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,
,
Departments of
*
Anesthesiology,
Surgery,
Medicine, and
Pathology, Washington University School of Medicine, St. Louis, MO 63110
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recent studies in animal models of sepsis as well as in patients who died of sepsis and multiple organ failure have shown that sepsis induces extensive loss of lymphocytes via apoptosis (8, 9, 10, 11). Because lymphocytes produce proinflammatory cytokines and activate macrophages, loss of lymphocytes may be beneficial to survival by down-regulating the excessive inflammatory response (10, 12). Alternatively, loss of lymphocytes in sepsis may be detrimental by impairing the ability of the immune system to combat pathogens (10, 12). In support of the concept that lymphocyte apoptosis is detrimental to host survival are a number of studies showing that patients with sepsis are immunologically impaired. Patients with sepsis become anergic, i.e., have no response to skin testing with Ags derived from microbes to which previous exposure would be expected (positive controls) (13, 14). There is also indirect evidence for an immunodeficient state in patients with sepsis. Studies show that trauma patients develop a decrease in circulating lymphocytes that is maximal at day 3. The lowest number of lymphocytes occurred in those trauma patients who developed infection or death (15). Furthermore, intensive care patients who develop a decreased lymphocyte count for >3 days are at a greatly increased risk of nosocomial sepsis (16).
Importantly, animal studies show that prevention of lymphocyte apoptosis either by overexpression of the antiapoptotic protein Bcl-2 or by administration of drugs that prevent activation of caspases (proteases that are activated in response to proapoptotic stimuli) improve survival in sepsis (17). To gain insight into the potential impact of lymphocyte apoptosis in sepsis, it is essential to determine both the extent of loss and type of lymphocytes that are being affected in the disorder. In the present study, the effect of sepsis on the various lymphocyte subsets, i.e., B cells, CD4 T cells, CD8 cytotoxic cells, and NK cells, was investigated. Defects in these lymphocyte subsets impair specific aspects of the host immune response and predispose to various pathogens. In addition to sepsis, other noxious stimuli such as ischemia/reperfusion or hypoxia can induce apoptosis (18). Therefore, nonseptic patients who were critically ill were also examined. Finally, we examined the role of active caspase-9 in lymphocyte apoptosis in sepsis. Apoptosis can proceed by two mechanistically distinct pathways, i.e., a receptor-mediated pathway that proceeds by activation of caspase-8 or a mitochondrial pathway that proceeds by caspase-9 (18, 19). Knowledge of the precise pathway of apoptosis will help identify stimuli that trigger cell death in sepsis and may allow for a more rational therapeutic approach.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The present work represents in part an extension of a previous study from this laboratory in which we reported the autopsy findings from 15 of the 27 patients with sepsis and 8 of the 25 patients with trauma who are included in the current examination (10). In the previous study, we investigated which of the two mechanisms of cell death (apoptosis or necrosis) occur in a variety of organs (brain, heart, lung, liver, spleen, colon, small intestine, kidney, and muscle) from patients dying of sepsis and multiple organ failure (10). As reported, spleen and colon were the two organs exhibiting the greatest degree of cell death, with apoptosis being the overriding mechanism of death in these organs (10). The aim of the current study was to characterize the immunologic status of septic patients by determining the extent of lymphocyte depletion and by identifying cell phenotypes that might be preferentially affected by apoptosis. In addition to immunohistochemical methods, flow cytometry was used to detect apoptosis and to corroborate immunohistochemical findings.
Spleen sampling in patients with sepsis
The method of obtaining spleen samples was described previously
(10) and is discussed briefly. Spleen samples were
obtained from 24 septic patients rapidly postmortem, whereas spleen
samples were obtained intraoperatively from 3 patients with sepsis
during a procedure to remove spleens with abscess formation. In the
spleen samples obtained postmortem, a protocol for immediate tissue
sampling allowed for tissue harvesting in the intensive care unit as
soon as informed consent could be obtained from next of kin. The spleen
sample was placed in 10% buffered formalin for 24 h. before
paraffin-embedding and sectioning. The length of time between onset of
death and tissue sampling ranged from 15 min to 6 h, with the vast
majority obtained between 30 and 90 min (10). In the three
patients with sepsis whose spleens were removed intraoperatively, the
sample was obtained from a grossly normal appearing section of the
spleen that was not directly contiguous with the site of infection.
Microscopic examination was used to confirm the lack of infectious
organisms and abscess formation in the spleen samples obtained
intraoperatively. Three other septic patients were excluded from the
study because they were taking chronic high doses of immunosuppressive
medication, i.e., corticosteroids and/or cyclosporin. Four patients
with sepsis who were being treated with corticosteroids were included
in the study (see Table I
). Two of these four patients had
corticosteroids initiated in the 24–36 h preceding their death. Low
physiologic doses of corticosteroids were used in the other two (see
Table I).
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Criteria of sepsis
Patients were classified as septic based on one of the three
following criteria: 1) positive blood, abdominal fluid, or tissue
cultures for bacteria or fungi (see Table I
); 2) intraoperative
evidence of infection, e.g., perforated large bowel with peritoneal
contamination, ischemic bowel with purulent peritoneal fluid; 3) a
histopathologic diagnosis of infection at postmortem examination (e.g.
bronchopneumonia, intraabdominal abscess). All patients also had
premortem clinical evidence of sepsis with signs and symptoms of sepsis
consisting of hypo- or hyperthermia, altered mental status, and
hemodynamic instability usually requiring vasopressors (see Table I).
Critically ill nonseptic patients
Spleens were examined from 16 patients who were critically ill
from a variety of nonseptic causes (Table II). Three patients were
examined prospectively, and 13 patients were examined retrospectively.
The 13 patients who were examined retrospectively were selected from
the autopsy files at Barnes Jewish Hospital. Patients were selected on
the following criteria: 1) no high clinical suspicion of sepsis and no
autopsy evidence of infection; 2) hospitalization in the medical or
surgical intensive care units; 3) no evidence of immunosuppression and
no immunosuppressive medication; 4) autopsy performed within 24 h
after death. We selected a time limit of 24 h for the postmortem
examination because previous studies from our laboratory demonstrated
adequate preservation of cellular morphology (without autolysis) and no
increase in histologic evidence of apoptosis (via light microscopic
examination) in spleens that were maintained at room temperature and
sampled at consecutive time points for 24 h (10).
Additional studies from our laboratory have shown no effect of a
24-h delay in tissue fixation on cell surface marker (CD) staining
(our unpublished observations). In the present study, the duration of
time between death and autopsy ranged from 0.5 to 21 h with a mean
delay of 12.5 h.
