Inhibition of complement and CD14 attenuates the Escherichia coli-induced inflammatory response in porcine whole blood

Abstract

The innate immune response is a double-edged sword in systemic inflammation and sepsis. Uncontrolled or inappropriate activation can damage and be lethal to the host. Several studies have investigated inhibition of downstream mediators, including tumor necrosis factor alpha (TNF-alpha) and interleukin-1beta (IL-1beta). Emerging evidence indicates that upstream inhibition is a better therapeutic approach for attenuating damaging immune activation. Therefore, we investigated inhibition of two central innate immune pathways, those of complement and CD14/Toll-like receptor 4 (TLR4)/myeloid differentiation protein 2 (MD-2), in a porcine in vitro model of Escherichia coli-induced inflammation. Porcine whole blood anticoagulated with lepuridin, which did not interfere with the complement system, was incubated with E. coli lipopolysaccharide (LPS) or whole bacteria. Inhibitors of complement and CD14 and thus the LPS CD14/TLR4/MD-2 receptor complex were tested to investigate the effect on the inflammatory response. A broad range of inflammatory readouts were used to monitor the effect. Anti-CD14 was found to saturate the CD14 molecule on granulocytes and completely inhibited LPS-induced proinflammatory cytokines in a dose-dependent manner. Anti-CD14 significantly reduced the levels of the E. coli-induced proinflammatory cytokines TNF-alpha and IL-1beta, but not IL-8, in a dose-dependent manner. No effect on bacterial clearance was seen. Vaccinia complement control protein and smallpox inhibitor of complement enzymes, two Orthopoxvirus-encoded complement inhibitors, completely inhibited complement activation. Furthermore, these agents almost completely inhibited the expression of wCD11R3, which is associated with CD18 as a beta2 integrin, on porcine granulocytes and decreased IL-8 levels significantly in a dose-dependent manner. As expected, complement inhibition reduced bacterial clearance. We conclude that inhibition of complement and CD14 attenuates E. coli-induced inflammation and might be used as a therapeutic regimen in gram-negative sepsis along with appropriate treatment with antibiotics.

FIG. 1.

Anti-CD14 (clone MIL-2) binds to and saturates CD14 on porcine granulocytes. (A) Whole blood from three pigs was incubated for 10 min at 37°C with different doses of unconjugated anti-CD14 (x axis). FITC-conjugated anti-CD14 was then added at a fixed concentration, and the samples were analyzed with a FACScan flow cytometer. The results of one of three experiments are shown. MFI, median fluorescence intensity. (B) Gating of the granulocytes as shown by a forward scatter-side scatter plot. (C) Histogram showing that there was a shift of fluorescence intensity (FI) from unsaturated CD14 (no unconjugated anti-CD14) (filled area) to saturated CD14 (62.5 μg/ml unconjugated anti-CD14). The results for an irrelevant detection control antibody (Ab) are also shown.

FIG. 2.

Effect of anti-CD14 on LPS-induced cytokine production. Whole blood from four pigs was preincubated with anti-CD14 or an isotype-matched control antibody for 5 min. LPS was then added to a final concentration of 100 ng/ml and incubated at 37°C for 2 h (TNF-α and IL-1β) (A and B) or 4 h (IL-8) (C). Cytokines were measured using ELISA. The results of one of four experiments are shown. T0, baseline sample; T120 and T240, negative control samples obtained after 120 and 240 min, respectively.

FIG. 3.

E. coli-induced complement activation and effect of complement inhibitors. (A) Whole blood from two pigs was incubated at 37°C with different amounts of E. coli for 2 h. The readout was the amount of the TCC determined using an ELISA. The means of the two experiments are shown. For an explanation of T0 and T120 see the legend to Fig. 2. AU, arbitrary units. (B) Whole blood from two pigs was preincubated for 5 min with different doses (x axis) of VCP, anti-CD14, or HSA as a control. A fixed dose of 10⁸ E. coli bacteria/ml whole blood was then added, and the samples were incubated for 30 min at 37°C. The readout was the amount of the TCC determined using an ELISA. The means and ranges of two separate experiments are shown. (C) Porcine whole blood was preincubated with VCP, SPICE, and a control (HSA) for 5 min. A fixed dose of 10⁸ E. coli bacteria/ml whole blood was then added, and the samples were incubated for 30 min at 37°C. The readout was the amount of TCC determined using an ELISA. The results of one representative experiment are shown.

FIG. 4.

Effect of complement and CD14 inhibition on wCD11R3 expression on granulocytes. (A) Different amounts of E. coli were added to porcine whole blood (n = 2) and incubated for 10 min at 37°C. The samples were analyzed for wCD11R3 expression using a FACScan flow cytometer. The means and ranges of two separate experiments are shown. MFI, median fluorescence intensity. (B) Gating of the granulocytes as shown by a forward scatter-side scatter plot. (C) Histogram showing the shift of the fluorescence intensity (FI) from 10⁵ E. coli bacteria/ml (filled area) or PBS alone to 10⁸ E. coli bacteria/ml. (D) Whole blood from two pigs was preincubated for 5 min with VCP, anti-CD14, or HSA as a control. A fixed dose of 10⁸ E. coli bacteria/ml whole blood was then added and incubated for 10 min at 37°C. The samples were analyzed to determine wCD11R3 expression using a FACScan flow cytometer. The results of one of two virtually identical experiments are shown. (E) Gating of the granulocytes, as shown in a forward scatter-side scatter plot. (F) Histogram showing the shift of fluorescence intensity from 10⁸ E. coli cells/ml with PBS (filled area), with HSA, and with anti-CD14 to VCP. The results for an irrelevant detection control antibody (Irr. control Ab) are also indicated.

FIG. 5.

Effect of anti-CD14 and SPICE on E. coli CFU. (A) Whole blood from two pigs was incubated with bacterial growth medium, and samples were taken at time zero (T0) and after 240 min (T240) (negative control). E. coli (10⁸ bacteria/ml whole blood) was incubated in bacterial growth medium, and samples were obtained at time zero and after 240 min (positive control). (B) Whole blood from two pigs was preincubated for 5 min with SPICE or anti-CD14, using HSA and an isotype-matched monoclonal antibody (IgG2b), respectively, as the controls. A fixed dose of 10⁸ E. coli bacteria/ml whole blood was then added and incubated for 240 min at 37°C. The data are the means and ranges (n = 2).

FIG. 6.

Effect of SPICE and anti-CD14 on E. coli-induced cytokine production. Whole blood from seven pigs (A and C) and from six pigs (B) was preincubated for 5 min with SPICE or anti-CD14, using HSA and an isotype-matched monoclonal antibody (IgG2b), respectively, as the controls. A fixed dose of 10⁵ E. coli bacteria/ml whole blood was then added and incubated for 2 h (TNF-α and IL-1β) or 4 h (IL-8) at 37°C. The data are the means ± standard errors of the means. A paired-sample t test was used to determine statistical significance (*, P < 0.05; **, P < 0.01). For an explanation of T0, T120, and T240 see the legend to Fig. 2.