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Ann Thorac Surg 2007;83:146-152
© 2007 The Society of Thoracic Surgeons

Original Articles: Cardiovascular

Leukocyte Effects of C5a-Receptor Blockade During Simulated Extracorporeal Circulation

Christine S. Rinder, MDa,b,*, Michael J. Smith, PhDc, Henry M. Rinder, MDb, Daniel N. Cortright, PhDa,b, Robbin M. Brodbeck, PhDa,b, James E. Krause, PhDd, Brian R. Smith, MDb

a Department of Anesthesiology, Yale University School of Medicine, New Haven, Connecticut
b Department of Laboratory Medicine, Yale University School of Medicine, New Haven, Connecticut
c Department of Biomedical Sciences, Quinnipiac University, Hamden, Connecticut
d Neurogen Corporation, Branford, Connecticut

Accepted for publication August 4, 2006.

* Address correspondence to Dr C.S. Rinder, Department of Anesthesiology, Yale University School of Medicine, 333 Cedar St, PO Box 208051, New Haven, CT 06520-8051. (Email: christine.rinder{at}yale.edu).


Drs Cortright, Brodbeck, and Krause disclose that they have a financial relationship with Neurogen Corp.

 

   Abstract
Top
 Abstract
Introduction
 Material and Methods
 Results
 Comment
 Acknowledgments
 References
 
BACKGROUND: Distinct pathways of leukocyte activation during simulated cardiopulmonary bypass are mediated by the complement C5a anaphylatoxin. We hypothesized that a human C5a receptor antagonist would specifically inhibit the inflammatory response of neutrophils to simulated extracorporeal circulation, while preserving the C5b-9 pathway for innate immunity.

METHODS: An in vitro extracorporeal circuit recirculated fresh heparinized whole blood through a membrane oxygenator with and without addition of a small molecule human C5a receptor antagonist. Samples were periodically drawn over 90 minutes for complement and leukocyte activation studies.

RESULTS: Addition of the C5a receptor antagonist to simulated extracorporeal circulation abrogated both neutrophil CD11b upregulation and interleukin 8 release (p < 0.01 for both), despite full generation of C3a and C5b-9; however, elastase release from neutrophils was unaffected. Although C5a receptor blockade only trended toward inhibiting monocyte CD11b upregulation (p = 0.09), circuit clearance of both monocytes (p = 0.04) and neutrophils (p = 0.01) was significantly decreased. In addition, the C5a receptor antagonist completely blocked both neutrophil–platelet and monocyte–platelet conjugate formation (p < 0.001 for both), without affecting platelet P-selectin expression.

CONCLUSIONS: C5a receptor blockade during simulated extracorporeal circulation completely blocked neutrophil ß2 integrin upregulation and induction of plasma interleukin 8, suggesting an acute downregulatory effect on neutrophil chemotaxis-related pathways, while preserving terminal complement generation and neutrophil elastase release. Inhibition of leukocyte–platelet conjugate formation suggests a novel function for leukocyte adhesive receptors, possibly related to preservation of elastase generation.

The systemic inflammatory response to cardiopulmonary bypass (CPB) activates components of innate immunity that, although vital for antimicrobial activity [1], contribute to post-CPB pulmonary, myocardial, renal, and neurocognitive dysfunction [2]. Complement activation is one such inflammatory component [3], but its link to specific peri-CPB inflammatory changes is still under investigation. Simulated extracorporeal circulation (SECC) has successfully modeled these in vivo inflammatory changes through activation of complement (C3a, C5a, and C5b-9 formation), circulating leukocytes (neutrophil ([PMN] and monocyte CD11b upregulation), and secretion of interleukin (IL) 8 and PMN elastase [4], as well as platelet activation, manifest by leukocyte–platelet conjugate formation [5].

