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Blood, Vol. 92 No. 11 (December 1), 1998:
pp. 4366-4374
In Vivo Treatment With Granulocyte Colony-Stimulating Factor
Results in Divergent Effects on Neutrophil Functions Measured In
Vitro
By
Patrick J. Leavey,
Karen S. Sellins,
Gail Thurman,
David Elzi,
Andrew Hiester,
Christopher C. Silliman,
Gary Zerbe,
J. John Cohen, and
Daniel R. Ambruso
From the Bonfils Blood Center and the Departments of Pediatrics,
Immunology and Biometrics, University of Colorado School of Medicine,
Denver.
 |
ABSTRACT |
We have studied the effects of granulocyte colony-stimulating factor
(G-CSF) administration to normal individuals on a variety of functional
and biochemical neutrophil characteristics that relate to host defense.
G-CSF adversely affected neutrophil (polymorphonuclear leukocyte
[PMN]) chemotaxis. While this could be partially
explained by reduced assembly of neutrophil F-actin, we also recognized an elevated cytosolic calcium mobilization and a normal upregulation of
neutrophil CD11b. G-CSF resulted in reduced PMN killing of Staphylococcus aureus with a 10:1 (bacteria:neutrophil) ratio and
normal killing with a 1:1 ratio. In association with this, we
demonstrated divergent effects on the respiratory burst of intact cells
and divergent effects on the content of marker proteins for neutrophil
granules. While G-CSF may have resulted in increased content of
cytochrome b558 in the cell membrane, it did not alter the
amounts of cytosolic oxidase components. After therapy, there was
normal content of the azurophilic granule marker, myeloperoxidase, decreased content of the specific granule marker, lactoferrin, and
normal content of lysozyme (found in both granules classes). Finally,
G-CSF therapy markedly reduced the apoptotic rate of the isolated
neutrophil. Therefore, considering disparate functional and biochemical
activities, the real benefit of G-CSF therapy may lie in enhanced
number and survival of neutrophils.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
GRANULOCYTE colony-stimulating factor
(G-CSF) is a lineage-specific hematopoietic cytokine widely used to
combat the risk of infection resulting from both congenital and
acquired neutropenias.1-4 G-CSF stimulates the
proliferation and differentiation of hematopoietic
precursors5 and modifies functional, biochemical, and
survival characteristics of mature phagocytic cells.6 It has been assumed, therefore, that the therapeutic benefits of G-CSF
relate both to a greater number of circulating neutrophils and to an
enhanced phagocyte function providing protection against microbial
invasion.
Neutrophil (polymorphonuclear leukocyte [PMN]) function is a
complicated synthesis of biochemical processes serving as a primary mechanism in defense of the host. PMN cellular activity includes motility, allowing the phagocyte to reach and ingest invading microbes,
and killing, which involves both oxygen-dependent and independent
mechanisms. Inherent in this process, the mature neutrophil has a short
life span during which time it either becomes involved in an
inflammatory process or undergoes spontaneous cell death by
apoptosis.7
G-CSF modulates many of these neutrophil activities. In vitro, G-CSF
acts as a chemotactic stimulus for isolated neutrophils,8 primes the neutrophil nicotinamide adenine dinucleotide phosphate (NADPH) oxidase for subsequent activation,9
and induces an increased content of neutrophil alkaline
phosphatase.10,11 Incubation of isolated neutrophils, with
G-CSF, prolongs survival as measured by a decrease in
neutrophil apoptosis.12,13 Therapy with G-CSF decreases the
bone marrow transit time for neutrophils,14 but may not
prolong neutrophil survival in vivo.14 G-CSF treatment modifies the neutrophil expression of  2
integrins6,15 and receptors for IgG,6,16
reduces neutrophil chemotaxis,6 and enhances microbicidal
killing3,17,18 and antibody-dependent cellular
cytotoxicity.19
Despite these data, the in vivo capacity of the neutrophil produced
during exogenous administration of G-CSF, to provide host defense, is
not well understood. A more complete description of the circulating PMN
is required to understand the mechanisms by which G-CSF therapy
influences the end products of myelopoiesis. We have undertaken a
systematic study of the effects of G-CSF administration to normal
individuals on a variety of neutrophil functional and biochemical
characteristics that relate to host defense. These results describe
novel changes in the circulating neutrophil produced during G-CSF
administration.
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MATERIALS AND METHODS |
Materials.
Human recombinant G-CSF was obtained from Amgen Inc, Thousand Oaks, CA.
