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. 1998 May;112(2):334–340. doi: 10.1046/j.1365-2249.1998.00559.x

Effects of granulocyte colony-stimulating factor (G-CSF) treatment on granulocyte function and receptor expression in patients with ventilator-dependent pneumonia

W N M Hustinx *,†,§, C P M Van Kessel †, E Heezius †, S Burgers ‡, J-W Lammers ‡, I M Hoepelman †,§
PMCID: PMC1904975  PMID: 9649199

Abstract

Considerable experimental evidence in animals suggests that treatment with G-CSF may have a beneficial effect in the management of severe infections in non-neutropenic hosts. This beneficial effect is attributed to an enhancement of granulopoiesis and neutrophil function, the latter possibly involving up-regulation of receptors on neutrophils that are involved in antibody-mediated cytotoxicity and killing of microorganisms. We compared neutrophil function and phenotype in blood and bronchoalveolar lavage fluid (BALF) of 10 patients with severe ventilator-dependent pneumonia, at baseline and following initiation of G-CSF treatment as adjunct to standard therapy. G-CSF treatment was associated with three-fold increased blood neutrophil counts at day 3 of treatment compared with baseline counts. Mean serum G-CSF concentration increased from 313 to 2007 pg/ml. After correction for lavage dilution effects, BALF G-CSF levels did not differ significantly from baseline, nor did neutrophil receptor expression (FcγRI, FcγRII, FcγRIII, CR3, and l-selectin) or indicators of neutrophil function such as respiratory burst activity, phagocytosis and killing of Candida albicans in BALF or blood. The mortality in this group of patients was 30% and compared favourably to the APACHE II-derived predicted mortality of 60%. We conclude that the possible therapeutic benefit of G-CSF administration in the early phase of severe bacterial pneumonia is not readily explained by its effect on baseline indicators of neutrophil function or receptor expression.

Keywords: ventilator-dependent pneumonia, G-CSF, granulocyte function

INTRODUCTION

Adjunctive treatment strategies are clearly needed to improve outcome in serious and complicated infections. To date, agents that have been evaluated clinically such as MoAbs against endotoxin, tumour necrosis factor (TNF), soluble TNF receptors, and IL-1 receptor antagonists, have all failed to confer a convincing beneficial effect [1]. G-CSF is a haematopoietic growth factor that promotes the growth of polymorphonuclear leucocytes. It is produced by vascular endothelial cells, fibroblasts, monocytes, and macrophages. In vivo, G-CSF production is stimulated by a variety of stimuli such as (endo-)toxins, bacteria, fungi and certain viruses. The prophylactic use of recombinant human (rh) CSF (including rhG-CSF) is currently recommended in the setting of cancer chemotherapy, among others to reduce the likelihood of and reduce infectious complications associated with febrile neutropenia in high risk patients. Their therapeutic use, as adjuncts to antibiotics, is to be reserved for neutropenic patients with prognostic factors that are predictive of clinical deterioration, such as pneumonia, hypotension, fungal infection, or sepsis [2]. In healthy volunteers, the function of rhG-CSF-induced neutrophils seems to be at least equal to that of normally produced neutrophils [3]. Apart from promoting granulopoiesis, rG-CSF has also been shown to increase neutrophil survival time [4], superoxide anion production, and chemotaxis [5,6], phagocytosis [5], and lipopolysaccharide (LPS) clearance. Up-regulation of the FcγRI (CD64) and RII (CD32), CR1 (CD35), CD14, TNF, and IL-1 receptors and of several receptors involved in neutrophil adherence like CR3 (CD11b/18) and l-selectin [7–9] occurs together with, and is perhaps in part responsible for, these functional changes. In vivo, rhG-CSF has been reported to have a beneficial effect in a variety of animal models of infection [10], including pneumonia models [11,12]. Together, these data are considered to justify clinical evaluation of rhG-CSF as an adjunctive treatment for serious infections in the non-neutropenic host [12,13].

