Published online before print May 22, 2003
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,
* Infection and Microbiology Unit and
Portex Unit Critical Care Group, Institute of Child Health, London, United Kingdom;
Department of Pathology and Microbiology, School of Medical Sciences, University of Bristol, United Kingdom; and
Department of Paediatrics, Imperial College School of Medicine (ICSM) at St. Mary’s, London, United Kingdom
Correspondence: Dr. M. J. Peters, Portex Unit Critical Care Group, Institute of Child Health, 30 Guilford Street, London, WC1N 1EH, UK. E-mail: m.peters{at}ich.ucl.ac.uk
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IIbß3 (GpIIb/IIIa) was seen. PNC were reduced on presentation and fell to very low levels after 24 h. Immunostaining of skin biopsies demonstrated that PNC appear outside the circulation in MD. In vitro studies of anticoagulated whole blood inoculated with Neisseria meningitidis supported these clinical findings with marked increases in PMNL CD11b/CD18 expression and activation but no detectable changes in platelet-activated IIbß3 or CD62P expression. In vitro PMNL activation with N. meningitidis (or other agonists) potentiated the formation of PNC in response to platelet activation with adenine diphosphate. Therefore, in severe MD, PMNL activation is likely to promote PNC formation, and we suggest that the reduced levels of PNC seen in established MD reflect rapid loss of PNC from the circulation rather than reduced formation.
Key Words: sepsis • adhesion • meningococcus • inflammation
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IIb/ß3 (GpIIb/IIIa). Other molecules such as CD40 and CD40L found on the platelet also act to link the haemostatic and inflammatory pathways [3
]. Circulating PNC can be observed in humans and may account for up to 25% of circulating PMNL [4 ]. These complexes appear to represent activated subpopulations of both cell types: Platelet activation with CD62P expression is required for PNC formation, and PMNL in PNC express higher levels of adhesion molecules, produce more superoxide, and phagocytose selected bacteria more readily than unbound neutrophils [5 , 6 ]. An increase in the formation of PNC, combining these platelet and PMNL properties, might be expected to contribute to the development of the haemostatic/inflammatory dysregulation, which underlies the systemic, inflammatory response.
Meningococcal disease (MD) is frequently associated with a severe systemic inflammatory response and progression to multiple organ failure characterized by shock, haemorrhagic skin lesions, and a marked coagulopathy. Platelets and PMNL are clearly implicated in the evolution of this process through the acceleration of thrombin production, cytokine and chemokine production, phagocytosis, and the release of reactive oxygen metabolites and proteases [7 , 8 ]. Thrombocytopenia and neutropenia are reliable signs of severe disease in patients with meningococcal septicaemia [9 ] and are thought to occur as a result of sequestration of both cell types in the microvasculature of the skin, lung, and other vital organs.
This disease is therefore an ideal scenario in which to investigate PNC formation and its relationship to platelet and PMNL activation and clinical severity. In this study, we have investigated the presence, time course, and mechanisms of PNC formation in 32 cases of severe pediatric MD requiring intensive care. We show that in the context of platelet and PMNL activation, PNC disappear from the circulation early in the course of meningococcemia and are detectable deep in the tissue lesions of these patients. In vitro studies of PNC formation with meningococci [10 ] lead us to suggest that the reduced numbers of circulating PNC seen in established MD may be explained by rapid loss from the circulation rather than reduced formation.
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IIbß3 (CD41/CD61; GpIIb/IIIa) mAb Pac-1 was purchased from the Cell Centre (Department of Human Genetics, University of Pennsylvania, Philadelphia). FITC-conjugated mouse mAb to CD62P (P-selectin) and to CD66b were obtained from Pharminogen (Cowley, UK). Isotype and fluorochrome-matched, negative control immunoglobulin G (IgG)1 and IgM mAb were purchased from Serotec. Fluorescence-activated cell sorter (FACS) lysing solution was purchased from Becton Dickinson (Oxford, UK). Thrombin, glycyl-L-propyl-L-arginyl-L-proline (GPRP), adenosine diphosphate (ADP), f-Met-Leu-Phe (fMLP), and Escherichia coli lipopolysaccharide (LPS), protein content <1%, serotype O111:B4, were obtained from Sigma (Poole, UK).
