Prevention of Lymphocyte Apoptosis in
Septic Mice with Cancer Increases Mortality
This information is current as
of June 15, 2017.
Amy C. Fox, Elise R. Breed, Zhe Liang, Andrew T. Clark,
Brendan R. Zee-Cheng, Katherine C. Chang, Jessica A.
Dominguez, Enjae Jung, W. Michael Dunne, Eileen M.
Burd, Alton B. Farris, David C. Linehan and Craig M.
Coopersmith
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The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2011 by The American Association of
Immunologists, Inc. All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2011; 187:1950-1956; Prepublished online 6 July
2011;
doi: 10.4049/jimmunol.1003391
http://www.jimmunol.org/content/187/4/1950
The Journal of Immunology
Prevention of Lymphocyte Apoptosis in Septic Mice with
Cancer Increases Mortality
Amy C. Fox,* Elise R. Breed,†,‡ Zhe Liang,†,‡ Andrew T. Clark,* Brendan R. Zee-Cheng,*
Katherine C. Chang,x Jessica A. Dominguez,{ Enjae Jung,* W. Michael Dunne,‖
Eileen M. Burd,# Alton B. Farris,# David C. Linehan,* and Craig M. Coopersmith†,‡
S
epsis is the leading cause of death among critically ill
patients in the United States, with .200,000 people dying
from the disease annually (1). Despite many advances in
understanding the pathophysiology of sepsis, mortality remains
unacceptably high (2).
Apoptosis is theorized to have a critical role in the pathophysiology of sepsis (3). Human autopsy studies of sepsis demonstrate increased apoptosis in the spleen and the intestinal
epithelium (4). In addition, apoptosis in circulating lymphocytes is
markedly increased in septic patients (5–7), and this is associated
with poor outcome (8). Animal models of sepsis replicate these
findings of increased sepsis-induced lymphocyte and intestinal
epithelial apoptosis (9–14). The functional significance of this
finding is demonstrated in animal models (predominantly
*Department of Surgery, Washington University School of Medicine, St. Louis, MO
63110; †Emory Center for Critical Care, Emory University School of Medicine,
Atlanta, GA 30322; ‡Department of Surgery, Emory University School of Medicine,
Atlanta, GA 30322; xDepartment of Anesthesiology, Washington University School
of Medicine, St. Louis, MO 63110; {Department of Anesthesiology, University of
Colorado at Denver School of Medicine, Aurora, CO 80045; ‖Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO
63110; and #Department of Pathology and Laboratory Medicine, Emory University
School of Medicine, Atlanta, GA 30322
Received for publication October 13, 2010. Accepted for publication June 8, 2011.
This work was supported by funding from the National Institutes of Health (Grants
GM66202, GM072808, GM008795, GM082008, and P30 DK52574).
Address correspondence and reprint requests to Dr. Craig M. Coopersmith, 101 Woodruff Circle, Suite WMB 5105, Atlanta, GA 30322. E-mail address: cmcoop3@emory.
edu
Abbreviations used in this article: BAL, bronchoalveolar lavage; MPO, myeloperoxidase; WT, wild type.
Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1003391
peritonitis-induced sepsis) demonstrating that prevention of apoptosis in lymphocytes, globally using knockout mice or siRNA or
in the intestinal epithelium, improves survival following sepsis
(15–35). Because apoptosis prevention has been repeatedly successful in improving survival when targeting a wide variety of
mediators by a number of investigative groups, there is significant
interest in translating these findings to the bedside (36–39).
There has been a longstanding disconnect between animal models
of sepsis and therapeutic trials in patients (40). There are complex
reasons why positive preclinical trials have not successfully translated into therapeutic benefit at the bedside, and one possibility is that
the populations studied are different. Typical animal models use mice
that were healthy prior to the onset of sepsis; however, the majority
of septic patients have one or more preexisting comorbidities (1).
Patients and animals subjected to a septic insult have increased
mortality in the setting of additional comorbidities (41–44). This is
consistent with a “two-hit” model of injury, where a chronic
comorbidity is the first insult and an acute septic injury represents the
second insult. Although each of these “hits” independently confers
some risk, their combined effects are disproportionately harmful
over what might have been predicted from either in isolation.
