Vol. 281, Issue 4, G957-G963, October 2001
Role of gut flora on intestinal group II
phospholipase A2 activity and intestinal injury in
shock
Ranna A.
Rozenfeld1,
Xueli
Liu1,
Isabelle
Deplaen1, and
Wei
Hsueh2
Departments of 1 Pediatrics and 2 Pathology,
Children's Memorial Hospital, Northwestern University Medical
School, Chicago, Illinois 60614
 |
ABSTRACT |
We previously showed
that group II phospholipase A2 (PLA2-II), a
secretory, bactericidal, and proinflammatory protein in intestinal crypts, is upregulated after lipopolysaccharide (LPS) and
platelet-activating factor (PAF) challenge. Here we examined whether
germ-free environment (GF) or antibiotic treatment (ABX) affects the
pathophysiological responses and intestinal PLA2-II
activity after PAF (1.5 µg/kg) or LPS (8 mg/kg) injection. We found
that LPS and PAF induced hypotension and mild intestinal injury in
conventionally fed (CN) rats; these changes were milder in ABX rats,
whereas GF rats showed no intestinal injury. PLA2-II enzyme
activity was detected in normal rat small intestine; the basal level
was not diminished in ABX or GF rats. PAF and LPS caused an increase in
PLA2-II activity, which was abrogated in GF and ABX rats.
Recolonization of GF rats by enteral contamination restituted their
PLA2-II response to PAF and LPS and susceptibility to bowel
injury. We conclude that PAF- and LPS-induced increases in
PLA2-II activity are dependent on gut bacteria, and ABX and
GF rats are less susceptible to LPS-induced injury than CN rats.
germ-free environment; mixed-antibiotic treatment; platelet-activating factor; lipopolysaccharide; bacteria
| |
INTRODUCTION |
GROUP II PHOSPHOLIPASE
A2 (PLA2-II) is a secretory, 14-kDa
protein (22, 38) that may play an important role in septic shock and inflammation (47). It is expressed in many
organs, including the lungs, gastrointestinal tract (21),
liver (12), and kidneys (18). In rats, the
largest amount of PLA2-II transcripts was found in the
ileum (21, 25). Morphological (23, 35) and
biochemical (40) studies revealed that PLA2-II
is preferentially localized in the Paneth cells in the crypts
(23, 35). PLA2-II is an acute phase protein
detected in sepsis (47) and inflammatory reactions
(24, 30). Aside from its well-established role in delayed
arachidonic acid release and prostaglandin production in inflammation
(31, 41), it also has an important function in innate
immunity, i.e., killing gram-negative (19, 50) as well as
gram-positive bacteria (11, 39).
Lipopolysaccharide (LPS) induces PLA2-II mRNA expression in
rat aorta, spleen, lungs, thymus (32), liver
(12), and kidneys (18) and activates enzyme
activity in the lungs (3, 26). Proinflammatory cytokines
such as interleukin-1 and tumor necrosis factor upregulate the mRNA
synthesis and secretion of PLA2-II in vitro (in mesangial
cells; Refs. 42, 48). Under intestinal inflammatory
conditions, such as ulcerative colitis and Crohn's disease (15,
16, 29), PLA2-II expression is increased.
PLA2-II has been recently subdivided into groups IIA, IIB,
and IIC (10, 31). Studies (25, 41) have shown
that the PLA2-IIA gene is constitutively expressed in rat
intestine and markedly elevated 24 h after LPS injection.
Platelet-activating factor (PAF) is an endogenous mediator of
intestinal injury in endotoxin shock (20, 43, 49). Direct injection of PAF into animals induces systemic pathophysiological responses, including shock, capillary leak, thrombocytopenia, neutropenia, pulmonary hypertension, bronchoconstriction (4, 5,
17), and intestinal injury (49). Previous studies
(45) showed that PAF also induces gene transcription and
enzyme activation of PLA2-II in the small intestine. Much
of the in vivo effect of LPS, including shock and bowel injury
(20, 49), could be largely abolished by PAF antagonists,
indicating that PAF is the endogenous mediator of LPS, and the two
agents may share a common final pathway. However, a previous study
(45) suggests that different pathways may exist for PAF
and LPS in the upregulation of intestinal PLA2-II, because
the LPS effect is not blocked by the administration of a PAF
antagonist. PAF-induced intestinal injury has been shown
(43) to be largely dependent on the presence of intestinal
flora (or their products), since germ-free environment (GF) and
antibiotic-treated (ABX) rats are protected from PAF-induced injury. It
is unclear whether PAF- or LPS-induced intestinal PLA2-II activation depends on intestinal flora and/or their products. The
purpose of this study is to examine the effect of GF or ABX on
1) basal PLA2-II activity in the intestine,
2) intestinal PLA2-II activity after PAF or LPS
challenge, and 3) the pathophysiological responses to PAF or
LPS challenge.