Clinical studies, patients, and specimens
The local research ethics committees approved the protocols, and written consent was obtained. The diagnosis of MD was based on a characteristic petechial or purpuric rash with clinical features of sepsis and was confirmed by culture of meningococci from blood or by detection of meningococcal antigens or DNA (by polymerase chain reaction) in blood (PHLS Meningococcal Reference Unit, Manchester, UK). Circulating platelet, neutrophil activation, and PNC formation were studied in patients with a clinical diagnosis of meningococcal sepsis by one of the investigators (M.J.P. or G.L.J.D.) who accompanied the emergency pediatric retrieval team during stabilization and transport to the Pediatric Intensive Care Unit (PICU) at Great Ormond Street Hospital for Children (London). These studies were repeated on arrival and during the ICU stay. All samples were collected through indwelling arterial catheters to minimize collection artifact. The first 2 mL was discarded, and the required volume was collected into sodium citrate to a final concentration of 0.38%. The Pediatric Index of Mortality (PIM) [12
] and the Glasgow Meningococcal Septicaemia Prognostic Score [13
], which were recorded on first contact with the ICU team, determined severity of illness. The ICU mortality by day 28 was noted. Tissue leukocyte–platelet complexes were studied using 3 mm punch skin biopsies taken from the edge of the purpuric lesions of five cases within 24 h of the first dose of parenteral antibiotics. Control subjects were selected on the basis of cases ventilated on the PICU with single-system failure, without evidence of a systemic inflammatory response (raised white cell count, temperature, or tachycardia) [14
].
Circulating PMNL activation
The levels of PMNL CD11b expression, CD11b/CD18 activation (as indicated by the binding of the mAb Mab24, which recognizes the active binding site of CD11b/CD18), and CD62L expression were measured by whole blood flow cytometry (Becton Dickinson FACScan and FASCalibur) as described previously [4
, 7
]. On a forward- and orthogonal light-scatter, two-dimensional dot-plot, the PMNL population was distinguished from lymphocytes and monocytes and was CD66b-positive. Five thousand events were analyzed.
Circulating platelet activation
Changes in free platelet-activated IIbß3 expression were measured by flow cytometry (Pac-1 antibody binding) using logarithmic light-scatter and fluorescence settings. Platelets were distinguished from other cells on the basis of their forward- and orthogonal light-scatter profile, and the accuracy of the gating was confirmed by staining with PE-labeled CD42b (>98%). Platelet responsiveness to ADP and thrombin (in the presence of GPRP to prevent fibrin cross-linking) was controlled for in each experiment.
PNC formation
As described previously [4
, 5
], dual immunostaining was undertaken with a directly conjugated antibody against a platelet antigen (CD42b) and a directly conjugated antibody against a PMNL antigen (CD11b). Samples were then treated with FACSlyse and fixed with 0.2% formaldehyde. Events in the neutrophil population staining positive for neutrophil and platelet antigens were considered to represent PNC and were distinguishable from events staining for a neutrophil marker alone. PNC were expressed as the percentage of the total neutrophil population with associated platelets.
Immunohistochemistry
Formalin-fixed, paraffin-embedded sections were immunostained for platelets (CD41), PMNL (neutrophil elastase), and monocyte (CD68) markers using a novel horseradish peroxidase-labeled polymer system (Envision++, Dako) [15
]. Microwave heat induction, antigen retrieval in citrate buffer, pH 6.0 (HDS05, SD Supplies, Aylesbury, UK), was required for optimal staining with the anti-CD41.
