Cathepsin S inhibitor prevents autoantigen presentation and autoimmunity (original) (raw)

Mice. Female NFS/N–strain mice carrying the mutant gene sld (29) were reared in our specific pathogen–free mouse colony, and given food and water ad libitum. Thymectomy was performed on day 3 after birth (3d-Tx) in NFS/sld mice. Nonthymectomied (non-Tx) NFS/sld mice and C56BL/6 mice purchased from Charles River Japan Inc. (Atsugi, Japan) were used as controls.

Measurement of endogenous cathepsin activities. Salivary glands, regional lymph nodes, and spleens from 3d-Tx NFS/sld SS model, non-Tx NFS/sld, and control C57BL/6 mice were used to assay for cathepsin B, L, and S activity. Lysosomes were isolated for the assay by gentle homogenization of samples using a Teflon homogenizer (Microtec Co. Ltd., Funabashi, Japan) pestle in 0.25 M cold sucrose. The suspension was centrifuged at 3,500 g for 10 minutes at 4°C. The supernatant was centrifuged at 25,000 g for 20 minutes at 4°C. The resulting pellet was resuspended with 50 mM acetate buffer (pH 5.0). The suspension fluid was frozen and thawed three times to disrupt lysosomal membranes. After three cycles of freezing and thawing, the fluid was centrifuged and the supernatant was used as a mitochondria and lysosome fraction. Cathepsin activities were assayed with Z-Arg-Arg-methyl coumarylamide (Peptide Institute, Osaka, Japan) as substrate at pH 5.0 for cathepsin B, with Z-Phe-Arg-methyl coumarylamide for cathepsin L, and using the method described by Inubushi et al. (30) for cathepsin S. The reaction was initiated by addition of substrate (10 μM final concentration) after preincubation with the test compound for 3 minutes at 37°C. The fluorescence of the liberated 7-amino-4-methylcoumarin was measured in a fluorescence spectrophotometer (Hitachi Science Systems Ltd., Hitachinaka, Japan). Emission at 460 nm was measured with excitation at 370 nm.

Cathepsin inhibitors. Specific inhibitors for cathepsin B (CA074), cathepsin L (Clik148), and cathepsin S (Clik60) have been developed with the help of computer-graphic modeling based on the stereo-structure as described previously (25–27). The common fragment, _N_-(L-trans-carbamoyloxyrane-2-carbonyl)-phenylalanine-dimethylamide, is required for specific inhibition of cathepsin L. Seven novel inhibitors of the cathepsin L inhibitor Katunuma (Clik) specifically inhibited cathepsin L at a concentration of 10–7 M in vitro, while almost no inhibition of cathepsins B, C, S, and K was observed. Four of the Cliks are stable and showed highly selective inhibition for hepatic cathepsin L in vivo. One of the Clik inhibitors contains an aldehyde group and specifically inhibits cathepsin S at 10–7 M in vitro (27).

In vivo treatment with cathepsin inhibitors. We examined the in vivo therapeutic effects of cathepsin S inhibitor (Clik60), cathepsin B inhibitor (CA074) and cathepsin L inhibitor (Clik148) in a murine model for SS. Each inhibitor (Clik60, CA074, and Clik148), dissolved in PBS, was administered intraperitoneally into model mice at doses of 0.1 mg/mouse/day from 4 weeks to 7 weeks (n = 10 for each), and then analyzed at 8 weeks, compared with untreated SS model mice (n = 7).

Histology. All organs were removed from the mice, fixed with 4% phosphate-buffered formaldehyde (pH 7.2), and prepared for histologic examination. The sections were stained with hematoxylin and eosin. Histological grading of the inflammatory lesions was done according to the method proposed by White and Casarett (31). These slides were scored by three independent, pathologists in a blinded manner.

Proliferation assay. Single-cell suspensions of spleen cells or regional lymph node cells (LNCs) from 3d-Tx, non-Tx NFS/sld, and C57BL/6 mice were cultured in 96-well flat-bottom microtiter plates (Nalge Nunc Intl. Co., Rochester, New York, USA) in RPMI-1640 containing 10% FCS, penicillin/streptomycin, and β-mercaptoethanol. For proliferation assay, a total of 5 × 105 cells per well were cultured for 72 hours under stimulation of recombinant α-fodrin protein (JS-1, 10 μg/ml) (10), ovalbumin (OVA) (10 μg/ml), and concanabalin A (ConA) (5 μg/ml), and pulsed with 1 μCi/well of [3H]thymidine (NEN Life Science Products Inc., Boston, Massachusetts, USA) during the final 20 hours of the culture. [3H]thymidine incorporation was evaluated using an automated β liquid scintillation counter. We further examined the in vitro preventive effects of cathepsin inhibitors (10–7 to 10–4 M CA074, Clik148, and Clik60) for antigen-specific proliferative T cell responses. T cell purification was done using CD4 mAb–bounded immunomagnetic beads (Dynal, Oslo, Norway).