Cancer is one of the most common comorbidities that can afflict
patients with sepsis. It is also associated with a high rate of
mortality, with ∼40% of septic patients with cancer dying from the
disease (1, 45). In addition, patients with malignancy are nearly
10-fold more likely to develop sepsis than the general population
(46). The factors that affect an individual’s susceptibility to developing sepsis can include tumor type, tumor size, presence of
metastatic disease, and host immunologic response. Our laboratory recently described an animal model of sepsis and cancer
in which mice that received a transplantable pancreatic adeno-
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Lymphocyte apoptosis is thought to have a major role in the pathophysiology of sepsis. However, there is a disconnect between
animal models of sepsis and patients with the disease, because the former use subjects that were healthy prior to the onset of infection while most patients have underlying comorbidities. The purpose of this study was to determine whether lymphocyte apoptosis
prevention is effective in preventing mortality in septic mice with preexisting cancer. Mice with lymphocyte Bcl-2 overexpression
(Bcl-2-Ig) and wild type (WT) mice were injected with a transplantable pancreatic adenocarcinoma cell line. Three weeks later,
after development of palpable tumors, all animals received an intratracheal injection of Pseudomonas aeruginosa. Despite having
decreased sepsis-induced T and B lymphocyte apoptosis, Bcl-2-Ig mice had markedly increased mortality compared with WT mice
following P. aeruginosa pneumonia (85 versus 44% 7-d mortality; p = 0.004). The worsened survival in Bcl-2-Ig mice was
associated with increases in Th1 cytokines TNF-a and IFN-g in bronchoalveolar lavage fluid and decreased production of the
Th2 cytokine IL-10 in stimulated splenocytes. There were no differences in tumor size or pulmonary pathology between Bcl-2-Ig
and WT mice. To verify that the mortality difference was not specific to Bcl-2 overexpression, similar experiments were performed
in Bim2/2 mice. Septic Bim2/2 mice with cancer also had increased mortality compared with septic WT mice with cancer. These
data demonstrate that, despite overwhelming evidence that prevention of lymphocyte apoptosis is beneficial in septic hosts without
comorbidities, the same strategy worsens survival in mice with cancer that are given pneumonia. The Journal of Immunology,
2011, 187: 1950–1956.
The Journal of Immunology
carcinoma cell line 3 wk prior to the onset of Pseudomonas aeruginosa pneumonia had a 24% increase in mortality compared
with septic mice that were previously healthy (44).
In light of 1) the extensive literature demonstrating a survival
benefit to preventing lymphocyte apoptosis in previously healthy
mice and 2) the knowledge that mice with cancer (or other
comorbidities) behave differently than previously healthy mice
subjected to the identical septic insult, this study examined whether
preventing lymphocyte apoptosis would improve survival in the
clinically relevant model of sepsis in the setting of cancer.
Materials and Methods
Mice
Cancer model
The transplantable mouse pancreas adenocarcinoma cell line Pan02 was
injected to induce cancer as described previously (51, 52). Pan02 cells were
cultured in RPMI 1640 medium supplemented with 1% glutamine, 1%
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 10% FBS, and 1%
penicillin–streptomycin (Cellgro, Herndon, VA). A total of 250,000 Pan02
cells were injected s.c. into each animal’s right inner thigh. Mice were then
housed for 3 wk in a barrier facility to allow tumors to grow prior to the
induction of sepsis. Animals given Pan02 cells developed well-defined,
circumscribed tumors at the injection site, but the tumors were not metastatic and did not cause mortality in the period studied in this set of
experiments. The longest axis of the tumor was used for measuring tumor
size. In a recent study from our laboratory (44), animals given Pan02 cells
3 wk earlier were compared with healthy mice. Animals had similar body
weights, liver function, kidney function, lung histology, wet-to-dry lung
ratios, blood cytokine levels (TNF-a, IL-6, IL-10, IL-12, and MCP-1),
bronchoalveolar lavage (BAL) cytokines (IL-6, IL-10, IL-12, and MCP-1),
gut apoptosis, intestinal villus length, intestinal proliferation, hematocrit,
WBC count, absolute neutrophil count, absolute lymphocyte count, and
platelet count. In contrast, the presence of cancer caused lower levels of
T lymphocyte and B lymphocyte apoptosis compared with healthy mice and
also induced higher levels of TNF-a in BAL fluid.
