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,
*
Department of Anesthesia and Perioperative Care,
Department of Medicine, and
Cardiovascular Research Institute, University of California, San Francisco, CA 94143;
Department of Anesthesiology and Intensive Care, Kyoto Prefectural University of Medicine, Kyoto, Japan; and
¶ InterMune, Inc., Brisbane, CA 94005
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and significant improvement in the hemodynamic parameters associated
with shock compared with animals treated in a similar manner with
nonspecific control IgG. The administration of anti-PcrV
F(ab')2 showed protective effects comparable to those of
whole anti-PcrV IgG. These results document that the therapeutic
administration of anti-PcrV IgG blocks the type III secretion
system-mediated virulence of P. aeruginosa and prevents
septic shock and death, and that these protective effects are largely
Fc independent. We conclude that Ab therapy neutralizing the type III
secretion system has significant potential against lethal P.
aeruginosa infections. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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P. aeruginosa lung infections are frequently associated with high mortality rates (4), particularly in patients who are mechanically ventilated (5). These poor outcomes appear to be due to the development of septic shock and multisystem organ dysfunction syndrome (4, 5, 6, 7). Our research suggests that the type III secretion system plays a critical role in the pathogenesis of P. aeruginosa-induced sepsis and in the mortality of infected animals and patients. We first showed that the type III secretion system was associated with the acute cytotoxicity of P. aeruginosa (8, 9, 10, 11). Depending on the type III secretion phenotype, P. aeruginosa caused acute organ damage (10, 11) and septic physiology (12, 13) in infected animals. We also recently documented that clinical isolates of P. aeruginosa from patients with respiratory or blood-borne infections expressed type III secreted proteins (14). Patients infected with P. aeruginosa strains producing type III secreted proteins had a 6-fold higher mortality rate and an increased incidence of bacteremia and organ failure than patients infected with P. aeruginosa strains not producing these proteins (14).
We also documented that translocation of the P. aeruginosa type III secreted toxins could be blocked by the administration of Ab to PcrV (15). PcrV is a homolog of LcrV, an Ag in the Yersinia type III secretion system (3, 16, 17). Ab against LcrV was shown to protect animals from a lethal dose of Yersinia pestis (18, 19, 20). PcrV and LcrV appear to be integral components of their type III toxin translocation processes (21), and Abs targeting PcrV or LcrV neutralize type III secretion. If this is correct, the neutralizing effects of F(ab')2 of Abs against PcrV should be comparable to those of whole IgG Abs.
Therefore, we investigated the therapeutic effects of anti-PcrV IgG and F(ab')2 of anti-PcrV IgG on septic shock in our animal models of P. aeruginosa pneumonia. Intravenous or intratracheal treatment with anti-PcrV IgG significantly decreased bacteremia, septic shock, and mortality in infected animals. In addition, anti-PcrV F(ab')2 had comparable therapeutic effects, indicating that these beneficial effects are due to the direct neutralization of the P. aeruginosa type III secretion system.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cytotoxic P. aeruginosa strain PA103 was used in this study (8, 10). Bacterial suspensions were prepared as previously described (11). Briefly, PA103 was subcultured on Vogel-Bonner minimal medium and inoculated into trypticase soy broth containing 10 mM nitrilotriacetic acid. The bacterial pellet was washed and diluted into the appropriate number of CFU per milliliter in lactated Ringer’s solution as determined by spectrophotometry. The number of bacteria was confirmed by plating dilutions of bacteria on sheep blood agar plates.