Simulated extracorporeal circulation studies from our laboratory have evaluated the utility of complement antagonists to blunt cellular responses. Monocyte activation can be blocked beginning at the C3 level [6], whereas neutrophil activation, critical to ischemia–reperfusion injury, was well-abrogated by interference at C5, with C3a still intact [5]; finally, platelet activation was C5b-9-dependent [7]. These ex vivo results prompted the use of C5 inhibition in human trials to reduce post-CPB complications [8]. However, blockade of terminal complement (C5b-9) formation, combined with CPB producing "adaptive" immunosuppression [3], could increase the risk for infection [1] and pulmonary complications [9]. Animal studies suggest that the host reaction to C5a anaphylatoxin can be effectively blunted with a specific C5a receptor (C5aR) antagonist [10], without blocking other complement components or formation of the C5b-9 membrane attack complex, the latter vital to innate immunity against pathogens.

We hypothesized that a human C5aR antagonist might effectively reduce neutrophil and cytokine (IL-8) inflammatory responses during in vitro SECC, while preserving C5b-9 formation. Any effect of C5aR blockade on monocytes would also be illuminating as recent SECC studies suggest that monocyte activation is affected at multiple complement levels [11]. During this study, two additional important pathophysiologic processes of the inflammatory response to SECC became evident: (1) there is a divergence of C5aR-dependent neutrophil activation pathways (elastase release versus ß2 integrin receptor upregulation and IL-8 release), and (2) C5aR blockade unexpectedly reduces leukocyte–platelet conjugate formation.


   Material and Methods
Top
 Abstract
 Introduction
 Material and Methods
Results
 Comment
 Acknowledgments
 References
 
Materials
Compound 4 [(benzyl{(1R)-[1-butyl-2-(3-fluorophenyl)-1H-imidazole-5yl]ethyl}amino) methyl]-3-chlorophenol, a small molecule human complement C5a receptor antagonist (henceforth referred to as C5aR antagonist), was supplied by Neurogen (Branford, CT).

Fluorochrome-conjugated monoclonal antibodies purchased from Pharmingen (San Diego, CA) included anti-CD41 (platelet glycoprotein IIb/IIIa), anti-CD62P (P-selectin), anti-CD45 (human leukocyte antigen), and anti-CD14 (monocyte receptor); anti-CD11b (ß2 integrin receptor) was obtained from ExAlpha (Boston, MA).

In Vitro Dose-Finding Studies
Affinity binding with [125I]-human C5a was performed as previously described [12] from purified P2 membranes prepared from baculovirus infected Sf9 cell pellets expressing the human C5aR, with a G-protein heterotrimeric complex of rat G{alpha}i2, human Gß1, and bovine G{gamma}2. The C5aR antagonist displaced [125I]-recombinant human C5a peptide binding with an effective concentration for 50% maximal response (EC50) value of 22.3 ± 5.0 nmol/L. Guanidine triphosphate-{gamma}35S binding to the human C5a receptor was then performed using a modification of a previously described method [13] with membranes obtained from Sf9 cells in the presence of 10 nmol/L recombinant human C5a; C5aR antagonist reversed the recombinant human C5a agonist effect (EC50 = 18 ± 1.7 nmol/L). In the absence of recombinant human C5a, C5aR antagonist did not stimulate a guanidine triphosphate-{gamma}35S binding response, indicating that its properties are consistent with that of an antagonist only.

Recombinant human C5a stimulated in vitro chemotaxis in purified PMN and U937 cells [14] with an EC50 of 0.5 nmol/L and 0.4 nmol/L, respectively, and C5aR antagonist effectively blocked recombinant human C5a-stimulated chemotaxis in both with an EC50 in the 20 nmol/L range and an EC90 estimated in the 0.5 to 1 µmol/L range. Because blood protein and biomaterial binding of small molecules can approach 90%, we chose a blood concentration of 5 µmol/L for SECC studies.

Simulated Extracorporeal Circuit Preparation
As previously described [5], circuits were assembled using a pediatric membrane oxygenator (VP CML Plus, Cobe Cardiovascular, Arvada, CO) and a roller pump (Cardiovascular Instruments Corp, Wakefield, MA), primed with Ringer’s lactate solution containing dextrose (4.0 g/L), mannitol (4.0 g/L), and porcine heparin (5 U/mL) and circulated at 1.5 L/min with sweep gas flow (95% oxygen, 5% carbon dioxide) at 0.25 L/min. The pH was maintained at 7.35 to 7.45 and the partial pressure of oxygen at greater than 150 mm Hg.

