Dr K. A. Brown, Department of Immunobiology, GKT, Guy's Hospital, 3rd Floor New Guy's House, London SE1 9RT, UK. E-mail: firstname.lastname@example.org
Evidence is growing to suggest that the multiple organ damage of the systemic inflammatory response syndrome (SIRS) arises from the untoward activity of blood polymorphonuclear cells (PMNs), which upon activation acquire the IgG high affinity receptor, CD64. In the current study, flow cytometry was used to assess the prevalence of CD64-bearing PMNs and the intensity of expression of CD64 in whole blood samples from 32 SIRS patients, 11 healthy normal subjects and from eight non-SIRS patients in the intensive care unit (ICU). The percentage of PMNs expressing CD64 was higher in SIRS patients (mean 65%) than in non-SIRS patients (mean 42%; P < 0·02) and in healthy controls (mean 19%; P < 0·001) and was particularly evident in patients with SIRS and sepsis (mean 71%; P < 0·02) as opposed to SIRS alone (mean 55%). There were more CD64 molecules expressed on PMNs from patients with SIRS (median 1331 molecules/cell) in comparison with PMNs from healthy subjects (median 678 molecules/cell; P < 0·01). The highest intensity of CD64 expression was associated with PMNs from patients with both SIRS and sepsis. Functional studies revealed that the supranormal binding of PMNs from patients with SIRS to endothelial monolayers treated with TNFα was impeded by anti-CD64 antibodies (mean 24% inhibition; P < 0·01). Monitoring the distribution of CD64+ PMNs and their level of CD64 expression could be of assistance in the rapid discrimination of patients with SIRS from other ICU patients and in the identification of PMNs which are likely to participate in the pathological manifestations of the disease.
The systemic inflammatory response syndrome (SIRS) is a major cause of mortality in medical and surgical intensive care units . The syndrome defines those patients in varying and usually co-existent states of organ failure, haemodynamic shock and sepsis for the purposes of prognosis, treatment and research [2,3]. Invasive infection is associated with most patients with SIRS and, despite a combination of technological support of organ function and antibiotic regimes, approximately 40–50% of patients die as a result of associated multiple organ failure . Patients with no classically defined evidence of infection have the same poor prognosis as others in whom severe infection is widely identified.
Although high levels of pro-inflammatory cytokines are present in the circulation of some patients with SIRS [5,6], antagonizing their activity has led to little improvement in clinical outcome . A disturbance in circulating PMN numbers is part of the SIRS definition [8,9] and the activation and sequestration of these cells in microvessels is likely to contribute to organ failure [8,10], either indirectly by interacting with vessel walls to increase vascular permeability or directly by infiltrating the tissue and releasing lytic factors [11,12]. Organ microcirculation could also be compromised by the increased stiffness of activated PMNs , which tend to form aggregates . The attachment of PMNs to endothelial cells is dependent upon the sequential intercedence of adhesion molecules on both cell types with opposing counter receptors . Stimulation of PMNs enhances interaction with endothelial cells  and PMNs in SIRS appear to possess an activated phenotype as judged by an increased expression of the adhesion molecule CD11b  and a decreased expression of L-selectin .
We recently noted that most PMNs that bind to endothelial monolayers express CD64 , which is the high affinity Fc receptor for IgG, and is considered to be an activation marker of PMNs . Normally, CD64 is present on the surface of a few circulating PMNs but its expression is up-regulated by incubation of the PMNs with IFNγ or by G-CSF acting on myeloid precursors in the bone marrow . This molecule, which contains three Ig-like domains in its extracellular region , binds both monomeric and aggregated IgG and contributes to antibody-dependent cellular cytotoxicity, phagocytosis and the clearance of immune complexes [24,25]. An increase in CD64 expression, which occurs on blood PMNs in patients with bacterial infections [26,27] and on PMNs in the synovial fluid of patients with rheumatoid arthritis , is considered to be a reliable indicator of acute inflammation .
