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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 7 (April 1), 1998:
pp. 2525-2535
Granulocyte Colony-Stimulating Factor Worsens the Outcome of
Experimental Klebsiella pneumoniae Pneumonia Through Direct
Interaction With the Bacteria
By
Thomas K. Held,
Martin E.A. Mielke,
Marcio Chedid,
Matthias Unger,
Matthias Trautmann,
Dieter Huhn, and
Alan S. Cross
From the Department of Hematology and Oncology and the Department of
Paidopathology und Placentology, Virchow-Klinikum, Humboldt-University;
the Institute for Infectious Diseases, Department of Medical
Microbiology and Infectious Diseases Immunology, Free University,
Berlin, Germany; the Laboratory of Cellular and Molecular Biology,
National Cancer Institute, National Institutes of Health, Bethesda, MD;
the Department of Microbiology, University of Ulm, Germany; and the
Division of Infectious Diseases and Program in Oncology, University of
Maryland Medical School, Baltimore, MD.
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ABSTRACT |
Besides its well-established effects on granulocytopoiesis,
granulocyte colony-stimulating factor (G-CSF) has been shown to have
direct effects on the recruitment and bactericidal ability of
neutrophils, resulting in improved survival of experimentally infected
animals. We studied the effect of G-CSF on the course of experimental
pneumonia induced by Klebsiella pneumoniae, an important
gram-negative bacillary pulmonary pathogen. Using a highly reproducible
murine model, we here show the paradoxical finding that mortality from
infection was significantly increased when animals received G-CSF
before induction of pneumonia. Administration of G-CSF promoted
replication of bacteria in the liver and spleen, thus indicating an
impairment rather than an enhancement of antibacterial mechanisms. By
contrast, a monoclonal antibody against Klebsiella K2 capsule
significantly reduced bacterial multiplication in the lung, liver, and
spleen, and abrogated the increased mortality caused by G-CSF. In vitro
studies showed a direct effect of G-CSF on K pneumoniae
resulting in increased capsular polysaccharide (CPS) production. When
bacteria were coincubated with therapeutically achievable
concentrations of G-CSF, phagocytic uptake and killing by neutrophils
was impaired. Western blot analysis showed three binding sites of G-CSF
to K pneumoniae. Binding of 125I-G-CSF to K
pneumoniae was displaced by an excess of unlabeled G-CSF, whereas
an unrelated cytokine, interleukin-1 , did not compete with G-CSF
binding to the bacteria. Thus, in this model, the direct effect of
G-CSF on a bacterial virulence factor, CPS production, outweighed any
beneficial effect of G-CSF on recruitment and stimulation of
leukocytes.
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INTRODUCTION |
GRANULOCYTE COLONY-stimulating factor
(G-CSF) is now widely used as a therapeutic agent for prevention or
treatment of neutropenia induced by myelotoxic agents.1,2
In addition, a number of experimental animal studies suggest its potential benefit in the treatment of infections by a variety of
microorganisms. For example, G-CSF was able to augment the recruitment
of polymorphonuclear leukocytes (PMN) into lungs of rats infected
intratracheally with Klebsiella pneumoniae and to enhance
intrapulmonary bactericidal activity against this pathogen, with an
enhanced survival of treated animals.3 Fatal pneumococcal pneumonia in rats could also be prevented by pretreatment with G-CSF.4 Splenectomized mice challenged with
Streptococcus pneumoniae aerosol had a
significantly greater survival rate when treated with
G-CSF.5 In Pseudomonas aeruginosa pneumonia after
experimental hemorrhage, G-CSF improved survival when administered
prophylactically.6 Finally, in neutropenic mice with
systemic infections caused by P aeruginosa, Escherichia coli,
Serratia marcescens, Staphylococcus aureus, or Caudida
albicans, G-CSF significantly enhanced survival,7,8 even when antibiotics failed to show therapeutic efficacy.8
K pneumoniae is one of the most frequently isolated
gram-negative bacterial pathogens in severe nosocomial
infections.9-11 The rapidly progressive clinical course of
Klebsiella pneumonia, which is often complicated by
multilobular involvement, lung abscesses and high
mortality,12,13 leaves little time to institute effective antibiotic treatment. In addition, an increasing proportion of nosocomial K pneumoniae isolates are resistant to multiple
antibiotics commonly used in intensive care units.14-16
Alternative approaches to the prophylaxis and supportive
treatment of Klebsiella respiratory infections are
therefore needed.