Superoxide dismutase (SOD), cytochrome c, Ficoll Hypaque, diisopropyl
fluorophosphate (DFP), phenylmethylsulfonyl fluoride (PMSF),
acetyl-leu-leu-arginine-AL (leupeptin),
formyl-methionyl-leucyl-phenylalanine (fMLP), phorbol myristate acetate
(PMA), platelet activating factor (PAF), bovine intestinal alkaline
phosphatase, -S-GTP, dimethyl sulfoxide (DMSO), sodium dodecyl sulfate
(SDS), and NADPH were purchased from the Sigma Chemical Co (St Louis,
MO). Rabbit antihuman lactoferrin (IgG) was purchased from US
Biochemicals (Cleveland, OH). Protein assay reagents and
standards were obtained from Pierce (Rockford, IL). Polyclonal
antibodies for the neutrophil NADPH oxidase components p67-phox,
p47-phox, and p40-phox were obtained from Dr Michael
Kleinberg (Greenebaum Cancer Center and University of
Maryland School of Medicine, Baltimore, MD), Dr Harry Malech (National
Institutes of Health [NIH], Bethesda, MD) and Dr Tony Segal (University of London Hospital, London, UK),
respectively.
Study population.
Healthy adult volunteers receiving no medications and with no
infections within the prior 2 weeks were enrolled into the study. All
subjects gave informed consent for this study through a protocol, which
was approved by the Colorado Multiple Institutional Review Board. As
experimental controls, we studied healthy adult volunteers not
receiving cytokine administration who also satisfied enrollment criteria. Each treatment volunteer [referred to as treated
volunteer(s)] was paired to a sex-matched control volunteer [referred
to as control(s)]. G-CSF was administered once daily by subcutaneous injection (10 µg/kg/d) for 7 consecutive days. Fourteen treated volunteers and controls were studied, nine men and five women, in each
study group. The administration of G-CSF was well-tolerated. Prominent
side effects included bone pain and headache in most treated volunteers
(grade I and II, mild and moderate severity). These symptoms were
responsive to nonnarcotic analgesics (ibuprofen and acetaminophen). In
one treated volunteer, G-CSF administration was discontinued on the
fifth day because of severe bone pain. All of the symptoms resolved
within 24 to 48 hours of discontinuing G-CSF. All treated volunteers
demonstrated a significant increase in total leukocyte count from a
mean of 4.87 ± 0.28 on day 0 to a mean of 31.61 ± 1.54 on day 4 (106 cells/mL; mean ± standard error of mean [SEM]),
a mean absolute neutrophil count of 2.64 ± 0.17 on day 0 to 24.92 ± 1.43 on day 4 (106 cells/mL; mean ± SEM) and a
mean percent band count of 1.1 ± 0.7 on day 0 (range, 0% to 7%)
to 18.6 ± 2.4 on day 4 (range, 5% to 39%). By day 1, the mean
percent band count was 15.3 ± 2; by day 2, 19.9 ± 3, and by day
3, 13.1 ± 2. Absolute neutrophil and band counts were based on
manual differential counts.
Cell isolation.
Heparinized (1 U/mL) peripheral blood samples were obtained before
cytokine administration on the first day of the protocol (day 0, D0)
and on the fifth day of administration (day 4, D4) from both study
groups (controls and treated volunteers). Blood samples were drawn
approximately 1 hour after the administration of G-CSF on day 4. Neutrophils were isolated by dextran sedimentation, Ficoll-Hypaque
centrifugation, and hypotonic cell lysis, as previously described.20 These cells were greater than 98% viable as
determined by the trypan blue exclusion test. Intact cells were used
immediately for functional and biochemical assays or stored at 2.5 × 107 cells/mL in 0.1% Triton X-100, at
 70°C for granule marker enzyme measurement.
Neutrophil chemotaxis, F-actin determination, and expression of
CD11b.
Neutrophil chemotaxis was determined by the leading front method using
a modified Boyden chamber technique, as previously described.21,22 Zymosan activated serum (2% by volume) was used as the chemotactic stimulus. To determine F-actin content, isolated neutrophils (1 × 106 cells), loaded with
NBD-phallacidin (Molecular Probes, Eugene, OR), were
incubated with fMLP (10 7 mol/L) in DMSO for 10 minutes at 37°C. F-actin content was then measured by
NBD-phallacidin staining, as previously published.23 Flow
cytometric analysis was performed using a BD FACScan (Becton Dickinson,
Mountain View, CA) calibrated with an excitation wavelength of 488 nm.