rhG-CSF was shown to be safe and well tolerated by patients with severe community-acquired pneumonia (CAP) [14]. In a recently concluded phase III study, involving 765 non-neutropenic patients with CAP, treatment with rhG-CSF was associated with a significant reduction in the incidence of ARDS, of diffuse intravascular coagulation, of empyema formation, and with a higher rate of complete resolution of chest x-ray abnormalities by day 28, compared with placebo. However, mortality was not significantly different between treatment arms [15]. Here, we report the results of an open study investigating the effect of rhG-CSF treatment on the expression of receptors and on the function of neutrophils isolated from blood and in bronchoalveolar lavage fluid (BALF) of 10 patients with severe ventilator-dependent pneumonia. The aim of this study was to document rhG-CSF-associated changes in baseline levels of neutrophil function and receptor expression that might explain some of the presumed beneficial effects of rhG-CSF in the study patients with severe CAP [15].

SUBJECTS AND METHODS

Patients

The study protocol was approved by the institutional review board of Utrecht University Hospital. Inclusion required written informed consent from patients or their relatives. Patients requiring mechanical ventilation for a presumed diagnosis of pneumonia, according to the criteria adopted by the Infectious Diseases Society of America [16], were eligible for inclusion. The following exclusion criteria applied: < 18 years of age, pregnancy, enrolment in any other investigational protocol, any concomitant end-stage disease, neutropenia ( < 500 neutrophils/ml), concurrent immunomodulatory treatment other than non-steroidal anti-inflammatory drugs or corticosteroids, documented hypersensitivity to rhG-CSF, or mechanical ventilation for more than 24 h following a presumed diagnosis of pneumonia. Healthy volunteers were donors of blood from which neutrophils were isolated for use as controls in the ex vivo experiments described below.

rhG-CSF

Commercially available recombinant-methionyl G-CSF (filgrastim; Neupogen) was used. Patients received once daily subcutaneous injections of 300 μg (if < 70 kg body wt) or 480 μg (if ≥ 70 kg body wt) until off-ventilator, with a maximum of 14 days. Administration of G-CSF was to be suspended once the leucocyte count exceeded 60 000 cells/ml. Administration of reduced (50%) doses was to be resumed when the leucocyte count had subsequently fallen to ≤ 30 000 cells/ml.

Bronchoalveolar lavage

Using a flexible fibreoptic bronchoscope wedged in the affected lung segment, bronchoalveolar lavage (BAL) was performed by instillment and subsequent recovery of an aliquot of 20 ml normal saline, followed by three consecutive aliquots of 50 ml normal saline. The mean recovery was 48% (range 28–86%). The first aliquot was discarded, whereafter all subsequently recovered BALF was pooled in a silicone-lined bottle. These samples were immediately centrifuged (300 g, 10 min) and supernatants were stored at −20°C until further use. Pellets were gently resuspended in a small volume of RPMI, containing 0.05% human serum albumin (HSA) (RPMI/HSA) and further processed for cell counting, differentiation, and neutrophil function tests as described below.

Cells

Blood neutrophils were isolated from heparinized blood by Ficoll density centrifugation as previously described by us [17].

Neutrophil chemiluminescence

The oxidative burst capacity and the priming state of neutrophils, as a measure of cell activation, were measured in whole blood and resuspended BALF cells, using a chemiluminescence assay. Tubes precoated with different priming agonists (C5a, platelet-activating factor (PAF), fMLP; ExOxEmis) were mixed with C3b-opsonized Zymozan and placed into the transport chain of a Berthold luminometer (Autolumat LB 953). EDTA blood or BALF cells were diluted 100-fold with PBS containing 5% glucose. Prewarmed luminol solution (Hanks' balanced salt solution (HBSS) with 0.05% HSA and 150 μm luminol), lucigenin solution (ExOxEmis), and diluted blood were automatically injected into the tubes, and each tube was repeatedly measured for 20 min at 37°C. The ct/min were recorded and, for each sample, calculated as total counts over a 20-min period. The values obtained were corrected for the absolute neutrophil counts (ANC), as determined by electronic counting (CoulterCounter) and a (Diff Quick-stained) blood smear or cytospin preparation.

