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* From the Louisiana State University Health Sciences Center, Department of Medicine, Section of Pulmonary/Critical Care Medicine, New Orleans, LA.
Correspondence to: Steve Nelson, MD, FCCP, Louisiana State University Health Sciences Center, Department of Medicine, Section of Pulmonary/Critical Care Medicine, 1901 Perdido St, Suite 3205, New Orleans, LA 70112; e-mail: snelso1{at}lsuhsc.edu
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Abstract |
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 TOP Abstract IntroductionCytokines and Host DefenseConclusionReferences |
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Key Words: cytokines • granulocyte colony-stimulating factor • inflammatory response • interleukins • pneumonia • tumor necrosis factor
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Introduction |
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TOPAbstractIntroductionCytokines and Host DefenseConclusionReferences |
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The primary function of these innate defenses is the elimination of bacterial organisms from the alveolus. A dual phagocytic system involving both alveolar macrophages and polymorphonuclear leukocytes (PMNs) mediates early bacterial clearance (Fig 1 ).1 The recruitment and activation of inflammatory cells at a site of infection involve the orchestrated expression of leukocyte and vascular adhesion molecules as well as the establishment of chemotactic gradients via the generation of chemokines and cytokines.
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Clearly, the cellular components of the innate immune response—the
alveolar macrophage and the neutrophil—need to communicate with each
other if an effective host defense is to be mounted. Mechanisms are
needed not only to initiate this response, but also to localize,
reinforce, and ultimately resolve it. One of the essential components
of the immune system that plays a critical role in these processes are
the cytokines.4
5
Cytokines are produced by a wide variety
of cells in the body, play an important role in many physiologic
responses, are involved in the pathophysiology of an extensive range of
diseases, and have therapeutic potential. Cytokines that have been
shown to fulfill an important role in the innate immunity of the lung
include tumor necrosis factor (TNF), IL-10, IL-12, the chemokines,
interferon 
(IFN-), and granulocyte colony-stimulating factor
(G-CSF).
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Cytokines and Host Defense |
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TOPAbstractIntroductionCytokines and Host DefenseConclusionReferences |
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TNF
Named for its ability to trigger the necrosis and involution of
certain tumors, TNF is now recognized as a central mediator of the
host’s response to infection.8
9
It is rapidly produced
following either antigen-specific or nonspecific stimulation and has,
therefore, been designated an early response, or "alarm," cytokine.
Lipopolysaccharide (LPS) is the best studied and most potent stimulus
for TNF production. In Gram-negative bacteria, LPS is the major
proinflammatory component of the cell walls, and the study of
LPS-induced TNF expression by alveolar macrophages is, accordingly,
very relevant to the role of TNF in the host defense response during
Gram-negative pneumonia.
In one of our earlier investigations,10 we determined the effects of LPS on TNF activity and the pulmonary inflammatory response in animals challenged with either IV or intratracheal LPS. In the unchallenged lung, BAL fluid contains no measurable TNF, and the cell population is composed almost exclusively of alveolar macrophages. Following the intratracheal administration of LPS, TNF rapidly rises within the BAL fluid, and a neutrophilic alveolitis develops within the lower respiratory tract.
These data suggest a role for TNF as a proximal factor in eliciting the recruitment of PMNs into the lung. This is supported by studies of the neutralization of LPS-induced increases in BAL fluid TNF by either an anti-TNF antibody or a replication-deficient adenovirus encoding a soluble TNF receptor. Both of these anti-TNF molecules suppress PMN influx into the lung.11 12 Most importantly, these studies show that LPS-induced TNF is largely confined to the LPS-challenged compartment. When LPS was administered IV, eliciting a large serum TNF response, TNF was not detected in the BAL fluid. Similarly, intratracheal LPS-induced increases in BAL fluid TNF did not result in an increase in serum TNF. Boujoukos and colleagues13 extended these observations to human volunteers administered IV LPS. In these normal subjects, despite high levels of circulating cytokines following systemic LPS administration, there was no significant increase in BAL fluid levels of TNF, IL-1, IL-6, or IL-8. Dehoux and colleagues14 studied lung and serum cytokine levels in 15 patients with unilateral community-acquired pneumonia (CAP). They too found that the inflammatory response was compartmentalized within the infected human lung and was limited to the site of the infection, with localized production of TNF, IL-1, and IL-6.
