Sepsis syndrome is frequently complicated by the development of nosocomial infections, particularly Gram-negative pneumonia. Although TNF-α (TNF) has been shown to mediate many of the pathophysiologic events in sepsis, this cytokine is a critical component of innate immune response within the lung. Therefore, we hypothesized that the transient transgenic expression of TNF within the lung during the postseptic period could augment host immunity against nosocomial pathogens. To test this, mice underwent 26-gauge cecal ligation and puncture (CLP) as a model of abdominal sepsis, followed 24 h later by intratracheal (i.t.) administration of Pseudomonas aeruginosa. In animals undergoing sham surgery followed by bacterial challenge, Pseudomonas were nearly completely cleared from the lungs by 24 h. In contrast, mice undergoing CLP were unable to clear P. aeruginosa and rapidly developed bacteremia. Alveolar macrophages (AM) recovered from mice 24 h after CLP produced significantly less TNF ex vivo, as compared with AM from sham animals. Furthermore, the adenoviral mediated transgenic expression of TNF within the lung increased survival in CLP animals challenged with Pseudomonas from 25% in animals receiving control vector to 91% in animals administered recombinant murine TNF adenoviral vector. Improved survival in recombinant murine TNF adenoviral vector-treated mice was associated with enhanced lung bacterial clearance and proinflammatory cytokine expression, as well as enhanced AM phagocytic activity and cytokine expression when cultured ex vivo. These observations suggest that intrapulmonary immunostimulation with TNF can reverse sepsis-induced impairment in antibacterial host defense.

Sepsis syndrome is a common and devastating illness, occurring in >500,000 patients in the United States annually (1). A common complication of sepsis syndrome is adult respiratory distress syndrome, which occurs in 25–45% of patients with sepsis associated with bacteremia (2, 3, 4). In addition, sepsis syndrome with or without adult respiratory distress syndrome is frequently complicated by the development of nosocomial pneumonia, which results in a mortality rate of 55–75% in mechanically ventilated patients (5, 6, 7, 8, 9).

In both humans with sepsis and animal models of sepsis, an imbalance between the expression of pro- and anti-inflammatory cytokines has been demonstrated (10, 11, 12, 13, 14, 15, 16). Proinflammatory cytokines, in particular TNF-α (TNF),3 are elevated during sepsis (17, 18), and levels of TNF have been associated with severity of sepsis and clinical outcome (19, 20, 21, 22). Moreover, most of the deleterious effects of sepsis can be mimicked by the administration of TNF and, to a lesser extent, IL-1 (23, 24, 25, 26). However, although attempts to neutralize TNF and other inflammatory cytokines have been shown to be beneficial in animal models of sepsis syndrome, inhibition of TNF in patients with sepsis has not been successful (27, 28, 29). In fact, TNF blockade has been shown to increase lethality in some patients with sepsis, particularly those with Gram-positive infection (30). A potential cause of increased lethality in these patients is that TNF is an integral component of effective innate immunity, and neutralization of TNF in the setting of infection may significantly impair antimicrobial host defense (31, 32, 33). Specifically, TNF has been shown to activate macrophage phagocytosis and microbicidal activity in vitro, as well as facilitate the site-directed recruitment of phagocytic cells in vivo (34, 35). Furthermore, inhibition of TNF results in decreased bacterial clearance and increased mortality in various animal models of infection (32, 36, 37).

Interestingly, while early responses in sepsis are characterized by the overzealous production of proinflammatory cytokines, later stages of the septic response are manifested by the elaboration of counterregulatory anti-inflammatory cytokines, including IL-10 and IL-13 (10, 12, 13, 14, 16). This later response is believed to result in a state of monocyte/macrophage “deactivation.” In animal models of peritoneal sepsis, macrophage display decreased Ag-presenting capability and ability to kill ingested organisms (15, 38). Moreover, monocytes obtained from septic animals or patients with sepsis have reduced capacity to produce proinflammatory cytokines, including TNF, upon ex vivo stimulation (10, 11). Finally, we showed that mice undergoing 26-gauge cecal ligation and puncture (CLP) demonstrated a markedly increased susceptibility to intratracheal (i.t.) challenge with Pseudomonas aeruginosa during the postseptic period (39).

The purpose of this study is to determine whether the increased susceptibility of mice to bacterial infection of the lung during the postseptic period is associated with an impaired ability to produce TNF by lung macrophages. Furthermore, rather than inhibiting the inflammatory response in sepsis, we have taken the novel approach of augmenting the expression of TNF in a compartmentalized fashion using intrapulmonary adenoviral gene therapy to reverse sepsis-induced impairment in lung antibacterial host defense.

