Cell culture
Chinese hamster ovary (CHO) cells stably expressing human AQP4-M23 (named CHO-AQP4 cells), as described [
16,
44], were cultured at 37 °C in 5% CO
2 95% air in F-12 Ham’s Nutrient Mixture medium supplemented with 10% fetal bovine serum, 200 μg/ml geneticin, 100 U/ml penicillin and 100 μg/ml streptomycin. Non-transfected CHO-K1 cells without AQP4 (called CHO-null cells) were cultured in the same medium but without geneticin. Human natural killer cells (NK cells) transfected to express the high-affinity 176 V variant of the Fcγ receptor IIIA [
66] were obtained from Fox Chase Cancer Center (Philadelphia, PA). NK cells were cultured in suspension in α-MEM (Sigma-Aldrich, St. Louis, MO) containing 0.1 mM 2-mercaptoethanol, 2 mM L-glutamine, 0.2 mM myoinositol, 10% FBS, 10% horse serum, 2.5 μM folic acid, 5 ml non-essential amino acids, 1 mM Na pyruvate, 1% penicillin/streptomycin, and 100 IU/ml of human recombinant interleukin-2 (GenScript, Piscataway, NJ). Primary astrocyte-neuron cocultures were generated from brains of embryonic day 18 Sprague-Dawley rats (neurons) or cerebral cortex of neonatal rats (astrocytes) as described [
19].
Neutrophils were isolated from human peripheral blood by a modified Ficoll-Hypaque method [
20,
40] using density gradient centrifugation and a commercial separation medium (Lympholyte-poly, CL5071, Cedarlane Labs, Burlington, NC) containing sodium metrizoate and dextran 500. After separation, red cell lysis buffer was added to the neutrophil layer to lyse residual red cells. The isolated neutrophils were resuspended in HBSS/HSA solution (2% HSA) at specified concentration and used within 2 h. Neutrophil purity was 97.0% as judged by immunofluorescence using neutrophil-specific CD66b antibody (1:200, 555723, BD Pharmingen, San Jose, CA).
Complement-dependent cytotoxicity
CHO-AQP4 and CHO-null cells were cultured individually or at specified cell number ratios (from 1:5 to 1:50) on coverslips in 24-well plates, and grown for 18-24 h until confluent. For assay of CDC, CHO cells were incubated for 2 h at 37 °C with 5% human complement and 10 μg/ml AQP4-IgG (or control human IgG) in Hank’s balanced salt solution (HBSS, pH 7.2; Invitrogen, Camarillo, CA).
Antibody-dependent cellular cytotoxicity
For assay of ADCC, CHO-AQP4 and CHO-null cell cocultures were generated as for CDC experiments. Primary neurons were grown on coverslips in 12-well plates and astrocytes were added to neurons at a 1:20 ratio before experiments. NK-92 cells overexpressing CD16 were used as the effector cells. Cells were pre-incubated with 5 μg/ml AQP4-IgG (or control IgG) for 30 min at 37 °C. In some experiments, cells preincubated with a 1:100 dilution of serum of an AQP4-IgG seropositive NMO patient. NK cells or neutrophils were then added to the AQP4-IgG-coated CHO cells at an effector: target cell ratio of 5:1, and incubated for an additional 1 h at 37 °C. Following treatments, cells were washed extensively with Hank’s Balanced Salt Solution (HBSS) to remove the remaining effector cells. In some studies, NK cells were pretreated with an inhibitor of the perforin cytotoxic pathway, concanamycin A (CMA, 10 nM, C9705, Sigma-Aldrich) [
66] for 2 h prior to AQP4-IgG addition. In some studies, a neutralizing anti-perforin antibody (δG9, 10 μg/ml, BD Pharmingen, San Jose, CA) was added together with NK cells onto the CHO cells. In some studies, the RGD-containing peptide RGDS (or control peptide RGES) (200 μM, Sigma-Aldrich) [
28] was pre-incubated with CHO cell cocultures for 1 h prior to AQP4-IgG addition.
Immunofluorescence
In some experiments, a fixable red-fluorescent dead-cell stain (L23102, amine-reactive dye, Invitrogen, Eugene, OR) at 1:1000 dilution was added 30 min prior to cell fixation. Cells were then rinsed in PBS and fixed with 4% paraformaldehyde (PFA) for 15 min. After fixation, cells were blocked for 1 h with 1% BSA and 0.1% Triton-X100 in PBS, and incubated with primary antibodies. Brains were cut as 7-μm-thick frozen sections, and blocked in the same buffer before immunostaining. Cultures and brain sections were incubated at 4 °C overnight with antibodies against AQP4 (sc-20812, 1:200, Santa Cruz Biotechnology, Dallas, TX), AQP4 (sc-9888, 1:200, Santa Cruz Biotechnology, Dallas, TX), C1q (ab71940, 1:50, abcam, Cambridge, MA), C5b-9 (ab55811, 1:200, abcam, Cambridge, MA), GFAP (AB5541, 1:1000; Millipore, Burlington, MA), MAP2 (PA5–17646, 1:100, Thermo Fisher Scientific, Rockford, IL), perforin (δG9, 556434, 1:100, BD Pharmingen, San Jose, CA), NeuN (ABN78, 1:200, Millipore, Burlington, MA), Olig2 (sc-48817, 1:100, Santa Cruz Biotechnology, Dallas, TX) or GFP (MA1–952, 1:20, Thermo Fisher Scientific, Rockford, IL) followed by 1 h incubation with appropriate species-specific Alexa Fluor-conjugated secondary antibody (5 μg/ml each, Invitrogen, Camarillo, CA). Sections and coverslips were mounted with ProLong Gold antifade reagent (P36931, Thermo Fisher Scientific, Rockford, IL), and immunofluorescence was visualized on a Nikon confocal microscope using a 20x/0.5 N.A., 60x/1.25 N.A., or 100x/1.4 N.A. oil objective lens.
Real-time imaging
For live-cell real-time imaging, CHO-AQP4 / CHO-null cell cocultures were imaged using a 20x, 0.45 NA objective lens on a Nikon Eclipse Ti microscope equipped with an environmental chamber at 37 °C and 5% CO2. CHO-AQP4 cells were loaded with the CellTracker™ Green CMFDA (C2925, 5 μM in HBSS) for 30 min at 37 °C, cell tracker was removed and cells were replated with unlabeled CHO-null cells overnight before experiments. Control experiments verified that 100% of CHO-AQP4 cells retained the CellTracker label overnight. Cells were then pre-incubated with 5 μg/ml AQP4-IgG for 30 min, which was then washed away prior to imaging. For imaging, cells were then incubated in HBSS containing ethidium homodimer-1 (1 μM, Invitrogen, Eugene, OR). Transmitted light (phase-contrast), green and red fluorescence images were obtained sequentially every 2 min for a 30 min baseline period and then for 1.5 h following addition of NK cells.