A total of 2,500 events was analyzed for each experiment. Results were
displayed on a log scale for FL1 mean channel fluorescence versus cell
count and were expressed as geometric mean channel fluorescence of
gated cells in FL1. The mean channel fluorescence was arbitrarily set
to 10 for unstimulated control. This produced a more useful
fluorescence curve allowing better interpretation of the shape of the
curve. Other samples were normalized accordingly, which allowed us to
compare changes in F-actin assembly on stimulation with greater
confidence; however, it did not allow a true interpretation of baseline
F-actin content in treated volunteers. The resting and stimulated
expression of neutrophil CD11b in response to three stimuli was also
determined. Briefly, isolated neutrophils (1 × 106/mL) were incubated with fMLP (106
mol/L), PMA (200 ng/mL) or PAF (2 × 106 mol/L)
for 5 minutes at 37°C. After stopping the reaction by adding
ice-cold Krebs-Ringer-Phosphate with Dextrose: 12.5 mmol/L Na2HPO4, 3 mmol/L
NaH2PO4, 4.8 mmol/L KCL, 120 mmol/L NaCl, 1.3 mmol/L CaCl2, 1.2 mmol/L
MgSO4·7H2O + 0.2% dextrose (KRPD), a phycoerythrin-labeled mouse antihuman CD11b antibody (Becton Dickinson) was added and allowed to incubate for 30 minutes at 4°C. The cells were then fixed with paraformaldehyde (1.6%). Cell surface CD11b was
detected with direct immunofluorescence using flow cytometry (Becton
Dickinson) by standard techniques. Results were displayed on log scale
for FL2 mean channel fluorescence versus cell count and expressed as
geometric mean channel fluorescence of gated cells in FL2. The mean
channel fluorescence was arbitrarily set to 10 for unstimulated control
CD11b and other samples were normalized accordingly.
Mobilization of cytosolic-free calcium.
The concentration of cytosolic calcium
[Ca2+]c was determined using the calcium
binding fluorometric dye indo-1,AM.24 Cells (2.5 × 107) were diluted to 5 × 106 cells/mL in
KRPD and incubated with 25 µg indo-1,AM (5 µmol/L final
concentration; Molecular Probes, Eugene, OR) for 10 minutes at
37°C. Cells were centrifuged for 8 minutes at 1,000g at
room temperature and resuspended at 1 × 106 cells/mL
in fresh KRPD at 37°C. Changes in the fluorescence ratio were
measured at the excitation wavelength of 355 nm and emission wavelength
of 485 nm and 405 nm in a Perkin-Elmer LS50B spectrofluorometer (Perkin-Elmer Corp, Norwalk, CT). For these measurements, 2 × 106 cells were diluted with 1 mL KRPD (final volume 3 mL)
in a standard cuvette with stirring at 37°C. Stimuli of 1 µmol/L
fMLP or 40 nmol/L PAF were added to the cells in the cuvette in the
spectrofluorometer. The Ca2+-saturated signal (r max) was
determined by lysing the cells with 0.1 mmol/L digitonin, and the
Ca2+-free signal (r min) was measured by subsequently
adding 5 mol/L EGTA, pH 7.35. [Ca2+]c was
calculated using Perkin Elmer Fluorescence Data Manager software and
the Grynkiewicz equation.25
Neutrophil bactericidal assay.
Bactericidal activity was measured as previously
described.22,26,27 Briefly, 1 × 106
neutrophils were incubated, at 37°C, with Staphylococcus
aureus (American Type Culture Collection [ATCC], Rockville, MD;
#2059) at 1:1 and, in some experiments, at 10:1 ratios
(bacteria:neutrophils) in the presence of 10% (by volume) pooled
normal serum. At 0, 30, 60, 90, and 120 minutes, 50-µL aliquots were
removed from the reaction tube and added to sterile H2O to
lyse the neutrophils. After serial dilutions appropriate to the initial
bacterial colony count, 50 µL of the resultant suspension was mixed
with trypticase soy agar in a sterile Petri dish. After incubation at
37°C overnight, bacterial colonies were counted and the surviving
bacteria at each sample time were expressed as a percent of initial
values.
Analysis of the neutrophil NADPH oxidase.
O2 production by intact neutrophils was
measured as the maximum initial rate of the SOD-inhibitable reduction
of ferricytochrome c.28 Neutrophils were stimulated by the
addition of 200 ng/mL PMA and 1 µmol/L fMLP. An additional
stimulation sequence included preincubation with 2 µmol/L PAF for 3 minutes at 37°C and then stimulation with 1 µmol/L fMLP.
