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* Center for Amyloidosis and Acute Phase Proteins, Department of Medicine, Royal Free and University College Medical School, and
Department of Infectious Diseases and Microbiology, Imperial College School of Medicine, Hammersmith Hospital, London, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-1
, or IL-6 after E. coli
infection nor susceptibility to LPS toxicity. Furthermore, protection
against infection was unaffected in IL-1R knockout mice, which have
deficient acute phase plasma protein responses, or after nonspecific
inhibition of acute phase protein synthesis by
D-galactosamine or specific depletion of complement C3 by
cobra venom factor. Increased production of G-CSF in the acute
phase response is thus a key physiological component of host defense,
and pretreatment with G-CSF to prevent bacterial infection in at-risk
patients now merits further study, especially in view of increasing
bacterial resistance to antibiotics. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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After most forms of tissue injury, infection, and inflammation, a complex cascade of cytokine production occurs in all endothermic species and mediates the nonspecific acute phase response (2). This comprises alterations in expression of genes encoding a wide range of plasma proteins, including proteinase inhibitors, clotting, complement, and transport proteins (3). The responses of serum amyloid A protein and of the pentraxins, C-reactive protein, and, in the mouse, serum amyloid P component are exquisitely sensitive, even to subclinical processes. This phenomenon and its stable evolutionary conservation strongly suggest that the acute phase response and its components have survival value. Indeed many of the cytokines and acute phase plasma proteins are postulated to have functions that may contribute to host defense and repair (4). Here, we show that the acute phase response induced by local sterile inflammation dramatically enhances resistance to acute pyogenic bacterial infections and that a single cytokine, G-CSF, is a key mediator.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BALB/c, C57BL/6, and CD1 mice were purchased from B & K Universal (Hull, U.K.). IL-1R-/- (BALB/c genetic background) mice were kindly provided by M. Kopf (Basel, Switzerland). A single s.c. injection of 0.5 ml of 10% (w/v) casein (ICN Pharmaceuticals, Basingstoke, U.K.) in 0.05 M NaHCO3 buffer was used to stimulate the acute phase response in mice as described previously (5). All control animals received s.c. injections of 0.5 ml of 0.05 M NaHCO3 buffer alone. D-galactosamine (10 mg; Sigma-Aldrich, Poole, U.K.) in saline was given i.p. at the same time as casein or control buffer injection to inhibit de novo hepatic protein synthesis (6). Cobra venom factor, 10 U, was injected i.p. at the same time as casein or control buffer injection to deplete complement component C3 (7). Recombinant murine G-CSF and rabbit polyclonal IgG Ab to mouse G-CSF were kindly provided by G. Senaldi (Amgen, Thousand Oaks, CA). Ab to G-CSF, 0.5 mg, was injected i.v. at the same time as casein or control buffer to neutralize endogenous circulating G-CSF. Normal rabbit IgG was used as a control for the anti-G-CSF Ab.
Bacterial infections and LPS toxicity
Groups of 10–20 weight- and sex-matched 8- to 10-wk-old mice received either casein or control buffer 24 h before infection. Escherichia coli O111:B4 (from B. J. Applemelk, Vrije Universiteit, Amsterdam, The Netherlands) or an M1T1 clinical isolate of Streptococcus pyogenes (8) was grown to log phase. The bacteria were then washed and resuspended in saline. Mice were infected with 5–10 x 107 E. coli by i.v. injection or with 1–5 x 107 S. pyogenes by i.m. or i.p. injection. Morbidity and mortality were then recorded until no further deaths occurred. In some experiments, CD1 mice infected with E. coli O111:B4 were partially treated with gentamicin as previously described (9). For investigation of LPS-induced toxicity, mice were injected with 10 mg/kg LPS (from E. coli O111:B4; Sigma-Aldrich) i.p. 24 h after casein or control buffer injection.