We have shown previously that a monoclonal antibody (MoAb III/5-1)
directed against the K2 capsular polysaccharide (CPS) of K
pneumoniae is protective in a mouse sepsis model as well as in
experimental K pneumoniae pneumonia in rats.17,18
However, antibodies may not be distributed equally within tissues and
therefore may not be present in effective concentrations in certain
organs. Moreover, to be effective, opsonophagocytic antibodies need the presence of phagocytic cells to enhance clearance of bacteria. We thus
aimed to extend our studies using G-CSF in addition to MoAb III/5-1 to
enhance the efficacy of nonantibiotic therapy against experimental
K pneumoniae pneumonia. Much to our surprise, we found a
dramatic increase in mortality when mice were pretreated with G-CSF
before induction of pneumonia. Subsequent studies showed that this
adverse effect of G-CSF was most likely a result of a direct action of
G-CSF on K pneumoniae. G-CSF bound specifically to the bacteria
and enhanced the production of cell-bound CPS, enabling the bacteria to
escape phagocytosis and to spread rapidly throughout the body.
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MATERIALS AND METHODS |
Animals.
Pathogen-free female BALB/c mice, 20 to 25 g, were obtained from the
breeding facility of the Institute of Infectious Diseases, Klinikum
Benjamin Franklin, Free University, Berlin, Germany, and used
throughout the study. They were provided free access to standard
laboratory animal diet and water and subjected to a 12-hour day/night
rhythm.
Bacteria.
K pneumoniae B5055 (serotype O1:K2)19 and F201
(serotype O1:K-)20 were used throughout the study. For
animal studies, we grew the bacteria to mid-log phase in trypticase soy
broth (TSB; Difco, Detroit, MI) for 4 hours at 37°C with gentle
shaking (100 rpm), washed them once with sterile physiological saline, and adjusted them spectrophotometrically to the desired concentrations, which were confirmed by plating serial 10-fold dilutions of the suspension on trypticase soy agar (TSA; Difco).
Reagents and injection protocol.
Sterile, pyrogen-free recombinant human G-CSF was obtained from AMGEN
(München, Germany). The MoAb III/5-1 is a mouse IgM directed
against K2-CPS and was described previously.17,18 Different
dilutions from both MoAb III/5-1 and G-CSF were made with
phosphate-buffered saline (PBS) under sterile conditions. All reagents
as well as the PBS itself were endotoxin free as assessed using the
limulus amebocyte lysate assay (Chromogenix, Mölndal, Sweden).
The assay detected E coli O111:B4 lipopolysaccharide (LPS) at
concentrations of 1 pg/mL and above. Animals were injected with either
50 µg/kg G-CSF subcutaneously (SC) and/or 10 mg/kg MoAb
III/5-1 intravenously (IV) as detailed in Table
1. Control animals received PBS only at all
time points at the same volume as the treated mice (0.1 mL).
In a preliminary experiment, we confirmed that G-CSF was active in this
mouse strain. Mice (three per group) were injected SC twice daily for 4 days with three different doses of G-CSF (15 µg/kg, 50 µg/kg, or
250 µg/kg, respectively), and blood was obtained from a tail vein
once daily beginning 4 days before G-CSF treatment and throughout the
treatment phase. Total leukocyte counts were determined with a Coulter
counter (Model DN; Coulter Electronics LTD, Harpenten Herts, UK) and
differential counts were obtained with smears stained with
May-Grünwald solution. Absolute numbers of each
cell type were calculated by multiplying the percentage obtained on
differential counting by the total white blood cell count. Data showed
a dose- and time-dependent increase in absolute neutrophil numbers as
well as in total leukocyte counts comparable to previous
reports,21 thus confirming that the G-CSF treatment used in
this study had the expected effects on leukocytes (data not shown).
Experimental pneumonia.
Animals were anesthetized with 8 mg/kg of xylacine (Bayer, Leverkusen,
Germany) and 80 mg/kg of ketamine hydrochloride (Parke, Davis & Co,
Munich, Germany) administered intraperitoneally. The bacterial inoculum
(total volume, 50 µL; containing 1 × 103
colony-forming units [CFU]) was instilled intranasally into the left
nasal opening while holding the mice upright. The animals were returned
to their cages, and survival as well as body weight were assessed every
12 hours. Surviving animals were killed at 72 hours after challenge.
Because both histology and culture of organ homogenates for
quantitative detection of bacteria are subject to sampling error if
only parts of the organs are examined, we decided not to perform both
histology and organ cultures in the same animal. Instead, at the start
of the study, three mice were randomly assigned to undergo histological
examination. If animals died during the observation period, necropsy
for histology was performed not later than 12 hours after death. All
surviving mice were examined bacteriologically at the end of the study
(72 hours after bacterial challenge). Quantitative culture of bacteria
in lungs, liver, and spleen, as well as histological examination were
performed as described.18 CFU detected in the organs and their log10 were standardized per 0.1 gram wet organ
weight. As a parameter of bacterial dissemination from the lung, an
index was computed for each investigated mouse according to the
following formula:
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Immunoblotting.
