Spontaneous Secretion of the Citrullination Enzyme PAD2 and Cell Surface Exposure of PAD4 by Neutrophils

Abstract

Autoantibodies directed against citrullinated epitopes of proteins are highly diagnostic of rheumatoid arthritis (RA), and elevated levels of protein citrullination can be found in the joints of patients with RA. Calcium-dependent peptidyl-arginine deiminases (PAD) are the enzymes responsible for citrullination. PAD2 and PAD4 are enriched in neutrophils and likely drive citrullination under inflammatory conditions. PADs may be released during NETosis or cell death, but the mechanisms responsible for PAD activity under physiological conditions have not been fully elucidated. To understand how PADs citrullinate extracellular proteins, we investigated the cellular localization and activity of PAD2 and PAD4, and we report that viable neutrophils from healthy donors have active PAD4 exposed on their surface and spontaneously secrete PAD2. Neutrophil activation by some stimulatory agents increased the levels of immunoreactive PAD4 on the cell surface, and some stimuli reduced PAD2 secretion. Our data indicate that live neutrophils have the inherent capacity to express active extracellular PADs. These novel pathways are distinguished from intracellular PAD activation during NETosis and calcium influx-mediated hypercitrullination. Our study implies that extracellular PADs may have a physiological role under non-pathogenic conditions as well as a pathological role in RA.

Keywords: PAD2; PAD4; citrullination; neutrophil; rheumatoid arthritis.

Figure 1

Immunofluorescence staining of PAD4 and PAD2 in resting neutrophils. Staining of permeabilized neutrophils with four different anti-PAD4 antibodies as indicated in panel (A) or PAD2 antibody in panel (B). DAPI staining was used to identify nuclear DNA. In parallel samples, recombinant PAD2 or PAD4 were preincubated with antibodies prior to cell staining. Results are representative of three different healthy donors.

Figure 2

Immunofluorescence and flow cytometric analysis of PAD4 and PAD2 in non-permeabilized neutrophils. (A) (Upper panels) Panel 1: staining of non-permeabilized neutrophils with an anti-PAD4 antibody; panel 2: phase-contrast image of panel 1; panel 3: overlay of anti-PAD4 staining and the phase-contrast image; panel 4: a parallel sample stained with anti-PAD4 incubated with recombinant PAD4 prior to cell staining. (Lower panels) Panels 1, 2, and 4: several different fields of anti-PAD4 staining overlaid with phase-contrast images; panel 3: magnified portion of the image above. (B) Flow cytometry of non-permeabilized neutrophils using an isotype-matched control antibody (gray shading), anti-PAD4 (red line), anti-PAD4 preincubated with recombinant PAD2 (black line), or anti-PAD4 preincubated with recombinant PAD4 (blue line). (C) Quantification of the mean fluorescence intensity (MFI) from the analysis of five separate donors analyzed as described in panel (B). (D) Similar experiment conducted with anti-PAD2 antibodies. (E) Quantification of the MFI from the analysis of five separate donors analyzed as described in (D). Data were analyzed by paired t-test (*p < 0.05, ^nsp > 0.05).

Figure 3

Flow cytometric analysis of surface PAD4 expression by stimulated neutrophils obtained from healthy donors. Flow cytometric analysis of surface PAD4 expression on non-permeabilized neutrophils from three healthy donors following treatment with various stimuli; medium alone (gray shaded) was used as a control. (A) Immune complexes (IC) (50 ng RNP plus 2% anti-RNP containing SLE serum) (red line) and the same IC diluted 1/3 (blue line), 1/9 (green line), 1/27 (purple line), and 1/81 (light blue line); (B) TNF-α, (C) PMA, (D) flagellin, (E) FSL-1, (F) IL-8. Concentrations of stimuli used in panels (B–F) were 10 ng/ml (red line), 3 ng/ml (blue line), and 1 ng/ml (green line), 0.3 ng/ml (purple line), and 0.1 ng/ml (light blue line). Bar graphs on the right shows the average mean fluorescence intensity (MFI) from the analysis of the three donors by paired t-test (*p < 0.05, **p < 0.01, ^nsp > 0.05).

Figure 4

Flow cytometric analysis of surface PAD4 levels in whole blood samples. Summary of mean fluorescence intensity (MFI) of PAD4 staining of different cells types from healthy donors, patients with rheumatoid arthritis (RA), and patients with SLE. (A) Neutrophils from healthy donors (n = 8) and patients from RA (n = 18). The difference between the two groups is not statistically significant by unpaired t-test. (B) Monocytes from the same donors in panel (A). (C) neutrophils from healthy donors (n = 5) and patients from SLE (n = 9), ^nsp > 0.05.

