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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 [...]

Discovery of a C3(H2O) uptake pathway has led to renewed interest in this alternative pathway triggering form of C3 in human biospecimens. Previously, a quantifiable method to measure C3(H2O), not confounded by other complement activation products, was unavailable. Herein, we describe a sensitive and specific ELISA for C3(H2O). We initially utilized this assay to determine baseline C3(H2O) levels in healthy human fluids and to define optimal sample storage and handling conditions. We detected ~500 ng/ml of C3(H2O) in fresh serum and plasma, a value substantially lower than what was predicted based on previous studies with purified C3 preparations. After a single freeze-thaw cycle, the C3(H2O) concentration increased 3- to 4-fold (~2,000 ng/ml). Subsequent freeze-thaw cycles had a lesser impact on C3(H2O) generation. Further, we found that storage of human sera or plasma samples at 4°C for up to 22 h did not generate additional C3(H2O). To determine the potential use of C3(H2O) as a biomarker, we evaluated specimens from patients with inflammatory-driven diseases. C3(H2O) concentrations were moderately increased (1.5- to 2-fold) at baseline in sera from active systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) patients compared to healthy controls. In addition, upon challenge with multiple freeze-thaw cycles or incubation at 22 or 37°C, C3(H2O) generation was significantly enhanced in SLE and RA patients' sera. In bronchoalveolar lavage fluid from lung-transplant recipients, we noted a substantial increase in C3(H2O) within 3 months of acute antibody-mediated rejection. In conclusion, we have established an ELISA for assessing C3(H2O) as a diagnostic and prognostic biomarker in human diseases. [ABSTRACT FROM AUTHOR]

Discovery of a C3(H 2 O) uptake pathway has led to renewed interest in this alternative pathway triggering form of C3 in human biospecimens. Previously, a quantifiable method to measure C3(H 2 O), not confounded by other complement activation products, was unavailable. Herein, we describe a sensitive and specific ELISA for C3(H 2 O). We initially utilized this assay to determine baseline C3(H 2 O) levels in healthy human fluids and to define optimal sample storage and handling conditions. We detected ~500 ng/ml of C3(H 2 O) in fresh serum and plasma, a value substantially lower than what was predicted based on previous studies with purified C3 preparations. After a single freeze-thaw cycle, the C3(H 2 O) concentration increased 3- to 4-fold (~2,000 ng/ml). Subsequent freeze-thaw cycles had a lesser impact on C3(H 2 O) generation. Further, we found that storage of human sera or plasma samples at 4°C for up to 22 h did not generate additional C3(H 2 O). To determine the potential use of C3(H 2 O) as a biomarker, we evaluated specimens from patients with inflammatory-driven diseases. C3(H 2 O) concentrations were moderately increased (1.5- to 2-fold) at baseline in sera from active systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) patients compared to healthy controls. In addition, upon challenge with multiple freeze-thaw cycles or incubation at 22 or 37°C, C3(H 2 O) generation was significantly enhanced in SLE and RA patients' sera. In bronchoalveolar lavage fluid from lung-transplant recipients, we noted a substantial increase in C3(H 2 O) within 3 months of acute antibody-mediated rejection. In conclusion, we have established an ELISA for assessing C3(H 2 O) as a diagnostic and prognostic biomarker in human diseases.