The purpose of the present study was to determine by using flow cytometric analysis if the prevalence of blood PMNs bearing CD64 and the number of CD64 molecules on the PMN surface were increased in patients with SIRS and whether there was differential expression of CD64 on PMNs from SIRS patients with sepsis when compared with SIRS patients without infection. Also, antibody blocking studies were undertaken to investigate whether CD64 contributed to the binding of patients' PMNs to endothelial cell monolayers.
Materials and methods
Subjects investigated, patient demographics and definition of SIRS
The diagnostic criteria for SIRS was temperature > 38°C < 36°C; heart rate > 90 beats/min; respiratory rate > 20 breaths/min or PaC03 > 32 mmHg and white blood cell (wbc) count > 12 000 cells/mm3 or < 4000 cells/mm3 or > 10% immature (band) forms . Patients from the intensive care unit (ICU) of Guy's and St Thomas's Hospital that were included in the investigation were allocated to one of three clinical groups: (a) patients not fulfilling SIRS criteria but needing intensive care admission for a variety of reasons unrelated to inflammatory processes and/or infection; (b) patients fulfilling SIRS criteria in whom neither clinical state nor microbiological findings suggested infection; and (c) SIRS patients with sepsis in whom the following additional criteria were met. These included either (i) isolation of an organism considered of pathogenic significance from an otherwise sterile site (blood, peritoneal cavity or lung via nondirected bronchial lavage), which led to directed antibiotic therapy, or (ii) isolation of an organism of recognized pathogenic potential from an intravascular catheter removed for reasons considered to be infection-related, and which led to improvement of physiological parameters consistent with that clinical suspicion, invariably associated with administration of antibiotics. In the latter case, antibiotic therapy was in many cases instituted before microbiology results were available. All patients were screened on a daily basis for the presence of pathogenic organisms by the culture of blood and urine and where indicated by the biopsy or aspiration of potentially infected sites. Where infections were associated with several organisms, microbiological data were reviewed in order to determine which organism was most likely to be responsible for disease on the basis of organism identity, density and anatomical source. Patients were designated as SIRS without sepsis when no positive cultures were obtained 72 h preceding and 48 h following the day of investigation. Healthy individuals with no obvious signs of infection were also studied to serve as a contemporaneous control group. Data concerning these three groups is shown in Table 1. At the time of the FACS analysis the operator of this technique was not aware of the clinical classification of patients included in the study.
Table 1. Details of patients and control subjects included in the study
Mortality refers to patients studied who later died in the ICU (Intensive Care Unit). *Some patients were infected with more than one organism. GNR, Gram negative rods; GPC, Gram positive cocci; Fungi, Candida spp. †Some patients were considered to harbour organisms of pathogenic significance from more than one site. Line, central venous catheter; blood, blood culture isolate; lung, isolate obtained from bronchoscopy specimen or nondirected bronchial lavage; sterile site, urine, peritoneal or pleural cavity; bone/soft tissue, osteomyelitis or deep tissue isolate. N/A, not applicable.
Normal healthy controls
43 (20 −66)
SIRS patients - sepsis
63 (44 −76)
20 (10 −29)
SIRS patients + sepsis
59 (21 −77)
21 (11 −33)
GNR – 11 GPC – 11 Fungi – 4
Line – 11 Blood – 5 Lung – 12 Sterile site – 4 Bone/soft tissue – 3
Whole blood analysis of CD64 expression
The method is based upon a rapid fixation procedure . Briefly, 1 ml of blood anticoagulated with sodium citrate was mixed with 1 ml LDS-751 (freshly made working solution) and cooled rapidly to 4°C. From each blood sample, two 20 µl aliquots were diluted in 100 µl of 0·1% bovine serum albumin/phosphate buffered saline (BSA/PBS). To the first aliquot was added 20 µl of FITC-labelled anti-CD64 antibodies (Medarex Inc, Annandale, NJ, USA), and to the second 20 µl of 0·1% BSA/PBS. Both were incubated for 5 min at room temperature in the dark before being treated with 2 ml of warmed Lyse solution (8 mg/ml NH4Cl, 1 mg/ml EDTA-NA3, pH 7·4) to remove contaminating erythrocytes. They were further incubated for at least 15 min, with mixing at 7 min, at room temperature in the dark. Samples were washed twice with 0·2% BSA/PBS at 400 g for 4 min. The final cell pellet was resuspended with 0·5 ml of fixative solution (1% formaldehyde with 0·1% BSA/PBS, pH 7·4) and stored at 4°C in the dark until flow cytometric analysis. All samples were analysed within 4 h poststaining.