Figure 5

Citrullination of extracellular substrates by intact neutrophils or neutrophil-conditioned media. (A) Upper panel, anti-citrullinated fibrinogen immunoblot of the supernatant from the incubation of fibrinogen for the indicated times in the presence of human neutrophils (lanes 1–7). Lane 7, fibrinogen was omitted. Lane 8 only contained fibrinogen. Lane 9, fibrinogen and recombinant PAD4. Lane 10, neutrophil-conditioned media. Lane 11, neutrophil with 2 mM EGTA. Lower panel, Coomassie Brilliant blue staining as a loading control. The bands corresponding to the α, β, and γ chains of fibrinogen are indicated. Note that α-fibrinogen is rapidly degraded by neutrophil-associated proteases. (B) Control immunoblot with anti-GAPDH, an intracellular protein, to demonstrate that significant lysis of neutrophils occurs only after 4 h of incubation with Triton X at 37°C under the experimental conditions. (C) Upper panel, anti-citrullinated histone H3 immunoblot of the supernatant from the incubation of histone H3 for the indicated time periods in the presence of human neutrophils (lanes 1–7). Lane 7, histone was omitted. Lane 8 only contained histone H3. Lane 9 contained histone H3 and recombinant PAD4. Lane 10, neutrophil-conditioned media. Lane 11, neutrophil with 2 mM EGTA. Note that citrullinated histone H3 is also cleaved by neutrophil-associated protease(s) to generate a slightly smaller protein. Lower panel, anti-histone H3 immunoblot as a loading control. This antibody does not recognize the proteolytically cleaved H3. (D) A similar experiment performed in the presence of an MMP inhibitor, a protease inhibitor cocktail, or both, as indicated. Lower panel, Coomassie Brilliant blue staining as a loading control. All data are representative of five independent experiments with different donors.

Figure 6

Western blot analysis of secreted PADs in neutrophil-conditioned media and determination of neutrophil PAD2/PAD4 extracellular activity. (A) Anti-PAD2 immunoblot of concentrated neutrophil-conditioned media from four different healthy donors (D1–D4). (B) Anti-PAD4 immunoblot of same samples with longer exposure time. (C) Characterization of the blocking anti-PAD2 and anti-PAD4 antibodies. Upper panel, anti-citrullinated histone H3 immunoblot of a reaction with recombinant PAD2 in the presence of control NIP228 IgG antibody (lane 1), blocking anti-PAD4 antibody (lanes 2–4), or a blocking anti-PAD2 antibody (lanes 5–7) at the indicated concentrations. Lower panel, similar reaction with recombinant PAD4 in the presence of the same antibodies. (D) Anti-citrullinated histone H3 immunoblot of the culture supernatant of human neutrophils incubated with histone H3 plus blocking anti-PAD2 or anti-PAD4 antibodies as indicated (lane 1–5); 2 mM EDTA was added to demonstrate the calcium-dependence of the reaction (lane 6); neutrophil-conditioned media, plus blocking antibodies as indicated (lanes 7–11). No antibodies were added in lanes 1 and 7, whereas control IgG NIP228 was added in lanes 2 and 8. (E) Anti-citrullinated fibrinogen immunoblot of the supernatant from the incubation of fibrinogen with neutrophils plus blocking antibodies. No antibodies were added in lane 1, whereas control IgG NIP228 was added in lane 2. Lower panel, Coomassie Brilliant blue staining as a loading control. All data are representative of five independent experiments with different donors.

Figure 7

ELISA-based protein citrullination analysis of kinetics and activity of PADs released from neutrophils. (A) Dose–response curves of fibrinogen citrullination mediated by recombinant human PAD2 and PAD4. (B) Kinetics of fibrinogen citrullination by neutrophil-conditioned media. (C) Citrullination of fibrinogen and (D) histone H3 by neutrophil-conditioned media can be blocked by the anti-PAD2 inhibitory antibody but not the anti-PAD4 antibody. All data are representative of four independent experiments with different donors.

Figure 8

ELISA-based histone H3 citrullination analysis of regulation of PAD2 secretion by neutrophil activators. Quantification of PAD2 activity in conditioned media from neutrophils stimulated with 50 ng RNP plus 2% anti-RNP containing SLE serum, 10 ng/ml PMA and 10 ng/ml lipopolysaccharide (LPS). Data represent the mean OD reading ± SD of four donors with statistical analysis by paired t-test (*p < 0.05).