The priming effect of C5a on monocytes is predominantly mediated by the p38 MAPK pathway

Abstract

The dysregulation of the inflammatory response after trauma leads to significant morbidity and mortality. Monocytes and macrophages play a central role in the orchestration of the inflammatory response after injury. Serum interleukin-6 (IL-6) concentration correlates with poor outcomes after injury. Tumor necrosis factor-alpha (TNF-alpha) is a proinflammatory cytokine that plays a crucial role in the pathogenesis of multiple organ dysfunction syndrome. Furthermore, in the presence of C5a, monocytes and macrophages have potentiated responses, but the mechanisms underlying this response remain largely unknown. Peripheral blood mononuclear cells (PBMCs) were isolated from healthy volunteers and pretreated with C5a (100 ng/mL) for 1 h before adding lipopolysaccharide (LPS) (10 ng/mL) for up to 20 h. Inhibitors for the mitogen-activated protein kinases (MAPKs) were added 1 h before adding C5a. C5a primes monocytes for LPS-induced IL-6 and TNF-alpha production. Treatment of PBMCs with C5a leads to a rapid activation of the 3 MAPK pathways. SP600125 (inhibitor of c-Jun NH2-terminal kinase MAPK) and PD98059 (inhibitor of extracellular signal-regulated kinase MAPK) did not affect the C5a priming of the LPS-induced IL-6 and TNF-alpha production, whereas SB203580, a specific inhibitor of p38 MAPK, did suppress the C5a priming effect. These results demonstrate that C5a primes adherent PBMCs and modulates LPS-induced IL-6 and TNF-alpha production. Results from extracellular signal-regulated kinase and c-Jun NH2-terminal kinase MAPK blockade suggest that these signaling pathways have minimal or no role in reprogramming LPS-mediated IL-6 and TNF-alpha production. On the contrary, in PBMCs, C5a activates the p38 cascade, and this pathway plays a major role in the C5a enhancement of LPS-induced IL-6 and TNF-alpha production.

Fig. 1. The C5a receptor (CD88) is present at the surface of human PBMCs.

A–D, Flow cytometric analysis of adherent PBMCs treated with C5a for 20 h from a representative healthy volunteer. A, Forward scatter/side scatter of peripheral blood leucocytes. Cells in the R3 region were regarded as CD14⁺ monocytes. B, Representative dot blot depicting log fluorescence of FL-2 (CD14-PE) on the y axis and FL-1 (FITC) on the x axis. Cells in the upper left quadrant are CD14-PE⁺. The percentage of positive cells represents the mean of 3 independent experiments. C, Representative dot blot depicting log fluorescence of FL-2 (PE) on the y axis and FL-1 [C5aR (CD88)-FITC] on the x axis. Cells in the lower right quadrant are C5aR (CD88)-FITC⁺. The percentage of positive cells represents the mean of 3 independent experiments. D, Representative dot blot depicting log fluorescence of FL-2 (CD14-PE) on the y axis and FL-1 [C5aR (CD88)-FITC] on the x axis. Cells in the upper right quadrant are CD14-PE⁺ and C5aR (CD88)-FITC⁺. The percentage of positive cells represents the mean of 3 independent experiments. E, Western blot of adherent PBMCs untreated (lane 1) or treated with C5a (100 ng/mL) for 20 h (lane 2). Membranes were probed with anti-CD14 and anti-C5aR (Cd88) antibodies.

Fig. 2. Effect of C5a on LPS-induced IL-6 and TNF-α production in human PBMCs.

The PBMCs were treated with or without C5a (100 ng/mL) for an hour and then stimulated with LPS (10 or 100 ng/mL) as indicated. White bars represent IL-6 levels; and black bars, TNF-α. Data are represented as mean ± SEM. Results were analyzed using a paired t test.

Fig. 3. Activation of the MAPK pathway by C5a.

Adherent PBMCs were treated with C5a (100 ng/mL) for different amounts of time, as indicated. Cell extracts were prepared and analyzed by Western blotting as described in “Materials and Methods.” Graphs represent densitometric data, normalized to ERK-2, expressed as fold increase from the controls (No LPS, No C5a) for (A) p-p38, (B) p-JNK, and (C) p-ERK1/2, respectively. Representative immunoblots probed with the respective phosphoantibody (D). The PBMCs were treated for 1 h with C5a and/or with LPS for 30 min (E). Downregulation of each pathway by their respective inhibitor was assessed by immunoblotting against (a) p-cJun for the JNK pathway, (b) pERK1/2 for the ERK pathway, and (c) p-CREB for the p38 cascade. The p-CREB antibody also recognizes the phosphorylated form of the CREB-related protein ATF-1.

Fig. 4. Inhibition of the C5a reprogramming effect on LPS-induced IL-6 and TNF-α production by SB203580, a p38 MAPK inhibitor.

The PBMCs were treated with or without SB203580 for an hour and with or without C5a (100 ng/mL) for an hour, then stimulated with LPS (10 ng/mL) as indicated. White bars represent IL-6 levels; and black bars, TNF-α. Data are represented as mean ± SEM. Results were analyzed using a paired t test.

Fig. 5. PD98059, an ERK MAPK inhibitor, does not block the C5a effect on LPS-dependent IL-6 and TNF-α production in human PBMCs.

The PBMCs were treated with or without PD98059 for an hour and with or without C5a (100 ng/mL) for an hour, then stimulated with LPS (10 ng/mL) as indicated. White bars represent IL-6 levels; and black bars, TNF-α. Data are represented as mean ± SEM. Results were analyzed using a paired t test.

Fig. 6. A JNK inhibitor, SP600125, does not abrogate the C5a effect on LPS-dependent IL-6 and TNF-α production in human PBMCs.

The PBMCs were treated with or without SP600125 for an hour and with or without C5a (100 ng/mL) for an hour, then stimulated with LPS (10 ng/mL) as indicated. White bars represent IL-6 levels; and black bars, TNF-α. Data are represented as mean ± SEM. Results were analyzed using a paired t test.