INTRODUCTION
Disturbances in microvascular flow play a major role in contributing to tissue hypoperfusion during septic shock (1–3). The inability to increase microvascular blood flow in the face of tissue hypoxia may be related to intravascular obstruction resulting from the endothelial inflammation and changes in cell rheology that occur during sepsis (4–6). Decreases in erythrocyte and neutrophil deformability, and increases in erythrocyte and neutrophil aggregation have been reported in septic shock (4,5,7). In particular, platelet-neutrophil interactions are increased in septic shock and may play an important role in impairing microvascular blood flow. Clinically, increases in the formation of platelet-neutrophil aggregates have been associated with the development of multiple organ failure in septic patients (8).
Receptor-ligand interactions are responsible for adhesive interactions between platelets and neutrophils. Contact between activated cells acts as a signal for ongoing activation and aggregation (9). Platelet membrane receptors P-selectin and glycoprotein complex IIb/IIIa are involved in platelet-neutrophil adhesion (10–12). In addition, intercellular adhesion molecule-2 (ICAM-2) has been demonstrated on platelets and may function as a neutrophil-binding receptor (13). β-2 integrins expressed on activated neutrophils bind platelet surface receptors and also play a role in the platelet-neutrophil aggregation (10,14). Accordingly, the purpose of this study was to examine the role of these individual platelet and neutrophil surface receptors on platelet-neutrophil aggregation in patients with septic shock. In addition, we examined the interaction between different platelet receptors, different neutrophil receptors, and combinations of platelet-neutrophil receptors on platelet-neutrophil aggregation during septic shock.
MATERIALS AND METHODS
Patient population
This study was approved by the Institutional Research Board of Saint Vincent's Hospital and Medical Center. Informed consent was obtained from either the patient or a surrogate. Normal healthy volunteers provided venous blood samples for isolation of neutrophils, platelets, and platelet poor plasma (NPPP), and to serve as control subjects. Septic platelet poor plasma (SPPP) was removed from 12 patients in septic shock. Septic shock patients met the following criteria: a positive blood culture or an identifiable site of infection; evidence of a systemic inflammatory response manifested by three of the following: temperature greater than 38.3°C or hypothermia less than 35.5°C, respiratory rate greater than 20 breaths/min, heart rate greater than 90 beats/min, and a leukocyte count >12,000/mm3; and systemic hypoperfusion defined by an arterial lactate greater than 2 meq/L or hypotension defined by a mean arterial pressure (MAP) <60 mmHg despite fluid infusion to a pulmonary arterial occlusion pressure ≥16 mmHg and requiring vasopressors to maintain a systolic arterial pressure greater than 90 mmHg (15). All measurements were taken within 24 h of meeting criteria for entrance into the study. Exclusion criteria included blood transfusions in the prior 48 h, presence of the human immunodeficiency virus, or corticosteroid administration.
Isolation of human neutrophils
Venous blood from normal volunteers was drawn into citrate anticoagulant and neutrophils were collected as previously described (7,16). Briefly, neutrophils were isolated by Dextran sedimentation followed by hypotonic lysis of erythrocytes and centrifugation over Percoll (Pharmacia, Piscataway, NJ). The resulting neutrophil pellet was suspended in Hank's solution (Life Technologies, Grand Island, NY). Trypan blue exclusion showed greater than 95% viability. The neutrophil-rich sample was analyzed for cell count by a Coulter counter and a neutrophil concentration of 5 × 103/mm3 was used for cell filtration.
Isolation of human platelets
Citrate anticoagulated blood from the same donor as used in neutrophil isolation was centrifuged at 290 g for 10 min and the platelet-rich plasma was collected. Platelets were resuspended at a concentration of 75 × 103/mm3.
Preparation of NPPP and SPPP
After whole blood from septic shock patients and control subjects was centrifuged at 290 g for 10 min, the supernatant was aspirated and centrifuged at 2,500 g for 10 min to yield SPPP and NPPP for platelet and neutrophil suspension.