All samples were analysed by a FACScan flow cytometer (Becton Dickinson) with Consort 32 Lysys version 1·02 software using the slow flow rate. Polymorphonuclear leucocytes were distinguished from other leucocytes and platelets by their characteristic side scatter. Results were displayed on an FITC and side-scatter histogram. In single parameter histograms of FITC fluorescence (log10 scale), the lower cursor was usually set so that no more than 1% of the cells in the negative control sample of each series stained positively with the isotype control antibody. The percentage of CD64-positive stained cells was obtained by constructing FL1 (FITC) histograms from gated PMN populations using both autofluorescence and labelled samples.
Calibration procedures for measuring the number of CD64 molecules
Prior to each experiment the flow cytometer was calibrated to ensure no changes had occurred in instrument sensitivity. The procedure involved the use of Quantum 26 beads (Flow Cytometry Standard Corp, San Juan, USA) which allowed detection limits to be expressed in molecules of equivalent soluble fluorochrome (MESF). Briefly, this technique makes use of five bead populations of equal size; four bead populations contain a known number of fluorescein molecules per bead, and the fifth acts as a negative control. A calibration plot was drawn whereby the five bead populations fell at specific fluorescence channel numbers. Samples of unknown fluorescence intensity were then measured and plotted against this calibration curve. Specifically, these calculations were made using QuickCal V.2 for WinList (Verity House Software Inc). The PMN population was gated by forward and side scatter criteria and redisplayed as a FL1 (FITC) histogram using a four-order log scale. The values for the fluorescence distribution obtained were plotted back onto a standardized linear regression line obtained using the quantum beads. The number of CD64 molecules was calculated directly, taking into account the fluorescence labelling ratio of the antibody, which was 2·3 : 1 FITC molecules per antibody molecule.
Anti-CD64 antibodies and the attachment of PMNs to endothelial monolayers
The method for measuring the binding of PMNs to endothelial cells is described in detail elsewhere . Briefly, endothelial cells were removed from human umbilical cord veins by treatment with 200 µl collagenase II solution (Sigma Chemical Co, Poole, UK) and resuspended in DMEM supplemented with 20% fetal calf serum (FCS), 2 mm glutamine, 200 U/ml penicillin, 100 U/ml streptomycin and gentamycin (Gibco, Paisley, UK). Endothelial cells were grown in gelatin (1% w/v)-coated flasks (Costar, Cambridge, MA, USA) in a 10% C02-humidified atmosphere at 37°C. When confluent, cells were detached with 3 ml trypsin-EDTA (Sigma), seeded onto gelatin-coated 96-well plates (Costar) and again grown to confluence. Identification of endothelial cells was confirmed by their characteristic morphology and by immunofluorescence staining with antibodies against human factor VIII-related antigen and human endothelial cells.
