Animals and antibodies
Female Lewis rats (8–16 weeks old; body weight 150–220 g) were obtained from Charles River (Sulzfeld, Germany) or from the Medical Experimental Center of the University of Leipzig. Female inbred Wistar–Prob rats (12–16 weeks old) were bred in the Medical Experimental Center. The animals were kept under standard conditions, two per cage, with food and water ad libitum and a 12 h/12 h light/dark cycle.
The mouse anti-rat-CD4 mAbs W3/25, OX35, and RIB5/2 (IgG1, IgG2a, and IgG2a, respectively) were used as ascites fluid for in vivo treatment or purified on Protein A–sepharose (for OX35 and RIB5/2) or Protein G–sepharose (for W3/25) columns (Pharmacia, Freiburg, Germany) for all in vitro experiments. The maximal endo-toxin content of the ascites was 58 IU/ml (W3/25), 17 IU/ml (RIB5/2), and 5 IU/ml (OX35); the final endotoxin concentrations for in vitro experiments were less than 1 IU/ml. For flow cytometry, directly FITC-labeled W3/25 and OX35 (gifts from P Kühnlein, Institute of Virology, University of Würzburg), and the mAbs G4.18 (anti-CD3, Pharmingen, Hamburg, Germany), OX8 (anti-CD8, Serotec, Oxford; UK), OX81 (anti-IL4, Pharmingen), A5-4 (anti-IL-10, Pharmingen), and DB-1 (anti-IFN-γ, Serotec) were used. MAbs MOPC21 (IgG1), UPC10 (IgG2a, both from Sigma, Deisenhofen, Germany) and mouse anti-glucose-oxidase FITC/phycoerythrin (PE) (IgG1, IgG2a, Dako, Hamburg Germany) served as isotype-matched control antibodies. The anti-TCRα/β mAb R73 was a kind gift of T Hünig (Institute of Virology, University of Würzburg). For sandwich ELISA analysis of cytokine concentrations, the following antibodies were used: DB-1 (anti-IFN-γ), biotinylated rabbit anti-rat IFN-γ (both Biosource, Ratingen, Germany); rabbit anti-rat IL-2, biotinylated A38-3 (anti-IL-2, both Pharmingen), A5-7, biotinylated A5-6 (both anti-IL-10, Pharmingen); OX81 (anti-IL-4), biotinylated rabbit anti-rat IL-4 (Biosource).
The mAbs were bound to a BIAcore surface via goat anti-mouse Ig-Fc and incubated with various concentrations of soluble rat CD4 (a gift of N Barclay, Oxford, UK). The data were analyzed in accordance with the method of Karlsson et al.[S1], and the association rate constant kass, the dissociation rate constant kdiss, and the affinity constant KA (kass/kdiss) were calculated.
Adjuvant arthritis and anti-CD4 treatment
Lewis rats (8–12 weeks old) were given 0.5 mg heat-inactivated M. tuberculosis (Difco, Detroit, MI, USA) in 100 μl paraffin oil (Riedel de Haën, Seelze, Germany), which was injected intradermally into the base of the tail. For preventive treatment, the rats (n = 6 in each group) received 3 mg of RIB5/2, 3 mg of W3/25, or 2 mg of OX35 intraperitoneally (corresponding to 17.1 and 11.4 mg/kg, respectively, on the basis of a mean body weight of 175 g) on days -1, 0, 3, and 6, i.e. 1 day before AA induction, on the day of induction (day 0), and thereafter. The lower dose of OX35 (2 mg) was chosen on the basis of previous experiments demonstrating its high clinical efficacy in AA [S2]. The control group received PBS [S3]. In addition, preventive treatment with an isotype-matched mAb from birth had shown no effect on rat AA in a previous study [S3].
For determination of the arthritis score, each paw was graded according to the extent of erythema and edema of the periarticular tissue, on a scale of 0–4, where 0=no inflammation, 1=unequivocal inflammation of one paw joint, 2=unequivocal inflammation of at least two paw joints or moderate inflammation of one paw joint, 3=severe inflammation of one or more paw joints, and 4=maximum inflammation of one or more paw joints [S4, S5]. The scores were then added to obtain the total score (maximal possible score of 16 for each animal).