Sepsis model
Three weeks after the injection of Pan02 cells, mice were made septic via
direct intratracheal injection of P. aeruginosa (American Type Culture
Collection 27853) (53). A midline cervical incision was made while using
isoflurane anesthesia, and 40 ml of a solution of bacteria diluted in 0.9%
NaCl (final concentration, 6 3 106 CFUs/ml) was injected directly into the
trachea using a 29-gauge insulin syringe. Animals were then held vertically for 10 s to enhance delivery of bacteria into the lungs. Mice received
a single 1-ml s.c. injection of 0.9% NaCl to replace insensible fluid losses
following incision closure. Animals were either sacrificed 24 h after induction of pneumonia for tissue harvest or followed 7 d for survival. Of
FIGURE 1. Effect of Bcl-2 overexpression on lymphocyte apoptosis in septic mice with cancer. T lymphocyte (A, B; n = 13–19 per group) and B
lymphocyte apoptosis (C; n = 14–19 per group) was decreased in Bcl-2-Ig mice compared with WT mice whether assayed by active caspase 3 staining (A,
C) or the TUNEL assay (B) by flow cytometry. Similar results were qualitatively seen on H&E-stained sections in which splenic apoptosis was elevated in
septic WT mice with cancer (D), but lower in Bcl-2-Ig mice subjected to the same insult (E). Representative histomicrographs are shown at original
magnification 3400. *p , 0.05.
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Nine- to 16-wk-old mice were used for all experiments. Transgenic mice
overexpressing human Bcl-2 in both T and B lymphocytes (Bcl-2-Ig mice)
were generated as previously described (17, 47). Of note, Bcl-2 expression
is significantly greater in the spleen than in the thymus in these animals.
Bim2/2 mice were initially generated at the Walter and Eliza Hall Institute
of Medical Research (Melbourne, VIC, Australia) (48), but were subsequently bred for over 3 y at Washington University for experiments
examining lymphocyte apoptosis and sepsis, where they were a gift from
Dr. Richard Hotchkiss (Washington University School of Medicine) (21).
Transgenic mice that overexpress Bcl-2 in the intestinal epithelium (FabplBcl-2 mice) were generated on an FVB/N background and were then
backcrossed to C57BL/6 mice for .20 generations (49, 50). For all studies
in this article, animals were compared with C57BL/6 littermates or controls bred in the same animal facility. Mice had free access to food and
water and were maintained on a 12-h light-dark schedule in a barrier facility. All studies complied with the National Institutes of Health Guidelines for the Use of Laboratory Animals and were approved by the Animal
Studies Committee of both Washington University and Emory University.
1951
1952
APOPTOSIS PREVENTION AND MORTALITY IN SEPSIS AND CANCER
Table II. Blood cytokines
Cytokine (pg/ml)
TNF-a
G-CSF
IL-10
IL-6
WT (n = 10–11)
3,445
14,200
187
3,197
6
6
6
6
392
1,236
41
584
Bcl-2-Ig (n = 9–10)
2,908
14,349
298
3,828
6
6
6
6
p Value
352
860
138
701
ns
ns
ns
ns
ns, not significant.
FIGURE 2. Effect of Bcl-2 overexpression in lymphocytes on survival
in septic mice with cancer. Mortality was nearly twice as high in Bcl-2-Ig
mice compared with WT mice 7 d after induction of P. aeruginosa
pneumonia. n = 20–27 per group. *p , 0.05.
Splenocyte apoptosis
Splenocyte apoptosis was quantified via flow cytometry using Abs against
active caspase-3 (Cell Signaling Technology, Beverly, MA) and via the
TUNEL assay (Phoenix Flow Apo-BrdU Kit, San Diego, CA) (21). T and
B cell populations were identified using fluorescein-labeled anti-mouse
CD3 (BD Pharmingen, Franklin Lakes, NJ) and PE-Cy5 conjugated antimouse CD45R/B220 (eBioscience, San Diego, CA), respectively. Flow
cytometric analysis (50,000 events per sample) was performed on FACScan (BD Biosciences, San Jose, CA) as described previously (5).
