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Originally published online as doi:10.1189/jlb.0906545 on August 3, 2007

Published online before print August 3, 2007
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(Journal of Leukocyte Biology. 2007;82:1115-1125.)
© 2007 by Society for Leukocyte Biology

Conformational activation of CD11b without shedding of L-selectin on circulating human neutrophils

Y. Orr*,{dagger}, J. M. Taylor*, S. Cartland*, P. G. Bannon{dagger},{ddagger}, C. Geczy§ and L. Kritharides*,||,¶,1

* Centre for Vascular Research and
§ Inflammatory Diseases Research Unit, School of Medical Sciences, The University of New South Wales, Kensington, NSW, Australia;
{ddagger} Department of Cardiothoracic Surgery, Royal Prince Alfred Hospital, NSW, Australia;
{dagger} The Baird Institute for Applied Heart and Lung Surgical Research, NSW, Australia;
|| The Heart Research Institute, NSW, Australia; and
Department of Cardiology, Concord Repatriation General Hospital, NSW, Australia

1 Correspondence: Centre for Vascular Research, School of Medical Sciences, 4th Floor Wallace Wurth Building, The University of New South Wales, Kensington, NSW, 2052, Australia. E-mail: l.kritharides{at}unsw.edu.au


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSION
 REFERENCES
 
Membrane-activated complex 1 (Mac-1; CD11b/CD18) is a β2 integrin implicated in the pathophysiology of neutrophil-mediated tissue injury whose functional capacity is determined by stimulus-induced conformational activation rather than up-regulation. Mac-1 up-regulation and conformational activation, together with shedding of L-selectin, are reported after in vitro neutrophil activation. However, their regulation on circulating human neutrophils during acute inflammation is unclear. Using flow cytometry, we investigated neutrophil expression of Mac-1, its activation-reporter neo-epitope CBRM1/5, and L-selectin during the inflammatory stimulus of cardiac surgery. A subpopulation of circulating neutrophils expressed CBRM1/5 (CBRM1/5+) under basal conditions (6.28±2.59%) and was persistently expanded (9.95±4.0%–15.2±4.2%; P<0.0001) peri-operatively, whereas total CD11b expression increased only transiently, intra-operatively. L-selectin expression was unchanged on CBRM1/5+ neutrophils, and soluble L-selectin levels decreased intra-operatively (P<0.01), indicating that L-selectin was not shed. Increased CBRM1/5 expression without L-selectin loss or CD11b up-regulation was replicated in vitro by neutrophil stimulation with IL-8, C3a, and platelet-activating factor. Heparin, a known CD11b ligand, which is administered during cardiac surgery, markedly reduced neutrophil expression of conformationally active CD11b in vivo and in vitro, identifying a potential mechanism for its anti-inflammatory properties. We conclude that conformational activation of CD11b occurs on circulating neutrophils in vivo and can occur in the absence of CD11b up-regulation and L-selectin shedding.

Key Words: inflammation • adhesion molecules • integrins • heparin


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSION
 REFERENCES
 
Neutrophil function is dependent on expression of an array of adhesion molecules, which enable interaction with endothelial and microbial ligands. L-selectin mediates neutrophil tethering and rolling on endothelium, whereas the β2 integrins LFA-1 (CD11a/CD18) and membrane-activated complex 1 (Mac-1; CD11b/CD18) mediate firm adhesion and arrest of neutrophils [1 ]. L-selectin and the β2 integrins are essential for the normal function of circulating neutrophils, as evidenced by the persistent neutrophilia, defective phagocytosis, and impaired endothelial adhesion in patients with an inherited deficiency of β2-integrin expression [2 ] or selectin function [3 ]. Ligation of CD11b activates multiple neutrophil effector functions, including transendothelial migration, homotypic aggregation, degranulation, phagocytosis, superoxide production, and the respiratory burst [4 , 5 ]. Elevated neutrophil CD11b levels and activation status positively correlate with indicators of disease severity [6 ] and adverse outcomes [7 , 8 ] in human acute inflammatory states. Inhibition of Mac-1 function in animal models supports a causative role in neutrophil-mediated inadvertent tissue injury associated with ischemia-reperfusion [9 , 10 ] and systemic complement activation [11 , 12 ]. However anti-Mac-1 strategies have consistently failed to improve outcome in humans [13 14 15 ], indicating that the targeted Mac-1 epitopes are not involved in neutrophil-mediated tissue injury.

Constitutively expressed Mac-1 cannot mediate efficient adhesion or effector functions [16 ], consistent with the quiescent, nonadherent state of circulating neutrophils. Stimulus-induced up-regulation can increase CD11b expression by five- to tenfold [17 , 18 ] but is not required for Mac-1-mediated adhesion [19 , 20 ], indicating that qualitative changes may be more relevant to CD11b functional capacity than total cell surface expression. Neutrophil stimulation induces a conformational change within the N-terminal ligand-binding I domain of CD11b, which improves adhesion competence [21 , 22 ] and facilitates subsequent effector functions [23 ]. This conformational activation of CD11b reveals a neo-epitope within the I domain known as CBRM1/5, expression of which facilitates Mac-1 functional capacity [21 ] and may provide a more functionally relevant measure of circulating neutrophil activation status in vivo than total CD11b levels. Although up-regulation of CD11b expression and L-selectin shedding typically accompany CD11b conformational activation in vitro [24 ], little is known about the regulation of CD11b conformational activation in vivo. The potential to regulate conformationally active CD11b may provide a more specific strategy to therapeutically modify neutrophil responses during acute inflammatory states, similar to that achieved with platelet GPIIb/IIIa antagonists in acute coronary syndromes [25 ].