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Due to the inability of obtaining normal human spleens, patients with blunt or penetrating abdominal trauma necessitating a splenectomy were used as a control population for comparison with the patients with sepsis. None of the trauma patients had significant comorbidities or was taking immunosuppressive medication; presumably, findings from this group are representative of those of a normal population. Spleens were removed rapidly after injury (usually within 3–8 h); therefore, values for the lymphocyte subsets should reflect those of a normal spleen.
Immunohistochemical staining for cell surface markers
An abbreviated description of the methods is provided. The complete description of immunohistochemical staining protocols is available on the website: http://elysium.wustl.edu/rhlab/
Abs against human CD3, CD8, CD20, and NK cell-like were from Dako (Carpinteria, CA). The mouse monoclonal anti-human CD4 Ab was from Vector (Burlingame, CA). Slides were heated and rinsed in Hemo De (Fisher Scientific, St. Louis, MO) and rehydrated. Endogenous peroxidase activity was blocked and Ag retrieval performed with Dako solution. CD4 Ag retrieval required microwave treatment in 1 mM EDTA, pH 8.0.
Anti-human CD3, CD8, and CD20 Abs were prediluted by the manufacturer and applied to sections. Next, HRP polymer (Dako EnVision) was added, and slides were developed with 3,3'-diaminobenzidine tetrahydrochloride (DAB).
Incubation with mouse anti-human NK cell-like Ab, diluted 1:75 in PBS, was done for 1 h after blocking with 10% rabbit serum in PBS. Slides were rinsed, incubated with a biotinylated rabbit anti-mouse IgM, rinsed, and incubated with an avidin-biotin peroxidase complex (VectaStain ABC Elite; Vector).
For CD4, primary Ab was diluted 1:75 and incubated for 1 h. After rinsing, secondary Ab (biotinyl-horse anti-mouse IgG; Vector) was added. Slides were rinsed, incubated with ABC, and developed with DAB.
Evaluation and image analysis of immunohistochemical stains
Slides were examined in a blinded fashion at 2x magnification to include as much of the tissue specimen for study as possible. Images were obtained using a Nikon (Melville, NY) COOLPIX 900 digital camera.
B cells and lymphoid follicles. The area of the spleen positively stained for B cells (CD20) was calculated using Image Pro (Media Cybernetics, Silver Spring, MD). Because expansion of B cell zones in splenic white pulp occurs in response to antigenic challenge, the number and area of the lymphoid follicles (which comprise the white pulp) are indications of the immunologic response/competence of the host. The number of lymphoid follicles was calculated and expressed per square centimeter of the spleen sample. The total area of the sample (red and white pulp) occupied by CD20-positive lymphoid tissue was also calculated.
CD4, CD8, CD3, and NK cells. The area of the spleen staining positive for CD4, CD8, CD3 or NK cells was calculated using Metamorph (Universal Imaging, West Chester, PA).
Immunohistochemical staining for active caspase-9
The affinity-purified rabbit anti-active caspase-9 Ab was a gift from Dr. Donald Nicholson (Merck Frosst Labs, Point Claire, Quebec, Canada). Briefly, tissues were deparaffinized, rehydrated, incubated in 3% H2O2, and rinsed. Ag retrieval was done in 0.1 M citrate buffer (pH 6.0) which was brought to a boil in a microwave oven. The tissues were blocked with a nonimmune serum (Zymed, San Francisco, CA). Primary Ab was diluted 1:1000 in PBS and incubated for 1 h at 37°C. Secondary Ab was added (Zymed). After incubation and washes, streptavidin complex was added (Zymed). Tissues were developed with metal-enhanced DAB (Pierce, Rockford, IL).
Flow cytometry of splenocytes from patients with sepsis or trauma
In addition to immunohistochemistry, spleens from five septic
and six trauma patients were examined by flow cytometry (20, 21). Two of the five spleens included in the septic group were
obtained intraoperatively during splenectomy; the three remaining
spleens were obtained postmortem. Spleens from the six trauma patients
were obtained intraoperatively. At the time of tissue harvesting, a
small piece of spleen (
250–500 mg.) was placed in PBS containing
glutamine (2 mM), glucose (10 mM), and 1% FCS. The tissue
section was promptly prepared for flow cytometry as previously
described (20, 21).
Generally, flow cytometry was performed within 4–8 h after obtaining tissue, and in no instances did the delay in examination exceed 24 h. Previous studies showed no differences in the degree of apoptosis in splenocytes examined immediately vs 24 h later providing that the splenocytes had been dissociated and stored at 4–8°C in appropriate enriched buffer as described above (our unpublished observations). Apoptosis was quantified using a commercially available fluorescein-labeled annexin V/propidium iodide kit (Apoptosis Detection Kit; R&D Systems, Minneapolis, MN) as described previously (20, 21). The various lymphocyte phenotypes were identified using fluorescently labeled mAbs directed against lymphocyte surface markers (PharMingen, San Diego, CA): B cells, CD20; CD4 T cells; CD8 T cells; CD3 T cells; NK cells.
Statistical analysis
Data are means ± SEM. Data were analyzed with a statistical software program, Prism (GraphPad Software, San Diego, CA). Data involving two groups only were analyzed by Student’s t test, whereas data involving more than two groups were analyzed using one-way ANOVA with Tukey’s multiple comparison test. Significance was accepted at p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Pertinent clinical and laboratory findings for the septic and
critically ill nonseptic patients are presented in Tables I and II,
respectively. With two exceptions (septic patient 16 and
critically ill nonseptic patient 10), the patients died. Twenty-three
of the 27 septic patients and 11 of 16 critically ill nonseptic
patients had a documented period of shock in which mean arterial
pressure was <60 mm Hg and/or vasopressor therapy (dopamine,
phenylephrine, and/or norepinephrine) was needed to maintain mean
arterial pressure of >60 mm Hg. (The time period immediately before
the patient’s demise (generally 30–90 min) was not included in the
assessment of mean arterial blood pressure.)
Patient circulating lymphocyte counts
Most of the septic patients had a persistently low absolute
lymphocyte count during their illness (data not shown). Values for the
lowest absolute lymphocyte count of the patients during the 48 h
preceding spleen harvesting are presented in Tables I and II.
Twenty-two of the 26 septic patients and 11 of 16 critically ill
nonseptic patients had an absolute lymphocyte count of
<1200/mm3 (the lower limit of normal at
Barnes Jewish Hospital) (Tables I and II).
Immunohistochemical staining
B cells (CD20). Several parameters were used to evaluate the effect of the disorders on B cells: 1) the total area of spleen (red and white pulp) occupied by B cells was determined; 2) the area of the spleen occupied by lymphoid follicles and the number of lymphoid follicles per cm2 of spleen were calculated. These two determinations provide complementary data on the effect of sepsis or critical illness on B cells.
