Dendritic cells genetically engineered to express IL-4 inhibit murine collagen-induced arthritis (original) (raw)

Mice. Male DBA/1 LacJ mice, 7–8 weeks old, were purchased from The Jackson Laboratory (Bar Harbor, Maine, USA). Mice were maintained in the Unit for Laboratory Animal Medicine of the University of Michigan. Animal use procedures have been approved by the University of Michigan Committee on the Use and Care of Animals.

Culture medium. Complete medium (CM) consisted of RPMI 1640 supplemented with 10% heat-inactivated FCS, 0.1 mM nonessential amino acids, 1 μM sodium pyruvate, 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 5 × 10–5 M 2-ME (all from Life Technologies Inc., Grand Island, New York, USA). Lymphocyte medium (LM) consisted of Click’s medium (Life Technologies Inc.) supplemented with 1% heat-inactivated mouse serum, penicillin, streptomycin, and 2-ME. LM was used to culture spleen cells or lymph node cells from mice that had received DC injections, to avoid responses to FCS components.

Retroviral vector construction. A retroviral vector construct (pRET6) that contains long terminal repeats derived from myeloproliferative sarcoma virus as well as a puromycin resistance gene was used in the present study. cDNA encoding murine IL-4 was obtained from pNGVL3-mIL-4 (Vector Core, University of Michigan, Ann Arbor, Michigan, USA). For construction of the mIL-4 retrovirus vector (pRET6-IL-4), a mIL-4 fragment of 443 bp was excised with EcoR V and Not I from pNGVL3-mIL-4, and subcloned into the pBluescript vector (Stratagene, La Jolla, California, USA). A Xho I and Not I fragment from pBluescript-mIL-4 was then subcloned into pRET6. Similarly, the cDNA encoding enhanced green fluorescent protein (EGFP, obtained from pEGFP-1; CLONTECH, Palo Alto, California, USA) was subcloned into pRET6 to make pRET6-EGFP. An amphotropic Phoenix packaging cell line (φNXA provided by G. Nolan, Stanford University, Palo Alto, California, USA) was transiently transfected with the pRET6-mIL-4 or pRET6-EGFP construct using calcium/phosphate precipitation. An ecotropic GP+E86 packaging cell line (24) was subsequently infected with filtered viral supernatants from φNXA in the presence of protamine. After selection with puromycin (4 μg/ml), these polyclonal GP+E86 cells were used as producer cell lines.

Generation of retrovirally transduced DCs from bone marrow cells. Bone marrow was flushed from femurs and tibias, and depleted of erythrocytes by lysis in hypotonic buffer. Bone marrow cells (106/ml) were plated on 9.5 × 106 irradiated (30 Gy) producer cells in 150 cm2 culture flasks (Becton Dickinson, Franklin Lakes, New Jersey, USA). Cells were cocultured for 2 days at 37°C, 5% CO2 in the presence of 10 ng/ml recombinant murine GM-CSF (rmGM-CSF; Immunex Corp., Seattle, Washington, USA), 10 ng/ml recombinant murine IL-4 (rmIL-4; Schering-Plough, Kenilworth, New Jersey, USA), and 8 μg/ml protamine sulfate in CM. For preparing control DCs (mock-transduced DCs), bone marrow cells were cocultured with nontransduced GP+E86 cell lines. On day 2, nonadherent cells were carefully removed from adherent producer cell lines and replated at 106/ml in CM with rmGM-CSF and rmIL-4. On day 5, nonadherent cells were harvested and DCs were enriched by centrifugation on 14.5% metrizamide (Sigma Chemical Co., St. Louis, Missouri, USA) gradients (25). DCs were washed in HBSS before injection.

Cell surface phenotype. DCs were analyzed by direct or indirect staining with mAb’s against cell surface markers. Cells were preincubated with 2.4G2, a rat anti-Fc receptor mAb, and incubated with PE-conjugated mAb’s against CD11c, CD40, CD80, or CD86 (all from BD PharMingen, San Diego, California, USA). For MHC class II staining, DCs were stained with anti–I-Eq (BD PharMingen), followed by PE-conjugated anti-mouse IgG, F(ab′)2 (Boehringer Mannheim, Indianapolis, Indiana, USA). Flow cytometric analysis was performed using an Elite cell sorter (Coulter Electronics Ltd., Hialeah, Florida, USA).

Retroviral transduction of primary T cells and NIH 3T3 cell line. T cells were enriched from splenocytes on a nylon wool column. The cells were activated for 2 days with immobilized anti-CD3 (145-2C11; BD PharMingen) and anti-CD28 (37.51; BD PharMingen) in CM, followed by culture for 2 days in CM with 300 IU/ml recombinant human IL-2 (rhIL-2; Chiron Corp., Emeryville, California, USA). For immobilized anti-CD3/anti-CD28, 24-well plates were coated overnight at 4°C with 600 μl/well of anti-CD3 and anti-CD28 (2 μg/ml in PBS). Retroviral transduction was performed using filtered retroviral supernatant in 24-well plates precoated overnight at 4°C with 500 μl/well of RetroNectin (80 μg/ml in PBS; Takara Shuzo Co., Shiga, Japan). Activated T cells, resuspended in viral supernatant at 106/ml, were added to the plates (2 ml/well) and centrifuged at 900 g for 90 minutes at 32°C. After centrifugation, the retroviral supernatants were replaced with CM containing rhIL-2 (300 IU/ml) and cells were incubated overnight at 37°C. The following day, T cells were replated and expanded for an additional 2 days in CM with rhIL-2. Transduced T cells were enriched by drug selection in puromycin (1 μg/ml) for 3 days. Puromycin-resistant T cells were activated for 24 hours with immobilized anti-CD3/anti-CD28, harvested, and then washed in HBSS before injection. Murine fibroblast NIH 3T3 cells were transduced with filtered retroviral supernatant and selected with puromycin (4 μg/ml) for 3 days.

