THE INFLAMMATORY RESPONSE OF GRAM-NEGATIVE PNEUMONIA
AND ITS RELATION TO CLINICAL DISEASE
Charles W. Frevert, D.V.M., Ph.D.
Research Assistant Professor, Department of Medicine
University of Washington, Pulmonary Research Laboratory
Seattle VA Medical Center
Pathogenesis of Gram-Negative Pneumonia
Despite the use of preventative measures, broad-spectrum antibiotics, and complex
supportive care, gram-negative pneumonias remain an important cause of morbidity and
mortality. In people, gram-negative bacteria have become the leading cause of hospitalacquired pneumonia accounting for 40-60% of the pathogens associated with
nosocomial pneumonia [1, 3]. In contrast, gram-negative pneumonias cause only a
small percentage (4-20%) of the community-acquired pneumonia diagnosed in humans
[1, 2, 4, 5]. In veterinary species, gram-negative bacteria are a common cause of
pneumonia in hospitalized patients and animals in their natural setting.
Three mechanisms predispose the lung to gram-negative pneumonia by allowing
bacteria access to the respiratory tract [1, 2]. These mechanisms are 1) the aspiration of
colonizing oropharyngeal gram-negative bacteria, 2) the bacteremic spread of gram
negative organisms to the respiratory tract, and 3) the aerosol spread of gram-negative
bacteria through aerosolization or nebulization of contaminated fluids. Once gramnegative organisms have gained access to the respiratory tract the development of
pneumonia is determined by the status of host defenses, the number of organisms that
gain acces to the respiratory tract and the virulence of the bacteria. In most instances,
pulmonary host defenses are able to clear the bacteria from the lungs with minimal
clinical signs. However, if host defenses are impaired, or if gram-negative bacteria
overwhelm host defense due to either excessive numbers or virulence factors, significant
clinical problems can develop.
Clinical manifestations of gram-negative pneumonia
When gram-negative pneumonia is not adequately controlled by pulmonary host
defenses a patient will develop a systemic inflammatory response. In rabbits with gramnegative pneumonia the intensity of the systemic inflammatory and physiological
response correlated with the inoculum size and whether the bacteria are cleared from or
proliferate in the lungs [6]. In this study, rabbits that cleared the gram-negative infection
developed a clinical picture (e.g., fever and leukocytosis) similar to systemic
inflammatory response syndrome (SIRS) in humans. These rabbits met the clinical
definition of sepsis, which includes the presence of SIRS plus tissue infection [7]. When
bacteria were instilled at much higher numbers or when bacteria began to proliferate in
the lungs, the investigators found that rabbits developed a clinical picture consistent with
severe sepsis, or septic shock [6].
Clinical studies suggest that the initial site of infection (e.g., lung) is an important
determinant on whether severe sepsis or septic shock develops. It has been shown that
patients with pneumonia and abdominal infections are far more likely to develop severe
sepsis when compared to infections at other sites (e.g., urinary tract or skin) or with
primary bacteremia. The importance of tissue infection has been further documented in
rabbits where septic shock developed following the airspace instillation but not the
intravenous infusion of Pseudomonas aeruginosa [8].
Pulmonary Host Defenses
The success or failure of pulmonary host defenses is a critical determinant for the
appearance of clinical lung disease and its systemic manifestations (e.g., sepsis and
septic shock). The
Ciliated
Epithelial Cells
lungs are an elaborate system
Type I
Epithelial Cell
and include physical defense
Gram Negative Bacteria
1
mechanisms (e.g., upper
TNF-
Type II
Epithelial Cell
2
AM
airway filtering, and cough)
and cellular defense
3
IL-8
PMN
antimicrobial defenses of the
IL-8
4
mechanisms (e.g., alveolar
macrophages, neutrophils and
Endothelial Cells
lymphocytes). The terminal
airways and alveoli are largely
Figure 1: Pulmonary Host Defenses in the alveolar space.
Key determinant in the clearance of gram-negative bacteria
from the lungs include: 1) bacterial recognition, 2) production
of pro- and anti-inflammatory cytokines, 3) production of
chemokines , and 4) PMN transmigration.
devoid of physical defense
mechanisms and therefore
rely primarily on pulmonary
cellular defenses to recognize,
ingest and kill bacteria that are deposited there. Of the cellular defenses, two phagocytic
cells, the alveolar macrophage (AM) and neutrophil (PMN) play a primary role in early
bacterial clearance. It has become apparent that the AM plays a pivotal role in
pulmonary host defenses and the clearance of gram-negative bacteria from the lungs.
