Properdin binding to complement activating surfaces depends on initial C3b deposition.

Two functions have been assigned to properdin; stabilization of the alternative convertase, C3bBb, is well accepted, whereas the role of properdin as pattern recognition molecule is controversial. The presence of nonphysiological aggregates in purified properdin preparations and experimental models that do not allow discrimination between the initial binding of properdin and binding secondary to C3b deposition is a critical factor contributing to this controversy. In previous work, by inhibiting C3, we showed that properdin binding to zymosan and Escherichia coli is not a primary event, but rather is solely dependent on initial C3 deposition. In the present study, we found that properdin in human serum bound dose-dependently to solid-phase myeloperoxidase. This binding was dependent on C3 activation, as demonstrated by the lack of binding in human serum with the C3-inhibitor compstatin Cp40, in C3-depleted human serum, or when purified properdin is applied in buffer. Similarly, binding of properdin to the surface of human umbilical vein endothelial cells or Neisseria meningitidis after incubation with human serum was completely C3-dependent, as detected by flow cytometry. Properdin, which lacks the structural homology shared by other complement pattern recognition molecules and has its major function in stabilizing the C3bBb convertase, was found to bind both exogenous and endogenous molecular patterns in a completely C3-dependent manner. We therefore challenge the view of properdin as a pattern recognition molecule, and argue that the experimental conditions used to test this hypothesis should be carefully considered, with emphasis on controlling initial C3 activation under physiological conditions.

The anti-inflammatory effect of combined complement and CD14 inhibition is preserved during escalating bacterial load.

Combined inhibition of complement and CD14 is known to attenuate bacterial-induced inflammation, but the dependency of the bacterial load on this effect is unknown. Thus, we investigated whether the effect of such combined inhibition on Escherichia coli- and Staphylococcus aureus-induced inflammation was preserved during increasing bacterial concentrations. Human whole blood was preincubated with anti-CD14, eculizumab (C5-inhibitor) or compstatin (C3-inhibitor), or combinations thereof. Then heat-inactivated bacteria were added at final concentrations of 5 × 10(4) -1 × 10(8) /ml (E. coli) or 5 × 10(7) -4 × 10(8) /ml (S. aureus). Inflammatory markers were measured using enzyme-linked immunosorbent assay (ELISA), multiplex technology and flow cytometry. Combined inhibition of complement and CD14 significantly (P < 0.05) reduced E. coli-induced interleukin (IL)-6 by 40-92% at all bacterial concentrations. IL-1ß, IL-8 and macrophage inflammatory protein (MIP)-1α were significantly (P < 0.05) inhibited by 53-100%, and the effect was lost only at the highest bacterial concentration. Tumour necrosis factor (TNF) and MIP-1ß were significantly (P < 0.05) reduced by 80-97% at the lowest bacterial concentration. Monocyte and granulocyte CD11b were significantly (P < 0.05) reduced by 63-91% at all bacterial doses. Lactoferrin was significantly (P < 0.05) attenuated to the level of background activity at the lowest bacterial concentration. Similar effects were observed for S. aureus, but the attenuation was, in general, less pronounced. Compared to E. coli, much higher concentrations of S. aureus were required to induce the same cytokine responses. This study demonstrates generally preserved effects of combined complement and CD14 inhibition on Gram-negative and Gram-positive bacterial-induced inflammation during escalating bacterial load. The implications of these findings for future therapy of sepsis are discussed.

Chimeric anti-CD14 IGG2/4 Hybrid antibodies for therapeutic intervention in pig and human models of inflammation.