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MATERIALS AND METHODS |
The stock solution of PAF (2 mg/ml
1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine,
Sigma Chemical, St. Louis, MO) in saline albumin (5 mg/ml) was stored
frozen in aliquots. The working solution was made fresh daily. LPS
(Salmonella typhosa) was purchased from Sigma Chemical. Male
GF Sprague-Dawley rats (80-120 g) were purchased from Taconic
(Germantown, NY); male normal Sprague-Dawley rats (80-120 g) were
purchased from Harlan Sprague Dawley (Indianapolis, IN). GF rats were
kept in a sterile institutional animal facility for no more than 2 days
before use. Neomycin, polymyxin B and metronidazole were obtained from
the local pharmacy. The specific PLA2-II inhibitor
LY-311727 was a generous gift from Eli Lilly (Indianapolis, IN).
Animal experiments.
Animals were anesthetized with Nembutal (65 mg/kg ip, Abbott
Laboratories, North Chicago, IL) and placed under warming lights. After
tracheotomy, the carotid artery and jugular vein were catheterized for
continuous blood pressure recording, blood sampling, and drug injection. The first part of the experiment consisted of two groups of
animals: conventionally fed (CN) and ABX rats. Each group was divided
into three subgroups: sham operated, PAF (1.5 µg/kg iv), and LPS (8 mg/kg iv). Each animal that was experimentally treated (PAF or LPS) was
always paired with a sham control. Preliminary experiments were done to
select doses of PAF and LPS that would avoid profound shock and gross
intestinal necrosis and thus loss of enzyme activity. The combined
antibiotic regimen (given in drinking water) included a mixture of
neomycin (250 mg · kg
1 · day1), polymyxin
B (9 mg · kg1 · day1), and
metronidazole (50 mg · kg1 · day1) for 3 days
before the experiment. This choice of ABX is based on previous findings
(43) showing protection against PAF-induced injury.
However, in the current study, animals were treated for 3 days instead
of 7 days (43), because preliminary data showed that rats
treated for 3 days have the lowest number of intestinal bacterial
colonies. The second part of the experiments consisted of GF rats with
or without recolonization by enteral contamination and CN
animals. The GF status of the animals was confirmed by negative
culture of the rectal swab at the time of experiment. Recolonization
was achieved by gavaging the GF rats with intestinal contents from CN
animals and then keeping them in regular cages for 3-4 days before
experiments. Rectal swab and bacterial culture were performed
to confirmed successful recolonization. The animals were injected with
LPS (8 mg/kg iv) or PAF (1.5 µg/kg iv). In addition, two groups of CN
and GF rats were pretreated with a specific PLA2-II
inhibitor, LY-311727 (2 mg/kg), given at time 
60 and
time 0 before LPS (8 mg/kg iv) (2).
Blood samples were collected at the beginning and the end of the
experiment (30 min in PAF experiments and 2 h for LPS experiments) for white blood cell count and assessment of hematocrit value. The
experimental period was chosen based on previous findings that the
enzyme activation peaked at 30 min and 2 h after PAF and LPS
administration, respectively. At the end of the experiment, the small
intestine was removed and examined for gross visible injury, sections
were taken for histological injury assessment, and the intestine was
frozen and stored for PLA2-II assay. In ABX, GF, and
recolonized GF (RGF) rats, the intestinal content from the terminal
ileum was cultured for aerobic and anaerobic bacteria.
Gross and microscopic injury score.
The severity score of gross injury was defined as follows: 1, mild
(slight reddish discoloration); 2, moderate (red discoloration often
with hemorrhage); and 3, severe (grossly necrotic, blackish red,
friable, and lusterless) (44). Sections were taken from the most severely affected areas and processed for paraffin embedding and subsequent sectioning and staining for histological confirmation of
necrosis. Multiple random sections were taken if the bowel appeared
normal. A pathologist recorded the histological changes in a blinded
fashion. The severity score for microscopic injury was defined as
follows: 0, no injury with intact surface epithelium; 0.5, minimal
injury involving epithelial cells at villus tips; 1, mild mucosal
injury confined to the top of the villi; 2, moderate injury involving
nearly one-half of the villi; and 3, severe injury with complete loss
of villi or extending to submucosa. The length of injured intestinal
segment was measured. Gross and histological scores were calculated by
multiplying the percentage of intestinal involvement (length of
abnormal intestine/total bowel length) by the severity score
(44).
