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* Division of Pulmonary, Critical Care and Occupational Medicine, Department of Internal Medicine,
Veteran’s Administration Medical Center,
Center for Gene Therapy, and
Department of Anatomy and Cell Biology, University of Iowa College of Medicine, Iowa City, IA 52242; and
¶ Department of Medical Biology and Genetics, College of Medicine, Akdeniz University, Antalya, Turkey
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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levels and
an increase in bacterial load. Finally, a profound degree of lymphocyte
apoptosis was observed in these animals. These observations suggest
that prior exposure to first-generation adenovirus gene therapy vectors
may worsen the outcome of some forms of sepsis. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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production (5, 6). We were interested in the use
of Ad vectors to transfer "therapeutic" genes in a sepsis model.
However, in our initial studies, we found that a control Ad vector
alone altered survival. Because of this finding, we altered the study
to evaluate the effect of the control Ad vector on survival in a murine
model of sepsis. The murine cecal ligation and puncture model (CLP) closely mimics the human sepsis syndrome (7). Therefore, we assessed the biosafety aspects of these constructs in the CLP model of sepsis. For this purpose, first-generation recombinant Ad vectors carrying two different reporter genes were delivered systemically to C57BL/6 mice. Here we report the effects of recombinant Ad vector infection on the septic mice. We found that the combination of Ad vectors with CLP resulted in a shortened survival time. This shortened survival time was accompanied by significant cell injury and an increase in bacterial load in a number of tissues, including liver, lung, thymus, and spleen.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Two recombinant Ad vectors expressing either enhanced green
fluorescent protein (Ad.EGFP; University of Iowa Vector Core, Iowa
City, IA), or an Escherichia coli 
-galactosidase protein
(Ad.LacZ) (8) were used for experimental studies. Both
vectors were E1/E3 deleted. First-generation recombinant adenoviral
stocks were generated as previously described and were stored in 10 mM
Tris with 20% glycerol at -80°C (9). Before use, viral
stocks were desalted through a PBS-Sephadex G-50 (Sigma-Aldrich, St.
Louis, MO) column to remove the glycerol. The particle titers of
adenoviral stocks were typically 1013 DNA
particles/ml. The functional titers of adenoviral stocks were
determined by plaque titering on 293 cells and expression assays for
encoded proteins. Typically the particle-PFU ratio was 25. C57BL/6 mice
received 2 x 1011 DNA particles of Ad
constructs through tail vein injections. In all cases, CLP procedures
and subsequent assays were performed 1 wk after the infection.
UV-inactivated first-generation adenoviral vectors
Ad.EGFP vectors were prepared as described above and then exposed to 2 J UV radiation as a thin layer in a Stratalinker (Stratagene, La Jolla, CA). Both stock and UV-inactivated vectors were incubated with RAW cells and then viewed by fluorescent microscopy to confirm inactivation. The vectors were injected in an identical manner. After CLP, mice were monitored for survival.
Second-generation adenoviral vectors
A second-generation recombinant E1-deleted, E2ats125 mutant adenoviral vector expressing the lacZ cDNA (10) was used to evaluate the contribution of adenoviral gene products to septic reactions to Ad. This Ad.LacZts125 vector harbors a temperature-sensitive mutation in E2a of the adenoviral genome, which significantly inhibits adenoviral gene expression. These second-generation adenoviral vectors were stored in glycerol, then desalted, and injected using techniques identical with those used with first-generation vectors. After CLP, mice were monitored for survival.
CLP procedure
CLP operations were performed as described previously
(11). C57BL/6 mice (female, age 6 wk at time of viral
injection; Harlan Breeders, Indianapolis, IN) were given general
anesthesia with ketamine-xylazine via i.p. injection. A midline
laparotomy was performed, and the cecum was identified. The cecum was
ligated first with a nylon suture and then punctured with a 19-gauge
needle. The abdomen was closed using a two-layer technique. Control
surgeries (sham) are performed in the same manner without the puncture
and ligation steps. Fluid support by the means of normal saline 0.5 ml
(
25 ml/kg) i.p. was provided during the procedure. Postoperative
checks were performed every 12 h for 72 h postoperatively.
Data including activity, piloerection, and position are monitored on
these checks. University of Iowa Animal Care Unit postprocedural care
and monitoring guidelines were followed. Lung permeability index (LPI)
was calculated from the formula LPI = (wet lung weight/total body
weight) x 100. Increase in LPI index indicates acute lung
injury.