Evaluation of spleens from septic patients showed a marked loss of B
cells compared with spleens from both trauma patients and critically
ill nonseptic patients (
Figs. 1–3; Table III). In many cases, spleens from septic
patients could be distinguished from spleens from trauma patients by
gross visual examination, i.e., with the naked eye, of the microscopic
slide (Fig. 1). There was a marked loss in the number and size of dark
stained foci representing the lymphoid follicles (Fig. 1). Microscopic
examination confirmed the gross visual examination and demonstrated a
43% decrease in area of lymphoid follicles (white pulp) and a 38%
decrease in total B cell area (red and white pulp) in septic vs trauma
patients (Figs. 2 and 3 and Table III).
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and Table III), there were individual
critically ill nonseptic patients who did demonstrate obvious decreases
in B cells (Fig. 4B).
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). As shown in Fig. 5A, there was a more profound
loss in B cells with increased duration of sepsis; compared with trauma
patients, there was a 25 and a 52% decrease in B cell area in patients
with sepsis for <7 and 
7 days, respectively
(p < 0.05 and p < 0.001,
respectively). The decrease in B cell area at 7 days was greater than
the decrease in B cell area at <7 days, p < 0.05
(Fig. 5A). In addition to the decrease in total B cell area,
sepsis caused a quantitatively similar progressive decrease in the area
of lymphoid follicles (Fig. 5A). Finally, the number of
lymphoid follicles was decreased in patients with sepsis by 40%
compared with trauma patients, and this difference did not change with
duration of sepsis, i.e., 101.6 ± 9.2, 58.7 ± 6.9, and
61.6 ± 11.6 lymphoid follicles/cm2 for
trauma patients, patients with sepsis for <7 days, and patients with
sepsis for 7 days, respectively (p < 0.01
and p < 0.05, respectively).
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, C and
D, and 7 and Table III). The
loss in CD4 T cells also was examined for the effect of duration of
sepsis. Compared with trauma patients, there was a 34% and a 46%
decrease in CD4 T cells in patients with sepsis for <7 days and 7
days, respectively (Fig. 5B; p < 0.001 for
both). However, there was no difference in percentage area of CD4 T
cells in patients in sepsis for <7 vs 7 days (Fig. 5B).
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). Similar to the B cell data,
individual critically ill nonseptic patients exhibited markedly
decreased CD4 T cell staining (Fig. 4, C and
D).
CD8 T cells and NK cells.
In contrast to the decrease in B cells and CD4 T cells, septic patients
had a 60% increase in CD8 T cells compared with trauma patients
(p < 0.05) (Table III). Similarly, critically
ill nonseptic patients had a 130% increase in CD8 T cells vs trauma,
(p < 0.05). There was a 44% increase in NK
cells in septic vs trauma patients, but this difference did not achieve
significance (p > 0.05) (Table III).
Interestingly, the CD4:CD8 ratio, a measure of immune status, was
decreased from 3:1 in trauma patients to 1:1 in septic patients
(p < 0.01 (Table III)).
CD3 T cells.
The CD3 surface marker is associated with the TCR in both CD4 and CD8 T
cells. In spleen, therefore, CD3 staining can be used to
corroborate CD4 and CD8 immunohistochemical findings, because the
number of CD3-positive T cells is an approximation of the sum of CD4
and CD8 T cells. In the present study, the sum of the areas of CD4 and
CD8 approximately equaled the area of CD3 in all three groups of
patients (Fig. 7 and Table III). Patients with sepsis had a 35%
decrease in the CD3 area compared with the critically ill nonseptic
patients (Fig. 7; p < 0.05).
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Previously, our laboratory has shown that patients with sepsis
(including some of the patients in the present study) have a
significant increase in caspase-3-mediated lymphocyte apoptosis in
spleen and other organs (10). In the former and current
studies, the characteristic morphologic changes of apoptosis, i.e.,
condensed and compacted nuclei with apoptotic bodies, were seen in
routine hematoxylin and eosin (H&E) stains of spleens from septic
patients (Fig. 8). In the present study,
slides were evaluated in a blinded manner at x400. With a photomask
eyepiece to overlay the area of interest, the number of mantle and
marginal cells in splenic white pulp that were positive for active
caspase-9 within the specified area (0.0072 mm2)
were counted.
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D). The number of cells that
were positive for active caspase-9 were 41.6 ± 15.4, 32.9 ±
15.4, and 5.8 ± 3.3 for critically ill nonseptic, septic, and
trauma patients, respectively. Both critically ill nonseptic and septic
patients were statistically different from trauma patients
(p < 0.05) but not from each other.
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Lymphocyte cell typing and quantitation of apoptosis by flow cytometry
No spleens were available from critically ill nonseptic
patients; therefore, only septic and trauma patients were compared. The
findings from flow cytometry closely paralleled the results from the
immunohistochemical staining studies. There was a 42% loss in B cells
and a 45% loss in CD4 T cells in septic patients compared with trauma
patients, p < 0.001 and p < 0.01,
respectively (Fig. 10A). In
contrast to the decrease in CD4 T cells and B cells, there was a 57%
increase in NK cells in patients with sepsis vs those with trauma,
p < 0.02. There was no difference in the percentage of
CD8 T cells in septic vs trauma patients (Fig. 10A). The percentage of
CD3 T cells was decreased by 38% in septic patients compared with
trauma patients, p < 0.003.
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B and
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). During
infection, B cells do not migrate to the site of pathogens but rather
proliferate in spleen and other organized lymphoid tissues and release
Abs that are transported to the focus of disease (22, 23).
Therefore, the decrease in B cells observed in the spleens of septic
patients has particular importance. The loss in CD4 T cells in septic
patients was also striking (Fig. 6). The loss in lymphocytes (both B
cells and CD4 T cells) is even more significant considering that it
occurs in the context of overwhelming infection, a condition in which
massive clonal expansion of B and T lymphocytes should be
occurring.
A hallmark of sepsis is immunosuppression that is characterized by loss
of delayed-type hypersensitivity, failure to eradicate a primary
infection, and propensity to acquire new secondary infections (2, 3). The documented loss in CD4 T and B cells (rather than the
anticipated increase in lymphocytes) may be contributing to
the immunosuppression by decreasing the number of immune cells
available to combat infection. In addition to causing a decrease in the
number of immune effector cells, lymphocyte apoptosis may contribute to
immunosuppression by other mechanisms as well. Recent research has
shown that apoptotic cells actively suppress the inflammatory response
(24, 25, 26). Voll et al. (24) demonstrated that
addition of apoptotic lymphocytes to endotoxin-stimulated PBMC caused a
shift from secretion of proinflammatory cytokines (TNF-
, IL-1
,
and IL-12) to antiinflammatory cytokines (IL-10). Barker et al.
