levels of DAF, and the levels of MCP and CD59 were unchanged. Thus, the response of ARPE-19 cells to hypoxia and oxidative stress are distinct.

18.3.2Oxidative Stress of Renal Tubular Epithelial Cells Does Not Alter Surface Expression of Crry by the Cells

Crry is the only membrane bound complement regulatory protein expressed by mouse renal PTECs (Thurman et al. 2006). To determine whether expression of Crry by PTECs is altered by oxidative stress, we treated PTECs with 0.5 mM H2O2 for 1 h and assessed surface levels of Crry by FACS analysis. Surface levels of Crry in the oxidatively stressed cells were not detectably different than levels in unmanipulated control cells (Fig. 18.2a). We also subjected PTECs to oxidative stress and then exposed them to 10% serum. Exposure of PTECs to 10% serum caused deposition of C3 onto the cell surface, but oxidative stress of the cells did not detectably alter the amount of C3 deposited on serum-exposed cells compared to unmanipulated control cells (Fig. 18.2b).

18.3.3Expression of MCP, CD55 and CD59 on the Surface

of Lung Epithelial Cells Increases After Oxidative Stress, but This Does Not Prevent Complement-Activation on the Cell Surface

We treated epithelial cells derived from human lung with the same dose of H2O2 and measured surface expression of the complement regulatory proteins MCP, CD55, and CD59 by FACS analysis. Cells treated with H2O2 demonstrated increased levels of all three proteins when compared to unmanipulated control cells (Fig. 18.2c–e). We also found that oxidative stress alone induced fixation of C3 to the cell surface even in the absence of exogenous serum (Fig. 18.2f). Based upon these results we suspect that oxidatively-stressed A549 cells secrete C3. Taken together, these data demonstrate that the A549 cells increase the surface expression of complement regulatory proteins in response to oxidative stress. This increased complement regulation may be offset by other cellular responses such as the release of C3 by the cells, however; and there is a net increase in complement deposition on the cell surface after treatment with H2O2.

18.4 Discussion

We have previously shown that oxidative stress of ARPE-19 cells alters the expression of complement regulatory proteins on the cell surface, resulting in greater deposition of C3 on the cell surface when they are subsequently exposed to complement sufficient serum (Rohrer and Renner 2008). In the current study we report

Fig. 18.2 Oxidative stress does not alter complement regulation on the surface of ptecs or A549 cells; Cells were grown to confluence and were treated with 0.5 mM H2O2 for 1 h. (a) PTECs were stained for Crry, and surface levels on oxidatively-stressed cells (shaded curve) were compared to unmanipulated controls (solid line) and an isotype control (gray line). (b) PTECs were treated with H2O2 and were then exposed to 10% mouse serum for 1 h. C3 deposition on the cells treated with H2O2 (shaded curve) was compared to that seen in unmanipulated controls (solid line) and an isotype control (gray line). Levels of (c) DAF, (d) MCP, and (e) CD59 on H2O2-treated A549 cells (solid lines) were compared with unmanipulated controls (shaded curves) and isotype controls (gray lines). (f) Levels of surface C3 were assessed from A549 cells treated with H2O2 (solid line) and were compared to unmanipulated cells (shaded curve) and isotype control antibody (gray line)

that chemical hypoxia of the ARPE-19 cells does not have a similar effect. Indeed, surface levels of DAF were increased after treatment of the cells with Antimycin A. We also tested the effects of oxidative stress on epithelial cells derived from kidney and lung. Treatment of PTECs with H2O2 did not alter the surface expression of the complement regulatory protein, Crry, and did not cause increased deposition of C3 on the cell surface after exposure to serum. Treatment of A549 cells with H2O2 increased the surface expression of MCP, CD55 and CD59. Thus, epithelial

cells from different tissues have distinct responses to oxidative stress. Oxidative stress reduces the ability of RPE cells to regulate complement on the cell surface. PTEC cells were not significantly affected by the treatment. Pulmonary epithelial cells increased their expression of complement regulatory proteins, but were still more susceptible to complement activation, possibly due to increased production of complement-activating proteins by the cells.

Pathologic complement activation on epithelial barrier layers is characteristic of inflammatory diseases in several organs, including the eye (Gehrs and Anderson 2006), the kidney (Thurman et al. 2006), and the lung (Taube and Thurman 2006). We initially hypothesized that a reduction in cell surface complement-regulation might be a generalized response of epithelial cells to infection or cellular stress. Such a response might evolve to help protect the host from invasive infection. Our current results, however, suggest that epithelial cells from different organs respond to cellular stress in idiosyncratic ways. Oxidative stress markedly increases the susceptibility of RPE cells to complement activation, but chemical hypoxia did not have this effect. On the other hand, hypoxia increases the susceptibility of renal epithelial cells to complement-mediated injury, but oxidative stress had very little effect.

The unique response of RPE cells to oxidative stress may reflect the environment in which these cells reside. The eye must be kept absolutely free of invasive pathogens, but it is also an immunologically privileged location. Resident cells must therefore perform surveillance functions, and generate proinflammatory factors if they sense pathogens or tissue damage. In this context, modulated regulation of the complement system at the RPE cell layer could be a rapid and potent defense mechanism against ocular infection. Epithelial cells in organs such as the lung, in contrast, are regularly exposed to inhaled pathogens and may have a higher threshold for induction of proinflammatory signals. Renal epithelial cells should also maintain a sterile environment, so it is not immediately clear why RPEs are sensitive to oxidative stress, but the renal cells are sensitive to chemical hypoxia. These responses may relate to the pathogens these cells have evolved to detect. The important role of complement activation in the development of AMD (Hageman and Luthert 2001; Gehrs and Anderson 2006), however, underscores the delicate balance that must be maintained to protect the RPE cell layer from autologous injury while also preventing infectious injury.

In summary, we have found that epithelial cells derived from the eye and the lung express DAF, CD59, and MCP. Epithelial cells from the mouse kidney express Crry in an analogous distribution to MCP in human renal epithelial cells (Ichida and Yuzawa 1994; Thurman et al. 2006). The expression of these proteins is altered by aseptic cellular stressors such as hypoxia and oxidative stress, but the response to these conditions differs from tissue to tissue. In RPE cells, oxidative stress reduces the expression of the cell surface complement regulators and sensitizes the cells to complement-mediated injury. This specific response is not seen in epithelial cells from the lung or kidney, and is not induced by hypoxia. These studies help explain the unique mechanisms by which uncontrolled complement activation may contribute to the development of AMD. A greater understanding of the molecular

events that cause AMD may help in the development of therapies to reduce tissue inflammation while minimizing the risk of infection.

Acknowledgments This work was supported in part by National Institutes of Health Grants DK077661 and DK076690 (JMT), DK035081 (MKP), EY13520 and EY017465 (BR) and HL082485 (ST), a vision core grant (EY014793), the Foundation Fighting Blindness, American Heart Association grant 0735101 N (VPF), a grant from the Sandler Program for Asthma Research (VMH), and an unrestricted grant to MUSC from Research to Prevent Blindness, Inc., New York, NY. BR is a Research to Prevent Blindness Olga Keith Weiss Scholar. The authors thank Luanna Bartholomew for editorial assistance. The authors declare the following disclosures. VMH is a cofounder of Taligen Therapeutics, Inc., which develops complement inhibitors for therapeutic use; JMT, VMH and BR are consultants for Taligen Therapeutics, Inc.

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