O2 was also measured after PMN exposure
to opsonized zymosan (OZ), 1 mg/mL.20 Neutrophil
subcellular fractions were prepared in the presence of protease
inhibitors DFP, PMSF, and leupeptin by the previously published
technique of nitrogen cavitation and discontinuous sucrose
centrifugation.28,29 The maximal rate of
O2 production over 5 minutes was assayed
in the SDS cell-free system in the presence of NADPH (200 µmol/L) and
 S-GTP (10 µmol/L) by the SOD-inhibitable reduction of cytochrome
c,28 and was normalized for the protein content of both the
cytosol and membrane fractions. Subcellular fractions (membrane and
cytosol) were added together (patient + patient) or were mixed with
control fractions (patient + control) to correct for any abnormalities
documented.28 Three cytosolic components of the neutrophil
oxidase (p40-phox, p47-phox, and p67-phox) were analyzed by Western
blot using an enhanced chemiluminescence (ECL) detection system
(Amersham Corp, Buckinghamshire, UK). The total content of cytochrome
b558 in specific granules was measured as the sodium
dithionite reduced-minus oxidized spectrum using the absorbance
coefficient of 160 mmol/L1 cm1 at
428 nm by spectrophotometric analysis.28,30
Granule proteins.
Myeloperoxidase (MPO), lysozyme, and alkaline phosphatase were measured
by previously described spectrophotometric techniques.29,31 Lactoferrin content was determined with a competitive enzyme-linked immunosorbent assay (ELISA).29,32 Whole cell and
subcellular protein concentration was measured with the BCA protein
assay (Pierce, Rockford, IL) and granule marker protein content and alkaline phosphatase were normalized for whole cell protein content.
Neutrophil apoptosis.
Polymorphonuclear leukocytes were isolated from heparinized whole blood
by centrifugation over polymorphprep gradients (GIBCO-BRL Life
Technologies, Grand Island, NY). On day 4, the PMN isolated from
treated volunteers tended to be less dense. After two washes in
RPMI-1640 (Sigma), PMN were cultured at 3 × 106cells/mL in tissue culture medium containing RPMI-1640,
10% fetal bovine serum (Gemini Bioproducts, Calabasas, CA), 2 mmol/L
l-glutamine (Sigma), and 50 µg/mL gentamicin (Sigma). PMN were
cultured in the presence or absence of G-CSF (25 ng/mL). Apoptosis was
assessed at regular intervals between 0 hours and 48 hours or until
there was greater than 90% apoptosis. Apoptosis was determined
morphologically, as described previously.33 The percent of
apoptotic cells was determined by counting a minimum of 100 cells.
Statistical methods.
Results were analyzed by analysis of variance appropriate for repeated
measures data34 using the computer statistical software package SAS (SAS Institute Inc, Cary, NC). Neutrophil apoptosis was
interpreted by logistic regression to predict the expected time to 50%
apoptosis and these results were used in an analysis of variance model
suitable for repeated measures data.34 The statistical
significance reported here represents the comparison (day 0 v
day 4 for treated volunteers) unless otherwise expressed.
| |
RESULTS |
Neutrophil motility.
Neutrophil directed migration measured in a modified boyden chamber was
significantly reduced in treated volunteers after G-CSF administration
compared with pretreatment values and controls values (*P = .0001) (Fig 1). Although nondirected
migration was also decreased, this was not statistically significant.
The ratio of directed to nondirected migration in treated volunteers
was significantly reduced from 2.21 ± 0.14 on day 0 to 1.66 ± 0.16 on day 4 (P = .02).
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| Fig 1.
Neutrophil chemotaxis. Neutrophil chemotaxis was measured
to zymosan-activated serum as described in Materials and Methods. There
was significantly reduced chemotaxis after 5 days of G-CSF therapy
(*P = .0001). There was also a trend toward less nondirected
migration (buffer) after G-CSF therapy, but this difference was not
significant (P = .18). Results are expressed as distance
migrated through filter (microns) and represent mean ± SEM, n = 8.
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To further evaluate the basis for the chemotactic defect,
stimulus-induced increase of neutrophil F-actin and expression of CD
11b were measured. F-actin assembly in response to fMLP was reduced
after G-CSF administration in vivo. The content of F-actin, in response
to fMLP, was 16.43 ± 0.99 on day 4 (mean channel fluorescence ± SEM, n = 6) compared with 18.81 ± 0.60 on day 0 (P = .04).
Results for PMNs from controls on day 0 and day 4 were 19.84 ± 0.29 and 18.33 ± 0.98, respectively. In two treated volunteers, the
baseline F-actin content was determined on day 4. In both, the baseline content of F-actin was increased (14.64 and 13.53; mean channel fluorescence).