(25) showed that macrophages that ingested apoptotic cells
had increased production of TGF-1, a potent
immunosuppressant and antiinflammatory cytokine. Fadok et al.
(26) reported that uptake of apoptotic vs necrotic cells
caused the macrophage to respond in an antiinflammatory or
proinflammatory manner, respectively.
Critically ill nonseptic patients
Although the critically ill nonseptic patients considered as a
group did not demonstrate loss of lymphocytes compared with the control
population (trauma patients), it was clear that certain individual
critically ill nonseptic patients did have significant loss of
lymphocytes (Fig. 4, A and B). The increase in
active caspase-9 in critically ill nonseptic patients relative to
trauma patients demonstrates that apoptosis of lymphocytes is occurring
and will result in cell loss. It is likely that studies examining
larger numbers of these patients would disclose subsets of critically
ill nonseptic patients, e.g., liver failure, renal failure, congestive
heart failure, who did have loss of lymphocytes that are comparable to
that in the septic patients. As indicated in septic patients (Fig. 5),
it is likely that the longer the duration of illness in the critically
ill nonseptic patient, the more likely there is to be loss of
lymphocytes. In this regard, the critically ill nonseptic patient who
had the most extensive loss of B cells (Fig. 4) was also the patient
who had the longest stay in the intensive care unit before death (8
days).
Although both critically ill nonseptic and septic patients had
comparable increases in the number of cells that were positive for
active caspase-9, many spleens of septic patients had a decreased
number of lymphocytes in the lymphoid follicle; i.e., there was a
decreased density of cells per unit area (see Fig. 9B). This
finding of depletion of splenic lymphocytes per unit area was reported
previously (10). It is likely that many of the lymphocytes
of septic patients had already undergone caspase-9-mediated apoptosis,
and this may have been a factor in the lack of difference in septic vs
critically ill nonseptic patients. Studies from our laboratory show
that there is a marked decrease in active caspase-9-positive cells in
patients who were septic for prolonged periods of time vs patients who
were septic for brief periods (data not shown).
Lymphocyte apoptosis: a key pathophysiologic mechanism in sepsis?
The present results establish an association between decreased lymphocytes and sepsis but do not establish causality between lymphocyte apoptosis and outcome in patients with sepsis. However, a growing body of animal studies supports the hypothesis that a major pathophysiologic mechanism in sepsis is lymphocyte apoptosis (8, 9, 10, 11, 27). Recently, Braun et al. (28) showed that administration of caspase inhibitors, drugs that block apoptosis, provided excellent neuroprotection in a rabbit model of pneumococcal meningitis. Ayala et al. (29) reported that septic mice deficient in FasLgld had decreased mucosal B lymphocyte apoptosis and improved survival compared with controls. Our laboratory has reported that prevention of lymphocyte apoptosis by a variety of compounds that inhibit caspases improves survival in a peritonitis animal model of sepsis (17, 20, 30). In addition to the efficacy of caspase inhibitors, mice in which the antiapoptotic protein Bcl-2 is overexpressed in T or B lymphocytes have decreased blood bacterial counts and improved survival in sepsis (17, 20, 30). Furthermore, adoptive transfer of T cells that overexpress Bcl-2 into nontransgenic mice improves survival in sepsis (30). Finally, caspase inhibition has also been reported to improve survival in an endotoxin model as well (31). Considered together, these studies provide strong supporting evidence in animal models that loss of lymphocytes in sepsis is a central pathogenic event.
Ischemia/reperfusion injury and other potential variables in sepsis-induced apoptosis
There are numerous metabolic and physiologic changes
in critically ill patients that may be confounding factors in the
sepsis-induced lymphocyte apoptosis. For example, the majority of
patients with severe sepsis have episodes of ischemia/reperfusion
injury and, in the present study, 23 of the 27 septic patients
required vasopressor therapy to maintain an adequate mean
arterial blood pressure (Table I). Animal studies and some human
data indicate that ischemia/reperfusion injury can induce
apoptosis in the gastrointestinal tract (32), heart
(33), kidney (34), and brain
(35).
Four of the patients with sepsis were receiving corticosteroids (see
Table I). It is possible that the corticosteroids contributed to
lymphocyte apoptosis. However, the dose of corticosteroids in two of
four septic patients (patients 23 and 27) was low (20–25 mg
hydrocortisone every 8 h) and much less than can be produced by
the body under severe stress. Furthermore, it is significant that all
four of the septic patients receiving corticosteroids were in the group
of patients who had sepsis for <7days. These patients had less loss of
B cells and CD4+ T cells than patients who were
septic for > 7 days.
Pathways of lymphocyte apoptosis in sepsis
There are two major pathways involved in initiation of apoptosis, i.e., a receptor initiated caspase-8-mediated pathway and a mitochondrial-initiated caspase-9-mediated pathway (18, 19). Activation of caspase-8 or caspase-9 subsequently leads to activation of caspase-3, an effector caspase that is in the final common pathway of the cell death program. Caspase-8 can be activated by a number of ligands including TNF and Fas (18, 36). The mitochondrial-mediated pathway can be activated by a diverse number of stimuli including, e.g., reactive oxygen species (36). Understanding the particular pathway of sepsis-induced apoptosis is important because it provides insight into potential factors responsible for initiating cell suicide and may allow for a more targeted therapy. The present studies showing activated caspase-9 in apoptotic lymphocytes of septic patients supports a mitochondrial-mediated pathway. Although demonstrating that caspase-9 is activated provides supporting evidence for a mitochondrial-initiated pathway, it is not conclusive proof because of recent studies which show that, in some instances, there is "cross-talk" between the various caspases (19, 36). However, a great deal of data in animal models of sepsis are consistent with the concept that sepsis-induced lymphocyte apoptosis proceeds by the mitochondria-initiated pathway. Sepsis-induced thymocyte and splenocyte apoptosis is not blocked in FasR-deficient mice (21) or in TNF p55 or TNF p75 receptor-deficient mice (our unpublished observations). Although controversial, most recent studies show that Bcl-2 inhibits the mitochondrial but not the receptor-mediated apoptosis pathway (19). Therefore, the published studies noting that overexpression of Bcl-2 in T or B cells prevents lymphocyte apoptosis in sepsis (17, 20) is also consistent with a mitochondrial-mediated death pathway in the disorder.