Cardiac surgery induces a highly proinflammatory, intravascular environment [26 , 27 ], which predictably induces tissue injury and myocardial ischemia/reperfusion. It thereby provides a useful model to investigate the regulation of CD11b conformational activation on circulating neutrophils during acute inflammation in vivo. Using whole blood flow cytometry, we identified a subpopulation of circulating neutrophils expressing the CD11b I domain activation-reporter neo-epitope CBRM1/5 in association with preserved L-selectin expression (CBRM1/5+/L-selectin+). This subpopulation was increased during cardiac surgery, reduced in the presence of heparin, and could be replicated by neutrophil stimulation in vitro.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 CONCLUSION
 REFERENCES
 
Fluorochrome-conjugated mAb
Anti-human CD11b mAb were CBRM1/5 (mouse IgG1K)-PE (eBioscience, San Diego, CA, USA); D12 (mouse IgG2a)-PE [BD Biosciences Immunocytometry Systems (BDIS), San Jose, CA, USA]; VIM12 (mouse IgG1K)-FITC (Caltag Laboratories, S. San Francisco, CA, USA); and 2LPM19c (mouse IgG1K)-PE (DakoCytomation, Sydney, Australia). The 2LPm19c mAb binds an epitope within the N-terminal I domain, which is present on all CD11b molecules and is distinct from that recognized by CBRM1/5, which binds a neo-epitope expressed only by a subpopulation of conformationally and functionally active CD11b molecules [21 ]. Similar to 2LPm19c, D12 and VIM12 mAb bind constitutively expressed epitopes within N- and C-terminal regions of CD11b, respectively. Other mAb were anti-human CD18 (6.7, mouse IgG1K)-PE, L-selectin [CD62 ligand (CD62L); Dreg-56, mouse IgG1K]-Cychrome (CyC), and IgG1K isotype control (MOPC-21)-PE, -FITC, and -CyC (all from BD Biosciences PharMingen, San Diego, CA, USA).

Reagents
Anticoagulants used were K3 EDTA and citrate-theophylline-adenosine-dipyradimole (CTAD) in sterile vacutainers (Becton Dickinson, Franklin Lakes, NJ, USA). The fluorescent nuclear dye Hoechst 33342 (Molecular Probes, Eugene OR, USA), 50 µM in 0.9% NaCl, was used to label leukocytes in whole blood. HBSS (Sigma Chemical Co., St. Louis, MO, USA) with 10 mM HEPES (Sigma Chemical Co.), 0.5% BSA (Sigma Chemical Co.), and 1 mM sodium azide (NaN3; HHBSS-BSA-N; Sigma Chemical Co.) was used to dilute samples. NaN3, 1 mM in 0.9% NaCl (Baxter Healthcare, Sydney, Australia), was used during antibody incubation. IL-8 in PBS/0.25% BSA and complement C3a in PBS (Calbiochem, Darmstadt, Germany); platelet-activating factor (PAF; β-acetyl-{gamma}-O-alkyl-L-{alpha}-phosphatidylcholine) in DMSO, diluted in 0.15 M NaCl/0.25% BSA; histamine and ATP, both in sterile water; and fMLF (all from Sigma Chemical Co.) and PF4 in PBS/0.25% BSA (R&D Systems, Minneapolis, MN, USA), were used for neutrophil stimulation. Unfractionated porcine heparin (Pharmacia, Perth, Australia) for analysis of the effect of heparin on adhesion molecule expression in vitro. All buffers and reagents were Zetapore (Cuno Filter Systems, Meriden, CT, USA)-filtered for sterilization and to minimize LPS contamination.

Patients
Patients, aged 40–80 years (Table 1A ), undergoing elective primary coronary artery bypass surgery (CABG) for chronic stable angina pectoris, were prospectively recruited from the Cardiothoracic Surgical Unit of Royal Prince Alfred Hospital (Sydney, Australia). Written, informed consent was obtained from all patients, and the study was approved by the institutional ethics committee.


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  		Table 1A. Patient Demographic Data

 
Surgical procedure
CABG with hypothermic cardiopulmonary bypass (CPB) was performed as described previously [28 ]. CPB was established after an i.v. dose of 400 U/kg, equivalent to ~5.7 U/ml blood vol unfractionated porcine heparin (Pharmacia), and was administered to maintain an activated clotting time greater than 350 s. The CPB circuit was primed with 2.5 L crystalloid and 10,000 U unfractionated porcine heparin (Pharmacia). At the completion of grafting, patients were weaned from CPB, and protamine sulfate (1 mg/100 U administered heparin) was given. Operative details and clinical outcome data are detailed in Table 1B .