Neutrophil chemotaxis

Directed migration of isolated neutrophils towards Zymosan-activated serum was determined using the under agarose technique [18].

Neutrophil receptor expression

The expression of several neutrophil receptors was studied in whole EDTA-treated blood and in resuspended BALF cells by direct immunofluorescence with specific MoAbs: MoAb 22–FITC (anti-CD64, FcγRI), IV.3–FITC (anti-CD32, FcγRII), and 3G8–FITC (anti-CD16, FcγRIII) were obtained from Medarex; MoAb 44a–FITC (anti-CR3) was prepared in our laboratory from an ATCC hybridoma; MoAb Leu-8–FITC (anti-l-selectin) was obtained from Becton Dickinson (Mountain View, CA). Fifty microlitres of blood or resuspended BALF cells were mixed with saturating amounts of MoAb and kept on ice for 30 min. Samples were then lysed and fixed with lysis buffer (Becton Dickinson), centrifuged (10 min, 300 g), washed once with ice cold RPMI/HSA, and again centrifuged. The cell pellet was resuspended in 0.5% paraformaldehyde and the sample was analysed on a FACScan flow cytometer (Becton Dickinson). Samples of 10 000 cells were analysed for neutrophil-associated fluorescence by appropriate gating of forward- and side-scatter parameters to exclude cell debris, large aggregates, and other cell types from the analysis.

Phagocytosis

The phagocytic capacity of isolated blood neutrophils and of neutrophils in BALF was measured with FITC-labelled Staphylococcus aureus, Cowan EMS, and flow cytometry, as previously described by us [17]. Bacteria and cells were mixed at a ratio of 10:1 in the presence of different concentrations of pooled human serum. After 15 min the reaction was stopped by fixation, and phagocytosis was expressed as the percentage of neutrophils with associated FITC-labelled bacteria. We did not discriminate between internalized and attached bacteria.

Killing of Candida albicans

Using flow cytometry, the killing capacity of isolated neutrophils was determined by the ability of Candida to retain a fluorescent probe. This technique was previously used by our laboratory for Cryptococcus neoformans [19]. Briefly, a clinical isolate of C. albicans was grown overnight in selective medium containing 6 μm 2,7-bis-(2-carboxyethyl)-5- (and-6)-carboxyfluorescein, acetomethyl ester (BCECF-AM), washed, and stored at −70°C in PBS containing 20% glycerol. An aliquot was thawed, washed, and spectrophotometrically adjusted to 1 × 107 cells/ml. Candida albicans was mixed with isolated neutrophils in a ratio of 3:1 and incubated for 30 min in the presence or absence of 3% normal serum at 37°C under shaking. An aliquot was taken from each tube and mixed with an equal volume of deoxycholate (25 mm) for 4 min at room temperature to solubilize the neutrophils. Then the samples were fixed with an equal volume of 1% paraformaldehyde and analysed in a flow cytometer for Candida-associated fluorescence. After exclusion of cell debris and cell aggregates, the fluorescence histogram of the Candida population was divided into viable, fluorescent cells and killed non-fluorescent cells (background fluorescence). The percentage of killing was corrected for the spontaneous loss of probe when C. albicans was incubated in medium alone.

Other laboratory assessments

Serum and BALF G-CSF levels were determined by a commercial ELISA system (Quantikine human G-CSF Immunoassay test kit; R&D Systems, Minneapolis, MN; lower limit of detection 39 pg/ml). Blood biochemistry and haematology parameters were measured by routine laboratory techniques. The BALF urea content was determined colourimetrically in urease-treated samples. To correct for lavage dilution effects, the results of BALF G-CSF assays were multiplied by the serum/BALF urea ratio [20].

Statistical analysis

Pretreatment and on-treatment continuous variables were compared by two-sample t-test. P≤ 0.05 was considered to denote a significant difference.