Other potential mechanisms of immunosuppression in sepsis
Although we speculate that sepsis-induced lymphocyte apoptosis is a central pathogenic event, it is possible that other mechanisms are involved in the immunosuppression that characterizes the disorder (7, 37). A large body of studies indicates that critically ill patients with trauma are anergic and have multiple defects in immune function (11, 37, 38, 39, 40, 41). Pellegrini et al. (39) demonstrated that T cells from trauma patients underwent apoptosis at an accelerated rate compared with normal volunteers. However, T cell anergy did not appear to correlate with lymphocyte apoptosis (39). Lederer et al. (38) and Wichman et al. (40) have shown that major injury and sepsis induce increased production of IL-10, a counterinflammatory cytokine that impairs resistance to infection and/or decreases the ability to combat infection. Puyana et al. (41) reported that both Th1- and Th2-type lymphokines are depressed in posttrauma anergy. Lyons et al. (42) noted that major injury induced increased production of IL-10 and thereby impaired resistance to infection. Thus, lymphocyte apoptosis may be only one of the many factors that are involved in compromising host defense.
Limitations and alternative hypotheses
The major focus of the present study was to determine the effect of sepsis on the various lymphocyte phenotypes. The best way to determine whether sepsis is having an effect is to compare spleens from a "normal" population. Because it is not possible to obtain spleens from healthy normal persons, we elected to use spleens removed acutely from patients who had no comorbidities but who had trauma to the spleen necessitating a splenectomy. Other than the fact that their spleens were acutely fractured and bleeding, the cellular composition and architecture should be normal (except for the area of injury).
A possible concern is whether the relatively longer delay in spleen
fixation in septic patients vs trauma patients (15-min to 6-h delay in
septic patients vs 5- to 30-min delay in trauma patients; see
Materials and Methods) could be responsible for some of the
experimental differences. Data suggest that the differences in time of
spleen fixation did not play a role in the results. Previous work from
our laboratory did compare spleens of septic patients to spleens
obtained from a group of patients after sudden cardiovascular death and
whose tissues were not obtained until 3–12 h postmortem
(10). Microscopic examination of H&E tissue sections from
these control spleens showed normal splenic architecture, no lymphocyte
depletion, and little evidence of apoptosis (10). In a
previous study, we showed that immunohistochemical staining for active
caspase-3, a key cell death protease, was also significantly less in
spleens from control vs septic patients (10). In the
present study, we demonstrated that active caspase-9 was not effected
by delay in tissue fixation (see Results on caspase-9).
Also, in the present study, spleens removed intraoperatively from three
septic patients (and formalin fixed immediately) exhibited some of the
most remarkable loss of lymphocytes (Fig. 1F). Finally, the
changes in lymphocyte phenotypes observed in spleens from septic
patients were not observed in critically ill nonseptic patients even
though the delay in spleen fixation was longer in the latter group.
A final limitation concerns interpretation of the effect of lymphocyte apoptosis on host survival in septic patients. Although we speculate that loss of lymphocytes is detrimental to survival in septic patients because of the resultant immunosuppression, it is possible that lymphocyte apoptosis may have beneficial effects (12). Apoptosis of lymphocytes may lead to decreased production of proinflammatory cytokines, which induce or contribute to the systemic inflammatory response syndrome and organ injury in sepsis. A recent study of Pseudomonas aeruginosa pneumonia showed that apoptosis of lung epithelial cells (lymphocyte apoptosis was not evaluated) helped to limit the spread of the infection systemically and was essential for survival (43).
The present results show that sepsis rather than inducing lymphocyte proliferation causes a profound and progressive decrease in B cells and CD4 T cells. It is possible that this loss in B cells and CD4 T lymphocytes impairs the ability of the patient to eradicate the infection and predisposes to other invading pathogens. If immunosuppression resulting from loss of lymphocytes is determined to be a key factor in patient survival in sepsis, therapy with caspase inhibitors (which have shown remarkable success in clinically relevant animal models of sepsis) (17, 30) may represent a novel approach in the treatment of this highly lethal disorder.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Richard S. Hotchkiss, Department of Anesthesiology, 660 South Euclid, St. Louis, MO 63110. E-mail address: hotch{at}morpheus.wustl.edu
3 Abbreviations used in this paper: DAB, 3,3'-diaminobenzidine tetrahydrochloride; H&E, hematoxylin and eosin.
Received for publication January 31, 2001. Accepted for publication March 23, 2001.
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  A. G. Cabrera, U. Dyamenahalli, J. Gossett, P. Prodhan, W. R. Morrow, M. Imamura, R. D.B. Jaquiss, and A. T. Bhutta Preoperative lymphopenia is a predictor of postoperative adverse outcomes in children with congenital heart disease J. Thorac. Cardiovasc. Surg., November 1, 2009; 138(5): 1172 - 1179. [Abstract] [Full Text] [PDF]
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 R. S. Hotchkiss, A. Strasser, J. E. McDunn, and P. E. Swanson Cell Death N. Engl. J. Med., October 15, 2009; 361(16): 1570 - 1583. [Full Text] [PDF]
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 J. Sharma, Q. Li, B. B. Mishra, M. J. Georges, and J. M. Teale Vaccination with an Attenuated Strain of Francisella novicida Prevents T-Cell Depletion and Protects Mice Infected with the Wild-Type Strain from Severe Sepsis Infect. Immun., October 1, 2009; 77(10): 4314 - 4326. [Abstract] [Full Text] [PDF]
