Ex Vivo Test for Measuring Complement Attack on Endothelial Cells: From Research to Bedside

Abstract

As part of the innate immune system, the complement system plays a key role in defense against pathogens and in host cell homeostasis. This enzymatic cascade is rapidly triggered in the presence of activating surfaces. Physiologically, it is tightly regulated on host cells to avoid uncontrolled activation and self-damage. In cases of abnormal complement dysregulation/overactivation, the endothelium is one of the primary targets. Complement has gained momentum as a research interest in the last decade because its dysregulation has been implicated in the pathophysiology of many human diseases. Thus, it appears to be a promising candidate for therapeutic intervention. However, detecting abnormal complement activation is challenging. In many pathological conditions, complement activation occurs locally in tissues. Standard routine exploration of the plasma concentration of the complement components shows values in the normal range. The available tests to demonstrate such dysregulation with diagnostic, prognostic, and therapeutic implications are limited. There is a real need to develop tools to demonstrate the implications of complement in diseases and to explore the complex interplay between complement activation and regulation on human cells. The analysis of complement deposits on cultured endothelial cells incubated with pathologic human serum holds promise as a reference assay. This ex vivo assay most closely resembles the physiological context. It has been used to explore complement activation from sera of patients with atypical hemolytic uremic syndrome, malignant hypertension, elevated liver enzymes low platelet syndrome, sickle cell disease, pre-eclampsia, and others. In some cases, it is used to adjust the therapeutic regimen with a complement-blocking drug. Nevertheless, an international standard is lacking, and the mechanism by which complement is activated in this assay is not fully understood. Moreover, primary cell culture remains difficult to perform, which probably explains why no standardized or commercialized assay has been proposed. Here, we review the diseases for which endothelial assays have been applied. We also compare this test with others currently available to explore complement overactivation. Finally, we discuss the unanswered questions and challenges to overcome for validating the assays as a tool in routine clinical practice.

C3‐dependent effector functions of complement

“…Detection of the C3 activation fragments in ex vivo models of complement activation and in tissues is important for clinical practice and for understanding disease pathogenesis. Analysis of C3 activation fragments and C5b‐9 on cultured endothelial cells incubated with serum from aHUS patients closely resembles the pathophysiological context of this disease 114 . It has shown elevated deposits from the sera of patients or model conditions of aHUS, malignant hypertension, elevated liver enzymes, low platelet syndrome (HELLP syndrome), sickle cell disease, preeclampsia, lupus nephritis, etc.…”

“…Analysis of C3 activation fragments and C5b‐9 on cultured endothelial cells incubated with serum from aHUS patients closely resembles the pathophysiological context of this disease. 114 It has shown elevated deposits from the sera of patients or model conditions of aHUS, malignant hypertension, elevated liver enzymes, low platelet syndrome (HELLP syndrome), sickle cell disease, preeclampsia, lupus nephritis, etc 109 , 115 , 116 , 117 , 118 In one case, it was used to adjust the therapeutic regimen with a complement‐blocking drug, as the test showed increased deposition of C5b‐9 before the relapse of the disease, encouraging further studies. 119 To make specific diagnoses, tissue staining for C3 activation fragments is critical in clinical practice, especially on kidney biopsies.…”

Anti-factor H autoantibody-associated hemolytic uremic syndrome in an Argentine pediatric cohort

“…Expression of terminal complement activation, C5b-9, in the bloodstream has not been validated as a reliable biomarker of aHUS activity [31] or relapse in patients with anti-FH antibodies [20]. However, Song et al [32] showed that C5b-9 levels increased during the acute phase and decreased during remission in a cohort of 33 positive for anti-FH patients. Determination of C5b-9 in two of our patients currently monitored to prevent possible relapse showed distinct C5b-9 expression profiles.…”

Complement Biosensors Identify a Classical Pathway Stimulus in Complement-Mediated Hemolytic Uremic Syndrome

“…Two principal techniques for in vitro diagnosis have been proposed, including the modified Ham assay (mHam) 9,10 and HMEC-1 staining for C5b-9 [11][12][13][14] , but neither is widely adopted. Both assays rely upon testing patient serum for abnormal cell-deposited complement activity.…”