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Ann Thorac Surg 2004;78:38-44
© 2004 The Society of Thoracic Surgeons
a Department of Medicine, Nordland Hospital, Bodø, and University of Tromsø, Tromsø, Norway
b Department of Immunology and Transfusion Medicine, Nordland Hospital, Bodø, and University of Tromsø, Tromsø, Norway
c Tanox, Inc, Houston, Texas, USA
d Carmeda AB, Stockholm, Sweden
e Institute of Immunology, Rikshospitalet University Hospital, Oslo, Norway
Accepted for publication February 3, 2004.
* Address reprint requests to Dr Lappegård, Department of Medicine, Nordland Hospital, N-8092 Bodø, Norway
e-mail: knut.lappegard{at}nlsh.no
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Abstract |
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 Top Abstract IntroductionMaterial and methodsResultsCommentAcknowledgmentsReferences |
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METHODS: Human whole blood was incubated in rotating loops of polyvinylchloride or heparin-coated polyvinylchloride tubing for 4 hours. Plasma concentrations of the cytokines tumor necrosis factor 
, interleukin (IL) 1ß, IL-6, IL-8, IL-10, and monocyte chemoattractant protein 1 (MCP-1) were quantified.
RESULTS: Polyvinylchloride induced a substantial increase in IL-8 and MCP-1, which was abolished by cycloheximide, indicating that they were synthesized during incubation. Interleukin 8 synthesis was completely complement-dependent since it was abolished by neutralizing antibodies to factor D and complement factor 5, as well as by a complement factor 5a receptor antagonist. Monocyte chemoattractant protein 1 synthesis was reduced by approximately half the amount by the complement inhibitors. Heparin-coated polyvinylchloride efficiently prevented synthesis of both IL-8 and MCP-1. Addition of recombinant human complement factor 5a to blood incubated in heparin-coated polyvinylchloride restored IL-8 and MCP-1 production completely and partly, respectively. In contrast to IL-8 and MCP-1, tumor necrosis factor , IL-1ß, interleukin 6 and IL-10 increased only marginally. A minor but significant increase in IL-1ß was complement-dependent, whereas a similar increase in IL-10 was completely prevented by heparin-coated polyvinylchloride. No significant changes were observed for tumor necrosis factor and IL-6.
CONCLUSIONS: Polyvinylchloride induced a marked increase in IL-8 and MCP-1, in contrast to a marginal increase in tumor necrosis factor , IL-1ß, IL-6, and IL-10. The increase in IL-8 and MCP-1 was prevented by heparin-coated polyvinylchloride. Interleukin 8 production was totally complement-dependent and mediated by complement factor 5a.
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Introduction |
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TopAbstractIntroductionMaterial and methodsResultsCommentAcknowledgmentsReferences |
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| Drs Fung and Mollnes disclose that they have a financial relationship with Tanox, Inc. Dr Riesenfeld discloses that he has a financial relationship with Carmeda AB.
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Cardiopulmonary bypass (CPB) induces a systemic inflammatory response involving activation of leukocytes, platelets, and plasma cascade systems [1, 2]. The procedure involves exposure of blood to artificial surfaces, but the relative contribution of the artificial surface itself in this response—in comparison with, for example, the membrane oxygenator, surgical trauma, or ischemia/reperfusion injury—is not well understood. Modification of the artificial surface by end-point attachment of heparin is known to attenuate several of the observed inflammatory responses both in vitro and in vivo [3–5]; and in some surgical centers the use of heparin-coated tubing is standard procedure, with excellent clinical results [6]. Similar in vivo observations have been made with inhibitors of the complement system [7, 8]. Complement activation is responsible for several of the other inflammatory reactions taking place during CPB, and the complement inhibitory properties of the heparin-coat may account for many of the beneficial effects of this surface. However, in an in vitro model, we have recently shown that various leukocyte responses differ in their dependency on complement and that surface modification with covalently attached heparin attenuates both complement-dependent and complement-independent reactions [9]. This model is restricted to study the effect of the surface and is based on a previously described technique using whole human blood anticoagulated with the recombinant hirudin analogue lepirudin, which does not interfere with complement activation [10]. The choice of anticoagulant in studies of complement involvement in inflammatory processes in full blood is crucial, as several anticoagulants interfere with complement activation and thus are unsuitable for this purpose. We suggest that hirudin at present is the best anticoagulant available for such studies.
The cytokine response in clinical CPB and in various laboratory models of this procedure has been studied previously [11–14]. However, the results from these studies are conflicting, both with respect to the role of surface heparin coating as well as complement inhibition on cytokine formation. This may be attributed in part to the lack of standardization of the procedures and models, or to the contribution from different surface-independent mechanisms of inflammation.