Resting and stimulated expression of neutrophil CD11b were also
examined. The agonists evaluated were PMA, fMLP, and PAF. No
significant effect of G-CSF administration on the upregulation of this
receptor was detected; the ratio of unstimulated:stimulated expression
was the same after administration as before
(Table 1). Thus, the impaired directed
migration of neutrophils demonstrated during treatment with G-CSF was
associated with diminished actin assembly, but normal 2
integrin expression.
Mobilization of cytosolic-free calcium.
We studied changes in [Ca2+]c after
incubation of neutrophils with fMLP and PAF to ascertain if
Ca2+ mobilization might explain the observed chemotactic
defect. Figure 2 presents a representative
study of changes in cytosolic calcium Ca2+ in response to
fMLP for one treated volunteer, before and after G-CSF administration.
There is a clear increase in the maximum [Ca2+]c reached after G-CSF administration,
while the rate of increase is the same compared with results obtained
before G-CSF administration began. Controls reached maximum
Ca2+ concentrations almost identical to results obtained
for the treated volunteer on day 0 (data not shown). Summary data for
the study groups (Table 2) show the
significant enhancing effect that treatment with G-CSF in vivo had on
the mean [Ca2+]c mobilization of isolated
neutrophils compared with day 0 values (P  .0005) and
compared with control values (P .003) for both PAF and fMLP.
There was a slight change in baseline values of [Ca2+]c for the PAF experiments (not
statistically significant) (Table 2). No changes were observed in
baseline values for fMLP, or in the rates of change for fMLP or PAF for
any group at any time of study.
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| Fig 2.
Neutrophil cytosolic calcium response. Cytosolic calcium
concentration (nmol/L) was measured over time in response to fMLP (1 µmol/L) as described in Materials and Methods. These results
demonstrate a representative study of one treated volunteer, of the six
pairs studied. There is a clear increase in peak
[Ca2+]c after G-CSF therapy in response to
fMLP compared with the response to fMLP before G-CSF.
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Neutrophil bactericidal activity.
The capacity to ingest and kill S aureus was studied to address
whether administration with G-CSF affected bacterial microbicidal function. Bactericidal activity was assayed at a bacteria:neutrophil ratio of 1:1 in five treated volunteers and was unaffected by G-CSF
administration (Fig 3A). However, on day 4 of G-CSF treatment, PMN bactericidal activity with ratios of 10:1
(bacteria:neutrophil) was reduced at all time points reaching
statistical significance at 30 minutes (*P = .0023) and 90 minutes (**P = .028) (Fig 3B).
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| Fig 3.
Neutrophil microbicidal activity. Neutrophil bactericidal
activity was measured as described in Materials and Methods. At a 1:1
ratio (bacteria:neutrophil) (A), the PMN bactericidal capacity was
normal after G-CSF therapy in vivo (normal, n = 15; treated
volunteers, n = 5). At 10:1 ratio (bacteria:neutrophil), the PMN
bactericidal capacity was reduced at all time points after G-CSF
treatment in vivo (B). The difference reached statistical significance
at 30 minutes (*P = .002) and at 90 minutes (**P = .028). Results are expressed as a percentage of the initial bacterial
count and represent mean ± SEM (normal, n = 13; treated volunteers,
n = 3).
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Neutrophil NADPH oxidase and granule marker proteins.
G-CSF has been shown to enhance the activity of the neutrophil NADPH
oxidase.2,35-37 We analyzed
O2 production in intact cells, as well
as in the cell free system to further define any changes induced by
G-CSF. NADPH oxidase activity of intact neutrophils in response to
several different stimuli is summarized in
Table 3. PMA-induced superoxide anion (O2) production was significantly
reduced on day 4 of G-CSF treatment (P .0001), as
was the PAF-primed/fMLP stimulated respiratory burst (P .0001). However, the production of O2
after fMLP stimulation was significantly higher on day 4 of G-CSF administration (P = .004). No differences were observed during G-CSF administration on the respiratory burst stimulated with opsonized
zymosan (Table 3).
On cellular activation, the membrane and cytosolic components of the
oxidase assemble in the plasma membrane or membrane of the
phagolysosome. We examined the activity of the neutrophil oxidase in
plasma membrane isolates in the cell-free system, as described in
Materials and Methods, to determine if any of the changes in oxidase
activity of intact cells reflect alterations in oxidase components.