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 J. Sharma, Q. Li, B. B. Mishra, C. Pena, and J. M. Teale Lethal pulmonary infection with Francisella novicida is associated with severe sepsis J. Leukoc. Biol., September 1, 2009; 86(3): 491 - 504. [Abstract] [Full Text] [PDF]
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J. Unsinger, J. S. McDonough, L. D. Shultz, T. A. Ferguson, and R. S. Hotchkiss Sepsis-induced human lymphocyte apoptosis and cytokine production in "humanized" mice J. Leukoc. Biol., August 1, 2009; 86(2): 219 - 227. [Abstract] [Full Text] [PDF]
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 J. Carcillo, R. Holubkov, J. M. Dean, J. Berger, K. L. Meert, K. J. S. Anand, J. Zimmerman, C. J. L. Newth, R. Harrison, D. F. Willson, et al. Rationale and Design of the Pediatric Critical Illness Stress-Induced Immune Suppression (CRISIS) Prevention Trial JPEN J Parenter Enteral Nutr, July 1, 2009; 33(4): 368 - 374. [Abstract] [Full Text] [PDF]
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 P. E. Marik Critical Illness-Related Corticosteroid Insufficiency Chest, January 1, 2009; 135(1): 181 - 193. [Abstract] [Full Text] [PDF]
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 H. Komura, M. Miksa, R. Wu, S. M. Goyert, and P. Wang Milk Fat Globule Epidermal Growth Factor-Factor VIII Is Down-Regulated in Sepsis via the Lipopolysaccharide-CD14 Pathway J. Immunol., January 1, 2009; 182(1): 581 - 587. [Abstract] [Full Text] [PDF]
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F. Pene, B. Zuber, E. Courtine, C. Rousseau, F. Ouaaz, J. Toubiana, A. Tazi, J.-P. Mira, and J.-D. Chiche Dendritic Cells Modulate Lung Response to Pseudomonas aeruginosa in a Murine Model of Sepsis-Induced Immune Dysfunction J. Immunol., December 15, 2008; 181(12): 8513 - 8520. [Abstract] [Full Text] [PDF]
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 A. Sinistro, C. Almerighi, C. Ciaprini, S. Natoli, E. Sussarello, S. Di Fino, F. Calo-Carducci, G. Rocchi, and A. Bergamini Downregulation of CD40 Ligand Response in Monocytes from Sepsis Patients Clin. Vaccine Immunol., December 1, 2008; 15(12): 1851 - 1858. [Abstract] [Full Text] [PDF]
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A. L. Gruver and G. D. Sempowski Cytokines, leptin, and stress-induced thymic atrophy J. Leukoc. Biol., October 1, 2008; 84(4): 915 - 923. [Abstract] [Full Text] [PDF]
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 J. L. Wynn, P. O. Scumpia, R. D. Winfield, M. J. Delano, K. Kelly-Scumpia, T. Barker, R. Ungaro, O. Levy, and L. L. Moldawer Defective innate immunity predisposes murine neonates to poor sepsis outcome but is reversed by TLR agonists Blood, September 1, 2008; 112(5): 1750 - 1758. [Abstract] [Full Text] [PDF]
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A. Pachot, M.-A. Cazalis, F. Venet, F. Turrel, C. Faudot, N. Voirin, J. Diasparra, N. Bourgoin, F. Poitevin, B. Mougin, et al. Decreased Expression of the Fractalkine Receptor CX3CR1 on Circulating Monocytes as New Feature of Sepsis-Induced Immunosuppression J. Immunol., May 1, 2008; 180(9): 6421 - 6429. [Abstract] [Full Text] [PDF]
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 H. Yasuda, A. Leelahavanichkul, S. Tsunoda, J. W. Dear, Y. Takahashi, S. Ito, X. Hu, H. Zhou, K. Doi, R. Childs, et al. Chloroquine and inhibition of Toll-like receptor 9 protect from sepsis-induced acute kidney injury Am J Physiol Renal Physiol, May 1, 2008; 294(5): F1050 - F1058. [Abstract] [Full Text] [PDF]
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O. M. Peck-Palmer, J. Unsinger, K. C. Chang, C. G. Davis, J. E. McDunn, and R. S. Hotchkiss Deletion of MyD88 markedly attenuates sepsis-induced T and B lymphocyte apoptosis but worsens survival J. Leukoc. Biol., April 1, 2008; 83(4): 1009 - 1018. [Abstract] [Full Text] [PDF]
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J. Tschop, A. Martignoni, H. S. Goetzman, L. G. Choi, Q. Wang, J. G. Noel, C. K. Ogle, T. A. Pritts, J. A. Johannigman, A. B. Lentsch, et al. {gamma}{delta} T cells mitigate the organ injury and mortality of sepsis J. Leukoc. Biol., March 1, 2008; 83(3): 581 - 588. [Abstract] [Full Text] [PDF]
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H. V. Groesdonk, F. Wagner, B. Hoffarth, M. Georgieff, and U. Senftleben Enhancement of NF-{kappa}B Activation in Lymphocytes Prevents T Cell Apoptosis and Improves Survival in Murine Sepsis J. Immunol., December 15, 2007; 179(12): 8083 - 8089. [Abstract] [Full Text] [PDF]
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 K. Takahashi, S. Satoi, H. Yanagimoto, N. Terakawa, H. Toyokawa, T. Yamamoto, Y. Matsui, S. Takai, A-H. Kwon, and Y. Kamiyama Circulating Dendritic Cells and Development of Septic Complications After Pancreatectomy for Pancreatic Cancer Arch Surg, December 1, 2007; 142(12): 1151 - 1157. [Abstract] [Full Text] [PDF]
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T. Tsuchiya, E. Mitsuo, N. Hayashi, Y. Hikita, H. Nakao, S. Yamamoto, K. Miyamoto, and H. Tsujibo Vibrio vulnificus Damages Macrophages during the Early Phase of Infection Infect. Immun., September 1, 2007; 75(9): 4592 - 4596. [Abstract] [Full Text] [PDF]
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 M. J. Delano, P. O. Scumpia, J. S. Weinstein, D. Coco, S. Nagaraj, K. M. Kelly-Scumpia, K. A. O'Malley, J. L. Wynn, S. Antonenko, S. Z. Al-Quran, et al. MyD88-dependent expansion of an immature GR-1+CD11b+ population induces T cell suppression and Th2 polarization in sepsis J. Exp. Med., June 11, 2007; 204(6): 1463 - 1474. [Abstract] [Full Text] [PDF]
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 A. Viardot, S. T. Grey, F. Mackay, and D. Chisholm Potential Antiinflammatory Role of Insulin via the Preferential Polarization of Effector T Cells toward a T Helper 2 Phenotype Endocrinology, January 1, 2007; 148(1): 346 - 353. [Abstract] [Full Text] [PDF]
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P. O. Scumpia, M. J. Delano, K. M. Kelly, K. A. O'Malley, P. A. Efron, P. F. McAuliffe, T. Brusko, R. Ungaro, T. Barker, J. L. Wynn, et al. Increased Natural CD4+CD25+ Regulatory T Cells and Their Suppressor Activity Do Not Contribute to Mortality in Murine Polymicrobial Sepsis J. Immunol., December 1, 2006; 177(11): 7943 - 7949. [Abstract] [Full Text] [PDF]