Simulated Extracorporeal Circuit Operation and Sampling
After approval by the institutional review board (last September 23, 2005) and informed consent, whole blood (500 mL) was drawn from healthy volunteers on no medications into a transfer pack (Baxter Healthcare Corp, Deerfield, IL) containing porcine heparin (5 U/mL). C5a receptor antagonist (5 µmol/L final concentration) or diluent was added to the transfer pack immediately before addition of the blood to the extracorporeal circuit. As blood was introduced to the circuit reservoir, prime fluid was withdrawn to yield a final circuit volume of 700 mL and a mean hematocrit of 26% ± 4% (standard deviation). Complete circuit mixing was accomplished within 2 minutes; this point was designated as time 0. The circuit flow rate was accelerated to 1.5 L/min, and the circuit was cooled to 28°C over the course of 5 minutes and maintained at that temperature for 60 minutes total. The circuit was then rewarmed to 37°C for an additional 30 minutes (90 minutes total recirculation). Five experiments were performed with C5aR antagonist and four with diluent. Whole blood samples at 0, 5, 15, 30, 45, 60, 75, and 90 minutes received a complete blood count and leukocyte differential and were fixed in 1% paraformaldehyde for flow cytometry. Plasma drawn at 0, 30, and 90 minutes was snap-frozen and stored at –70°C until assayed for C3a, C5b-9, IL-8, neutrophil elastase, and soluble P-selectin.

Flow Cytometry
Fixed whole blood was washed and resuspended in buffer with fluorescent monoclonal antibody at 4°C for 20 minutes, then washed and resuspended for flow cytometry (FACScan, Becton-Dickinson, Mountain View, CA). Leukocyte activation and the percentage of leukocytes with bound platelets was determined using (1) fluorescein isothiocyanate (FITC) anti-CD45, phycoerythrin (PE) anti-CD11b, and PE–Cy5–anti-CD14, and (2) FITC–anti-CD45, PE–anti-CD41a, and PE–Cy5–anti-CD14, respectively. Platelet CD62P expression was determined using FITC–anti-CD41a and PE–anti-CD62P. CD11b fluorescence, P-selectin expression, and leukocyte–platelet conjugates were determined as previously described [15].

Plasma Assays
C3a and C5b-9 levels were measured using enzyme-linked immunoassay kits (Quidel, San Diego, CA), as were IL-8 (R&D, Minneapolis, MN) and neutrophil elastase (EM Sciences, Gibbstown, NJ). Mixing studies using control and C5aR antagonist–treated samples were incorporated into all immunoassays to confirm the absence of direct interference by the C5aR antagonist.

Statistics
All results (mean ± standard error of the mean) are expressed as a percentage of the individual experiment’s time 0 value to minimize interdonor variability, with the exception of IL-8 in which baseline levels were uniformly undetectable. This technique is particularly critical for flow cytometry measurements because multiple variables affect absolute fluorescence intensity. Indeed, valid interpretation of SECC studies generally involves changes in a given variable, and thus, all flow cytometric testing from a single SECC experiment is performed on the same day to obviate any variability attributable to fluctuations in laser intensity or antibody fluorescence. Because values did not meet criteria for normal distribution, the Mann-Whitney test was used to assess single variable changes between baseline and end recirculation. The effect of the C5aR antagonist on variables measured at multiple time points was evaluated with repeated measures two-way analysis of variance to evaluate the effects of the drug, time on SECC, and any interaction of drug as a function of time (Prism GraphPad software, San Diego, CA).


   Results
Top
 Abstract
 Introduction
 Material and Methods
 Results
Comment
 Acknowledgments
 References
 
ß2 Integrin Effects of the C5a Receptor Antagonist
Control SECC activated leukocytes, with PMN and monocyte CD11b levels increasing at 90 minutes to 134% ± 9% (Fig 1) and 179% ± 19% (Table 1) of baseline, respectively (p < 0.04 for both), similar to previous studies [12]. The C5aR antagonist had divergent effects; PMN CD11b upregulation during SECC was abrogated, reaching only 93% ± 10% of baseline (p = 0.003 compared with control; Fig 1), whereas monocyte CD11b showed only a trend toward inhibition with C5aR antagonist during SECC (p = 0.09; Table 1).