Local, systemic, and stimulated cytokine analysis
BAL and whole blood were used to assess local and systemic cytokine
levels, respectively (44). The trachea was lavaged with 1 ml sterile 0.9%
NaCl to obtain BAL fluid. Whole blood was collected retro-orbitally and
centrifuged at 5000 rpm for 5 min in serum separator tubes. BAL and
whole blood cytokine concentrations were then evaluated using the BioPlex Pro Mouse Cytokine Standard Group I Kit (Biorad, Hercules, CA)
according to manufacturer’s protocol. All samples were run in duplicate.
Stimulated cytokines were evaluated by harvesting the spleen 24 h after
septic injury. Lymphocytes contained in splenocyte suspensions were cultured
in vitro and stimulated with anti-CD3 and anti-CD28 (BD Biosciences, San
Jose, CA) as described previously (19). The supernatant was removed 24 h
later, and cytokine levels were measured using the Bio-Plex cytokine kit.
Pneumonia severity
H&E-stained lung sections were evaluated by a pathologist (A.B.F.)
blinded to sample identity to determine the severity and distribution of
pneumonia. Samples were first graded to determine the percent of tissue
with inflammation.
An inflammation score ranging from 0 to 5 (0 = none; 1 = ,5%; 2 =
5–10%; 3 = 10–19%, 4 = 20–49%, 5 = .50%) was then calculated. Next,
the degree of atelectasis was assessed as follows: none, focal representing
,10%; moderate, 10–50%; and severe, .50%. Vascular congestion was
then assessed using the same scale as atelectasis. Finally, a subjective
analysis of each individual section was performed.
Myeloperoxidase activity
Bacterial cultures
Blood was collected from the inferior vena cava, and BAL fluid was
obtained as described above. BAL samples were serially diluted in sterile
saline and plated on sheep blood agar plates. Plates were incubated
overnight at 37˚C and examined for colony counts 24 h later.
Intestinal epithelial apoptosis
Apoptotic cells were quantified in 100 contiguous, well-oriented crypt-villus
units using H&E staining and active caspase-3 staining (53). Apoptotic cells
were identified on H&E-stained sections by morphologic criteria. Cells with
characteristic nuclear condensation and fragmentation were considered to be
apoptotic. For active caspase-3 staining, intestinal sections were deparaffinized, rehydrated, and incubated in 3% hydrogen peroxide for 10 min. Ag
retrieval was performed by placing slides in Ag Decloaker (Biocare Medical, Concord, CA) and heating them in a pressure cooker for 45 min. Slides
were then blocked with 20% goat serum (Vector Laboratories, Burlingame,
CA) and incubated with rabbit polyclonal anti-active caspase-3 (1:100; Cell
Signaling Technology, Beverly, MA) overnight at 4˚C. The following day,
slides were incubated with goat anti-rabbit biotinylated secondary Ab
(1:200; Vector Laboratories) for 30 min at room temperature followed by
Vectastain Elite ABC reagent (Vector Laboratories) for 30 min. Slides were
developed with diaminobenzidine and counterstained with hematoxylin.
Statistics
All data were tested for Gaussian distribution using the Shapiro-Wilk
normality test. If data were found to have a Gaussian distribution, comparisons were performed using the Student t test, and results were presented
as mean 6 SEM. If data did not have a Gaussian distribution, comparisons
were performed using the Mann–Whitney U test and were presented as
median 6 range. Survival studies were analyzed using the log-rank test.
Data were analyzed using the statistical software program Prism 4.0
(GraphPad, San Diego, CA). A p value ,0.05 was considered statistically
significant.
Results
In all experiments, mice were injected with the transplantable
pancreatic adenocarcinoma cell line Pan02. Three weeks later,
animals were given P. aeruginosa pneumonia. As a result, all
animals developed sepsis in the setting of preexisting cancer.
Bcl-2 overexpression in lymphocytes increases mortality
despite preventing sepsis-induced lymphocyte apoptosis
BAL fluid was obtained after tracheal instillation with 1 ml sterile saline.
BAL fluid was collected and centrifuged at 5000 rpm for 5 min. Following
Transgenic mice overexpressing human Bcl-2 in both T and B
lymphocytes (Bcl-2-Ig mice) had decreased levels of T lymphocyte apoptosis compared with wild type (WT) littermates by both
Table I. BAL cytokines
Table III. Stimulated splenocyte cytokines
Cytokine (pg/ml)
TNF-a
IFN-g
IL-10
IL-6
ns, not significant.