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  		Table 1B. Operative and Outcome Data

 
Blood (9 ml) was collected from the radial arterial line of n = 16 CABG patients at the following stages (Table 1C ): 1, Radial arterial line insertion prior to anesthetic induction; 2, after sternotomy and conduit harvesting, pre-heparin administration; 3, 10 min post-heparin, prior to institution of CPB; 4, during myocardial ischemia and CPB, just before aortic cross-clamp (AXC) release; 5, 15–20 min after myocardial reperfusion (AXC release); 6, 6 h postoperatively; and 7, 24 h postoperatively. Samples (1 ml) were anticoagulated with EDTA and CTAD, loaded with 50 µM Hoechst 33342 at 30°C for 10 min, and then placed on ice in the dark, and cells were labeled for flow cytometry. Residual blood (8 ml) from each sample was anticoagulated with EDTA and placed immediately on ice, and the plasma fraction was isolated by centrifugation (3000 rpm for 20 min at 4°C) and then stored at –80°C until batch analysis of soluble L-selectin (sL-selectin) levels.


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  		Table 1C. Sampling Stages

 
Neutrophil activation in whole blood in vitro
In preliminary experiments, neutrophils in samples collected by 21 G needle venepuncture were markedly activated; the proportion of CBRM1/5+ neutrophils increased 15.8 ± 2.7-fold compared with samples aspirated from 20 G radial arterial lines (data not shown). To avoid this, in vitro studies were performed exclusively on blood samples collected via a 20-G indwelling radial artery cannula from a separate cohort of elective cardiac surgery patients (n=8) preoperatively. Samples were anticoagulated with EDTA and CTAD, and 1 ml aliquots were dispensed into prewarmed (30°C), sterile glass vacutainers containing 50 µM Hoechst 33342, with each agonist in duplicate for simultaneous Hoechst loading and stimulation of leukocytes. The agonists tested were IL-8 (10 ng/ml), C3a (5 µg/ml), PF4 (1 µg/ml), histamine (5 µg/ml), PAF (10 ng/ml), ATP (5 µM), and PBS/0.25% BSA (40 µl) as the negative control. Samples stimulated in the absence of heparin were incubated for 10 min at 30°C in the dark and then placed on ice and subsequently labeled for flow cytometry. Heparin-exposed samples were incubated with agonists for 5 min at 30°C in the dark, and then 10 U/ml unfractionated porcine heparin (Pharmacia) was added, and incubation continued for a total of 10 min. Samples were then placed on ice immediately, maintained in the dark, and labeled for flow cytometry.

Flow cytometry
Antibody labeling of whole blood was a modification of a previously published method [29 ]. Aliquots (5 µL) of Hoechst-labeled blood were incubated with 1 µL each mAb (predetermined saturating concentrations) in 1 mM NaN3 on ice in the dark for 15 min and then diluted to 1 ml with HHBSS-BSA-N and stored at 4°C in the dark for up to 6 h. Labeling of live neutrophils in whole blood within 60 min of sampling, without isolation, erythrocyte lysis, or fixation was performed to minimize potential ex vivo alteration of the cell surface membrane components measured [30 , 31 ].

Flow cytometry was performed using an LSR bench-top flow cytometer (BDIS); Cell Quest Pro software (BDIS, Version 4.0.2) was used to acquire and store data files. The validity of data over time was confirmed by daily calibration of the flow cytometer using Calibrite Rainbow beads (BDIS). A total of 1–1.5 x 106 events per sample was acquired, and leukocytes, identified by positive Hoechst 33342 fluorescence, and neutrophil events within the leukocyte gate, identified on the basis of their characteristic forward- and side-angle light-scatter, were analyzed for mAb-related fluorescence. This granulocyte-gating strategy was based on a previously validated technique for identification of neutrophils in whole blood flow cytometry samples [29 , 32 ]. The percentage of neutrophil events with positive mAb fluorescence was determined for each sample (isotype controls defined parameters for negative fluorescence), and logarithmically acquired data for positive populations were linearly converted to mean fluorescence intensity (MFI).

Full blood counts
Differential full blood counts on EDTA-CTAD-anticoagulated blood from CABG patients were performed with a Sysmex SF-3000 (Roche Diagnostics, Sydney, Australia) automatic full blood-count analyzer. Neutrophil counts after baseline were corrected for hemodilution using the formula: (baseline hematocrit/observed hematocrit) x observed neutrophil count = corrected count.

Neutrophil adhesion assay
Neutrophil adhesion to the CD11b I domain ligand fibrinogen was assayed using a dilute whole blood technique. Sterile, 24-well tissue-culture plates precoated overnight with fibrinogen (45 µg/well in PBS, Sigma Chemical Co.) at 4°C were blocked for 1 h with 1% BSA (Sigma Chemical Co.). Blood was collected from healthy volunteers (n=3) by modified venepuncture (drip collection using 21 G butterfly needle and tourniquet) to minimize CD11b activation, anticoagulated with EDTA, and stimulated with PAF (10 ng/ml) or an equal volume of DMSO (Sigma Chemical Co.; negative control) ± heparin (100 U/ml) for 10 min at 37°C. Samples were diluted 1:100 with HHBSS, 0.5 ml aliquots were placed into fibrinogen-coated wells and incubated for 1 h at 37°C and then washed five times with HHBSS and stained with Diff Quick, and adherent neutrophils, identified by their characteristic staining and morphology, were counted.

