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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([H.sub.2]O), the hydrolytic product of C3, and not native C3, from the extracellular milieu. Uptake was rapid, saturable, and sensitive to competition with unlabeled C3([H.sub.2]O), indicating a specific mechanism of loading. Under steadystate conditions, approximately 80% of incorporated C3([H.sub.2]O) was returned to the extracellular space. These studies identify an ICS recycling pathway for C3([H.sub.2]O). The loaded C3([H.sub.2]O) represents a source of C3a, and its uptake altered the cytokine profile of activated [CD4.sup.+] 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., Introduction The complement system's component C3 is the most abundant complement protein in blood and the central player in all three activation pathways (classical, alternative, and lectin). Complement engagement resulting [...]