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. 2009 Sep 15;106(37):15861-6.
doi: 10.1073/pnas.0903613106. Epub 2009 Aug 26.

Human Genetic Deficiencies Reveal the Roles of Complement in the Inflammatory Network: Lessons From Nature

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Human Genetic Deficiencies Reveal the Roles of Complement in the Inflammatory Network: Lessons From Nature

Knut Tore Lappegård et al. Proc Natl Acad Sci U S A. .
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Abstract

Complement component C5 is crucial for experimental animal inflammatory tissue damage; however, its involvement in human inflammation is incompletely understood. The responses to gram-negative bacteria were here studied taking advantage of human genetic complement-deficiencies--nature's own knockouts--including a previously undescribed C5 defect. Such deficiencies provide a unique tool for investigating the biological role of proteins. The experimental conditions allowed cross-talk between the different inflammatory pathways using a whole blood model based on the anticoagulant lepirudin, which does not interfere with the complement system. Expression of tissue factor, cell adhesion molecules, and oxidative burst depended highly on C5, mediated through the activation product C5a, whereas granulocyte enzyme release relied mainly on C3 and was C5a-independent. Release of cytokines and chemokines was mediated to varying degrees by complement and CD14; for example, interleukin (IL)-1beta and IL-8 were more dependent on complement than IFN-gamma and IL-6, which were highly dependent on CD14. IL-1 receptor antagonist (IL-1ra) and IFN-gamma inducible protein 10 (IP-10) were fully dependent on CD14 and inversely regulated by complement, that is, complement deficiency and complement inhibition enhanced their release. Granulocyte responses were mainly complement-dependent, whereas monocyte responses were more dependent on CD14. Notably, all responses were abolished by combined neutralization of complement and CD14. The present study provides important insight into the comprehensive role of complement in human inflammatory responses to gram-negative bacteria.

Conflict of interest statement

Conflict of interest statement: J.D.L. is the inventor of patent applications related to the use of Compstatin and C5aR antagonist as therapeutic complement inhibitors. T.E.M. is an inventor of a patent application related to the use of combined inhibition of complement and CD14. The other authors have no competing financial interest to declare.

