Reactive oxygen species inhibit catalytic activity of peptidylarginine deiminase

<p>Protein citrullination catalysed by peptidylarginine deiminase (PAD) may play an important pathogenic role in several chronic inflammatory diseases and malignancies. PAD2, PAD4, and citrullinated proteins are found in the synovium of rheumatoid arthritis patients. PAD activity is dependent on calcium and reducing conditions. However, reactive oxygen species (ROS) have been shown to induce citrullination of histones in granulocytes. Here we examine the ability of H₂O₂ and leukocyte-derived ROS to regulate PAD activity using citrullination of fibrinogen as read-out. H₂O₂ at concentrations above 40 µM inhibited the catalytic activity of PAD2 and PAD4 in a dose-dependent manner. PMA-stimulated leukocytes citrullinated fibrinogen and this citrullination was markedly enhanced when ROS formation was inhibited by the NADPH oxidase inhibitor diphenyleneiodonium (DPI). In contrast, PAD released from stimulated leukocytes was unaffected by exogenously added H₂O₂ at concentrations up to 1000 µM. The role of ROS in regulating PAD activity may play an important part in preventing hypercitrullination of proteins.</p

Serum complement promotes the binding of MBP to normal B cells.

<p>PBMCs from healthy donors were incubated for 30 min with or without 30 µg/ml biotinylated MBP in medium containing normal serum (30% v/v), or in pure medium. (A) Histogram plot depicting MFI values of MBP binding to B cells in one representative healthy donor. B) The binding of MBP to B cells from 7 healthy donors is shown, expressed as percentage MBP-positive B cells. C) Before addition to the culture media, serum was treated in one of three ways: heat-inactivated (h.i.) by heating to 56°C for 30 min, or supplemented with EDTA or sodium polyanethole sulphonate (SPS) in different concentrations. The resulting MFI values from 5–7 healthy donors are shown. Bars and error bars represent means and SEM *p<0.05 **p<0.01 and ***p<0.001.</p

Uptake and Presentation of Myelin Basic Protein by Normal Human B Cells

<div><p>B cells may play both pathogenic and protective roles in T-cell mediated autoimmune diseases such as multiple sclerosis (MS). These functions relate to the ability of B cells to bind and present antigens. Under serum-free conditions we observed that 3–4% of circulating B cells from healthy donors were capable of binding the MS-associated self-antigen myelin basic protein (MBP) and of presenting the immunodominant peptide MBP85-99, as determined by staining with the mAb MK16 recognising the peptide presented by HLA-DR15-positive cells. In the presence of serum, however, the majority of B cells bound MBP in a complement-dependent manner, and almost half of the B cells became engaged in presentation of MBP85-99. Even though complement receptor 1 (CR1, CD35) and CR2 (CD21) both contributed to binding of MBP to B cells, only CR2 was important for the subsequent presentation of MBP85-99. A high proportion of MBP85-99 presenting B cells expressed CD27, and showed increased expression of CD86 compared to non-presenting B cells. MBP-pulsed B cells induced a low frequency of IL-10-producing CD4+ T cells in 3 out of 6 donors, indicating an immunoregulatory role of B cells presenting MBP-derived peptides. The mechanisms described here refute the general assumption that B-cell presentation of self-antigens requires uptake via specific B-cell receptors, and may be important for maintenance of tolerance as well as for driving T-cell responses in autoimmune diseases.</p></div

Correlation between MBP-induced Th-cell proliferation and production of IL-17 and IL-5.

<p>The MBP-induced IL-5 responses (A) and IL-17 responses (B) were compared with the CD4+ T cell proliferation of seven multiple sclerosis patients who were heterozygous for the minor A-allele of rs5743291. Closed circles (n = 4) represent patients who also carried the minor T-allele of the rs2066842 polymorphism. Spearman's rank correlation coefficient was used to determine association between data sets.</p

Influence of complement on the presentation of MBP85-99 by DR15+ B cells.