WT (n = 10)
436
17
179
11,813
6
6
6
6
48
1
54
3,632
Bcl-2-Ig (n = 11)
p Value
Cytokine (pg/ml)
6
6
6
6
0.03
0.008
ns
ns
IL-10
TNF-a
IFN-g
IL-6
734
43
82
13,469
115
17
33
2,325
ns, not significant.
WT (n = 9)
187
56
10,492
1,922
6
6
6
6
15
4
1,375
189
Bcl-2-Ig (n = 9)
p Value
6
6
6
6
0.004
ns
ns
ns
117
52
12,493
1,509
15
5
1,364
209
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note, mortality is nearly twice as high in mice given Pan02 cells 3 wk prior
to the onset of P. aeruginosa pneumonia compared with previously healthy
mice given the identical insult (44). The rationale for studying P. aeruginosa pneumonia following cancer, as opposed to a more commonly studied
model of sepsis such as cecal ligation and puncture, was a recent study
extensively characterizing the effect of pneumonia superimposed on cancer
(44).
the addition of substrate buffer containing O-dianisidine and 0.0005%
hydrogen peroxide, myeloperoxidase (MPO) activity was measured at 470nm wavelength over 6 min (Bio-Tek Instruments-mQuant Microplate
Spectrophotometer, Winooski, VT). MPO activity was calculated as ΔOD/
minute per microliter of BAL fluid.
The Journal of Immunology
1953
Bcl-2 overexpression in lymphocytes does not alter pneumonia
severity
FIGURE 3. Effect of Bcl-2 overexpression in lymphocytes on tumor
size.Bcl-2-Ig mice and WT mice were both injected with 250,000 Pan02
cells and then given sepsis with P. aeruginosa 3 wk later. Overexpression
of Bcl-2 in lymphocytes had no affect on the growth of tumors. n = 9–10
per group.
Bcl-2 overexpression in lymphocytes in septic mice with cancer
induces an upregulation of Th1 cytokines in BAL fluid but not
plasma
To determine whether the local host response played a role in the
elevated mortality in septic Bcl-2-Ig mice, BAL cytokines were
measured in transgenic and WT mice (Table I). Concentrations of the
Th1 cytokines TNF-a (p = 0.008) and IFN-g (p = 0.03) were significantly higher in Bcl-2-Ig than WT mice. In contrast, when plasma
cytokines were assayed, no significant differences were detected
between septic Bcl-2-Ig and WT mice with cancer (Table II).
Bcl-2 overexpression in lymphocytes in septic mice with cancer
decreases production of IL-10 in stimulated lymphocytes
Lymphocytes isolated from the spleens of septic Bcl-2-Ig mice that
were incubated ex vivo with anti-CD3/28 had lower production of
the Th2 cytokine IL-10 compared with WT littermates (p = 0.004;
Table III). Other stimulated cytokines were similar in septic mice
with cancer, regardless of whether they overexpressed Bcl-2 in
their lymphocytes.
Bcl-2 overexpression in lymphocytes does not alter tumor size
To determine whether overexpression of an antiapoptotic protein
in lymphocytes caused a secondary effect on cancer growth, tumor
size was measured in Bcl-2-Ig and WT mice. No difference in
tumor size was identified, regardless of whether an animal overexpressed Bcl-2 in its lymphocytes (Fig. 3).
Septic Bim2/2 mice with cancer have increased mortality
compared with WT mice
To determine whether the alteration in mortality was specific to
Bcl-2-Ig mice or potentially more generalizable, survival studies
were repeated in Bim2/2 mice that were injected with Pan02 cells
as described above and made septic via P. aeruginosa pneumonia
3 wk later. Septic Bim2/2 mice with cancer had an 18% increase in mortality compared with WT mice with cancer (p =
0.046; Fig. 5).
