The DEK nuclear autoantigen is a secreted chemotactic factor

Abstract

The nuclear DNA-binding protein DEK is an autoantigen that has been implicated in the regulation of transcription, chromatin architecture, and mRNA processing. We demonstrate here that DEK is actively secreted by macrophages and is also found in synovial fluid samples from patients with juvenile arthritis. Secretion of DEK is modulated by casein kinase 2, stimulated by interleukin-8, and inhibited by dexamethasone and cyclosporine A, consistent with a role as a proinflammatory molecule. DEK is secreted in both a free form and in exosomes, vesicular structures in which transcription-modulating factors such as DEK have not previously been found. Furthermore, DEK functions as a chemotactic factor, attracting neutrophils, CD8+ T lymphocytes, and natural killer cells. Therefore, the DEK autoantigen, previously described as a strictly nuclear protein, is secreted and can act as an extracellular chemoattractant, suggesting a direct role for DEK in inflammation.

FIG. 1.

Intracellular DEK is not an exclusively nuclear protein. Human peripheral blood monocytes were isolated and cultured using a glass chamber slide system and were maintained in 40% human serum. Cells were placed in serum-free medium overnight before staining the next day. Day 3 MDM (panel a), day 5 MDM (panel c), or day 12 MDM (panel e) were incubated with DEK polyclonal antiserum and stained with FITC-conjugated goat anti-rabbit antibody for immunodetection by confocal microscopy. Nuclear staining with 4′,6′-diamidino-2-phenylindole (DAPI) is shown in panels b, d, and f (magnification, ×100). The white arrowheads in panel e show MDM with minimal or no nuclear staining for DEK. The corresponding nuclei are again indicated by the white arrowheads in panel f.

FIG. 2.

DEK is secreted in a CK2-dependent manner as monocytes differentiate into MDM. Monocytes incubated in 40% human serum for 3, 7, and 12 days were washed and maintained in serum-free medium for 12 h prior to collection of cell supernatants. A total of 30 μg of protein was subjected to Western blot analysis using anti-DEK monoclonal antibody. The arrows in panels A and B indicate the 50- and 35-kDa forms of DEK. (A) Western blot of supernatants and nuclear extracts. Total nuclear protein loaded is shown by Ponceau S staining. (B) Day 8 MDM treated with a 50 μM concentration of the highly specific CK2 blocker TBB (+) or with the dimethyl sulfoxide control (−) for 3 h were washed and maintained in serum-free medium for the indicated time. Supernatants were then collected and analyzed by Western blotting using mouse monoclonal anti-DEK antibody, polyclonal CD81 antibody, or antivimentin antibody. (C) Increasing amounts of concentrated supernatant from day 12 MDM were collected and analyzed for the presence of histones by silver staining. For controls, purified histones and cell extract were run on the same gel.

FIG. 3.

DEK is secreted in exosomes and in an exosome-free form from differentiated MDM. (A) Colocalization of DEK and the exosomal marker CD81 by confocal microscopy. Day 13 MDM cultured in glass chamber slides as described in Materials and Methods were incubated with mouse anti-CD81 antibody and stained with an Alexa Fluor-conjugated rabbit anti-mouse antibody to detect CD81 (panel b) or incubated with goat polyclonal DEK antiserum and stained with a FITC-conjugated rabbit anti-goat antibody to detect DEK (panel c). Colocalization of DEK and CD81 (panel d, shown in yellow and indicated by white arrowheads) was detected in vesicular structures throughout the cells. Cell nuclei were stained with 4′,6′-diamidino-2-phenylindole (DAPI) (blue) (panel a). Magnification for all four panels, ×200. (B) Western blot analysis of exosomes in supernatants from day 15 MDM. Human peripheral blood monocytes were isolated and maintained in 40% serum for 15 days. Cells were placed in serum-free medium overnight before supernatant collection. Supernatants were centrifuged at 500 × g, 10,000 × g, and 70,000 × g as described in Materials and Methods. The pellets from specific fractions were solubilized in sample buffer for Western blot analysis. Exosomes are expected to be present in the 70,000 × g fraction (9). (C) Western blot analysis of the exosome-containing and exosome-free fractions from day 15 MDM supernatants. Concentrated supernatant fractions, with exosomes (70,000 × g) and without exosomes, were probed with mouse monoclonal anti-DEK antibody or goat polyclonal anti-CD81 antibody. (D) The 70,000 × g, exosome-containing fraction was used in a proteinase K protection assay. The sample in lane 1 was not treated, that in lane 2 was treated with proteinase K (prot K) alone, and that in lane 3 was treated with proteinase K and Triton X-100. Samples were analyzed for the presence of DEK and CD81 under reducing conditions by Western blot analysis, using specific goat polyclonal anti-DEK antibody or anti-CD81 antiserum. DEK is detected at 50 kDa and 35 kDa, and CD81 is detected at 20 kDa.

FIG. 4.