For determination of cell depletion and modulation of surface molecules, blood samples (100 μl) were taken via retro-orbital puncture on day 8 or day 13 after induction of AA. Whole-blood cells were stained with saturating amounts of directly FITC-labeled W3/25, OX35, G4.18, OX8, or respective isotype, followed by erythrocyte lysis. Ten thousand events were analyzed using an Epics XL flow cytometer (Coulter, Krefeld, Germany) and the results displayed as histograms.
To assess the DTH, either 10 μg of the arthritogen M. tuberculosis in 50 μl PBS, or 50 μl PBS only, were injected intradermally into the pinna of the left or the right ear, respectively, on day 13 after induction of AA. One day after injection, the swelling of the ears was determined with a gauge (Hahn & Kolb, Stuttgart, Germany). Swelling was expressed as the difference (mm) between the thickness of the left and the right ear.
T-cell reactivity in vitro
T cells were purified from spleens as described elsewhere [S6]. Briefly, the spleens were passed through a stainless steel sieve and the resulting suspension was centrifuged through Lymphoprep (Pharmacia). Cells contained in the interphase were then washed twice and loaded onto a 10-ml syringe with 1.2 g nylon wool (Polyscience, Eppelheim, Germany). After incubation for 1 hour at 37°C, 5% CO2, the cells were eluted with RPMI 1640/GlutaMaxI con- taining 10% FCS, 15 mM HEPES, 100 U/ml penicillin, and 100 μg/ml streptomycin (thereafter called R10F, all from GIBCO-BRL, Eggenstein, Germany). The resulting total T-cell suspension was ≥ 95% CD3+, as evaluated by flow cytometry. Either purified total T cells were directly used for proliferation assays, or CD4+ T cells were first negatively purified by adding 5 μg/1×107 cells OX8 mAb for 30 min on ice and, after washing, 75 μl Dynabeads-M450 coupled to goat anti-mouse IgG (Dynal, Hamburg, Germany). The suspension was again incubated for 30 min on ice and then separated by a magnetic particle concentrator (MPC®, Dynal). The purity of CD4+ T-cell populations was always ≥ 95%, as assessed by flow cytometry. For enrichment of dendritic cells (DC), suspensions of spleen cells from healthy rats were subjected to an overnight adhesion step on Petri dishes (Falcon®, Becton Dickinson, Heidelberg, Germany) and then centrifuged through 2 ml of a 14.5% metrizamide solution (Sigma) in R10F. DC were then irradiated with 15 Gy to prevent their proliferation in subsequent assays. For proliferation assays, 1 × 105 purified total or CD4+ spleen T cells per well of 96-well round-bottom plates were incubated with 1 × 104 DC per well and 1 μg/ml ConA (Sigma) for 72 hours. Then 1 μCi/well 3H-thymidine (Amersham-Buchler, Braunschweig, Germany) was added. After an additional incubation for 16 hours, cells were harvested onto fiber filters and cell-bound radioactivity was measured by β-scintillation counting (Matrix96®, Canberra Packard, Dreieich, Germany).
Primary allogeneic mixed lymphocyte culture
Total T cells or purified CD4+ T cells from spleens of healthy Lewis rats, and allogeneic DC from spleens of Wistar–Prob rats, were prepared as described above. Total or CD4+ T cells were seeded at 4 × 105 cells per well in 96-well flat-bottom plates, together with 1, 2, or 4 × 104 DC per well and 1, 5, or 10 μg/ml of the respective purified anti-CD4 mAb (or isotype control mAbs). After incubation for 72 hours (37°C, 5% CO2), bromodeoxyuridine (BrdU) was added and incubation was continued for an additional 16 hours. The proliferation was determined by a BrdU cell-proliferation ELISA (Boehringer Mannheim, Mannheim, Germany) in accordance with the supplier's recommendations.
For anti-CD4 preincubation, spleen CD4+ T cells were incubated with the anti-CD4 mAbs or isotype control mAbs (10 μg per 1 × 107 cells) for 30 min at 4°C and washed once. The bound mAbs were then cross-linked with goat anti-mouse IgG (Jackson Lab, 20 μg/1 × 107 cells) for 1 hour at 37°C. After washing, the cells (1 × 106 per ml and per well) were seeded in 24-well plates previously coated with R73 and harvested after 24 and 48 hours.
