Animals
Male inbred Lewis rats were obtained from the University of Limburg (Maastricht, The Netherlands) and were used between 7 and 10 weeks of age. The animals were kept under conventional conditions and had access to standard pelleted rat chow and acidified water ad libitum. The Utrecht University Animal Ethics Committee approved all animal experiments.
Antigens
Heat-killed Mt, strain H37RA, was obtained from Difco Laboratories (Detroit, Michigan, USA). For immunization, Mt was suspended in incomplete Freund's adjuvant (Difco Laboratories). Peptides Mt HSP60176–190 (EESNTFGLQLELTEG; one-letter amino acid codes) (HSP60 stands for heat shock protein 60), Mt HSP60211–225 (AVLEDPYILLVSSKV) and OVA323–339 (ISQAVHAAHAEINEAGR) (OVA stands for Ovalbumin) were obtained from Isogen Bioscience (Maarssen, The Netherlands).
mAbs and second-step reagents
The anti-CD134 (OX40) and anti-CD25 (OX39) hybridomas were obtained from the ECACC (Salisbury, UK) [9]. The 12CA5 hybridoma producing IgG2b isotype control mAb was kindly provided by Dr GJ Strous (Department of Cell Biology and Institute of Biomembranes, University Medical Center, Utrecht, The Netherlands). mAbs were isolated from hybridoma supernatant by affinity chromatography with GammaBind Plus Sepharose (Roche Pharmacia, Uppsala, Sweden). For ease of flow cytometric detection, some purified mAbs were biotinylated with D-biotinoyl-ε-aminohexanoic acid-N-hydroxy-succinimide ester (Roche Molecular Biochemicals, Basel, Switzerland). Fluorescein isothiocyanate (FITC)-conjugated anti-CD4 (OX35) and anti-CD45RA (OX33), phycoerythrin (PE)-conjugated goat-anti-mouse immunoglobulin, PE-conjugated streptavidin, peridinin chlorophyll protein (PerCP)-conjugated anti-T-cell antigen receptor (anti-TCR)-αβ (R73) and IgG1 isotype control (A112), and allophycocyanin-conjugated streptavidin were purchased from BD Pharmingen (San Diego, California, USA).
Culture of rat CD4+T cell clone A2b
The isolation, maintenance, and properties of rat CD4+ T cell clone A2b have been described previously [10]. The arthritogenic T cell clone A2b recognizes the 180 to 188 epitope of Mycobacterium tuberculosis HSP60 [11]. Cells were cultured in medium (Iscove's modified Dulbecco's medium (Invitrogen, Merelbeke, Belgium), supplemented with L-glutamine (2 mM), 2-mercaptoethanol (50 μM), penicillin (50 U/ml) and streptomycin (50 μg/ml)) with 2% heat-inactivated normal rat serum.
Induction of AA
Rats were injected intradermally with 100 μl of Mt in incomplete Freund's adjuvant at the base of the tail. For studying cell-surface marker expression, CD4+ subset specificity during AA and liposome binding in vivo, 10 mg/ml Mt was used. For AA treatment studies, rats were immunized with 5 mg/ml Mt (yielding 100% disease incidence, but lower maximum disease scores in comparison with 10 mg/ml Mt). Rats were weighed and examined for clinical signs of arthritis in a semi-blinded set-up. Severity of arthritis was scored by grading each paw from 0 to 4 based on erythema, swelling and immobility of the joints, resulting in a maximum score of 16 per animal [12].
Ex vivoanalysis of cell-surface marker expression
Before Mt immunization or 7, 10, 14, 21 or 35 days afterwards, rats were killed and popliteal lymph nodes (PLN), inguinal lymph nodes (ILN), spleen, and peripheral blood were isolated. Single-cell suspensions were prepared by mechanically forcing the organs through a 70 μm mesh; erythrocytes were removed from the splenocyte and blood suspensions by Ficoll-Isopaque gradient centrifugation. Cells (2 × 105 per sample) were labeled with anti-CD134 for 30 min on ice, followed by incubation with PE-conjugated goat anti-mouse immunoglobulin and subsequently with anti-CD4-FITC. The cells were incubated and washed (between each labeling step) in blocking buffer (PBS (Cambrex Bio Science, Verviers, Belgium) containing 4% heat-inactivated rat serum, 1% fraction V BSA (Sigma-Aldrich Chemie, Zwijndrecht, The Netherlands) and 0.1% NaN3). Finally, cells were washed in PBS, fixed in 2% paraformaldehyde and stored at 4°C in the dark. Cell-associated fluorescence was analyzed within 10 days on a FACSCalibur using Cell Quest software (Becton Dickinson, Brussels, Belgium).