Intestinal PLA2-II assay.
Intestinal tissue was homogenized in buffer solution containing HEPES,
sucrose, EDTA, EGTA, leupeptin, pepstatin, and phenylmethylsulfonyl chloride. The debris and nuclei were removed by centrifugation at 1,000 g for 5 min (33), and the supernatant was then
centrifuged at 200,000 g for 1 h. The
PLA2-II assay was performed following method A
(33) or B (7, 13) after
preliminary kinetic study. In method A, the enzyme (100 µg
protein) was added to the sonicated substrate, 1-palmitoyl,
2-[14C]arachidonoyl-sn-3-glycerophosphoethanolamine,
and incubated at 37°C for 30 min. The reaction was terminated with
ethanol containing arachidonic acid and acetic acid (20°C) and then
centrifuged. Lipids were extracted, separated by TLC, developed in
benzene-diethyl ether-ethyl acetate-glacial acetic acid (80:10:10:0.2),
scraped, and counted in a scintillation counter (33). In
method B, the enzyme and substrate, autoclaved
14C-labeled Escherichia coli, were incubated at
37°C for 10 min. The reaction was terminated with chloroform-methanol
(2:1) followed by acidified water. Phospholipids were extracted by
Folch's method (7, 13); TLC was performed as described
for method A above. Method A gives an absolute
quantity of PLA2-II activity. Method B is
simpler and more rapid than method A. PAF experiments were performed using both method A and method B, which
yielded similar results. Thus only method B was used in the
LPS experiments.
Statistical analysis.
Statistical analyses were performed using Wilcoxon's multivariate
analysis. Data are presented as means ± SE. P < 0.05 was considered significant.
| |
RESULTS |
Bacterial cultures of intestinal content from ABX rats showed
results similar to previous findings (43), namely,
complete elimination of E. coli and marked reduction of
other gram-negative bacteria. Lactobacillus was the
predominant bacteria after 3 days of treatment, and this was only
present in one-third of the colony-forming units of sham controls.
Bacterial cultures of GF rats confirmed the sterile state of the
animals. RGF animals had similar intestinal flora to CN rats, as shown
by bacterial culture.
PAF, at the dose used, induced immediate, marked hypotension with the
nadir reached by 5 min (Fig. 1) in all
groups of rats (CN, ABX, GF, and RGF), which slowly recovered within 30 min in all except RGF rats. These rats showed a persistent significant hypotension at 30 min, significantly more severe than in other groups.
(Fig. 1B). PAF induced peripheral leukocytosis in all groups
of rats, but not significantly so in GF and RGF groups (see Fig.
3A). PAF also induced hemoconcentration (indicated by increased hematocrit) in all groups of rats (see Fig. 4A).
GF (see Fig. 4A) partially ameliorated the
hemoconcentration. PAF, at the dose used, did not cause lethality
and only resulted in minimal gross and
microscopic injury (Table 1) of the intestine in CN rats and minimal
microscopic injury in ABX rats. GF rats had no gross or microscopic
injury. RGF rats had minimal gross and microscopic injury, similar to
CN rats.
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Fig. 1.
Systemic blood pressure (in mmHg) over time after
injection of platelet-activating factor (PAF; 1.5 µg/kg iv) in
conventionally fed (CN) and antibiotic-treated (ABX) rats
(A) or germ-free (GF) and recolonized GF (RGF) rats
(B). Values are means ± SE. All 4 groups had
significant hypotension at 5 min compared with sham controls. RGF still
had significant hypotension at 30 min compared with other groups.
* P < 0.05, compared with sham rats;
$ P < 0.05, compared with GF rats.
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The blood pressure slowly decreased after LPS administration with the
first nadir reached at 15-20 min (Fig.
2). In all groups of animals, there
appeared to be some recovery of blood pressure, with a second nadir
reached at 90 min. At 2 h, all groups of rats remained hypotensive
(Fig. 2). LPS-induced shock was partially abrogated by ABX pretreatment
(Fig. 2A). RGF rats showed more severe hypotension than CN
and GF rats (Fig. 2B). LPS caused peripheral leukopenia (Fig. 3B) and
hemoconcentration (increase in hematocrit) (Fig.