Induction and treatment of arthritis in mice. Mice were injected intradermally with 100 μg of chicken collagen type II (CII; Chondrex, Redmond, Washington, USA) in complete adjuvant (Chondrex) at the base of the tail on day 0. In randomized treatment groups, mice received subcutaneous, intravenous, or intraperitoneal injections of 3 × 105 mock-transduced, EGFP-transduced, or IL-4-transduced DCs on day 15. The subcutaneous injection was divided equally into the right and left flanks with 1.5 × 105 cells per site. In a separate experiment, mice received intraperitoneal injections of the indicated numbers of IL-4–transduced DCs, T cells, or NIH 3T3 cells. Limbs were individually scored every other day on a scale of 0–3 by a blinded observer. Scores were assigned based on the amount of erythema, swelling, or joint rigidity present in each limb, giving a maximum score of 12 per mouse.

Functional DC migration assay. IL-4–transduced DCs were incubated overnight with 50 μg/ml of keyhole limpet hemocyanin (KLH). The KLH-pulsed DCs were washed in HBSS and injected in mice subcutaneously, intravenously, or intraperitoneally at a dose of 3 × 105 cells per mouse, as already described here. Spleen and inguinal lymph nodes were harvested 5 days after DC injection, and KLH-specific T-cell responses were determined by proliferation assays and IL-2 release. The isolated cells from spleen and lymph nodes were resuspended in LM. A total of 5 × 105 cells were cultured with or without KLH (50 μg/ml) in 96-well flat-bottom plates. Proliferation was measured in triplicate in a 72-hour assay, with 0.8 μCi [3H]thymidine added during the last 16 hours of culture. [3H]thymidine incorporation was determined using a liquid scintillation counter. Supernatants from duplicate 48-hour cultures were stored at –80°C until determination of IL-2 levels by ELISA.

Direct enumeration of in vivo migration of EGFP-transduced DC. A total 3 × 106 EGFP-transduced DCs were injected subcutaneously, intravenously, or intraperitoneally into mice. The subcutaneous. injection was performed in the right and left flanks with 1.5 × 106 cells, respectively. At the indicated time after injection, mice were sacrificed and spleens were harvested. The spleens were then cut into small fragments with scissors and digested with collagenase and DNase for 30 minutes at 37°C. After depletion of erythrocytes, flow cytometric analysis was performed to detect fluorescent cells within the single-cell suspension of spleen cells. At least 2 × 106 living cells were analyzed. Spleen cells from untreated mice were used as a negative control. In parallel experiments, EGFP+ cells within the samples were directly counted using fluorescence microscopy. By this method, cells with both green and red fluorescence were regarded as autofluorescent cells and were not counted as EGFP+ cells.

Spleen cell cultures. Spleen cells were cultured in 96-well plates at a density of 5 × 106 cells/ml (200 μl/well) in LM. Cells were cultured for 72 hours with anti-CD3 (5 μg/ml, 145-2C11), or for 96 hours with medium alone or heat-denatured CII (100 μg/ml). Culture supernatants were stored at –80°C until analyzed for cytokines by ELISA.

Cytokine assays. Murine IFN-γ, IL-2, and IL-4 were assayed by ELISA using paired antibodies (BD PharMingen) according to the manufacturer’s instructions. The lower limits of detection were 40 pg/ml for IFN-γ and IL-2, and 10 pg/ml for IL-4.

Anti-CII antibody titers. The titers of anti-CII antibodies in serum samples were determined by ELISA. All samples were measured in duplicate. Microtiter plates were coated with 5 μg/ml native CII (ELISA grade; Chondrex) overnight at 4°C and then blocked by incubation with PBS containing 10% FCS. After washing with PBS/0.05% Tween 20, 100 μl of diluted serum samples were added to the wells and the plates were incubated at 4°C overnight. Plates were washed and incubated at room temperature for 2 hours with biotinylated rat anti-mouse IgG1 or IgG2a (BD PharMingen). Plates were washed thoroughly and incubated with 2.5 μg/ml avidin-peroxidase (Sigma Chemical Co.) for 30 minutes. After the last wash, plates were developed with ABTS (Sigma Chemical Co.). Optical densities were determined at 405 nm. Dilutions of a single serum sample from a CII-immunized mouse were used to generate a standard curve.

Statistical analysis. The statistical analysis was performed using χ2 test for values of arthritis mice and arthritis limbs or the Mann-Whitney _U_-test for clinical severity and antibody levels.