Cell sorting and ex vivo proliferation of CD4+T cell subsets
At 7 and 10 days after Mt immunization, PLN, ILN, and spleens of 10 to 15 rats were isolated and each organ type was pooled. Single-cell suspensions were prepared as described above. Cells were washed and incubated in PBS containing 4% heat-inactivated rat serum, and stained with anti-CD134-biotin/streptavidin-allophycocyanin and anti-CD4-FITC. CD4+, CD4+CD134- and CD4+CD134+ cells were sorted with a FACS Vantage and Cell Quest software (Becton Dickinson), resulting in fractions that were 87 to 97% pure.
Sorted cells were washed and incubated for 72 hours in medium with 2% heat-inactivated normal rat serum in flat-bottomed 96-well plates (Corning-Costar, Schiphol, The Netherlands) at 5 × 104 cells per well in the presence of 30 Gy-irradiated thymocytes as antigen-presenting cells (APC) (106 cells per well) and concanavalin A (Con A; 2.5 μg/ml) or antigen (20 μg/ml Mt HSP60176–190, 20 μg/ml Mt HSP60211–225). Finally, cells were pulsed for 18 to 20 hours with [3H]thymidine, 0.4 μCi per well (specific radioactivity 1 Ci/mmol; Amersham Biosciences, Roosendaal, The Netherlands), after which [3H]thymidine incorporation was measured. Results are presented as the mean stimulation index (SI, defined as [3H]thymidine incorporation in the presence of antigen or Con A divided by [3H]thymidine incorporation in the absence of antigen or Con A) of triplicate wells. For logistic reasons, ILN single-cell suspensions were kept overnight on ice and were washed, stained and sorted on the following day.
Preparation of (mAb-coupled) PEG-liposomes
Liposomes were composed of egg phosphatidylcholine, cholesterol, poly(ethyleneglycol)2000-distearoylphosphatidylethanolamine (PEG2000-DSPE) and maleimide-PEG2000-DSPE in a molar ratio of 2:1:0.075:0.075. Egg phosphatidylcholine was kindly provided by Lipoid (Ludwigshafen, Germany), PEG2000-DSPE was purchased from Avanti Polar Lipids (Birmingham, Alabama, USA), cholesterol from Sigma-Aldrich Chemie, and Maleimide-PEG2000-DSPE from Shearwater Polymers (Huntsville, Alabama, USA). Liposomes used for investigating liposome binding in vivo contained 0.1 mol% 1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt (DiD; Molecular Probes Europe, Leiden, The Netherlands). Liposomes used for investigating in vitro binding and internalization contained 0.1 mol% Texas red-phosphatidylethanolamine (Molecular Probes Europe). Liposomes used for studying drug delivery in vitro and for the treatment of AA contained 2 mol% 5'-fluoro-2'-deoxyuridine dipalmitate (FUdR-dP; that is, 0.06 mol FudR-dP per 3 mol main lipid constituents) (synthesized as described previously [13]).
Lipids (and FUdR-dP or DiD) were dissolved in chloroform/methanol (9:1) and mixed. A lipid film was prepared through rotary evaporation under vacuum and dried under nitrogen. The lipids were hydrated with HN buffer (4-(2-hydroxyethyl)-1-piperazine ethanesulphonic acid (HEPES) and 135 mM NaCl) at pH 6.7. The resulting vesicles were sized by repeated extrusion through 100 nm polycarbonate filters. Particle size and size distribution were determined by dynamic laser light scattering with an Autosizer 4700 Spectrometer (Malvern Instruments, Malvern, Worcestershire, UK). Liposome preparations had a mean particle diameter ranging from 100 to 200 nm (polydispersity between 0.1 and 0.2). Typically, the mean liposomal diameter varied by less than 20% within any given experiment. The anti-CD134 or IgG2b isotype control mAbs were coupled to liposomes by a thiol-maleimide method described previously [13]. In brief, free thiol groups were introduced in the mAbs using the heterobifunctional reagent N-succinimidyl-S-acetylthioacetate (SATA; Sigma-Aldrich Chemie). Free SATA was separated from the derivatized mAbs by gel permeation chromatography, resulting in ATA-derivatized mAbs dissolved in HN buffer at pH 7.4. mAbs with reactive thiol groups, induced by deacetylating the ATA-protein, were incubated with liposomes at 4°C overnight at a ratio of 0.05 to 0.1 mg of mAbs per μmol lipid. N-ethylmaleimide (8 mM in HN buffer, pH 7.4) was added to cap unreacted thiol groups. Unconjugated mAbs were removed by gel-permeation chromatography or by centrifugation at 100,000 g. The liposomal protein content was determined as described previously [14]. Liposomes contained 25 to 125 μg of mAbs per μmol of lipid. Typically, the mAb content of the different liposome preparations within any given experiment varied by less than 20%.
