Maturation of dendritic cells depends on proteolytic cleavage by cathepsin X

Abstract

The maturation status of dendritic cells (DCs) is crucial for effective antigen presentation and initiation of the primary immune response. Maturation stimuli cause the adhesion of immature DCs to the extracellular matrix, which is accompanied by recruitment of the CD11b/CD18 [macrophage antigen-1 (Mac-1)] integrin receptor, cytoskeleton reorganization, and podosome formation. Cathepsin X, a cysteine protease expressed in DCs and other APCs, is involved in Mac-1 activation. We have shown that during maturation, cathepsin X translocates to the plasma membrane of maturing DCs, enabling Mac-1 activation and consequently, cell adhesion. In mature DCs, cathepsin X redistributes from the membrane to the perinuclear region, which coincides with the de-adhesion of DCs, formation of cell clusters, and acquisition of the mature phenotype. Inhibition of cathepsin X activity during DC differentiation and maturation resulted in an altered phenotype and function of mature DCs. It reduced surface expression of costimulatory molecules, increased expression of inhibitory Ig-like transcripts 3 and 4 (ILT3 and ILT4), almost completely abolished cytokine production, diminished migration, and reduced the capacity of DCs to stimulate T lymphocytes. These results stress the importance of cathepsin X in regulating DC adhesion, a crucial event for their maturation and T cell activation.

Figure 1

Inhibition of cathepsin X prevents maturation-induced changes in DC morphology. Monocyte-derived DCs were imaged in tissue-culture flasks using phase-contrast (at 10× original magnification) 48 h after inducing DC differentiation by the addition of rhGM-CSF (500 U/ml) and rhIL-4 (400 U/ml), as well as 4 h and 48 h after promoting DC maturation by the addition of rhGM-CSF (500 U/ml) and 20 ng/ml LPS in the presence or absence of the cathepsin X inhibitor (2F12 mAb). Differential interference contrast images of control and 2F12 mAb-treated DCs after the indicated time are shown. Arrows mark the loosely adherent clumps.

Figure 2

Phenotypic characteristics of DC maturation. Surface marker expression was determined by FACS analysis of mature DCs stimulated for 48 h with LPS (20 ng/ml; A). Broken line shows staining with an isotype control, dotted line the staining of mature DCs, and solid line staining of DCs matured in the presence of the cathepsin X inhibitor. The results are representative of three independent experiments, and the average MFI for control and mAb 2F12-treated DCs is given in the right top corner in histograms. Surface expression of cathepsin X (solid line) was evaluated in adherent maturing and floating mature DCs (B and C). Immature DCs were stimulated with 20 ng/ml LPS for 48 h or TNF-α for 5 days and analyzed for cathepsin X (katX) membrane (nonpermeabilized, DC) or intracellular (permeabilized, DC) expression. Broken lines represent isotype controls (B). Confocal images of cathepsin X translocation to the plasma membrane in maturing, adherent DCs (C). Original scale bars represent 20 μm. Fluorescence microscopy was performed using a Carl Zeiss LSM 510 confocal microscope. Images were analyzed using Carl Zeiss LSM image software 3.0.

Figure 3

Cathepsin X enables podosome formation. Immature DCs on Day 5 of differentiation were centrifuged with cytospin (Cytofuge) for 6 min at 1000 rpm onto glass cover slides. Actin was labeled with phalloidin-tetramethylrhodamine B isothiocyanate conjugate (500 ng/ml) for 30 min at room temperature. Podosome formation, present in control, immature DCs (A), is prevented by inhibition of cathepsin X during DC differentiation (B). Adhesion of maturing DCs coincides with β₂-integrin activation and colocalization with actin (C). Immature and mature DCs were labeled by centrifugation with cytospin (Cytofuge), whereas maturing, adherent DCs were labeled by seeding immature DCs onto glass coverslips in 24-well plates in the presence of 20 ng/ml LPS and allowing adherence for 20 h. The active form of β₂ integrin was labeled with mAb 24 (green fluorescence) and colocalized with actin (red fluorescence) in adherent, mature DCs. Meanwhile, formation of typical dendrites in mature DCs (D) was not inhibited by cathepsin X inhibition (E). Original scale bars represent 20 μm. Fluorescence microscopy was performed using a Carl Zeiss LSM 510 confocal microscope. Images were analyzed using Carl Zeiss LSM image software 3.0.