Figure 1
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  		Fig 1. Leukocyte CD11b upregulation during simulated extracorporeal circulation. CD11b expression was measured on neutrophils (PMN) at the time points shown (expressed as a percentage of baseline). The mean ± standard error of the mean from five experiments performed after addition of the C5aR antagonist ({diamondsuit}) and four control experiments using diluent ({blacktriangleup}) are shown. The C5aR antagonist significantly inhibited neutrophil CD11b upregulation (p = 0.003).

 

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  		Table 1. Effects of Simulated Extracorporeal Circulation on Leukocytes
 
Preservation of Circulating Leukocytes by the C5a Receptor Antagonist
Control SECC showed PMN clearance by the circuit, with circulating PMN decreasing to 57% ± 5% of baseline (p= 0.01; Table 1) at 90 minutes; monocyte clearance was even more dramatic, with the monocyte nadir at 36% ± 11% of baseline (p < 0.01; Table 1). The C5aR antagonist significantly blunted clearance of both circulating PMN (p = 0.01; Table 1), preserving counts at 72% of baseline, and monocytes (p = 0.04; Table 1), with nadir levels at 75% of baseline.

Effects of the C5a Receptor Antagonist on Leukocyte Secretion
Neutrophil elastase secretion was induced by control SECC, with plasma levels increasing to 1498% ± 1004% of baseline at 90 minutes (p < 0.01; Table 2). Interleukin 8 elaboration was similarly upregulated; IL-8 levels were undetectable (<8 pg/mL) at the start of control SECC, but reached 72.5 ± 11.0 pg/mL after 90 minutes (p = 0.005; Table 2). In contrast to CD11b, neutrophil elastase release was not inhibited by the C5aR antagonist, with levels rising comparably to control SECC, reaching 2115% ± 1467% of baseline (p = 0.7; Table 2). However, the increase in IL-8 was eliminated by the C5aR antagonist, with IL-8 levels remaining undetectable out to 90 minutes (p = 0.002; Table 2).


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  		Table 2. Effect of C5a Receptor Antagonist on Leukocyte Secretion, Platelets, and Complement Activation a
 
Effect of C5a Receptor Antagonist on Leukocyte–Platelet Conjugates
During control SECC, the percentage of circulating PMN and monocytes with bound platelets, a sensitive marker of ongoing platelet activation [16], increased significantly, reaching 277% ± 54% and 232% ± 43% of baseline, respectively (p = 0.01 for both; Fig 2). Addition of the C5aR antagonist to SECC abrogated the increase in PMN–platelet conjugates (96% ± 10%; Fig 2A), and significantly blunted the increase in monocyte–platelet conjugates (119%± 13%; Fig 2B; p < 0.01 for both).


Figure 2
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  		Fig 2. Leukocyte–platelet conjugates during simulated extracorporeal circulation. The percentage of neutrophil (PMN)–platelet (A) and monocyte–platelet (B) conjugates is expressed as a percentage of baseline. The mean ± standard error of the mean from five experiments performed after addition of the C5aR antagonist ({diamondsuit}) and four control experiments using diluent ({blacktriangleup}) are shown. The C5aR antagonist significantly inhibited both neutrophil–platelet (A) and monocyte–platelet conjugate formation (B; p < 0.001 for both).

 
Effect of C5a Receptor Antagonist on Platelets
During control SECC, the percentage of circulating CD62P+ platelets and total platelet number did not change significantly with time (p = 0.58 and p = 0.34, respectively), nor was there an increase in plasma P-selectin levels (p = 0.4). Addition of the C5aR antagonist did not significantly change any platelet variable compared with control (Table 2).

Complement Activation
Control SECC resulted in significant complement activation, with C3a and sC5b-9 rising to 1,737% ± 958% (p = 0.005) and 7,369% ± 5,156% (p = 0.03) of baseline, respectively. The C5aR antagonist did not block either C3a generation or sC5b-9 formation (Table 2).