4B) in all four groups of
rats, but not significantly so in GF rats. Antibiotic pretreatment
partially ameliorated LPS-induced changes in the white blood cell count
and hematocrit (Figs. 3B and 4B).
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Fig. 2.
Systemic blood pressure (in mmHg) over time after
injection of lipopolysaccharide (LPS; 8 mg/kg iv) in CN and ABX rats
(A) or GF and RGF rats (B). Values are means ± SE. Compared with sham controls, all 4 groups showed significant
hypotension at 15 min and also at 2 h. LPS-induced hypotension was
partially abrogated in ABX rats. RGF rats had more severe hypotension
compared with CN or GF rats.* P < 0.05, compared
with sham controls; # P < 0.05, compared with
CN-LPS rats.
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Fig. 3.
Change in peripheral white blood cell (WBC) count
(expressed as % of values at time 0) after PAF
(A) or LPS injection (B). Values are means ± SE. PAF caused leukocytosis in CN and ABX rats; LPS caused
leukopenia in CN and ABX rats. After LPS, ABX and GF rats had less
severe leukopenia compared with CN rats. * P < 0.05, compared with respective control; # P < 0.05 compared with CN LPS-treated rats.
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Fig. 4.
Change in hematocrit value (expressed as % of values at
time 0) after PAF (A) or LPS injection
(B). Values are means ± SE. PAF caused
hemoconcentration in all groups. LPS caused hemoconcentration in CN,
ABX, and RGF groups. After PAF, GF rats had less severe
hemoconcentration compared with CN rats. RGF rats had more severe
hemoconcentration compared with GF rats. After LPS, ABX and GF rats had
less severe hemoconcentration compared with CN rats.
* P < 0.05, compared with respective control;
# P < 0.05, compared with CN PAF- or LPS-treated
rats; $ P < 0.05, compared with GF PAF-treated
rats.
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LPS induced mild gross and microscopic injury of the intestine in CN
rats (Table 1). GF rats had minimal gross (congestion) and no
microscopic injury after LPS. ABX rats had statistically less
microscopic injury after LPS than CN rats. RGF rats had mild gross and
microscopic injury. LPS caused no mortality at the dose used.
PLA2-II enzyme activity was detected in the small intestine
of unstimulated normal animals (Fig. 5).
Neither ABX nor GF diminished the basal level of PLA2-II.
Both PAF and LPS increased PLA2-II activity (to 2-fold
baseline) in CN rats. The effect of PAF peaked at 30 min. The LPS
effect was slower than PAF, just beginning to plateau at 2 h. Thus
30 min was chosen as the end point for PAF and 2 h for LPS
experiments. The increase in PLA2-II activity in response
to PAF (Fig. 5, A and B) and LPS (Fig.
5C) was abrogated in GF and ABX rats. PAF and LPS did not
significantly cause an increase in the activity of PLA2-II
in ABX and GF groups. The responses of RGF rats were similar to those
of CN rats to PAF and LPS.
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Fig. 5.
Group II phospholipase A2
(PLA2-II) activity in the small intestine. A:
activity expressed in nmol · mg
protein1 · h1 using method
A (see MATERIALS AND METHODS) for PLA2-II
assay after PAF injection. B: PLA2-II activity
expressed as %conversion/min, as assayed by method B (see
MATERIALS AND METHODS) after PAF injection. C:
enzyme activity expressed as %conversion/min after LPS injection.
Values are means ± SE. PAF increases PLA2-II
activity. The effect of PAF is abrogated in GF and ABX rats. RGF rats
have a normal PLA2-II response to PAF. LPS increases
PLA2-II activity. The effect of LPS is abrogated in GF and
ABX rats. RGF rats have a normal PLA2-II response to LPS.
* P < 0.05, compared with respective control;
# P < 0.05, compared with CN PAF- or LPS-treated
rats; $ P < 0.05, compared with GF PAF- or
LPS-treated rats.