Neutrophil membrane isolated from treated volunteers, when mixed with
autologous cytosol, produced 7.4 ± 1.9 nmol
O2-/min/mg protein on day 0 and 11.1 ± 1.5 nmol O2-/min/mg protein on day 4 (mean ± SEM, n = 8) (not statistically different). However, neutrophil membrane
isolated from treated volunteers, when mixed with cytosol isolated from
control neutrophils, produced 5.0 ± 0.7 nmol
O2/min/mg protein on day 0 and 10.7 ± 1.1 nmol O2/min/mg protein on day
4 (P = .01). These data suggest that the capacity of membrane
to support oxidase activity had increased after G-CSF therapy.
Additional studies evaluated cytosolic oxidase components.
Figure 4 is a representative study of two
treated volunteers and an untreated control and demonstrates similar
amounts of p67-phox, p47-phox, and p40-phox in cytosol before and after the administration of G-CSF. Despite an apparent decrease in p40-phox in #7 D0, there was no statistically significant effect
on the quantity of any oxidase protein studied, as measured by
densitometry (data not shown). This experiment was completed in eight
treated volunteers (data not shown).
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| Fig 4.
Neutrophil cytosolic NADPH oxidase components. Neutrophil
cytosolic oxidase components were examined as described in Materials
and Methods. (A) Results with antibodies to p47-phox and p67-phox. (B)
Results with an antibody to p40-phox. Despite an apparent decrease in
p40-phox in #7 D0, there was no statistically significant effect on the
quantity of the oxidase proteins studied, as measured by densitometry
(data not shown). These results demonstrate a representative study of
two treated volunteers and a control. C, control; #7 and #8 denote
treated volunteers, on day 0 and day 4.
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Neutrophil lactoferrin, lysozyme, and myeloperoxidase were measured in
whole cell lysates as markers for two major granule classes. G-CSF
administration resulted in a significantly reduced whole cell
lactoferrin content (P = .0004)
(Table 4), while no effect of G-CSF
administration was detected on the quantity of lysozyme or
myeloperoxidase. Neutrophil alkaline phosphatase, which is located in
secretory vesicles and neutrophil plasma membrane, was significantly
increased after five doses of G-CSF (P = .0001) (Table 4).
Content of cytochrome b558, the membrane associated component of the NADPH oxidase, was measured in the specific granule subcellular fraction (as described in Materials and Methods). Cytochrome b558 was significantly reduced on day 4 at 0.43 ± 0.06 compared with 0.68 ± 0.10 on day 0 (nmol/mg specific
granule protein; mean ± SEM, n = 7; P = .026) and 0.70 ± 0.07 in controls (n = 7).
Neutrophil apoptosis.
G-CSF added to neutrophils in vitro prolongs neutrophil
survival.12,13 We addressed the survival of neutrophils
after the in vivo administration of G-CSF. Neutrophils isolated from
untreated controls and G-CSF-treated volunteers were placed in culture
and assayed for apoptotic characteristics at multiple time points. Neutrophil survival was significantly longer after G-CSF
administration. The impact of G-CSF administration on the survival of
PMN was apparent by 9 hours of incubation
(Fig 5). Using logistic regression, as
described in Materials and Methods, we predicted the time to 50%
apoptosis for all study groups (Table 5).
Neutrophils isolated from treated volunteers on day 4 had a time to
50% apoptosis of 30.8 ± 2.9 hours, which was nearly twice that
measured before administration (P = .007; Table 5). When G-CSF
was added to cultures in vitro, there was a further enhancement of
survival of PMN obtained after 5 days of cytokine administration when
compared with day 0 survival (P = .02) (Table 5).
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| Fig 5.
Neutrophil apoptosis. Neutrophil apoptosis was determined
as described in Materials and Methods. Apoptosis of isolated
neutrophils, after 5 days of G-CSF therapy, was significantly reduced
by 9 hours of culture and remained lengthened throughout the assay time
(30 hours). Results are expressed as percent apoptosis and represent
mean ± SEM, n = 8 (matched pairs); C, control; P, treated
volunteer.
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| |
DISCUSSION |
Many clinical benefits of G-CSF have been documented. G-CSF therapy
reduces the length of neutropenia after chemotherapy38 and
bone marrow transplantation.39 It reduces the incidence of
hospital admissions for chemotherapy-related fever and
neutropenia.38 Its impact on the quality of life in
patients with congenital neutropenias is also well
described.3,40,41 G-CSF has specificity for
polymorphonuclear neutrophils with demonstrated effects on their
morphology,42 activities,35,43 and
survival.44 Interpretation and correlation of currently
available data regarding the effect of G-CSF on neutrophils are
difficult because information gained from in vitro studies may not
correlate with results obtained during administration of the drug.
