Microparticles from patients with systemic lupus erythematosus induce production of reactive oxygen species and degranulation of polymorphonuclear leukocytes

Abstract

Background: The interaction of circulating microparticles (MPs) with immune cells in systemic lupus erythematosus (SLE) is sparsely investigated. We examined the ability of MPs from SLE patients to induce production of reactive oxygen species (ROS) and degranulation of polymorphonuclear leukocytes (PMNs).

Methods: Plasma MPs, leukocytes and sera isolated from 20 SLE patients and 10 healthy controls were mixed in different combinations, with or without lipopolysaccharide (LPS), and incubated for 30 min. Dihydrorhodamine 123 was used to measure ROS production by flow cytometry. The ability of immunoglobulin G (IgG) isolated from five SLE patients to increase MP-induced production of ROS by PMNs was tested. Cell supernatants were analysed for content of primary, secondary and tertiary granule components by Luminex assays.

Results: MPs from SLE patients promoted ROS production by PMNs, and enhanced LPS-induced ROS production and release of primary granules by PMNs, when added to samples of autologous leukocytes and serum. In a similar autologous setting, MPs from healthy controls enhanced LPS-induced ROS production by PMNs. When leukocytes from a healthy control were stimulated with autologous MPs in the presence of various sera, SLE patient serum promoted ROS production and release of primary and secondary granules by PMNs. A role for antibodies in this respect was indicated by the observation that supplementation of normal serum with IgG from SLE patients promoted MP-induced ROS production by healthy PMNs. Moreover, when various MPs were incubated with leukocytes and serum from a healthy control, patient-derived MPs induced more ROS production by PMNs than did healthy control-derived MPs.

Conclusions: SLE patients display increased ROS production and degranulation by PMNs in response to MPs, which partly depends on serum components, including antibodies, MP properties and hyper-responsiveness of the PMNs per se.

Fig. 1

PMN ROS production and degranulation induced by autologous MPs and/or LPS. Leukocytes from 20 SLE patients and 10 healthy controls were incubated with (a) autologous MPs, (b) LPS, (c) a combination of autologous MPs and LPS or (d) PMA for 30 min in the presence of autologous serum and DHR, a probe for H₂O₂ production. Fluorescent oxidation product rhodamine 123 contained in PMNs staining positive for CD15 and negative for the dead cell marker near infrared was measured by flow cytometry and expressed as median fluorescence intensity (MFI) units. (e) Increase in ROS production after addition of MPs or MPs in combination with LPS. PMN production of ROS after stimulation with (f) MPs or (g) PMA, adjusted for background activity (of unstimulated cells), is shown for healthy controls and SLE patients with high and low levels of circulating complement C3, respectively. (h) Concentration of the primary granule protein myeloperoxidase (MPO) released into the cell supernatants. HC healthy control, LPS lipopolysaccharide, MP microparticle, ROS reactive oxygen species, SLE systemic lupus erythematosus, Unstim unstimulated

Fig. 2

Influence of serum on MP-induced ROS production by SLE PMNs. Purified leukocytes from eight SLE patients, six of whom had SLEDAI > 5, were incubated with DHR and stimulated for 30 min at 37 °C with autologous MPs alone or in combination with LPS, or with PMA, in a medium containing 25% v/v autologous serum or a pool of NHS. The resulting ROS production by PMNs was analysed by flow cytometry, and the ratio between median fluorescence intensity (MFI) values obtained in the presence of SLE serum above that obtained in the presence of NHS was calculated after adjustment for background (unstimulated cells). Ratios from three experiments were excluded because either the numerator or denominator was negative. (a) Ratios between MFI values observed after stimulation with MPs and LPS in the presence of autologous serum above that observed in the presence of NHS. Bars represent median ratios. (b) Correlation between the ratio and circulating levels of C3 in samples stimulated with a combination of MPs and LPS. Dotted line represents the lower limit of the normal range. LPS lipopolysaccharide, MP microparticle, PMA phorbol-12-myristate-13-acetate, SLE systemic lupus erythematosus, Unstim unstimulated

Fig. 3

MP-induced ROS production and degranulation of PMNs in the presence of heterologous sera. Leukocytes from a healthy blood group 0 donor were suspended in a medium containing 25% v/v of serum from 20 SLE patients and 10 healthy controls (see Table 1, frozen samples). DHR was used as probe for H₂O₂, and the cells were stimulated with autologous MPs in combination with LPS for 30 min at 37 °C before flow cytometry. (a) Median fluorescence intensity (MFI) after subtraction of background fluorescence (unstimulated cells). SLE patients were divided into two groups with circulating C3 levels > 1 g/l and ≤ 1 g/l, respectively. (b) Correlation between MFI values and anti-dsDNA antibody levels of patient sera. (c–e) Degranulation measured in the corresponding cell supernatants as the content of myeloperoxidase (MPO) from primary granules, neutrophil gelatinase-associated lipocalin (NGAL) from secondary granules and matrix metallopeptidase 9 (MMP-9) from tertiary granules. Bars represent median values. dsDNA double-stranded DNA, HC healthy control, SLE systemic lupus erythematosus

Fig. 4

Influence of exogenously added IgG on MP-induced ROS production by PMNs. Leukocytes purified from three healthy blood group 0 donors were stimulated with autologous MPs, alone (open symbols) or in combination with LPS (closed symbols), in the presence of autologous serum supplemented with various amounts of IgG from SLE patients (SLE IgG) or healthy donors (IVIg). DHR was used as probe for H₂O₂ production. Shown is the resulting median fluorescence intensity (MFI) of PMNs, as measured by flow cytometry. Symbols represent average and error bars represent range. IgG immunoglobulin G, LPS lipopolysaccharide, MP microparticle, SLE systemic lupus erythematosus

Fig. 5

Influence of SLE and healthy control MPs on MP-induced ROS production and degranulation of PMNs. Leukocytes from a healthy control were suspended in a medium containing 25% v/v of normal human serum. DHR was added to the suspensions, and the cells were stimulated with LPS in combination with MPs from 20 SLE patients and 10 healthy controls for 30 min at 37 °C before flow cytometry. (a) The resulting median fluorescence intensity (MFI) after subtraction of background fluorescence (unstimulated cells). Bars represent median values. Comparisons between groups using Mann–Whitney U test (b) Content of neutrophil gelatinase-associated lipocalin (NGAL) in the supernatants of leukocytes stimulated with MPs and LPS in combination shown as median values after subtraction of the background (unstimulated cells). HC healthy control, SLE systemic lupus erythematosus