Serum alanine aminotransferase (ALT), TNF-, and soluble TNFR1
(sTNFR1) measurements
Sera were collected by retro-orbital bleeding of C57BL/6 mice at
different time points after CLP. Serum ALT measurements (an indication
of liver damage) were performed as previously described
(8). For serum TNF- and sTNFR1 measurements, ELISA kits
from R&D Systems (Minneapolis, MN) were used according to
manufacturer’s instructions.
Bacterial load assay
Twenty-four hours after CLP, animals were anesthetized, and blood samples were obtained by retro-orbital bleeding. Lung and liver tissues were harvested next. Tissues were homogenized and directly plated on Luria-Bertani plates in the absence of any antibiotics. Colony counts were obtained 24 h after plating and normalized to gram of tissue. EDTA (5%) was used to prevent blood coagulation before plating.
Immunocytochemistry procedure
Immunocytochemistry assays for visualization of Kupffer cells were performed according to a procedure modified from that of Zhou et al. (12). Labeling of Kupffer cells was performed using biotin-labeled mAb to mouse F4/80 (macrophages, Ly-71; Caltag Laboratories, Burlingame, CA). Biotin was detected with Texas red-conjugated streptavidin under fluorescent microscopy using a Texas Red filter (Boehringer-Mannheim, Indianapolis, IN).
In situ cell death detection assay
C57BL/6 animals were perfused with 2% paraformaldehyde before harvesting liver, lung, spleen, and thymus tissues. These tissues were soaked in 30% sucrose before embedding in OCT compound (Tissue-Tek, Elkhart, IN) and sectioning. Sections were treated with 2% paraformaldehyde for 30 min followed by a 15-min postfixation in ice-cold methanol. Sections were then permeabilized in 0.01% Triton X-100. The in situ cell death detection kit, TMR red, was used to detect apoptotic cells in tissue sections, and results were analyzed by fluorescent microscopy. Image quantification was performed using Image-Pro Plus 4.1 program from Media Cybernetics (Silver Spring, MD) to determine the percent apoptotic area.
Statistical analysis
Statistical analyses (survival, etc.) were performed using the Prism program from GraphPad Software (San Diego, CA) and are specifically stated in the figure legends.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Efficient in vivo gene delivery is one of the most desired features of gene therapy. To assess the effectiveness of in vivo gene delivery in the CLP model, C57BL/6 mice were injected with first-generation Ad.EGFP through the tail vein. Liver sections were analyzed under fluorescent microscopy to assess the efficiency of in vivo gene delivery and the transgene expression. In accordance with the previous reports (1), first-generation recombinant Ad constructs efficiently transduced hepatic cells such that >98% of the hepatic cells were positive for transgene expression (data not shown).
After successful delivery of the first-generation Ad vectors, in vivo,
mortality rates for the CLP model of sepsis were evaluated after using
different sized needles to perforate the cecum. In the absence of an Ad
infection, a 50% death rate was observed at 24 h post-CLP with an
18-gauge needle (data not shown), and at 48 h post-CLP with a
19-gauge needle (Fig. 1
). Based on the
need for a flexible monitoring time, the 19-gauge needle technique was
chosen to compare the effects of first-generation Ad constructs on
survival after CLP. Ad.EGFP-infected animals exhibited a 50% death
rate by 24 h after CLP compared with 48 h with CLP alone. To
rule out transgene toxicity, animals were infected with an Ad construct
expressing a different type of transgene, namely -galactosidase
(Ad.LacZ). However, Ad.LacZ-infected animals also displayed a 50%
death rate by 24 h post-CLP compared with 48 h with CLP alone
(Fig. 1). On the contrary, no adverse effect of adenovirus on the
survival rates was observed in Ad-infected sham operated animals.
Second-generation adenoviral vectors (ts125LacZ) did not have an effect
on early mortality after subsequent CLP (Fig. 1). ts125LacZ vectors,
and UV-inactivated first-generation vectors had an identical effect on
post-CLP survival (data not shown). These results suggested that the
first-generation recombinant adenovirus constructs accelerate
CLP-induced mortality in C57BL/6 mice.
