1 Infectious Disease and
2 Nephrology Sections, Department
of Medicine, Veterans Affairs Medical Center, University of Minnesota
School of Medicine, Minneapolis, Minnesota 55417;
3 Naval Medical Research Institute
Detachment, Vibrio cholerae induces massive
intestinal fluid secretion that continues for the life of the
stimulated epithelial cells. Enhanced regional blood flow and
peristalsis are required to adapt to this obligatory intestinal
secretory challenge. Nitric oxide (NO) is a multifunctional molecule
that modulates blood flow and peristalsis and possesses both cytotoxic
and antibacterial activity. We demonstrate that, compared with those in
asymptomatic control subjects, levels of stable NO metabolites
(
epithelial cells; mucosal response to infection; innate intestinal
immunity
VIBRIO CHOLERAE is a common and
serious cause of diarrheal disease and dehydration in developing
countries. These effects are mediated in large part by the actions of
cholera toxin (CT), which irreversibly stimulates intestinal epithelial
cell secretion (7, 11, 31). The massive fluid loss often associated
with V. cholerae diarrhea requires
increased regional blood flow. CT may also elicit the production of
intestinal neurotransmitters, particularly vasoactive intestinal
peptide (VIP), which promote peristalsis (13). The mutually enhanced
release of VIP and nitric oxide (NO) provides powerful mediators of
relaxation in both intestinal and vascular smooth muscle (14, 22, 33).
Thus NO may participate in the regulation of regional blood flow and
intestinal motility (8, 18, 19). This multifunctional molecule (35) may
also affect the integrity of mucosal epithelial cells in the intestine
(3, 10, 36, 37, 40) and exert antibacterial activity (6). Therefore, we
determined whether acute cholera infection elicited NO production in
humans. Because these studies of both natural and experimental
infection in humans strongly suggested that V. cholerae-induced diarrhea was accompanied by increased
NO generation, we characterized the mechanisms and localization of NO
production in the intestine using a rabbit ileal loop model in vivo and
the bactericidal activity of NO products against V. cholerae.
Human Subjects
Natural infection.
In March 1995, during the cholera season in Lima, Peru, we collected
sera from 10 asymptomatic adults and 26 age- and sex-matched patients
(mean age 40 ± 17.3 yr, range 18-80; 14 female, 12 male) with
acute watery diarrhea on presentation to the hospital. These symptomatic patients had been ill (>6 liquid stools/day) for a mean
of 2.1 ± 0.8 days; 11 had moderate and 15 had severe dehydration by
World Health Organization criteria. Food intake was limited in the
24-48 h before presentation in the cholera group due to nausea and
vomiting. Levels of serum creatinine were similar in both symptomatic
and healthy groups (1.0 vs. 0.8 mg/dl).
Experimental infection.
At the United States Army Medical Research Institute of Infectious
Diseases, 10 healthy volunteers aged 18-40 yr with no underlying medical illnesses, recent travel, intestinal symptoms, or history of
cholera were challenged orally with 5 × 106 colony forming units (cfu) of
live wild-type V. cholerae 01 (El Tor
Inaba 9N16961; Center for Vaccine Development, Baltimore, MD) in
bicarbonate-ascorbic acid buffer. Of these 10 volunteers, five had been
immunized 1 mo earlier with a live attenuated oral vaccine, the Peru-15
strain, a genetically attenuated strain of V. cholerae 01 El Tor Inaba in which the CT genetic
element (ctx, zot, and
ace) had been replaced with a CT-B
subunit gene, as previously described (16). Volunteers were challenged
with wild-type V. cholerae 01 and
closely monitored for vital signs, diarrhea [ Rabbit Ileal Loop Assay
ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References
/
) are significantly increased in sera from acutely ill Peruvian patients
with natural cholera infection as well as from symptomatic volunteers
from the United States infected experimentally with V. cholerae. In a rabbit ileal loop model in vivo, cholera
toxin (CT) elicited fluid secretion and dose-dependent increases in levels of
/
in the fluid (P < 0.01). In
contrast, lipopolysaccharide (LPS) elicited no such effects when
applied to the intact mucosa. NO synthase (NOS) catalytic activity also
increased in toxin-exposed tissues (P < 0.05), predominantly in epithelial cells. The CT-induced NOS activity was Ca2+ dependent and
was not suppressed by dexamethasone. In conclusion, symptomatic
V. cholerae infection induces NO
production in humans. In the related animal model, CT, but not LPS,
stimulated significant production of NO in association with increases
in local Ca2+-dependent NOS
activity in the tissues.
INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References
METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References
2 loose or watery
stools in 48 h (or
300 ml)], and fever (>100°F) (16).
Whole blood, plasma, and 12-h urine samples were collected each morning
on days 0,
2, 4,
and 7 postchallenge and stored at
20°C until tested. All volunteers were offered the same diet throughout the course of the study. Written informed consent was obtained from all patients as approved by the Human Subjects Research Review Board of the Office of the U.S. Army Surgeon General.
/
in each loop. Samples of intestinal fluid were centrifuged at 3,000 g for 5 min and stored at
20°C until tested. Loop fluid was also cultured for both
aerobic and anaerobic bacteria. All protocols were approved by the
Animal Subjects Committee at the Veterans Affairs Medical Center.
Measurement of
/
Assay for NO Synthase Activity in the Intestinal Wall
After 8 h, washed ileal loop tissues were frozen with liquid nitrogen and stored atNO synthase (NOS) activity was determined by the conversion of L-[14C]arginine (Amersham, Arlington Heights, IL) to L-[14C]citrulline, as previously described (17, 30, 32). Ca2+-dependent activity was determined as the difference between the quantity of L-[14C]citrulline produced from control samples and samples containing 3 mM EGTA to bind Ca2+. Ca2+-independent activity was determined as the difference between the activity in samples containing 3 mM EGTA and samples containing both 3 mM EGTA and 1 mM NG-nitro-L-arginine (L-NNA), an inhibitor of NOS. NOS activity was expressed as picomoles of L-[14C]citrulline formed per gram protein per minute (2).
/
Production by Rabbit Intestinal Tissue In Vitro
Tissue Staining for NADPH Diaphorase
To localize NOS activity in the intestine, we stained Formalin-fixed tissues for NADPH diaphorase, a sensitive marker of NOS activity (4, 12), as previously described (38, 39).Antibacterial Activity of NO
To determine whether the NO produced locally in the intestine may have had direct effects on the organism, the susceptibility of five V. cholerae (El Tor; 2 Inaba and 3 Ogawa) and five E. coli H7:0157 strains was tested using a microbroth dilution technique in M9 medium (thiol-free) as described by De Groote et al. (6) and Reimer et al. (28). Briefly, log phase growth of each isolate was washed in M9 medium and inoculated to a final concentration of 3.8 × 104 cfu/well. 2,2'-(Hydroxynitrosohydrazono)bis-ethanamine (DETA-NO; Research Biochemicals International, Natick, MA), diethylamine diazeniumdiolate ion (DEA-NO; Cayman Chemical, Ann Arbor, MI), 3-morpholinosydnonimine hydrochloride (SIN-1; Cassela, Frankfurt, Germany), hydrogen peroxide (Sigma), hypoxanthine (HX; Sigma), and xanthine oxidase (XO; EC 1.1.3.22; Sigma) were diluted to stock concentrations, filter sterilized, and added at the following final concentrations: 0.0625-4 mM DETA-NO, 0.0625-4 mM DENO, 0.125 mM SIN-1, 6.2-100 µM hydrogen peroxide, 6.2-100 µM HX, and 7.5 × 10Statistics
Values for ![]() |
RESULTS |
---|
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Clinical Response to Live V. Cholerae Infection in Humans
Patients with natural V. cholerae infection in Peru were more ill and more dehydrated than those with experimental cholera infection in the U.S. After experimental challenge of volunteers in the U.S. with live V. cholerae, 6 of 10 volunteers developed abdominal pain and diarrhea (2 of 5 vaccinees and 4 of 5 control subjects) (16). Total intestinal fluid loss in symptomatic patients ranged from 300-3,830 ml, and symptoms were most prominent on days 2-4. Mean blood pressures remained stable throughout the course of infection, independent of the presence or absence of diarrhea.Levels of
/
|
In U.S. volunteers, mean levels of
/
in plasma were also increased significantly in patients with diarrhea
compared with preinfection values but remained unchanged in subjects
without diarrhea (Fig. 2). Among symptomatic patients, peak levels of
/
on day 4 were significantly higher
than their baseline values (182 vs. 125 nmol/ml;
P < 0.001) and compared
with those in asymptomatic subjects (P < 0.04; Fig. 2). Previous immunization had no independent effect on
plasma levels of
/
.