Figures

Fig. 1.
Fig. 1.
Characterization of the complement deficiencies. (A) Molecular characterization of the C2 deficiency by gel electrophoresis of PCR fragments generated with primers flanking the 28 bp genomic deletion. A 174-bp fragment was generated in individuals without C2 deficiency [lane 1, C5 control; lane 2, C5-deficient patient (C5D), lane 3, C2 control]. A 146-bp fragment was generated in the C2-deficient patient (C2D) (lane 4) and a positive control with a homozygous C2 deletion (lane 6). Both fragments were generated in a heterozygous carrier of the deletion (lane 5). Lane M: molecular weight markers. (B) Detection of C2, corresponding to a molecular size of 102 kDa by Western blot analysis. Lane 1, a pool of normal human serum, lane 2, C2D, lane 3, C2-depleted serum control, lane 4, normal human serum control. The 150-kDa band represents IgG, visualized as a result of species cross-reactivity of the anti-IgG antiserum. (C) PCR amplification of C5 cDNA with primers located in exons 25 and 29 resulted in fragments of 594 bp in individuals without C5 deficiency (lane 2, C5 control; lane 3, C2 control; lane 4, C2D), while two aberrant fragments were found in C5D (lane 1). Lane M: molecular weight markers. (D) Cloning and sequencing of the aberrant C5 fragments from the patient showed that the longest fragment of 498 bp was from cDNA with a deletion of exon 27, while the short fragment of 338 bp was from cDNA with a deletion of exons 26 and 27. The arrows indicate the joining sites. The stop codon generated by the exon 25–28 join is underscored. (E) Detection of C5, corresponding to a molecular size of 190 kDa, by western blotting. Lane 1, a pool of normal human serum; lane 2, C5-depleted serum control; lane 3, C5D; lane 4, C5D reconstituted with purified C5, lane 5, purified C5. (F) Functional complement activity was analyzed using Wielisa, which is based on pathway-specific activation of serum with final common deposition of the terminal C5b-9 complex as the readout. Lower reference values are 40% (gray zone, 40–70%) for the classical pathway (CP), 10% for the lectin pathway (LP) and 10% (gray zone, 10–30%) for the alternative pathway (AP). The lower detection limit for all pathways was 5%. The C2D patient was deficient in the CP and LP, and the C5D patient was deficient in the CP, LP, and AP; the C5 control was deficient in the LP, and the C2 control was normal. (G) Reconstitution of C2D and C5D blood was accomplished by adding highly purified and functionally active C2 and C5 proteins in increasing doses to the deficient samples. Normal functional activity was obtained at doses corresponding to the reference values for human serum. Thus, for the experiments performed with whole blood in the rest of this study, C2 and C5 were added to obtain plasma concentrations of 24 and 80 mg/L, respectively. Fully restored activity of the components was verified in lepirudin plasma prepared from the blood used in these experiments. (H) MBL genotypes and concentration. MBL in the C2D patient and C2 control were normal, with the genotype YA/YA (associated with high expression of MBL) and no structural defects. The C5D patient and C5 control displayed MBL defects with the genotypes XA/C and XA/B, respectively. XA is associated with low expression of MBL and B and C are structural defects in the gene. These two genotypes are molecularly regarded as equivalent. Thus, the role of MBL in the experiments performed in the present study could be clearly dissected from the C5 defect by using the genotypically MBL-matched C5 control individual, comparing the results with those for the MBL-sufficient C2D control. Individuals with MBL concentrations <100 μg/L are considered MBL-deficient, 100–500 μg/L is considered low, and >500 μg/L is normal. All data depicted in this figure were repeated with virtually identical results.
Fig. 2.
Fig. 2.
Bacterial growth and phagocytosis. (A) Growth of E. coli in complement-deficient (CD) (open circles), complement-reconstituted (CD+R) (closed circles) and control (closed triangles) blood. Live E. coli (1 × 105/mL) was added to whole blood and incubated for 0, 10, 60, 120, 180, or 240 min. The blood was then seeded onto agar plates and the colony-forming units (CFU)/mL blood were calculated. C5D blood (open circles) had no bactericidal ability (Left). Addition of purified C5 to C5D blood restored the bactericidal capacity. In contrast to the C5D blood, the absence of C2 did not interfere with bacterial killing (Right). Data are presented as mean and range of two experiments performed on separate days. (B) Phagocytosis of heat inactivated E. coli by granulocytes in complement-deficient, complement-reconstituted and control blood with or without the addition of a C5a receptor antagonist. (Left) C5D patient (C5D) and control (C5 Ctr). (Right) C2D patient (C2D) and control (C2 Ctr). Results are expressed as median fluorescent intensity (MFI). BGR = background of reconstituted blood without E. coli. BG = background of control blood without E. coli. D = complement-deficient blood incubated with E. coli. DR and Ctr = reconstituted and control blood incubated with E. coli. RA = reconstituted or control blood incubated with E. coli in the presence of a C5a receptor antagonist. Data are presented as mean and range of two experiments performed on separate days. In the C5 panel, three of the columns represent single experiments because of missing values.