Polyclonal anti-murine TNF and IL-12 Abs used in ELISAs were produced by immunization of rabbits with murine recombinant cytokines in multiple intradermal sites with CFA. Carrier-free murine recombinant TNF and IL-12 were purchased from R&D Systems (Minneapolis, MN). Abs were purified over an endotoxin-free protein A column.

Construction and characterization of a human type 5 adenoviral vector used to generate recombinant vectors was performed as described previously (40, 41, 42, 43, 44). This vector, pAdBglII, is a replication-defective adenovirus that has deletions in the E1 region and partial deletions in the E3 region of the viral genome. An expression cassette was inserted into the E1 position of the viral genome and consisted of the human cytomegalovirus (hCMV) promoter, the 1.1 kb cDNA for murine TNF (mTNF; 63169; American Type Culture Collection, Manassas, VA), and a transcription termination signal supplied by the bovine growth hormone gene polyadenylation sequence (33). Briefly, to insert the mTNF cDNA, aAdBglII was linearized using the restriction enzyme EcoR V, incubated with mTNF cDNA, and ligated, then candidate clones were screened for the presence and orientation of mTNF cDNA by PCR. For comparison, a control adenovirus vector was used that was identical with Ad5 mTNF, but contained the Escherichia coli gene encoding the protein β-galactosidase (LacZ) inserted into the E1 region of the viral genome. Both vectors were propagated using the permissive 293 cell line from which viral lysates were made (42). Virus was twice purified by cesium chloride density gradient centrifugation and desalted on Sephadex G50 columns (Pharmacia, Uppsala, Sweden) eluted with PBS. Evidence of TNF expression in cell-free supernatants was confirmed by specific ELISA. Adenovirus containing either mTNF cDNA or LacZ, as control, were administered to mice 24 h after CLP, i.t. 2.5 × 108 PFU or i.p. 7 × 108 PFU.

Specific pathogen-free CD-1 mice (6- to 12-wk-old females; Charles River Breeding Laboratories, Wilmington, MA) were used in all experiments. All mice were housed in specific pathogen-free conditions within the animal care facility at the University of Michigan (Unit for Laboratory Animal Medicine) until the day of sacrifice.

The CLP model was used as a model of systemic sepsis syndrome as previously described (12). In distinct contrast to CLP models using larger gauge cecal punctures (19-gauge and larger) in which most animals rapidly develop bacteremia due to enteric organisms and death occurs as a result of polymicrobial sepsis (45), CLP using a 26-gauge needle results in the development of bacteremia in only 10–15% of animals (data not shown). However, this insult induces a marked septic response with death occurring in ∼10–20% of animals. To induce sepsis syndrome, pathogen-free female CD-1 mice were anesthetized with pentobarbital (Butler, Columbus, OH) 50 mg/kg i.p. followed by inhaled methoxyflurane (Metafane; Pitman-Moore, Mundelein, IL) as needed. In these mice, a 1–2 cm longitudinal incision to the lower right quadrant of the abdomen was performed and the cecum was exposed. The distal one-third of the cecum was ligated with 3-0 silk suture and punctured through with a 26-gauge needle. The cecum was then replaced into the peritoneal cavity and the incision was closed with surgical staples. In sham control animals, the cecum was exposed but not ligated or punctured, then returned to the abdominal cavity. All mice were administered 1 ml of sterile saline s.c. for fluid resuscitation during the immediate postoperative period.

Bronchoalveolar lavage (BAL) was performed to obtain alveolar macrophages (AM) in pure culture for ex vivo studies. The trachea was exposed and intubated using a 1.7-mm outer diameter polyethylene catheter. BAL was performed by instilling PBS containing 5 mM EDTA in 1-ml aliquots. Fifteen milliliters of PBS was instilled per mouse, with ∼10 ml of lavage fluid retrieved. Lavaged cells from each group of animals were pooled and counted after hypotonic lysis, and cytospins for determination of BAL differentials were prepared. Lavaged cells consisted of >95% AM for each of the groups examined (data not shown). Cells were resuspended in RPMI 1640 medium (Life Technologies, Paisley, PA) to give a concentration of 500,000 cells/ml. AM were isolated by adherence, and 250,000 cells were placed in each well of a 24-well cell culture plate (Corning Glass, Corning, NY) and washed with 500 μl of RPMI 1640 after 45 min. LPS was then added to selected wells and incubated for 18 h. Supernatants were collected and the production of TNF was determined by ELISA.

Alveolar macrophage phagocytic assay was performed as a modification of a previously described method (46). Briefly, murine AM (105 cells) were incubated with 5% normal murine serum (as source of opsonin) for 5 min at 37°C in 8-well Labteks (Nunc, Naperville, IL). P. aeruginosa (106 bacteria) were added and incubated for 60 min at 37°C. The supernatants were removed, and the cells were washed three times with sterile HBSS. The gasket was removed and slides were allowed to air dry. Diff-Quick stain (Baxter, Miami, FL) was performed and 200 cells per well were counted to determine the number of intracellular P. aeruginosa per AM and the percentage of AM containing intracellular bacteria. Phagocytic index (PI) was determined as follows (47): PI = [(number of AM with bacteria/total cells) × 100] × [total number of bacteria/total cells].