CD14 is a key recognition molecule of innate immune responses, interacting with several TLRs. TLR signaling cross-talks extensively with the complement system, and combined CD14 and complement inhibition has been proved effective in attenuating inflammatory responses. Pig models of human diseases have emerged as valuable tools to study therapeutic intervention, but suitable neutralizing Abs are rare. Undesired Fc-mediated functions, such as platelet activation and IL-8 release induced by the porcine CD14-specific clone Mil2, limit further studies. Therefore, an inert human IgG2/IgG4 hybrid C region was chosen for an rMil2. As revealed in ex vivo and in vivo pig experiments, rMil2 inhibited the CD14-mediated proinflammatory cytokine response similar to the original clone, but lacked the undesired Fc-effects, and inflammation was attenuated further by simultaneous complement inhibition. Moreover, rMil2 bound porcine FcRn, a regulator of t1/2 and biodistribution. Thus, rMil2, particularly combined with complement inhibitors, should be well suited for in vivo studies using porcine models of diseases, such as sepsis and ischemia-reperfusion injury. Similarly, the recombinant anti-human CD14 IgG2/4 Ab, r18D11, was generated with greatly reduced Fc-mediated effects and preserved inhibitory function ex vivo. Such Abs might be drug candidates for the treatment of innate immunity-mediated human diseases.

Combined inhibition of complement C5 and CD14 markedly attenuates inflammation, thrombogenicity, and hemodynamic changes in porcine sepsis.

Complement and the TLR family constitute two important branches of innate immunity. We previously showed attenuating effects on inflammation and thromogenicity by inhibiting the TLR coreceptor CD14 in porcine sepsis. In the present study, we explored the effect of the C5 and leukotriene B4 inhibitor Ornithodoros moubata complement inhibitor (OmCI; also known as coversin) alone and combined with anti-CD14 on the early inflammatory, hemostatic, and hemodynamic responses in porcine Escherichia coli-induced sepsis. Pigs were randomly allocated to negative controls (n = 6), positive controls (n = 8), intervention with OmCI (n = 8), or with OmCI and anti-CD14 (n = 8). OmCI ablated C5 activation and formation of the terminal complement complex and significantly decreased leukotriene B4 levels in septic pigs. Granulocyte tissue factor expression, formation of thrombin-antithrombin complexes (p < 0.001), and formation of TNF-α and IL-6 (p < 0.05) were efficiently inhibited by OmCI alone and abolished or strongly attenuated by the combination of OmCI and anti-CD14 (p < 0.001 for all). Additionally, the combined therapy attenuated the formation of plasminogen activator inhibitor-1 (p < 0.05), IL-1ß, and IL-8, increased the formation of IL-10, and abolished the expression of wCD11R3 (CD11b) and the fall in neutrophil cell count (p < 0.001 for all). Finally, OmCI combined with anti-CD14 delayed increases in heart rate by 60 min (p < 0.05) and mean pulmonary artery pressure by 30 min (p < 0.01). Ex vivo studies confirmed the additional effect of combining anti-CD14 with OmCI. In conclusion, upstream inhibition of the key innate immunity molecules, C5 and CD14, is a potential broad-acting treatment regimen in sepsis as it efficiently attenuated inflammation and thrombogenicity and delayed hemodynamic changes.

The role of properdin in zymosan- and Escherichia coli-induced complement activation.

Properdin is well known as an enhancer of the alternative complement amplification loop when C3 is activated, whereas its role as a recognition molecule of exogenous pathogen-associated molecular patterns and initiator of complement activation is less understood. We therefore studied the role of properdin in activation of complement in normal human serum by zymosan and various Escherichia coli strains. In ELISA, microtiter plates coated with zymosan induced efficient complement activation with deposition of C4b and terminal complement complex on the solid phase. Virtually no deposition of C4b or terminal complement complex was observed with mannose-binding lectin (MBL)-deficient serum. Reconstitution with purified MBL showed distinct activation in both readouts. In ELISA, normal human serum-induced deposition of properdin by zymosan was abolished by the C3-inhibiting peptide compstatin. Flow cytometry was used to further explore whether properdin acts as an initial recognition molecule reacting directly with zymosan and three E. coli strains. Experiments reported by other authors were made with EGTA Mg²âº buffer, permitting autoactivation of C3. We found inhibition by compstatin on these substrates, indicating that properdin deposition depended on initial C3b deposition followed by properdin in a second step. Properdin released from human polymorphonuclear cells stimulated with PMA did not bind to zymosan or E. coli, but when incubated in properdin-depleted serum this form of properdin bound efficiently to both substrates in a strictly C3-dependent manner, as the binding was abolished by compstatin. Collectively, these data indicate that properdin in serum as well as polymorphonuclear-released properdin is unable to bind and initiate direct alternative pathway activation on these substrates.