Flow cytometric analysis. Spleen cells and regional LNCs from 3d-Tx and non-Tx NFS/sld mice were analyzed by flow cytometry. Single-cell suspensions were stained with antibodies conjugated to phycoerythrin (anti-CD4; Cedarlane Laboratories Ltd., Hornby, Ontario, Canada) and FITC (anti-CD8 from Cedarlane Laboratories Ltd.; anti-CD44, anti-CD45RB, anti–Mel-14, and anti–I-A_q_ from Pharmingen, San Diego, California, USA), and analyzed with an EPICS (Beckman Coulter, Miami, Florida, USA). Apoptotic or necrotic cells were detected by flow cytometry with an EPICS (Beckman Coulter) using the Annexin V–FITC Apoptosis Detection Kit (Genzyme Pharmaceuticals Co. Ltd., Cambridge, Massachusetts, USA). Viable cells (1 × 105) were gated according to size and scatter to eliminate dead cells and debris from analysis.

Measurement of fluid secretion. Detection of tear and saliva volume of the treated and untreated SS animal model of NFS/sld mice was done according to a method as described (32). Five mice in each group were analyzed.

Measurement of cytokine production. Cytokine production from spleen cells of 3d-Tx and non-Tx NFS/sld mice was tested by two-step sandwich ELISA using a mouse IL-2, IL-4, and IFN-γ kit (Genzyme Pharmaceuticals). In brief, culture supernatants from spleen cells activated with immobilized anti-CD3 mAb (10 μg/ml) (Cedarlane Laboratories Ltd.) for 3 days were added to microtiter plates precoated with anti–IL-2, –IL-4, and –IFN-γ capture antibody and incubated overnight at 4°C. After addition of biotinylated detecting antibody and incubation at room temperature for 45 minutes, avidin-peroxidase was added and incubated at room temperature for 30 minutes. Plates were washed extensively with 1% Tween in PBS between each step. Finally, 2,2′-azino-di-3-ethylbenzthiazoline sulfonate (ABTS) substrate containing H2O2 was added and the colorimetric reaction was read at an absorbance of 450 nm using an automatic microplate reader (BioRad Laboratories Inc., Hercules, California, USA). The concentrations (in pg/ml) of IL-2, IL-4, and IFN-γ were calculated according to the standard curves produced by various concentrations of recombinant cytokines.

Primary culture of mouse salivary gland cells. Mouse salivary gland (MSG) epithelial cells were prepared as previously described (33). Briefly, MSGs were minced into 1-mm2 pieces, washed with HBSS without Ca2+ and Mg2+, and placed in a 60-mm dish containing HBSS with 0.76 μg/ml EDTA, 4.9 μg/ml L-ascorbic acid, and 4.9 μg/ml reduced glutathione. Fragments were washed with DMEM/STI and placed in a mixture of collagenase (type I, 75 U/ml) and hyaluronidase (type IV, 500 U/ml) dissolved in DMEM/F12 containing 10% FBS. The first digest suspension was passed through sterile 100-μm nylon mesh filter and were redigested for 30 minutes by the same digestion procedure, and then the digest suspension was passed through a 100-μm nylon mesh filter. Adherent cells, after culture in MEM containing 10% FBS for 24 hours at 37°C, were isolated as salivary gland epithelial cells. We confirmed that the cells over 95% were positively stained with anti-keratin polyclonal antibody.

Detection of serum autoantibodies against 120-kDa α-fodrin. Serum autoantibody production against 120-kDa α-fodrin was analyzed by immunoblotting and ELISA as described previously (6, 7, 10).

Confocal immunofluorescence analysis. IFN-γ–stimulated and nonstimulated MSG cells were fixed with 1% paraformaldehyde and were incubated with mAb to I-A_q_ molecule (Pharmingen) and FITC-labeled AFN303–318 peptide (described below). In vitro effects of cathepsin inhibitors were examined by the preincubation with each inhibitor (10–5 M) for 6–24 hours. The labeled second antibody was Texas red–conjugated goat anti-mouse IgG (Molecular Probes Inc., Eugene, Oregon, USA). For microscopy, a Leica TCS-NT laser scanning microscope (Leica Microsystems Nussloch GmbH, Nussloch, Germany) was used.