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Journal of Peking University(Health Sciences) ›› 2018, Vol. 50 ›› Issue (6): 968-974. doi: 10.19723/j.issn.1671-167X.2018.06.005

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Increased receptor activator of nuclear factor kappa B ligand expressed on B10 cells in rheumatoid arthritis

Hong jiang LIU1,Xiao feng GUO1,Fan lei HU2,Cui ping YAN1,Xiang jun CUI1,Xue liang YAN1,Zhan guo LI2,Yuan JIA2,△(),Shu lin ONG1,△()   

  1. 1. Department of Rheumatology and Immunology,the People’s Hospital of China Three Gorges University,Yichang 443000, Hubei, China
    2. Department of Rheumatology and Immunology, Peking University People’s Hospital, Beijing 100044, China
  • Received:2018-07-02 Online:2018-12-18 Published:2018-12-18
  • Contact: Yuan JIA,Shu lin ONG E-mail:jiayuan1023@sina.com;sslin0717@sina.com
  • Supported by:
    Supported by the National Natural Science Foundation of China(81701614)

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Abstract:

Objective: To detect receptor activator of nuclear factor kappa B ligand (RANKL) expressed on B10 cells in rheumatoid arthritis (RA) and to evaluate the correlation between RANKL-producing B10 cells in RA and clinical features and laboratory parameters, trying to reveal the possible role of B10 cells in the pathogenesis of RA and the potential mechanism of impaired immunosuppressive capacities.Methods:25 RA patients and 20 healthy volunteers were enrolled. These RA patients did not received treatment with glucocorticoids, disease-modifying anti-rheumatic drug and biologics during the recent half of a year. The levels of RANKL-producing B10 cells were measured by flow cytometry (FCM) and polymerase chain reaction (PCR). The correlation between the frequencies of RANKL-producing B10 cells in RA and clinical data, laboratory parameters were analyzed. The role of tumor necrosis factor α (TNF-α) and interleukin 1β (IL-1β) in inducing RANKL expression in B10 cells was evaluated by in vitro stimulation assay. Independent samples t test, Pearson and Spearman correlation were used for statistical analysis.Results:B10 cells were capable of producing RANKL at a low level in health controls. The frequencies of RANKL-producing B10 cells were markedly higher in RA patients than in health controls (3.65%±1.59% vs. 2.25%±0.68%, P<0.01). The frequencies of these cells correlated po-sitively with RA tender joint counts, swollen joint counts and disease activity score in 28 joints (DAS28) (r=0.479, P=0.035;r=0.519, P=0.008;r=0.526, P=0.019). However, no correlation was found between these cells and RA patient age, disease duration, or the levels of erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), rheumatoid factor (RF) and anti-citrullinated peptide antibody (ACPA). After in vitro stimulation by TNF-α, but not IL-1β, B10 cells isolated from healthy donors demonstrated fundamentally upregulated expression of RANKL.Conclusion:Our studies showed the frequencies of RANKL-producing B10 cells were markedly higher in RA patients, and their frequencies were positively correlated with RA tender joint counts, swollen joint counts and DAS28. These findings suggested that B10 cells might be involved in RA bone destruction.

Key words: Arthritis,rheumatoid, B10 cells, RANK ligand, Bone destruction

CLC Number: 

  • R593.22

Figure 1

Healthy individual B10 cells and non B10 cells were capable of producing RANKLSSC, side scatter; FSC, forward scatter; RANKL, receptor activator of nuclear factor kappa B ligand; PBMC, peripheral blood mononuclear cell; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. A, representative flow cytometry plot depicting the gating strategy for RANKL expression in healthy individual B10 cells and non-B10 cells; B, CD19+ CD24hi CD270+ and CD19+ CD24loCD27- B cells were sorted by flow cytometry and subjected to PCR analysis of RANKL. The representative gel result was shown. PBMC was used as the positive control."

Figure 2

Increased RANKL expressed on B10 cells in RA patientsAbbreviations as in Figure 1.A,the frequencies of RANKL-producing CD19+CD24hiCD27+ B10 cells in rheumatoid arthritis patient (RA, n=25) and healthy controls (HC, n=20) were analyzed by flow cytometry;B,the frequencies of RANKL-producing non-B10 cells in RA and healthy controls were analyzed by flow cytometry. CD19+CD24hiCD27+ B10 cells from 5 healthy volunteers and 5 RA patients were sorted by flow cytometry,then were subjected to PCR (C) and real-time PCR (D) analyses of RANKL expression."

Table 1

Clinical and laboratory parameters of RA patients"

Items RA patients (n=25)
Age/years, x-±s 54.16±11.82
Gender (female/male) 21/4
Duration/years, x-±s 11.85±7.60
Tender joint counts of 28 joints, x-±s 9.20±6.51
Swollen joint counts of 28 joints, x-±s 9.04±5.90
DAS 28, x-±s 5.04±1.64
ESR/(mm/h), x-±s 48.96±32.30
CRP/(mg/L), median (minimum,maximum) 17.40 (1.57,119)
RF/(IU/mL), median (minimum,maximum) 44.1 (9, 832)
ACPA/(U/mL), x-±s 184.58±114.12

Figure 3

Correlation analysis of RANKL-producing B10 cells with RA patient clinical manifestationsAbbreviations as in Figure 1 and Table 1. The frequencies of RANKL-producing CD19+CD24hiCD27+ B10 cells were positively correlated with RA patient tender joint counts (A),swollen joint counts (B) and DAS 28 (C). No correlation was found between these cells and RA patient age (D),disease duration (E),the levels of ESR (F),CRP (G),ACPA (H) and RF (I)."

Figure 4

TNF-α induced the expression of RANKL in B10 cellsTNF-α,tumor necrosis factor α;IL-1β,interleukin 1β;other abbreviations as in Figure 1. Flow cytometry-sorted CD19+CD24hiCD27+ B10 cells from healthy donors (n=4) were stimulated with TNF-α or IL-1β in vitro for 4 days. Then the relative RANKL expression was evaluated by PCR (A) and real-time PCR (B)."

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