K pneumoniae B5055 was grown in TSB at 37°C overnight and
collected by centrifugation (1,560g, 15 minutes, 4°C). The
pellet was lysed in Tris/EDTA buffer (50 mmol/L Tris, 1 mmol/L EDTA; pH
8.0 [Sigma Chemical Co, St Louis, MO]) containing 1 mmol/L phenylmethylsulfonyl fluoride, 100 µg/mL Aprotinin, and 0.1 mmol/L Leupeptin (all Sigma) by three cycles of freeze-thawing. Samples were
then reduced by heating the lysate for 10 minutes at 80°C in the
presence of 0.5% sodium dodecyl sulfate and 1.25% mercaptoethanol (both from Sigma), subjected to polyacrylamide gel electrophoresis (PAGE; 12%) and blotted onto PVDF membranes (Millipore, Bedford, MA).
An aliquot of the lysate was subjected to proteinase K treatment (Boehringer Mannheim, Indianapolis, IN) before reduction (60 minutes at
60°C). After blocking nonspecific binding sites with PBS containing 0.1% TWEEN 20 and 3% bovine serum albumin (BSA;
Sigma), individual lanes were incubated in a Hoefer
Decafold apparatus (Pharmacia Biotech, Piscataway, NJ) as indicated
(4°C, overnight) with G-CSF, rmIL-1 (Genzyme, Cambridge, MA), or
G-CSF which was preabsorbed with rabbit anti-human G-CSF-IgG (a kind
gift from Dr P. Stevens and Ed Shatzen, AMGEN, Thousand Oaks, CA).
After washing, bound G-CSF was detected by incubation (4°C, 4 hours)
with a murine anti-human G-CSF MoAb (IgG1, also kindly provided by Dr
P. Stevens and Ed Shatzen) which was preabsorbed with reduced bacterial
lysate to reduce nonspecific binding (37°C, 1 hour, at a ratio of 1:1 [vol/vol]). After additional washing steps, bound MoAb was detected with a horseradish peroxidase-labeled goat anti-mouse IgG (Kierkegaard & Perry, Gaithersburg, MD) and subsequent enhanced chemiluminescence. Identical replicate samples were also stained after PAGE using conventional silver-staining procedures as described.22
Binding of 125I-G-CSF to K pneumoniae.
Single colony isolates of K pneumoniae B5055 or K
pneumoniae F201 were grown to late log-phase and assessed for their
ability to bind 125I-G-CSF at either 37°C or 4°C.
Bacteria (1 × 108 CFU) were incubated in 0.5 mL of PBS
containing 1% heat-inactivated fetal calf serum plus various
concentrations of labeled and/or unlabeled G-CSF or
interleukin-1 (IL-1). Preliminary experiments showed that there
was no difference in binding between samples with and those without
sodium azide or between samples incubated at 37°C or 4°C. Thus, the
final binding experiments were performed in 1.5-mL
Eppendorf tubes at 4°C in the absence of sodium
azide. The tubes were slowly rotated for 40 minutes, centrifuged for 5 minutes (14,250g), and the supernatants immediately removed. Bound radioactivity in the pellets as well as in the supernatants was
counted. Measurements of bound and free ligand were converted to molar
concentrations according to standard methods.23
In vitro production of CPS in the presence of G-CSF.
After incubation of bacteria overnight in TSB (37°C, 120 rpm), we
transferred 2 × 105 CFU into 250 mL of fresh TSB with
either G-CSF (10 ng/mL), recombinant murine IL-1 (2 ng/mL), or in
TSB alone to obtain an initial concentration of approximately 850 CFU/mL (ie, a 1:230 dilution). Bacteria were incubated again (37°C,
120 rpm), and 50-mL samples were withdrawn at 2, 4, 6, 8, and 10 hours
and immediately placed on ice for subsequent analysis. Colony counts,
quantification of cell-bound CPS by means of a previously described
enzyme-linked immunosorbent assay,18,24 as well as the cell
disruption before the determination of total bacterial cell
protein19 were performed exactly as described. Total
bacterial cell protein was determined according to the method of Lowry
et al25 by using different concentrations of BSA in
distilled water as a standard. To account for possible increase in cell
mass, CPS amounts were expressed as the ratio of cell-bound CPS/total
protein rather than cell-bound CPS per colony counts.19 For
use in the microphagocytosis and PMN killing assays, samples at 6 hours
of incubation were used.
Preparation of PMN, serum, and microphagocytosis and PMN killing
assays.
Heparinized (50 U/mL) venous blood was obtained from one healthy donor
in all tests. PMN were isolated and residual erythrocytes were lysed as
described earlier.26 Serum was obtained from one healthy
volunteer under conditions that preserved complement activity, stored
in small aliquots at  80°C, and used throughout all experiments at
a final concentration of 60% vol/vol.
We performed the killing assay as described previously.17
Briefly, PMN at a final concentration of 2.5 × 105
cells/well, normal human serum as a complement source, bacteria (prepared as described above) at a final concentration of 2.5 × 104 CFU/well (thus, yielding a final PMN:bacteria ratio of
10:1), and Hanks' Balanced Salt Solution with Ca2+ and
Mg2+ were incubated in 96-well round-bottom tissue culture
plates (Costar, Cambridge, MA) in a total volume of 150 µL.
Incubation was performed at 37°C with shaking (1,000 rpm).