Production of rabbit anti-PcrV IgG and anti-ExoU IgG
The coding sequences for PcrV and ExoU were amplified from the
chromosome of P. aeruginosa PA103 by PCR. The PCR fragments
were then ligated into the E. coli expression vector
pGEX-2TK (Amersham Pharmacia Biotech, Piscataway, NJ) to create a GST
fusion protein construct. E. coli nontagged rPcrV and rExoU
were produced as follows. Recombinant proteins were induced by
isopropylthio-
-galactoside and purified from E. coli
milieu using GST purification modules (Amersham Pharmacia Biotech). The
bound recombinant proteins were digested overnight with thrombin to
cleave the GST tag. The proteins were then eluted with reduced
glutathione, dialyzed overnight against PBS, and applied to a
detoxification column (Detoxi-Gel; Pierce, Rockford, IL) to remove
endotoxin. The endotoxin level of the final product was <2 endotoxin
units/ml as measured by the Limulus amebocyte lysate assay
(Pyrochrome; Associates of Cape Cod, Falmouth, MA). The purities of
rPcrV and rExoU were evaluated using SDS-PAGE and silver staining (Fig. 1
a, left). An
intense single band of rPcrV and rExoU with minor degradates appears in
the stained gel.
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a, right),
and among various proteins derived from the PA103 lysate (Fig. 1b, left) only native PcrV or ExoU was recognized
by anti-PcrV IgG or anti-ExoU IgG (Fig. 1b,
right). The control nonimmune IgG was purified from a naive rabbit by the method described above. Anti-PcrV F(ab')2 was prepared from rabbit antiserum by pepsin digestion and protein A-agarose chromatography by Ab Solutions (Palo Alto, CA). Neither whole IgG nor nonprotein A binding fragments were detected in the F(ab')2 preparation derived from the antiserum.
Bactericidal activity test
PA103 (257 ± 38 CFU/ml) was suspended in PBS alone, PBS with 3% fresh rabbit serum, or PBS with 3% heat-inactivated rabbit serum. Control IgG (rabbit preimmune serum-derived), anti-PcrV IgG, anti-PcrV F(ab')2, or gentamicin was added to the suspension at a concentration of 10 µg/ml. After incubation for 1 h at 37°C, 100 µl of suspension was mixed with 300 µl of sterile saline and spread on sheep blood agar plates. Plates were grown overnight at 37°C, and the CFUs were counted.
Protocols for animal investigations
The protocols for all animal experiments were approved by the animal research committee of the University of California (San Francisco, CA). Pathogen-free male BALB/c mice, 8–12 wk old (Charles River Laboratories, Wilmington, MA), were used to analyze the efficacy of anti-PcrV IgG on survival. Specific pathogen-free male New Zealand White rabbits (range of body weight, 3.6–4.4 kg; Western Oregon Rabbit) were used for experiments analyzing the effect of anti-PcrV IgG on sepsis.
Mouse survival studies and histological analysis of infected lungs
The mice were housed in cages with filter tops in specific pathogen-free conditions. They were briefly anesthetized with inhaled sevoflurane (Ultane; Abbott Laboratories, Abbott Park, IL) in an oxygenated chamber and placed in a supine position with their heads elevated approximately 30°. Bacterial inoculums (5 x 105 CFU of PA103 in 50 µl of lactated Ringer’s solution) were instilled slowly into the left lung of each animal using a gavage needle (24-gauge modified animal feeding needle; Popper & Sons, New Hyde Park, NY) as previously described (13). Once awake, the mice were returned to their cages, monitored regularly, and allowed access to food and water. The mice received an i.v. injection of nonspecific control IgG, anti-PcrV IgG, anti-PcrV F(ab')2, or anti-ExoU IgG at a designated time point after the bacterial instillation. The survival of each mouse was monitored over the next 7 days. In each experimental group designated mice were euthanized for histological analysis of the infected lungs 20 h after the airspace instillation of PA103 (5 x 105 CFU). The lungs were perfused with 10% buffered formalin phosphate for fixation and were embedded in paraffin. Mounted sections were stained with H&E.
