Dendritic cells (DC) comprise a heterogeneous network of professional antigen-presenting cells, directly linking innate and adaptive immunity. While implicated in the pathogenesis of different chronic inflammatory arthritides, the analysis of DC subsets has been hampered by a lack of specific DC markers and reliable quantitation. Previously, we have described the significant reduction of circulating peripheral blood (PB) myeloid DC (mDC) in rheumatoid arthritis (RA) patients and the significant reduction of circulating PB plasmacytoid DC (pDC) in both RA and psoriatic arthritis patients. Furthermore, we have shown that both of these DC subsets are present in synovial fluid (SF) from RA and psoriatic arthritis patients, although mDC significantly exceed pDC.
This present study characterises the immunophenotype and functional characteristics of RA-derived mDC and pDC in order to assess their potential pathogenic role in arthritis.
pDC and mDC were sequentially purified by magnetic cell sorting (MACS) using anti-neuropilin-conjugated magnetic microbeads (pDC) and biotin-conjugated anti-CD1c followed by positive selection using anti-biotin-conjugated magnetic microbeads (mDC), from RA PB (n = 3) and SF (n = 3) and compared with normal healthy controls (mDC, n = 4; pDC, n = 5). Purified cells were phenotyped using CD1c-FITC (mDC only), BDCA-2-FITC (pDC only), CD123-PE (pDC only), CCR5-PE (pDC only), CD11c-PE (mDC only), CD14-PE (mDC only), CD40-APC, CD62L-FITC, CD80-FITC, CD83-APC, CD86-APC, CCR7-PE pre and post 24-hour stimulation with TLR2 agonist S. aureus peptiodoglycan (mDC) or TLR9 agonist CpG oligonucleotide (ODN) 2216 (pDC). Supernatants were harvested from all cultures and cytokine concentrations analysed using Luminex.
Circulating RA PB-derived mDC and pDC display a typical immature DC phenotype compared with mDC and pDC from healthy control subjects; however, L-selectin (CD62L) expression was significantly decreased on RA pDC (P = 0.0369) but not mDC (P = 0.1573). Conversely, while RA SF-derived pDC displayed an immature phenotype directly comparable with their normal PB-derived counterparts, RA SF-derived mDC displayed increased CD80 and CD86, but decreased CD62L, suggesting a less immature phenotype. Moreover, RA SF-derived mDC had a higher CD14 expression, suggesting that these cells may be monocyte-derived mDC. FACScan analysis revealed that both SF-derived DC subsets upregulated maturation markers following Toll-like receptor stimulation. Cytokine analysis revealed that while pDC from both normal PB and RA SF produced large levels of interferon gamma and tumour necrosis factor alpha in response to TLR9 stimulation, only RA SF pDC produced low levels of IL-10 and interferon gamma. Similarly, mDC from both normal PB and RA SF produced large levels of tumour necrosis factor alpha; however, RA SF mDC produced more IL-10 than their normal PB counterparts.
We report the ex vivo immunophenotype and functional characteristics of circulating RA PB-derived and SF-derived mDC and pDC.