RESULTS

Patients

Fourteen of 16 consecutive patients requiring mechanical ventilation for severe pneumonia were eligible for inclusion. Ten patients consented to enter the study. Their mean (range) age was 54 years (39–74 years), their mean pretreatment APACHE II score was 27 (15–40), the mean baseline PaO2/FiO2 ratio 200 (104–356), and the mean number of days until off-ventilator was 10 (5–21). Nine patients had premorbid underlying conditions considered to increase their risk of acquiring serious infections. Three patients developed septic shock with multiple organ dysfunction. The all-cause mortality at day 28 after initiation of rhG-CSF treatment was 30%. Treatment was withdrawn in two patients. The mean APACHE II-derived predicted 28-day mortality [21] was calculated to be 60%. Bacterial pneumonia was culture-documented in all patients and considered hospital-acquired in five (50%) of them. Sputum cultures of six patients yielded single isolates of S. aureus (n = 3) and Pseudomonas aeruginosa (n = 3). Patients received rhG-CSF for a mean of 10 days (range 5–14 days). There were no noticeable adverse effects of rhG-CSF treatment in any patient. In only one patient was rhG-CSF administration suspended because of a neutrophil count exceeding 60 000 cells/ml. Details of the clinical and demographic characteristics are shown in Table 1.

Table 1.

Clinical characteristics of 10 patients with severe ventilator-dependent pneumonia, treated with recombinant human (rh)G-CSF

graphic file with name cei0112-0334-t1.jpg

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Blood and BALF neutrophil counts and G-CSF levels

Mean blood neutrophil counts had tripled by day 3 of G-CSF treatment (27.9 × 106 cells/ml, range 7.4–57.2 × 106 cells/ml) and peaked, virtually without exception, by day 7 (mean 45 × 106 cells/ml, range 28.6–96.5 × 106/ml) (Fig. 1). The mean percentage (range) of band forms was 12.6 (1–30) at baseline and 11.9 (1–37) on day 3. The mean BALF neutrophil count on day 3 (1.83 × 106 cells/ml) did not significantly differ from that at baseline (2.2 × 106 cells/ml) (Fig. 1). Mean (range ± s.e.m.) serum G-CSF concentrations on day 0 (n = 5) and day 3 (n = 6) were 313 pg/ml (182–599 pg/ml; ± 165 pg/ml) and 2007 pg/ml (39–4325 pg/ml; ± 1764 pg/ml), respectively (P < 0.05). In BALF, these concentrations were 4690 pg/ml (937–10 016 pg/ml; ± 3133 pg/ml) and 4432 pg/ml (339–15 719 pg/ml; ± 3757 pg/ml), respectively, after correction for dilution effects (Table 2).

Fig. 1.

Fig. 1

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Mean blood and bronchoalveolar lavage fluid (BALF) neutrophil counts in 10 patients with ventilator-dependent bacterial pneumonia before (day 0; □) and following the start of recombinant human (rh)G-CSF treatment (day 3; ▪). Error bars indicate s.e.m. *P < 0.001. ANC, Absolute neutrophil counts.

Table 2.

G-CSF levels in serum and bronchoalveolar lavage fluid (BALF) of patients with ventilator-dependent pneumonia before and on the third day after the start of treatment with recombinant human G-CSF

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Phagocytosis, oxidative burst, and chemotaxis

Without exception, phagocytosis by blood neutrophils of patients was increased, but not significantly, compared with that of neutrophils from untreated healthy controls, both at baseline and at day 3. Compared with baseline, no significant change in the level of phagocytosis by neutrophils in blood or BALF was observed on day 3 (Fig. 2). However, at baseline and on day 3, in the presence of low opsonin concentrations, blood and BALF neutrophils from patients showed higher levels of phagocytosis than did neutrophils from controls. The oxidative burst activity of neutrophils (both in blood and BALF) did not differ significantly between day 0 and day 3 of G-CSF treatment (not shown). The mean chemotactic activity of neutrophils from the blood of patients decreased significantly (P < 0.05) from baseline to day 3, but was not significantly different from that of neutrophils from healthy controls, either at baseline or on day 3 of rhG-CSF treatment. Polymorphonuclear neutrophils (PMN) in BALF did not show chemotactic activity.

