining serum calcium, especially in patients with secondary hyperparathyroidism? Vitamin D deficiency is described as endemic[5]; additionally people with rheumatoid arthritis (RA) have consistently low levels[6], and agents used, such as corticosteroids deplete vitamin D levels. In turn calcium cannot be absorbed and instead must be taken from the bone and the increase in PTH is the mechanism by which this occurs. In one study of 850 men with RA[6] the prevalence of insufficiency (below 75 nmol/L (30 ng/mL)) was 84% and deficiency (below 50 nmol/L (20 ng/mL)) was 43%. Also, after adjustment, hypovitaminosis D was more common in those with anti-cyclic citrullinated peptide antibody positivity and those who are non-white; deficiency was independently associated with greater joint tenderness and higher high sensitivity CRP. Indeed, in a recent study[7] of rituximab patients, all were vitamin D deficient with levels below 50 nmol/L (20 ng/mL). The study apparently did not measure PTH or calcium levels.

The calcium aspect of rituximab does not appear to have been studied, but a concern is that hypocalcaemia may result and cardiac or skeletal muscle problems could follow. As far as the author is aware, calcium measurement is not a part of any routine blood draws in rituximab therapy in RA. The mere prospect that hypocalcaemia may occur should induce us to think of preventive strategies. One approach may be similar to that used with bisphosphonates: to ensure calcium levels are adequate and that the patient is taking a source of vitamin D before therapy. To be more accurate, 25-hydroxy vitamin D can be measured and we can ensure it is above 75 nmol/L (30 ng/mL). This is because it takes levels between 75-80 nmol/L (30-32 ng/mL) to suppress PTH and a level above 85 (34 ng/mL) for intestinal calcium absorption to be optimised[5]. In the elderly, levels above 122 nmol/L (49 ng/mL) may be required to suppress PTH to what is considered normal in young adults[8].

Even if further studies show the inhibition of osteoclastogenesis by rituximab is negligible and of little consequence in the development of hypocalcaemia, the approach remains sound. This is because by ensuring adequate levels of vitamin D and calcium, patients will be on the strongest ground in ensuring bone health, along with the many benefits that may result from improved vitamin D levels[9].

In sum, at a minimum we should consider measurement of calcium levels and ensure a basic level of vitamin D input; alternatively, 25 (OH) D levels should be measured to ensure a level above at least 75 nmol/L (30 ng/mL) and preferably above 100 nmol/L (40 ng/mL).

References

1. Aloia JF Feuerman M Yeh JK. Reference range for serum parathyroid hormone. Endocr Pract 2006;12: 137-144.

2. Jacome-Galarza CE Lee SK Lorenzo JA et al. Parathyroid hormone regulates the distribution and osteoclastogenic potential of hematopoietic progenitors in the bone marrow. J Bone and Miner Res 2011;26: 1207-1216.

3. Wallis DE Penckofer S Sizemore GW. The "sunshine deficit" and cardiovascular disease. Circulation 2008;118: 1476-1485.

4. Boumans MJ Thurlings RM Yeo L et al. Rituximab abrogates joint destruction in rheumatoid arthritis by inhibiting osteoclastogenesis. Ann Rheum Dis 2012;71: 108-113.

5. Cannell JJ Hollis BW Zasloff M et al. Diagnosis and treatment of vitamin D deficiency. Expert Opin Pharmacother 2008;9: 107-118.

6. Kerr GS Sabahi I Richards JS et al. Prevalence of vitamin D insufficiency/deficiency in rheumatoid arthritis and associations with disease severity and activity. J Rheumatol 2011;38: 53-59.

7. Hasan E Olusi S Al-Awadhi A et al. Effects of rituximab treatment on the serum concentrations of vitamin D and interleukins 2, 6, 7, and 10 in patients with rheumatoid arthritis. Biologics 2012;6: 31-35.

8. Kinyamu HK Gallagher JC Rafferty KA et al. Dietary calcium and vitamin D intake in elderly women: effect on serum parathyroid hormone and vitamin D metabolites. Am J Clin Nutr 1998;67: 342-348.

9. Wilding PM Cardiovascular disease, statins and vitamin D. Br J Nurs 2012;21: 214-220.

Conflict of Interest:

None declared

Dear Editor,

A recent report by Hot and colleagues investigated the possible link between the increased expression of IL-17 in rheumatoid diseases and the high incidence of cardiovascular disorders in patients suffering these pathologies.

To address this point the authors used an elegant approach that combined in vitro cell functional studies with gene fingerprint analysis by microarray. Consistent with several previous studies, including our own (1), the authors showed that IL-17A per se does not cause a significant inflammatory response in HUVEC while it greatly enhance the inflammatory properties of TNF?.

To investigate the functional consequences of these results the authors used a wide range of tests including endothelial cell migration and invasion. Most interestingly, they also showed that the conditioned medium of HUVEC stimulated with IL-17A with or without TNF? caused a significant increase in platelet aggregation.

These results further support our previous studies showing a proaggregant effect of IL-17A on both murine and human platelet aggregation (2). Like for endothelial cells, IL-17A alone did not influence platelet activation while it amplified the inflammatory effect of ADP. More specifically, IL-17A increased the primary reversible phase of ADP-induced platelet aggregation in a concentration-dependent manner.

The results presented by Hot et al. in Figure 6A show no difference between the conditioned medium of HUVEC treated with IL-17A or IL-17A + TNF? while in most of the other assays the combination of these two cytokines provided significant differences compared to the effects of IL- 17A alone. Thus it might be possible to hypothesize that the proaggregant effect observed might be due, al least in part, to a direct effect of IL- 17A still present in the medium. This could be easily addressed by adding an anti-IL-17A neutralizing antibody to the conditioned medium before the test on platelet aggregation.

References

1. Maione F, Paschalidis N, Mascolo N, et al. Interleukin 17 sustains rather than induces inflammation. Biochem Pharmacol 2009;77(5):878-87.

2. Maione F, Cicala C, Liverani E, et al. IL -17A increases ADP-induced platelet aggregation. Biochem Biophys Res Commun 2011;408(4):658-62.

Conflict of Interest:

None declared