Interpretation of available data into routine therapeutic strategies
needs to recognize the baseline function of the neutrophil population
studied6 and the dose, timing, and route of G-CSF
administration. Furthermore, many studies have focused on a limited
number of evaluation parameters. This report presents a comprehensive
evaluation of neutrophils from normal individuals treated with G-CSF.
We describe functional and biochemical properties of the circulating
neutrophil produced during G-CSF administration in vivo and suggest a
rationale for some of the unique characteristics, which differentiate
the neutrophil produced during exogenous G-CSF administration from the
neutrophil produced under standard in vivo conditions.
In this report, one of the most significant findings was the reduction
in directed migration of isolated neutrophils. Associated with this
defect, we recognized a reduced ability to upregulate F-actin and an
increased peak [Ca2+]c in response to the
same chemotactic stimulus. Baseline CD11b expression has been reported
as decreased after five doses of G-CSF administration.45
However, the data presented here demonstrate a normal ability to
upregulate neutrophil CD11b after the administration of five doses of
G-CSF. Overall evidence6,14 is in agreement with our
motility data after G-CSF exposure in vivo, but the reason for this
abnormality remains unexplained. Assembly of actin to filamentous form
is reflective of neutrophil chemotactic potential.23 A
neutrophil with increased F-actin may be less mobile and have less
reserve for motion in response to chemotactic stimuli, as in newborn
neutrophils46 and neutrophils from patients with severe
congenital neutropenia.1 Thus, our demonstration of reduced
assembly and increased baseline content (in only two volunteers) of
F-actin, may partially account for some reduced neutrophil chemotaxis.
The impact of G-CSF administration on mobilization of cytoplasmic
calcium was studied because [Ca2+]c is
involved in receptor-linked activation of a variety of functions, including chemotaxis.1 Our observation of an enhanced
stimulus-induced calcium mobilization after G-CSF is in agreement with
previous data in neutrophils from patients with glycogenosis Ib as
described by McCawley.47 However, the enhanced calcium
mobilization in these PMN has not been associated with any improvement
in neutrophil chemotaxis.47,48 The association between an
enhanced stimulus-induced calcium mobilization and reduced neutrophil
chemotaxis and altered F-actin content remains unclear.
Using S aureus, we examined neutrophil microbicidal
activity27 in standard and stress conditions by varying
bacteria:neutrophil ratios. Microbicidal activity remained intact at
1:1 (bacteria:neutrophil) ratio, but was mildly diminished in three
treated volunteers under the stress conditions of 10:1 ratio. Contrary
to reports by Liles18 and Fossat,17 our results
suggest a normal to mildly deficient killing activity after G-CSF
administration, albeit we used different organisms and different
organism:neutrophil ratios. Killing of an ingested microbe results from
the simultaneous and synergistic action of toxic oxygen species
(O2, H2O2, .OH,
HClO),49 with multiple oxygen
independent microbicidal mechanisms supplied by the contents of the
specific and azurophilic granules.50,51 Oxygen-dependent
killing reflects the ability of the neutrophil NADPH oxidase to produce
O2-. G-CSF has been reported to enhance oxidase
activity.6,36,43 In our study, however, we found divergent
effects depending on the stimulus used. The response to fMLP was
enhanced in cells, which developed under the influence of G-CSF. The
physiological stimulus OZ produced a normal response after G-CSF
therapy. We recognized diminished PAF priming for the fMLP response,
while the PMA-induced respiratory burst was also reduced after G-CSF therapy, in agreement with Macey.52 These last two findings may relate to a reduction in overall neutrophil responsiveness after
partial activation by in vivo cytokine administration, as described by
Khwaja.53 Furthermore, immature neutrophils have higher
levels of protein kinase C (PKC) type III, an isoenzyme that is not responsible for cellular activity.54 If the
neutrophils we described are less mature than normal, PMA, an activator
of PKC, may induce a less than maximal respiratory burst.
These divergent effects on oxidase activity in intact cells could, of
course, be the result of alterations of NADPH oxidase constituents; and
to explore this, we examined the oxidase in more detail. We
demonstrated overall normal generation of
O2 in the cell-free assay; however, in
cross-mixing experiments, an increased amount of
O2 was generated when neutrophil
membrane from treated volunteers (day 4) was mixed with control cytosol
(day 4). Increased cytochrome b558 in PMN membrane after
G-CSF administration might be inferred from this finding. However,
membrane content of this heterodimer was not measured. NADPH oxidase
cytosolic component proteins were unaffected by G-CSF treatment. Thus,
the oxidase components remain intact with G-CSF therapy, although there
appears to be indirect evidence for increased membrane cytochrome
b558. Accordingly, the differing oxidase activities in
response to various agonists may be related to accessory pathways
required to transform stimulus coupled signals to assembly and
activation of the oxidase.