Bcl-2 overexpression in the intestinal epithelium has no effect
on mortality
To determine whether the effect of altering sepsis-induced apoptosis was lymphocyte specific, a survival study was performed on
transgenic mice that overexpressed Bcl-2 in the intestinal epithelium (Fabpl-Bcl-2 mice). There was no difference in survival
between septic Fabpl-Bcl-2 mice with cancer and septic WT mice
with cancer (Fig. 6A). However, unlike either septic Bcl-2-Ig mice
with cancer or previously healthy septic Fabpl-Bcl-2 mice, no
protection against sepsis-induced apoptosis was identified in the
cell type where the transgene was expressed in this set of experiments (Fig. 6B, 6C).
Discussion
This study demonstrates that prevention of sepsis-induced lymphocyte apoptosis in mice with cancer increases mortality. These
results contrast significantly from extensive literature demonstrating
that prevention of lymphocyte apoptosis in a peritonitis model of
sepsis improves survival in mice that were healthy prior to their
septic insult. In addition, animals with the identical genetic alteration
in apoptosis signaling respond to sepsis in contradictory manners
depending on whether an animal has cancer prior to the onset of
sepsis and the model of sepsis used. Both Bcl-2-Ig mice and Bim2/2
FIGURE 4. Effect of Bcl-2 overexpression on pulmonary pathology. Bcl-2-Ig mice and WT mice were both injected with 250,000 Pan02 cells and then
given sepsis with P. aeruginosa 3 wk later. Overexpression of Bcl-2 in lymphocytes had no affect on pneumonia severity (A) or percentage of lung tissue
with inflammation (B). MPO activity in BAL fluid was also unaffected (C). n = 6 per group.
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the active caspase-3 staining and TUNEL assay as measured by
flow cytometry (Fig. 1A, 1B). Bcl-2-Ig mice also had decreased
levels of B lymphocyte apoptosis (Fig. 1C). A similar decrease in
apoptosis was also observed on H&E-stained splenic sections
(Fig. 1D, 1E).
A different set of Bcl-2-Ig and WT cancer mice were then
followed for survival after the induction of sepsis. Despite the fact
that Bcl-2-Ig mice had lower lymphocyte apoptosis, 85% of Bcl2-Ig mice died 7 d after the onset of sepsis compared with 44% of
WT mice (p = 0.004; Fig. 2).
In light of the differences identified in BAL cytokines, pneumonia
severity was assessed to determine whether alterations in pulmonary pathology were responsible for the mortality difference
between Bcl-2-Ig and WT mice. Both pneumonia severity and percentage of lung tissue with inflammation was similar between
Bcl-2-Ig and WT mice (Fig. 4A, 4B). No differences were detected
between the groups in atelectasis (ranging from none to moderate
in Bcl-2-Ig mice, ranging from focal to moderate in WT mice) or
vascular congestion (focal to moderate in all animals, without
differences between groups). In addition, there were no differences in neutrophil infiltration and degranulation in BAL fluid as
measured by MPO activity between Bcl-2-Ig and WT mice (Fig.
4C). Bacteria were not detectable in BAL fluid 24 h after tracheal
instillation of P. aeruginosa (n = 5–6 mice per group).
1954
APOPTOSIS PREVENTION AND MORTALITY IN SEPSIS AND CANCER
FIGURE 5. Effect of knocking out the proapoptotic factor Bim on
survival in septic mice with cancer. Mortality was higher in Bim2/2 mice
than in WT mice (n = 11–14 per group) 7 d after induction of P. aeruginosa
pneumonia. *p , 0.05.
FIGURE 6. Effect of Bcl-2 overexpression on intestinal epithelial apoptosis and survival in septic mice with cancer. No difference in mortality was
observed between Fabpl-Bcl-2 mice and WT mice 7 d after induction of P. aeruginosa pneumonia (A, n = 27–29 per group). Despite the fact that Bcl-2 was
overexpressed in the intestinal epithelium in transgenic mice, intestinal epithelial apoptosis was similar by both H&E staining (B) and active caspase-3
staining (C) in Fabpl-Bcl-2 mice and WT mice. n = 12–13 per group.
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mice have increased survival if peritonitis is initiated in previously
healthy mice (17, 21, 54), but have decreased survival if pneumonia
is initiated in mice with preexisting cancer.