Immunosuppressive agents block the secretion of DEK. (A) Synovial fluid samples obtained from a patient with a noninflammatory Baker's cyst (lane 1) or patients with JIA (lanes 2 and 3) were analyzed for the presence of DEK by Western blotting after the fluid was separated from cells by centrifugation as described in Materials and Methods. DEK was detected by Western blotting using the mouse monoclonal anti-DEK antibody. (B) Day 11 serum-activated MDM were incubated in serum-free medium for 12 h alone or in the presence of 0.5 μM or 1 μM dexamethasone (Dex). (C) Day 12 serum-differentiated MDM were incubated in serum-free medium alone for 12 h or in the presence of 10 μM CCCP or 1 μg/ml CsA. In panels B and C, secreted DEK was again detected in the supernatant by Western blotting using DEK-specific monoclonal antibody.

FIG. 5.

DEK is a chemoattractant for monocyte-depleted peripheral white blood cells. (A) IL-8 induces the secretion of DEK from MDM. Day 12 MDM grown in 10% serum were maintained for 12 h in serum-free conditioned medium alone or with 10 ng/ml of recombinant IL-8. Supernatants were then harvested, and secreted DEK was detected in the supernatant by Western blotting using DEK-specific monoclonal antibody. (B) DEK is a chemoattractant. Monocyte-depleted peripheral white blood cells were prepared as described in Materials and Methods. A total of 1 × 10⁵ cells in 100 μl of RPMI 1640 medium were placed in the upper chambers of a 24-well chemotaxis microchamber plate with a pore size of 5 μm. The lower chambers contained RPMI 1640 medium with various concentrations of recombinant DEK (made in baculovirus), recombinant GLI-2 control protein (also made in baculovirus), or no protein control (C′). After 1 h, the number of fluorescently labeled migrating cells in the lower chamber was determined at 485- or 535-nm wavelengths by a Tecan GENios plate reader (Phenix, Austria). The results are expressed as the average increase ± standard deviation (error bar) of the number of cells migrating toward DEK or GLI-2 compared to control wells containing medium only. Each assay was run in duplicate. Data shown using 34 nM and 68 nM DEK are the averages from three different donors, while data using 136 nM DEK are the averages from six different donors. The increase in the migration of monocyte-depleted peripheral white blood cells toward 136 nM DEK versus GLI-2 or no-protein control is statistically significant (P = 0.006). (C) DEK attracts CD8⁺ T cells and CD56⁺ natural killer cells. Monocyte-depleted peripheral white blood cells were placed in the upper chamber of a 24-well chemotaxis microchamber plate with a pore size of 5 μm. Lower chambers contain either 79 nM DEK in RPMI 1640 medium or medium alone. Cells were collected 1 h after migration and double stained with immunofluorescent antibodies to CD8⁺ (PE) and CD4⁺ (FITC) or CD56 (PE) and CD19 (FITC). Fluorescence profiles were recorded by FACS analysis. The results are expressed as the average increase ± standard deviation (error bar) of the percentage of cells migrating toward DEK versus control wells containing medium only. Migration of CD8⁺ cells and CD56⁺ cells toward DEK is statistically significant (*, P = 0.034; **, P = 0.0028). The results shown represent the averages of three experiments using cells from three different donors.

FIG. 6.

DEK is a chemoattractant for neutrophils. (A) Neutrophils purified as described in Materials and Methods were placed in the upper chambers of a 24-well chemotaxis microchamber plate with a pore size of 3 μm as described in the legend to Fig. 5. The lower chambers contained RPMI 1640 medium with various concentrations of recombinant DEK, as indicated. The bar labeled “control” contained only medium in the lower chamber. The bar labeled “DEK up” had DEK in the upper chamber and medium in the lower chamber. Data shown represent the averages ± standard deviations (error bars) for two experiments with cells from two different donors. The increase in neutrophil migration toward 79 nM DEK protein versus control is statistically significant (*, P = 0.0067). (B) Groups of mice received intraperitoneal injections of either PBS or 50 μg of either LPS, recombinant DEK, or a recombinant protein control prepared in parallel to DEK in baculovirus (an isoform of human GLI-2). Twenty mice were tested in each of the four groups. Half of the mice in each group were sacrificed after 4 h (white bars), and the other half were sacrificed at 8 h (black bars) after intraperitoneal injection and peritoneal lavage. The bars represent the average numbers ± standard deviations (error bars) of neutrophils migrating into the peritoneum of each group of mice. DEK significantly increased the infiltration of neutrophils compared to the control protein human GLI-2 (P = 0.011). PMNs, polymorphonuclear leukocytes.

FIG. 7.

Schematic model of DEK secretion by MDM. DEK is mainly a DNA-binding nuclear protein in undifferentiated monocytes/macrophages (step 1). With the acquisition of a more differentiated phenotype by MDM, DEK is incorporated in secretory vesicles in the multivesicular compartment and secreted via exosomes (step 2). Possibly through an intermediate step involving exosomal pickup by antigen-presenting cells (APC) (step 3), B cells produce autoantibodies against DEK (step 4). Alternatively, via an incompletely defined, Golgi apparatus-independent pathway (step 5), DEK is directly secreted into the extracellular milieu, where it acts as a chemoattractant for leukocytes (step 6). PMNs, polymorphonuclear leukocytes.