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.
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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

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Figure 2

Loading of C3(H2O) is specific.

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Loading of C3(H2O) is specific. Uptake of C3(H2O) versus native C3 was i...
Uptake of C3(H2O) versus native C3 was investigated by incubating Farage cells with (A) 20 μg/ml of a commercially available C3 preparation (C3) or a freshly purified C3 preparation (fresh C3) or (B) 5 μg/ml commercially available C3 or C3(MA). Cell lysates were prepared and analyzed for uptake by WB. GAPDH, loading control. C3, 20 ng (lane 1); cell lysates, 2.4 × 105 cell equivalents. (C) Labeled C3 (C3-DL488) was utilized to visualize C3 loading into cells and determine whether uptake is sensitive to competition with unlabeled protein. ARPE-19 cells were loaded for 5 minutes without (top panel) or with (bottom panel, 10-fold) 2-, 5-, or 10-fold excess concentrations of unlabeled C3(H2O) followed by C3-DL488 (50 μg/ml) for an additional 15 minutes and visualized by confocal microscopy. Original magnification, ×60. C3-DL488 uptake per cell is quantified in part D. n = 3 representative cells per condition. Blue, DAPI; green, C3-DL488. *P < 0.05 compared with no excess of unlabeled C3(H2O) by 1-way ANOVA with Dunn’s multiple comparison test. (E) To determine whether denatured C3(MA) is internalized, Farage cells were incubated with 10 μg/ml C3(MA) or heat-denatured C3(MA) (hD-C3[MA]). C3, 30 ng (lane 1); cell lysates, 4 × 105 cell equivalents (lanes 3–5). (A, B, and E) Blots are representative of 2 independent experiments.