Frequency of Th17 CD4+ T cells in early rheumatoid arthritis: A marker of anti-CCP seropositivity

Objective: To examine the frequency and phenotype of Th17 cells in the peripheral blood of early RA (eRA) patients. Methods: CD4+ T cells were isolated from the peripheral blood of 33 eRA patients, 20 established RA patients and 53 healthy controls (HC), and from the synovial fluid of 20 established RA patients (RASF), by ficoll-hypaque gradient and magnetical negative selection. After polyclonal stimulation, the frequency of Th17 and Th1 cells was determined by flow cytometry and concentrations of IL-17, IFN-γ, TNF-α and IL-10 were measured by ELISA in cell-free supernatants. Results: When all of our eRA patients were analyzed together, a significantly lower percentage of circulating Th17 cells and a lower CD4-derived IL-17 secretion were observed in comparison with HC. However, after stratifying by anti-CCP antibody status, circulating Th17 cells were decreased in anti-CCP(+) but not in anti-CCP(-)-eRA. All Th17 cells were CD45RO+CD45RA- and CCR6+. Dual Th17/Th1 cells were also exclusively decreased in anti-CCP(+)-eRA. Circulating Th17 and Th17/Th1 cells were negatively correlated with anti-CCP titres. When anti-CCP(+)-eRA patients were retested one year after initiating treatment with oral methotrexate, their circulating Th17 frequency was no longer different from HC. Of note, the percentage of circulating Th1 cells and the secretion of CD4-derived IFN-γ, TNF-α and IL-10 were not different between eRA patients and HC. In established RA patients, circulating Th17 and T17/Th1 cell frequencies were comparable to HC. In RASF, both Th17 and Th1 cells were increased when compared with blood of eRA patients, established RA patients and HC. Conclusion: Decreased circulating Th17 levels in eRA seem to be a marker of anti-CCP seropositivity, and return to levels observed in healthy controls after treatment with methotrexateThis work was supported by Ministerio de Ciencia e Innovación grant SAF 2009-07100, (http://www.idi.mineco.gob.es/portal/site/MICINN) and by RETICS Program, RD08/0075 (RIER) from ‘‘Instituto de Salud Carlos III’’ (ISCIII) (http://www.isciii.es/

High incidence of clinical fragility fractures in postmenopausal women with rheumatoid arthritis. A case-control study.

Objectives: To estimate the incidence of clinical fragility fractures in postmenopausal women with rheumatoid arthritis (RA) and analyze risk factors for fracture. Methods: Incidence of clinical fragility fractures in 330 postmenopausal women with RA was compared to that of a control population of 660 age-matched postmenopausal Spanish women. Clinical fractures during the previous five years were recorded. We analyzed associations with risk factors for fracture in both populations and with disease-related variables in RA patients. Results: Median age of RA patients was 64 years; median RA duration was eight years. Sixty-nine percent were in remission or on low activity. Eighty-five percent had received glucocorticoids (GCs); 85 %, methotrexate; and 40 %, ≥1 biologic DMARD. Fifty-four patients and 47 controls had ≥1 major osteoporotic fracture (MOF). Incidence of MOFs was 3.55 per 100 patient-year in patients and 0.72 in controls (HR: 2.6). Risk factors for MOFs in RA patients were age, previous fracture, parental hip fracture, years since menopause, BMD, erosions, disease activity and disability, and cumulative dose of GCs. Previous fracture in RA patients was a strong risk for MOFs (HR: 10.37). Conclusion: Of every 100 postmenopausal Spanish women with RA, 3-4 have a MOF per year. This is more than double that of the general population. A previous fracture poses a high risk for a new fracture. Other classic risk factors for fracture, RA disease activity and disability, and the cumulative dose of GCs are associated with fracture development

Acidos grasos poliinsaturados "omega"-3 y su papel como mediadores de la inflamación en la artritis reumatoide

Tesis doctoral inédita leída el 10-10-1994 en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Cirugí

Expression of IL-17A by RA CD4+ T cells.