Septic shock experiments using anesthetized rabbits
Surgical preparation. Rabbits were anesthetized with an i.v. injection of sodium pentobarbital (25 mg/kg). A tracheotomy was performed, and an endotracheal tube (3.5-mm inner diameter) was inserted and connected to a volume-cycled ventilator (Harvard Apparatus, Holliston, MA). Mechanical ventilation was delivered at a tidal volume of 10 ml/kg body weight, with a positive end-expiratory pressure of 3–4 cm H2O. The respiratory rate and inspired oxygen fraction (FIO2)3 were controlled to keep the arterial carbon dioxide pressure between 35 and 45 mm Hg and the arterial oxygen partial pressure at approximately 150 mm Hg. Volatile anesthetics (0.5–1.0% of halothane) were administered throughout the experiment. Muscle paralysis was achieved with i.v. pancuronium bromide (initial dose, 1 mg/kg, followed by 0.3 mg/kg/h).
A polyethylene tube (0.86-mm inner diameter) was inserted through the endotracheal tube into the left lower lung for subsequent bacterial instillation. Two arterial catheters were placed: one in the right carotid artery for continuous blood pressure monitoring and the other in the right femoral artery for blood sampling. A balloon-tipped, thermodilution catheter (4-French size; Arrow International, Reading, PA) was placed in the pulmonary artery via the right femoral vein. All animals received lactated Ringer’s solution i.v. at a rate of 4 ml/kg/h throughout the 9-h experimental interval. Arterial blood was sampled every 30 min for analysis.
Bacterial instillate. The instillate solution for rabbits contained a volume of 1.5 ml/kg lactated Ringer’s solution/5% BSA with 0.5 µCi 131I-labeled human albumin (Merck Frosst Labs, Kirkland, Canada) as an alveolar protein tracer and 3 mg of anhydrous Evans blue to identify the instilled lung. Each rabbit received an inoculum of 3.6 x 109 CFU PA103 with this instillate. The radioactivity of the instillate was measured in a gamma counter (Autogamma model 5550; Packard Instrument, Downers Grove, IL). For the rabbits of the noninfected control group, the instillate contained everything except P. aeruginosa.
Infection and therapeutic interventions.
Experimental groups are listed in Table I. Three groups of rabbits received an
i.v. administration of control IgG, anti-PcrV IgG, or anti-PcrV
F(ab')2 (5 mg/kg) 1 h after the airspace
instillation of bacteria. Another three groups of rabbits received
control IgG, anti-PcrV IgG, or anti-PcrV
F(ab')2 (3 mg/kg) intratracheally 1 h after
infection. Three additional rabbits were used for a sham control group.
These rabbits received airspace instillates that did not contain
bacteria and received i.v. lactated Ringer’s solution without IgG. We
also tested i.v. anti-PcrV IgG without bacterial instillation in
one rabbit.
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Quantification of lung injury. The severity of lung epithelial injury was quantified by the efflux of 131I-labeled albumin (the alveolar protein tracer) across the alveolar epithelial barrier into the circulation as we previously reported (10, 13). The efflux of 131I-labeled albumin into the bloodstream was calculated by multiplying the counts measured in the blood sample (per milliliter) by the systemic blood volume (body weight x 0.07). Nine hours after bacterial instillation, the rabbits were deeply anesthetized and exsanguinated. Each lung was excised. Lungs were individually homogenized in a sterile manner, placed in preweighed aluminum pans, and dried to constant weight in an oven at 80°C for 3 days. The water to dry weight ratio of the lung was used as an index of lung edema.
Bioassay for TNF-.
The biological activity of TNF- in plasma samples was analyzed by
using mouse sarcoma cells (WEHI-13VAR; American Type Culture
Collection, Manassas, VA) as reported previously (22).
Bacterial cultures. For the quantification of bacteremia, 100 µl of blood was obtained in a sterile manner and streaked onto a sheep blood agar plate. Lung homogenates were diluted in sterile PBS and streaked onto a sheep blood agar plate for the quantification of bacteria in the lungs.