P. aeruginosa was administered i.t. to CD-1 mice 24 h post-CLP or sham surgery. We chose to use P. aeruginosa (strain UI-18; Parke-Davis, Ann Arbor, MI) in our studies, as this organism is a common respiratory pathogen in patients with sepsis, and immunocompetent mice are generally resistant to infection with this particular strain when administered via the intrapulmonary route (39). P. aeruginosa was cultured in tryptic soy broth (Difco, Detroit, MI) for 18 h at 37°C. The concentration of bacteria in broth was determined by measuring the amount of absorbency at 600 nm. A standard of absorbencies based on known CFU was used to calculate inoculum concentration. Bacteria were pelleted by centrifugation at 3000 rpm for 15 min, washed two times in saline, and resuspended at the desired concentration. Animals were anesthetized with weight-based phenobarbital i.p. The trachea was exposed, and 30 μl inoculum or saline was administered via a sterile 26-gauge needle. The inoculum in all studies contained 5 × 104 CFU P. aeruginosa. The skin incision was closed with surgical staples.

At designated time points, the mice were anesthetized with inhaled carbon dioxide, blood was collected by right ventricular puncture with heparinized syringes, and the animals were sacrificed. Whole lungs were then harvested for assessment of TNF and IL-12 protein expression. After removal, lungs were homogenized in 1 ml of protease inhibitor (Boehringer-Mannheim, Mannheim, Germany) in sterile saline solution using a tissue homogenizer. One-milliliter aliquots of sterile 1× PBS was added, and the homogenates were sonicated for 30 s. Homogenates were centrifuged at 2500 rpm for 10 min at 4°C. Supernatants were collected, passed through a 0.45-micron filter (Gelman Sciences, Ann Arbor, MI), then stored at −20°C for assessment of cytokine levels. Lungs for histologic examination were excised en bloc without perfusion and inflated with 1 ml of 4% paraformaldehyde in PBS to improve resolution of anatomic relationships.

At the time of sacrifice, plasma was collected, the right ventricle perfused with 1 ml PBS, then lungs were removed aseptically and placed in 2 ml sterile saline. The tissues were then homogenized with a tissue homogenizer under a vented hood on ice. Serial 1:5 dilutions of both lung homogenates and plasma were made. Ten microliters of each dilution was plated on soy base blood agar plates (Difco, Detroit, MI.). Plates were incubated for 24 h at 37oC, after which colonies were counted.

Lung MPO activity (as assessment of neutrophil influx) was quantitated by a method described previously (48). Briefly, whole lungs were homogenized in 2 ml of a solution containing 50 mM potassium phosphate, pH 6.0, with 5% hexadecyltrimethylammonium bromide and 5 mM EDTA. One hundred microliters of the resulting homogenate was sonicated and centrifuged at 12,000 × g for 15 min. The supernatant was mixed 1:15 with assay buffer and read at 490 nm. MPO units were calculated as the change in absorbency over time.

mTNF and IL-12 were quantitated using a modification of a double ligand method as previously described (12). Briefly, flat-bottom 96-well microtiter plates (Immuno-Plate I 96-F; Nunc, Roskilde, Denmark) were coated with 50 μl/well of rabbit Ab against the various cytokines (1 μg/ml in 0.6 M NaCl, 0.26 M H3BO4, and 0.08 M NaOH, pH 9.6) for 16 h at 4°C and then washed with PBS, pH 7.5, 0.05% Tween-20 (wash buffer). Microtiter plate nonspecific binding sites were blocked with 2% BSA in PBS and incubated for 90 min at 37°C. Plates were rinsed four times with wash buffer and diluted (neat and 1:10) cell-free supernatants (50 μl) in duplicate were added, followed by incubation for 1 h at 37°C. Plates were washed four times, followed by the addition of 50 μl/well biotinylated rabbit Abs against the specific cytokines (3.5 μg/ml in PBS, pH 7.5; 0.05% Tween 20; and 2% FCS), and plates were incubated for 30 min at 37°C. Plates were washed four times, streptavidin-peroxidase conjugate (Bio-Rad, Richmond, CA) was added, and the plates were incubated for 30 min at 37°C. Plates were washed again four times, and chromogen substrate (Bio-Rad) was added. The plates were incubated at room temperature to the desired extinction, and the reaction was terminated with 50 μl/well of 3 M H2SO4 solution. Plates were read at 490 nm in an ELISA reader. Standards were one-half log dilutions of recombinant murine cytokines from 1 pg/ml to 100 ng/ml. This ELISA method consistently detected murine cytokine concentrations above 25 pg/ml. The ELISA did not cross-react with IL-1, IL-2, IL-4, or IL-6. In addition, the ELISA did not cross-react with members of the murine chemokine family, including murine JE/MCP-1, RANTES, keratinocyte-derived chemokin, MIP-2, growth-related oncogene-α, or epithelial neutrophil-activating protein-78.