IL-8
While these proinflammatory mediators are not thought to have
direct chemoattractant activity, both TNF and IL-1 are potent inducers
of IL-8 production by several cell types, including alveolar
macrophages, type II epithelial cells, and lung
fibroblasts.15
Among this family of chemotactic cytokines,
or chemokines, IL-8 has been identified as the major chemotactic factor
for PMN in the lung. Boutten and colleagues16
measured
serum and BAL fluid IL-8, as well as the number of PMNs in the BAL
fluid, in 17 patients with unilateral CAP. IL-8 was found to be
elevated in the BAL fluid but not in the serum, and the BAL fluid IL-8
concentrations correlated positively with the numbers of PMN in the BAL
fluid. In a model of Klebsiella pneumoniae, Greenberger
and colleagues17
showed that in vivo
neutralization of macrophage inflammatory protein 2, the functional
murine homolog of IL-8, resulted in a 60% reduction in PMN influx, a
fourfold increase in the numbers of viable bacteria recovered from the
lungs, and early dissemination of K pneumoniae to both
the blood and the liver.
This selective increase in proinflammatory cytokines may be a critical factor in compartmentalizing the host inflammatory response within a specific tissue site in an attempt to maintain homeostasis. Clearly, it would not be advantageous to the patient infected with pneumonia to activate a generalized systemic response to an initially localized insult. Furthermore, these data suggest that specific mechanisms exist to insulate the lower respiratory tract from the effects of systemic endotoxin and high circulating levels of cytokines. However, in pathologic states, certain cytokines may circulate to act beyond the organ of origin.
Therefore, the local production of cytokines is a normal part of the host response to a bacterial infection, and the presence of cytokines under these conditions does not equate with tissue injury or organ failure. If cytokines are important mediators in pulmonary host defense, then an impaired cytokine response should be associated with an increased susceptibility to infections. Neutralization of the TNF response has been shown to impair pulmonary host defenses against a wide range of other pathogens, including Pseudomonas aeruginosa, Legionella pneumophila, K pneumoniae, Staphylococcus aureus, Streptococcus pneumoniae, and Mycobacterium tuberculosis.4 11 12 18 19 20 21 22
It is now widely recognized that TNF is a pivotal mediator in
determining the outcome of a broad array of infectious diseases in the
host. Its potential role in immunotherapy would be obvious, except that
systemic administration of this proinflammatory cytokine is associated
with significant toxicity and poor penetration into the lung. An
alternative approach to the direct instillation of TNF into the lung is
to prime alveolar macrophages with IFN-, so that they secrete
greater amounts of TNF and other mediators in response to an infectious
stimulus. IFN- is a cytokine produced by T cells and natural killer
cells that is instrumental in cell-mediated immunity and has been shown
to enhance several macrophage effector cell functions, including the
respiratory burst, macrophage-derived TNF release, and antimicrobial
activity.23
IFN-, like TNF, appears to be largely
confined to the compartment in which it is endogenously released or
experimentally administered.24
In a recent study by Maus
and colleagues,25
alveolar macrophages obtained by BAL
from patients with severe CAP showed a significantly reduced TNF
response to LPS stimulation in vitro. Stimulation of
alveolar macrophages from these patients with LPS plus IFN-
augmented the TNF response to near-normal levels. Similar findings have
been reported in patients with sepsis.26
Whether these
findings are relevant for therapeutic strategies to overcome states of
impaired host defense in patients with pneumonia awaits further
investigation.
IL-10
IL-10 is a cytokine that was first recognized for its role in
promoting Th2-type immune responses through the inhibition of
cell-mediated (Th1) immune responses. It is now clear that IL-10 is
also important in the innate immune response to bacterial pathogens.
This anti-inflammatory cytokine downregulates the production of TNF,
IFN, and certain chemokines. van der Poll and colleagues27
showed that intranasal administration of S pneumoniae
results in a marked increase in IL-10 in the lungs of normal mice, and,
as has been shown for other cytokines, this response is largely
compartmentalized. In this model, intrapulmonary administration of
IL-10 2 h prior to the S pneumoniae challenge
reduced the lung TNF and IFN- responses. These animals experienced
higher lung and blood bacterial counts and increased early mortality.
Not surprisingly, treatment of mice with anti-IL-10 antibody prior to
the pneumococcal lung challenge was associated with a significant
increase in lung TNF, a sixfold decrease in lung bacterial count, and
improved survival. Greenberger and colleagues28
similarly
reported that administration of a polyclonal anti-IL-10 antiserum in a
murine model of K pneumoniae resulted in improved host
defense and prolonged survival.