Plasma anti-PcrV IgG titer. In the rabbits receiving anti-PcrV IgG or anti-PcrV F(ab')2, we quantified the plasma anti-PcrV titer by the ELISA against rPcrV. Immunoplates for the ELISA were coated with rPcrV protein (10 µg/ml) and incubated overnight at 4°C. Plasma samples were added to the plates, followed by application of anti-rabbit monoclonal IgG conjugated with alkaline phosphatase (Sigma-Aldrich, St. Louis, MO) as a secondary Ab. Plates were washed two to six times with PBS/Tween 20 after the addition of each Ab. OD was measured at 405 nm after adding phosphatase substrates (Sigma 104; Sigma-Aldrich). The concentration of IgG in each sample was calculated by comparing the absorbance to a standard curve made from anti-PcrV IgG solution.
Statistical analysis
The difference between the control IgG-treated group and the anti-PcrV IgG- or anti-PcrV F(ab')2-treated group was analyzed. The Mantel-Cox rank test was used for assessment of mouse survival. Repeated measure ANOVA followed by Student’s t test or Mann-Whitney U test were used for comparison of serial data. Student’s t test was used for comparison of the other data. Data are presented as the mean ± SEM. p < 0.05 or 0.01 was considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Rabbit polyclonal anti-PcrV IgG and anti-PcrV
F(ab')2 did not show any bactericidal activity
against P. aeruginosa PA103, even in the presence of serum.
In contrast, gentamicin significantly decreased the number of bacteria
(Table II).
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To examine the therapeutic effects of rabbit polyclonal
anti-PcrV IgG on the survival of P. aeruginosa
infection, we performed studies in mice with lethal pulmonary P.
aeruginosa infections. Nonspecific control IgG (100 µg),
anti-PcrV IgG (10, 50, or 100 µg), or anti-ExoU IgG (100
µg) was administered i.v. after the airspace instillation of a lethal
dose of P. aeruginosa strain PA103 (5 x
105 CFU/mouse; Fig. 2). Fewer than 20% of mice receiving
control IgG either 1 or 4 h after infection survived >3 days. All
mice treated with 100 µg of anti-ExoU IgG 1 h after
infection died in 2 days. All mice receiving 50 µg or more of
anti-PcrV IgG 4 h after airspace instillation survived. While
10 µg of anti-PcrV IgG given 4 h after infection did not
improve mortality, mice receiving the same dose administered 1 h
after infection had a survival rate of 80%.
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).
Increased recruitment of neutrophils with destruction of the alveolar
structure was observed in the lungs of mice treated with nonspecific
control IgG, while anti-PcrV IgG significantly decreased
inflammation and lung injury even when given 4 h after the
infection.
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To analyze the therapeutic effects of anti-PcrV IgG in a lung
infection associated with sepsis, we used a rabbit model of acute
septic shock induced by the airspace instillation of P.
aeruginosa. Rabbits receiving an airspace instillation of PA103
consistently exhibit septic physiology within 8 h
(13). By monitoring parameters of septic shock, including
hemodynamics, metabolic acidosis, bacteremia, and the production of
inflammatory cytokines, we were able to evaluate the therapeutic
effects of the anti-PcrV IgG in detail. In these experiments we
administered anti-PcrV IgG or control IgG, either i.v. or
intratracheally, 1 h after the airspace instillation of PA103.
Rabbits receiving control IgG developed septic shock regardless of the
route of administration of the Ab. These rabbits had a decrease in
cardiac output to <70% of baseline (data not shown), mean arterial
pressures decreased to approximately 60% of baseline, and they
developed a severe metabolic acidosis (<10 mEq/l; Fig. 4). Two rabbits that received control IgG
intratracheally died at 8 h from septic shock and severe hypoxemia
(Fig. 5a). Lung edema, as
assessed by the lung water:dry weight ratio, was severe (Fig. 5b), and progressive lung epithelial injury, quantified by
the efflux of alveolar protein tracer, was detected in the rabbits that
received control IgG (Fig. 5c). Significant increases in
bacteremia (Fig. 6a) and
plasma TNF- concentrations were also observed in the rabbits that
received control IgG (Fig. 6b).