Ratio scale data were evaluated by ANOVA with Bonferroni’s multiple comparison test follow-up, whereas survival curves were analyzed by the log-rank and Kaplan-Meier tests. Lung and serum CFU data were analyzed using unpaired t test. All calculations were performed by the Prism 3.0 statistical program (GraphPad Software, San Diego, CA).

To assess bacterial clearance in the postseptic period, CD-1 mice underwent either CLP or sham surgery, followed 24 h later by the administration of 5 × 104 CFU of P. aeruginosa. The mice were sacrificed, and blood and lung Pseudomonas CFU were determined. The 24-h time point was chosen because postseptic effects were greatest at this time point; later time points such as 48 h and 72 h, result in prohibitive mortality in CLP mice challenged with Pseudomonas. As shown in Fig. 1, total lung P. aeruginosa CFU was significantly greater in the CLP mice 24 h postchallenge as compared with sham animals. Specifically, all of the CLP mice had recoverable P. aeruginosa, with a mean log CFU of 6.84 ± 1.17, as compared with only 60% of sham mice, with a mean log CFU of 1.98 ± 0.63 (p < 0.01). In addition, the CLP group had high-grade P. aeruginosa bacteremia 24 h after i.t. administration of the bacteria, whereas none of the sham mice were bacteremic (p < 0.01). Finally, 71% of CLP mice administered P. aeruginosa died by 48 h, whereas none of the sham mice challenged with Pseudomonas died out to 10 days postinfectious challenge (data not shown).

FIGURE 1.

Effect of CLP on lung and plasma P. aeruginosa CFU after i.t. challenge. Mice underwent either CLP or sham surgery, followed 24 h later by i.t. administration of 5 × 104 CFU of P. aeruginosa. ∗, p < 0.01 as compared with sham animals. n = 7–9 per condition.

FIGURE 1.

Effect of CLP on lung and plasma P. aeruginosa CFU after i.t. challenge. Mice underwent either CLP or sham surgery, followed 24 h later by i.t. administration of 5 × 104 CFU of P. aeruginosa. ∗, p < 0.01 as compared with sham animals. n = 7–9 per condition.

Close modal

Because TNF is an important component of effective antibacterial host defense and AM are a major cellular source of TNF in the lung, studies were performed to evaluate the ability of AM to produce TNF in the postseptic period when cultured ex vivo. BAL was performed on CLP and sham mice 24 h after surgery, then AM were isolated by adherence and incubated for 18 h in the presence or absence of LPS (1 μg/ml). As shown in Fig. 2, resting AM recovered from sham animals produced small but detectable quantities of TNF. However, TNF production from AM isolated from CLP mice was decreased significantly as compared with AM from sham animals (p < 0.05). Furthermore, AM from CLP mice produced 60% less TNF after LPS stimulation as compared with similarly treated AM recovered from sham animals (p < 0.001).

FIGURE 2.

Effect of CLP on TNF production by AM isolated from sham or CLP mice 24 h postsurgery. AM were cultured in the presence or absence of LPS (5 μg/ml), and supernatant TNF levels were assessed after 18 h in culture. ∗, p < 0.001; #, p < 0.05, as compared with TNF production by AM recovered from sham animals. n = 6 per condition.

FIGURE 2.

Effect of CLP on TNF production by AM isolated from sham or CLP mice 24 h postsurgery. AM were cultured in the presence or absence of LPS (5 μg/ml), and supernatant TNF levels were assessed after 18 h in culture. ∗, p < 0.001; #, p < 0.05, as compared with TNF production by AM recovered from sham animals. n = 6 per condition.