Dissecting the effects of lipopolysaccharides from nonlipopolysaccharide molecules in experimental porcine meningococcal sepsis.

OBJECTIVE: To dissect the in vivo responses to lipopolysaccharide compared with nonlipopolysaccharide structures of whole meningococci. DESIGN: Comparative experimental study. SETTING: University hospital with an animal intensive care unit and laboratory. SUBJECTS: Twenty-four anesthetized healthy Norwegian landrace pigs of 30 kg (+/- 2.5 kg) grouped into two test groups and one control group. INTERVENTIONS: Exponentially increasing numbers of Neisseria meningitidis H44/76 (NmLPS+) or a knockout mutant of H44/76 completely lacking lipopolysaccharide (NmLPS-) were infused intravenously to the pigs. MEASUREMENTS AND MAIN RESULTS: Physiological and hematologic parameters were continuously recorded and biochemical analyses were performed in batch after completion. Systemic vascular resistance, cardiac index and lactate changed significantly more in the NmLPS+ than in the NmLPS- group (p < .05). Mean pulmonary artery pressure increased early in the NmLPS+ and late in the NmLPS- group, but finally reached equally high values. Capillary leakage (fluid requirement, plasma albumin loss, organ wet/dry ratio) was more prominent in the NmLPS+ group (p < .05). Leukocytes were depleted in a highly lipopolysaccharide-dependent manner (p < .001). Thrombin-antithrombin complexes and plasminogen activator inhibitor-1 increased 2.5 to five times more in the NmLPS+ group (p < .05). Maximum cytokine concentrations in plasma were markedly higher in the NmLPS+ group (p < .05): tumor necrosis factor-alpha (40 times), interleukin-1beta (40 times), interleukin-6 (13 times), and interleukin-10 (four times). Interleukin-12 increased only in the NmLPS+ group. CONCLUSION: This large animal model, which simulates human disease well, confirms the potency of lipopolysaccharide but provides clear evidence that nonlipopolysaccharide molecules induce cardiovascular and hematologic changes quite similar to those caused by lipopolysaccharide. In general, 10- to 20-fold higher doses of the lipopolysaccharide-deficient mutant were required to induce the same degree of pathophysiological changes. Endotoxic activity of Gram-negative bacteria should no longer be attributed solely to the activity of lipopolysaccharide.

Organ inflammation in porcine Escherichia coli sepsis is markedly attenuated by combined inhibition of C5 and CD14.

Sepsis is an infection-induced systemic inflammatory syndrome, potentially causing organ failure. We previously showed attenuating effects on inflammation, thrombogenicity and haemodynamics by inhibiting the Toll-like receptor co-factor CD14 and complement factor C5 in a porcine Escherichia coli-induced sepsis model. The present study explored the effect on organ inflammation in these pigs. Tissue samples were examined from the combined treatment group (n = 8), the positive (n = 8) and negative (n = 6) control groups after 4h of sepsis. Inflammatory biomarkers were measured using ELISA, multiplex and qPCR analysis. Combined inhibition of C5 and CD14 markedly attenuated IL-1ß by 31-66% (P < 0.05) and IL-6 by 54-96% (P < 0.01) in liver, kidney, lung and spleen; IL-8 by 65-100% in kidney, lung, spleen, and heart (P < 0.05) and MCP-1 by 46-69% in liver, kidney, spleen and heart (P < 0.05). Combined inhibition significantly attenuated tissue factor mRNA upregulation in spleen (P < 0.05) and IP-10 mRNA upregulation in four out of five organs. Finally, C5aR mRNA downregulation was prevented in heart and kidney (P < 0.05). Combined inhibition of C5 and CD14 thus markedly attenuated inflammatory responses in all organs examined. The anti-inflammatory effects observed in lung and heart may explain the delayed haemodynamic disturbances observed in septic pigs receiving combined inhibition of C5 and CD14.