Plasma sL-selectin levels
Plasma sL-selectin levels were analyzed at sample stages 1–7 (n=16 patients) using a commercial EMSA (ELISA) kit (R&D Systems), according to the manufacturer’s instructions. sL-selectin levels, obtained from samples after baseline, were corrected for hemodilution using the formula: (baseline hematocrit/observed hematocrit) x observed sL-selectin level = corrected sL-selectin level. Standard curves and sample concentrations were determined in duplicate using SoftMax Pro (Version 4.0, Molecular Devices, VIC, Australia) software.

Statistical analysis
Results were analyzed using Prism statistical software (GraphPad Prism Version 4.00 for Windows, GraphPad Software, San Diego, CA, USA). Pair-wise comparisons were performed using Wilcoxon signed rank test or paired t-tests as appropriate. Multiple comparisons were performed using Friedman test with Dunn’s post-test or one-way repeated measures ANOVA with Tukey’s post-test as appropriate. Statistical significance was defined as P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 CONCLUSION
 REFERENCES
 
Total Mac-1 and conformationally active CD11b are differentially expressed on circulating neutrophils
Neutrophils were identified by the combination of positive Hoechst 33342 fluorescence (Fig. 1A ) and their characteristic forward- and side-angle light-scatter (Fig. 1A and 1B) . CBRM1/5 expression was analyzed in combination with L-selectin, shedding of which is a conventional marker of neutrophil activation [33 ] (Fig. 1C and 1E) . At baseline, a small proportion of circulating neutrophils (6.28±2.59%; mean±SEM, n=16) bound the CBRM1/5 mAb (CBRM1/5+, Figs. 1C and 1D , and 2A ). The proportion of CBRM1/5+ neutrophils increased slightly, although not significantly, with surgical trauma alone (Fig. 2A) and decreased with systemic heparin administration (discussed below). Subsequently, during CPB/myocardial ischemia, the proportion of circulating neutrophils, which were CBRM1/5+, increased significantly (P<0.0001) to a maximum of 15.2 ± 4.2% (Figs. 1E and 1F and 2A) and remained persistently elevated relative to post-heparin values during reperfusion and postoperatively (Fig. 2A) .


Figure 1
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  		Figure 1. A subpopulation of circulating neutrophils expresses conformationally active CD11b during basal and acute inflammatory conditions in vivo. Flow cytometry was used to determine neutrophil expression of the CD11b activation-specific neo-epitope CBRM1/5 in combination with L-selectin in patients undergoing cardiac surgery. (A–C and E) Dot-plots from a representative patient demonstrate total leukocyte events with neutrophils gated (A, R1); characteristic forward- and side-angle light-scatter of gated neutrophils (B, R2); neutrophil CBRM1/5 (y-axis) and L-selectin (CD62L, x-axis) expression at baseline (C) and during CPB at peak myocardial ischemia (E). Events in the upper-right quadrant represent CBRM1/5+ neutrophils at each stage. (D and F) Histograms from the same representative patient demonstrate neutrophil CBRM1/5-PE expression (bold lines) relative to that of the PE-conjugated isotype control mAb (dashed lines) at baseline (D) and during peak myocardial ischemia on CPB (F). (F) Light line represents CBRM1/5 expression at baseline.

 

Figure 2
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  		Figure 2. CBRM1/5+ and Mac-1+ neutrophil populations are influenced differentially by cardiac surgery. The proportion of circulating neutrophils expressing the CBRM1/5 neo-epitope (%CBRM1/5+), CD11b, and CD18 at baseline and in response to CABG was determined by flow cytometry at sample stages 1–7 (Table 1C) . Data represent mean ± SEM %CBRM1/5+ (A), %CD11b+ (B), and %CD18+ (C) neutrophils for n = 16 patients. ***, P < 0.001; **, P < 0.01; *, P < 0.05, versus post-heparin. PMNs, Polymorphonuclear neutrophils.

 
Total CD11b expression was analyzed using mAb directed against the N (D12)- and C (VIM12)-terminal domains, as heparin-binding to the CD11b I domain [34 ] may obscure N-terminal epitopes. In contrast to CBRM1/5 neo-epitope expression, CD11b and CD18 were expressed by >99.4% of circulating neutrophils at all operative stages (Fig. 2B and 2C) . The quantity of CD11b (CD11b MFI) on these Mac-1+ neutrophils increased mildly and transiently during CPB/myocardial ischemia (D12 mAb: 0.9±0.2-fold, Fig. 3B and 3E ; VIM12 mAb: 0.7±0.2-fold, Fig. 3C and 3F ) and reperfusion (VIM12 only), relative to post-heparin values. The intensity of CBRM1/5 expression on CBRM1/5+ neutrophils (in those patients, n=14, who had detectable CBRM1/5+ neutrophils at all sample stages) was similarly up-regulated during CPB/myocardial ischemia and reperfusion relative to baseline and to post-heparin samples (Figs. 1F and 3A) . CD18 levels on Mac-1+ neutrophils increased slightly, although not significantly, intra-operatively (Fig. 3D and 3G) , coincident with the rise in CD11b. These results indicated that the relative size of the CBRM1/5+ neutrophil subpopulation was more sensitive to the stimulatory effects of cardiac surgery than total Mac-1 expression.