Close modal

Given that there is impaired ability to produce TNF by AM in the postseptic period, we attempted to restore TNF expression within the lung using i.t. TNF gene therapy. To determine whether adenoviral TNF gene therapy could enhance proinflammatory cytokine levels in the postseptic period, CD-1 mice underwent CLP followed 24 h later by administration of P. aeruginosa (5 × 104 CFU) in combination with either 2.5 × 108 PFU of type 5 adenovirus containing the mTNF cDNA inserted into the E1 region of the viral genome (adTNF), or control LacZ adenoviral vector (adCTL), or no treatment. This dose of adenovirus was used as it was shown to produce maximal benefit in our model, and the administration of adTNF at this dose resulted in significant expression of TNF within the lung by 24 h and lasting out to 14 days after i.t. administration (33). Lungs were harvested 24 h later for whole lung cytokine measurements as determined by ELISA. As shown in Table I, a significant increase in lung TNF was observed in sham animals challenged with P. aeruginosa. In contrast, no induction of TNF was observed in Pseudomonas-infected CLP animals receiving control vector or vehicle. However, a 6-fold increase in TNF was observed in Pseudomonas-infected CLP mice receiving adTNF (p < 0.001). Interestingly, administration of adTNF also resulted in a substantial induction of IL-12 at 24 h (p < 0.001). The induction of cytokine was selective for TNF and IL-12, as treatment with adTNF did not alter the expression of the CXC chemokines MIP-2 or kenotinocyte-derived chemokine (data not shown).

Table I.

Lung cytokine levels in CLP or sham mice challenged with P. aeruginosa (5 × 104 CFU) alone or in combination with either adTNF (2.5 × 108 PFU), adCTL, or no treatment

Lung TNF (ng/ml ± SEM)Lung IL-12 (ng/ml ± SEM)
SHAM 0.559 ± 0.075 2.451 ± 0.138 
CLP 0.725 ± 0.092 3.499 ± 0.169 
SHAM+ PA-7 1.907 ± 0.448a Not done 
CLP+ PA-7 0.397 ± 0.397 6.337 ± 0.436 
CLP+ PA-7+ adCTL 0.417 ± 0.061 4.308 ± 0.285 
CLP+ PA-7+ adTNF 3.109 ± 0.537# 14.690 ± 0.959# 
Lung TNF (ng/ml ± SEM)Lung IL-12 (ng/ml ± SEM)
SHAM 0.559 ± 0.075 2.451 ± 0.138 
CLP 0.725 ± 0.092 3.499 ± 0.169 
SHAM+ PA-7 1.907 ± 0.448a Not done 
CLP+ PA-7 0.397 ± 0.397 6.337 ± 0.436 
CLP+ PA-7+ adCTL 0.417 ± 0.061 4.308 ± 0.285 
CLP+ PA-7+ adTNF 3.109 ± 0.537# 14.690 ± 0.959# 
a

, p < 0.05; #, p < 0.001 when comparing adTNF group to adCTL or no treatment group; n = 6–9 per condition. PA-7 = P. aeruginosa.

Initial experiments indicated that AM isolated from CLP mice had an impaired ability to produce TNF constitutively and in response to endotoxin. However, TNF levels in the lung can be up-regulated by the i.t. administration of adenoviral TNF gene therapy (Table I). Therefore, we next attempted to improve survival by enhancing the expression of TNF within the lung in the setting of intrapulmonary bacterial challenge. In these studies, CLP mice were administered either 2.5 × 108 PFU of adTNF or adCTL or vehicle concomitant with P. aeruginosa (5 × 104 CFU). Treatment of CLP animals with P. aeruginosa with vehicle or control adenovirus resulted in long-term survival in only 33 and 25% of animals, respectively (Fig. 3). In contrast, treatment with adTNF resulted in significant increase in survival, with 91% of animals surviving long-term, p < 0.05. Concomitant treatment with adTNF was required as the protective effect of adTNF was lost if administered 24 h after Pseudomonas administration (data not shown).

FIGURE 3.

Effect of i.t. adTNF administration on survival in mice undergoing CLP followed 24 h later by i.t. administration of P. aeruginosa (5 × 104 CFU) in combination with adTNF (2.5 × 108 PFU), adCTL, or no treatment. ∗, p < 0.05 when compared with adCTL and Pseudomonas alone groups. n = 11–15 per group.

FIGURE 3.

Effect of i.t. adTNF administration on survival in mice undergoing CLP followed 24 h later by i.t. administration of P. aeruginosa (5 × 104 CFU) in combination with adTNF (2.5 × 108 PFU), adCTL, or no treatment. ∗, p < 0.05 when compared with adCTL and Pseudomonas alone groups. n = 11–15 per group.

Close modal

To determine whether protective effects of TNF could be observed by the systemic overexpression of TNF in the postseptic period, CLP mice were challenged 24 h post-CLP with Pseudomonas i.t. in combination with adTNF or adCTL (7 × 108 PFU) administered i.p. This dose of adTNF was chosen because it resulted in significant blood levels of TNF (2.49 ± 0.79 μg/ml) at 24 h after i.p. administration. In contrast to the beneficial effects observed with intrapulmonary adTNF administration, no significant difference in short- or long-term survival was observed in animals receiving adTNF i.p. as compared with animals receiving adCTL or no adenoviral treatment (Fig. 4). In fact, a trend toward increased mortality was observed in the animals receiving adTNF i.p.

FIGURE 4.

