Biosynthesis of C3 by human mesangial cells. Modulation by proinflammatory cytokines.

Deposits of complement (C) components are found in the glomeruli of patients with various glomerulonephritides without detectable immunoglobulins, thus suggesting a pathogenetic role of the locally produced proteins of this system. In the present study, we have examined human mesangial cells (HMC) for their ability to secrete C3. Three different cell lines were examined and all showed a basal production of C3, which was up-regulated following stimulation with IL-1 beta. IL-6 had no direct stimulatory effect on its own, but synergized with IL-1 to induce an increased production of C3 in the culture supernatant and its relative amount was confirmed by SDS-PAGE and immunoblot. Another agonist such as lipopolysaccharide was not able to induce any significant C3 synthesis. Analysis of C3 HMC gene expression, performed by both reverse transcription-polymerase chain reaction of isolated RNA and Northern blot, confirmed the parallel increase of the specific transcript under IL-1 beta and IL-1 beta + IL-6 stimulation. From these data we conclude that production of C3 in the mesangium could have a pathophysiologic relevance.

Effect of cytokines on the secretion of the fifth and eighth complement components by HepG2 cells.

Liver cells can be induced by interleukin-1, tumor necrosis factor and interleukin-6 to secrete higher amounts of complement components. Information, so far available only for the early components, indicates that these cytokines exhibit different effects on various complement proteins. For instance, they promote the biosynthesis of C3 and B but have no effect on that of C4 and C2. These observations led us to evaluate the ability of interleukin-1, tumor necrosis factor and interleukin-6 to modulate the secretion of the late complement components by HepG2 cells, a human hepatoma-derived cell line known to produce several complement proteins. The amount of complement components in the culture supernatant was evaluated by a sensitive enzyme-linked immunosorbent assay revealing picogram levels of these proteins. The HepG2 cells were found to secrete a substantial amount of C3 (approximately 1 microgram/10(6) cells), easily detectable C5 (approximately 150 ng/10(6) cells) and C8 (approximately 10 ng/10(6) cells) and a low amount of C6 (approximately 0.5 ng/10(6) cells), whereas the levels of both C7 and C9 could not be measured. The addition of interleukin-1, tumor necrosis factor and interleukin-6 to the cell culture resulted in an enhanced secretion of C8, whereas that of C5 was only marginally increased. None of these cytokines had a clear effect on the secretion of C6 nor induced the production of C7 and C9.(ABSTRACT TRUNCATED AT 250 WORDS)

Study on the synthesis of complement components by human renal mesangial cells in culture. Assessment of the effect of cytokines.

Renal mesangial cell (MC) cultures are easily established and widely used. MC produce some complement (C) regulatory proteins. We studied whether MC synthesize C components (C3, C5, C8). MC cultures were established from normal portions of cortices of nephrectomies for renal cancer. After growing to near-confluence in RPMI/17% FBS and resting for 24 h in RPMI/0.5% FBS, MC were stimulated up to 72 h with IL-1 beta or IL-6 (10, 100, 1000 U/ml). Neither C5 nor C8 were detected by ELISA. While C3 was present in supernatant under basal conditions (15.5-107.6 ng/10(6) cells/24h) in different MC lines. IL-1 beta up-regulated the synthesis by 2.4-4.5 folds, whereas IL-6 did not show any effect. C3 synthetic rate was 1.76 ng/h/10(6) cells under IL-1 stimulation versus basal rate of 0.37 ng/h/10(6) cells. MC production of C3, especially induced by IL-1 may have pathogenetic relevance in glomerulonephritis.

Study on the synthesis of complement components by human renal mesangial cells in culture. Assessment of the effect of cytokines.

Renal mesangial cell (MC) cultures are easily established and widely used. MC produce some complement (C) regulatory proteins. We studied whether MC synthesize C components (C3, C5, C8). MC cultures were established from normal portions of cortices of nephrectomies for renal cancer. After growing to near-confluence in RPMI/17% FBS and resting for 24 h in RPMI/0.5% FBS, MC were stimulated up to 72 h with IL-1ß or IL-6 (10, 100, 1000 U/ml). Neither C5 nor C8 were detected by ELISA. While C3 was present in supernatant under basal conditions (15.5-107.6 ng/10(6) cells/24h) in different MC lines. IL-1ß up-regulated the synthesis by 2.4-4.5 folds, whereas IL-6 did not show any effect. C3 synthetic rate was 1,76 ng/h/10(6) cells under IL-1 stimulation versus basal rate of 0,37 ng/h/10(6) cells. MC production of C3, especially induced by IL-1 may have pathogenetic relevance in glomerulonephritis.

Monoclonal antibodies as probes to investigate the molecular changes of C5 associated with the different stability of the molecule on sheep erythrocytes and Escherichia coli 0111:B4.

The fifth C component (C5) exhibits a different stability when bound to sheep E or Escherichia coli 0111:B4, being fairly stable on the bacterial intermediate sensitized E. coli 0111:B4 coated with C components up to C5 (BAC1-5) and extremely labile on the RBC intermediate sensitized sheep E coated with C components up to C5 (EAC1-5). We examined the possibility that molecular changes of membrane-bound C5 might be responsible for the different functional behavior of the two intermediates using mAb to C5 and sensitive immunoassays to detect bound C5. The decay of EAC1-5 over 30 min of incubation at 37 degrees C was associated with a significant drop in the reactivity of bound C5 with three of four mAb used. These results contrasted with those obtained with BAC1-5, which showed unchanged reactivity with all mAb tested over the same period of incubation. The effect of mAb on the activity of C5 was then investigated in an attempt to relate the change of the reactivity pattern of EAC1-5 with the functional modification of bound C5. MAb 1.5 and 1.6 were the only antibodies that interfered with the functional activity of C5, although through a different mechanism. In particular, mAb 1.5 was active both on fluid-phase and on membrane-bound C5 and is therefore likely to interact with the binding site for the late components on C5. Conversely, mAb 1.6 was only effective on fluid-phase C5 and acted by promoting a decay of BAC1-5 similar to the spontaneous decay of EAC1-5. We suggest that the bacterial outer membrane may protect C5 from functional decay and that mAb 1.6 interferes with the stabilizing effect of the bacteria in an as yet unclear manner.