Figure 3
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  		Figure 3. Cardiac surgery induces similar up-regulation of CBRM1/5 and Mac-1 on the respective neutrophil populations. (A) CBRM1/5 MFI was determined on CBRM1/5+ neutrophils at sample stages 1–7 (Table 1C) . Data represent mean ± SEM CBRM1/5 MFI for n = 14 CABG patients who had detectable CBRM1/5+ neutrophils at all sample stages; **, P < 0.01, versus baseline; ###, P < 0.001; #, P < 0.05, versus post-heparinzation. (B–D) Neutrophil CD11b (B and C) and CD18 (D) expression was determined by flow cytometry on blood collected from CABG patients (n=16) at sample stages 1–7. CD11b expression was determined using mAb to the N (B, D12)- and C (C, VIM12)-terminal domains. Data represent mean ± SEM CD11b (B and C) or CD18 (D) MFI; ***, P < 0.001; **, P < 0.01; *, P < 0.05, versus post-heparin. (E–G) Flow cytometry histograms from a representative patient demonstrate neutrophil CD11b (E, D12 mAb; F, VIM12 mAb) and CD18 (G) mAb-binding at baseline (light lines) and during peak myocardial ischemia/CPB (bold lines); dashed lines are the isotype control-related fluorescence.

 
Systemic heparinization decreases the proportion of neutrophils expressing the CBRM1/5 neo-epitope
Heparin binds the CD11b I domain on activated but not resting neutrophils [34 ], raising the possibility that the active conformation of the I domain is involved. CBRM1/5, total CD11b, CD18, and L-selectin expression was assessed immediately prior to and 10 min following administration of 400 U/kg heparin (i.e., pair-wise analysis of Samples 2 and 3).

The proportion of circulating neutrophils expressing the CBRM1/5 neo-epitope decreased markedly (4.1±0.9-fold) following systemic heparinization (Fig. 4A and 4B ), whereas proportions of CD11b+ and CD18+ neutrophils (≥99.4%) were unaffected (data not shown). The intensity of CBRM1/5 expression on residual CBRM1/5+ neutrophils was unexpectedly not affected by heparin (Fig. 4C) . In contrast, the intensity of CD11b expression on Mac-1+ neutrophils was reduced mildly by heparin; N- terminal mAb-binding decreased by 14.9 ± 4.5% (Fig. 4D and 4E) and C-terminal mAb binding, by 17.1 ± 2.6% (Fig. 4F and 4G) . CD18 (Fig. 4H and 4I) and L-selectin (Fig. 4J and 4K) levels were unaltered by heparin administration.


Figure 4
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  		Figure 4. Systemic heparinization decreases the proportion of neutrophils exhibiting CBRM1/5 neo-epitope expression. The %CBRM1/5+ neutrophils (A and B), CBRM1/5 MFI on CBRM1/5+ neutrophils (C), N (D12, D and E)- and C (VIM12, F and G)-terminal domain total CD11b, CD18 (H and I), and L-selectin (J and K) expression were assessed on neutrophils in blood collected from CABG patients (n=16) pre- and post-heparin administration using flow cytometry. Data represent mean ± SEM %CBRM1/5+ neutrophils (A), MFI for mAb binding (C, n=14 patients), and (D, F, H, J, n=16 patients) or flow cytometry histograms for pre (light lines)- and post (bold lines)-heparin samples from a single representative patient (B, E, G, I, K, dashed lines represent isotype control-related fluorescence; *, P < 0.05; **, P < 0.01; ***, P < 0.001, for pre-heparin versus post-heparin. (Data for CBRM1/5 MFI represent n=14 CABG patients with detectable CBRM1/5+ neutrophils at all sample stages.)

 
L-selectin is preserved on circulating neutrophils during increased CBRM1/5 expression
Neutrophil L-selectin expression was maintained intra-operatively but decreased significantly early postoperatively (Fig. 5A ). L-selectin levels were similar on CBRM1/5+ and CBRM1/5– neutrophils and remained constant on the CBRM1/5+ subpopulation intra-operatively (Fig. 5A) . L-selectin increased during CPB/myocardial ischemia and reperfusion on the CBRM1/5– subpopulation (P<0.001 relative to baseline for CBRM1/5– neutrophils, Fig. 5A ) and tended to be higher on CBRM1/5– neutrophils at this stage (Fig. 5A and 5B) . Plasma sL-selectin levels decreased during CPB/myocardial ischemia and reperfusion in all patients and subsequently increased nonsignificantly during the early, postoperative phase (Fig. 5C) . Thus, under conditions of peak CBRM1/5 expression, there was no evidence of L-selectin shedding.


Figure 5
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  		Figure 5. L-selectin was not shed during cardiac surgery. (A) L-selectin MFI was analyzed separately on CBRM1/5+ (•) and CBRM1/5– ({blacktriangleup}) neutrophils at sample stages 1–7. Data represent mean ± SEM MFI for n = 14 CABG patients with detectable CBRM1/5+ neutrophils at all stages; ***, P < 0.001, versus baseline; ###, P < 0.001; #, P < 0.05, versus post-heparin and CPB/myocardial ischemia samples. L-selectin expression on CBRM1/5+ versus CBRM1/5– neutrophils was not significantly different (P=0.19, two-way repeated measures ANOVA). (B) Flow cytometry histogram from a representative patient illustrating L-selectin expression on CBRM1/5+ (bold line) and CBRM1/5– (light line) neutrophils during myocardial ischemia/CPB. The dashed line represents the isotype control mAb. (C) Plasma sL-selectin levels were determined at sample stages 1–7 using a commercial ELISA kit and results after baseline corrected for hemodilution as described in Materials and Methods. Data represent mean ± SEM sL-selectin (ng/ml) for n = 16 patients; **, P < 0.01, versus baseline.