Effect with i.p. TNF gene therapy in CLP mice challenged with i.t. P. aeruginosa (5 × 104 CFU) in combination with i.p. adTNF, adCTL (7 × 108 PFU), or no treatment, 24 h after CLP. n = 8–12 per group.

FIGURE 4.

Effect with i.p. TNF gene therapy in CLP mice challenged with i.t. P. aeruginosa (5 × 104 CFU) in combination with i.p. adTNF, adCTL (7 × 108 PFU), or no treatment, 24 h after CLP. n = 8–12 per group.

Close modal

To determine whether the improved survival in Pseudomonas-infected CLP mice treated with adTNF was due to enhanced bacterial clearance, lungs from CLP mice challenged with P. aeruginosa concomitant with either adTNF or control vector were harvested 24 h after i.t. administration of Pseudomonas. The 24-h time point was selected based on the significant difference in survival between the two groups at that time. All of the mice in the control vector-treated group had P. aeruginosa CFU isolated from their lungs 24 h posttreatment, with a log mean value of 5.21 ± 0.31 CFU. In contrast, the adTNF group had 2.72 ± 0.84 Pseudomonas CFU recovered at 24 h, with 50% of the animals having completely cleared P. aeruginosa at this time point, p < 0.05 (Fig. 5).

FIGURE 5.

Effect of intrapulmonary TNF gene therapy on lung P. aeruginosa CFU in CLP mice administered P. aeruginosa (5 × 104 CFU) concomitant with adTNF or adCTL (2.5 × 108 PFU) 24 h post-CLP. ∗, p < 0.05 as compared with group receiving adCTL. n = 8–14 per condition.

FIGURE 5.

Effect of intrapulmonary TNF gene therapy on lung P. aeruginosa CFU in CLP mice administered P. aeruginosa (5 × 104 CFU) concomitant with adTNF or adCTL (2.5 × 108 PFU) 24 h post-CLP. ∗, p < 0.05 as compared with group receiving adCTL. n = 8–14 per condition.

Close modal

To assess whether intrapulmonary TNF-transgenic expression increased lung neutrophil influx in CLP mice challenged with i.t. P. aeruginosa, total lung MPO levels were measured 12 and 24 h after Pseudomonas challenge. As shown in Fig. 6, there was a modest increase in lung MPO in CLP mice at 12 and 24 h post- surgery as compared with sham animals. In response to i.t. Pseudomonas administration, lung MPO increased by ∼6-fold in both the CLP and sham animals, p < 0.05, with the increase in lung MPO being similar in both groups. Furthermore, lung MPO in Pseudomonas-infected CLP mice treated with adTNF did not differ from that observed in CLP mice receiving either control vector or no treatment at 12 and 24 h after Pseudomonas administration.

FIGURE 6.

Lung MPO levels in CLP mice treated with either adTNF or adCTL (2.5 × 108 PFU) i.t. concomitantly with P. aeruginosa i.t. challenge (5 × 104 CFU). A, Lung MPO at 12 h after P. aeruginosa and adTNF or adCTL administrations. B, Lung MPO at 24 h after P. aeruginosa and adTNF or adCTL administration. ∗, p < 0.05. n = 4–8 per condition.

FIGURE 6.

Lung MPO levels in CLP mice treated with either adTNF or adCTL (2.5 × 108 PFU) i.t. concomitantly with P. aeruginosa i.t. challenge (5 × 104 CFU). A, Lung MPO at 12 h after P. aeruginosa and adTNF or adCTL administrations. B, Lung MPO at 24 h after P. aeruginosa and adTNF or adCTL administration. ∗, p < 0.05. n = 4–8 per condition.

Close modal

TNF production by AM isolated from CLP animals was shown to be significantly decreased as compared with AM from sham animals. To assess the efficacy of intrapulmonary adTNF gene therapy on reversing this defect, mice underwent CLP followed 24 h later by the i.t. administration of either adTNF, adCTL, or no treatment. At 48 h post-CLP, BAL was performed, and AM was isolated by adherence and incubated in the presence or absence of LPS (1 μg/ml) for 18 h. As previously shown, unstimulated AM from CLP mice produced minimal amounts of TNF (Fig. 7). When stimulated with LPS, AM from CLP alone and adCTL-treated animals produced moderately increased amounts of TNF as compared with AM not stimulated by LPS. However, AM from CLP animals treated with adTNF in vivo produced ∼2.6-fold more TNF than CLP mice alone or treated with adCTL in vivo (p < 0.05).

FIGURE 7.

TNF production by AM from CLP mice harvested 24 h after i.t. administration of adTNF, adCTL, or no treatment. TNF production in supernatants was determined after 18 h in culture in the presence or absence of LPS (1 μg/ml). ∗, p < 0.05 as compared with all other groups. n = 6–9 per condition.