Fig. 2.

Fig. 2

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Phagocytosis by neutrophils isolated from blood or in bronchoalveolar lavage fluid (BALF) obtained from 10 patients with ventilator-dependent bacterial pneumonia before (day 0) (a) and following the start of G-CSF treatment (day 3) (b). Neutrophils were incubated with FITC-labelled Staphylococcus aureus, in the absence or presence of graded concentrations of human pooled serum (HPS). Phagocytosis was quantified by the number of fluorescent neutrophils. Neutrophils isolated from blood of healthy untreated donors served as control. Error bars indicate s.e.m. No significant differences were observed.

Killing of Candida albicans

Neutrophils isolated from the blood or in BALF of patients had a (non-significant) decreased ability to kill C. albicans compared with that of neutrophils from controls, both before and during G-CSF treatment (Fig. 3).

Fig. 3.

Fig. 3

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Killing of Candida albicans by neutrophils in blood or bronchoalveolar lavage fluid (BALF) obtained from 10 patients with ventilator-dependent bacterial pneumonia before (day 0) and following the start of G-CSF treatment (day 3). Neutrophils isolated from blood of healthy untreated donors served as control. Error bars indicate s.e.m. No significant differences were observed between day 3 and baseline in corresponding samples. *P < 0.05 control versus BALF on day 3.

Neutrophil receptor expression

Isolated neutrophils from patients and healthy controls were evaluated for receptor expression before (day 0) and during (day 3) rhG-CSF treatment. The following receptors were studied: FcγRI, FcγRII, FcγRIII, CR3, and l-selectin. No significant differences were observed on day 3 compared with baseline levels. Baseline expression of FcγRI, FcγRII, and CR3 receptors tended to be higher in neutrophils isolated from BALF than in neutrophils isolated from the blood of patients and healthy donors (controls), probably indicating that the cells were primed. Expression of the CR3 receptor on BALF neutrophils was significantly higher than that on blood neutrophils from patients and healthy controls on day 3 of treatment. On day 3, FcγRI receptor expression on neutrophils in BALF and blood from patients was significantly increased compared with that of controls (P < 0.05). The expression of FcγRIII and l-selectin receptors tended to be lower in neutrophils from patients (both in BALF and blood) than in neutrophils from controls. These differences were not significant (Fig. 4).

Fig. 4.

Fig. 4

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Mean expression of FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), CR3 (CD11b) and l-selectin (CD62L) receptors on neutrophils isolated from blood and in bronchoalveolar lavage fluid (BALF) from 10 patients with ventilator-dependent pneumonia, before (day 0,(a)) and during (day 3, (b)) treatment with recombinant human (rh)G-CSF. Neutrophils isolated from blood of healthy untreated donors served as control. Error bars indicate s.e.m. No significant differences were observed between pretreatment and treatment levels of expression of individual receptors. *P < 0.05 versus control; **P < 0.01 versus blood neutrophils of controls and patients.

