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Originally published online as doi:10.1189/jlb.0806538 on March 27, 2007

Published online before print March 27, 2007
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(Journal of Leukocyte Biology. 2007;81:1404-1413.)
© 2007 by Society for Leukocyte Biology

The role of complement C3 opsonization, C5a receptor, and CD14 in E. coli-induced up-regulation of granulocyte and monocyte CD11b/CD18 (CR3), phagocytosis, and oxidative burst in human whole blood

Ole-Lars Brekke*,{dagger},1, Dorte Christiansen*, Hilde Fure*, Michael Fung{ddagger} and Tom E. Mollnes*,§

* Department of Laboratory Medicine, Nordland Hospital, Bodø, Norway;
{dagger} Institute of Medical Biology, University of Tromsø, Tromsø, Norway;
{ddagger} Tanox Inc., Houston, Texas, USA; and
§ Institute of Immunology, Rikshospitalet University Hospital and University of Oslo, Oslo, Norway

1 Correspondence: Department of Laboratory Medicine, Nordland Hospital, N-8092 Bodø, Norway. E-mail: ole.lars.brekke{at}nlsh.no


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The relative role of complement and CD14 in Escherichia coli-induced leukocyte CD11b up-regulation, phagocytosis, and oxidative burst in human whole blood was examined. The highly specific thrombin inhibitor lepirudin was used as anticoagulant, as it does not affect complement activation. Complement inhibition at the level of C3 (anti-C2 and anti-factor D) and C5 (C5a receptor antagonist and anti-C5/C5a) efficiently inhibited CD11b up-regulation, phagocytosis, and oxidative burst in granulocytes. Monocyte activation was generally less complement-dependent, but when C3 activation was blocked, a pronounced inhibition of phagocytosis and oxidative burst was obtained. Only the combination of anti-C2 and antifactor D blocked E. coli C3 opsonization completely. Whole E. coli, disrupted E. coli, and the C3-convertase activator cobra venom factor up-regulated CD11b rapidly on both cell types, proportional to their complement activation potential in the fluid phase. In comparison, purified LPS at concentrations comparable with that present in the E. coli preparations did not activate complement. Oxidative burst was induced only by whole bacteria. Finally, the combination of complement inhibition and anti-CD14 completely blocked E. coli-induced granulocyte and monocyte CD11b up-regulation and quantitatively, virtually abolished phagocytosis. The results indicate that complement and CD14, despite differential effects on granulocytes and monocytes, are the two crucial, quantitative factors responsible for E. coli-induced CD11b, phagocytosis, and oxidative burst in both cell types.

Key Words: sepsis • endotoxin • complement receptor 3


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Complement plays an essential role in inflammation and innate immunity against infectious disease. Sepsis caused by Escherichia coli and other gram-negative bacteria is still a serious disease despite the presence of a range of antibiotics [1 ]. One main reason for this is the huge and sometimes inappropriate inflammatory response induced by the bacteria themselves or by released bacterial components such as LPS, especially when the bacteria enters the blood stream. This inflammation participates in the tissue damage and multi-organ failure, which are hallmarks of severe sepsis. High levels of secondary inflammatory mediators such as C5a, the terminal complement complex, cytokines, different arachidonic acid metabolites, and acute-phase reactants such as C-reactive protein have been reported in the blood from septic patients [2 , 3 4 5 ]. The elevated C5a levels during sepsis impair immune function through stimulation of thymocyte apoptosis [6 ] and inhibition of the bactericidal function of neutrophils [7 ].

E. coli entering the bloodstream are opsonized rapidly by complement including C1q, mannose-binding lectin (MBL), C4b, and C3b/iC3b [8 ], in addition to Igs [9 ]. The terminal C5b-9 complex (TCC) induces lysis of certain complement-sensitive bacterial strains. The opsonization of bacteria by complement also facilitates the binding of the bacteria to the adherence complement receptor 1 (CR1) and the integrin and phagocytosis receptor CR3 (CD11b/CD18) on blood leukocytes [10 ]. Complement activation in the fluid phase with the release of C5a up-regulates CD11b rapidly and induces oxidative burst [11 ]. C5a is also involved in the defect phagocytosis and oxidative burst in granulocytes observed in the cecal ligation/puncture (CLP)-induced sepsis in rats [12 ]. Furthermore, C5a mediates a number of other detrimental effects during sepsis including enhanced synthesis of inflammatory mediators and degranulation of granulocytes [13 ]. Thus, complement is a double-edged sword and plays a dual role during sepsis.

LPS is a major constituent of the membrane in gram-negative bacteria and may be released from the bacteria when they multiply or lyse [14 ]. LPS induces a septic-like condition in vivo including fever, increased body temperature, and synthesis of pro- and anti-inflammatory cytokines by different cell types including macrophages [14 ]. LPS binds to soluble or membrane CD14, which functions as a receptor for LPS [15 ], after binding of LPS to a LPS-binding protein [16 ]. CD14 in monocyte/macrophage cell membranes are attached through a GPI linkage [16 ], and the transmembrane signal is mediated through TLR4 [17 ] and MyD88 [18 ]. The nonreceptor-mediated signaling by LPS is also mediated through CD14 [19 ]. LPS and CD14 are therefore important molecules involved in the inflammation induced during gram-negative sepsis. However, blocking of CD14 using mAb in animal models inhibits LPS effects [20 ] but has no effect on the mortality during the CLP model of sepsis in mice [21 ]. It is interesting that LPS has been reported to activate the transcription factor NF-{kappa}B through CR3, indicating a possible link between complement and LPS signaling [22 ].