 
Stimulus-induced up-regulation of CBRM1/5 expression can occur independently of changes in total CD11b and L-selectin
Our in vivo studies unexpectedly indicated that neutrophil CBRM1/5 neo-epitope expression was compatible with concurrent L-selectin expression. We investigated CBRM1/5, total CD11b, CD18, and L-selectin expression during neutrophil stimulation in vitro to determine potential mediators of this response. Agonists studied (IL-8, C3a, PAF, histamine, ATP, and PF4) were chosen on the basis of their reported systemic release during cardiac surgery [26 , 35 36 37 ] and suspected direct or indirect effects on neutrophil function within the vascular space. Incubations were performed in whole blood at 30°C to replicate the hypothermic, intravascular environment during cardiac surgery; an incubation duration of 10 min was used, as CBRM1/5 is maximally up-regulated within this interval [21 ].

IL-8, C3a, and PAF significantly increased the proportion of CBRM1/5+ neutrophils and the intensity of neo-epitope expression on the CBRM1/5+ subpopulation relative to the negative control, whereas histamine and PF4 were ineffective (Table 2A and Fig. 6A 6B 6C ). In contrast, total CD11b expression did not increase under these conditions (Table 2B ). Whole blood incubation with histamine, ATP, and PF4 significantly reduced CD11b expression relative to unstimulated neutrophils, whereas the other agonists provoked a mild but nonsignificant increase in total CD11b (Fig. 6J 6K 6L) . CD18 expression increased in response to IL-8 and PAF (Table 2B) , whereas the other agonists induced only minimal changes. Unlike CBRM1/5, which was expressed only by a subpopulation of neutrophils, ≥99.5% of neutrophils were Mac-1+ and L-selectin+ for all conditions tested (Fig. 6J 6K 6L) ; L-selectin expression was unaffected by neutrophil stimulation at 30°C (Table 2C and Fig. 6 , A–C and J–L).


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  		Table 2A. Effect of Stimulation and Heparin on Neutrophil CBRM1/5 Neo-Epitope Expression

 

Figure 6
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  		Figure 6. Stimulus-induced up-regulation of CBRM1/5 expression can occur independently of changes in total CD11b and L-selectin. Neutrophils in whole blood were stimulated with agonists or PBS/0.25% BSA in the absence or presence of heparin (10 U/ml). CBRM1/5 neo-epitope (CBRM1/5 FI; A–F, y-axis; G–I, x-axis), total CD11b (CD11b FI; J–L, y-axis; M–O, x-axis), and L-selectin (CD62L FI; A–F and J–L, x-axis) expression was determined by flow cytometry, and flow cytometry dot-plots and histograms from one donor representative of results obtained in n = 8 demonstrate the response to PBS/0.25% BSA (A and J), IL-8 (B and K), and histamine (C and L) and the effect of heparin on detection of CBRM1/5+ events (D and G, PBS/0.25% BSA; E and H, IL-8; F and I, histamine) and CD11b expression (M, PBS/0.25% BSA; N, IL-8; O, histamine).

 

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  		Table 2B. Effect of Stimulation and Heparin on Neutrophil CD11b and CD18 Expression

 

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  		Table 2C. Effect of Stimulation and Heparin on Neutrophil L-Selectin Expression

 
Conventional neutrophil stimulation with fMLF at 37°C was performed to determine whether previously reported, simultaneous CD11b up-regulation and conformational activation associated with L-selectin shedding [24 ] could also be demonstrated. To replicate conventional sampling techniques, blood was collected via 21 G needle venepuncture from normal individuals (n=2) and neutrophils in whole blood exposed to fMLF (10–6 M) at 37°C for up to 20 min. The proportion of CBRM1/5+ neutrophils increased from 25.9 ± 1.2% to 94.3 ± 0.7% (Fig. 7A and 7B ), total CD11b increased almost fivefold (Fig. 7D and 7E) , and the proportion of L-selectin+ neutrophils decreased markedly from 99.95 ± 0.06% to 33.8 ± 17.6% (Fig. 7B and 7E) . No further up-regulation of CBRM1/5 or total CD11b expression or loss of L-selectin was evident at 20 min, indicating that maximal responses occurred within 10 min (data not shown).


Figure 7
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  		Figure 7. Neutrophil stimulation with fMLF at 37°C. Neutrophils in whole blood collected by 21 G venepuncture were incubated at 37°C with 10–6 M fMLF for 10 min in vitro (A, B, D, E) and then exposed to heparin (20 U/ml) for a further 10 min at 37°C (C and F). CBRM1/5 (A–C, y-axis), total CD11b (D–F, y-axis), and L-selectin (A–F, x-axis) expression was determined by flow cytometry. Dot-plots are from a single donor representative of n = 2; events in right-sided quadrants represent L-selectin+ neutrophils, whereas those in left-sided quadrants are L-selectin-negative.