Quantitative bacteriology, cytokine assays, and acute phase protein assays
Mice were sacrificed at sequential time points after infection, and viable bacteria were quantified by colony counts from serial dilutions of heparinized blood. Immunofluorescence staining was used to visualize bacteria within tissues. Sections, 30 µm, of liver and spleen from mice 12 h after infection with E. coli were incubated with a rabbit polyclonal Ab to the o-polysaccharide side chain of LPS from E. coli O111:B4 (kindly provided by D. Heumann, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland) and subsequently with a fluorescein-conjugated goat anti-rabbit Ab (Jackson ImmunoResearch Laboratories, West Grove, PA) before conventional fluorescence microscopy using a Nikon Eclipse E600 (Nikon, Kingston, U.K.). ELISA immunoassays (R&D Systems Europe, Rennes, France) were used for quantification of cytokines, and electroimmunoassays were used to measure serum amyloid P component (SAP)4 and C3 in mouse serum, as previously described (5).
Neutrophil function assays
Neutrophil phagocytosis and respiratory burst responses were
measured by a modified flow cytometric assay (10) using
E. coli O111:B4, which was transformed with the plasmid
pFPV25.1 containing gfp-mut 3a under the constitutive S13
ribosomal protein promoter (from D. Holden, Imperial College School of
Medicine, London, U.K.) to express green fluorescent protein
(GFP) (3). Dihydroethidine (Sigma-Aldrich), 0.1 µg/ml,
was added to whole heparinized mouse blood, followed by
107 GFP-expressing bacteria in early log phase
(
10 bacteria per neutrophil). Phagocytosis was allowed to take place
for 15 min, rotating at 37°C, and then was stopped by FACS Lysing
Solution (BD Biosciences, San Jose, CA). Ingestion of GFP-expressing
bacteria and respiratory burst responses, indicated by oxidation of
dihydroethidine to red fluorescent ethidium bromide, were quantified by
flow cytometry (FACSCalibur flow cytometer with CellQuest software, BD
Biosciences). Granulocytes, 104, identified by
light scatter parameters were analyzed in each sample. Relative mean
cellular green and red fluorescence in cells that were positive for
both fluorochromes quantified phagocytosis and respiratory burst
responses. Neutrophil recruitment to tissues was measured by a
myeloperoxidase assay (11). Livers from mice 3 h
after infection were washed with PBS, homogenized mechanically
(Ultramax T25; IKA Labortechnik, Staufen, Germany) in 0.5% (w/v)
hexadecyltrimethylammonium bromide in 50 mM
K2PO4, pH 6.0, and then
sonicated on ice for 20 s, and cell suspensions were further lysed
by three freeze-thaw cycles. Tissue debris was removed by
centrifugation and samples were standardized for protein content using
a detergent-compatible protein assay (Bio-Rad Laboratories, Hemel
Hempstead, U.K.). Relative myeloperoxidase activity in samples was
compared in microtiter plates by incubating equal volumes of sample and
substrate (0.1% (w/v) o-dianisidine dihydrochloride in
homogenization buffer with 0.001% (v/v)
H2O2), stopping the
enzymatic reaction with 1 M
H2SO4 and reading the OD at
460 nm using a microplate reader. Samples were diluted to give ODs in
the linear range.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In mice, s.c. injection of casein, a classical sterile
inflammatory stimulus (5), induces a rise in serum levels
of the acute phase proteins that is maximal at 24 h (Fig. 1
). Mice that received an s.c. injection
of casein 24 h before i.v. infection with E. coli or
i.p. or i.m. infection with S. pyogenes had markedly reduced
morbidity and improved survival compared with control animals injected
with solvent alone (Fig. 2). The same
results were obtained in outbred CD1 as well as inbred C57BL/6 and
BALB/c mice, indicating that this is a general phenomenon.
Casein-pretreated mice had significantly fewer viable bacteria in the
blood within 1 h of infection and at least 1000-fold fewer live
bacteria by 12 h (Fig. 3A). Estimation of bacterial
load by immunofluorescence showed accelerated clearance of bacteria
from the tissues of "acute phase" mice with strikingly greater
numbers of organisms in the livers and spleens of control animals (Fig. 3B). In contrast, pretreatment with casein did not improve
the survival of mice receiving a lethal parenteral dose of
Gram-negative bacterial LPS (Fig. 3C). The improved survival
of casein-pretreated mice after infection with E. coli thus
does not result from prior exposure to LPS, which may contaminate the
casein preparation and could potentially increase tolerance to LPS
toxicity (12). This observation also suggests that
pretreatment with casein does not protect against the proinflammatory
host response, which has been widely implicated in the pathogenesis of
sepsis (13). Indeed, we found that after i.v. infection of
mice with E. coli there were no significant differences
between casein-pretreated and control groups in the serum
concentrations of the major proinflammatory cytokines TNF-, IL-1,
and IL-6, (Fig. 4).