We have shown previously that a specific C5a receptor antagonist (C5aRa) reduced the E. coli-induced expression of the adhesion molecule CD11b dose-dependently [11 ]. It is notable that the C5aRa did not inhibit E. coli-induced CD11b up-regulation completely, especially in monocytes, suggesting that other signaling pathways might be involved.

We hypothesized that a combined inhibition of complement and CD14 inhibition would reduce leukocyte activation. Thus, in the present study, the role of complement C3 opsonization, C5a, and LPS/CD14 on E. coli-induced CD11b up-regulation, phagocytosis, and oxidative burst was studied using a novel human whole blood model, where anticoagulation does not interfere with complement activation [11 ]. The results showed that the E. coli-induced granulocyte activation was more dependent on complement, and monocyte activation was more dependent on CD14. It is interesting that the activation of both cell types was blocked efficiently by combining complement and CD14 inhibition.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Equipment and reagents
All equipment, including tips, tubes, and buffers used in the experiments, were endotoxin-free. Cobra venom factor (CVF; Quidel, San Diego, CA, USA) was incubated overnight with END-X®B15 (Associates of Cape Cod Inc., East Falmouth, MA, USA) to remove LPS. Recombinant human C5a (rhC5a), EDTA, paraformaldehyde, BSA, and purified E. coli LPS were purchased from Sigma-Aldrich (St. Louis, MO, USA). Polypropylene tubes were from Nalgene Nunc (Roskilde, Denmark). PBS, with or without calcium and magnesium, was from Life Technologies (Paisley, UK) and lepirudin (Refludan®) was obtained from Hoechst (Frankfurt am Main, Germany) or Schering (Cambridge, UK). Tryptone and yeast extract were from Becton Dickinson and Co. (Sparks, MD, USA). Flow cytometry was performed using a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA, USA). LDS-751 was obtained from Molecular Probes (Leiden, The Netherlands). The Burst test and Phago test kits were obtained from Orpegen Pharma (Heidelberg, Germany). OD was measured on a MRX microplate reader (Dynex Technologies, Denkendorf, Germany).

Antibodies and inhibitors
The murine IgG1 mAbs to human C2 (clone 175-62), Factor D (Clone 166-32) [23 ], anti-C5 (137-30), blocking the cleavage of C5, anti-C5/C5a (clone 137-26) [24 ], and the isotype-matched control anti-HIV-1 gp120 (clone G3-519) were from the laboratory of M. Fung (Tanox Inc., Houston, TX, USA). Murine anti-human CD14 (clone 18D11) F(ab')2 and a control F(ab')2 were obtained from Diatec AS (Oslo, Norway). The C5aRa and a corresponding control peptide were from the laboratory of M. Fung. The maximal inhibitory dose of each mAb and inhibitor was determined in initial dose-response experiments. All antibodies were checked for LPS contamination using an endotoxin assay described below and purified using END-X®B15 (Associates of Cape Cod Inc.) if necessary.

Bacterial strains and counting
Heat-inactivated E. coli and FITC-labeled E. coli Strain LE392 were from Orpegen Pharma. In some experiments, E. coli Strain LE392 (ATCC 33572) from American Type Culture Collection (Manassas, VA, USA) was grown in Luria-Bertani medium (1% Tryptone, 0.5% yeast extract, 1% NaCl in H2O) overnight and then washed once in PBS (3220 g, 10 min) at +4°C. After resuspension in PBS, E. coli was heat-inactivated at 60°C for 60 min. E. coli was then washed at +4°C in PBS without Ca2+ and Mg2+ and stored at –70°C in PBS. E. coli was counted after staining with Syto BC using a bacteria counting kit (Molecular Probes). To obtain absolute counts, an aliquot of the bacteria was counted on the flow cytometer using Trucount tubes from Becton Dickinson, and 2500 beads were acquired. Disrupted E. coli bacteria were obtained by pulse sonication on ice (0°C) using a Bandelin Sonopuls HD2070 sonicator equipped with a MS 72 probe from Bandelin Electronic Gmbh and Co. (Berlin, Germany). After sonication, bacterial counting on flow cytometry was used to verify that all bacteria were disrupted.

Whole blood model of sepsis
Blood from healthy donors was obtained after informed consent according to guidelines from the local ethics committee. Experiments with each blood donor were performed as single experiments at different time-points. In brief, fresh human whole blood was collected in polypropylene tubes containing lepirudin (50 mg/L). Aliquots of whole blood in sterile polypropylene tubes were then preincubated for 4 min in a water bath at 37°C with PBS, antibodies, or inhibitors in PBS (14.3% of total vol, v/v) as indicated. E. coli or PBS (14.3% of total vol, v/v) was then added, and samples were incubated 10, 20, or 30 min further as indicated. After incubation, samples were processed immediately for flow cytometry. Aliquots for assay of complement activation products were placed on ice (0°C) immediately and supplemented with EDTA (10 mM final concentration) to stop further activation. Samples were centrifuged (3220 g, 15 min at 4°C), and plasma was stored at –70°C until further analysis.