Immediately after mixing the ingredients, and after 60 and 90 minutes,
samples (10 µL) were taken from each well and placed on ice into a
glass tube containing sterile distilled H2O with 0.1% BSA
(wt/vol) to lyse the PMN without killing the bacteria. Viable counts
were determined by plating serial dilutions on TSA. We calculated the
percentage of surviving bacteria according to the following
formula:
Controls
in each experiment included incubation of PMN and bacteria without
serum; bacteria and serum without PMN; and PMN, bacteria and
heat-inactivated serum (56°C, 30 minutes, to show that there were no
antibodies or nonspecific serum activity against K2-CPS), each of which
showed no killing of bacteria.
To assess phagocytosis, we incubated bacteria, PMN, and normal human
serum as described above. After 60 minutes, samples (10 µL) were
taken from each well and processed as described. Immediately after the
samples were withdrawn, the 96-well plates were centrifuged (250g, 5 minutes) to separate the neutrophils out of the
PMN-bacteria suspensions. Samples (10 µL) were withdrawn again and
processed as above. The numbers of bacteria remaining in suspension
after pelleting the PMN are given as percent of the total number of bacteria counted in the samples before centrifugation.
Statistical analysis.
Using a software package (Statistica for Windows, version 4.5;
StatSoft, Tulsa, OK), we compared survival by the Cox-Mantel test, and
all other parameters (body weights, bacterial counts, CPS/protein
ratios, percentage of surviving bacteria) by the two-tailed Mann-Whitney U test.
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RESULTS |
Pretreatment with G-CSF worsened the outcome of experimental K
pneumoniae pneumonia in mice.
Non-neutropenic mice were infected intranasally with 0.5 × LD50 of K pneumoniae. When G-CSF was administered
before induction of pneumonia, significantly fewer mice survived 72 hours than in the control group (Fig 1).This deleterious effect of G-CSF was not observed when treatment was
started 24 hours after the bacteria were inoculated, but there was no
improved survival compared with control animals (Fig 1). The enhanced
lethality observed when mice were pretreated with G-CSF was completely
abolished by cotreatment with MoAb III/5-1, which is specific for
K2-CPS of K pneumoniae (Fig 1).
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| Fig 1.
Survival after pulmonary infection via the intranasal
route with 1 × 103 CFU of K pneumoniae B5055.
Numbers in parentheses denote surviving animals/total number of
animals. *, P = .0094 of treatment with G-CSF/pre compared
with mice treated with MoAb III/5-1 (both groups) and P = .011 of treatment with G-CSF/pre compared with control animals and
animals receiving G-CSF after induction of pneumonia (Cox-Mantel
test).
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Because the body weight is a reliable parameter for the degree of
illness in this type of pneumonia,18 we measured the change in body weight to obtain information about the course of infection in
animals which did not die. Neither pretreatment with G-CSF nor the
initiation of G-CSF treatment at 24 hours after bacterial challenge led
to any improvement in weight compared with control animals (total
weight loss [in grams] of control mice [median, 4.6; quartiles,
5.4 and 4.3]; total weight loss [in grams] of mice pretreated
with G-CSF [median, 5.1; quartiles, 5.5 and 4.4]; total
weight loss [in grams] of mice treated with G-CSF at 24 hours after
bacterial challenge [median, 4.4; quartiles, 4.7 and 3.9]).
However, weight loss could be reduced significantly by the IV
administration of MoAb III/5-1 (total weight loss in grams [median,
2.9; quartiles, 4.4 and 1.1], P = .0045 as compared with control mice). The best outcome was observed when the treatment with MoAb III/5-1 was combined with pretreatment of the mice with G-CSF
(total weight loss in grams [median, 1.9; quartiles, 2.4 and
0.9], P = .0003 as compared with control mice).
Pretreatment with G-CSF promoted bacterial spread to liver and
spleen.
In addition to survival and degree of illness, we examined the
bacterial spread in K pneumoniae pneumonia treated with or without G-CSF in our model. When organs were investigated in surviving animals 72 hours after bacterial challenge, the group pretreated with
G-CSF had no significant decrease in colony counts in the lungs (Fig 2). However, pretreatment with
G-CSF led to an increased colony count in liver and spleen compared
with control animals. In contrast, MoAb III/5-1 administered with
G-CSF-pretreatment was able to significantly decrease colony counts in
all three organs investigated, thus reversing the effect of
G-CSF-pretreatment. If treatment with G-CSF was started at 24 hours
after bacterial challenge, there was no indication of enhanced
bacterial spread (Fig 2). The presence of bacteria in other organs that
originated from the lungs, indicative of spread of infection from a
primary to a secondary site, can be expressed as an index. Table
2 shows this effect of G-CSF more directly:
only pretreatment with G-CSF showed a ratio of bacterial counts in
distant organs to lung greater than 1. Thus, in G-CSF-pretreated
animals, bacterial dissemination was promoted from the lung to other
sites.
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| Fig 2.