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Peritoneal infection results in early liver toxicity so we
evaluated cell viability in sections from treated and untreated
animals. Liver tissue was examined by TUNEL assay to determine the
amount of apoptosis in hepatic cells (Fig. 2A). CLP surgery alone did not
produce apoptosis in the liver, which is consistent with previous
reports (13, 14). Interestingly, a limited degree of
hepatic apoptosis was observed in Ad-infected sham operated animals.
CLP did not seem to increase the amount of liver apoptosis in
Ad-infected animals. Therefore, hepatic apoptosis was attributed to the
Ad infection itself.
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B). CLP procedure further raised serum ALT levels. Serum
ALT levels were maximal by 6 h after CLP in both CLP and Ad.CLP
animals. Nonetheless, a greater degree of liver damage occurred in
animals exposed to both Ad and CLP compared with CLP alone as
determined by ALT levels. These observations suggest that the
combination of Ad and CLP is more toxic to liver cells than either
insult alone.
The lung is often a target during sepsis, so we also evaluated cell
viability in lung sections (15). Our apoptosis assays
indicated a limited degree of apoptosis in the lungs of animals that
underwent CLP alone (Fig. 3A).
Ad infection alone did not produce detectable levels of lung apoptosis
compared with sham controls. However, Ad-infected CLP animals exhibited
a greater degree of lung apoptosis than did the CLP group alone. As
reported previously, the lung damage in bacterial infections is
correlated with an increase in the lung permeability (16).
Therefore, the LPI was used as an indication of acute lung damage.
Animals in the CLP and Ad.CLP groups lost 5% of total body weight
in 24 h, but there was no difference in total body weight between
the two groups (data not shown). Interestingly, the increase in the LPI
of the CLP group compared with that of sham controls was not
statistically different at 24 h. The same was true with the
Ad-infected sham group (Fig. 3B). However, Ad-infected CLP animals had
a greater LPI than did the other groups. CLP increased LPI of animals
by 48 h after the operation (data not shown). This suggested that
lung damage occurred earlier in the Ad-infected CLP animals than in the
CLP alone or Ad alone group; this accelerated lung injury correlated
with accelerated mortality.
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The severity of sepsis in humans is correlated with the degree of
lymphocyte apoptosis (17). To determine the extent of
apoptosis, TUNEL assays were performed on tissue sections isolated from
either thymus (Fig. 4A) or
spleen (Fig. 4B). As seen in Fig. 4A, both CLP
alone and Ad-infected sham groups manifested some degree of thymic
apoptosis. However, a profound degree of thymic apoptosis was observed
in Ad-infected CLP animals. Apoptosis is also observed in the spleens
of critically ill septic humans (18) and in septic mice
(19). Our TUNEL assays indicated that both CLP and
Ad-infected sham groups exhibited apoptosis in the spleen (Fig. 4B). The degree of apoptosis was greater in the Ad-infected
CLP group than in the rest of the groups. These results suggest that Ad
and CLP alone induce some degree of apoptosis and that the greatest
amount of apoptosis is found in the spleen and thymus of the
Ad-infected CLP animals.
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is reduced and the bacterial load are increased in
Ad-infected CLP animals
Cytokines play crucial roles in the development of sepsis syndrome
(11). A strong correlation between serum TNF- levels
and the severity of sepsis has been reported in septic patients
(20). In addition, TNF- has also been shown to play an
important role in the development of multiple organ dysfunction
syndrome (21). These results emphasize the importance of
TNF- in the establishment of the sepsis syndrome. Serum TNF-
levels were analyzed by ELISA. We did not observe any detectable levels
of TNF- in the blood of sham operated animals or Ad-infected sham
operated animals by ELISA. There was an increase in the serum TNF-
levels of CLP animals (Fig. 5A), which is consistent with
previous studies of the CLP model (22, 23, 24). Interestingly,
Ad-infected CLP animals exhibited a smaller rise in the serum TNF-
levels after CLP than did the CLP alone group. Therefore, in these
studies, an inverse correlation was observed between the serum TNF-
levels and the mortality rates. This observation suggests that the
increased mortality in Ad-infected CLP animals might be due to an
increase in bacterial infection and not to increased amounts of
TNF.
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B).
Interestingly, Ad-infected CLP animals had much greater levels of
bacterial load in the blood, liver, and lung. Among these sources,
liver had the highest degree of bacterial load. This observation
suggests a defect in bacterial clearance in Ad-infected CLP
animals.