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A specific PLA2-II inhibitor (LY-311727) was given to CN
and GF rats before LPS injection to evaluate the effect on
pathphysiological changes. The PLA2-II inhibitor had
minimal protection against hypotension in GF rats (not statistically
significant) but none in CN rats (data not shown). The
inhibitor had no effect on hematocrit or white blood cell count. There
was no mortality in the inhibitor-treated groups. The CN animals had
similar gross and microscopic injury compared with controls.
| |
DISCUSSION |
Intestinal injury is important in the perpetuation of septic
shock, multiple organ dysfunction syndrome (MODS), and acute respiratory distress syndrome (ARDS). Circulating PLA2 has
been recognized as a mediator of cardiovascular collapse in septic shock (46). Circulating PLA2 is vasoactive,
causing hypotension, increased vascular permeability, and acute lung
injury (14). Excessive release and/or activation of
PLA2 appears to be a pivotal event in the development of
sepsis, septic shock, and MODS (14). Release of
PLA2 into the circulation occurs in states of profound illness, including sepsis, shock, severe injury, and pancreatitis, all
of which are linked to the development of ARDS and MODS. Experimental and clinical evidence (1) suggests that PLA2
may serve a primary regulatory role in the development of these
inflammatory disorders. The concentration of PLA2-II is
increased (up to 100- to 150-fold) in the sera of patients suffering
from inflammatory diseases (34). The role of
PLA2-II in the pathogenesis of sepsis is further supported by the observation that administration of PLA2-II
inhibitors prolonged survival in a murine model of LPS shock
(27).
PAF, an endogenous mediator of endotoxin shock (20, 49),
induces intestinal necrosis (49), which may augment the
development of MODS. A previous study (43) showed that PAF
causes elevation of serum LPS levels (indicating bacterial
translocation), and PAF-induced intestinal injury and lethality are
reduced in GF and ABX rats. PAF also upregulates the gene expression
and enzyme activity of intestinal PLA2-II, even at doses
insufficient to cause prolonged shock and intestinal necrosis
(45). The PAF-induced increase in PLA2-II
activity was abrogated in GF and ABX rats, suggesting that the effect
of PAF on this enzyme is largely mediated by "indigenous"
intestinal bacteria or their products.
Previous studies (41) have shown that intravenous
injection of LPS induces PLA2-II mRNA expression in various
organs, including the intestine. In the present study, we have shown
that the increase in PLA2-II activity induced by LPS was
abrogated in GF and ABX rats. This observation suggests that LPS
activation of this enzyme is also largely mediated by indigenous
intestinal bacteria and/or their products. In other words,
bacterial adherence to the intestinal mucosa and/or subsequent invasion
of the tissue is required for the enzyme activation. Indeed, endotoxin
has been shown to cause bacterial translocation in mice (8,
9). However, our preliminary experiments showed that bacterial
translocation does not occur up to 6 h after the injection of PAF
or LPS, much longer than our experimental periods, suggesting that
bacterial products such as LPS, rather than bacteria themselves, are
responsible for the injurious effects observed in CN rats. Although
enterocytes do not express the LPS receptor CD14 on their cell surface,
they express Toll-like receptors (TLR) (6) and could be
directly stimulated by LPS in vitro. It is possible that LPS binds to
TLR, resulting in a change in mucosal permeability and loss of barrier function. The role of enteral bacteria or their products in this model
is further supported by the observation that GF and ABX reduce
LPS-induced hemoconcentration and bowel injury. ABX also ameliorates
LPS-induced hypotension. The impaired response in GF animals may be
partly explained by a deficient inflammatory response, because GF
animals have fewer intestinal mast cells (28) and impaired
production of cytokines (36) and superoxide (37). Furthermore, because ABX animals are also protected
from the adverse effects of PAF and LPS, an abnormal inflammatory
response need not be invoked as the sole cause of the observed
protection. Thus it appears that gut flora and/or their products are
necessary for the injurious effect of PAF and LPS. Hence, the important clinical implication that ABX, by reducing gut bacteria and their products, may ameliorate local as well as systemic inflammation and
therefore prevent the development of shock and bowel necrosis in septic patients.
| |
ACKNOWLEDGEMENTS |
We thank Wei Huang for providing technical assistance and Tianyue
Chen for statistical assistance.
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FOOTNOTES |
This work was supported by National Institute of Diabetes and Digestive
and Kidney Diseases Grant DK-34574 and a Society of Critical Care
Medicine Founders Grant.
Address for reprint requests and other correspondence: W. Hsueh, Children's Memorial Hospital, Dept. of Pathology, 2300 Children's Plaza, Chicago, IL 60614 (E-mail: w-hsueh{at}nwu.edu).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 24 July 2000; accepted in final form 24 May 2001.
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