The aim of the present study was to apply the novel human whole blood model to investigate the role of complement in the cytokine response to the clinically frequently used artificial surface polyvinylchloride.
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Material and methods |
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Complement inhibitors
The cyclic hexapeptide AcF[OPdChaWR], a complement factor 5a receptor antagonist (C5aRA), was synthesized as previously described [15]. The monoclonal antibodies 166-32 (anti-factor D; immunoglobulin G1; blocks factor D function) and 137- 30 (anti-C5; immunoglobulin G1; blocks cleavage of C5) have been described earlier [10, 16]. The C5aRA, the monoclonal antibodies, as well as an isotype-matched control monoclonal antibody and a control peptide were all produced in the laboratory of one of the authors (M.F.).
Experimental model
The model has previously been described in detail [17] but was modified on the critical point of anticoagulation. Blood was drawn from healthy laboratory volunteers using lepirudin, a recombinant form of hirudin, instead of heparin as anticoagulant. Hirudin is a highly specific thrombin inhibitor and has no effect on the complement system. This is in contrast to heparin, which can either potentiate or attenuate complement activation, depending on the concentration used [10]. Samples of blood were supplied with specific complement inhibitors (antifactor D [166-32] 10 µg/mL, anti-C5 [137-30] 50 µg/mL, or C5aRA 5 µmol/L), cycloheximide 50 µg/mL, recombinant human C5a 1 µg/mL or equal volumes of saline. A volume of 750 µL blood was then transferred to segments of PVC or H-PVC tubing (length 30 cm, internal diameter 3 mm). Each segment was closed end-to-end and incubated by rotating slowly in an incubator at 37°C for 4 hours, if not otherwise stated. Complement inhibitors and cycloheximide were tested in blood circulated in PVC tubing whereas recombinant human C5a was incubated in blood circulated in H-PVC tubing. The blood was processed immediately after collection; and in order to reduce delay in handling, the experimental setup did not allow for testing of all the different inhibitors in the same experiment. However, baseline values as well as blood circulated in PVC tubing without inhibitor and blood circulated in H-PVC tubing were included in all experiments. All inhibitors were tested in blood from 6 different donors; and as baseline values as well as values from PVC tubing and H-PVC tubing were determined in every experiment, each donor served as his or her own control. After incubation, the blood was centrifuged at 3,220g for 15 minutes at 4°C. The plasma was frozen in aliquots at –70°C for later analysis of cytokines.
Preliminary experiments showed that incubation of blood in PVC tubing for 0.5, 1, or 2 hours gave no measurable increase for the various cytokines tested. After 4 hours of incubation, there were differences for all the mediators compared with baseline. Thus, 4 hours was chosen as standard incubation time for subsequent experiments.
Enzyme immunoassays
Assays for tumor necrosis factor (TNF-), interleukin (IL) 1ß, IL-6, and IL-10 were from R&D Systems (Minneapolis, MN); assays for IL-8 were from PeliKine, Sanquin Reagents (Amsterdam, Netherlands) and for monocyte chemoattractant protein 1 (MCP-1) from BioSource International (Camarillo, CA). All assays were performed according to the manufacturers instructions. Where available from the manufacturer, high-sensitivity kits were used.
Activation of the terminal complement pathway was determined in an enzyme immunoassay using the monoclonal antibody aE11 specific for a C9 neoepitope in the sC5b-9 complex using a modification of an assay described in detail previously [18].
Statistics
The Wilcoxon test for paired observations was used for comparison within groups, and the Mann-Whitney test was used for between group comparisons. Statistical significance was defined as p less than 0.05 (two-tailed). All complement inhibitors were tested in six separate experiments with blood from different donors. Baseline values for the various cytokines, as well as control PVC and H-PVC loops were included in every experimental set-up, and thus represent a much larger number of experiments with narrower confidence intervals than for each of the inhibitors tested. Data in the figures are expressed as median with 95% nonparametric confidence intervals.
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Results |
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TopAbstractIntroductionMaterial and methodsResultsCommentAcknowledgmentsReferences |
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Effect of heparin coating and complement inhibition on PVC-induced production of IL-8 and MCP-1
The H-PVC reduced the PVC-induced increase in IL-8 from 513 to 79 pg/mL (Fig 2,
p < 0.001) and the PVC-induced increase in MCP-1 from 233 to 48 pg/mL (Fig 3,
p < 0.001). The inhibition was similar for IL-8 and MCP-1, reaching 80% to 85% reduction compared with values obtained in PVC tubing.