Post challenge inhibition of C3 and CD14 attenuates Escherichia coli-induced inflammation in human whole blood.

Combined inhibition of CD14 and complement, two main inducers of the inflammatory response, have proved particularly effective in attenuating Gram-negative bacteria-induced inflammation. Approaching possible clinical relevance, we investigated the effect of such inhibition in a post-challenge setting. Human whole blood was anti-coagulated with lepirudin. Anti-CD14, compstatin (C3 inhibitor) and the combination thereof were added 5 min prior to or 5, 15 or 30 min after adding Escherichia coli. Total incubation time with Escherichia coli was 120 min. Cytokines, myeloperoxidase (MPO) and the terminal complement complex (TCC) were measured using multiplex technology and ELISA. Delayed combined inhibition significantly attenuated the inflammatory response. IL-1ß, IL-8 and TNF-α were significantly inhibited in the range of 20-40%, even when adding the inhibitors with up to 30 min delay. IL-6 was significantly inhibited with 15 min delay, and MIP-1α and MPO with 5 min delay. Complement activation (TCC) was blocked completely at each time point compstatin was added, whereas the cytokines and MPO increased steadily between the time points. The combined regimen was significantly more effective than single inhibition in the pre-challenge setting. The attenuation of Escherichia coli-induced inflammation in a post-challenge setting suggests a potential therapeutic window for this treatment in sepsis.

Selective inhibition of TNF-alpha or IL-1 beta does not affect E. coli-induced inflammation in human whole blood.

Inhibition of the inappropriate and excessive inflammatory response has been a main issue in sepsis-related research. Historically, TNF-alpha and IL-1 beta have been postulated as key mediators in sepsis, but selective inhibition of these cytokines has failed in clinical trials. Recently it was found that inhibition of upstream recognition by complement and CD14 could efficiently reduce Escherichia coli (E. coli)-induced inflammation. An ex vivo model with lepirudin-anticoagulated human whole blood was used to explore the significance of selective inhibition of TNF-alpha and IL-1 beta in E. coli-induced inflammation. The effect of TNF-alpha, IL-1 beta, complement and CD14 on the inflammatory response was assessed by adding highly specific neutralizing agents to these mediators. Proinflammatory cytokines, expression of CD11b and oxidative burst were measured. The controls included relevant isotype-matched immunoglobulins and peptides. Selective inhibition of TNF-alpha or IL-1 beta had no impact on E. coli-induced release of proinflammatory cytokines, CD11b-upregulation or oxidative burst. In contrast, the combined inhibition of complement and CD14 virtually abolished these responses. These data suggest that both TNF-alpha and IL-1 beta are downstream mediators and as single mediators play a limited role within the complex inflammatory reactions induced by E. coli.

The down-stream effects of mannan-induced lectin complement pathway activation depend quantitatively on alternative pathway amplification.