Effect of different treatments on the growth and spread
of 1 × 103 CFU of K pneumoniae B5055 instilled
into the lungs. Treatments are as outlined in Table 1. The number of
animals investigated is given within the bars. Data are given as median ± quartiles. *, P = .017 compared with control animals; **,
P = .0074 compared with control animals and .0019 compared
with treatment with G-CSF/pre; ***, P = .014 compared with
treatment with G-CSF/pre (Mann-Whitney U test).
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Histology further supported this observation. Control animals showed
well-developed pneumonia with abscess formation (Fig 3A) in their lungs, but in their
livers, only small areas of inflammation with scattered microabscesses
and foci of hepatic necrosis were observed (Fig 3B). In contrast, mice
pretreated with G-CSF had only mild peribronchitic alterations in their
lungs without signs of containment of inflammation such as abscess
formation (Fig 4A). Livers and spleens of
mice pretreated with G-CSF were severely altered, showing large
abscesses which contained massive amounts of bacteria (Fig 4B). In
addition, large necrotic areas surrounded by granulocytes were observed
in the livers (Fig 4C). When MoAb III/5-1 was administered in addition
to pretreatment with G-CSF, almost all changes observed in animals
pretreated with G-CSF were reversed: there were only minor bronchial
and peribronchial infiltrations by granulocytes (minimal change focal
pneumonia), and the livers showed small microabscesses as observed in
control mice. When treatment with G-CSF was started at 24 hours after
bacterial challenge, a mixed pattern was observed. In the lungs, there
was a beginning pneumonic reaction and a moderate perivascular and
septal edema. However, in the liver there was almost no difference to
the severe alterations observed in mice pretreated with G-CSF: huge
abscesses loaded with bacteria joined extended necrotic areas.
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| Fig 3.
(A) Micrograph of lung tissue 72 hours after intranasal
infection with 1 × 103 CFU of K pneumoniae B5055.
Lung abscess with severe tissue destruction in a PBS-treated control
animal. Hematoxylin & Eosin (H&E); original magnification ×150. (B)
Small foci of hepatic necrosis. H&E; original magnification ×600.
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| Fig 4.
(A) Micrograph of lung tissue 48 hours after intranasal
infection with 1 × 103 CFU of K pneumoniae B5055
and pretreatment with G-CSF (50 µg/kg sc) at 48 hours, 36
hours, 24 hours, and 12 hours before infection. Peribronchiolar neutrophils without destruction of lung parenchyma. H&E; original magnification ×150. (B) Splenic abscesses in the red
pulp containing numerous gram-negative bacilli are found. H&E; original
magnification ×370. (C) Confluent hepatic necroses. H&E; original
magnification ×150.
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Coincubation of K pneumoniae B5055 with G-CSF resulted in
enhanced capsular polysaccharide production and increased resistance
against killing by neutrophils.
The enhanced spread to secondary sites in animals pretreated with G-CSF
suggested that K pneumoniae in these mice were better able to
evade phagocytic host defenses. This could be caused by either enhanced
growth of the bacteria or enhanced production of antiphagocytic
virulence factors. Therefore, we first investigated whether the
presence of G-CSF had any effect on the growth kinetics of K
pneumoniae. Bacteria grown in the presence of clinically relevant
concentrations of G-CSF (10 ng/mL)27 showed no difference in the growth rate compared with controls grown in TSB only (Fig 5). Second, we investigated the influence
of G-CSF on the production of cell-bound CPS, the main mechanism by
which K pneumoniae evades phagocytic host
defenses.28 There was a marked increase in the production
of cell-bound CPS when bacteria were incubated in the presence of G-CSF
as compared with controls (Fig 6). In
contrast, IL-1 used at a concentration previously shown to
significantly bind to other gram-negative bacteria29 had no
effect on the growth rate (Fig 5) or on the production of cell-bound
CPS (Fig 6). A nonvirulent bacterial control strain (F201), producing
only minimal amounts of K2-CPS, showed no increase in cell-bound CPS production when incubated under the same conditions (Fig 6, inset).
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| Fig 5.
Growth of K pneumoniae B5055 in the absence or
presence of either G-CSF (10 ng/mL) or IL-1 (2 ng/mL) as
described in Materials and Methods. Data are from three independent
experiments, each performed in duplicate, and are given as median ± quartiles.
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| Fig 6.
Enhancement by G-CSF of the production of cell-bound CPS.
K pneumoniae B5055 was grown in the absence or presence of
either G-CSF (10 ng/mL) or IL-1 (2 ng/mL) as described in Materials and Methods. Data are from four independent experiments, each performed
in duplicate, and are given as median ± quartiles. *, P = .02 of bacteria grown in the presence of G-CSF as compared with
controls (Mann-Whitney U test). Inset: Effect of G-CSF on the
production of cell-bound CPS in the avirulent strain K
pneumoniae F201. Bacteria were grown in the absence or presence of
G-CSF (10 ng/mL) as described in Materials and Methods. Data are from four independent experiments, each done in duplicate, and are given as
median ± quartiles. Note different scale on the y axis.