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J. Unsinger, J. M. Herndon, C. G. Davis, J. T. Muenzer, R. S. Hotchkiss, and T. A. Ferguson The Role of TCR Engagement and Activation-Induced Cell Death in Sepsis-Induced T Cell Apoptosis J. Immunol., December 1, 2006; 177(11): 7968 - 7973. [Abstract] [Full Text] [PDF]
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 A. Sarkar, M. W. Hall, M. Exline, J. Hart, N. Knatz, N. T. Gatson, and M. D. Wewers Caspase-1 Regulates Escherichia coli Sepsis and Splenic B Cell Apoptosis Independently of Interleukin-1beta and Interleukin-18 Am. J. Respir. Crit. Care Med., November 1, 2006; 174(9): 1003 - 1010. [Abstract] [Full Text] [PDF]
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J. A. Russell Management of Sepsis N. Engl. J. Med., October 19, 2006; 355(16): 1699 - 1713. [Full Text] [PDF]
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 K. Laudanski, C. Miller-Graziano, W. Xiao, M. N. Mindrinos, D. R. Richards, A. De, L. L. Moldawer, R. V. Maier, P. Bankey, H. V. Baker, et al. Cell-specific expression and pathway analyses reveal alterations in trauma-related human T cell and monocyte pathways PNAS, October 17, 2006; 103(42): 15564 - 15569. [Abstract] [Full Text] [PDF]
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G. Suntharalingam, M. R. Perry, S. Ward, S. J. Brett, A. Castello-Cortes, M. D. Brunner, and N. Panoskaltsis Cytokine Storm in a Phase 1 Trial of the Anti-CD28 Monoclonal Antibody TGN1412 N. Engl. J. Med., September 7, 2006; 355(10): 1018 - 1028. [Abstract] [Full Text] [PDF]
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H. Xiao, J. Siddiqui, and D. G. Remick Mechanisms of Mortality in Early and Late Sepsis Infect. Immun., September 1, 2006; 74(9): 5227 - 5235. [Abstract] [Full Text] [PDF]
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S. J. Schwulst, M. H. Grayson, P. J. DiPasco, C. G. Davis, T. S. Brahmbhatt, T. A. Ferguson, and R. S. Hotchkiss Agonistic Monoclonal Antibody Against CD40 Receptor Decreases Lymphocyte Apoptosis and Improves Survival in Sepsis J. Immunol., July 1, 2006; 177(1): 557 - 565. [Abstract] [Full Text] [PDF]
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Y.-E. Claessens, M. Fontenay, F. Pene, J.-D. Chiche, M. Guesnu, C. Hababou, N. Casadevall, J.-F. Dhainaut, J.-P. Mira, and A. Cariou Erythropoiesis Abnormalities Contribute to Early-Onset Anemia in Patients with Septic Shock Am. J. Respir. Crit. Care Med., July 1, 2006; 174(1): 51 - 57. [Abstract] [Full Text] [PDF]
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R. S. Hotchkiss, K. W. McConnell, K. Bullok, C. G. Davis, K. C. Chang, S. J. Schwulst, J. C. Dunne, G. P. H. Dietz, M. Bahr, J. E. McDunn, et al. TAT-BH4 and TAT-Bcl-xL Peptides Protect against Sepsis-Induced Lymphocyte Apoptosis In Vivo J. Immunol., May 1, 2006; 176(9): 5471 - 5477. [Abstract] [Full Text] [PDF]
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S. B. Flohe, H. Agrawal, D. Schmitz, M. Gertz, S. Flohe, and F. U. Schade Dendritic cells during polymicrobial sepsis rapidly mature but fail to initiate a protective Th1-type immune response J. Leukoc. Biol., March 1, 2006; 79(3): 473 - 481. [Abstract] [Full Text] [PDF]
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M. Soller, A. Tautenhahn, B. Brune, K. Zacharowski, S. John, H. Link, and A. von Knethen Peroxisome proliferator-activated receptor {gamma} contributes to T lymphocyte apoptosis during sepsis J. Leukoc. Biol., January 1, 2006; 79(1): 235 - 243. [Abstract] [Full Text] [PDF]
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 R. G. Wunderink Nosocomial Pneumonia, Including Ventilator-associatedPneumonia Proceedings of the ATS, December 1, 2005; 2(5): 440 - 444. [Abstract] [Full Text] [PDF]
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 J.-M. Cavaillon, C. Adrie, C. Fitting, and M. Adib-Conquy Reprogramming of circulatory cells in sepsis and SIRS Innate Immunity, October 1, 2005; 11(5): 311 - 320. [Abstract] [PDF]
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 A. Tefferi, C. A. Hanson, and D. J. Inwards How to Interpret and Pursue an Abnormal Complete Blood Cell Count in Adults Mayo Clin. Proc., July 1, 2005; 80(7): 923 - 936. [Abstract] [PDF]
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R. S. Hotchkiss, S. B. Osmon, K. C. Chang, T. H. Wagner, C. M. Coopersmith, and I. E. Karl Accelerated Lymphocyte Death in Sepsis Occurs by both the Death Receptor and Mitochondrial Pathways J. Immunol., April 15, 2005; 174(8): 5110 - 5118. [Abstract] [Full Text] [PDF]
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B. C. Urban, T. T. Hien, N. P. Day, N. H. Phu, R. Roberts, E. Pongponratn, M. Jones, N. T. H. Mai, D. Bethell, G. D. H. Turner, et al. Fatal Plasmodium falciparum Malaria Causes Specific Patterns of Splenic Architectural Disorganization Infect. Immun., April 1, 2005; 73(4): 1986 - 1994. [Abstract] [Full Text] [PDF]
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K. A. Felmet, M. W. Hall, R. S. B. Clark, R. Jaffe, and J. A. Carcillo Prolonged Lymphopenia, Lymphoid Depletion, and Hypoprolactinemia in Children with Nosocomial Sepsis and Multiple Organ Failure J. Immunol., March 15, 2005; 174(6): 3765 - 3772. [Abstract] [Full Text] [PDF]
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 T. Kashimoto, S. Ueno, H. Hayashi, M. Hanajima, K. Yoshioka, K. Yoshida, K. Mutoh, and N. Susa Depletion of lymphocytes, but not neutrophils, via apoptosis in a murine model of Vibrio vulnificus infection J. Med. Microbiol., January 1, 2005; 54(1): 15 - 22. [Abstract] [Full Text] [PDF]
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A. K. Nussbaum and J. L. Whitton The Contraction Phase of Virus-Specific CD8+ T Cells Is Unaffected by a Pan-Caspase Inhibitor J. Immunol., December 1, 2004; 173(11): 6611 - 6618. [Abstract] [Full Text] [PDF]
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S. Falcone, C. Perrotta, C. De Palma, A. Pisconti, C. Sciorati, A. Capobianco, P. Rovere-Querini, A. A. Manfredi, and E. Clementi Activation of Acid Sphingomyelinase and Its Inhibition by the Nitric Oxide/Cyclic Guanosine 3',5'-Monophosphate Pathway: Key Events in Escherichia coli-Elicited Apoptosis of Dendritic Cells J. Immunol., October 1, 2004; 173(7): 4452 - 4463. [Abstract] [Full Text] [PDF]