FIGURE 7.

TNF production by AM from CLP mice harvested 24 h after i.t. administration of adTNF, adCTL, or no treatment. TNF production in supernatants was determined after 18 h in culture in the presence or absence of LPS (1 μg/ml). ∗, p < 0.05 as compared with all other groups. n = 6–9 per condition.

Close modal

We have previously shown that CLP results in significant impairment in the ability of AM to ingest Gram-negative bacteria ex vivo (46). To assess whether adTNF treatment enhanced AM phagocytic function, mice underwent CLP, followed 24 h later by the administration of adTNF or adCTL i.t. After an additional 24 h, AM were obtained by BAL, and incubated with P. aeruginosa ex vivo; then, AM phagocytosis activity determined. Importantly, treatment with adTNF in vivo resulted in significantly enhanced phagocytic activity of AM (PI, 92.3 ± 3.7), as compared with AM isolated from mice treated with adCTL (PI, 42.9 ± 10.8, p < 0.05) (Fig. 8).

FIGURE 8.

Phagocytic activity of AM from CLP mice harvested 24 h after i.t. administration of adTNF or adCTL. ∗, p < 0.01 when adTNF treated group is compared with the adCTL group (mean of three separate experiments).

FIGURE 8.

Phagocytic activity of AM from CLP mice harvested 24 h after i.t. administration of adTNF or adCTL. ∗, p < 0.01 when adTNF treated group is compared with the adCTL group (mean of three separate experiments).

Close modal

Sepsis syndrome results in an imbalance in the expression of pro- and anti-inflammatory cytokines, which predisposes the host to pulmonary infections, particularly the development of nosocomial pneumonia. Previous sepsis studies have shown TNF to be the mediator of many of the adverse systemic effects and hemodynamic instability in septic patients and in animal models of sepsis (29, 49). However, attempts to block TNF in clinical studies did not necessarily improve outcome (28, 50, 51). In fact, mice deficient in p55TNF receptor have been shown to have a much higher mortality than normal control mice when they undergo CLP (52). One of the reasons may be because of the key role TNF plays in innate host immunity (31, 32, 53). We used the CLP model to induce sepsis, followed by the i.t. administration of P. aeruginosa to mimic the development of Gram-negative nosocomial pneumonia in patients during the peri-septic period.

As we and others have demonstrated, the 26-gauge CLP model is characterized by an initial rise in proinflammatory cytokines in blood and peritoneal fluid, which then subsides 24 h following the original septic insult (12, 13, 16). Measurements of cytokines after 24 h demonstrated a decline in TNF, IL-12, and IFN-γ levels, but persistence in the expression of IL-10. This phase of the sepsis syndrome, which is dominated by the expression of anti-inflammatory molecules, has been termed the compensatory anti-inflammatory response syndrome (18). We observed a substantial impairment in the ability to clear P. aeruginosa from the lung during the postseptic period, which resulted in the development of high grade Pseudomonas bacteremia and markedly increased lethality. Sepsis-induced impairment in innate immunity occurred in association with a significant impairment in the ability of AM to produce TNF constitutively and in response to LPS (54). Our findings are similar to that observed in blood monocytes isolated from septic patients, which also display an inability to produce TNF, as well as other activating cytokines (55, 56, 57). This immunologically refractory state is similar to that of LPS tolerance, whereby exposure to LPS results in subsequent blunted responses to rechallenge with LPS. Interestingly, inhibition of cytokine production observed in monocytes isolated from septic patients or LPS-desensitized monocytes has been shown to be partially reversed by in vitro incubation of monocytes with anti-IL-10 Abs (10, 58). Furthermore, we have shown that neutralization of IL-10 24 h after CLP can partially reverse sepsis-induced impairment in the clearance of Pseudomonas from the lung, suggesting that IL-10 is a major mediator of this effect (39).

Given the importance of TNF in innate host immunity, balanced by its detrimental effects, particularly if produced in large quantities systemically, compartmentalized expression of TNF at the site of infection represents an attractive approach to immunotherapy. Nelson et al. have previously demonstrated that TNF levels increased in a compartmentalized fashion in response to i.t. administered LPS (59). Similarly, human subjects with unilateral community-acquired pneumonia were found to have increased TNF levels and increased total cell counts in the involved lung as compared with the uninvolved lung and serum and control subjects (55). These findings support a compartmentalized host response to infectious insults. Indeed, CLP mice inoculated with P. aeruginosa concomitant with the administration of adTNF i.t. had significantly increased survival as compared with control mice. Moreover, when adTNF was given i.p., there was no survival benefit in CLP mice challenged with Pseudomonas (and possibly a detrimental effect), suggesting that local, compartmentalized TNF treatment is essential. The lack of benefit with systemic adTNF may be partially explained by the inability to achieve appreciable expression of TNF in the lung after i.p. adTNF administration (<30 pg), despite ample blood levels of TNF, peaking at 24 h after i.p. adTNF administration. In addition, enhanced blood TNF levels may potentiate the deleterious effects of sepsis syndrome during the development of Pseudomonas bacteremia. These results highlight the importance of local control of infection (i.e., within the lung), thus preventing the development of bacteremia and the associated manifestations of the septic response.