Whole blood model of meningococcal bacteraemia
This was performed as described previously using the following Neisseria meningitidis strains: NCTC 10025 (A: 4.NT); NCTC 10026 (B: NT.NT); NCTC 8554 (C: NT.P1.5); and strain 107770 [nongroupable (unencapsulated): 15.NT; 10]. In addition, N. meningitidis wild-type (WT) H44/76 (B: 15:P1.7,16) and a LPS-deficient lpxA– mutant [16
] were used to further characterize PNC formation and neutrophil activation. Stationary-phase meningococci grown overnight on GC agar (Difco, Detroit, MI) supplemented with 1% Vitox (Oxoid, Basingstoke, Hampshire, UK) were used [10
]. To minimize variation between experiments, a single, healthy donor was used. Subsequent experiments to confirm our findings were conducted using blood obtained from further six healthy, adult volunteers. Blood was collected without stasis; the first 2 mL drawn was discarded, and the remainder was anticoagulated with 3.8% sodium citrate. Aliquots (5 mL) of whole blood were then inoculated with meningococci (at final concentration, 106–108 cfu/mL) and were then incubated on a rocking platform at 9 rpm at 37°C [17
]. At specific time intervals, aliquots of blood were removed for analysis of platelet activation and PNC formation as described above. Red blood cell and platelet counts did not fall over the 60-min period of observation, and haemolysis was not observed in separated samples. Beyond 60 min, significant PMNL and platelet activation occur in unstimulated, control samples [4
, 10
]; therefore, this model was not examined after longer incubations.
Statistics
Experiments were performed on at least four occasions. Data are presented as median and interquartile ranges (IQR) or mean and standard errors when normally distributed. The differences between the groups were analyzed by a Wilcoxon-sum ranked tests or paired t-tests as appropriate.
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Table 1. Characteristics of 27 Cases of Acute MD Studied on the PICU at Great Ormond Street Hospital
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Figure 1. Circulating PMNL activation in 27 cases of acute MD. (A) CD11b expression, (B) activation of CD11b/CD18 (Mab24 binding), and (C) CD62L expression are compared with control values (mean and 95% confidence intervals) observed in 20 control PICU patients. Solid circles represent nonsurvivors and open circles, survivors.
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IIbß3 was assessed in 18 subjects (including five nonsurvivors) with MD within 24 h of presentation (Fig. 2
). Compared with controls, individuals had a greater than 50% increase in activated IIbß3. However, overall, the median value was no different from PICU controls (median, range), there was a trend toward lower levels of IIbß3 activation in fatal cases, and there is no clear pattern observed with the time course of the disease.
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Figure 2. Circulating platelet IIbß3 activation in acute MD. Circulating platelets (n=18) are not significantly more activated than control PICU cases (n=8, median and IQR shown). There is a trend toward lower levels of IIbß3 activation in fatal cases, and no clear pattern is observed with the time course of the disease. Solid circles represent nonsurvivors and open circles, survivors.
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Figure 3. PNC in 23 cases of acute MD. Control values for PNC (mean and 95% confidence intervals are shown) were observed in PICU-control patients (n= 20). Solid circles represent nonsurvivors and open circles, survivors. There was a reduced level of PNC on presentation, which reduced further between 24 and 48 h.
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IIbß3 expression staining (P=0.5; Fig. 4
) were observed. This was despite a significant increase in circulating platelet count (P=0.018).
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Figure 4. The effect of platelet transfusion on platelet count, % PNC, and IIbß3 (Pac-2 MFI) activation. Paired observations in seven patients in before and after platelet transfusion are shown. There is a significant increase in platelet count but no change in % PNC or IIbß3 activation. These data support the interpretation of the fall in PNC shown above (Fig. 3)
as a true change rather than an artifact of altered platelet counts.
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Figure 5. Platelet–neutrophil complexes in meningococcal disease. Immunoperoxidase staining of skin biopsies from five children with MD (examples shown in A and B) showing evidence of a perivascular acute inflammatory cell infiltrate of PMNL and monocytes. Leukocyte–platelet complexes (CD41-positive) were observed (arrowed). These complexes were rarely seen inside blood vessels and were often observed deep in the periphery.
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Figure 6. PMNL CD11b expression and CD11b/CD18 activation in the whole blood model of MD. (A) CD11b expression is significantly increased following 30 min incubation with WT N. meningitides in a dose-dependent manner across a range of concentrations from 106 to 108 cfu/mL. Means and standard errors of eight experiments are shown. *, P < 0.05, and **, P < 0.01, in paired t-test versus control samples at 30 min. Incubation with the LPS-deficient mutant (LPXa) N. meningitides induces an increase in CD11b at 108 cfu/mL only. (B) CD11b/CD18 activation indicated by Mab24 binding follows an identical pattern.