LPS activation of complement in lepirudin plasma and serum
Lepirudin-anticoagulated whole blood and clotted whole blood were centrifuged at 3220 g for 15 min at 4°C. Plasma or serum from three or four different donors were pooled and frozen at –70°C until the experiments were performed. Plasma or serum was preincubated with PBS for 5–6 min and then incubated with E. coli LPS in tenfold titrations from 1000 to 0.001 mg/L for 30 min at 37°C. E. coli bacteria were similarly added in equivalent LPS concentrations. Immediately after incubation at 37°C, EDTA (10 mM final concentration) was added to stop further complement activation, the samples were centrifuged at +4°C, and the supernatants were stored at –70°C. The experiment was performed three times using three different plasma and serum pools.

Flow cytometric analysis
Granulocyte and monocyte CD11b expression was analyzed using anti-CD11b PE (Becton Dickinson, San Jose, CA, USA) after fixation of the cells with 0.5% (v/v) paraformaldehyde as described previously [11 ]. Whole blood was stained with the nuclear dye LDS-751 [fluorescence 3 (FL3)] to separate leukocytes from RBC. Monocytes and granulocytes were gated separately in a plot displaying side-scatter versus FL1 [anti-CD14 FITC (Becton Dickinson, San Jose, CA, USA)]. Phagocytosis of FITC-labeled E. coli was analyzed by flow cytometry after 10 min incubation using the Phago test kit (Orpegen Pharma) and quenching solution to avoid interference of surface-bound bacteria as described previously [11 ]. Phagocytosis was expressed as median fluorescence intensity (MFI) of the whole granulocyte or monocyte population. In some experiments, phagocytosis was also expressed as percent-positive cells using a marker set on the upper fluorescence limit in the negative control consisting of E. coli Strain LE392 without FITC labeling incubated 10 min in whole blood. Cells with a higher fluorescence were regarded as positive cells.

Oxidative burst
Oxidative burst was measured using the Burst test (Orpegen Pharma). Samples were processed immediately (baseline sample T0) or after 10 min incubation by transferring 100 µL blood to 5 mL polypropylene tubes (Becton Dickinson, Franklin Lakes, NJ, USA), and 20 µL substrate solution containing dihydrorhodamine 1,2,3 was added. All tubes were then incubated 10 min at 37°C, and samples were lysed and washed according to kit instructions. Within 1 h, monocytes and granulocytes in PBS were gated separately in a forward/side-scatter dot-plot using flow cytometry. Results were expressed as MFI of the whole granulocyte or monocyte population.

Analysis of E. coli C3 opsonization in lepirudin and EDTA plasma
Lepirudin-anticoagulated whole blood and EDTA whole blood were centrifuged at 3220 g for 15 min at 4°C, and plasma was separated from blood cells. EDTA plasma and lepirudin plasma were preincubated 4 min at 37°C with PBS or antibodies, respectively. E. coli was added, and samples were further incubated 10 min, washed twice (3220 g, 10 min, 4°C), and resuspended with PBS containing 0.1% (w/v) BSA.

C3 opsonization of whole E. coli bacteria was detected by flow cytometry using FITC-conjugated rabbit anti-human C3c (F0201, Dako, Glostrup, Denmark) and FITC-conjugated rabbit anti-mouse Ig (F0261, Dako) as isotype control. Samples were incubated 15 min at 20°C, and PBS was added before analysis on the flow cytometer. Results were expressed as MFI.

Deposition of donor serum IgG, IgM, C1q, and C3 on E. coli
Heat-inactivated E. coli was incubated 10 min at 37°C in heat-inactivated serum from the healthy blood donors. E. coli antibodies were analyzed by flow cytometry using FITC-conjugated rabbit anti-human IgG (F0056, Dako) or anti-human IgM (F0058, Dako) and an isotype control (X0929, Dako). Serum from a patient with newly diagnosed E. coli sepsis using standard blood culture techniques was used as positive control and was obtained after informed consent according to guidelines from the local ethics committee. Results were expressed as MFI.

The correlation between anti-E. coli IgM and IgG antibodies and complement activation was investigated in the same sera. Heat-inactivated E. coli was incubated 10 min at 37°C in serum from eight healthy blood donors. PBS or heat-inactivated serum was used as a negative control. C1q and C3 opsonization of whole E. coli bacteria was detected by flow cytometry using FITC-conjugated rabbit anti-human C1q (F0254, Dako) or C3d (F0323, Dako). FITC-conjugated rabbit anti-mouse Igs (F0261, Dako) were used as isotype control. Results were expressed as MFI.

Endotoxin assay
Measurements of LPS in buffers, reagents, E. coli, and E. coli supernatant were carried out as an endpoint test using a Limulus amoebocyte lysate-Pyrochrome® kit (Associates of Cape Cod Inc.) or a QCL-1000 kit from Bio-Whittaker (Walkersville, MD, USA). Before the total LPS concentration in whole E. coli bacteria was measured, they were disrupted by sonication as described above. When the free LPS concentration was measured in the supernatants from whole E. coli, the bacteria were centrifuged at 2300 g for 10 min, and the supernatant was filtered through a 0.2-µm Pall filter (Pall Corp., Ann Arbor, MI, USA) to remove whole bacteria.