Improved survival observed with intrapulmonary transgenic TNF expression was due to improved bacterial clearance from the alveolar space. We found that 50% of the treated mice had completely cleared the organisms 24 h after adTNF treatment, whereas all of the mice given control vector remained infected at that time point. Of the animals in the adTNF treatment that did not eradicate P. aeruginosa, the number of CFU was similar to that observed in animals receiving control vector, indicating there was a subgroup of animals that did not benefit from adTNF treatment. The reason for enhanced clearance in some but not all animals is unclear. This is likely attributable to inherent variability in the degree of sepsis-induced immunosuppression and response to adenoviral gene therapy (particularly given that an outbred strain of mice was used in these studies, which greatly increased genetic heterogeneity). Alternatively, augmented intrapulmonary TNF production may have resulted in heightened lung injury in some animals, partially negating beneficial effects on bacterial clearance.

Mechanism(s) for increased bacterial clearance from the alveolar space in animals treated with adTNF does not appear to be due to enhanced recruitment of neutrophils to the lung. Lung MPO level increased after intrapulmonary Pseudomonas challenge, but there was no significant difference in the levels postadenoviral TNF treatment at time zero, 12 h, and 24 h. This was confirmed histologically (data not shown), as in all three groups there was evidence of a vigorous neutrophilic infiltrate, suggesting the neutrophils were recruited secondary to the infectious insult rather than TNF overexpression. However, we cannot exclude the distinct possibility that TNF may enhance neutrophil phagocytic and microbicidal functions of recruited neutrophils, which has been demonstrated in vitro (35, 60). Furthermore, our studies indicated that intrapulmonary TNF expression in vivo enhanced the ability of AM to ingest P. aeruginosa when cultured ex vivo. Similar effects of TNF on macrophage phagocytic activity have been shown by others (61). The mechanism for enhanced phagocytic function in response to in vivo expression of the TNF transgene remains unclear, as we observed no significant change in the expression of several AM cell surface molecules involved in the phagocytic response, including CD54, CD11b, CD11c, and CD16 (data not shown).

Cytokine measurements in lung homogenates of adTNF-treated mice demonstrated significant increases in TNF, without detectable increases in serum levels. This confirms compartmentalized expression of TNF after i.t. gene delivery. We have previously shown that airway epithelial cells are the primary cells that express the TNF transgene after i.t. adTNF administration (33). We also observed the AM cultured ex vivo also produced increased quantities of TNF after in vivo adTNF treatment. Enhanced production of TNF by AM may reflect the priming effect of TNF and potentially other activating cytokines on AM cytokine production. Alternatively, increased expression adTNF by AM may reflect transfection of these cells and subsequent expression of the transgene, which has been demonstrated in vivo and in vitro (62, 63, 64). In addition to TNF, we also observed a significant increase in the production of IL-12 in the lung. TNF has been shown to enhance IL-12 production from NK and T cells previously (65, 66). Given that IL-12 is a potent inducer of IFN-γ, it is quite likely that beneficial effects of adTNF in vivo are in part attributable to activating effects of cytokine networks initiated by TNF. Interestingly, even though TNF has been shown to be an inducer of CXC and CC chemokines in vitro, we observed no induction of these chemokines by 24 h posttransfection.

In summary, our studies demonstrated that sepsis syndrome results in a profound defect in innate host defense, which occurs in association with an impaired ability of AM to produce TNF. Compartmentalized transgenic TNF expression improves survival, bacterial clearance, production of other important activating cytokines (i.e., IL-12), and restores several AM effector cell activities. In distinct contrast to failed approaches of TNF neutralization in sepsis, augmenting cytokine-mediated host innate immune responses in the lung during the postseptic period may serve as an effective and important adjuvant to the treatment of life-threatening Gram-negative pneumonia in critically ill patients.

1

This research was supported by National Institutes of Health Grants HL58200, HL57243, and P50HL60289.

3

Abbreviations used in this paper: TNF, TNF-α; CLP, cecal ligation and puncture with 26-gauge needle; AM, alveolar macrophage(s); mTNF, murine TNF; adTNF, type 5 adenovirus containing the mTNF; BAL, bronchoalveolar lavage; MPO, myeloperoxidase; LacZ, β-galactosidase; adCTL, control LacZ adenoviral vector; i.t., intratracheal(ly); PI, phagocytic index.

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