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IIbß3 expression or up-regulation of CD62P expression (Fig. 7
). Platelet degranulation, as measured by release of immunoreactive ß-thromboglobulin, also did not increase following whole blood inoculation with live meningococci (data not shown). Marked changes in platelet activation were seen following stimulation with the agonists ADP or thrombin, indicating that platelet function was not affected by the experimental conditions. Neither purified LPS nor the bioactive peptide fMLP caused detectable increases in CD62P expression or IIbß3 activation (data not shown).
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Figure 7. Lack of detectable platelet activation in the whole blood model of MD. The mean and standard error of four experiments are shown. (A) IIbß3 (GpIIb/IIIa) and (B) CD62P expression remains unchanged despite incubation with a range of serogroups of N. meningitidis.
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IIbß3 activation (data not shown).
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Figure 8. PNC formation in vitro; the whole blood model of MD: the relative contributions of platelet and PMNL activation. (A) WT N. meningitidis at 106–108 organisms/mL caused no increase in PNC. As expected, the platelet agonist ADP induced increased PNC formation under all conditions, but this effect was significantly potentiated by coincubation with N. meningitidis or purified LPS. (B) The potentiation of PNC formation in response to ADP is also seen in response to non-LPS-mediated neutrophil activation with the peptide fMLP or the LPXa strain of LPS-deficient meningococci (at 108 organisms/mL only). The mean and standard errors of at least four experiments are shown. The results of paired t-test against paired control levels of PNC in samples stimulated with ADP alone are shown.
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Platelet activation and consumption are key features of severe MD to the extent that thrombocytopenia is considered a clinically useful prognostic indicator, and thrombosed vessels are prominent on histological examination [18 , 20 ]. In spite of this clear evidence of platelet involvement in MD, we were unable to demonstrate any significant activation of circulating platelets in patients with this condition. The lack of platelet activation in a relevant clinical setting is a frequent finding and is thought to be largely a result of consumption of the most activated platelets, leaving only the quiescent cells available for sampling [4 , 21 ]. This view is supported by recent studies in baboons in which labeled thrombin-activated platelets could only be detected briefly in the circulation following a platelet transfusion [20 ]. Taken together, the results from this and other studies indicate that detection of platelet activation in vivo is dependent on the balance between activation and recruitment. This balance will vary with the underlying disease process, age of patients, and timing of sampling [22 , 23 ].
Although it is apparent that platelet activation is occurring in MD patients, even if this is difficult to demonstrate in vivo, the stimuli required for platelet activation in this disease are unclear. One possibility is that N. meningitidis itself could directly activate platelets. Despite in vitro studies implicating endotoxin as stimulant of platelets [24 25 26 27 28 ], our own studies of direct stimulation of whole blood with high concentrations of meningococci failed to induce changes in platelet adhesion molecule expression or activation. This was independent of meningococcal serogroup, bacterial concentration, and blood donor. Our data would support a view that platelet activation in MD occurs through other mechanisms.
The mechanisms of the coagulopathy of severe systemic inflammation have been extensively investigated, and known key elements include activation of the tissue factor (TF) pathway by endothelial and monocyte TF expression; consumption or inhibition of endogenous anticoagulant (e.g., antithrombin III, protein C); and fibrinolytic capacity by increased plasminogen activator inhibitor-1 and possibly reduced activation of protein C [29 , 30 ]. All these processes lead to increased and prolonged thrombin production and in the absence of a direct bacterial effect, are more likely to be the initial stimulants for platelet activation in MD. This is consistent with the clinical observation that the most extreme thrombocytopenia occurs after the most profound neutropenia in severe MD [9 ].
The role of PMNL in sepsis is complex [31 ]. Although PMNL phagocytosis of microorganisms is fundamentally important in the initial response to invasive bacterial infection, there is also evidence that these cells may be responsible for much of the tissue injury evident in multiorgan failure following sepsis [32 33 34 ]. Neutrophils are constitutively short-lived, undergoing apoptosis in a matter of hours. Factors that delay this process are associated with more severe organ failure [32 ].