Microbicidal activity is also dependent on the integrity of neutrophil
granules. We examined the status of azurophilic and specific granules
indirectly by measuring several marker proteins. G-CSF is known to
increase the transcription and amount of neutrophil alkaline
phosphatase,11,55 an enzyme stored in secretory vesicles and membrane.51 This characteristic feature of
G-CSF-exposed neutrophils was reconfirmed here. A reduced specific
granule marker, lactoferrin, and a normal myeloperoxidase are
consistent with reduced specific granules possibly due to
degranulation. However, degranulation alone is unlikely to fully
explain the apparent changes in lactoferrin for several reasons. We
recognized equal amounts of neutrophil lysozyme before and after
cytokine treatment, which would be expected to be lower in states of
specific granule degranulation.26 Transcobalamin 2 binding
protein, another indicator of secondary granule content, is reported to
be normal during G-CSF therapy.56 Also, PMN isolated after
G-CSF administration continue to recruit normal amounts of CD11b/CD18
(also stored in specific granules) to the cell surface in response to
cell stimuli, as we and others6 have demonstrated. Lastly,
if reduced cytochrome b558, as we described here, were the
exclusive result of degranulation, then the amount of cytochrome
b558 per mg of specific granule protein should remain the
same. This is not the case in these experiments. Hence, a specific
effect of G-CSF on production of neutrophil lactoferrin and possibly
other proteins remains possible and merits further exploration.
Neutrophils die by apoptosis. In vitro exposure of neutrophils to
inflammatory cytokines interleukin-1 (IL-1), tumor necrosis factor (TNF), IL-6, interferon-, and GM-CSF, as well as
lipopolysaccharide (LPS) all decrease neutrophil
apoptosis.13 G-CSF also has significant effects on
neutrophil apoptotic rates in vitro7,12 and has some
documented effects on neutrophil apoptosis after in vivo administration.57 However, G-CSF therapy in vivo is
reported not to alter the circulation half-life of
neutrophils.14 Neutrophils, which express
bcl-2,58 also demonstrate normal neutrophil
survival kinetics, despite the inhibiting effect this proto-oncogene
has on neutrophil apoptosis. We have shown here that in vivo
administration of G-CSF markedly decreased the apoptotic rate of the
isolated peripheral blood neutrophil, in agreement with results by
Adachi et al.57 Also, there was an additive effect of
exogenous G-CSF, reducing apoptotic rate in PMN collected after G-CSF
therapy. In additional studies not reported here, we have demonstrated a protective effect of G-CSF administration on neutrophil apoptosis when neutrophils are exposed to cycloheximide.59 The in
vivo and in vitro effects delaying apoptosis, exhibited by G-CSF, have implications for the prolonged survival of neutrophils at an
inflammatory site.
Early in vitro studies describing the effects of G-CSF on the
neutrophil led to the concept that G-CSF administration resulted in
cells with markedly enhanced function. Our studies demonstrate that
G-CSF administration imparts only modest effects on neutrophil function
and biochemistry and results in a diverse pattern of activities not
always consistent with increased function. Inherent in enhancing the
activity of neutrophils is a risk for tissue injury and this risk is
presumably avoided by G-CSF, given the modest effects we describe.
Given the diverse functional and biochemical effects on neutrophils,
the clinical benefit of G-CSF most likely lies in an increased absolute
number of neutrophils with enhanced survival characteristics providing
improved host defense.
| |
ACKNOWLEDGMENT |
We are grateful to Dr Richard B. Johnston, Jr, and Dr Arthur Verhoeven
for their critical comments on this project and to Flo Usechek for her
secretarial assistance in preparation of this manuscript.
| |
FOOTNOTES |
Submitted December 15, 1997;
accepted July 21, 1998.
Supported by the Margery Wilson Transfusion Medicine Fellowship,
Bonfils Blood Center, The Stacy Marie True Memorial Trust, a
Transfusion Medicine Academic Award, National Heart, Lung and Blood
Institute, National Institutes of Health (K07-HL02036), Public Health Services research Grant No. 5 01 RR00051 from the Division of Research Resources, a grant from Amgen, Inc, Thousand Oaks,
CA, and a Clinical Associate Physician Award (M01-RR00069) from the
General Clinical Research Centers Program, National Centers for
Research Resources, National Institutes of Health.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Daniel R. Ambruso, MD, Bonfils Blood
Center, 4200 E Ninth Ave, B-128, Denver, CO 80262.
| |
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Abstract
Introduction