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A number of acute phase proteins have well-described roles in host
defense against bacterial infections (4). Indeed, in
previous studies pretreatment of mice with IL-1, which was found to
enhance survival from subsequent Klebsiella pneumoniae or
Pseudomonas aeruginosa infection, was attributed to the
synthesis of acute phase proteins, because inhibition of hepatic
protein synthesis by D-galactosamine completely
abrogated the protective effect of IL-1 (6). Therefore, we
investigated the possible role of IL-1 in our casein model by using
mice with targeted deletion of the IL-1 receptor
(IL-1R-/-) (14). As expected, the
serum rise in acute phase proteins in response to casein was blunted
compared with that of wild-type controls (Fig. 5A). Nonetheless, pretreatment
of IL-1R-/- mice with casein still led to
significant protection against lethal i.v. infection with E.
coli (Fig. 5B). Clearly, therefore, neither IL-1 nor
acute phase protein synthesis induced by IL-1 make physiologically
significant contributions to host defense against bacterial infection
in this model. To further explore the contribution of major acute phase
plasma proteins in our model, we investigated the role of global de
novo hepatic acute phase protein synthesis in wild-type mice and the
role of the complement system in particular. The complement system
plays an important role in host defense against bacteria, and many
complement proteins, including the pivotal C3 component, are acute
phase reactants. However, specific depletion of circulating C3 by prior
administration of cobra venom factor (7) (Fig. 5C) did not abrogate the protective effect against infection
conferred by prior induction of the acute phase response (Fig. 5D). Furthermore, the protective effect remained intact even
when mice received D-galactosamine (Fig. 5F), a specific inhibitor of de novo hepatic protein
synthesis (6), at a dose that did not cause significant
hepatic necrosis but still globally reduced the acute phase plasma
protein response by 50% (Fig. 5E). Higher doses of
D-galactosamine, which completely block the acute
phase response, cause severe liver damage that compromises the
infection models and therefore could not be used (data not shown).
Nevertheless, these results indicate that the enhanced resistance to
acute lethal systemic bacterial disease, displayed by animals mounting
an acute phase response at the time of infection, is not dependent on
increased production of the classical acute phase plasma proteins.
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The major effect of an existing acute phase response was clearly
to enhance host capacity to kill and clear virulent bacteria.
Neutrophils are the major cell type primarily involved in defense
against pyogenic bacteria, but there was no difference in circulating
neutrophil counts between control mice and those receiving casein 30
min, 12 h, or 24 h previously, nor was there a difference
between these two groups at 1, 3, 6, or 12 h after i.v. infection
with E. coli (data not shown). However, the
neutrophils from casein-pretreated mice showed significantly enhanced
phagocytosis of bacteria in vitro and greater respiratory burst
responses than neutrophils from controls, suggesting that they may have
increased capacity for killing bacteria (Fig. 6, A and B).
Furthermore, there was significantly enhanced neutrophil recruitment
and accumulation within the tissues of the casein-pretreated animals in
vivo (Fig. 6C), suggesting that whole body neutrophil
numbers may have been higher.
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This critical priming of neutrophil function was most likely to be
mediated by cytokines. Therefore, we measured serum levels of candidate
cytokines after casein injection. As expected, there was a rise in
IL-6, which is the major mediator of increased production of acute
phase proteins by the liver (15). Serum IL-6 values peaked
at 6 h and normalized by 24 h (Fig. 7A). The circulating
concentration of G-CSF also rose and, in contrast to IL-6, detectable
levels were sustained at 24 h. However, no TNF-, IL-1,
IFN-
, or GM-CSF were detected in the serum at the sensitivities of
the assays used, nor were any cytokines detected in the serum of
control mice receiving buffer alone. The G-CSF response after casein
injection was intact in IL-1R-/- mice,
indicating that IL-1 activity is not required.