Clinical studies of patients with severe sepsis such as this are limited by the wide variability in the presentation, age, ethnic origin, and timing of sample collection. Despite this, we observed a consistent pattern of early, marked PMNL activation with high levels of CD11b expression and activation and reduced CD62L expression. Activated PMNL are likely to attach to endothelial cells and to transmigrate from the circulation, but clearly the rate at which this adhesion takes place will affect the detectable levels of activation in the residual circulating population. In spite of the variety of potential fates for an activated PMNL in a septic blood vessel, we found increased PMNL CD11b expression and Mab24 binding early in the course of clinical MD. Indeed, significantly higher levels of CD11b expression and activation were seen in eventual nonsurvivors. This may reflect inhibition of normal PMNL chemotactic function in the severely ill [35
, 36
]. Some suggestion that the more activated PMNL are most rapidly recruited is provided by the observed, inverse correlation between the log of the neutrophil count and CD11b expression (r=–0.7; P=0.01) or activation (Mab24 binding, r=–0.7; P<0.001). As was the case with platelets, a dynamic relationship between activation and recruitment exists for PMNL and will influence the properties of any remaining, circulating neutrophils. ß2 Integrin expression activation declined rapidly in MD patients surviving more than 24 h. This may be explained by a reduction in the levels of agonists such as tumor necrosis factor or LPS. However, the role of PMNL in the pathogenesis of sepsis is not limited to the first 24 h [37
], and an alternative explanation of this fall in neutrophil activation is a shift from early disease when there may be a large pool of PMNL circulating in an activated state but with a relative deficiency of endothelial-adhesive counter-receptors toward a "higher flow" state with greater marrow output and rapid recruitment of the more activated PMNL to the now very widespread, activated endothelial cells. In our whole blood model, there was an increase in CD11b and Mab24 binding within minutes of stimulation with bacterial agonists, as expected from previous studies [17
, 23
, 38
]. In contrast to the in vitro studies, the activated PMNL adhesion molecule profile persisted in this model [17
, 23
, 38
]. Although care must be taken when comparing findings in the brief (60-min) in vitro model with the more complex and slowly evolving clinical scenario, the whole blood model contains no endothelium, and therefore, the potential for recruitment of the more activated neutrophils must be reduced.
In this context, our observation that in patients with MD, PNC were almost undetectable by 24 h is pertinent. Our previous studies have shown that PNC represent a subpopulation of activated PMNL and platelets [5
]. As such, they are likely to be particularly adhesive, as they are able to bind through PMNL and platelet adhesion systems. In our whole blood experiments, we were able to show that PNC formation occurs at a significantly enhanced rate in the presence of N. meningitidis and platelet activation. The role of PMNL ß2 integrin and platelet IIbß3 in this enhancement is the subject of ongoing work.
In contrast, PNC formation in vivo appears to be rapidly followed by PNC loss from the circulation. Similar observations have been made in baboons in which PNC numbers were only increased for a median of 5 min after labeled, activated platelet transfusion [5 , 20 ]. Similarly, changes in PNC numbers in patients are not explained by a coincident fall in platelet count because of the lack of effect of platelet transfusion on PNC numbers. Therefore, it would appear that PNC are formed in the presence of stimuli to PMNL and platelets but are then recruited, probably to sites of vascular injury. We have previously demonstrated that PNC are reduced in the presence of transiently injured endothelium (as shown by loss of endothelial vasodilator function) in a human model of ischaemia-reperfusion injury [21 ]. The acute, inflammatory states of cardiopulmonary bypass [39 ] and multiple organ failure [22 , 40 ] have also been associated with reduced PNC. The presence of PNC in skin biopsy specimens from patients with MD provides further support for the formation and recruitment of PNC in disease states.
The relationship among PMNL, platelets, bacteria, and cytokines is considered to be central to our understanding of sepsis. It is interesting that recent data indicate that platelet-derived cytokines delay PMNL apoptosis [41 ], which is a key feature in the generation of sepsis-induced organ injury [32 ]. This study provides further support for the interaction of activated platelets to PMNL in PNC having a pathophysiological role in acute, severe inflammation. Future studies are needed to confirm PNC as a central point of interaction between the inflammatory and haemostatic pathways.
Received October 26, 2002; revised February 12, 2003; accepted February 19, 2003.
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