For the isolation of PMNs, 20 ml of heparinized blood were diluted with 50 ml of nine parts 0·16 mol/l NH4Cl (BDH, Poole, UK): 1 part 0·17 mol/l Tris (BDH), pH 7·4, and allowed to stand for 10 min at room temperature. Following centrifugation at 400 g for 10 min the supernatant was discarded and the lysis stage repeated on two occasions. The PMN pellet was washed three times (50 g for 10 min) in Hanks's balanced salt solution without calcium and magnesium (Sigman). Enriched preparations of PMNs (purity > 96%) were radiolabelled with Na251CrO4 (Amersham International plc, Aylesbury, UK) for 45 min at 37°C at a concentration of 3 µCi/106 cells, washed three times with DMEM plus 5% FCS and resuspended in DMEM with 10% FCS to 2 × 106 cells/ml. Prior to the adherence assay many of the endothelial monolayers were treated for 5 h with 10 U/ml recombinant TNFα (supplied by Dr A. Meager, National Institute for Biological Standards and Control, UK). To assess the contribution of CD64 to adhesion, radiolabelled PMNs (4 × 106 in 350 µl medium) were pretreated either with a 1 : 50 dilution of anti-CD64 monoclonal antibodies (clone 197; Cambio, Cambridge, UK) or with isotype control immunoglobulin (IgG2a) for 30 min at 4°C. The cells were washed before being overlaid onto the endothelial monolayers. All wells were washed twice in DMEM without serum prior to the introduction of 100 µl labelled PMNs (2 × 105 cells/well). After incubation at 37°C for 1 h the non-adherent leucocytes were removed by five washings, the monolayer disrupted with 200 µl of 0·1 m NaOH and the lysate removed and counted in an auto-gamma scintillation counter. Each test involved the use of at least six randomly allotted wells. Leucocyte adhesion was expressed in terms of the percentage of leucocytes originally dispensed onto the endothelial monolayers. It was calculated as follows:
The percentage of PMNs expressing CD64 from the three groups of subjects was normally distributed and the results are therefore presented as the mean ± standard deviation. In the assessment of the number of CD64 molecules on PMNs the data was not normally distributed and the results were therefore expressed as median values. Comparisons between more than two groups of normally distributed data were addressed by analysis of variance (anova) using Bonferroni's correction for multiple testing. This was undertaken by a commercial software package (Graphpad Prism 2·01) where P < 0·01 was considered as significant. Differences between two groups of normally distributed data were assessed by the two-tailed Student's t-test. When the data were not normally distributed comparisons between three groups of variables was examined by the Kruskal–Wallis statistic with inclusion of the Dunn's multiple comparison test and for the comparison of two groups of variables the Mann–Whitney test was used.
Distribution of PMNs bearing CD64
Figure 1 shows the distribution of blood PMNs expressing CD64 in patients who fulfilled the SIRS criteria, in patients who were in the ICU but had no evidence of SIRS and in normal healthy subjects (controls). There were far more CD64-positive PMNs in the blood of patients with SIRS (mean 65%, range 23–98%) than in the blood of non-SIRS patients in the ICU group (mean 42%, range 17–69%; P < 0·02) and in healthy subjects (mean 19%, range 3–38%; P < 0·001). Although the percentage of PMNs bearing CD64 in the ICU control patients was twice that of the healthy control subjects this difference was not statistically significant (P < 0·05). Further examination of the SIRS group (Fig. 2) revealed that patients with sepsis had a greater number of circulating CD64+ PMNs (mean 71%; P < 0·02) than patients without sepsis (mean 55%). Figure 2 also shows that the high prevalence of CD64+ PMNs in SIRS patients with sepsis was not restricted to fungal infections or to patients infected by either Gram-negative or Gram-positive organisms.
Number of CD64 receptors on PMNs
In the second stage of the investigation the PMNs from all of the above subjects were also analysed for CD64 receptor expression density. Figure 3 shows that the number of CD64 receptors is higher on PMNs from SIRS patients with sepsis (median 1331 molecules/cell) than PMNs from healthy control subjects (median 678 molecules/cell; P < 0·01). The median number of CD64 molecules on PMNs from ICU control patients was 971 molecules/cell and this distribution was not different from the PMNs of healthy subjects (P > 0·05) and of patients with SIRS (P > 0·05). Of the 10 SIRS patients who had in excess of 2000 CD64 receptors on their PMNs, only one was diagnosed as having SIRS without sepsis. This point is further illustrated in Fig. 4, which shows that the number of CD64 receptors is higher on PMNs from SIRS patients with sepsis (median 1905) than PMNs from SIRS patients without sepsis (median 963; P < 0·01). Interestingly, the density of CD64 receptors on the SIRS patients without sepsis is very similar to the values obtained from healthy subjects and ICU control patients. The PMNs bearing large numbers of CD64 receptors were present in the blood of patients with fungal, Gram-negative and Gram-positive infections.