Monoclonal blocking antibodies
Murine blocking monoclonal antibodies (PharMingen, San Diego, CA) were used against the platelet and neutrophil surface receptors. On the platelet, monoclonal antibodies anti-CD41 and abciximab recognize the glycoprotein complex IIb/IIIa; monoclonal antibody anti-CD62P blocks P-selectin; and monoclonal antibody anti-CD11a/LFA blocks ICAM-2. On the neutrophil, monoclonal antibody anti-CD11b blocks the β2 integrin CD11b, which associates to form the CD11b/CD18 complex; and monoclonal antibody anti-CD18 blocks the β2 integrin CD18, which associates with the α-chains of CD11a, Cd11b, or CD11c. In a separate set of cell filtration experiments, an irrelevant control antibody, IgG (Immunotech, Westbrook, ME), was used to assess for a nonspecific antibody-mediated response. During flow cytometry experiments, fluorescein isothiocyanate- (FITC) labeled IgG and phycoerythrin- (PE) labeled IgG (Immunotech) were used as control antibodies to rule out nonspecific binding. CD63 fluorescence was compared to background staining with IgG control antibody and absolute values of antibody positive platelets were expressed after adjusting for nonspecific fluorescence.
Cell filtration apparatus
Cell filtration through polycarbonate 5-μm pore filters is an in vitro experimental model for the in vivo situation of capillary plugging. The recording of the filtration pressure (Pi) with time during constant flow allows for the analysis of plugging of filter pores by blood cells (17). Pi (mmHg) reflects the maximal pressure achieved for each cell suspension at a constant volume of 1 mL and a fixed flow rate of 1 mL/min. Changes in Pi (ΔPi) for each subject was the difference in Pi between cell suspensions in NPPP and cell suspensions in SPPP after the addition of monoclonal blocking antibodies. The filtration apparatus was prepared as described previously (7,17). Polypropylene filter chambers (Millipore, Bedford, MA) and sterile plastic tubing were incubated with 1% organosilane (PCR Inc., Gainesville, FL) for 5 min at 20°C. Immediately prior to use, the chambers and tubing were incubated with 20% heat-inactivated serum for 2 h at 37°C. Polycarbonate filters (Costar, Cambridge, MA) with a pore size of 5 μm were then placed into the filter chambers. The infusion system consisted of an adjustable-flow infusion pump (KDS Model 210; Stoelting, Wood Dale, IL) attached to siliconized tubing. A pressure transducer and digital monitor coupled to a strip recorder were linked downstream.
Flow cytometry
Platelet activation and platelet-neutrophil interactions were assessed using murine monoclonal antibodies that were all fluorescein conjugated to either PE or isothiocyanate. Anti-CD41-PE (PharMingen) is a platelet-specific monoclonal antibody that recognizes the glycoprotein complex IIb/IIIa on resting and activated platelets. Anti-CD63-PE monoclonal antibody (PharMingen) recognizes a lysosomal glycoprotein expressed on activated platelets and monocytes. Anti-CD66b-FITC monoclonal antibody (Immunotech) recognizes a glycosyl-phosphatidylinositol-anchored glycoprotein strongly expressed by neutrophils. Anti-CD14-PE monoclonal antibody recognizes monocytes. The purity of neutrophil preparations was monitored using anti-CD14 monoclonal antibody to exclude monocytes and anti-CD66b monoclonal antibody to include activated neutrophils.
Platelet activation was assessed as follows. Platelet-rich plasma was obtained during the isolation of neutrophils. Thereafter, 5 μL of platelet-rich plasma aliquots was added to polypropylene tubes containing 45 μL of conjugated monoclonal antibody (anti-CD63-PE or anti-CD41-PE) in Tyrodes buffer (0.1% bovine serum albumin [BSA], 0.1% glucose, 2 mmol/L MgCl2, 137.5 mmol/L NaCO3, and 2.6 mmol/L KCl, pH 7.4). After a 15-min incubation period at room temperature (23°C) in the dark, the sample was diluted to 0.5 mL in Tyrodes buffer. FACS analysis was done within 6 h of sampling with a FACS Caliber flow cytometer (Becton-Dickinson, Mountain View, CA) and the Cell Quest (Becton-Dickinson) software. Platelets were identified by forward and side scatter plot and CD-41 positivity. Platelet activation was assessed by CD-63 positivity.
Platelet-neutrophil interactions were assessed as previously described (7,8). Fifty microliters of platelet-neutrophil aliquots was added to tubes containing saturating concentrations of fluorescein-conjugated monoclonal antibodies (anti-CD63-PE and anti-CD66b-FITC) in 25 μL of Tyrodes buffer and was then incubated for 30 min at room temperature (23°C) in the dark. For flow cytometric analysis, neutrophil subgroups were identified by size and granularity in the forward vs. side scatter plot and by binding of a subgroup-specific monoclonal antibody, anti-CD66b. The mean intensity of the anti-CD63-PE immunofluorescence of the neutrophil subgroup was used as an index of activated platelet adhesion to the neutrophil.