Assay of complement activation
Activation of the classical pathway was measured as C1rs-C1 inhibitor complexes and analyzed in an enzyme immunoassay (EIA) as described previously [25 ]. The alternative convertase complex C3b-Bb-properdin was measured by EIA as described previously [11 ]. Activation of the final common pathway was quantified by the C3 activation product C3bc [26 ]. C5a was analyzed in an EIA using the neo-epitope-specific mAb 4A2E10E2 as capture antibody and 3G3C4 as secondary antibody as described previously [27 ]. The TCC was measured by EIA using the specific mAb aE11 as described previously [28 ].

Statistical analysis
The statistical analysis was performed using SigmaStat Version 3.1 for Windows from SPSS Science Software GmbH (Erkrath, Germany). Friedman repeated measures ANOVA on ranks was used followed by multiple comparison versus E. coli plus PBS using Dunn’s method. P < 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of different complement inhibitors on E. coli-induced CD11b up-regulation, phagocytosis, and oxidative burst
To study the role of complement activation in E. coli-induced leukocyte activation, the effect of anti-C5 mAb 137-30 (blocking C5 cleavage), a C5aRa, anti-C2, and anti-factor D mAb on C5a formation, CD11b up-regulation, phagocytosis, and oxidative burst was examined. E. coli enhanced C5a formation rapidly in human whole blood after 10 min incubation (Fig. 1 ). Anti-C2 and anti-factor D mAb in combination efficiently blocked E. coli-induced C5a formation, phagocytosis, and oxidative burst in granulocytes (Fig. 1) . In comparison, the C5aRa or mAb against C5 reduced E. coli-induced oxidative burst in granulocytes slightly less efficiently than anti-C2 and anti-factor D mAb in combination, and the effect on E. coli-induced CD11b up-regulation was similar. The results indicate that the combined inhibition of C3 opsonization and C5a formation most efficiently blocked E. coli-induced phagocytosis and oxidative burst in granulocytes and that C5a is the main mediator of complement-mediated granulocyte CD11b up-regulation.


Figure 1
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  		Figure 1. Effect of complement inhibition on granulocyte activation in human whole blood. The effect of a C5aRa and mAbs to C5 (a-C5), C2 (a-C2), and factor D (a-D) on E. coli-induced C5a formation, CD11b up-regulation, phagocytosis, and oxidative burst in granulocytes after 10 min incubation was examined. Complement activation measured as plasma C5a formation was analyzed using EIA and expressed as arbitrary units (AU)/mL. CD11b up-regulation, phagocytosis, and oxidative burst were measured by flow cytometry and expressed as MFI. The inhibitor concentrations used were C5aRa (7.1 mg/l), anti-C5 137-30 blocking C5 cleavage (71 mg/L), anti-C2 (71 mg/L), anti-factor D (36 mg/L), and combined anti-C2 and anti-factor D mAb (a-C2+a-D; 71+36 mg/L). Controls were an isotype-matched control mAb (Ctr. ab; 71 mg/L) and a control peptide (Ctr. pep.; 7.1 mg/L). Results are given as the median and 25–75 percentile of six different blood donors. *, P < 0.05, compared with E. coli alone (71x106/mL) using Friedman repeated measures ANOVA on ranks followed by one-way multiple comparison versus E. coli using Dunn’s method. There was no statistical difference between the negative controls T0 (baseline sample) and T10 (baseline sample incubated for 10 min without E. coli), and the latter was used for statistical comparison with test samples.

 
The effects of complement inhibition on monocytes differed from that in granulocytes (Fig. 2 ). Anti-C2 and anti-factor D mAb in combination again effectively reduced E. coli-induced phagocytosis and oxidative burst, whereas the effect on CD11b up-regulation was less pronounced. The C5aRa and anti-C5 mAb reduced E. coli-induced CD11b up-regulation and oxidative burst much less efficiently than in the granulocytes. Detectable levels of E. coli IgG and lower levels of IgM antibodies were found in the serum from the six blood donors (results not shown). However, no correlation between oxidative burst and the presence of E. coli antibodies or C1q deposition on the E. coli surface was found, indicating that activation of the classical complement pathway through such antibodies cannot explain our findings.


Figure 2
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  		Figure 2. Effect of complement inhibition on monocyte activation in human whole blood. The effect of complement inhibition on E. coli-induced CD11b up-regulation, phagocytosis (n=5), and oxidative burst in monocytes in the same whole blood samples as described for granulocytes with identical experimental conditions, inhibitors, concentrations, readouts, data presentation, and statistics as described in the legend to Figure 1 . *, P < 0.05 compared to E. coli alone (71 x 106/mL) using Friedman repeated measures analysis of variance on ranks followed by one way multiple comparison versus E. coli using Dunn’s method.

 
When phagocytosis of E. coli in human granulocytes and monocytes was expressed as percent-positive cells and as MFI, interesting differences were found (Fig. 3 ). Only the combination of anti-C2 and anti-factor D mAb completely inhibited phagocytosis in granulocytes expressed as percent-positive cells. In comparison, blocking of C5 less efficiently reduced phagocytosis measured as percent-positive cells but reduced it effectively expressed as MFI. Thus, in granulocytes, C5a is quantitatively responsible for the phagocytosis (MFI), but blocking of C3 is required for complete inhibition of phagocytosis expressed as percent-positive cells. In monocytes, however, only the combination of anti-C2 and anti-factor D mAbs reduced phagocytosis significantly, measured as MFI and percent-positive cells, indicating the importance of C3 opsonization for monocyte phagocytosis.