IL-12
IL-12 is a heterodimeric protein consisting of two subunits (p35
and p40) and was initially recognized for its ability to promote
Th1-type immune responses. Numerous studies have shown that IL-12 can
enhance cell-mediated host resistance to a wide range of intracellular
pathogens.29
Utilizing immunohistochemical techniques in
animals infected with K pneumoniae, researchers have
found that IL-12 production appears to be localized primarily to
alveolar macrophages, pulmonary epithelial cells, and PMN.
Greenberger and colleagues30
studied the role of
IL-12 in innate immunity in mice intratracheally challenged with
K pneumoniae. This bacterial challenge resulted in a
time-dependent increase in IL-12 messenger RNA and protein in the lung.
Passive immunization with anti-IL–12 serum at the time of the
bacterial challenge suppressed the bactericidal activity of the lung
and decreased survival. Intratracheal administration of a
nonreplicating adenoviral vector containing a cytomegalovirus promoter
and complementary DNAs coding for both the p35 and p40 subunits
of IL-12 improved survival. These survival benefits were reduced if
either endogenous TNF or IFN- was neutralized in vivo
in these animals transfected with this adenoviral vector.
G-CSF
For more than a century, physicians have recognized the
relationship of the WBC count to the occurrence, severity, and outcome
of many infectious diseases. Leukocytosis and neutrophilia were noted
as frequent markers of infectious processes in Osler’s first textbook
of medicine.31
However, the physiologic principles
underlying these responses were poorly understood. Colony-stimulating
factors are a family of acidic glycoproteins that are required for the
proliferation and differentiation of hematopoietic progenitor cells. Of
this cytokine family, which includes granulocyte macrophage
colony-stimulating factor, macrophage colony-stimulating factor,
IL-3, and G-CSF, it is G-CSF that plays an important role in
maintaining the normal blood PMN count and enhancing the functional
properties of PMN, including chemotaxis, phagocytosis, and bactericidal
activity (reviewed in Dale et al32
in 1995).
Examination of the lung may be particularly useful in determining the role of endogenous G-CSF in infectious states. Mononuclear phagocytes, including alveolar macrophages, are known to produce G-CSF when stimulated by bacterial products or cytokines. Tazi and colleagues33 reported that alveolar macrophages recovered from patients with pneumonia produce G-CSF spontaneously, whereas alveolar macrophages from healthy control subjects produce G-CSF only after LPS stimulation. Furthermore, we have also shown that G-CSF, in contrast to TNF and other cytokines, is not compartmentalized, and intrapulmonary instillation of G-CSF results in a significant increase in the number of circulating PMNs.34 We hypothesize that one of the major roles of mononuclear phagocytes (such as the alveolar macrophage) during infection is the local production of cytokines (such as TNF and IL-1), which subsequently induce the alveolar macrophage population to produce G-CSF. Under this hypothesis, G-CSF acts locally to activate and recruit PMNs into the infected lung and functions systemically to stimulate the formation of additional PMNs, thus reinforcing the host’s response until the infection is resolved (Fig 2 ).
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In several models of severe pneumonia, G-CSF improves survival. Splenectomy is a known risk factor for increased morbidity and mortality resulting from pneumococcal pneumonia. In a murine model, G-CSF administered from 24 h before challenge to 3 days after the challenge improved survival among splenectomized mice exposed to an aerosol challenge with S pneumoniae.35 To study the effect of G-CSF in a rabbit model of Gram-negative pneumonia and sepsis, Smith and colleagues36 inoculated rabbits transtracheally with Pasteurella multocida and treated them 24 h later with penicillin G and either G-CSF or placebo once daily for up to 5 days. All rabbits underwent careful histologic examination at the time of septic death or when killed on day 6. In this study, sepsis-induced leukopenia was a predictor of significantly improved survival with G-CSF therapy (57%, vs 39% in control animals). Interestingly, the majority of the survival benefit occurred within the first 24 h of G-CSF treatment, which was prior to the onset of G-CSF-induced neutrophilia. Histologic examination of these animals did not demonstrate evidence of organ toxicity related to G-CSF therapy.