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The increase in cell-bound CPS by the virulent strain B5055 grown in
the presence of G-CSF resulted in a significant resistance to uptake
and killing by neutrophils in vitro (Fig
7). After coincubation for 6 hours with
G-CSF, about 80% of the initial bacterial inoculum survived as
compared with 23% of bacteria grown in the absence of G-CSF. This
difference was still observed after 90 minutes of exposure to
phagocytosis and killing by PMN.
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| Fig 7.
Inhibition by G-CSF of in vitro phagocytosis and killing
of Klebsiella by neutrophils. K pneumoniae B5055 was
grown for 6 hours in the absence or presence of G-CSF (10 ng/mL) and
subsequently subjected to phagocytosis of PMN in the presence of 60%
normal human serum as described in Materials and Methods. Data are from four independent experiments, each performed in duplicate, and are
given as median ± quartiles. *, P = .014 compared
with the corresponding time point of controls grown in the absence of
G-CSF (Mann-Whitney U test).
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To investigate whether this increased survival was caused by an
increase in resistance to adherence and ingestion by the neutrophils, we measured the number of bacteria remaining in the cell-free supernatants of PMN-bacterial suspensions following centrifugation after 60 minutes of coincubation. Eighty-four percent (median; 82.7%
and 85.5%, quartiles) of the total bacteria were recovered when K
pneumoniae was grown in the presence of G-CSF. In contrast, only
53.9% (median; 52.9% and 59.2%, quartiles) of the total bacteria were recovered from the bacteria grown in TSB only
(P = .014), suggesting that less bacteria were taken up by
neutrophils when G-CSF was present during bacterial growth before the
phagocytosis assay.
G-CSF bound to K pneumoniae B5055.
If G-CSF altered the virulence properties of K pneumoniae, it
should bind to the bacteria via a specific binding site. The question
of a possible binding of G-CSF to K pneumoniae was investigated by two independent methods. First, immunoblotting of whole bacterial cell lysates showed three binding sites with a molecular weight of 41, 25, and 21 kD, respectively (Fig 8, lane
1). The absence of binding to lysates
pretreated with proteinase K indicates that the binding sites were, at
least in part, of protein nature (Fig 8, lane 2). Controls included
incubation of the blotted lysate with G-CSF preincubated with rabbit
anti-G-CSF-IgG, rmIL-1 (thus omitting G-CSF), and incubation with
mouse IgG1, all of which showed no binding (Fig 8).
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| Fig 8.
Binding of G-CSF to K pneumoniae B5055 whole cell
lysates as assessed by immunoblotting. L, bacterial cell lysate; PK-L,
bacterial cell lysate pretreated with proteinase K; +/p, G-CSF
preabsorbed with rabbit anti-human G-CSF-IgG. Lanes 1 through 5 are
from a representative blot. Lanes 6 through 8 are from a silver-stained identical replica of the gel. Binding of G-CSF to specific
bands is indicated by arrows; molecular weight (in kD) is indicated on
the left.
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We also analyzed specific binding using 125I-labeled G-CSF.
As shown in Fig 9A, binding of
125I-G-CSF to the virulent strain B5055 was dose dependent
and saturable. 125I-G-CSF also bound to the avirulent
strain F201, although to a lesser extend. Specific binding could be
inhibited in a dose-dependent manner by increasing amounts of competing
unlabeled G-CSF suggesting the presence of specific binding sites for
G-CSF (Fig 9B). Competition with a different cytokine, IL-1, did not
displace specific binding of 125I-G-CSF (Fig 9C).
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| Fig 9.
Binding of 125I-G-CSF to K pneumoniae
B5055. (A) Binding curve of 125I-G-CSF after 40 minutes at
4°C with K pneumoniae B5055 or K pneumoniae F201.
Results are representative of at least three independent experiments.
(B) Competition of G-CSF with 125I-G-CSF for binding to
K pneumoniae B5055. 125I-G-CSF (50 ng) was
incubated (4°C, 40 minutes) with K pneumoniae, along with
various concentrations of unlabeled G-CSF. The results, plotted as
specific binding relative to results of an assay with no added
competitor, are from four independent experiments, each done in
duplicate and are shown as mean ± standard deviation. (C) Competition
of IL-1 with 125I-G-CSF for binding to K
pneumoniae B5055. 125I-G-CSF (50 ng) was incubated
(4°C, 40 minutes) with K pneumoniae, along with various
concentrations of unlabeled IL-1. The results, plotted as specific
binding, are from four independent experiments, each performed in
duplicate, and are shown as mean ± standard deviation.
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DISCUSSION |
In this study, we show that the administration of G-CSF to a
non-neutropenic host may worsen a subsequent pulmonary infection by
K pneumoniae because of a direct effect of the cytokine on the
pathogen. Although some studies point to an effect of cytokines from
eukaryotes on procaryotic cells in vitro,29-32 to our
knowledge there are no reports showing the potential in vivo
significance of this observation. Here, we report for the first time
that G-CSF not only binds to and modifies bacterial cells in vitro, but
that these effects have fatal consequences in a relevant model of
bacterial pneumonia and sepsis.