CD64 and PMN binding to endothelium
To ascertain whether CD64 expression contributed to the attachment of PMNs to endothelium, PMNs from three SIRS patients with sepsis and three patients without sepsis were treated with anti-CD64 antibodies prior to their overlaying onto untreated and TNFα-treated endothelial monolayers in a quantitative radiometric adherence assay. Adherence values were compared with PMNs incubated with a similar concentration of isotype-matched control antibodies. Figure 5 shows that in each experiment the anti-CD64 antibodies impeded PMN attachment to TNFα-treated endothelium with an overall mean 24% inhibition of adhesion (P < 0·01). The antibodies did not significantly modify the binding of PMNs to untreated endothelial monolayers. To investigate if the PMNs from SIRS patients were exhibiting an abnormal binding to endothelial monolayers, each of the above 6 experiments also included PMNs from age- and sex-matched healthy individuals. The PMNs from the patients were found to be more adherent than control cells to both untreated (mean 34% increase; P < 0·05) and TNF-treated endothelial cells (mean 51% increase; P < 0·01).
The high mortality of SIRS may arise from an aberrant interaction of PMNs with blood vessel walls that leads to microvascular injury, tissue infiltration and multiple organ damage [31,32]. Activation of PMNs promotes aggregation  and binding to endothelium , both of which could result in microcirculatory obstruction, increased tissue extravasation and tissue damage by the extracellular release of lysosomal enzymes and oxygen reaction species. The high affinity Fc receptor for IgG, CD64, which is absent from most blood PMNs, is considered to be a marker of cell activation . In the present study the distribution of blood PMNs bearing CD64 was shown to be far higher in patients with SIRS than in healthy subjects and in patients needing ICU admission for causes unrelated to recognized inflammatory processes, infection or both. Within the patients fulfilling SIRS criteria, high levels of CD64-bearing PMNs and an increase in the number of CD64 molecules on the PMN surface, was particularly associated with sepsis. From the demonstration that anti-CD64 antibodies impeded the binding of PMNs from patients with SIRS to TNF-treated endothelium it appears that, in this disorder, CD64 is contributing to the interaction of PMNs with blood vessel walls.
Earlier reports described an increased expression of CD64 on blood PMNs in patients with bacterial infections [26,27] although it was not apparent whether the effect related to an increased density of the surface molecule, to more PMNs expressing CD64 or a combination of the two. The current study reveals a supranormal increase not only in the number of CD64 molecules expressed on the PMN surface of SIRS patients but also in the prevalence of PMNs bearing CD64. Since these observations were associated predominantly with SIRS patients with sepsis it appears that within this patient subgroup there has been both quantitative and qualitative changes in PMN expression of CD64. We found no association between the distribution and level of CD64 expression on PMNs and the type of organism responsible for the sepsis, unlike an earlier study which noted that infections induced by Gram-negative bacilli were more effective than streptococci or staphylococci in eliciting increased expression of CD64 on monocytes . Large variations in the distribution of CD64-positive PMNs in SIRS patients with sepsis may be due to the patients being at different stages of the infectious process, especially as resolution of infection results in a decline in the percentage of CD64-bearing blood PMNs  or that the site and the extent of tissue invasion by infectious organisms influences the character and magnitude of the PMN response. The patients included in the current investigation were not homogeneous in relation to either the type or the location of infectious organisms. To address the consideration of whether measurement of CD64 expression on PMNs could be used to discriminate SIRS patients with infection from those without will require an investigation of more tightly defined patient groups.