A differential host response appears to play a role in explaining
this discrepancy. Bcl-2-Ig mice with cancer have increases in their
Th1 cytokines TNF-a and IFN-g in BAL fluid 24 h after the onset
of pneumonia compared with WT septic mice with cancer. In
addition, when their splenocytes are stimulated ex vivo, they have
decreased production of the Th2 cytokine IL-10. These findings
suggest that there is an exaggerated inflammatory response in
septic mice with cancer when lymphocyte apoptosis is prevented.
This increased proinflammatory response is both local and systemic in nature given the findings in BAL fluid and stimulated
splenocytes.
The inflammatory response following sepsis is complex. Either
too much or too little inflammation can damage the host by causing
unintended organ failure or leaving the host susceptible to secondary infections, respectively. Some degree of inflammation is
beneficial, because immunoparalyzed hosts are at risk for fatal
infectious complications. However, the appropriate amount of
inflammation varies with both the clinical scenario and the length of
time elapsed after the initial insult. Too much inflammation can be
as harmful as too little inflammation, and it is likely that the increased inflammatory state in the lungs and the decreased ability of
splenocytes to secrete compensatory anti-inflammatory cytokines
played a role in the excessive mortality seen in this study.
In our prior description of why septic mice with cancer were
more likely to die than septic mice that were previously healthy
(both WT C57BL/6 mice), we found that sepsis induced an increase
in T and B lymphocyte apoptosis in all animals (44). However,
septic mice with cancer had decreased T and B lymphocyte apoptosis compared with previously healthy septic mice. These findings imply that some degree of lymphocyte apoptosis may be
beneficial in a subset of septic hosts in light of the fact that 1) mice
with cancer have lower levels of sepsis-induced apoptosis than
previously healthy mice despite higher mortalities, and 2) preventing this apoptosis leads to a further increase in mortality.
Thus, an important implication of this study is that prevention
of lymphocyte apoptosis may not always be a beneficial therapeutic strategy, and it may be a harmful strategy in select patient
groups with a specific disease process. This issue is directly relevant because of the large amount of interest in translating antiapoptotic therapies to the bedside of patients with sepsis based on
overwhelming evidence in animal models and observational human studies suggesting that lymphocyte apoptosis is harmful in
sepsis.
One possible explanation for the discrepancy between our results
and those published in the existing literature is the difference in the
model used. The vast majority of mouse studies demonstrating the
benefit of preventing lymphocyte apoptosis use the cecal ligation
and puncture model, and there is overwhelming evidence supporting apoptosis prevention in previously healthy mice with
peritonitis. In contrast, this study uses a pneumonia model. To
our knowledge, there is only one published study examining
lymphocyte apoptosis prevention in pneumonia, in which overexpression of Bcl-2 in lymphocytes resulted in a trend toward
improved survival in P. aeruginosa pneumonia (54). A similar
experiment performed for this study also demonstrates a nonsignificant 16% improvement in survival in Bcl-2-Ig mice subjected to P. aeruginosa pneumonia compared with WT mice (data
not shown). Based on this finding, it is possible that Bcl-2 overexpression confers either a small survival benefit or has no
meaningful benefit in previously healthy mice subjected to
pneumonia. Each of these results would be at least somewhat
different from previously healthy mice subjected to peritonitis,
where apoptosis prevention confers a marked survival benefit.
Importantly, the results also differ significantly from mice with
cancer that subsequently are given pneumonia, demonstrating that
the addition of cancer as a comorbidity alters the host response by
changing an intervention (i.e., lymphocyte apoptosis prevention)
that may ordinarily be either beneficial or neutral into one that is
detrimental to host survival. Although both peritonitis and pneumonia models induce sepsis, they do not induce an identical inflammatory response. Both Bcl-2-Ig mice and Bim2/2 mice have
increased basal numbers of lymphocytes compared with WT
animals. In a pneumonia model, this baseline increase in effector
cells could lead to profound changes in a stressed host. As such,
this increase in effector cells might be more physiologically significant than prevention of lymphocyte apoptosis and functionally
overwhelm it, resulting in the observed proinflammatory response.
This explanation could help to explain the differential result
The Journal of Immunology
Despite these limitations, these results lead to a new paradigm in
understanding lymphocyte apoptosis in sepsis. There continues to
be overwhelming evidence that lymphocyte apoptosis is harmful in
hosts that were healthy prior to the onset of septic peritonitis.