Figure 3
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  		Figure 3. Effect of complement inhibition on the degree of phagocytosis of E. coli expressed using percent-positive cells as read-out. Granulocyte (left panel) and monocyte (right panel) phagocytosis of FITC-labeled E. coli was measured by flow cytometry after 10 min incubation and calculated as percent-positive cells. Experimental conditions, inhibitors, concentrations, and controls are as described in Figure 1 . Results are given as the median and 25–75 percentile of six and five different blood donors for granulocytes and monocytes, respectively. *, P < 0.05, compared with E. coli alone.

 
Effect of complement inhibitors on E. coli C3 opsonization
We then examined the effect of anti-C2 and anti-factor D mAbs on E. coli C3 opsonization using flow cytometry (Table 1 ). Anti-C2 and anti-factor D mAbs in combination blocked the C3 opsonization of E. coli bacteria completely after 10 min incubation in lepirudin plasma. In comparison, anti-C5 and anti-C2 and anti-factor D mAbs alone had no effect. The IgG opsonization was unaffected by the same mAbs (data not shown). This indicates that the classical/lectin (C2) and the alternative (factor D) pathways contribute significantly to C3 opsonization and that combined inhibition of these is necessary for completely blocking E. coli C3 opsonization.


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  		Table 1. Effect of Complement Inhibition on E. coli C3 Opsonization in Lepirudin Plasma

 
Time course of E. coli-induced leukocyte activation in whole blood
The time course of leukocyte activation including CD11b up-regulation and oxidative burst in human whole blood is shown in Figure 4 . Whole E. coli bacteria, sonication-disrupted E. coli, and E. coli LPS were added in LPS equivalent concentration (0.5 mg/L). As a control for the contribution of sole fluid-phase complement activation, CVF, which activates the C3 convertase directly, was included. The CVF concentrations used gave TCC levels, comparable with stimulation with whole E. coli (Fig. 5 ). Whole E. coli bacteria, disrupted E. coli bacteria, and CVF up-regulated CD11b rapidly on granulocytes (Fig. 4 , upper left panel). LPS had no effect on granulocytes after 10 min incubation but enhanced CD11b expression after 20 min incubation. In comparison, all stimuli up-regulated monocyte CD11b rapidly (Fig. 4 , upper right panel). It is interesting that only whole E. coli enhanced the oxidative burst in granulocytes and monocytes (Fig. 4 , lower left and right panels, respectively), indicating that oxidative burst is closely related to phagocytosis of whole bacteria.


Figure 4
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  		Figure 4. Time course of E. coli-, disrupted E. coli-, and purified LPS-induced leukocyte CD11b up-regulation and oxidative burst in human whole blood, which was incubated for 10, 20, or 30 min with whole E. coli ({blacksquare}), disrupted E. coli ({square}amp;), purified LPS L-8274 ({triangleup}), CVF ({blacktriangledown}), and PBS control ({diamondsuit}). Granulocyte (left panels) and monocyte (right panels) CD11b expression (upper panels) and oxidative burst (lower panels) were examined using flow cytometry. Whole E. coli (71x106/mL), disrupted E. coli, and purified LPS were added in equivalent LPS concentrations (0.5 mg/L). CVF (5 U/mL), which activates the C3 convertase directly, was included as control for the effect of pure fluid-phase complement activation. Data from one of three experiments with virtually identical results are shown.

 

Figure 5
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  		Figure 5. Dose-response effect of whole E. coli, disrupted E. coli, and purified LPS on complement and leukocyte activation in human whole blood, which was incubated for 10 min with whole E. coli (left panels), equivalent amounts of disrupted E. coli (second panels from left), and purified E. coli LPS L-8274 (second panels from right). CVF (right panels) was included as a control for pure, fluid-phase complement activation. The former three preparations all contained 0.5 mg LPS/L at the highest concentration. Complement activation was measured as TCC formation (top panels; {blacksquare}) and is expressed as AU/mL. CD11b expression (middle panels) and oxidative burst (bottom panels) in granulocytes ({circ}) and monocytes ({triangleup}) were measured using flow cytometry, and results are given as MFI. Data from one of three experiments with virtually identical results are shown.

 
Dose-response effect of whole E. coli, disrupted E. coli, LPS, and CVF on complement and leukocyte activation
To further examine the role of complement activation on the bacterial surface and in the fluid phase, we incubated increasing concentrations of whole E. coli bacteria, E. coli disrupted by sonication, or purified LPS (all with equivalent concentration of LPS) or CVF to human whole blood for 10 min. The effect on complement activation (TCC) and leukocyte CD11b expression and oxidative burst is shown in Figure 5 . Whole and disrupted E.coli bacteria as well as CVF activated complement dose-dependently, as revealed by increased TCC formation (Fig. 5 , top panels). It is notable that purified LPS did not enhance TCC formation, indicating that LPS is not up-regulating CD11b through activation of complement at these LPS concentrations.

All four preparations up-regulated monocyte CD11b after 10 min incubation (Fig. 5 , middle panels). In comparison, LPS did not up-regulate CD11b in granulocytes, in contrast to the other three preparations, where a dose-dependent effect was observed. The effect of whole bacteria and disrupted E. coli bacteria was comparable on CD11b up-regulation on monocytes and granulocytes.