<p>PBMCs from healthy HLA-DR15+ donors were incubated for 18 h with MBP in media containing normal serum. Cells were stained with FITC anti-CD19 and biotinylated MK16, followed by streptavidin-PE. (A) The binding of MK16 at different serum concentrations is shown as mean fluorescence (MFI) values normalised to that of 10% serum, (N = 4). B) Before addition of serum (30% v/v), different concentrations of the complement inhibitory compound sodium polyanethole sulphonate (SPS) were added. MFI values are shown, normalised to samples without SPS, (N = 6). (C) The PBMCs were pre-incubated with the anti-CR1 mAb3D9 or polyclonal sheep anti-human CR2, or both, before addition of serum (30% v/v) and MBP. Anti-glycophorin (GP)-A was used as negative control. Data are shown as means±SEM, (N = 4–6). **p<0.01.</p

Patient characteristics.

<p>Minor alleles are marked in bold.</p>1<p>RRMS: relapsing-remitting MS, CIS: clinically isolated syndrome.</p>2<p>Positivity was defined as more than 1% dividing CD4+ T cells after allowance for background proliferation.</p

Influence of NOD2-polymorphisms on MBP-induced Th cell responses.

<p>CFSE-labeled mononuclear cells from twenty-nine patients were incubated for 10 days in medium containing (30% v/v) autologous serum and 30 µg/ml human MBP. (A) The contents of IL-10 in the culture supernatants were assessed after one day of incubation, and the contents of (B) IL-5 and (C) IL-17 were quantified after seven days. After 10 days of incubation, division of CD4+ T cells (D) was tracked by flow cytometry as cells having undergone more than one division. The data were grouped according to the patients' NOD2-polymorphisms: Nine were homozygous wildtypes for both rs2066842 and rs5743291 (WT; open circles), 13 carried the minor allele of rs2066842 only (light shaded circles), three carried the minor allele of rs5743291 (dark shaded circles), and four carried the minor allele of both polymorphisms (closed circles). Net values, after subtractions of background activities, are shown. Horizontal lines show the medians, and P-values were calculated using the Mann-Whitney U-test.</p

Presentation of MBP85-99 by HLA-DR15+ B cells.

<p>PBMCs from HLA-DR15+ individuals were incubated with MBP (whole protein) in the presence or absence of normal serum (30% v/v) for 18 h. Biotinylated mAb MK16 and streptavidin-PE were used as markers of MBP85-99 presentation. A) Representative histogram plot of 5 healthy donors showing binding of MK16 to live (7AAD-negative) B cells in the absence or presence of serum and MPB. B) The percentages of MK16-positive live B cells in 5 healthy HLA-DR15+ donors are shown; background values (no MBP added) have been subtracted. C) MK16 staining of B cells incubated with 30 µg/ml thyroglobulin (Tg), tetanus toxoid (TT), myelin basic protein (MBP) or no antigen (-Ag) in 4 healthy HLA-DR15+ donors (black bars) and 4 healthy HLA-DR15/16 negative donors (white bars). Means and SEM are shown. *p<0.05.</p

Phenotype of MBP85-99-presenting B cells.

<p>PBMCs from healthy HLA-DR15+ donors were incubated with MBP (30 µg/ml) in RPMI containing 30% (v/v) normal serum for 18 h. The presentation of MBP85-99 by CD19+ B cells was assessed using biotinylated MK16 and streptavidin-PE. A) Representative dot plot showing a subset of B cells that present MBP58-99 (MK16+) and a subset that do not (MK16−). Expression of various surface markers was evaluated in these subsets. B) The percentages of MK16+ (black bar) or MK16− (white bar) B cells expressing CD27 are shown (N = 4). B-cell expression of the co-stimulatory molecules C) CD86 and D) CD80 is shown as mean fluorescence intensity (MFI) values (N = 5). 7-AAD was used to exclude dead cells. Data are shown as means and SEM.</p

Influence of the rs5743291 polymorphism on MBP-induced CD4+ T cell responses.

<p>TNF-alpha was produced in all cases and IL-10 in 28 out of 29 cases. These cytokines were not included in the table.</p>1<p>G-allele  =  major frequency allele.</p>2<p>Fisher's exact test.</p>3<p>CD4+ T cell proliferation measured after 10 days incubation.</p>4<p>Cytokines measured in cell cultures supernatants after seven days stimulation with MBP.</p>5<p>Cytokine measured in cell cultures supernatants after one day of stimulation with MBP.</p