However, prevention of lymphocyte apoptosis by chronic genetic
alterations in the mitochondrial pathway is harmful in mice with
cancer that subsequently develop sepsis from pneumonia. Thus, the
results do not support the view that sepsis-induced apoptosis is
a maladaptive response independent of the host or insult; instead,
they suggest a response that is modifiable by pre-existing host
comorbidities and a model of sepsis used in which a basal level
of lymphocyte cell death may be beneficial under the correct
circumstances. These results should be repeated using other types
of tumors, other comorbidities, other models of sepsis, and other
apoptosis inhibitor strategies to determine their generalizability.
These results should also be considered in designing entry criteria
for future clinical trials aimed at preventing lymphocyte apoptosis
in sepsis.
Disclosures
The authors have no financial conflicts of interest.
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between mice subjected to peritonitis versus those subjected to
pneumonia, although it would not explain the difference in survival between Bcl-2 overexpression in mice that were healthy
prior to the onset of pneumonia versus those that had cancer prior
to the onset of pneumonia.
Although the vast majority of preclinical studies use healthy, 6–
12-wk-old mice, these animals actually model a patient population
that almost never gets septic and is unlikely to die of the disease in
the rare instance that it develops. Sepsis is a disease that most
commonly affects the elderly or patients with comorbidities, or
both; however, the typical study using young, previously healthy
mice is the equivalent of studying the disease in a 13 y old (55, 56)
without any medical history. Interventions that may be beneficial
in this age group may lose their efficacy or actually be harmful in
either aged patients or those with chronic comorbidities, who have
markedly different inflammatory milieus at baseline.
The findings in septic Fabpl-Bcl-2 mice reinforce the importance of cancer as a comorbidity. Previously healthy Fabpl-Bcl-2
mice do not have the increase in sepsis-induced gut epithelial
apoptosis seen in WT animals subjected to the same insult and
have improved survival following sepsis (24, 57). In contrast,
when these animals have cancer prior to the induction of sepsis,
they neither prevent sepsis-induced apoptosis nor improve survival. Although the finding that Bcl-2 was ineffective in preventing sepsis-induced gut apoptosis was surprising, we have
previously described that these animals fail to prevent sepsisinduced intestinal apoptosis in the absence of lymphocytes (58),
which suggests that the strength of apoptotic signaling (in the
intestine at least) may be modifiable by host factors outside of the
local environment. Furthermore, although the loss of the survival
benefit conferred by gut-specific Bcl-2 was not as striking as the
worsening of survival conferred by lymphocyte-specific Bcl-2 in
septic mice with cancer, the results are consistent in that both the
host response to sepsis and attempts to alter this response may be
determined, at least in part, prior to the onset of infection.
This study has a number of limitations. Because all nonsurvival
studies were performed at a single time point (24 h), the experimental design did not allow for a dynamic assessment of the
temporal changes in the host response. Next, although WT C57BL/
6 mice were subjected to the same model of sepsis, mortality was
significantly higher in experiments examining the effect of Bim2/2
mice than in those examining the effect of Bcl-2-Ig mice. There is
no clear explanation for this finding, because the experiments
were performed by the same person (A.C.F.). Next, the decrease in
sepsis-induced lymphocyte apoptosis in Bcl-2-Ig mice was small.
We believe this decrease is small because sepsis-induced lymphocyte apoptosis is significantly lower in mice with cancer than
in previously healthy mice (44). Previous work from our laboratory has shown that T lymphocyte apoptosis in Pan02-bearing
septic mice is 10.4% and that T lymphocyte apoptosis in sham
mice with cancer is 3.5% (44). In a comparison of these prior
results with data shown in Fig. 1B, Bcl-2 decreased sepsis-induced
T lymphocyte apoptosis to levels seen in sham mice. The fact that
mortality was decreased in Bcl-2-Ig mice and Bim2/2 mice, both
of which have improved survival following cecal ligation and
puncture in previously healthy mice (17, 21, 54), strengthens our
findings. However, all transgenic and knockout mice used in this
study have lifelong genetic alterations that could lead to chronic
changes in other immune effector cells, which could affect the
outcome when mice are subjected to pneumonia. As such, an alternate strategy initiating either Abs or drugs that inhibit apoptosis
after the onset of pneumonia might have more clinical relevance
and in theory a different effect on mortality.
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