Figure 4

Cathepsin X colocalizes with the Mac-1 integrin receptor in maturing, adherent DCs. Cathepsin X was labeled with Alexa Fluor 488-labeled mouse 2F12 mAb that recognizes the mature, active form. Absence of colocalization of cathepsin X (green fluorescence) and Mac-1 integrin receptor (red fluorescence) in immature DC (iDC; A) and mature DCs (C) is shown. Differential interference contrast images are shown, respectively. Original bars, 20 μm. Maturing, adherent DCs were labeled by seeding immature DCs onto glass coverslips in 24-well plates in the presence of 20 ng/ml LPS and allowing adherence for 20 h. The translocation of cathepsin X in maturating, adherent DCs to the plasma membrane, where it colocalizes with the Mac-1 integrin receptor, is demonstrated (B). Original scale bars represent 20 μm. Fluorescence microscopy was performed using a Carl Zeiss LSM 510 confocal microscope. Images were analyzed using Carl Zeiss LSM image software 3.0.

Figure 5

Cathepsin X inhibitor abrogates cytokine production in DCs, which were differentiated and subsequently activated (20 ng/ml LPS) in the presence or absence of the cathepsin X inhibitor. Cell-free supernatants were collected after 48 h and after the addition of LPS and then analyzed using ELISA. Student’s t-tests were calculated for control DCs versus mAb 2F12-treated DCs. Mean control DC responses from three independent experiments are shown.

Figure 6

Cathepsin X enables adhesion of immature DCs upon addition of maturation stimulus. Immature DCs were matured with 20 ng/ml LPS on a plastic (A)- or fibrinogen (B and C)-coated surface in the absence (A) or presence of cathepsin X inhibitor mAb 2F12 (B) and AMS36 (C). The percentage of adherent DCs was estimated with the MTS assay. Data are as diagrams showing the time course of DC adhesion to a plastic- or fibrinogen-coated surface, respectively (see also Supplementary Videos 1 and 2 for adhesion in the presence or absence of the cathepsin X inhibitor 2F12 mAb to a fibrinogen-coated surface). (Lower) Photomicrographs of adherent DCs on a plastic (A)- or fibrinogen-coated surface (B) after the indicated time (2 h or 5 h) of LPS stimulation are shown for control and mAb 2F12-treated cells.

Figure 7

Mature DCs, differentiated and matured in the presence of the cathepsin X inhibitor, exhibit reduced migration and T cell-stimulatory capacity. The capacity of mature DCs to migrate was evaluated in 5 μm transwell plates. The chemotactic response of mature DCs to fibrinogen or fibronectin + MCP-1 (50 ng/ml) is presented as the percentage of transmigrated DCs in the bottom compartment of a transwell plate. Mature DCs, differentiated and matured in the presence of the cathepsin X inhibitor, 0.5 μM, show reduced migration ability compared with untreated, mature DCs, irrespective of chemotactic stimulus (A). The cathepsin X inhibitor was not present in the migration assay. Bars represent mean percentage of migrated DCs ± sd. Mature DCs were pretreated with mitomycin C and subsequently cocultured with purified, allogeneic T cells or mononuclear cells (MNC; B). Samples were treated in quadruplicate. Results are the means ± sd of at least three independent assays.

Figure 8

HPLC analysis of the β₂-integrin cytoplasmic domain cleaved with recombinant cathepsin X. The noncleaved cytoplasmic domain (cytoplasmic domain NPKFAES⁷⁶⁹) and corresponding cleaved cytoplasmic domain (cytoplasmic domain NPK⁷⁶⁵), detected after treatment with recombinant cathepsin X, were identified with electron-mass spectrometry in the fraction eluted between 8.6 and 9.5 min. Other peaks did not contain any cleaved peptides. Inhibition with 2F12 mAb completely abolished cathepsin X proteolytic activity, and the peak was identical to that of noncleaved peptide.