Serum soluble TNF-R1 is increased in Ad-infected CLP animals
To determine possible causes of the decrease in serum TNF- in
Ad-infected CLP animals, ELISA was used to measure sTNFR1 in serum
after CLP. There was an increase in serum sTNFR1 within 24 h of
CLP (Fig. 6), which was consistent with
prior studies of bacterial sepsis (25, 26). This
response was significantly greater in mice previously infected with
Ad.LacZ vectors (Fig. 6). These data correlate with the decreased serum
TNF- response seen in Ad-infected CLP animals.
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Liver has been shown to be an important target organ in the CLP
model of murine sepsis (14, 27). Kupffer cells are the
resident macrophages of the liver and play an essential role in the
outcome of sepsis (28). To modify the behavior of Kupffer
cells by gene therapy, Kupffer cells likely would be targeted by these
vectors. An immunocytochemistry analysis using a Kupffer cell-specific
marker (Ly-71) (29) was performed in Ad-infected animals
to determine whether first-generation recombinant adenovirus vectors
can transduce Kupffer cells. As seen in Fig. 7, A and B,
recombinant Ad constructs successfully transduced Kupffer cells. To
determine whether there is a loss of Kupffer cells in Ad-infected CLP
animals, in situ TUNEL assay was performed before labeling cells with a
Kupffer cell-specific marker. Image quantification determined that 28%
of the Kupffer cells exhibited nuclear labeling with the TUNEL assay.
This was imaged in 6 animals, with 20 scans per animal; representative
images are shown in Fig. 7, E and F. This is in
contrast to animals undergoing CLP alone, where no apoptosis was seen
in the liver (Fig. 2A). Although it was difficult to
quantitate the percentage of Kupffer cells that were EGFP positive, it
did appear the majority of these cells expressed the transgene. This
result suggested that Kupffer cell loss is at least in part responsible
for the decreased clearance of bacteria in Ad-infected CLP animals.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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activity (30),
which may result in decreased bactericidal activity. As an aggregate,
these observations suggest that Ad gene therapy constructs worsen
sepsis by a number of important, distinct mechanisms.
During the course of sepsis, several proinflammatory cytokines,
including TNF- are produced (31). Interestingly, a
strong correlation between the serum TNF- level and the severity of
sepsis has been reported in septic patients (20). As
previously reported, limited amounts of TNF- are sufficient to
control most bacterial infections (32, 33). Yet,
overproduction of TNF- leads to a lethal inflammatory response and
increased mortality (34, 35). Therefore, modulation of the
magnitude of the TNF- response appears to play a critical role in
the final outcome of a bacterial insult (31). For this
reason, serum TNF- levels were monitored in our studies. A
significant increase in the serum TNF- levels is detected with CLP.
Intriguingly, Ad-infected CLP animals exhibited lower levels of TNF-
than did the CLP group alone. The decreased TNF- seen in Ad-infected
CLP animals may be due to several factors. It has been suggested that
serum cytokines represent an overflow from the most affected organs
(36); therefore, the observed loss of Kupffer cells in the
livers of Ad-infected CLP animals may lead to decreased serum levels of
TNF-. Previous studies have shown mast cells have an important role
in the TNF- response to CLP (37). It is possible that
adenoviral vectors adversely affected mast cell function in our
studies. Finally, our observation of decreased serum TNF- may be due
to an increase in sTNFR1. There may also have been a loss of
macrophages in other sites in these animals.
It was not surprising to show that the mortality of CLP was worsened by
previous viral infection. First-generation adenoviral vectors have been
shown to stimulate TNF- production (38). One might
hypothesize that two infectious insults might stimulate excessive
TNF- and therefore increase organ injury. However, it was intriguing
that the increased mortality was associated with a decrease in serum
TNF-. Recent studies have shown that adenoviral constructs aimed at
decreasing TNF production have led to improved mortality in
zymosan-induced multisystem organ failure (3). We
therefore sought an alternate explanation for the increased mortality
in the CLP animals exposed to Ad constructs. One explanation of the
accelerated mortality in the CLP animals exposed to Ad constructs is a
greater severity of sepsis. This is supported by observations showing
increased bacterial load in these animals. We were also able to show
that this adenoviral construct exposure had greater toxicity to
macrophages than did the controls both in vivo and in vitro.
Macrophages, especially in the liver, are important for bacterial
clearance.