   Comment
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
Acknowledgments
 References
 
Blockade of the C5aR during SECC successfully abrogated PMN CD11b upregulation despite uninhibited generation of C3a and C5b-9. By contrast, monocyte CD11b upregulation was only partially blocked by C5aR blockade, consistent with findings [6] that C5a is not the sole activator of monocyte integrins during SECC. These results establish that receptor blockade, rather than complement generation inhibition, is a viable approach to future studies, and they demonstrate additional significant findings. Despite inhibition of PMN CD11b upregulation, C5aR antagonism did not decrease PMN elastase release, suggesting differential regulation as has been noted in the dialysis setting [17]. Similar to a study of artificial surface–induced leukocyte activation [18], C5aR blockade abrogated IL-8 secretion, suggesting that release of this preformed cytokine during SECC is mediated through C5aR. Despite the weak effect of C5aR antagonism on monocyte integrins, clearance of both monocytes and PMN was blunted, as was formation of both PMN–platelet and monocyte–platelet conjugates during SECC. This latter finding is distinct from that of Lappegard and colleagues [19], who found that only PMN-platelet conjugates were decreased by a C5aR inhibitor. This discrepancy may be attributable to their different SECC system, which excluded the membrane oxygenator and used lepirudin as the anticoagulant.

Activation of PMN integrins by C5a during CPB is associated with organ injury [20]; CD11b-mediated PMN adhesion is explicitly linked [21] to post-CPB pulmonary dysfunction and cardiac reperfusion injury [22]. The selective ability of the C5aR antagonist to abrogate PMN CD11b upregulation during SECC, while preserving C5b-9 formation, may be advantageous, especially in the setting of organ transplantation, in which blockade of complement-mediated graft injury [23] would be coupled with preserved antimicrobial activity. Abrogation of IL-8 release by the C5aR antagonist might also confer a clinical benefit because of the association between IL-8 and adverse post-CPB cardiopulmonary outcomes [24, 25]. Glucocorticoids [26] have been successful at blunting IL-8 release during SECC; C5aR blockade may have the same effect without broad immunosuppression or hyperglycemia.

In contrast to PMN CD11b, the C5aR antagonist did not block PMN elastase release induced by SECC. Preservation of elastase release may also be an advantage of the C5aR antagonist. Although elastase can, under some circumstances, contribute to host injury, abrogation of elastase release in clinical trials was associated with increased mortality [27].

Two well-described physiologic processes may explain how PMN CD11b upregulation is prevented by the C5aR antagonist, while elastase release is unaffected. Complement inhibition at the level of C3 cleavage inhibits PMN elastase release during SECC [6, 28] and in vivo [29]. C3a may directly stimulate PMN elastase release [30], and PMN elastase is contained within azurophilic granules, which are differentially regulated from the tertiary granules responsible for CD11b mobilization to the cell surface [31]. Dialysis studies have confirmed that late complement activation and neutrophil elastase release are not linked [32]. Thus, PMN elastase release may be attributed to C3a through a pathway distinct from C5a, the latter critical for CD11b upregulation.

An alternative explanation for differential regulation of elastase release and CD11b is suggested by Wachtfogel and associates [33], namely that kallikrein formation during SECC induces PMN elastase release but does not regulate CD11b. Formed kallikrein and C1 are both bound to C1 inhibitor, with the C1–C1 inhibitor complex outcompeting kallikrein–C1 inhibitor formation by tenfold. Complement inhibition at C3a, by diminishing production of activated complement components capable of binding to C1 inhibitor, frees up more C1 inhibitor to block kallikrein activity, thereby diminishing kallikrein-induced elastase release. By contrast, when complement inhibition targets only the C5a receptor, early complement components are free to bind C1 inhibitor, leaving sufficient unbound kallikrein to stimulate PMN elastase release. Thus, both kallikrein and C3a may contribute to elastase release despite C5aR blockade during SECC.