The deleterious effect on mortality from experimental pneumonia was
observed when G-CSF was administered before the induction of pneumonia
(Fig 1). Bacterial load in lungs, livers, and spleens of pretreated
animals indicated that, in this situation, bacteria spread more easily
from the lung to distant organs and that there was a decreased
eradication of the bacteria from these secondarily infected organs (Fig
2, Table 2). Histological analysis showed the development of severe
necroses and abscesses within liver and spleen (Fig 4B and C). When
treatment with G-CSF was started 24 hours after bacterial challenge, we
observed a mixed picture: although mortality and histological changes
in the lungs were not different from control animals, we found the same
type and size of abscesses and confluent hepatic necrosis in the liver as in mice pretreated with G-CSF. A recent study using a highly virulent strain of Pasteurella multocida to
induce gram-negative bacterial pneumonia and sepsis in rabbits showed a
similar histology: treatment with G-CSF led to significantly increased
inflammation in liver and spleen compared with the placebo
group.33 In addition, rats pretreated with G-CSF showed a
significant decrease in survival when challenged subsequently with
E coli.34 However, the basis for this adverse
outcome was not investigated.34 A history of treatment with
G-CSF was also a highly significant risk factor for the development of
subsequent disseminated Mycobacterium avium
complex infection in patients with acquired immunodeficiency syndrome.35
The impaired uptake and killing of the bacteria observed here was
surprising, because G-CSF has been shown to promote phagocytosis and
subsequent elimination of microbial organisms.36 We
therefore hypothesized that G-CSF might have a direct action on the
bacteria resulting in increased bacterial virulence, which outweighs
the well-known effects of G-CSF on host defenses. To investigate this question, we asked whether G-CSF binds to K pneumoniae. Using two independent methods, we could show that G-CSF binds to at least
three binding sites (Fig 8). These binding sites are, in part, of
protein nature because pretreatment of the bacterial cell lysates with
proteinase K abolished binding (Fig 8). Studies with
125I-G-CSF showed dose dependency and saturability of
binding of G-CSF to K pneumoniae (Fig 9A). Also, specificity
could be shown by competition of unlabeled G-CSF with
125I-G-CSF for the binding to K pneumoniae, again
in a dose-dependent manner (Fig 9B). Finally, another
cytokine, IL-1, which binds to E coli,29 did not
replace bound 125I-G-CSF from its receptor(s), even when
present in a 500-fold excess (Fig 9C).
Having established the binding of an eukaryote growth factor to
prokaryote cells, the next question to be asked was whether this
binding of G-CSF had any consequences on the virulence of K
pneumoniae. Earlier reports of effects of cytokines on bacteria showed mainly an effect on the growth, but those differences were at
most 1 log only in vitro29 and are thus questionable as to whether they play a role in vivo. No other experiments were performed to investigate whether the binding of a cytokine to a bacterium had any
consequences with regard to bacterial virulence or outcome from
infection.29 Only one paper showed enhancement of
invasiveness of Shigella flexneri to HeLa cells
when the bacteria were coincubated with TNF-,31
providing some functional data in terms of virulence, but there was no
identification of possible virulence factors being altered because of
the presence of the cytokine.31 Hence, we first looked at
the growth of K pneumoniae in the presence or absence of G-CSF.
For these and subsequent experiments, we chose a concentration of G-CSF
shown to be within therapeutically achievable concentrations (10 ng/mL).27 We could not show any differences in the growth
rate of the bacteria (Fig 5). However, the fact that the virulence of
Klebsiella strains in humans and animals is strongly correlated
with the degree of encapsulation is well
established.28,37-40 We therefore investigated whether the
coincubation with G-CSF of the strain used in this study, K
pneumoniae B5055, would result in an altered production of
cell-bound CPS. Indeed, K pneumoniae B5055 produced
significantly more cell-bound CPS when grown in the presence of G-CSF
than when grown in media alone (Fig 6). To confirm the functional
importance of this finding, we subjected the bacteria grown either in
the presence or absence of G-CSF to a PMN killing assay in which
killing of the bacteria by neutrophils in vitro depends on
phagocytosis. As expected of bacteria with increased amounts of
cell-bound CPS, bacteria coincubated with G-CSF were significantly more
resistant to killing by neutrophils than untreated controls (Fig 7). In
separate assays, we could show that this enhanced resistance was caused
by decreased phagocytosis of the bacteria, which can be explained most
easily by the increased production of cell-bound CPS.17,28
Because other cytokines like IL-1 have been described to increase
virulence of gram-negative bacteria29 and because this cytokine plays a major role in gram-negative bacterial infection and
sepsis,41 we investigated the possibility that it may
affect the virulence of K pneumoniae as well. Interestingly, a
500-fold excess of IL-1 did not compete with binding of
125I-G-CSF to K pneumoniae (Fig 9C), whereas
unlabeled G-CSF did in a dose-dependent manner (Fig 9B). Accordingly,
the coincubation of the bacteria with IL-1 did not lead to increased
bacterial growth or enhanced production of cell-bound CPS (Figs 5 and
6).
