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Department of Medicine, Division of Pulmonary and Critical Care Medicine, University of Michigan Medical School, Ann Arbor, MI 48109; and
Department of Microbiology, Toho University, Tokyo, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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(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. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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-
(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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
Adenoviral vectors
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 
-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 x
108 PFU or i.p. 7 x
108 PFU.
Animals
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.
Animal model
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
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.
Phagocytic assay
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) x 100] x [total number of bacteria/total cells].
P. aeruginosa inoculation
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 x 104 CFU P. aeruginosa. The skin incision was closed with surgical staples.
Lung harvesting for cytokine analysis and histologic examination
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 1x 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.
Determination of lung and plasma P. aeruginosa CFU
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 myeloperoxidase (MPO) assay
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 x 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.
Murine cytokine ELISAs
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-, or
epithelial neutrophil-activating protein-78.
Statistical analysis
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).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 x 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).
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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).
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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 x
104 CFU) in combination with either 2.5 x
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).
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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 x 108 PFU of adTNF or adCTL or
vehicle concomitant with P. aeruginosa (5 x
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).
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). In
fact, a trend toward increased mortality was observed in the animals
receiving adTNF i.p.
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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).
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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.
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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).
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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).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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-
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-,
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.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Theodore J. Standiford, The University of Michigan Medical Center, Department of Internal Medicine, Division of Pulmonary and Critical Care Medicine, 6301 Medical Science Research Building III 1150 West Medical Center Drive, Ann Arbor, MI 48109-0642.
3 Abbreviations used in this paper: TNF, TNF-; 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, -galactosidase; adCTL, control LacZ adenoviral vector; i.t., intratracheal(ly); PI, phagocytic index.
Received for publication June 28, 2000. Accepted for publication September 15, 2000.
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