Monocytes during SECC react strongly to C3a [34] and upregulate surface CD11b, whereas C5a drives monocytes to bind and migrate into tissues, a process that includes CD11b [35] Thus, it is not surprising that addition of the C5aR antagonist to SECC did not completely block monocyte CD11b upregulation. Alternatively, it is possible that higher doses of the C5aR antagonist would have provided additional protection and enabled monocyte CD11b downregulation by the C5aR antagonist to reach statistical significance. A second caveat is that a greater number of repetitions of both control and C5aR antagonist–treated SECC might have shown significance by obviating type 2 error. Still, previous work strongly suggests that complete blockade of monocyte CD11b upregulation is best achieved by interventions that target both C3a and C5a [6, 7]. By contrast, monocyte IL-8 production is known to be primarily affected by C5a [18], and the C5aR antagonist abrogated IL-8 production. Thus, our findings suggest divergent anaphylatoxin effects on monocyte ß2 integrin regulation (C3a and C5a) versus cytokine production (C5a).

The C5aR antagonist abrogated formation of leukocyte–platelet conjugates during SECC. Graded platelet activation during unstable angina [36] or CPB [5] is demonstrated by conjugate increases [16]. In the cardiac setting, leukocyte–platelet aggregates are associated with poor outcome [36]; SECC uniformly increases these conjugates, which are dependent on binding of platelet P-selectin to leukocyte P-selectin glycoprotein ligand-1. Elastase cleaves P-selectin glycoprotein ligand-1 on unactivated PMN, rendering the PMN unable to bind P-selectin. By contrast, activated leukocytes resist cleavage [37]; this may occur through C5a-dependent P-selectin glycoprotein ligand-1 clustering [38] and increased surface CD11b binding of elastase [39]. During SECC, the C5aR antagonist blunted leukocyte stimulation by C5a, but elastase release was uninhibited. Hence, this may result in less P-selectin glycoprotein ligand-1 clustering, and with fewer CD11b surface receptors, P-selectin glycoprotein ligand-1 would be more vulnerable to elastase, resulting in less P-selectin binding to PMN and fewer conjugates [37]. The vulnerability of monocyte P-selectin glycoprotein ligand-1 to elastase cleavage has not been studied, nor is the role of monocyte CD11b upregulation in protecting P-selectin glycoprotein ligand-1 known. Because monocytes showed some inhibition of CD11b upregulation to the C5aR antagonist, monocyte P-selectin glycoprotein ligand-1 may be somewhat vulnerable to elastase-mediated cleavage. Given the recently identified role of P-selectin and P-selectin glycoprotein ligand-1binding in thrombosis, stroke, and myocardial infarction [40], abrogation of conjugate formation during cardiac surgery may be desirable.

Adhesion of leukocytes to the circuit is dependent on leukocyte–biomaterial interaction and adhesion of leukocytes to platelets that have attached to the passivated biomaterial [41]. Production of leukocyte–platelet conjugates is a surrogate for the latter, and hence the reduction in circulating conjugates may account for the majority of the reduced leukocyte loss to the circuit. CD11b is also important for leukocyte–biomaterial interaction [42], and reduction of PMN CD11b upregulation likely contributes to preservation of circulating PMN. However, preservation of monocyte numbers, despite incomplete inhibition of CD11b upregulation, suggests that binding of activated platelets to monocytes contributes to monocyte clearance by the circuit.

In conclusion, C5aR blockade reveals a possible divergence between neutrophil (elastase release versus integrin upregulation and IL-8 release) and monocyte (integrin upregulation versus IL-8 release) activation pathways during SECC. This blockade, possibly through these divergent effects, is efficacious at inhibiting leukocyte–platelet aggregate formation and loss of inflammatory cells to the CPB circuit.


   Acknowledgments
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
 Acknowledgments
References
 
This work was supported by the National Institutes of Health HL-47193. The C5aR antagonist was supplied by Neurogen Corporation. The authors thank Robin Meade and A.P. Kieltyka for technical assistance.


   References
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
 Acknowledgments
 References
 

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Knut Tore Lappegard
Ann. Thorac. Surg. 2007 83: 152. [Extract] [Full Text] [PDF]



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Ann. Thorac. Surg., January 1, 2007; 83(1): 152 - 152.
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