DISCUSSION

In the present study we have shown that rhG-CSF treatment of patients requiring mechanical ventilation for severe pneumonia was well tolerated and associated with a three-fold increased mean blood neutrophil count on day 3 of treatment, compared with baseline counts. The number of neutrophils in BALF was not affected by rhG-CSF treatment. Neither the expression of neutrophil FcγRI, -RII, and -RIII, CR3, or l-selectin receptors on neutrophils, nor the studied indicators of neutrophil function (phagocytosis, oxidative burst, chemotaxis, killing of C. albicans) on day 3 of rhG-CSF treatment were significantly different from corresponding baseline values. These findings contrast with the significantly increased levels of FcγRI receptor expression following rhG-CSF treatment reported by others [22,23]. However, they investigated neutrophils in healthy volunteers or in non-infected patients, which may explain the much larger difference between pretreatment and treatment levels of FcγRI expression. This hypothesis is supported by the fact that baseline (endogenous) G-CSF concentrations (313 ± 165 pg/ml) were significantly elevated in our patients compared with values reported for healthy volunteers and non-infected patients [24]. The higher G-CSF concentrations in BALF compared with blood, as found at baseline, may be related to the local production of G-CSF by alveolar macrophages [25]. Elevated endogenous G-CSF concentrations have been reported to accompany the acute phase of bacterial infection [26]. We previously reported similar findings in a study with murine anti-TNF MoAb in patients with sepsis syndrome and septic shock [27]. In this study, baseline mean (± s.d.) endogenous G-CSF concentrations ranged from 1507 ± 934 pg/ml in patients with negative blood cultures to 175 607 ± 171 103 pg/ml in patients with documented Gram-negative bacteraemia. Elevated endogenous G-CSF concentrations may have caused the high(er) baseline levels of neutrophil receptor expression and functional activity of neutrophils isolated from our patients, and may explain why no significant differences were observed between values at baseline and on day 3. We do not know if this lack of difference is related to the timing (day 3 of G-CSF treatment) chosen for comparison with baseline. Spiekerman et al. [23] reported an enhancement of receptor expression and neutrophil function within 2–4 days of rhG-CSF treatment in patients undergoing chemotherapy for malignancy. Allen et al. [28], studying the effect of G-CSF administration on neutrophil function and receptor expression in healthy volunteers, reported significantly increased PMN function by day 3 with a maximum effect by day 5 of G-CSF treatment. Our findings suggest that the mechanism of any added protection conferred by concomitant rhG-CSF treatment of patients with acute infections does not involve receptor up-regulation and enhancement of neutrophil function. This leaves the question open by what mechanism the benefit of adjunctive rhG-CSF treatment, as reported in a range of experimental studies under non-neutropenic conditions (reviewed in [10]), is achieved.

In these studies, rG-CSF was efficacious, whether administered prophylactically or therapeutically. Enhanced neutrophil recruitment to the site of infection [29], attenuation of the systemic TNF inflammatory response [29–31] and, possibly, synergism with antibiotic therapy [13,32] are suggested mechanisms for the protective or therapeutic action of rG-CSF. It was initially feared that administration of rhG-CSF to patients with sepsis might lead to or aggravate ARDS, through rhG-CSF-induced enhancement of neutrophil recruitment to sites of infection. However, studies in pneumonia models have provided evidence to the contrary. In our patients, BALF neutrophil counts did not increase with rhG-CSF treatment, although we do not know to what extent such counts adequately reflect changes in the interstitium. A similar finding was reported in bone marrow transplant patients [33]. In a study of neonatal sepsis, rhG-CSF administration was not associated with pulmonary or other organ toxicity [34]. The recent consensus statement by the American Thoracic Society (ATS) [35] has indicated that hospital-acquired pneumonia (HAP) continues to be the number one cause of death from nosocomial infection. On average, the attributable mortality of HAP is estimated to be 25–35%. The associated mortality may, however, increase dramatically to levels of ≥ 70% when HAP is complicated by signs and symptoms consistent with sepsis.

A better understanding of the pathogenesis of severe HAP (and sepsis, for that matter) and additional therapeutic options are clearly needed to improve the outcome of severe HAP and CAP. The ATS consensus statement supports the clinical evaluation of cytokines such as rhG-CSF and interferon-gamma as possible adjuncts to standard therapy for severe HAP. This may also apply to other cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF) [13]. The mortality in the group of patients described here was 30% and compared favourably with the APACHE II-derived mean predicted mortality of 60%. We conclude that the possible therapeutic benefit of rhG-CSF administration in the early phase of severe ventilator-dependent pneumonia is not readily explained by its effect on baseline indicators of neutrophil function or receptor expression. Clearly, a blinded placebo controlled study is needed to substantiate this seemingly beneficial effect.

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