Elsevier

Molecular Immunology

Volume 118, February 2020, Pages 142-152
Molecular Immunology

Complement fragments are biomarkers of antibody-mediated endothelial injury

https://doi.org/10.1016/j.molimm.2019.12.011Get rights and content

Highlights

Abstract

Antibody-mediated rejection (AbMR) adversely affects long-term graft survival in kidney transplantation. Currently, the diagnosis of AbMR requires a kidney biopsy, and detection of complement C4d deposition in the allograft is one of the diagnostic criteria. Complement activation also generates several soluble fragments which could potentially provide non-invasive biomarkers of the process. Furthermore, microvesicles released into the plasma from injured cells can serve as biomarkers of vascular injury. To explore whether soluble complement fragments or complement fragments bound to endothelial microvesicles can be used to non-invasively detect AbMR, we developed an in vitro model in which human endothelial cells were exposed to anti-HLA antibodies and complement sufficient serum. We found that complement fragments C4a and sC5b-9 were increased in the supernatants of cells exposed to complement-sufficient serum compared to cells treated complement-deficient serum. Furthermore, complement activation on the cell surface was associated with the release of microvesicles bearing C4 and C3 fragments. We next measured these analytes in plasma from kidney transplant recipients with biopsy-proven acute AbMR (n = 9) and compared the results with those from transplant recipients who also had impaired allograft function but who did not have AbMR (n = 30). Consistent with the in vitro results, complement fragments C4a and Ba were increased in plasma from patients with AbMR compared to control subjects (P < 0.001 and P < 0.01, respectively). Endothelial microvesicle counts were not increased in patients with AbMR, however, and the number of microvesicles with C4 and C3 bound to the surface was actually lower compared to control subjects (both P < 0.05). Our results suggest that plasma complement activation fragments may be useful as non-invasive biomarkers of antibody-mediated complement activation within the allograft. Complement-opsonized endothelial microvesicles are decreased in patients with AbMR, possibly due to enhanced clearance of microvesicles opsonized with C3 and C4 fragments.

Introduction

Kidney transplantation is the optimal treatment for patients with end stage renal disease (ESRD) (Wolfe et al., 1999). However, acute and/or chronic immunologic injury mediated by donor-specific antibodies (DSAs) against donor human leukocyte antigens (HLA) is a major cause of kidney graft loss and is a significant barrier to long-term graft survival (Sellares et al., 2012). AbMR is characterized pathologically by microvascular injury and inflammation. Even in the absence of graft dysfunction, these pathologic changes on surveillance biopsies (subclinical AbMR) are associated with inferior graft outcomes (Loupy et al., 2015; Orandi et al., 2015). The diagnosis of AbMR also requires evidence of DSA interaction with the endothelium, usually detected as C4d deposition in the peritubular capillaries (Haas, 2014). During antibody-mediated classical pathway activation C4 is covalently attached to target tissues, providing a durable marker of this process. Although there are cases of C4d-negative AbMR, complement activation is associated with a poorer prognosis and is generally considered an important contributor to graft injury in AbMR (Feucht et al., 1993; Kikic et al., 2015).

Because of the impact of AbMR, with or without concurrent graft dysfunction, on short- and long-term graft outcomes, better methods for diagnosing and monitoring its presence are needed. Currently, a kidney biopsy is required for diagnosis of AbMR, and the only tools available for surveillance are protocol biopsy and serially monitoring for DSA. While DSA monitoring is an attractive option because it is noninvasive and widely available, it has low specificity for subclinical AbMR and frequently does not reflect active inflammation and injury (Parajuli et al., 2017; Schinstock et al., 2017). In addition to a potential pathogenic role in allograft injury, complement activation also generates several soluble fragments that can serve as clinical biomarkers of inflammation (Frazer-Abel et al., 2016). For example, every molecule of C4b that is attached to target cells is accompanied by release of a C4a fragment. Classical pathway activation within the allograft may, therefore, increase C4a levels in the plasma.