Thus, our in vitro studies correlated well with our in vivo results
which show enhanced bacterial spread to distant sites only when G-CSF
was present before infection. Also, the data obtained using the MoAb
against K2-CPS, MoAb III/5-1, lent further support to our hypothesis,
because its administration in an optimal dose17,18 together
with G-CSF not only reversed the increased mortality observed with
pretreatment with G-CSF alone (Fig 1), but also ameliorated the massive
necrotic changes observed in animals receiving only G-CSF. The MoAb
against K2-CPS, III/5-1, presumably directly counteracted the effect of
G-CSF on K pneumoniae, which resulted in the enhanced
production of CPS. In fact, those animals receiving both antimicrobial
therapy (MoAb III/5-1) and therapy to enhance host defenses (G-CSF)
showed the best outcome with respect to survival, body weight,
bacterial colony counts, and histology (Figs 1 and 2).
At first glance, these observations seem to contrast with a prior study
which showed a beneficial effect of G-CSF (administered at the same
dose, ie, 50 µg/kg) on survival in a similar pneumonia model using
rats instead of mice, but also with K pneumoniae.3 However, there are marked differences between this study and our observations. Most importantly, the bacterial strain used by Nelson et
al3 seems to be rather avirulent because approximately 5 × 107 CFU were needed to elicit a lethal pneumonia.
Consequently, mortality induced by this level of bacterial challenge
may have been caused by lethal intoxication, perhaps by LPS, and not by
replicating and disseminating bacteria, attributable to the CPS. In
contrast, the strain used in our study is even more virulent based on
differences in CPS production17 than a strain used in one
of our previous reports (K pneumoniae
Caroli)17,18 in which a 1000-fold fewer amount of CFU
(3 × 104) was able to provoke a fulminant pneumonia
in rats.18 To test this hypothesis, we incubated an
avirulent strain of K pneumoniae (F201, producing only minimal
amounts of K2-CPS) with G-CSF under the same conditions as the virulent
strain (B5055). Although G-CSF bound to this avirulent strain F201 (Fig
9A), there was no increase in cell-bound CPS production (Fig 6, inset).
When administered intranasally into mice, no lethality was observed
with K pneumoniae F201 (up to a dose of 5 × 107
CFU) in contrast to K pneumoniae B5055
(LD50 = 1.3 × 103 CFU; T.K. Held, M. Trautmann, and A.S. Cross, manuscript in preparation). Such differences in the response of various bacterial strains have also
been shown in other studies: IL-1, IL-2, and GM-CSF enhanced growth
only of virulent strains of E coli, but not of avirulent
strains.29,30 Indeed, in all reports showing a beneficial effect of treatment of bacterial infections with G-CSF, a relatively high number of organisms had to be administered to
elicit a lethal effect: 5 × 106 CFU of
P aeruginosa42; 2 × 107 CFU of P
aeruginosa6; 5.9 × 106 CFU of
P aeruginosa7; 5 × 1010 CFU of E
coli43; 1 × 106 CFU of S
pneumoniae4; 4.5 × 107 CFU of S
aureus; 1.2 × 106 CFU of E coli; or 1 × 106 CFU of S marcescens8; or a fecal
inoculum containing up to 42 different bacterial species.44
G-CSF enhances the number and function of
neutrophils36,45-48 and is recently considered as a
prophylactic agent against sepsis, even in non-neutropenic
patients.49-52 However, we here show that G-CSF, in
addition to its known effects on the host, may also act directly on the
invading bacteria by increasing the virulence of K pneumoniae.
Our results show that this dual action under some conditions may
actually shift the balance in favor of the bacteria and cause
deleterious effects. The potential of this latter outcome should be
considered, especially in situations in which G-CSF is administered
without concomittant antimicrobial therapy such as antiinfective agents
or protective antibodies.
| |
FOOTNOTES |
Submitted June 20, 1997;
accepted November 19, 1997.
Supported in part by a grant from AMGEN GmbH, München,
Germany.
Presented in part at the 96th General Meeting of the American
Society for Microbiology, New Orleans, LA, May 19-23, 1996 (Abstract E-18).
Address reprint requests to Thomas K. Held, MD, Abteilung für
Innere Medizin m.S. Hämatologie und Onkologie,
Virchow-Klinikum der Humboldt-Universität,
Augustenburger Platz 1, 13353 Berlin, Germany.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
We are grateful to Drs H. Hahn and R. Chang for critical discussion.
The invaluable help of Dr Stephanie Treiber-Held is deeply appreciated.
We also thank Ed Shatzer and Dr P. Stevens for generously supplying
cytokines and various antibodies.
| |
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Abstract
Introduction
Abstract