Anti-CD4-preincubated cells were washed, fixed with 4% paraformaldehyde in PBS, permeabilized (0.5% saponin in PBS, 1% FCS, 0.01% NaN3; for this step, supplemented with 10% rat serum), and incubated with 1 μg anti-IFN-γ FITC, anti-IL-4 PE, anti-IL-10 PE, or directly labeled isotype-control mAbs for 30 min at 4°C. FACS analysis was performed using a FACScan® flow cytometer (Becton Dickinson).
Detection of TNF-α was performed using a bioassay based on the lysis of the WEHI164/13 cell line after exposure to TNF-α. Assay specificity was ensured by adding a neutralizing mAb to rat TNF-α (100 μg/ml; clone 45418.111; R&D Systems, Wiesbaden, Germany).
Culture supernatants were analyzed for IFN-γ, IL-2, IL-4, and IL-10 by sandwich ELISA with the above-mentioned capture and detection antibodies. Recombinant cytokine standards were as follows: IFN-γ (Laboserv), IL-2, IL-10, and IL-4 (Pharmingen); range for IFN-γ, IL-2, and IL-10 was 39–5000 pg/ml; range for IL-4 was 9.85–1250 pg/ml. Cytokine concentrations in the culture supernatants were calculated from a standard curve using the software EasyFit (SLT, Crailsheim, Germany).
Electrophoretic mobility shift assays
Purified spleen CD4+ T cells were preincubated with the anti-CD4 mAbs as above and then either stimulated on anti-CD3 mAb-precoated 6-well plates (1 × 107 cells per well) or with a combination of phorbol myristoyl acetate (PMA; 10 ng/ml) and ionomycin (250 ng/ml) for 4 hours at 37°C. After stimulation, the cells were washed once with PBS and microcentrifuged (10,000 × g) at 4°C for 1 min. The pellet was resuspended in 400 μl ice-cold buffer A (10 mM HEPES, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 0.5 mM PMSF, 1 mM dithiothreitol, and 1 mM Na3VO4) and placed on ice for 15 min. Subsequently, 25 μl of a 10% Nonidet P-40 solution (Boehringer Mannheim) were added to the sample and the cells were homogenized by vortexing for 30 s followed by microcentrifugation for 1 min. The nuclear pellets were resuspended in 50 μl of buffer B (20 mM HEPES, 400 mM NaCl, 0.1 mM EDTA, 0.1 mM EGTA, 2 mM PMSF, 1 mM dithiothreitol, and 1 mM Na3VO4) and shaken for 15 min at 4°C. After microcentrifugation for 5 min at 4°C, the supernatants were aliquoted and stored at -70°C until further use. The nuclear extracts (10 μg total protein) were than incubated with 2 × 108 cpm of 32P-labeled, double-stranded oligonucleotide probe (sense strand only; AP-1: 5'-CGC TTG ATG AGT CAG CCG GAA-3' ; NF-κB: 5'-AGT TGA GGG GAC TTT CCC AGG C-3' ; both from Promega, Mannheim, Germany; NF-AT: 5'-CGC CCA AAG AGG AAA ATT TGT TTC ATA-3' ; Santa Cruz, Heidelberg, Germany) in 25 μl binding buffer (1 M Tris, 1 M boric acid, 0.02 M EDTA, 5% glycerol) supplemented with poly [dI-dC] (0.16 mg/ml Pharmacia) and 2 mM dithiothreitol. The samples were separated on a 4% polyacrylamide gel at 200 V. After scanning in a phosphor imager (BAS-1000, Fuij Photo Film Co. Ltd, Japan), the bands were quantified using the PCBAS 2.09g software (Fuij Photo Film Co. Ltd).
Differences between experimental groups were evaluated with the two-tailed nonparametric Mann–Whitney (U) test. The Spearman rank correlation test was used to verify whether the ex vivo 3H-thymidine uptake of CD4+ T cells correlated with the arthritis score of individual rats. Statistical significance was accepted at P ≤ 0.05.