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Mice with targeted deletion of the G-CSF gene are severely neutropenic
(17) and cannot be used in the present models of infection
to analyze underlying mechanisms. Therefore, we used specific
neutralizing Ab to G-CSF to block its effects in vivo after injection
of casein. This had no effect on neutrophil counts or the rise in serum
acute phase proteins (data not shown), but it completely abrogated the
protective effect against i.v. infection with E. coli (Fig. 7
B). The key role of G-CSF itself in mediating protection
was then directly confirmed by pretreating mice with a single s.c.
injection of 1 µg of recombinant murine G-CSF or control buffer
24 h before infection. Pretreatment with G-CSF alone, which
produced no stimulation of acute phase plasma protein production,
provided the same significant survival benefit, as the casein induced
acute phase response (Fig. 7C). G-CSF was similarly
effective when given at 3 or 6 h before infection, but not 48–72
h before, immediately before, or at 2 h after infection (Fig. 8).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1-acid glycoprotein
(18). However, in our model, nonspecific inhibition of
acute phase protein synthesis did not diminish the protective effect of
pretreatment with casein. Use of nonspecific proinflammatory stimuli in previous reports has not led to the development of a therapeutic strategy to increase the resistance of patients at risk of bacterial infections. Our study demonstrates clearly that G-CSF is a cytokine component of the acute phase response and is the critical mediator therein responsible for enhancing nonspecific host resistance to bacterial infection. Our results confirm and extend previous studies in which pretreatment with supraphysiological doses of exogenous G-CSF provided nonspecific protection against bacterial infection. These include rodent models of P. aeruginosa (19, 20), K. pneumoniae (21), and S. pneumoniae (22) infection and polymicrobial sepsis after cecal ligation and puncture (23). However, the present model significantly demonstrates that endogenous production of G-CSF can induce this protective effect, and it highlights the physiological importance of this cytokine as part of the acute phase response.
Our results are of particular importance because they suggest an immediately applicable strategy for increasing host resistance to infection in clinical practice. Although pretreatment here with G-CSF effectively improved survival in subsequent bacterial infection, administration of G-CSF at the time of infection or afterward provided no benefit. This is consistent with clinical studies in a number of different contexts where concurrent treatment of infection with G-CSF has been of little benefit. For example, G-CSF is widely used after cytotoxic chemotherapy (24), but although it is effective at reducing the duration of neutropenia, it has had no impact on morbidity or mortality (25). G-CSF has also recently been evaluated as an adjunctive agent for treatment of infection in nonneutropenic patients, but with mixed results. The addition of G-CSF to standard therapy in severe community-acquired pneumonia had no beneficial effect (26), although in diabetic patients with foot infections, administration of G-CSF accelerated resolution of cellulitis, reducing antibiotic use and hospital length of stay (27). Our findings suggest that pretreatment with G-CSF before infection becomes manifest, particularly in patients at risk such as those undergoing surgical procedures, may be an effective adjunct or alternative to antibiotic prophylaxis in preventing bacterial infection.
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Acknowledgments |
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Footnotes |
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2 J.C. and M.B.P. contributed equally to this paper.
3 Address correspondence and reprint requests to Dr. Mark B. Pepys, Department of Medicine, Center for Amyloidosis and Acute Phase Proteins, Royal Free and University College Medical School, Rowland Hill Street, London NW3 2PF, U.K. E-mail address: m.pepys{at}rfc.ucl.ac.uk
4 Abbreviations used in this paper: SAP, serum amyloid P component; GFP, green fluorescent protein.
Received for publication January 24, 2002. Accepted for publication May 3, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in experimental Gram-negative shock. J. Infect. Dis. 162:421.[Medline]
1-acid glycoprotein in nonspecific resistance to a lethal Gram-negative infection. J. Biol. Chem. 275:14903.This article has been cited by other articles:
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) or control
buffer (
) in groups of five mice sacrificed at times shown; each
point represents the mean ± SD.
) were significantly
greater than those from controls (
). C, Neutrophil
accumulation into liver tissue 3 h after infection of BALB/c mice
with E. coli demonstrated significantly greater
neutrophil recruitment in casein-pretreated mice (
) 6 h
before infection with E. coli had significantly greater
survival than those receiving G-CSF 2 h after infection (
) or
control mice (
). B, BALB/c mice receiving a single
s.c. injection of recombinant murine G-CSF (