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 6

C3(H2O) processing by cofactor proteins.

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C3(H2O) processing by cofactor proteins. Whole cell lysates (A and B) an...
Whole cell lysates (A and B) and subcellular fractions (B) were prepared from Farage cells incubated with C3(MA), with or without FH and FI, and analyzed by WB with a goat anti-FI pAb (A) or a goat anti-FH pAb (B). Nonreducing conditions. FH, 20 ng; cell equivalents, 3 × 105. Representative of 2 independent experiments. (C) Farage cells were incubated for 15 minutes with 15 μg/ml C3(MA) alone or plus FI (I) (2 μg/ml), or FI and FH (H) (25 μg/ml). The resulting cell lysates were analyzed by WB with a goat anti-C3 pAb (top panel) or a rabbit anti-C3a pAb (bottom panel). Under reducing conditions. C3, iC3b and iC3(MA), 20 ng; cell lysates, 4 × 105 cell equivalents loaded. Representative of 3 independent experiments. (D) Lysates prepared from Farage cells incubated with C3(MA) alone or with FH and FI for the times indicated were analyzed for uptake and CA by WB with a rabbit anti-C3a pAb. Reducing conditions. C3, 70 ng; C3a, 30 ng; cell lysates, 4 × 105 cell equivalents loaded. Representative of 2 independent experiments. (E) Schematic depicting the cleavage and inactivation of C3(H2O) resulting from CA. These experiments were also performed with fibroblasts, and the same results were obtained.