A C3(H20) recycling pathway is a component of the intracellular complement system
Michelle Elvington, … , Hrishikesh S. Kulkarni, John P. Atkinson
Michelle Elvington, … , Hrishikesh S. Kulkarni, John P. Atkinson
Published February 13, 2017
Citation Information: J Clin Invest. 2017;127(3):970-981. https://doi.org/10.1172/JCI89412.
View: Text | PDF
Research Article Immunology Inflammation

A C3(H20) recycling pathway is a component of the intracellular complement system

Abstract

An intracellular complement system (ICS) has recently been described in immune and nonimmune human cells. This system can be activated in a convertase-independent manner from intracellular stores of the complement component C3. The source of these stores has not been rigorously investigated. In the present study, Western blotting identified a band corresponding to C3 in freshly isolated human peripheral blood cells that was absent in corresponding cell lines. One difference between native cells and cell lines was the time absent from a fluid-phase complement source; therefore, we hypothesized that loading C3 from plasma was a route of establishing intracellular C3 stores. We found that many types of human cells specifically internalized C3(H2O), the hydrolytic product of C3, and not native C3, from the extracellular milieu. Uptake was rapid, saturable, and sensitive to competition with unlabeled C3(H2O), indicating a specific mechanism of loading. Under steady-state conditions, approximately 80% of incorporated C3(H2O) was returned to the extracellular space. These studies identify an ICS recycling pathway for C3(H2O). The loaded C3(H2O) represents a source of C3a, and its uptake altered the cytokine profile of activated CD4+ T cells. Importantly, these results indicate that the impact of soluble plasma factors should be considered when performing in vitro studies assessing cellular immune function.

Authors

Michelle Elvington, M. Kathryn Liszewski, Paula Bertram, Hrishikesh S. Kulkarni, John P. Atkinson

×

Figure 5

Characterization of C3(H2O) recycling.

Options: View larger image (or click on image) Download as PowerPoint
Characterization of C3(H2O) recycling. (A) The amount of C3(H2O) uptake ...
(A) The amount of C3(H2O) uptake was assessed by WB and quantified by densitometry. Farage cells were incubated for 1 hour with increasing concentrations of C3(MA) in 10% C3-depleted serum. Densitometry was used to determine ng of C3(MA) taken up per 105 cell equivalents. (B) The kinetics of loading were determined by incubating Farage cells with 5 μg/ml C3(MA) for increasing lengths of time and analyzed by WB. Densitometry was used to assess the amount of C3(H2O) present in the cells at each time point. Inset is a representative WB. (C–E) The kinetics of C3(H2O) loss from established stores was assessed in Farage (C), Jurkat (D), and ARPE-19 (E) cells. Cells were incubated for 1 hour with 20 μg/ml C3, washed, and plated in the absence of C3 (normal growth media) for the indicated time points. Densitometric scanning of WB was used to determine the amount of C3(H2O) remaining in the cells compared with cells lysed immediately after loading. (F) The amount of C3(H2O) released from loaded (C3 loaded) or unloaded (unloaded) Farage cells into the supernatant was assessed by WB at the indicated time points. (G) The kinetics of C3(H2O) loss from freshly isolated CD4+ T cells that contained C3(H2O) stores loaded in vivo was assessed. The isolated CD4+ T cells were plated in complete media containing 10% FBS and 25 U/ml rhIL-2, and cell lysates were prepared at the indicated time points. Densitometric scanning was used to determine the amount of C3(H2O) remaining in the cells compared with cells lysed immediately after isolation. (A–G) Data (mean ± SD) are from 2 independent experiments.