Experimental protocol
The addition of blocking monoclonal antibodies to the desired cell fraction occurred 1 h after the addition of septic plasma and 30 min before the coincubation of cells. First, single blocking monoclonal antibodies were added to desired cell lines. Then, using the SPPP from the same septic shock patients, combinations of blocking monoclonal antibodies were used.
Platelet-neutrophil suspensions from control subjects and septic shock patients, suspended in NPPP and SPPP, were filtered with and without the presence with blocking monoclonal antibodies at a flow rate of 1 mL/min. After the addition of blocking monoclonal antibodies to desired cell lines, fluorescein-conjugated monoclonal antibodies were added to 100 μL of the resultant platelet-neutrophil suspensions for analysis by flow cytometry.
Statistical analysis
Statistical analysis of cell filtration and flow cytometry data was performed using the Student's t test on paired samples. Results were considered significant at P < 0.05. Results are expressed as means ± standard error.
RESULTS
The mean age and APACHE II score for septic shock patients was 62 ± 4.0 years and 28 ± 2.0 years, respectively. All septic shock patients required vasopressors, lactate levels were 3.8 ±1.0 mmol/L, and hospital mortality was 60%. The mean age of volunteers was 35.8 ± 2.0 years with a male-to-female ratio of 1:1.
The Pi of platelets and neutrophils suspended in SPPP was significantly greater than that of cells suspended in NPPP (24 ± 1.0 mmHg vs. 14 ± 1.0 mmHg;P < 0.05). The addition of either platelet-or neutrophil-blocking monoclonal antibodies significantly decreased the Pi of platelets and neutrophils suspended in SPPP, but did not normalize these values.
ΔPi for each subject between cell suspensions in NPPP and SPPP and in NPPP and SPPP after the addition of single blocking monoclonal antibodies were examined. Suspension of platelet-neutrophil combinations in SPPP as compared to NPPP resulted in an increment (ΔPi SPPP-NPPP) of 11.4 ± 1.6 mmHg. Antibodies to CD41, CD62P, CD11a, CD11b, CD18, and abciximab all significantly decreased the ΔPi SPPP-NPPP of the platelet-neutrophil suspensions (P < 0.01, Fig. 1). There was no significant effect on filtration pressures of cells suspended in SPPP in the presence of the nonspecific binding antibody, IgG (Fig. 1).
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Fig. 1: Difference in ΔPi for individual subjects between P-PMN suspensions in NPPP and SPPP, and in NPPP and SPPP after the addition of single blocking monoclonal antibodies. * P < 0.01 vs. ΔPi SPPP-NPPP.
ΔPi for each subject between cell suspensions in NPPP and SPPP and in NPPP and SPPP after the addition of combinations of blocking monoclonal antibodies were examined (Fig. 2). Platelet-neutrophil suspensions containing the combinations of blocking monoclonal antibodies anti-CD41/CD62P, anti-CD41/CD11b, and anti-CD62P/CD11b significantly decreased the ΔPi SPPP-NPPP. When platelet receptor P-selectin, CD62P, was blocked simultaneously with the CD11b receptor on the neutrophil, the ΔPi SPPP-NPPP, 1.6 ± 0.5 mmHg, was lower than that of all other single or combinations of blocking monoclonal antibodies.
Fig. 2: Difference in ΔPi for individual subjects between P-PMN suspensions in NPPP and SPPP, and in NPPP and SPPP after the addition of combinations of blocking monoclonal antibodies. * P < 0.01 vs. ΔPi SPPP-NPPP.
Platelet-neutrophil interactions in septic and normal plasma suspensions, with and without the addition of monoclonal antibodies, were assessed by flow cytometry. The mean fluorescence of platelet CD63-PE binding to the neutrophil suspended in SPPP was significantly greater than the CD63-PE binding to neutrophil suspended in NPPP (mean fluorescence 780 ± 130 lfu vs. 295 ± 35 lfu, respectively, P < 0.05;Figs. 3 and 4). This was associated with a significantly greater percentage of activated platelet binding to neutrophils when suspended in SPPP as compared to activated platelet binding to neutrophils suspended in NPPP (45 ± 5.5 vs.15.5 ± 3, respectively, P < 0.05). Mean fluorescence of CD63-PE binding to neutrophils was significantly attenuated in all cell suspensions in SPPP with single monoclonal antibodies as compared to cells suspended in SPPP alone (P < 0.05;Fig. 3). This was associated with a significant decrease in percentage of activated platelet binding to neutrophils in the presence of anti-CD41, anti-CD62P, abciximab, and anti-CD11b monoclonal antibodies (P < 0.05).