Liposome binding to CD4+ T cells in vivo
On day 7 after Mt immunization, rats received saline or 5 μmol (lipid) DiD-labeled liposomes subcutaneously (s.c.) in each hind paw. After 30 min the rats were killed, and the PLN, ILN, and spleens were isolated. Single-cell suspensions were prepared as described above. Subsequently, cells were stained with anti-CD4-FITC, anti-CD134-biotin/streptavidin-PE and anti-TCR-αβ-PerCP, or with anti-CD45RA-FITC, anti-CD134-biotin/streptavidin-PE and anti-TCR-αβ-PerCP. Cell-associated fluorescence was measured by flow cytometry.
Liposomal drug delivery to T cells in vitro
A2b T cells were activated in vitro to induce CD134 expression by stimulation overnight with 2.5 μg/ml Con A (Sigma-Aldrich Chemie) in the presence of 30 Gy-irradiated Lewis thymocytes as APC (ratio of T cells to APC = 1:25). Alternatively, a spleen cell suspension (at 2 × 105 cells/ml) was stimulated for 3 days with 2.5 μg/ml Con A to induce CD134 expression on splenic T cells. Next, viable cells were collected from the culture by Ficoll-Isopaque gradient centrifugation and transferred to round-bottomed 96-well plates at 105 cells per sample.
For studying anti-CD134 liposome binding to T cells in vitro by flow cytometry, A2b cells were incubated with 5 nmol (lipid) of anti-CD134 liposomes or IgG2b isotype control liposomes, or anti-CD134 mAb or IgG2b isotype control mAb for 30 min on ice. Cells were then washed and incubated on ice with FITC-conjugated goat anti-mouse immunoglobulin to label the cell-bound mAbs or liposomes. Finally, cell-associated fluorescence was measured. For analysis of the interaction of CD4+ T cells and liposomes by confocal microscopy, activated A2b cells were incubated with 50 nmol of the different liposomal formulations in medium for 30 min on ice, washed and cultured in medium with 2% heat-inactivated normal rat serum at 37°C in 5% CO2. Activated spleen cells were incubated with 100 nmol of liposomes. At the indicated time points, cell-associated fluorescence was assessed.
For assessment of in vitro drug delivery by anti-CD134 liposomes, activated A2b cells were incubated at 37°C in 5% CO2 without or with 1 nmol (lipid) of the different liposomal formulations per well or with an equal concentration (100 nM) of free FUdR (Sigma-Aldrich Chemie) in 200 μl of medium without serum. After 30 min, cells were washed three times in medium and cultured for 48 hours in 200 μl of conditioned medium (medium supplemented with 10% heat-inactivated fetal calf serum (Bodinco, Alkmaar, The Netherlands), 10% culture supernatant of the EL-4 lymphoma (containing murine IL-2) and 1% non-essential amino acids (Invitrogen)). Finally, cells were pulsed for 18 to 20 hours with [3H]thymidine as described above, after which [3H]thymidine incorporation was measured. Results are expressed as the mean percentage of inhibition of proliferation of duplicate cultures relative to the incubation without liposomes (defined as 0%).
Treatment of AA with liposomes and ex vivoproliferation assay of LN cells after liposomal treatment
AA was induced in Lewis rats as described above. Rats received 5 μmol of the different liposome formulations s.c. in each hind paw or HN buffer (see below) as a control on days 3 and 7 or on days 3, 7, and 10 after Mt immunization. Rats were followed for arthritis development as described above.
Proliferation of lymphocytes from liposome-treated animals was measured in quadruple cultures of 2 × 105 cells per well without additional APC. Cells were cultured in 96-well flat-bottomed plates in 200 μl of medium containing 2% heat-inactivated normal rat serum in the absence or presence of antigen (20 μg/ml Mt HSP60176–190 or 20 μg/ml OVA232–339) or Con A (2.5 μg/ml). After 72 hours, cells were pulsed for 18 to 20 hours with [3H]thymidine as described above, after which [3H]thymidine incorporation was measured.
Statistical evaluation
The statistical significance of differences was evaluated with GraphPad Prism 3.02 (GraphPad Software, San Diego, California, USA). For statistical analysis of CD134 expression in vivo and liposomal drug delivery in vitro, a one-way analysis of variance with Dunnett's post-hoc test was used. For analysis of differences in the development of AA, a Mann-Whitney test was used for arthritis scores and an unpaired Student's t-test for body weight.