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The increase in MCP-1 was reduced by approximately half the amount using anti-factor D or anti-C5 (p < 0.001 and p < 0.01 for the two antibodies versus PVC; the median values were reduced from 233 to 148 and 169 pg/mL, respectively; Fig 3). The C5aRA attenuated the MCP-1 response significantly and to the same extent as the other complement inhibitors (p < 0.001 versus PVC, median value reduced from 233 to 96 pg/mL; Fig 3). Addition of recombinant C5a to blood circulated in H-PVC tubing significantly increased MCP-1 levels compared with baseline values and with H-PVC tubing without C5a (p < 0.01 for both; Fig 3). The amount of recombinant C5a that completely restored the IL-8 synthesis, only partially, but still significantly, restored the MCP-1 synthesis (p < 0.01 versus PVC; Fig 3). The differences observed for IL-8 and MCP-1 are consistent with a differential effect of complement activation in induction of these cytokines.
PVC-induced production of IL-1ß and IL-10
Interleukin-1ß
The median level of IL-1ß increased modestly from 0.03 to 3.83 pg/mL after 4 hours of incubation in PVC tubing (p < 0.001 versus baseline; Fig 4).
The increase in H-PVC tubing as well as in samples incubated with anti-factor D antibody was significantly lower (p < 0.05 versus PVC, 2.19 and 1.54 pg/mL, respectively; Fig 4). The C5aRA also inhibited IL-1ß production, but the number of experiments was too low for statistical calculation (data not shown).
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and interleukin-6 and IL-6 after 4 hours incubation in PVC tubing.
PVC-induced complement activation
The terminal complement complex increased significantly in the PVC tubing, confirming that uncoated PVC activates complement. This activation was markedly reduced by H-PVC, also confirming previous data that this heparin coating prevents complement activation. Antifactor D and anti-C5 reduced terminal complement complex to the same extent as H-PVC, whereas the C5aRA had no effect on terminal complement complex formation, as expected (data not shown).
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Comment |
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TopAbstractIntroductionMaterial and methodsResultsCommentAcknowledgmentsReferences |
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Circulation of blood in PVC tubing induces complement activation primarily through the alternative pathway [9]. Anticoagulation may directly affect complement, either by activation, for example, by low doses of heparin, or by inhibition as seen with calcium binders and high doses of heparin. Thus, conflicting results from different experimental models may in part be due to the use of different anticoagulants. In our model lepirudin, a recombinant form of hirudin, is used as anticoagulant since it has been shown to have no effects on complement activation [10]. We therefore suggest that this model is superior for studies of complement involvement in artificial surface-induced inflammatory responses.
By using specific complement inhibitors it can be determined to what extent the different inflammatory reactions are secondary to complement activation. We have recently reported that variuos inflammatory responses in such a model indeed vary in their dependency on the complement system [9]. It should be noted, however, that results from our model are not always readily comparable to results from in vivo or different in vitro models as we selectively study the interaction between blood and the artificial surface of the tubing.
A group of structurally similar cytokines which in addition to cytokine properties also are highly chemotactic and participate in the recruitment of monocytes and granulocytes to sites of inflammation are referred to as chemokines. Interestingly, the two chemokines included in the present study, IL-8 and MCP-1, differed principally from the other cytokines in that they were substantially increased by the PVC surface. IL-8 has been shown to increase in different settings of CPB [19–21]. In the light of our results, the increases in IL-8 reported in these studies may well be induced through interaction between blood and the artificial surface of the tubing. We found a 100-fold increase in plasma levels of IL-8 after 4 hours of incubation in PVC loops. The increase was dependent on protein synthesis as it was substantially reduced by the addition of cycloheximide. Furthermore, the IL-8 synthesis was totally complement-dependent as the increase could be blocked by anti-factor D, anti-C5 and a C5aRA. The results are consistent with a previous study [16] using an in vitro hypothermic CPB model with unmodified PVC circuits. The increase in IL-8 occured in the later phase of CPB and was inhibited by anti-factor D (clone 166-32). In our study, addition of recombinant human C5a to the blood in the heparin-coated loops completely restored IL-8 synthesis, further supporting the notion that this synthesis is C5a dependent.
The PVC-induced increase in IL-8 was almost completely inhibited in the heparin-coated tubing. Ashraf and associates [19] found no differences in IL-8 between ordinary and heparinized circuits in vivo. However, as mentioned earlier, the relative contribution of the various parts of the CPB system in such models is largely unknown. Furthermore, we have recently shown that even though the heparin coat is effective in preventing surface complement activation, it cannot inhibit or attenuate reactions taking place in the fluid phase [9]. Thus, complement being activated in parts of the CPB circuit other than at the tubing surface, will not be affected by the heparin coat and can therefore trigger secondary reactions such as IL-8 synthesis.