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). Lung damage was also significantly less severe in
these rabbits, particularly in those given IgG intratracheally (Fig. 5). Bacteremia was significantly reduced 10- to 100-fold compared with
the control IgG groups and developed later in the course of infection
(Fig. 6a). The plasma TNF- concentration was
significantly lower (10–20 pg/ml) compared with >100 pg/ml in the
control IgG groups (Fig. 6b). Finally, the number of
bacteria cultured from the instilled lung was also significantly
decreased by i.v. anti-PcrV IgG (Fig. 6c). These
findings indicate that anti-PcrV IgG not only had significant
therapeutic effects against acute lung injury, but also decreased
systemic bacteremia and TNF- concentrations and prevented septic
shock.
As control experiments, three rabbits received airspace instillates
that did not contain bacteria. Hemodynamics did not change
significantly, and metabolic acidosis did not occur throughout the
experimental period (Fig. 4). These rabbits had no apparent lung edema
(Fig. 5b), epithelial injury (Fig. 5c),
bacteremia (Fig. 6a), or plasma TNF- elevation (Fig. 6b). In one additional rabbit that received a nonbacterial
instillate, we administered anti-PcrV IgG i.v. and observed no
effect on any of the measured parameters.
Anti-PcrV F(ab')2 protect animals comparable to whole IgG
To further explore the mechanism of the anti-PcrV IgG, we
examined the administration of anti-PcrV
F(ab')2 in infected animals. We administered
anti-PcrV F(ab')2 i.v. to mice infected with
a lethal dose of PA103. One hundred micrograms of anti-PcrV
F(ab')2 administered i.v. 1 h after airspace
instillation of PA103 resulted in an 80% survival rate at 1 wk (Fig. 2a). The histology of lungs of animals that received
anti-PcrV F(ab')2 therapy revealed
significantly decreased inflammation and lung injury, similar to that
seen after anti-PcrV IgG (Fig. 3a).
Next, we tested the effects of anti-PcrV
F(ab')2 in our rabbit model of septic shock. We
administered anti-PcrV F(ab')2 (3 mg/kg)
intratracheally 1 h after the instillation of PA103. This
treatment provided statistically significant protection from lung
damage (Fig. 5), bacteremia (Fig. 6a), and septic shock
(Fig. 4). These effects were comparable to those seen with whole
anti-PcrV IgG molecules. Next, we administered anti-PcrV
F(ab')2 (5 mg/kg) i.v. 1 h after the
instillation of PA103 (3.6 x 109 CFU). This
also improved lung damage and bacteremia, although the effects on
septic shock were not as potent as those of whole anti-PcrV IgG
molecules. In rabbits treated with i.v. anti-PcrV
F(ab')2, mean arterial pressure decreased to
75 ± 3% of baseline (p = 0.18 compared
with control IgG group), and base excess was -4 ± 1 mEq/l at
8 h (Fig. 4).
Plasma anti-PcrV IgG titer
We measured the anti-PcrV titers in the plasma of the rabbits
receiving either anti-PcrV IgG or anti-PcrV
F(ab')2. In rabbits receiving i.v. anti-PcrV
IgG (whole molecules of IgG), anti-PcrV titers were consistently
elevated during the experimental period. In rabbits receiving i.v.
anti-PcrV F(ab')2, a rapid decrease in the
plasma titer was observed over 8 h (Fig. 7, left). In rabbits receiving
intratracheal administration of anti-PcrV IgG or anti-PcrV
F(ab')2, the plasma anti-PcrV titer was
trivial, although there was a slight gradual increase during the
experimental period (Fig. 7, right).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The word secretion describes the process by which toxins are transferred from the bacterial cytosol across the inner and outer bacterial membranes (2, 24). This process requires a secretion apparatus involving many protein components encoded by the type III secretion system regulatory gene locus. The word translocation describes the process of toxin transfer directly into the eukaryotic cytosol across the eukaryotic plasma membrane (2, 24). This process is mediated by proteins termed translocators, and Yersinia LcrV is essential for the process of translocation (25, 26). P. aeruginosa PcrV, a homolog of Yersinia LcrV, is also essential for the process of toxin translocation, as the isogenic mutant of P. aeruginosa missing the PcrV gene (pcrV) is unable to intoxicate eukaryotic cells (15). Ab against PcrV appears to decrease type III secretion-mediated virulence by blocking the translocation process.