Damaged endothelial cells also release microvesicles. Microvesicles are sub-micrometer-sized membrane-bound vesicles (0.05−1 μm) shed from a variety of cells, including endothelial cells, both constitutively and in response to activation, injury, and apoptosis (Anderson et al., 2010; Beyer and Pisetsky, 2010). Studies have demonstrated that endothelial microvesicles increase in diseases associated with endothelial injury, and they can be isolated from peripheral blood as circulating markers of events at the cellular level (Boulanger et al., 2007; Chironi et al., 2009; Hsu et al., 2013). It was also reported that AbMR increases the number of C4-opsonized endothelial microvesicles, possibly reflecting complement-mediated endothelial injury (Tower et al., 2016).

We hypothesized that antibody-mediated complement activation would lead to increased generation of soluble complement activation fragments and release of C3 or C4-bearing endothelial microvesicles that could serve as plasma-derived biomarkers of AbMR in renal transplant recipients. To test this hypothesis, we developed and characterized an in vitro model of AbMR using immortalized human endothelial cells and collected pilot data with samples from renal transplant recipients with impaired graft function in the absence of AbMR and from patients with biopsy-proven AbMR.

Section snippets

Complement activation, deposition, and clearance on endothelial cells

Confluent HMEC-1 cells grown in 6-well plates were incubated with a saturating concentration of mAb W6/32 (3 μg/mL) for 30 min at 37 °C (see Supplementary Fig. 1). Cells were subsequently washed with PBS and incubated with culture medium with or without 10 % normal human serum (NHS; Quidel, San Diego, CA) at 37 °C. Higher NHS concentrations were examined but did not significantly alter the results using this model. The degree of C4 deposition per cell was greater when subconfluent cells were

Complement activation on endothelial cells in culture

HMEC-1 cells were incubated with a saturating concentration of anti-HLA class I antibody (W6/32) (Supplemental Fig. 1) and subsequently exposed to NHS to elicit complement activation and deposition on the cell surface (Fig. 1). Early after serum exposure, active C4 in the form of C4b (recognized by an anti-C4c detection antibody) is abundant but subsequently declines at later time points (Fig. 1A). This decline is presumably due to conversion to the inactive form C4d that is detected early

Discussion

In this study we developed an in vitro model of AbMR using immortalized human microvascular endothelial cells and a monoclonal anti-class I HLA antibody to test whether complement activation fragments and endothelial microvesicles could serve as biomarkers of AbMR. Our model approximates the pathophysiology of AbMR in transplant recipients as demonstrated by the deposition of complement fragments on the cell surface, analogous to C4d fixation on endothelial cells in transplant biopsies with

Conclusion

Our in vitro model and pilot clinical data show that soluble complement fragments generated by antibody-mediated complement activation on the endothelium can be detected and give insight to events at the cellular level. Our data also suggests that circulating microvesicles are not a reliable biomarker of AbMR in contrast to a previous report. Current methods for surveillance and diagnosis of AbMR rely on detection of DSA and analysis of various antibody characteristics (i.e. titer, sub-class,

Authors’ contributions

JT, ES and ML designed research. ES, JL, BR, ML performed experiments. ZY, BF, JC, and DL helped analyze the results. ES and JT wrote the manuscript, and all authors critically read and commented on manuscript drafts.

Conflict of interest

JMT receives royalties from Alexion Pharmaceuticals, Inc., and is a consultant for AdMIRx, Inc., a company developing complement inhibitors. He also holds stock and will receive royalty income from AdMIRx.

CRediT authorship contribution statement

Erik Stites: Conceptualization, Investigation, Writing - original draft. Brandon Renner: Methodology, Investigation. Jennifer Laskowski: Methodology, Investigation. Moglie Le Quintrec: Conceptualization, Investigation, Resources. Zhiying You: Formal analysis. Brian Freed: Resources. James Cooper: Resources. Diana Jalal: Conceptualization, Resources. Joshua M. Thurman: Conceptualization, Methodology, Writing - review & editing, Funding acquisition.

Acknowledgements

This work was supported by National Institutes of HealthT32 DK007135 (ES), DK113586 and DK076690 (JMT). Imaging experiments were performed in the University of Colorado Anschutz Medical Campus Advance Light Microscopy Core supported in part by NIH/NCATS Colorado CTSI Grant Number UL1 TR001082. We would like to acknowledge the assistance of the University of Colorado Flow Cytometry Shared Resource, as well as Ronald P. Taylor and Margaret A. Lindorfer who generously provided the 3E7 and 1H8

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