The increase in IL-8 synthesis in the PVC tubing and the effect of complement inhibition and heparin coating led us to investigate whether these effects also applied to MCP-1, another member of the chemokine family. This protein is mainly synthesized in endothelial cells and to some extent in adipose tissue, but it is likely that it can be produced by most cell types. Monocyte chemoattractant protein 1 has been shown to be a strong predictor for coronary events in people with arteriosclerosis [22], and is thought to be an important factor in the atherosclerotic process itself. In mice with a genetic susceptibility to arteriosclerosis, inhibition of MCP-1 was shown to attenuate the atherosclerotic process [23, 24]. The cytokine MCP-1 has not previously been studied in in vitro models of CPB. We found a significant increase after blood was circulated 4 hours in PVC loops demonstrating that leukocytes themselves can produce MCP-1 upon stimulation. The fact that cycloheximide to a large extent could inhibit this increase indicates a substantial de novo synthesis, and not just release of preformed protein. Heparin coating efficiently prevented the increase in MCP-1 whereas complement inhibition only partly blocked MCP-1. This finding might indicate that there are several different stimuli for MCP-1 increase, acting through complement-dependent as well as complement-independent mechanisms. Furthermore, the addition of doses of recombinant C5a sufficient to completely restore the IL-8 synthesis, only partly restored the MCP-1 increase, further supporting the notion that the PVC-induced IL-8 is totally complement-dependent whereas that of MCP-1 is partly complement-dependent.
Previous studies have usually reported increased levels of IL-6 after CPB [19, 21]. Our data indicate that the PVC surface per se is not responsible for IL-6 increase, as we were unable to demonstrate any differences between baseline plasma levels and levels after incubation for 4 hours in PVC tubing. In vivo studies with heparin-coated loops have shown reduction in IL-6 compared with conventional PVC-loops [14] whereas others have shown similar increases in both groups [25]. The membrane oxygenator, surgical trauma, ischemia/reperfusion or other factors such as endotoxin, could contribute to the increased IL-6 seen in clinical settings. Like IL-6, TNF- did not increase in our model, which is in accordance with previous results [25]. In contrast, a modest, but reproducible and statistically significant increase in IL-1ß was observed in our study. However, the levels after incubation were still very low, and similar in magnitude to data reported previously [25]. Collectively, our data indicate that the PVC surface itself hardly induces any increase in the traditional proinflammatory cytokines, which is in contrast to the marked increase seen for the chemokines IL-8 and MCP-1.
Interleukin-10 is mainly an antiinflammatory cytokine and has therefore been regarded as beneficial in the inflammatory response. On the other hand it could be argued that as long as it reflects a biological response, its increase in an in vitro model of CPB would indicate a bioincompatible property of the surface. Interleukin-10 also possesses proinflammatory properties [26], making the interpretation of a possible beneficial or harmful effect of inhibiting this cytokine difficult. The effect of heparin coating on IL-10 release has been a subject of debate based on conflicting data in the literature. Butler and associates [12] found an increase in IL-10 in patients undergoing CPB with no difference between heparin-coated (Duraflo II) and unmodified circuits. In patients undergoing heart or heart-lung transplantation, a procedure with longer CPB than coronary artery bypass graft, Wan and associates [27] found lower levels of IL-10 after aortic declamping in the heparin-coated group. In contrast, using Bioline heparinized circuits, Harig and associates [28] found elevated IL-10 levels compared with control circuits. Finally, Giomarelli and associates [29] found reduced IL-10 release from stimulated monocytes harvested from heparin-coated circuits of CPB-patients compared with unmodified circuits. In our model, we found a modest but significant increase in IL-10 by the PVC surface, which was prevented by the heparin coating, consistent with the attenuating effect of this surface on all mediators tested. One possible explanation for the conflicting IL-10 results is that heparin and heparan sulfate are able to bind IL-10 [30]. Thus, various heparinized circuits may have different IL-10 binding properties, and differences in plasma concentration may reflect differences in binding capacity rather than differences in induction of synthesis or release. Our data on IL-10 are in accordance with the general notion that the biocompatibility of the heparin coat used in this study is optimal, in the sense that it protects against any biological response, whether it is proinflammatory or anti-inflammatory.
In conclusion, our data show that contact between blood and a PVC surface induces a substantial increase in the chemokines IL-8 and MCP-1, in contrast to a very modest release of the other cytokines tested. The various products differ in their dependency on the complement system, IL-8 being totally complement-dependent, whereas heparin coating efficiently inhibits both the complement-dependent and complement-independent cytokine responses.
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Acknowledgments |
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TopAbstractIntroductionMaterial and methodsResultsCommentAcknowledgmentsReferences |
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References |
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TopAbstractIntroductionMaterial and methodsResultsCommentAcknowledgmentsReferences |
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rd KT, Fung M, Bergseth G, et al. Effect of complement inhibition and heparin coating on artificial surface-induced leukocyte and platelet activation. Ann Thorac Surg 2004;77:932–41
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