Fig. 3: Mean fluorescence of P-PMN aggregates (CD63-positive neutrophils) in NPPP and SPPP and in SPPP after the addition of single monoclonal antibodies. * P < 0.05 vs. P + PMN + SPPP.
Fig. 4: Mean fluorescence of P-PMN aggregates (CD63-positive neutrophils) in NPPP and SPPP and in SPPP after the addition of combinations of monoclonal antibodies. * P < 0.05 vs. P + PMN + SPPP.
Mean fluorescence of CD63-PE binding to neutrophils was significantly attenuated in all cell suspensions in SPPP with combinations of blocking monoclonal antibodies as compared to cells suspended in SPPP without blocking monoclonal antibodies (P < 0.05;Fig. 4). This was associated with a significant decrease in percentage of activated platelet binding to neutrophils in the presence of anti-CD41/CD62P, anti-CD41/CD11b, CD11a/CD11b, and anti-CD62P/CD11b (P < 0.05). The greatest attenuation in mean fluorescence occurred by blocking platelet receptor P-selectin, CD62P, and neutrophil receptor CD11b.
Two examples of FACS histograms have been included (Figs. 5 and 6). They represent the mean fluorescence of activated platelet CD63-PE binding to neutrophils in SPPP with and without the addition of blocking monoclonal antibodies to receptors P-selectin and CD11b.
Fig. 5: Analysis of P-PMN adhesion without the addition of blocking monoclonal antibodies. Platelet fluorescence histogram of neutrophil region. M1 defines the region of interest identifying the activated platelet fluorescence CD63 binding to neutrophils when isolated cell lines were stimulated with SPPP and then combined.
Fig. 6: Analysis of P-PMN adhesion with the addition of blocking monoclonal antibodies. Platelet fluorescence histogram of neutrophil region. M1 defines the region of interest identifying the activated platelet fluorescence CD63 binding to neutrophils when isolated cell lines were stimulated with SPPP before the addition of blocking monoclonal antibodies to platelet receptor P-selectin and neutrophil receptor CD11b.
DISCUSSION
Interactions between platelets and neutrophils have been implicated in the pathophysiology of impaired microvascular blood flow and thrombosis in a number of clinical settings, including myocardial infarction, stroke, vascular graft occlusion, and cardiopulmonary bypass (18–20). The deposition of platelet-neutrophil aggregates has also been postulated to play a major role in the development of organ failure in patients with sepsis (8). In a previous study we demonstrated that the formation of platelet-neutrophil aggregates was increased in patients with septic shock (7). More importantly, the formation of these aggregates appeared to be the major factor limiting cellular filtration. In experimental studies, a correlation has been established between decreased cellular filtration and in vivo impairment of microvascular blood flow (21). This study confirms our prior observation that septic shock is associated with increased formation of platelet-neutrophil aggregates that impair cellular filtration and may impair microvascular blood flow.
Stimulation of either isolated platelets or neutrophils has been utilized to study platelet-neutrophil interactions. Commonly used stimulants for platelets have included thrombin, epinephrine, and adenosine diphosphate, while neutrophils have been stimulated with N-formyl-methionyl-leucyl-phenylalanine (fMLP) and complement (10–13). Stimulation of either neutrophils or platelets increases platelet-neutrophil aggregation, with the largest number of platelets bound per neutrophil being observed when the neutrophil is the stimulated cell (11). Cell-specific stimulation of both platelets and neutrophils produces greater adhesion than is observed with single-cell stimulation and causes large numbers of neutrophils to bind to large numbers of platelets (11). In our study, the use of septic serum stimulated both neutrophils and platelets, thereby producing a degree of aggregation similar to that observed with combinations of stimulants are utilized.