Although live first-generation adenoviral vectors significantly accelerated mortality following subsequent CLP, both UV inactivation and ts125LacZ negated this effect. Therefore, the effects are likely a result of viral gene expression. First-generation vectors are E1 and E3 depleted, rendering them unable to replicate, but some viral protein expression still occurs (10). First-generation vectors are commonly contaminated with replication-competent virus (arising through recombination between vector and producer cell DNA sequences) (39), which induces further inflammatory responses in recipients of gene therapy. This has stimulated many investigators to create advanced generations of adenoviral vectors for potential gene therapy. Indeed, the second-generation vectors used in our studies did not accelerate CLP induced mortality. This implies that E2a-regulated adenoviral gene expression stimulated the changes observed in sepsis after adenoviral infection. This finding may provide further support for the use of advanced-generation adenoviral vectors in clinical trials using gene therapy in the setting of sepsis.
It has also been suggested that lymphocyte depletion via apoptosis may also be a major determinant of sepsis-induced mortality in critically ill patients (17). In accordance with these reports, our results showed greater apoptosis of lymphocytes in thymus and spleen in CLP animals exposed to adenoviral constructs. Intriguingly, Ad infection itself generated a low level apoptosis in these tissues. These observations suggest that one mechanism, for the increased mortality found in CLP animals infected with adenoviral constructs, might be a profound loss of lymphocytes. Finally, CLP animals exposed to Ad had greater evidence of lung and liver parenchymal injury, suggesting another mechanism for increased mortality. The specific mechanism of the increased cellular killing with Ad vectors and CLP is not addressed in this study.
The relationships among TNF-, sTNFR1, and bacterial load are
complex. TNF- is a major stimulus for the production of sTNFR1
(26, 40). In low concentrations, sTNFR1 may stabilize
TNF-; whereas at higher concentrations, the receptor acts mainly as
an inhibitor of TNF function (30). Furthermore, it has
been suggested that serum levels of TNF- may not reflect tissue
levels of TNF- (41, 42). Subsequently, systemic
inflammation may be represented more accurately by sTNFR1 than by
TNF- (43). However, our findings of increased sTNFR1
and decreased TNF- clearly represent a state of immunosuppression,
as evidenced by the increase in bacterial load.
The stimulus for this state of immunosuppression is not clear but is
affected by the previous Ad vector infection. Ad is a potent stimulator
of TNF- (44) and could therefore indirectly stimulate
sTNFR1. However, sTNFR1 is not constitutively increased after Ad
infection, as evidenced by low levels at early time points after CLP.
However, with prior adenoviral infection, sTNFR1 markedly increases
after CLP. Whether this represents increased transcription,
translation, or shedding of cellular receptors into the circulation is
not yet known. Many studies have focused on techniques to reduce
systemic TNF- in the setting of sepsis, in that it is likely an
early contributor to systemic injury (41, 45). However,
techniques that limit the immune response in sepsis may render the
patient more susceptible to infection (46, 47).
Overall, these studies suggest that first-generation adenoviral gene therapy vectors accelerate mortality associated with sepsis in an animal model. These studies did not address toxicity of adenoviral vectors that are delivered locally to various tissues. They do suggest, however, that caution might be necessary if these agents are delivered systemically to human subjects.
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Acknowledgments |
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Footnotes |
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2 K.D. and S.S. contributed equally to this article.
3 Address correspondence and reprint requests to Dr. Kevin Doerschug, College of Medicine, University of Iowa, 200 Hawkins Drive, C-33, GH, Iowa City, IA 52242. E-mail address: kevin-doerschug{at}uiowa.edu
4 Abbreviations used in this paper: Ad, adenovirus; CLP, cecal ligation and puncture; EGFP, enhanced green fluorescent protein; Ad.EGFP, Ad vector expressing EGFP; Ad.LacZ, Ad vector expressing Escherichia coli -galactosidase protein; LPI, lung permeability index; ALT, alanine aminotransferase; sTNFR1, soluble TNFR1.
Received for publication February 22, 2002. Accepted for publication September 30, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) and Ad-infected CLP group (
) were determined as
described in Materials and Methods. The
y-axis represents CFU per milliliter of blood or CFU per
gram of liver or lung tissue. Data represent the average of (±SEM) of
six independent animals. *, Statistically significant differences in
serum TNF levels between CLP and Ad-infected CLP (Ad.CLP) groups, as
determined by ANOVA and Tukey’s multiple comparison test
(p < 0.05); ***, p <
0.001.
