|
|
Blood, Vol. 113, Issue 15, 3485-3493, April 9, 2009
C1q enhances IFN- production by antigen-specific T cells via the CD40 costimulatory pathway on dendritic cells
Blood Baruah et al.
113: 3485
Supplemental materials for: Baruah et al
Mice and reagents
C1q knockout mice were generated as described earlier1 and backcrossed onto C57BL/6 for ten generations. Age and sex matched C57BL/6 mice were used as controls. Rag2−∕− Marilyn mice (TCR transgenic for the HYAbDby peptide NAGFNSNRANSSRSS) were a kind gift from Dr O Lantz (Paris, France).2 Animals were kept under specific pathogen-free conditions. All animal care and procedures were conducted according to institutional guidelines. All cell cultures were performed in RPMI 1640 (Invitrogen Life Technologies, Paisley, UK) supplemented with 100U/ml penicillin (Invitrogen), 100 µg/ml streptomycin (Invitrogen), 1.5mM L-glutamine (Invitrogen) and 10% heat inactivated fetal calf serum (FCS) (Biosera, East Sussex, UK). Human C1q isolated from human serum (purity >90% by SDS-PAGE) was purchased from Calbiochem (San Diego, California). Anti-Calreticulin antibodies for the staining experiments were purchased from Novus Biologicals. For functional studies, anti-Calreticulin antibodies were kindly provided by Dr D. Mitchell (Warwick, UK). Human DCs
Peripheral blood mononuclear cells (PBMCs) obtained from healthy donors and C1q deficient patients were differentiated into DCs as previously described.3,4 Briefly, monocytes were sorted from PBMCs using CD14 magnetic beads, and cultured in the presence of GM-CSF (100ng/ml) and IL-4 (10ng/ml) (R&D Systems) for 5–7 days. Maturation of DCs was induced by stimulation with LPS (0.1–1 µg/ml) or soluble CD40L (1–2 µg/ml, Alexis-Axxora, UK). After 48 hours the cells were harvested and the expression of CD40, CD80, CD83, CD86 and HLA-DR assessed by flow cytometry. The supernatants were collected and stored frozen until cytokine quantification by ELISA. The experiments using blood samples were approved by the institutional review board and all human participants gave written informed consent. Induction of apoptosis
Thymocytes were isolated from male and female C57/BL6 mice and apoptosis was induced by gamma-irradiation at the dose of 10–12 Gy followed by incubation at 37°C for 24 hours. Typically 70–80% of cells were positive for annexin V-FITC (AxV, BD Pharmingen) alone while about 20% of cells were positive for both AxV and propidium iodide (PI, 1 µg/ml, BD Pharmingen). Phagocytosis assay
Apoptotic thymocytes were incubated with murine DCs at 37°C for varying periods of time from 1 to 24 hours. The ratio of apoptotic cells to DCs was routinely 2:1. Cytospins of the cells were prepared and stained with Diff-Quik II (Dade Behring) and assessed under oil microscopy. The number of DCs with completely engulfed apoptotic bodies was counted and the phagocytic index (the number of ingested apoptotic bodies per 100 dendritic cells) calculated as described previously.5 ELISA
IL-12p70 and IL-10 levels in culture supernatants were measured using commercial Duoset ELISA kits (R&D Systems, UK) according to the manufacturer’s instructions. A sandwich ELISA for IFN-γ was performed using purified capture and biotinylated detection antibodies from BD-Pharmingen. REFERENCES 1. Botto M. C1q knock-out mice for the study of complement deficiency in autoimmune disease. Exp Clin Immunogenet. 1998;15:231–234.
2. Braun MY, Grandjean I, Feunou P, et al. Acute rejection in the absence of cognate recognition of allograft by T cells. J Immunol. 2001;166:4879–4883.
3. Castellano G, Woltman AM, Nauta AJ, et al. Maturation of dendritic cells abrogates C1q production in vivo and in vitro. Blood. 2004;103:3813–3820.
4. Baruah P, Dumitriu IE, Peri G, et al. The tissue pentraxin PTX3 limits C1q-mediated complement activation and phagocytosis of apoptotic cells by dendritic cells. J Leukoc Biol. 2006;80:87–95.
5. Taylor PR, Carugati A, Fadok VA, et al. A hierarchical role for classical pathway complement proteins in the clearance of apoptotic cells in vivo. J Exp Med. 2000;192:359–366.
Files in this Data Supplement:
- Figure S1. C1qa−∕− DCs respond to LPS and engulf apoptotic cells comparably to WT DCs (JPG, 75.2 KB)
-
(A) WT or C1qa−∕− GM-CSF DCs were cultured alone or in the presence of LPS (100 ng/ml) for 48 hrs and the expression of CD40, CD80 and CD86 was evaluated by flow cytometry. Shown is a representative routine experiment. (B) DCs from WT or C1qa−∕− mice were cultured alone or stimulated with LPS (0.1–100 ng/ml) or CpG (1 µm) or CpG random sequence (CpG ctrl) (1 µm). The production of IL-12p70 was evaluated by ELISA. Results depicted are from one experiment out of three performed with DCs from different mice (mean ± SD of duplicate samples). C. DCs from WT and C1qa−∕− mice were cultured with apoptotic cells for various time intervals. The cells were fixed and stained and phagocytosis was evaluated using light microscopy.
|
|