Platelet activation results in the surface expression of P-selectin (CD62P), which in turn interacts with its ligand the P-selectin glycoprotein ligand-1 on the surface of neutrophil to induce the formation of platelet-neutrophil aggregates (9,10,22). Subsequent firm adhesion involves interaction between the neutrophil CD11b/CD18 receptor and glycoprotein Ibalpha, a component of the glycoprotein Ib-IX-V complex, the platelet von Willebrand receptor (23). In our study we observed that antibodies to P-selectin, CD11b, and CD18 all decreased sepsis-induced platelet-neutrophil aggregation as determined by both filtration and flow cytometry. These data are consistent with prior reports demonstrating that incubation with antibodies to P-selectin significantly attenuates platelet-neutrophil aggregation (9,11,14). In contrast, earlier studies have observed that antibodies to either CD18 or CD11b have variable effects on platelet-neutrophil aggregation, depending on the antibodies and model used (11,14,24).
The decrease in platelet-neutrophil aggregation following exposure to antibodies to CD11a differs from previous studies that have not observed any decrease in platelet-neutrophil aggregation when neutrophils are exposed to antibodies to CD11a (11,14,24). This difference in results may reflect the fact that we used septic plasma, as compared with thrombin, to stimulate platelets. ICAM-2, the counter receptor for neutrophil CD11a, is expressed on platelets (13). The use of septic plasma as a stimulus for platelet activation may have increased platelet ICAM-2 expression, thereby forming the basis for the decrease in platelet-neutrophil aggregation that we observed with antibodies to CD11a.
Platelet activation results in increased expression of glycoprotein complex IIb/IIIa (CD41a), which plays a fundamental role in platelet aggregation. In our study, both a monoclonal antibody to CD41a and abciximab, the Fab fragment of a human-murine monoclonal antibody glycoprotein IIb/IIIa receptor, decreased sepsis -induced platelet-neutrophil aggregation. This observation is similar to an earlier study that found that antibodies to glycoprotein IIb/IIIa decreased the adhesion of activated platelets to neutrophils (12). The postulated mechanism of glycoprotein IIb/IIIa-mediated neutrophil adhesion involves fibrinogen that is bound to aggregated platelets and is a ligand for neutrophil CD11b and CD11c (12). In the case of abciximab, there are also data to suggest that it is not selective for glycoprotein IIb/IIIa, but also cross reacts with the leukocyte integrin CD11b/CD18, thereby providing another potential mechanism for the decrease in platelet-neutrophil aggregation that we observed (25). Indeed, abciximab has recently been reported to decrease circulating platelet-neutrophil aggregates in patients undergoing coronary angioplasty (26).
When combination of monoclonal antibodies were studied, the combination of antibodies to P-selectin and CD11b demonstrated the greatest reduction in platelet-neutrophil aggregation as measured both by filtration and flow cytometry. This observation is consistent with the central role of these receptors in platelet-neutrophil interactions and with previous reports that have demonstrated that this combination has an additive effect in decreasing platelet-neutrophil aggregation (11,14,24). Combining antibodies against glycoprotein IIb/IIIa and CD11b did not demonstrate the same additive effect in reducing platelet-neutrophil aggregation.
This study involves the formation of platelet-neutrophil aggregates in freely suspended cells. Another form of platelet-neutrophil interaction that plays an important role in vascular occlusion is the binding of neutrophils to platelets that have already been deposited on an inflamed or injured microvascular surface (9). Although this latter process was not examined in this study, P-selectin mediates the initial rolling adhesion of neutrophils to bound activated platelets. Subsequent immobilization and attachment are mediated by neutrophil CD11b/CD18 (27,28). Accordingly, the observations in our study may apply to this in situ form of platelet-neutrophil interaction.
Increases in platelet aggregation and increases in neutrophil aggregation may also limit cellular filtration. We have previously demonstrated that blocking the neutrophil CD11b/CD18 receptor decreases septic neutrophil aggregation and increases septic neutrophil filtration (5). In this study, it is possible that some of the improvement in cellular filtration with the different monoclonal antibodies was related not only to the effect of decreasing platelet-neutrophil aggregation, but also decreases in isolated platelet and neutrophil aggregates. In addition, in sepsis, decreases in neutrophil deformability may contribute to increases in resistance to cell filtration (5,7). However, the demonstrated improvement in cell filtration coupled with demonstrated decreases in the formation of platelet-neutrophil aggregates by flow cytometry support the importance of platelet-neutrophil interactions in limiting cell filtration.
In summary, platelet-neutrophil aggregation is increased in septic shock. This aggregation is mediated by the interaction of multiple platelet and neutrophil surface receptors. However, the platelet surface receptor P-selectin and the neutrophil receptor CD11b/CD18 appear to play the most important role in these interactions.
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