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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 Article has an altmetric score of 5

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

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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 1

Human cell lines demonstrate uptake of C3 from an exogenous source.

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Human cell lines demonstrate uptake of C3 from an exogenous source.
(A) ...
(A) Whole cell lysates from peripheral blood B lymphocytes (B cell) and a human B cell line (Farage) were prepared and analyzed for C3 under reducing (R) and nonreducing (NR) conditions by WB. Lane 5 is a 4-fold longer exposure of lane 3. C3, 30 ng; C3b, 30 ng; B cells, 7.4 × 104 cell equivalents; Farage, 6.3 × 105 cell equivalents. (B) Two human cells lines, ARPE-19 and Farage, were incubated in 10% NHS for 30 minutes to assess whether they take up C3. C3, 30 ng; cell lysates, 2.4 × 105 cell equivalents. (C) Farage cells were incubated for 15 minutes with commercially available purified C3 and washed 3 (lane 3), 6 (lane 4), or 9 (lane 5) times prior to preparation of cell lysates. Cell lysates (top panel), final wash supernatants (bottom panel). C3, 30 ng; cell lysates, 2.4 × 105 cell equivalents; wash supernatants, 20 μl (of 100 μl). Representative blot of 4 (A and B) or 2 (C) independent experiments.

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ISSN: 0021-9738 (print), 1558-8238 (online)

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