Based on the favorable results obtained from preclinical studies, clinical trials have begun using G-CSF as an adjuvant to antibiotics in the treatment of patients with pneumonia. A phase III, double-blind, placebo-controlled trial of recombinant human G-CSF for the treatment of hospitalized patients with CAP has recently been concluded.37 This was a multicenter trial involving 756 patients enrolled in 71 centers in the United States, Canada, and Australia. Participants in this study were randomized to receive 300 µg/d G-CSF (380 patients) or placebo (376 patients) in addition to conventional antibiotic therapy. Treatment duration was for up to 10 days, and the length of the study observation period was 28 days or until death. The primary objectives of the study were to determine safety and the effect of G-CSF on the time to resolution of morbidity, which was defined as an index of several clinical variables that are useful in determining whether a patient with pneumonia is benefiting from therapy.38
Mortality was low (6%) in this study, and length of stay was only 7 days. Both variables were unaffected by G-CSF treatment. Similarly, the time to resolution of morbidity was 4 days in each treatment group. Treatment with G-CSF was safe and well tolerated. In the intent-to-treat analysis, G-CSF increased blood neutrophil levels threefold, significantly accelerated radiographic resolution of pneumonia, and reduced serious complications. Post hoc analyses showed that these benefits were more pronounced in patients with multilobar pneumonia. In this study, there were 261 patients with multilobar pneumonia, and 28% of these patients were admitted to an ICU at study entry.
Based on the favorable trends seen in these studies, additional trials in patients with multilobar pneumonia and in patients with severe pneumonia and sepsis were initiated and recently completed. The primary end point in the study of G-CSF in patients with multilobar pneumonia was a reduction in the development of organ dysfunction (ARDS, disseminated intravascular coagulation, acute renal failure, shock). A reduction in mortality was the primary efficacy end point in the pneumonia/sepsis trial. Although analyses of these studies are ongoing, the primary end point in both trials was not significantly different for the G-CSF and placebo treatment groups.
What mechanisms are responsible for the beneficial effects of G-CSF therapy that have been observed in these nonneutropenic infected hosts? Are these benefits related more to G-CSF-induced neutrophilia than to enhanced neutrophil function? Several possibilities have been proposed. One of these that we find particularly intriguing is the effect of G-CSF on neutrophil-antibiotic interactions.
Certain antibiotics are known to concentrate in neutrophils, and since G-CSF enhances many of the functions of these cells, we hypothesized that G-CSF might increase the uptake of certain antibiotics into this cell. Furthermore, since G-CSF increases the number of cells responding to an infection, neutrophil-mediated transport of antibiotics to the site of infection might also be increased. These effects may be particularly important in parts of the body where delivery of antibiotics is difficult and/or in the treatment of pathogens that are relatively resistant to treatment. To test this hypothesis, we incubated human neutrophils with G-CSF and ciprofloxacin.39 It is known that concentrations of ciprofloxacin within PMNs are normally three to four times greater than extracellular concentrations of the drug. In these studies, G-CSF increased the intracellular-to-extracellular concentration of ciprofloxacin approximately 10-fold.
Yasuda and colleagues40 studied the correlation between the number of PMNs in the blood at the time of infection and the therapeutic efficacy of an antibiotic. In these experiments, the therapeutic effect of netilmicin, an aminoglycoside, was not significantly affected by the number of PMNs at the time of infection. In contrast, however, the therapeutic effect of ceftazidime, a ß-lactam, was strikingly affected by the number of PMNs in the blood at the time of infection. A 75% decrease in number of blood PMNs at the time of infection resulted in a 99% decrease in therapeutic efficacy of ceftazidime, while a 10-fold increase in PMNs resulted in a 10-fold increase in the anti-infection activity of ceftazidime.
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Conclusion |
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TOPAbstractIntroductionCytokines and Host DefenseConclusionReferences |
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Footnotes |
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= interferon
; IL = interleukin; LPS = lipopolysaccharide;
PMN = polymorphonuclear leukocyte; TNF = tumor necrosis factor |
References |
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TOPAbstractIntroductionCytokines and Host DefenseConclusionReferences |
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antibody suppresses pulmonary antibacterial defenses [abstract]. Am Rev Respir Dis 143S,A393
impairs pulmonary host defenses to Legionella pneumophila. J Infect Dis 176,1019-1028[ISI][Medline]
impairs host defense during pneumococcal pneumonia in mice. Am J Respir Crit Care Med 155,603-608[Abstract]
is required in the protective immune response against Mycobacterium tuberculosis in mice. Immunity 2,561-572[CrossRef][ISI][Medline]
interferon. J Infect Dis 155,574-577[ISI][Medline]
to the human lung. J Clin Invest 88,279-302
treatment. Nat Med 3,678-681[CrossRef][ISI][Medline]
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