Elsevier

Immunobiology

Volume 212, Issue 2, 5 April 2007, Pages 129-139
Immunobiology

Use of monoclonal antibodies to assess expression of anaphylatoxin receptors in tubular epithelial cells of human, murine and rat kidneys

https://doi.org/10.1016/j.imbio.2006.11.003Get rights and content

Abstract

To assess published evidence of anaphylatoxin receptor expression in renal tubular epithelial cells, monoclonal antibodies (mAbs) against human, mouse and rat receptors for C5a and C3a (C5aR, C3aR) were raised using receptor-expressing transfectants as immunogens. Applying these reagents in immunohistochemistry, we observed that mAbs with reactivities against three distinct epitopes of human C5aR N-terminus recognized tissue macrophages but not at all renal tubular epithelial cells. These findings were surprising, as strong tubular staining had been previously demonstrated by mAbs raised against a synthetic N-terminal C5aR peptide. To extend our study to mammalian kidneys, renal specimens from normal rats as well as LPS-treated Balb/c and MRL/lpr mice, which suffered from lupus-type nephritis, were examined. Similar to humans, mAbs against murine or rat C5aR strongly recognized infiltrating leukocytes in situ whereas tubular epithelial cells remained negative. As a mAb has been previously used to document C3aR expression in renal tubular epithelial cells, kidney specimens were examined using newly established mAbs against different epitopes of human, murine and rat C3aR. In contrast to published evidence, C3aR was detectable exclusively in interstitial leukocytes but not in epithelial tubular cells of normal and diseased tissues. Taken together, our findings question a direct involvement of tubular epithelial cells in anaphylatoxin-mediated renal inflammation. Furthermore, as we demonstrate in the case of anaphylatoxin receptors, cross-reactivities of mAbs may constitute as yet underestimated pitfalls in immunohistochemical antigen detection.

Introduction

Complement can be activated through the classical, alternative and lectin pathways. C3 is cleaved upon complement activation with generation of C3a and C3b, followed by activation and production of C5a and C5b (Walport, 2001). The anaphylatoxins C3a and C5a are responsible for attracting and activating leukocytes, especially phagocytic cells such as granulocytes and monocytes/macrophages. Anaphylatoxin receptors are closely related members of the rhodopsin family of seven transmembrane-spanning G protein-linked receptors (Ames et al., 1996).

Evidence has accumulated that non-myeloid cells in various tissues display abundant anaphylatoxin receptor expression. In human and murine lungs, C5aR and C3aR were identified in a variety of resident cell types such as bronchial epithelial cells, alveocytes, bronchial and vascular smooth muscle cells and endothelial cells (Drouin et al., 2001). In addition, it was shown that inflammatory conditions up-regulate pulmonary anaphylatoxin receptor expression (Drouin et al., 2001; Fregonese et al., 2005).

In human and murine renal tissues, anaphylatoxin receptors were detected predominantly in tubular epithelial cells (Abe et al., 2001; Braun et al., 2004; Fayyazi et al., 2000). However, functional consequences of renal tubular anaphylatoxin receptor expression remained elusive or not well characterized (Braun et al., 2004; Peake et al., 1999). Expression of anaphylatoxin receptors in renal tubular cells was shown to be up-regulated in lupus nephritis in humans and mice (Abe et al., 2001; Bao et al., 2005a, Bao et al., 2005b) as well as in murine models of renal ischemia-reperfusion injury (de Vries et al., 2003) and sepsis (Riedemann et al., 2002).

Furthermore, anaphylatoxins are involved in the promotion of experimental lupus nephritis in MRL/lpr mice and tubulointerstitial injury in a murine model of immune complex-mediated glomerulonephritis (Bao et al., 2005a, Bao et al., 2005b; Welch et al., 2002; Wenderfer et al., 2005). In all these experimental settings; however, evidence for non-myeloid anaphylatoxin receptor expression to be instrumental in mediating anaphylatoxin-induced tissue injuries was not presented. It is also noteworthy that the view of a widespread expression of anaphylatoxin receptors in pulmonary non-myeloid cells has been challenged (Thangam et al., 2005; Zwirner et al., 1999a, Zwirner et al., 1999b). Therefore, a reexamination of renal anaphylatoxin receptor expression appeared warranted.

To achieve this goal we raised monoclonal antibodies (mAbs) against human, rat and murine anaphylatoxin receptors and applied these reagents in immunohistochemical examinations of normal as well as diseased renal tissues. Beside an abundant expression of anaphylatoxin receptors in myeloid cells, we detected no evidence for the presence of C5aR and C3aR in renal tubular epithelial cells in situ under normal and inflammatory conditions. Thus, our findings stress the importance of a critical evaluation of antibody reactivities and may have important implications for the supposed expression of anaphylatoxin receptors in non-myeloid cells of other organs.

Section snippets

Monoclonal antibodies

Monoclonal Ab hC3aRZ8 (mouse IgG2b) (Soruri et al., 2003a, Soruri et al., 2003b) as well as mAb hC3aRZ3 (mouse IgG1) (Zwirner et al., 1999b) are reactive against human C3aR. Monoclonal Abs hC5aRZ1 (mouse IgG1), P12/1, S5/1 (both mouse IgG2a), and W17/1 (mouse IgG1) bind to human C5aR (Schakel et al., 2002; Werfel et al., 1996; Oppermann et al., 1993). Monoclonal Ab R63 (mouse IgG1) recognizes rat C5aR (Rothermel et al., 2000) and mAb 10/92 (rat IgG2a) mouse C5aR (Soruri et al., 2003b).

Monoclonal antibodies against human C5a receptor

We raised novel rat mAbs against native human C5aR molecules by immunizing rats with RBL-2H3 transfectants. These mAbs reacted with RBL-2H3 transfectants expressing human C5aR but were unreactive with parental RBL-2H3 cells or RBL-2H3 transfectants expressing mouse C5aR or human C3aR. Antibodies were further tested for reactivity against two chimeric C5aR molecules expressed in RBL-2H3 cells (Rothermel et al., 2000). Chimera 1 consisted of a human C5aR N-terminus (aa 1–40) on top of a rat C5aR

Discussion

Anaphylatoxin receptor expression has been described in many different cell types in various tissues. In the kidney, tubular cells have been identified as a main reservoir of anaphylatoxin receptors. At the protein level, receptor expression in human kidneys has been detected using mono- as well as polyclonal Abs. The different mAbs (P12/1, W17/1) against hC5aR used in these immunohistochemical studies (Abe et al., 2001; Fayyazi et al., 2000) as well as mAb S5/1 used herein were generated in

Acknowledgements

This work was supported by a Grant from the Deutsche Forschungsgemeinschaft (ZW 38/3-3). We thank Ms. Olga Walter and Ms. Sandra Henke for expert technical assistance.

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