Fig. 3.
Fig. 3.
Leukocyte expression of tissue factor and adhesion molecules. (A and B) Expression of tissue factor (TF) on monocytes as indicated by median fluorescent intensity (MFI) in flow cytometry. Blood was challenged with N. meningitidis 5 × 106/mL or 5 × 107/mL in separate experiments on two consecutive days. The top (A) and bottom (B) panels show the C5D patient and C2D patient with their respective controls. Column designations at the bottom are common for lower and upper panels: BGR = background of reconstituted blood without N. meningitidis. BG = background of control blood without N. meningitidis. D = complement-deficient blood incubated with N. meningitidis. DR and Ctr = reconstituted or control blood incubated with N. meningitidis. CS = reconstituted or control blood incubated with N. meningitidis in the presence of the C3 convertase inhibitor compstatin. RA = reconstituted or control blood incubated with N. meningitidis in the presence of a C5a receptor antagonist. Data are presented as mean and range of the two experiments. (C–F) Expression of CD62L and CD11b was determined by flow cytometry. Blood was challenged with N. meningitidis 1 × 107 or 2.5 × 107/mL in separate experiments on 2 consecutive days. Data are presented as mean and range of the two experiments. BG = background of C5D blood without bacteria added. BGR = background of reconstituted C5D blood without bacteria added. D = C5D blood challenged with bacteria. DR = C5D blood reconstituted with purified C5 and challenged with bacteria. CS = C5D blood reconstituted with purified C5 and challenged with bacteria in the presence of compstatin. RA = C5D blood reconstituted with purified C5 and challenged with bacteria in the presence of a C5a receptor antagonist. aCD14 = C5D blood reconstituted with purified C5 and challenged with bacteria in the presence of an anti-CD14 antibody. aCD14+CS = C5D blood reconstituted with purified C5 and challenged with bacteria in the presence of compstatin and an anti-CD14 antibody. (C) CD62L shedding from monocytes was complement-independent and relied on CD14. (D) CD62L shedding from granulocytes was completely dependent on C5. (E) CD11b up-regulation on monocytes was both complement and CD14-dependent. (F) CD11b up-regulation on granulocytes was completely dependent on C5.
Fig. 4.
Fig. 4.
Activation of granulocytes. The designations given for the columns at the bottom of the panels are as described in Fig. 3. The “Ctr” column in panel A is deficient blood reconstituted with purified complement factor and challenged with bacteria in the presence of control antibody and control peptide. (A) Oxidative burst in granulocytes. The oxidative burst in granulocytes was expressed as median fluorescent intensity (MFI) determined by flow cytometry. Blood was challenged with E. coli at 5 × 107/mL and mean and range of two experiments performed on two consecutive days are shown. Panels show results from C5D (Left) and C2D (Right), respectively. The granulocyte oxidative burst was abolished in the absence of C5, but only partially in the absence of C2. The effect was mediated through C5a. (B) Results from the C2D patient are presented. Release of granulocyte enzymes (lactoferrin, elastase, and myeloperoxidase) is shown as mean and range of two experiments from consecutive days with two different concentrations of E. coli (1 × 106/mL or 5 × 106/mL, respectively). Notably, this release reaction was completely dependent on C3 and independent of C5.
Fig. 5.
Fig. 5.
Release of cytokines. Results from the C5D patient are presented. Blood was challenged with E. coli at 1 × 106 or 5 × 106/mL in separate experiments on two consecutive days. Data are presented as mean and range of these two experiments. The designations given for the columns at the bottom of the figure are as described in Fig. 4. (A) Proinflammatory cytokines (TNF-α = tumor necrosis factor-alpha, IL-1β = interleukin-1 beta, IL-6 = interleukin 6, IFN-γ = IFN-gamma) and chemokines (IL-8 = interleukin 8, MIP-1α = macrophage inflammatory protein 1-alpha) were induced by bacteria largely in a CD14-dependent manner with various levels of dependence on complement. (B) IP-10 (IFN-γ inducible protein 10 or CXCL10) and the anti-inflammatory cytokine IL-1ra (interleukin 1 receptor antagonist) displayed a pattern opposite to the cytokines described in A. Complement C5 protected against IP-10 and IL-1ra release and inhibition of complement enhanced the release. Both IP-10 and the IL-1ra release were totally blocked by anti-CD14. (C) A number of cytokines, including IL-10 as shown here, were not induced by E. coli during the incubation period.
Fig. 6.
Fig. 6.
The role of complement and CD14 in the inflammatory reactions induced by Gram-negative bacteria. Crucial dependence on C3 and C5 are shown in blue and red, respectively. CD14-dependence is shown in yellow, and the relative dependence on C5 and CD14 is illustrated with merging colors. An inverse complement-dependence, that is, an enhanced release in the absence of complement, was CD14-mediated and seen for two markers.

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