A value <0.05 was considered significant. Results Mouse hepatocytes constitutively express DAF, CD59 and Crry To systematically examine the distribution of intrinsic cell surface complement inhibitors, DAF, CD59 and Crry_on mouse hepatocytes, we isolated murine primary hepatocytes by collagenase digestion with high purify (> 95%, data not shown) following an established protocol (17). primary hepatocytes were first loaded with 3 M BCECF-AM (Invitrogen, CA) in MEM medium at 37C for 1 hr. After washing, labeled hepatocytes were incubated at 37C with 50 g/ml rabbit anti-OVA IgG Rabbit polyclonal to ACTN4 and 30% mouse serum in 100 l GVB/Ca2+ Mg2+ buffer (veronal-buffered saline supplemented with 0.1% gelatin, 5 mM CaCl2 and 3 mM MgCl2) for another 30 min. 1 mM EDTA was included to inhibit complement activation in the controls. Following incubation, complement mediated cell injury was assessed by measuring levels of converted BCECF released into the supernatants using a fluorescence microtiter plate reader (Molecular Devices, CA) with excitation and emission wavelengths of 485 and 538 nm. To calculate the percentage of BCECF release after complement mediated cellular injury, the following equation was used: percentage of BCECF release = [(ACB)/(CCB)] 100%; where A represents the mean experimental BCECF release, B represents the mean spontaneous BCECF release and C represents the mean maximum BCECF released which was induced by incubating cells with 0.1% SDS. The cells were also collected and assessed for C3b deposition by staining with an anti-mouse C3 mAb followed by flow cytometry analysis as described before (19). Induction of autoimmune hepatitis 0.5 mg of the rabbit anti OVA IgG was injected into Hep OVA Tg mouse through the tail vein. Livers and sera were collected 4 hrs later. Serum ALT levels were measured by an automatic biochemical analyzer in the Clinical Core Laboratory of University PI-103 Hydrochloride Hospital Case Medical Center, and livers were sectioned and analyzed by H&E staining and immunohistochemical staining. Flow cytometry analysis and immunohistochemical staining To examine the distribution of intrinsic cell surface complement regulators, 2105 freshly isolated primary hepatocytes were incubated with 5 g/ml of mAbs against mouse DAF, CD59 or Crry, respectively, or the same concentration of non-relevant rat IgG as unfavorable controls. Mouse erythrocytes known to express all the three intrinsic cell surface complement regulators were included as positive controls. For immunohistochemical stainings, liver tissues were snap frozen in liquid nitrogen, then 7 micron cryosections were cut and stained with mAbs against rabbit IgG (rabbit anti-OVA IgG), mouse C3, mouse C5b-9 and mouse CD11b using a Vectastain ABC kit (Vector Labs, CA) following the manufacturer provided protocol. Non-relevant isotope IgGs were used as controls. Complement depletion by CVF To deplete complement, 20 g of purified cobra venom factor (CVF) (Sigma, MO) was injected in each mouse. Serum samples were collected from the tail vein before and after CVF injection for standard EshA C3b uptake assays (20) to verify the depletion of complement. In brief, 5105 EshA were incubated at 37C with 10% of the serum samples collected before and after CVF injection in 100 l GVB/Ca2+ Mg2+ buffer for 30 min, then stained with 5 g/ml FITC labeled anti-mouse C3 mAb followed by flow cytometry analysis on a flow cytometer (LSR I, BD Biosciences, CA). Recombinant soluble mouse DAF PI-103 Hydrochloride preparation and treatment Yeast expressing soluble mouse DAF CCP 1C4 with a C-terminus 6 his tag was developed in the lab (20). For soluble mouse DAF preparation, recombinant yeast was cultured in YPD media and induced with 1% methanol for 2 days. Secreted soluble mouse DAF protein was purified from the supernatants by Ni2+ affinity chromatography (Qiagen, CA) and dialyzed against PBS. The concentrations of the resultant mouse DAF was measured using a Bio-Rad protein assay kit (Bio-Rad, CA) following the manufacturer PI-103 Hydrochloride provided protocol. For DAF-based treatment, 200 g of purified recombinant mouse DAF protein was injected per mouse 40 min before anti- OVA IgG administration. Sera and livers were collected 4 hr after induction of hepatitis. Inhibition of systemic serum complement by administrated DAF protein was assessed by C3b uptake assays using antibody sensitized sheep erythrocytes (EshA) as described above. Serum ALT levels and PI-103 Hydrochloride liver histopathology were assessed as described above. Statistics All experiments were repeated at least twice. Results were compared using the ANOVA test. A value <0.05 was considered significant. Results Mouse hepatocytes constitutively express DAF, CD59 and Crry To systematically examine the distribution of intrinsic cell surface complement inhibitors, DAF, CD59 and Crry_on mouse hepatocytes, we isolated murine primary hepatocytes by collagenase digestion with high purify (> 95%, data not shown) following an established protocol (17). After the isolation, we stained the hepatocytes with respective mAbs followed by flow cytometry analysis. These assays showed that mouse primary hepatocytes constitutively express all intrinsic cell surface complement regulator DAF, CD59 and Crry (Fig. 1). Open in a separate window Physique 1 Mouse primary.