Oxidative burst was induced only by whole E. coli bacteria (Fig. 5 , bottom panels), more pronounced in granulocytes than in monocytes, indicating that complement activation in the absence of whole E. coli bacteria is not sufficient to stimulate oxidative burst. Furthermore, complement activation in the fluid phase induced by CVF is associated with CD11b up-regulation but is not sufficient to stimulate oxidative burst in the absence of whole E. coli bacteria.

Complement activation induced by purified LPS and whole E. coli bacteria
The effect of purified LPS and whole E. coli bacteria on complement activation in the fluid phase measured as TCC formation was then investigated (Fig. 6 ). Whole E. coli bacteria at equivalent LPS concentrations above 0.1 mg/L enhanced TCC formation rapidly in plasma and serum. In comparison, three different E. coli LPS preparations did not activate complement at concentrations up to 1 mg/L. However, LPS at concentrations from 10 mg/L induced TCC formation in plasma and serum. In conclusion, LPS at concentrations present in whole E. coli bacteria is not responsible for E. coli-induced complement activation. LPS at high concentrations activates the complement system, but the free LPS concentrations measured in the whole E. coli preparations used in the present study were 3–5 µg/L, which is far below the complement-activating level.


Figure 6
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  		Figure 6. Effect of whole E. coli and three different E. coli LPS preparations on complement activation in human plasma and serum. Human lepirudin-anticoagulated, pooled plasma (upper panel) or pooled serum (lower panel) was incubated for 60 min with whole E. coli ({blacksquare}) or equivalent amounts of purified E. coli LPS L-8274 ({circ}), L-2880 ({triangledown}), or L-2630 ({triangleup}). Complement activation was measured as TCC formation and is expressed as AU/mL. Data from one of three experiments with virtually identical results are shown.

 
Effect of neutralizing C5a and CD14 on CD11b up-regulation and phagocytosis
The effect of neutralizing C5a and CD14 on CD11b up-regulation was examined (Fig. 7 ). The C5a neutralizing mAb 137-26 completely inhibited the CVF-induced CD11b up-regulation in granulocytes and monocytes. Whereas 137-26 markedly inhibited the whole and disrupted E. coli-induced CD11b up-regulation on granulocytes, the effects on monocytes were less pronounced. Conversely, the anti-CD14 mAb completely inhibited the LPS-induced up-regulation of CD11b in monocytes and markedly inhibited the disrupted E. coli-induced CD11b up-regulation on monocytes and granulocytes, although the effect on the latter was less pronounced. The effect of anti-CD14 on CD11b expression by whole bacteria was modest for granulocytes and monocytes.


Figure 7
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  		Figure 7. Effect of inhibition of C5a and CD14 and a combination thereof on leukocyte CD11b up-regulation in human whole blood. Dose-response effect of the anti-C5a neutralizing mAb 137-26 (left panel), F(ab')2 fragments of an anti-CD14 mAb (middle panel), and a combination of these (right panel) on CD11b up-regulation in granulocytes (upper panels) and monocytes (lower panels) was examined using flow cytometry. Human whole blood was stimulated with whole E. coli ({blacksquare}), disrupted E. coli ({square}amp;), purified LPS (0.5 mg/L; {triangleup}), or CVF ({blacktriangledown}) for 10 min. Data from controls incubated 10 min with PBS only are also indicated ({diamondsuit}). Data from one of three experiments with virtually identical results are shown.

 
It is most important that by combining the anti-C5a and anti-CD14 mAb, E. coli- and disrupted E. coli-induced CD11b up-regulation was abolished completely on granulocytes and monocytes (Fig. 7 , right panels). In addition, the LPS-induced CD11b expression on monocytes (not observed on granulocytes) was abolished completely, similar to that observed with anti-CD14 alone. It is interesting that combining the anti-C5a and anti-CD14 mAb also almost completely blocked E. coli-induced phagocytosis expressed as MFI, but not as percent-positive cells measured using flow cytometry (data not shown). The data indicate that fluid-phase activation of complement with release of C5a plays a key role in the initial E. coli-induced up-regulation of granulocyte CD11b. In contrast, monocyte CD11b up-regulation is mediated mainly through CD14 and is less dependent on complement. The combination of anti-C5 and anti-CD14 mAb completely blocked E. coli-induced CD11b up-regulation in both cell types, indicating that these to mediators are crucially involved in the initial E. coli-induced leukocyte CD11b up-regulation.

The role of C5a in up-regulation of CD11b and oxidative burst
The effect of rhC5a on CD11b up-regulation in human whole blood was examined. RhC5a up-regulated CD11b rapidly and dose-dependently on granulocytes and monocytes (Fig. 8 , left panel). The rhC5a-induced CD11b up-regulation was blocked completely by the C5aRa in both cell types, supporting the essential role of C5a in CD11b expression (Fig. 8 , right panel). It is interesting that rhC5a did not induce any oxidative burst in granulocytes or monocytes (data not shown). This further supports that complement activation in the fluid phase with release of C5a is necessary, but not sufficient, in E. coli-induced oxidative burst, whereas C3 opsonization of whole E. coli bacteria is necessary for phagocytosis and the subsequent oxidative burst.