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 D H Wyllie, I C J W Bowler, and T E A Peto Relation between lymphopenia and bacteraemia in UK adults with medical emergencies J. Clin. Pathol., September 1, 2004; 57(9): 950 - 955. [Abstract] [Full Text] [PDF]
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 J. Frank, K. Witte, W. Schrodl, and C. Schutt CHRONIC ALCOHOLISM CAUSES DELETERIOUS CONDITIONING OF INNATE IMMUNITY Alcohol Alcohol., September 1, 2004; 39(5): 386 - 392. [Abstract] [Full Text] [PDF]
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P. A. Efron, A. Martins, D. Minnich, K. Tinsley, R. Ungaro, F. R. Bahjat, R. Hotchkiss, M. Clare-Salzler, and L. L. Moldawer Characterization of the Systemic Loss of Dendritic Cells in Murine Lymph Nodes During Polymicrobial Sepsis J. Immunol., September 1, 2004; 173(5): 3035 - 3043. [Abstract] [Full Text] [PDF]
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 J. G. R. WEAVER, M. S. ROUSE, J. M. STECKELBERG, and A. D. BADLEY Improved survival in experimental sepsis with an orally administered inhibitor of apoptosis FASEB J, August 1, 2004; 18(11): 1185 - 1191. [Abstract] [Full Text] [PDF]
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U. Bommhardt, K. C. Chang, P. E. Swanson, T. H. Wagner, K. W. Tinsley, I. E. Karl, and R. S. Hotchkiss Akt Decreases Lymphocyte Apoptosis and Improves Survival in Sepsis J. Immunol., June 15, 2004; 172(12): 7583 - 7591. [Abstract] [Full Text] [PDF]
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A. Wellmer, M. von Mering, A. Spreer, R. Diem, H. Eiffert, C. Noeske, S. Bunkowski, R. Gold, and R. Nau Experimental Pneumococcal Meningitis: Impaired Clearance of Bacteria from the Blood Due to Increased Apoptosis in the Spleen in Bcl-2-Deficient Mice Infect. Immun., June 1, 2004; 72(6): 3113 - 3119. [Abstract] [Full Text] [PDF]
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Y. Le Tulzo, C. Pangault, L. Amiot, V. Guilloux, O. Tribut, C. Arvieux, C. Camus, R. Fauchet, R. Thomas, and B. Drenou Monocyte Human Leukocyte Antigen-DR Transcriptional Downregulation by Cortisol during Septic Shock Am. J. Respir. Crit. Care Med., May 15, 2004; 169(10): 1144 - 1151. [Abstract] [Full Text] [PDF]
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N. Methot, J. Huang, N. Coulombe, J. P. Vaillancourt, D. Rasper, J. Tam, Y. Han, J. Colucci, R. Zamboni, S. Xanthoudakis, et al. Differential Efficacy of Caspase Inhibitors on Apoptosis Markers during Sepsis in Rats and Implication for Fractional Inhibition Requirements for Therapeutics J. Exp. Med., January 20, 2004; 199(2): 199 - 207. [Abstract] [Full Text] [PDF]
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P. S. Grutkoski, Y. Chen, C. S. Chung, and A. Ayala Sepsis-induced SOCS-3 expression is immunologically restricted to phagocytes J. Leukoc. Biol., November 1, 2003; 74(5): 916 - 922. [Abstract] [Full Text] [PDF]
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N. Jiang, C. F. Reich III, and D. S. Pisetsky Role of macrophages in the generation of circulating blood nucleosomes from dead and dying cells Blood, September 15, 2003; 102(6): 2243 - 2250. [Abstract] [Full Text] [PDF]
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K. W. Tinsley, M. H. Grayson, P. E. Swanson, A. M. Drewry, K. C. Chang, I. E. Karl, and R. S. Hotchkiss Sepsis Induces Apoptosis and Profound Depletion of Splenic Interdigitating and Follicular Dendritic Cells J. Immunol., July 15, 2003; 171(2): 909 - 914. [Abstract] [Full Text] [PDF]
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R. S. Hotchkiss, K. C. Chang, M. H. Grayson, K. W. Tinsley, B. S. Dunne, C. G. Davis, D. F. Osborne, and I. E. Karl Adoptive transfer of apoptotic splenocytes worsens survival, whereas adoptive transfer of necrotic splenocytes improves survival in sepsis PNAS, May 27, 2003; 100(11): 6724 - 6729. [Abstract] [Full Text] [PDF]
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R. S. Hotchkiss and I. E. Karl The Pathophysiology and Treatment of Sepsis N. Engl. J. Med., January 9, 2003; 348(2): 138 - 150. [Full Text] [PDF]
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K. Doerschug, S. Sanlioglu, D. M. Flaherty, R. L. Wilson, T. Yarovinsky, M. M. Monick, J. F. Engelhardt, and G. W. Hunninghake First-Generation Adenovirus Vectors Shorten Survival Time in a Murine Model of Sepsis J. Immunol., December 1, 2002; 169(11): 6539 - 6545. [Abstract] [Full Text] [PDF]
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R. Salomao, M. K.C. Brunialti, E. G. Kallas, P. S. Martins, O. Rigato, and M. Freudenberg Lipopolysaccharide--cell interaction and induced cellular activation in whole blood of septic patients Innate Immunity, October 1, 2002; 8(5): 371 - 379. [Abstract] [PDF]
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A. Oberholzer, C. Oberholzer, K. S. Bahjat, R. Ungaro, C. L. Tannahill, M. Murday, F. R. Bahjat, Z. Abouhamze, V. Tsai, D. LaFace, et al. Increased Survival in Sepsis by In Vivo Adenovirus-Induced Expression of IL-10 in Dendritic Cells J. Immunol., April 1, 2002; 168(7): 3412 - 3418. [Abstract] [Full Text] [PDF]
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R. S. Hotchkiss, K. W. Tinsley, P. E. Swanson, M. H. Grayson, D. F. Osborne, T. H. Wagner, J. P. Cobb, C. Coopersmith, and I. E. Karl Depletion of Dendritic Cells, But Not Macrophages, in Patients with Sepsis J. Immunol., March 1, 2002; 168(5): 2493 - 2500. [Abstract] [Full Text] [PDF]
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A. Byrne and D. J. Reen Lipopolysaccharide Induces Rapid Production of IL-10 by Monocytes in the Presence of Apoptotic Neutrophils J. Immunol., February 15, 2002; 168(4): 1968 - 1977. [Abstract] [Full Text] [PDF]
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 R. S. Hotchkiss, W. M. Dunne, P. E. Swanson, C. G. Davis, K. W. Tinsley, K. C. Chang, T. G. Buchman, I. E. Karl, H. Grassme, S. Kirschnek, et al. Role of Apoptosis in Pseudomonas aeruginosa Pneumonia Science, November 30, 2001; 294(5548): 1783a - 1783. [Full Text] [PDF]
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