 
Heparin reduces neutrophil expression of the CBRM1/5 neo-epitope
As systemic heparinization transiently reduced the proportion of CBRM1/5+ circulating neutrophils and the intensity of CD11b expression on Mac-1+ neutrophils in vivo, its effects on recognition of Mac-1 epitopes on neutrophils in vitro were investigated to assess if this were a direct effect of heparin. As it was not ethically feasible to include a control group of CABG patients not receiving heparin, as exposure to CPB strongly activates coagulation, we investigated the effect of heparin on CBRM1/5 expression in vitro under conditions similar to the intravascular environment during CPB. In an attempt to replicate the in vivo environment during CPB and based on previously documented concentrations of heparin required for binding to CD11b [34 ], a dose of 10 U/ml was used. To compare the effect of heparin on mAb binding to conformationally active versus the total CD11b I domain, the I domain-specific 2LPm19c mAb was used to detect total CD11b in vitro.

Irrespective of the stimulus, the proportion of neutrophils expressing the CBRM1/5 neo-epitope was decreased significantly by heparin; a similar, marked reduction was evident for unstimulated neutrophils or those activated by specific agonists (Table 2A and Fig. 6 , D–I). In samples incubated with IL-8, C3a, PAF, and histamine, heparin reduced the intensity of CBRM1/5 expression by 13.4 ± 1.9% on those neutrophils, which remained CBRM1/5+ (Table 2A) . Although ≥99.2% of neutrophils remained CD11b+ and CD18+ (data not shown), the intensity of CD11b expression was reduced by 17.2 ± 1.6% (Table 2B and Fig. 6 , M–O) and of CD18 by 14.8 ± 1.4% (Table 2B) on these Mac-1+ neutrophils. Heparin did not significantly reduce CBRM1/5, CD11b, or CD18 expression on neutrophils activated by fMLF (Fig. 7C and 7F) or ATP or CD18 levels induced by PF4 (Table 2A and 2B) . The proportion of CBRM1/5+ neutrophils induced by stimulation with fMLF (≥94%) was unaffected by heparin concentrations of up to 1000 U/ml (Table 2D ). L-selectin levels were unaffected by heparin, regardless of the stimulus (Table 2C) .


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  		Table 2D. Heparin Does Not Suppress CBRM1/5 Up-Regulation by fMLF

 
Stimulation with PAF promotes neutrophil adhesion
Neutrophil adhesion to fibrinogen, which is mediated by conformationally active CD11b [21 ], was analyzed to determine the effect of PAF and heparin on CD11b functional capacity. PAF significantly increased neutrophil adhesion to fibrinogen relative to basal levels (Table 2E ). Heparin significantly reduced PAF-stimulated neutrophil adhesion to fibrinogen but did not alter basal adhesion (Table 2E) .


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  		Table 2E. Effect of PAF and Heparin on Neutrophil Adhesion

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
CONCLUSION
 REFERENCES
 
We have shown for the first time that conformational activation of CD11b can occur without alterations in L-selectin expression on a subpopulation of circulating neutrophils under relatively quiescent conditions in vivo and is up-regulated by the acute inflammatory stimulus of cardiac surgery. As CBRM1/5 expression identifies functionally active CD11b [21 , 23 ], the CBRM1/5+/L-selectin+ neutrophil subpopulation may be an important target for anti-inflammatory strategies. Although total CD11b increased mildly and transiently on Mac-1+ neutrophils, the CBRM1/5+ subpopulation was substantially and persistently enlarged peri-operatively. This indicates that CD11b conformational activation on neutrophils and its translocation from intracellular reservoirs can be regulated independently on neutrophils in vivo and that CBRM1/5 neo-epitope expression is a more sensitive marker of circulating neutrophil activation than total Mac-1 levels. Our in vitro results implicate IL-8, PAF, and C3a in the pathophysiology of CBRM1/5 up-regulation during cardiac surgery and suggest that heparin may reduce detection of conformationally active CD11b.

Neutrophil CD11b expression is up-regulated by diverse systemic, inflammatory stimuli [38 39 40 ]. Although this correlates positively with indicators of disease severity [6 ] and adverse outcomes [7 , 8 ], increased CD11b expression does not necessarily equate to augmented CD11b-adhesive capacity [19 , 20 ], which is requisite for potentially injurious, neutrophil-endothelial interactions [41 ]. However, Mac-1 adhesion competence [21 ] and activation of Mac-1-dependent neutrophil effector functions [23 ] are facilitated by conformational activation of CD11b. Increased expression of conformationally active CD11b is seen on circulating leukocytes during systemic acute inflammatory states [8 , 42 , 43 ] and given its functional significance in vitro, may be more relevant to the pathophysiology of neutrophil-mediated tissue injury. Circulating monocytes with increased CBRM1/5 expression in vivo exhibit increased Mac-1-mediated endothelial adhesion in vitro [43 ], supporting the functional relevance of this response. The CBRM1/5+/L-selectin+ phenotype we have identified characterizes a circulating neutrophil subpopulation with likely heightened potential for rolling and firm adhesive endothelial interactions, which may be implicated in inadvertent tissue injury associated with acute inflammatory states. Although the exact functional significance of increased CBRM1/5 expression during cardiac surgery remains to be defined, the inability of anti-Mac-1 strategies to improve outcomes in human anti-inflammatory trials may be a result of the failure of particular antibodies to specifically inhibit conformationally active CD11b on circulating leukocytes. The effectiveness of the anti-platelet GPIIa/IIIb mAb 7E3 in acute coronary syndromes may be partially attributed to its ability to bind conformationally active CD11b and inhibit Mac-1-mediated leukocyte adhesion [44 ]. The expanded CBRM1/5+ circulating neutrophil subpopulation, which we demonstrate, peri-operatively identifies a potential target for such selective, anti-integrin strategies.