Figure 8
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  		Figure 8. Effect of rhC5a on granulocyte and monocyte CD11b expression in human whole blood. rhC5a up-regulated CD11b (left panel) dose-dependently on granulocytes ({circ}) and monocytes ({triangleup}) after 10 min incubation. CD11b up-regulation was measured by flow cytometry and is expressed as MFI. The effect of adding increasing concentrations of C5aRa to human whole blood stimulated with rhC5a (5 mg/L) is shown in the right panel with the same symbols as described for the left panel. Data from one of three experiments with similar results are shown.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present paper indicates a critical role of complement C3 opsonization, C5a, and CD14 in E. coli-induced CD11b up-regulation, phagocytosis, and oxidative burst in granulocytes. In monocytes, the phagocytosis and oxidative burst were highly dependent on C3 opsonization but less dependent of C5a. Based on the inhibition experiments, C5a/C5aR and LPS/CD14 seem to be the essential two factors quantitatively responsible for E. coli-induced CD11b up-regulation in granulocytes and monocytes. It is interesting that purified E. coli LPS did not activate complement at concentrations found in the E. coli preparations but up-regulated monocyte CD11b in a CD14-dependent manner. Finally, our data indicate that opsonization by complement C3 is a critical event in the initial E. coli-induced phagocytosis and oxidative burst in both cell types and that a combined inhibition of C3 and CD14 blocks these events most efficiently. To our knowledge, this is the first time the relative role of complement and CD14 has been studied in E. coli-induced leukocyte activation in fresh human whole blood, where all mediators studied are able to interact mutually. This was obtained using the thrombin-specific inhibitor lepirudin as anticoagulant, which has no adverse effects on complement [11 ].

Inhibition of complement C3 opsonization and C5a formation using anti-C2 and anti-factor D mAb in combination effectively inhibited E. coli-induced phagocytosis and oxidative burst. Although the C5aRa, anti-C5, and anti-C5a mAbs effectively reduced phagocytosis of E. coli measured as MFI, they were less effective than the combination of anti-C2 and anti-factor D mAb when the data were expressed as percent-positive cells, underscoring the critical role for bacterial C3 opsonization in this context. Previous studies using isolated granulocytes support that serum is required for efficient phagocytosis of E. coli and E. coli-induced oxidative burst [29 , 30 ]. The serum factors required for efficient complement opsonization and phagocytosis of E. coli include complement C1q, C3, and the neutrophil bactericidal/permeability-increasing protein [30 ]. Our study extends previous data showing that complement C3 and C5a are critical in the oxidative burst in neutrophils [7 ]. However, the bactericidal function of neutrophils is mediated by enzymes in the phagosomes and not by the oxidative burst itself [31 ], as previously suggested. Our data indicating the important role of complement C3 opsonization in the phagocytosis of E. coli in human whole blood do not rule out the greater importance of LPS in E. coli binding by adherent macrophages through the receptor family CR3/LFA-1/p150,95 in the absence of complement [32 ]. A previous study also indicates that polymorphonuclear neutrophils (PMNs) in solution have a different distribution of CR3 and CD14 compared with those adsorbed to a surface, as only PMNs in solution showed CR3 and CD14 close to each other [33 ].

Complement inhibition with the combination of anti-C2 and anti-factor D mAb blocks the classical, alternative, and MBL pathways. Anti-C2 inhibits the classical and MBL/lectin pathways, and both pathways can be activated by E. coli. In the serum, from our blood donors, anti-E. coli antibodies of IgM and IgG classes were detected. However, no correlation between the antibody levels—C1q deposition on the E. coli surface and oxidative burst—was found, supporting that the classical/MBL pathways have less importance in E. coli-induced complement activation compared with the alternative pathway in our model [11 ]. The significant effect of anti-C2 and anti-factor D could in part be a result of inhibited complement-mediated lysis of E. coli with correspondingly less liberation of several soluble, inflammatory mediators such as LPS. Microvesicles of Neisserria meningitidis induce complement activation [34 ], indicating that activation of the complement system is not limited to whole bacteria. C3b binds covalently to the bacterial surface and is converted rapidly to iC3b [35 ]. As the effect of anti-C2 combined with anti-factor D blocks C3 activation and thereby bacterial opsonization, as we documented in our experiments, the consequent binding to C3 receptors—C3b to CR1 and iC3b to CR3—is abolished. Furthermore, the downstream activation of complement with release of the anaphylatoxins C3a and C5a is blocked simultaneously by these mAbs. A previous report about the phagocytosis and oxidative burst in isolated human granulocytes stimulated with zymosan particles also supports the critical role of C3 opsonization and CD11b [36 ]. It is interesting that E. coli strains with K capsular polysaccharides, which activated complement at a slow rate, were opsonized ineffectively by C3 and were resistant to phagocytosis of human granulocytes, further supporting the important role of C3 opsonization [9 ].

Although the combination of anti-C2 and anti-factor D mAbs blocks complement C3 opsonization completely on the bacterial surface, the bacteria are still opsonized by C1q, MBL, C4b (C4 is upstream to C2), and Igs. It is surprising that anti-C2 and anti-factor D, when used separately, had no effect on E. coli C3 opsonization (Table 1) , and the same inhibitors reduced C5a formation slightly in the fluid phase (Fig. 1) . This may indicate that the classical/lectin (C2) and the alternative (factor D) pathways are sufficient to give maximally detectable C3 deposition on the bacterial surface. However, the relative role of the classical pathway in E. coli-induced complement activation cannot be elucidated from these experiments, as anti-C2 blocks the classical and MBL/lectin pathways.