Complement activation plays an important role in human pathophysiology. The effect of classical pathway activation is largely dependent on alternative pathway (AP) amplification, whereas the role of AP for the down-stream effect of mannan-induced lectin pathway (LP) activation is poorly understood. In normal human serum specific activation of LP was obtained after exposure to a wide concentration range of mannan on the solid phase. Reaction mechanisms in this system were delineated in inhibition experiments with monoclonal antibodies. Direct mannose-binding lectin (MBL) independent activation of AP was not observed even at high mannan concentrations since addition of the inhibiting anti-MBL mAb 3F8 completely abolished generation of the terminal C5b-9 complex (TCC). However, selective blockade of AP by anti-factor D inhibited more than 80% of TCC release into the fluid phase after LP activation showing that AP amplification is quantitatively responsible for the final effect of initial specific LP activation. TCC generation on the solid phase was distinctly but less inhibited by anti-fD. C2 bypass of the LP pathway could be demonstrated, and AP amplification was also essential during C2 bypass in LP as shown by complete inhibition of TCC generation in C2-deficient serum by anti-fD and anti-properdin antibodies. In conclusion, the down-stream effect of LP activation depends strongly on AP amplification in normal human serum and in the C2 bypass pathway.

Acidosis during reoxygenation has an early detrimental effect on neuronal metabolic activity.

We recently showed that acidosis is protective during hypoxia and detrimental during reoxygenation. We hypothesized that the detrimental effect of acidosis during reoxygenation was due to a negative effect on mitochondrial function. Human postmitotic NT2-N neurons were exposed to 3 h of hypoxia and glucose deprivation and then reoxygenated for 0, 1, 4, 9, or 21 h. The detrimental effect of acidotic reoxygenation on metabolic activity was evident already after 1 h of reoxygenation, when MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] reduction (percentage of normoxic controls) was significantly higher in cells reoxygenated with neutral compared with acidotic medium both after acidotic hypoxia (83+/-26% versus 67+/-27%, p=0.006) and after neutral hypoxia (51+/-12% versus 41+/-7%, p=0.005). Hypoxanthine, a marker of cellular energy failure, increased more with acidotic compared with neutral reoxygenation both after acidotic hypoxia (after 21 h: 7.7+/-2.7 versus 3.1+/-1.9 microM, p<0.001) and after neutral hypoxia (10.4+/-2.6 versus 7.9+/-2.8 microM, p=0.001). During hypoxia and reoxygenation, there was an earlier reduction in the activity of complex IV compared with complexes II+III, and the ratio between these complexes fell during the first hour of reoxygenation. The reduction in complex IV activity was alleviated with acidotic hypoxia. Acidosis during reoxygenation, however, had no effect on the activity of either complex IV or complexes II+III. We conclude that acidosis during hypoxia increases neuronal survival and preserves complex IV activity. Acidosis during reoxygenation has an early detrimental effect on metabolic activity, but this is not mediated through an effect on the mitochondrial complexes IV or II+III.

Acidosis has opposite effects on neuronal survival during hypoxia and reoxygenation.

To study the effect of extracellular acidosis on apoptosis and necrosis during ischemia and reoxygenation, we exposed human post-mitotic NT2-N neurones to oxygen and glucose deprivation (OGD) followed by reoxygenation. In some experiments, pH of the cell medium was lowered to 5.9 during either OGD or reoxygenation or both. Staurosporine, used as a positive control for apoptosis, caused Poly(ADP-ribose)-polymerase (PARP) cleavage and nuclear fragmentation, but no PARP cleavage and little fragmentation were seen after OGD. Low molecular weight DNA fragments were found after staurosporine treatment, but not after OGD. No protective effect of caspase inhibitors was seen after 3 h of OGD and 21 h of reoxygenation, but after 45 h of reoxygenation caspase inhibition induced a modest improvement in 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) cleavage. While acidosis during OGD accompanied by neutral medium during reoxygenation protected the neurones (MTT: 228 +/- 117% of neutral medium, p < 0.001), acidosis during reoxygenation only was detrimental (MTT: 38 +/- 25%, p < 0.01). We conclude that apoptotic mechanisms play a minor role after OGD in NT2-N neurones. The effect of acidosis on neuronal survival depends on the timing of acidosis, as acidosis was protective during OGD and detrimental during reoxygenation.