In our animal model of sepsis, proinflammatory cytokines such as
TNF- are produced in the alveolar airspace of infected lungs in
response to bacteria (12, 13). These mediators
progressively leak into the circulation across the damaged lung
epithelium (13), contributing to the development of a
systemic inflammatory reaction that induces acute septic physiology
(27). Bacteria disseminate into the circulation across the
same injured epithelial barrier. Translocation of the cytotoxic type
III secreted toxins by virulent P. aeruginosa induces lung
epithelial necrotic injury, severely compromising the epithelial-blood
barrier (11). Notably, the airspace instillation of large
quantities of endotoxin does not cause epithelial injury
(28). Therefore, blockade of the bacterial type III
secretion system should prevent lung epithelial injury and similarly
prevent the dissemination of bacteria and mediators from infected
lungs.
Animals receiving anti-PcrV IgG therapy had significantly less lung epithelial injury, bacteremia, and hypercytokinemia. The intratracheal administration of anti-PcrV IgG almost completely protected the lung epithelium. i.v. administered anti-PcrV IgG also reduced lung damage, probably by acting at the lung interstitium or by migrating into the infected lung airspace across the already injured lung epithelium. This protective effect against lung injury was also associated with decreased dissemination of bacteria and inflammatory mediators, reducing the severity of the septic reaction and preventing shock and death. The inhibition of bacterial proliferation in the lung may also have contributed to the reduced lung damage and systemic response. This effect was probably the result of preserved alveolar macrophage phagocytic function (15, 29), since the anti-PcrV Abs did not have direct bactericidal activity.
Anti-PcrV F(ab')2 administered intratracheally after the initiation of lung infection also protected animals against septic shock. These results indicate that blockade of bacterial type III secretion, not an Fc-dependent mechanism (30), is the primary mechanism for protection against sepsis, although Fc-dependent mechanisms might still contribute to the full effect. It is not surprising that i.v. administered anti-PcrV F(ab')2 did not lead to complete protection. The clearance of anti-PcrV F(ab')2 from the circulation is much faster than that of whole IgG (31), as shown in our results, resulting in eventual sepsis and bacteremia. These results document the superiority of whole molecular IgGs as i.v. therapeutic agents.
To date, the pathogenesis of sepsis caused by Gram-negative bacteria has largely been explained as a systemic inflammatory response induced by the interaction between bacterial endotoxin and host pattern recognition systems, including Toll-like receptors, CD14, and LPS-binding proteins (32). However, therapeutic trials of Abs (HA-1A or E5) against endotoxin have failed to show significant clinical improvement in patients with Gram-negative bacteremia and severe sepsis (33, 34). Although sepsis appears to be a heterogeneous syndrome involving a multifactorial and complex pathogenesis, our research suggests that a bacterial virulence mechanism such as the type III secretion system, distinct from endotoxin, plays a critical role in the pathogenesis of P. aeruginosa-induced sepsis. This implies that the pathogenesis of sepsis caused by other Gram-negative pathogens, such as Serratia, Proteus, Klebsiella, etc., may involve similar type III secretion systems. Further investigations, including the development of an mAb against PcrV, defining homologous targets in other Gram-negative bacteria, and rapid identification of virulent bacteria in infection, should yield valuable new approaches to the prevention and treatment of bacterial sepsis.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Jeanine P. Wiener-Kronish, Box 0542, Department of Anesthesia and Perioperative Care, University of California, San Francisco, CA 94143-0542. E-mail address: wienerkj{at}anesthesia.ucsf.edu
3 Abbreviation used in this paper: FIO2, inspired oxygen fraction.
Received for publication May 21, 2001. Accepted for publication September 6, 2001.
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