<p>CD4+ T cells were isolated from the peripheral blood of healthy controls (HCPB) (n = 53), the peripheral blood of early RA patients (eRAPB) (n = 33), the peripheral blood of established RA patients (RAPB) (n = 20) and the synovial fluid of established RA patients (RASF) (n = 20), and stimulated with PMA/Ionomycin for 16 h or with anti-CD3/CD28/CD49d for 4 days. A, B. Percentage of CD4+ T cells expressing IL-17A (Th17 cells) after a 16 h stimulation, as determined by flow cytometry. Because the CD4 molecule is downregulated upon stimulation with PMA, shown is CD3 expression on isolated CD4+ T cells. C. Representative flow cytometry dot-plots showing expression of CD45RO, CD45RA and CCR6 versus IL-17A in isolated CD4+ T cells. D. Secretion of IL-17A to the culture medium of CD4+ T cells stimulated for 16 h with PMA/ionomycin or for 4 days with anti-CD3/CD28/CD49d. Box-plots represent the median and interquartile range of all studied subjects, whiskers represent the maximum and minimum values. *p<0.05 vs HCPB; † p<0.05 vs RAPB; ¥ p<0.05 vs eRAPB.</p

Proportion of Th17/Th1 cells in RA.

<p>CD4+ T cells were isolated from the peripheral blood of healthy controls (HCPB) (n = 53), the peripheral blood of early RA patients (eRAPB) (n = 33), the peripheral blood of established RA patients (RAPB) (n = 20) and the synovial fluid of established RA patients (RASF) (n = 20). A, B. Percentage of CD4+ T cells expressing both IL-17A and IFN-γ (Th17/Th1 cells) after a 16 h stimulation with PMA/ionomycin, as determined by cytometry. Box-plots represent the median and interquartile range of all studied subjects, whiskers represent the maximum and minimum values. *p<0.05 vs HCPB; † p<0.05 vs RAPB; ¥ p<0.05 vs eRAPB.</p

Expression of IFN-γ, TNF-α and IL-10 by RA CD4+ T cells.

<p>CD4+ T cells were isolated from the peripheral blood of healthy controls (HCPB) (n = 33), the peripheral blood of early RA patients (eRAPB) (n = 33) and the synovial fluid of established RA patients (RASF) (n = 20), and stimulated with PMA/Ionomycin for 16 h or with anti-CD3/CD28/CD49d for 4 days. A, B. Percentage of CD4+ T cells expressing IFN-γ (Th1 cells) after a 16 h stimulation, as determined by flow cytometry. Because the CD4 molecule is downregulated upon stimulation with PMA, shown is CD3 expression on CD4+ T cells. C. Secretion of IFN-γ by CD4+ T cells stimulated for 16 h with PMA/ionomycin or for 4 days with anti-CD3/CD28/CD49d. D. Secretion of TNF-α and of IL-10 by CD4+ T cells stimulated for 16 h with PMA/ionomycin. Box-plots represent the median and interquartile range of all studied subjects, whiskers represent the maximum and minimum values. *p<0.05 vs HCPB; † p<0.05 vs eRAPB.</p

Relation of Th17 and Th17/Th1 frequencies with anti-CCP antibodies and with erosions in eRA.

<p>A. Percentage of Th17 and Th17/Th1 cells among CD4+ T cells isolated from the peripheral blood of healthy controls (HCPB) (n = 53), the peripheral blood of anti-CCP+ eRA patients (ACCP+) (n = 15) and the peripheral blood of anti-CCP- eRA patients (ACCP-) (n = 18). *p<0.05 vs HCPB. B. Linear correlation between the percentage of circulating Th17 or Th17/Th1 cells and the anti-CCP antibody titre in anti-CCP+ eRA patients. C. Frequencies of Th17 and Th17/Th1 cells in the peripheral blood of eRA patients with erosive (n = 9) or non-erosive disease (n = 24) at initial presentation. *p<0.05 vs patients with erosive disease. D. Left panel: Percentage of patients with erosive disease at initial presentation among anti-CCP+ and anti-CCP- eRA subjects; Right panel: anti-CCP antibody titres in anti-CCP+ eRA patients with erosive or non-erosive disease at initial presentation. *p<0.05 vs patients with erosive disease. Box-plots represent the median and interquartile range of all studied subjects, whiskers represent the maximum and minimum values.</p

In vivo effect of treatment on the circulating Th17 and Th17/Th1 frequencies.

<p>Eight patients with anti-CCP+ eRA donated blood for a second time, one year after the first visit, and while receiving treatment with oral MTX with or without low-dose prednisone. Four out of these patients had achieved remission and 4 of them demonstrated persistent disease activity. Shown are the frequencies of circulating Th17 and Th17/Th1 cells in patients who achieved remission (ACCP+ remission) (n = 4), in patients who did not achieve remission (ACCP-non-remission) (n = 4) and in their age and sex-matched controls (HCPB) (n = 8), observed at first evaluation (“before treatment”) and at the one-year follow-up visit (“after treatment”). Box plots represent the median and interquartile range of all studied subjects, whiskers represent the maximum and minimum values. *p<0.05 vs HCPB.</p