Following the recognition of a bacterial pathogen, the AM must eliminate the
microorganism from the alveolus. The AM is capable of killing and digesting low doses
of Staphylococcus aureus on its own, however when gram negative bacteria are
deposited into the lung the AM becomes an effector cell, initiating PMN migration and
activation (Figure 1) [9-11]. Depletion of AMs from the airspaces of the lungs prior to the
instillation of gram-negative bacteria has been shown to cause impaired pulmonary
recruitment of PMN, decreased bacterial clearance and increased mortality [12, 13].
Neutrophil migration into the lung is the result of endogenous (chemokines, and C5a)
and exogenous (fMLP) chemotactic factors. Of the endogenous chemotactic factors,
chemokines have been shown to be important chemotactic factors for the pulmonary
recruitment of PMNs in gram-negative pneumonia [14, 15]. Neutropenia has been
shown to decrease the clearance of bacteria and increase mortality rates in animal [16]
and clinical studies of gram-negative pneumonia [17].
Bacterial Recognition: The innate immune system has evolved a set of germlineencoded receptors that recognize common motifs on microbial pathogens. These
receptors, commonly referred to as pattern recognition receptors, include the mannose
receptor, scavenger receptors, and CD14 [18-20]. CD14 is an important recognition and
signaling receptor for lipopolysaccharide (LPS) and gram negative bacteria. The
recognition and signaling through CD14-dependent pathways requires 3 proteins, LPS
binding protein (LBP), CD14, and toll-like receptors (TLR) [21, 22]. LPS-binding protein,
a plasma lipid transfer protein, acts on LPS aggregates or bacterial membranes and
presents LPS monomers to binding sites on CD14 [23]. CD14, a
glycosylphosphatidylinositol (GPI)-linked protein found on the surface of myeloid cells
binds to LPS, resulting in cellular activation, production of pro-inflammatory cytokines
(e.g., TNF-, IL-6) and chemokines (e.g., IL-8) and activation of inducible nitric oxide
synthase (iNOS) [24, 25]. While LPS recognition is mediated by CD14, another receptor
is required for intracellular signaling [26]. Toll-like receptor-4 is a likely candidate for
CD14-dependent signal transduction because TLR-4 initiates NFLPS-resistant mice have a critical mutation in the intracellular portion of TLR-4 [27-29].
Because CD14-LPS interactions result in cellular signaling and production of
proinflammatory cytokines, this recognition pathway is considered a critical component
of pulmonary host defenses toward gram-negative bacteria.
Chemokines: The term chemokine has been used to describe a group of structurally
similar chemotactic cytokines that attract and activate leukocytes [30]. The chemokines
are separated into 4 families which are structurally defined (i.e., CXC-, CC-, CX3C- and
C hem otactic Index
C-chemokine families). The CXC1.0
chemokines are peptides that have 1
0.8
amino acid separating the first two
0.6
cysteines while the first two cysteines
0.4
of the CC-chemokine family are
0.2
Interleukin-8
E N A -78
G R O -
G R O -
G R O -
0.0
-0.2
1e-11 1e-10 1e-9
1e-8
1e-7
1e-6
1e-5
Log 10 [C hem oattractant] (M )
Figure 2: Neutophil migration towards 5 CXC
chemokines in vitro.
adjacent. In general, CXC-chemokines
are chemoattractants for PMN while
CC-chemokines are chemoattractants
for monocytes, eosinophils and
lymphocytes. Interleukin-8 is the
prototype of a family of cytokines
known as the CXC-chemokines. This family of chemotactic cytokines are potent and
effective PMN attractants and are believed to be primarily responsible for the pulmonary
recruitment of PMNs in response to gram-negative bacteria [14, 31-33] (Figure 2).
Following the recognition of gram-negative bacteria by AM (Step 1, Fig. 1), IL-8 is
produced directly by AM (Step 2, Fig.1) or by the activation of other cells in the lungs
through cytokine networks (Step 3, Fig. 1) [34].
Therapeutic Strategies
Despite significant advances in the understanding of the pathogenesis of gramnegative pneumonia, antibiotic therapy and intensive supportive care remain our primary
means to treat pulmonary infections. However, due to difficulties in identifying
appropriate pathogens and an increase in antibiotic resistant bacteria there is an
increased interest in the development of novel therapeutic strategies for the treatment of
gram-negative pneumonia and its clinical sequelae. Currently, therapeutic strategies are
being developed for the treatment of patients with gram-negative pneumonia using two
different approaches. The first strategy has focused on improving pulmonary host
defenses to improve the clearance of bacteria from the lungs. Therapeutic interventions
with granulocyte-colony stimulating factor (G-CSF) and interferon-
-
examples of this strategy. The second strategy has focused on the exuberant systemic
inflammatory response that often occurs in patients with gram-negative pneumonia and
has attempted to block or minimize this response. Some of these therapeutic strategies
have focused on blocking TNF-
-LPS interactions.
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