The initial E. coli-induced CD11b up-regulation in granulocytes after 10 min was mainly C5a-dependent, supporting previous studies [11 , 37 ]. However, CD14 also contributed to the CD11b up-regulation in granulocytes, as only the combination of anti-C5 and anti-CD14 mAbs blocked the initial CD11b up-regulation completely. The role of LPS/CD14 in neutrophil CD11b up-regulation is also supported by previous studies [38 ]. Furthermore, Gordon et al. [37 ] showed that E. coli-induced CD11b up-regulation in isolated granulocytes incubated in buffer in the absence of serum was inhibited completely by polymyxin B, suggesting that LPS mediates E. coli-induced CD11b up-regulation solely in the absence of serum. However, one could not expect a role of complement in serum-free models using isolated cells, emphasizing the advantage of using whole blood models when studying the relative role of candidates in the inflammatory cross-talk. In contrast to granulocytes, monocytes are highly dependent on CD14 for up-regulation of CD11b at low LPS concentrations [39 ]. It is interesting that we found that C5a also contributed significantly to E. coli-induced monocyte up-regulation of CD11b, as only the combination of anti-CD14 and anti-C5a mAb blocked this effect completely. Collectively these data indicate that C5a/C5aR interaction and activation of CD14 are required and sufficient for CD11b up-regulation on granulocytes and monocytes, although the relative importance of these two differs significantly between the two cell types.

C5a was important, not only for E. coli-induced CD11b up-regulation but also for phagocytosis and oxidative burst, as supported by the effect of anti-C5a mAb 137-26, which inhibits the effect of C5a by binding to the C5a moiety of C5 without preventing C5 cleavage [24 ]. It is interesting that the inhibitory effect of anti-C5a mAb on phagocytosis of E. coli was more pronounced when it was given as MFI and less pronounced when phagocytosis was expressed as percent-positive cells, suggesting that C5a is involved in the quantitatively most efficient part of phagocytosis, most likely a result of the up-regulation of CD11b, whereas blocking of C3 opsonization was required for complete inhibition. Furthermore, we documented a direct effect of CVF and rhC5a in the expression of CD11b, which was blocked by the C5aRa. Consistently, Sprong et al. [40 ] have shown that neutralization of C5a inhibited CD11b up-regulation and phagocytosis of N. meningitides. Marder et al. [41 ] showed that leukotriene B4 (LTB4) up-regulates granulocyte CD11b, and this up-regulation can be inhibited by a LTB4 receptor antagonist. It is therefore possible that LTB4 acts as a second messenger between C5a and CD11b.

The role of soluble E. coli-derived molecules such as LPS in inflammation has been studied in detail previously [14 ]. The present study shows that LPS, at concentrations present in the whole E. coli bacteria preparations, do not activate complement. Similar results were found with N. meningitidis LPS [34 , 42 ]. However, LPS at high concentrations induced TCC formation, supporting previous data that LPS might activate complement [43 ]. We speculate that the LPS-induced complement activation at high LPS concentrations may be a result of the formation of LPS in micellar forms or binding of LPS to plastic surfaces, as the latter phenomena have been reported to activate the alternative complement pathway [44 ]. However, as LPS only induces complement activation at concentrations above the levels found in vivo in E. coli sepsis [44 ], it is unlikely that LPS is an important complement activator in vivo. However, LPS might sensitize blood mononuclear cells to the activation by C5a, as previously shown for IL-1ß and TNF-{alpha} synthesis [45 ]. Furthermore, blocking of CD14 and CD18, the latter being the common ß-chain of CR3/CD11b and CR4/CD11c, inhibits group B streptococci-induced TNF synthesis in monocytes, supporting a possible link between CD14 and ß-integrins [46 ].

In conclusion, the present data indicate that activation of CD14 and complement, including C3 opsonization and C5a release, are crucial events, which are both required and sufficient for E. coli-induced activation of granulocytes and monocytes, as judged by CD11b up-regulation, phagocytosis, and subsequent oxidative burst in an in vitro human whole blood model of sepsis. Provided that the bacteria are treated properly with antibiotics, we propose that a combined inhibition of complement and CD14 might be a therapeutic approach to reduce the uncontrolled, inflammatory response in sepsis.


   ACKNOWLEDGEMENTS
 
This work was supported by grants from the Family Blix Foundation, Sigval Bergesen d.y. and Wife Nanki’s Foundation, the Sonneborn Charitable Trust, the Research Council of Rikshospitalet, Anders Jahre’s Fund for the Promotion of Science, Odd Fellow Foundation, the Norwegian Rheumatism Association, and the Norwegian Foundation for Health and Rehabilitation. M. F. is an employee of Tanox Inc., the company producing anti-C5a, anti-C2, and anti-factor D mAb. We kindly thank Prof. Kaare Bergh at the Norwegian University for Science and Technology (Trondheim, Norway) for the supplement of the antibodies used for C5a quantification.

Received August 29, 2006; revised December 23, 2006; accepted January 7, 2007.


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