Abstract
Autoantibodies against citrullinated proteins are diagnostic for rheumatoid arthritis. However, the molecular mechanisms driving protein citrullination in patients with rheumatoid arthritis remain poorly understood. Using two independent western blotting methods, we report that agents that trigger a sufficiently large influx of extracellular calcium ions induced a marked citrullination of multiple proteins in human neutrophils, monocytes, and, to a lesser extent, T lymphocytes and natural killer cells, but not B lymphocytes or dendritic cells. This response required 250–1,000 μM extracellular calcium and was prevented by EDTA. Other neutrophil activating stimuli, such as formyl-peptides, GM-CSF, IL-6, IL8, TNFα, or phorbol ester, did not induce any detectable increase in protein citrullination, suggesting that receptor-induced calcium mobilization is insufficient to trigger hypercitrullination. We conclude that loss of membrane integrity and subsequent influx of high levels of calcium, which can be triggered by perforin released from cytotoxic cells or complement mediated formation of membrane attack complexes in the joints of rheumatoid arthritis patients, are sufficient to induce extensive protein citrullination in immune cells, notably neutrophils. This mechanism may provide the citrullinated autoantigens that drive autoimmunity in this devastating disease.
Highlights
Rheumatoid arthritis is a common form of arthritis, second only after osteoarthritis in prevalence, and is still for most patients a severe, painful, and debilitating disease [1]
Plasmacytoid dendritic cells, myeloid dendritic cells, and natural killer cells (NK cells) were isolated by negative immunoselection from fresh leukopacks obtained from healthy donors (Stemcell Technology, negative selection markers for NK cells were CD3, CD4, CD14, CD19, CD20, CD36, CD66b, CD123, HLA-DR, and glycophorin A; pDC and mDC selection markers were not disclosed by manufacturer) following manufacturer’s instructions with a purity of over 95%
Reactivity to both antibodies was essentially undetectable in untreated human neutrophil samples but increased in a time-dependent manner in cells treated with ionomycin (Figure 1(a))
Summary
Rheumatoid arthritis is a common form of arthritis, second only after osteoarthritis in prevalence, and is still for most patients a severe, painful, and debilitating disease [1]. Beginning in the earliest stages of RA, the synovial fluid has a high content of neutrophils [2]. Their presence correlates with joint stiffness, changes in the lubricating properties of the synovial fluid [5], and the escalating inflammation of the surrounding joint tissue referred to as synovitis. Single-nucleotide polymorphisms in the gene for PAD4, PADI4, are consistently associated with RA [7, 8] in Asian population and possibly in Caucasian populations [9], further strengthening the notion that granulocytederived PAD4 may be a key player in the citrullination of proteins in the synovial fluid, which subsequently acts as autoantigens to trigger an immune response resulting in anticitrulline-peptide antibodies [6]
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