Our observation that the SIRS patients without sepsis also possessed more CD64-positive PMNs than healthy controls suggests that in the absence of infection an expansion in the number of CD64-bearing PMNs could arise from as yet undefined responses to trauma or inflammatory insult, which have the potential to induce a clinical state fulfilling SIRS criteria. Such non-infectious stimuli could be responsible for the increased incidence of CD64-bearing PMNs in the synovial fluid of patients with rheumatoid arthritis  and in the blood of sickle cell patients during the crisis phase of the disease . Alternatively, it is conceivable that increased numbers of CD64-positive PMNs in the SIRS patients without sepsis arise in response to localized infections or catheter or peg sites, which are often associated with intensive care patients. Additional studies are planned to ascertain whether sepsis-related induction of CD64 relate to infection per se rather than to the increased inflammation arising therefrom.
Two cytokines are known to upregulate CD64 expression on PMNs: IFNγ by a direct effect on circulating cells  and G-CSF by its activity on myeloid precursors in the bone marrow . In response to invasion by microbial pathogens both cytokines are generated by components of the immune system and blood levels of G-CSF are increased during the leucocytosis accompanying bacterial infections . High concentrations of the pro-inflammatory cytokines IL-1 and TNFα are associated with tissue damage and multiple organ failure [34,35], but there is no uniform agreement as to whether circulating levels of these cytokines and others, including G-CSF, are consistently elevated in patients with sepsis [36–40]. It seems unlikely that CD64-bearing PMNs originate from an environment with high local concentrations of IFNγ since it is generally thought that PMNs do not re-enter the circulation after tissue extravasation, even though lymph from thoracic ducts of patients with SIRS contains a disproportionately high number of PMNs . Some investigators question the pathological significance of IL-1 and TNFα to sepsis. Pretreatment of animals with IL-1 and TNFα antagonizes tissue damage arizing from lethal infection by intracellular bacteria or Gram-negative bacilli [42–44] and plasma from patients with severe sepsis, known to contain high levels of pro-inflammatory cytokines and LPS, fails to activate monocytes because of their antagonism by naturally occurring inhibitors . Against this background it seems unlikely that the high prevalence of CD64-positive PMNs in SIRS is due to the activity of circulating cytokines.
It is the animal models of sepsis that provide the most convincing evidence of the pathological role of PMNs in microvascular injury. Activation of both PMNs and endothelia is associated with microvascular injury ; mice rendered neutropenic survive an otherwise lethal intravenous injection of endotoxin and disease progression is inhibited by the administration of antibodies against adhesion molecules which promote PMN attachment to endothelium . As stated earlier, PMN acquisition of CD64 expression is associated with cell stimulation and the high expression of CD11b on PMNs from SIRS patients (17) combined with a low expression of L-selectin (18) provides additional evidence of PMN activation. Both CD11b and L-selectin contribute to the binding of PMNs to blood vessel walls and their abnormal expression on PMNs from SIRS patients is in accord with the increased adhesiveness to endothelial monolayers of PMNs following activation . In the present study the attachment of PMNs from SIRS patients to cultured endothelial cells stimulated with TNFα was inhibited by anti-CD64 antibodies. This observation is particularly pertinent to the pathology of SIRS as TNFα activation of endothelial cells up-regulates the expression of vascular adhesion molecules that appear on blood vessel walls adjacent to inflammatory lesions. The inhibitory activity of the antibodies may have arisen from an indirect interference with the expression of known adhesion molecules in close proximity to CD64 or a direct masking of adhesion-promoting epitopes on the CD64 molecule itself. Experiments are in progress to resolve this issue. The PMNs from SIRS patients were more adherent to untreated and TNFα-treated endothelial cells than control PMNs, which is in accord with the observation that upon activation PMNs increase their attachment to endothelium . Circulating PMNs of SIRS patients exhibit an increased production of intracellular H2O2 and phagocytosis  and this enhanced functional status is supported by the acquisition of CD64 molecules whose cross-linking leads to antibody-dependent cytotoxicity, phagocytosis, degranulation, production of oxygen-reactive species and release of cytokines [24,49]. Our recent observation that most PMNs which adhere to cultured endothelial cells express CD64  advances the view that the pathological manifestations of SIRS may arise from the untoward interaction of CD64+ PMNs with blood vessel walls and prompts the consideration of using CD64 expression as an objective marker of sepsis in the context of SIRS.