2006). We recently reported that oxidative stress of ARPE cells alters the expression of cell surface complement regulatory proteins and permits increased activation of complement when the cells are subsequently exposed to complement-sufficient serum (Rohrer and Renner 2008). In a separate set of studies we have previously demonstrated that tissue ischemia alters complement regulation on the surface of renal epithelial cells (RPE) (Thurman et al. 2006). These findings suggest that epithelial cells from different organs may respond to cellular stressors by altering their expression of complement regulatory proteins, and that this response may contribute to autologous complement-mediated injury in diseases such as AMD.

Cells control complement activation on their surface through the expression of complement regulatory proteins. The three cell surface complement regulatory proteins are membrane cofactor protein (MCP; CD46), decay accelerating factor (DAF; CD55), and CD59. Crry is a rodent complement regulatory protein that is a homologue of MCP and DAF (Molina and Wong1992). Local complement activation is likely a result of the balance of complement proteins, activating molecules, and inhibitory molecules. As the interface of the internal milieu with the outside world, epithelial cells must be able to protect themselves from autologous complement-mediated injury while also fostering effective clearance of pathogens by complement proteins present in the serum. Retinal pigmented epithelial (RPE) cells likely perform surveillance functions, possibly triggering an inflammatory response when pathogens are detected.

We hypothesized that RPE cells respond to cellular stress as if it is infection, and reduce their surface expression of complement regulatory proteins to foster the local immune response. In addition to AMD, uncontrolled activation of the alternative pathway on barrier epithelial layers occurs in ischemic acute kidney injury (Thurman and Ljubanovic 2003) and in asthma (Taube and Thurman 2006); so we further hypothesized that the mechanisms of altered complement regulation that we have described for RPE cells might also be seen in epithelial cells derived from the kidney and lung. We have, therefore, examined whether oxidative stress induces changes in the expression of complement regulatory proteins on the surface of renal and pulmonary epithelial cells, similar to what we have observed in RPE cells.

18.2 Material and Methods

18.2.1 Reagents

The reagents used in these studies included pooled normal human serum (NHS; Quidel) as a source of complement proteins, or complement-sufficient mouse serum obtained by bleeding C57Bl/6 mice. Primary antibodies to DAF (Chemicon International), CD59 (Chemicon International), MCP (Monosan), Crry (BD Biosciences), and C3 (ICN Pharmaceuticals) were used. Species-specific secondary antibodies were from Jackson ImmunoResearch and MP Biomedicals, Inc.

18.2.2 Cell Culture

We used three different epithelial cell lines: the retinal pigment epithelial cell line, ARPE-19; the BUMPT, renal proximal tubular epithelial cell (PTEC) line; and the pulmonary epithelial cell line, A549. The ARPE-19, human RPE cell line (Dunn and Aotaki-Keen 1996; Dunn and Marmorstein 1998), was grown in DMEM-F12 (Gibco) with 10% fetal bovine serum (FBS) and penicillin-streptomycin (PenStrep). Upon reaching confluence, FBS was removed completely for 2 weeks for cells to form a monolayer with tight junctions. Monolayer barrier function was confirmed in parallel cultures grown on transwell inserts that allow for measurements of transepithelial resistance. The PTEC line was originally derived from the Immortmouse (Sinha and Wang 2003). These cells were grown in DMEM medium supplemented with 10% FBS, Pen-Strep, and 0.2 U/mL interferon-gamma (IFN-γ; Peprotech). The IFN-γ in this medium permits expression of the H-2Kb-tsA58 transgene in these cells (Sinha and Wang 2003). After the cells reached confluence they were changed to 1:1 DMEM/Ham’s F12 supplemented with 5 mg/L transferrin (Invitrogen Life Technologies), 50 nM hydrocortisone (Sigma-Aldrich), and 5 mg/L insulin for 2 days to suppress expression of the transgene. Finally, the A549 cell line (Lieber and Smith 1976), was grown in DMEM (Gibco) with 10% FBS and Pen-Strep. To induce chemical hypoxia, cells were treated with 1 μM antimycin A (Sigma-Aldrich) for 1 h. In some experiments the cells were treated with 0.5 mM H2O2 for 1 h. This dose falls within the range also used by other investigators to induce oxidative stress in these cell types (Panayiotidis and Stabler 2008). For experiments in which complement activation on the surface of the cells was examined, the cells were subsequently incubated in 10% normal human serum (for the A549 and ARPE-19 cell lines), or complement-sufficient mouse serum (for the murine BUMPT cell line), at 37◦C for 1 h

18.2.3 Flow Cytometry

The surface expression of the complement regulatory proteins DAF, MCP, and CD59, was examined by flow cytometry. Cells were released from the culture plates by treatment with Accutase (Innovative Cell Technologies, Inc.), and washed in PBS. The expression of surface proteins was examined by incubating the cells with primary antibody to the proteins, at 4◦C for 1 h and washing the cells in PBS, and repeating this step with secondary antibodies when necessary. Cells were then washed and resuspended in 500 μL of PBS, run on a FACSCalibur machine (BD Biosciences), and analyzed with CellQuest Pro software (BD Biosciences). To measure complement activation on the cell surface, the cells were washed in PBS, incubated with a FITC-conjugated polyclonal antibody to C3 for 1 h at 4◦C, and analyzed by FACS as described above.