Protein synthesis of the pro-inflammatory S100A8/A9 complex in plasmacytoid dendritic cells and cell surface S100A8/A9 on leukocyte subpopulations in systemic lupus erythematosus

Abstract

Introduction: Systemic lupus erythematosus (SLE) is an autoimmune disease with chronic or episodic inflammation in many different organ systems, activation of leukocytes and production of pro-inflammatory cytokines. The heterodimer of the cytosolic calcium-binding proteins S100A8 and S100A9 (S100A8/A9) is secreted by activated polymorphonuclear neutrophils (PMNs) and monocytes and serves as a serum marker for several inflammatory diseases. Furthermore, S100A8 and S100A9 have many pro-inflammatory properties such as binding to Toll-like receptor 4 (TLR4). In this study we investigated if aberrant cell surface S100A8/A9 could be seen in SLE and if plasmacytoid dendritic cells (pDCs) could synthesize S100A8/A9.

Methods: Flow cytometry, confocal microscopy and real-time PCR of flow cytometry-sorted cells were used to measure cell surface S100A8/A9, intracellular S100A8/A9 and mRNA levels of S100A8 and S100A9, respectively.

Results: Cell surface S100A8/A9 was detected on all leukocyte subpopulations investigated except for T cells. By confocal microscopy, real-time PCR and stimulation assays, we could demonstrate that pDCs, monocytes and PMNs could synthesize S100A8/A9. Furthermore, pDC cell surface S100A8/A9 was higher in patients with active disease as compared to patients with inactive disease. Upon immune complex stimulation, pDCs up-regulated the cell surface S100A8/A9. SLE patients had also increased serum levels of S100A8/A9.

Conclusions: Patients with SLE had increased cell surface S100A8/A9, which could be important in amplification and persistence of inflammation. Importantly, pDCs were able to synthesize S100A8/A9 proteins and up-regulate the cell surface expression upon immune complex-stimulation. Thus, S100A8/A9 may be a potent target for treatment of inflammatory diseases such as SLE.

Figure 1

Increased cell surface S100A8/A9 in patients with active disease compared with inactive disease and healthy controls. The cell surface S100A8/A9 was determined by flow cytometry on A) CD14⁺⁺CD16⁺, B) CD14⁺⁺CD16^-, C) CD3⁺, D) CD19⁺, E) CD16+ PMNs, F) BDCA-1⁺ and G) BDCA-2⁺ cells. The expression is defined as the mean fluorescence index (MFI) ratio between the S100A8/A9 antibody and its control isotype antibody in each experiment. The line represents the median-value. Active disease was defined as SLEDAI > 4 (SLE active).

Figure 2

S100A8/A9 mRNA expression in different cell populations, serum levels and cell surface S100A8/A9 upon pDC activation. A) The relative expression of S100A8 and S100A9 mRNA in different cell populations. PBMCs were isolated and sorted by flow cytometry before determining the mRNA levels of S100A8 and S100A9 by real-time PCR. B) Serum levels of S100A8/A9 measured by an in-house ELISA in SLE patients and healthy controls. The line represents the median value. C) Purified pDCs were stimulated with immune complexes for 20 h and analyzed for cell surface S100A8/A9 by flow cytometry. The data are presented as the mean fluorescence index (MFI) ratio with one standard deviation as compared to unstimulated cells for each experiment.

Figure 3

Analysis of S100A8/A9 staining in different leukocyte populations by confocal microscopy. Cells were isolated, fixed, permeabilized and stained for S100A8/A9 with the 27E10 antibody. T cells, B cells and mDCs (A, C and E, respectively) had no detectable S100A8/A9 expression while PMNs, pDCs and monocytes (B, D and F, respectively) had membrane-associated, as well as intracellular, S100A8/A9.