Changes in the proportion of CBRM1/5+ neutrophils in response to cardiac surgery, stimulation in vitro, and exposure to heparin suggest that CD11b conformational activation follows a threshold effect on each cell, whereby a certain proportion of CD11b molecules is activated [21 ], causing the cell to become CBRM1/5+. The intensity of neo-epitope expression on CBRM1/5+ cells is likely determined by levels of total CD11b expression, as demonstrated by the similar changes in CD11b and CBRM1/5 MFIs during cardiac surgery and exposure to heparin. Stimulation of neutrophils in vitro with soluble plasma mediators known to be released during cardiac surgery [26 , 35 36 37 ] confirmed that conformational activation of CD11b could be induced independently of changes in total Mac-1 or L-selectin levels. Stimulation with PAF also increased neutrophil adhesion to the CD11b I domain ligand fibrinogen, indicating that conformational activation of CD11b is functionally relevant. Hypothermia limits stimulus-induced changes in CD11b [19 ] and L-selectin expression [45 ], without inhibiting enhanced, CD11b-mediated adhesion [19 ] and may provide an environment conducive to apparently independent regulation of CBRM1/5 expression. Our in vitro results suggest that C3a, which has not been implicated previously as a regulator of CD11b activation, may contribute to the increased CD11b conformational activation evident intra-operatively. In contrast, platelet release products (ATP, PF4) and histamine do not appear to contribute to this response.

Heparin substantially decreased proportions of CBRM1/5+ neutrophils apparent in vivo and in vitro and reduced CD11b-mediated adhesion, whereas proportions of Mac-1+ neutrophils were unaffected, and CD11b expression on these neutrophils was only reduced mildly. This suggests that heparin preferentially interacts with conformationally active CD11b, obscures the CBRM1/5 neo-epitope, and inhibits conformationally active I domain adhesive function under conditions of mild activation such as stimulation with PAF. Under conditions of more severe activation, such as that achieved with fMLF, heparin does not suppress CD11b conformational activation, suggesting a qualitative difference between the mild activation induced by agents such as PAF and that induced by fMLF. This may also indicate that the heparin effect is not explained completely by epitope masking alone. It was not feasible to investigate neutrophil CBRM1/5 expression during CABG in the absence of heparin because of excessive activation of coagulation, so effects of heparin were investigated on CBRM1/5 mAb-binding in vitro under conditions similar to the intravascular environment during CPB. These results support our proposal that heparin interacted with CBRM1/5 on neutrophils from patients pre-CPB. The failure of heparin to reduce the intensity of CBRM1/5 expression on residual CBRM1/5+ neutrophils in vivo is more difficult to interpret, although a subtle reduction in the intensity of CBRM1/5 expression, to a degree similar to that achieved with CD11b, may not have been detectable on this small (2.1±0.6%) neutrophil population. This is supported by the in vitro findings, which revealed reduced CBRM1/5 intensity of similar magnitude to the decreases in CD11b and CD18 expression on a larger population (>12%, Table 2A ) of residual CBRM1/5+ neutrophils upon exposure to heparin. Preferential binding of conformationally active CD11b identifies a potential mechanism by which heparin binds CD11b [34 ] and inhibits binding of I domain ligands [46 ] on activated but not resting neutrophils. It may also contribute to the ability of heparin to inhibit CD11b-mediated, neutrophil-endothelial interactions and transendothelial migration in vivo [47 ].


   CONCLUSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSION
REFERENCES
 
The presence of a CBRM1/5+/L-selectin+ neutrophil subpopulation in vivo and its expansion peri-operatively confirm circulating neutrophil heterogeneity [48 ], indicate conformational activation of CD11b can occur on circulating neutrophils, and identify a potential target for integrin-based, anti-inflammatory strategies. Our in vivo and in vitro findings demonstrate that the usual, inverse regulation of CD11b and L-selectin expression [33 ] is not invariable and that CD11b up-regulation and conformational activation can be regulated independently.


   ACKNOWLEDGEMENTS
 
This work was supported by the National Heart Foundation of Australia, the National Health and Medical Research Council of Australia, the Royal Australasian College of Surgeons, the Australasian Society of Cardiothoracic Surgeons Research Foundation, and NSW Ministry for Science and Medical Research Infrastructure Grant. The authors thank flow cytometrist Leonie Gaudry for excellent technical assistance and Professor Colin Chesterman and the Departments of Haematology and Flow Cytometry, Prince of Wales Hospital (Sydney, Australia), for use of equipment and resources invaluable to this project.

Received September 4, 2006; revised June 12, 2007; accepted June 25, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSION
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