|
As in plasma,
/
levels in urine increased significantly from preinfection values only
in U.S. patients with diarrhea (1.1 nmol
/
/µg creatinine preinfection vs. 2.8 and 2.5 nmol
/
/µg creatinine on days 2 and
4, respectively;
P < 0.03) (Fig.
3). These values in urine were higher on
day 2 than those in asymptomatic subjects (P < 0.001), among whom, as
in serum, levels did not change. In patients with diarrhea, despite
increased levels of
/
in plasma and urine, mean arterial pressure remained stable, and only
two developed mild fever. These data suggested a limited systemic
inflammatory response to this mucosal infection and a local intestinal
source of NO in humans.
|
Secretory Response to CT and LPS in Rabbits
We further characterized and localized the effects of V. cholerae infection on the L-arginine-NO pathway in an in vivo rabbit ileal loop model (5, 15). This model permitted us to examine the independent effects of purified CT and LPS from V. cholerae on intestinal fluid secretion and NO production.The fluid response to CT in isolated rabbit ileal loops (expressed as
volume-to-length ratio) was dose dependent, with doses as low as 10 ng/ml producing detectable fluid accumulation (Fig. 4). In pilot experiments, fluid was present
as early as 6-8 h after inoculation. Levels of
/
in the fluid also showed a dose-dependent response to CT (Fig. 4).
|
In contrast to the effects of CT alone, purified V. cholerae LPS (1-100 µg/ml) elicited no
appreciable fluid accumulation over 18 h when injected alone into the
lumen of the ileal loops of live animals. Moreover, LPS induced no
additive or synergistic accumulation of fluid or
/
when added to CT (0.01, 0.1, and 1 µg/ml) in the loops (data not shown).
The
/
measured in the lumen was not due to a food source, as the animals were
not fed during the trial and loops were sealed. In addition, bacterial contaminants in the loops at the time of harvest were few in number and
present in low concentrations (data not shown).
NOS Activity in Tissue
Intestinal tissue specimens obtained from loops stimulated with CT in vivo showed a twofold increase in NOS activity compared with levels in control tissues, as detected by the oxidation of L-arginine to L-citrulline (Table 1; P < 0.05). The activity measured was consistently inhibitable by a specific NOS inhibitor (L-NNA). Moreover, the unstimulated NOS activity, as well as that induced by CT, was predominantly Ca2+ dependent (79-97% inhibitable by 3 mM EGTA).
|
We localized the production of CT-induced NOS in the intestine by
staining for NADPH diaphorase, a sensitive indicator of NOS activity,
which is normally present in intestinal neuronal and endothelial cells
(23, 24, 33). NADPH diaphorase staining was dramatically enhanced in
rabbit ileal loops exposed to CT compared with that in control loops.
However, the dense staining was restricted primarily to the epithelial
cells (Fig. 5). Expression of
NADPH diaphorase staining observed in intestinal neurons and vessels
did not change discernibly in rabbits with or without CT exposure. Very
few acute inflammatory cells were seen by light microscopy either
within the epithelium or lamina propria in tissue stained with
hematoxylin-eosin in the absence or presence of CT. Moreover, no
mononuclear cells stained with NADPH diaphorase in the lamina propria.
Thus CT-induced increases in luminal
/
levels were associated with a concomitant rise in mucosal NOS activity
in the epithelial cells, rather than in neurons, endothelial or smooth
muscle cells, or lamina propria macrophages.
|
We further characterized this local NO synthesis by incubating finely
minced intestinal tissue from healthy control rabbits in vitro with CT
and LPS. We found that CT elicited high levels of
/
in the culture supernatants (Fig. 6),
whereas denatured CT did not. The CT-induced
/
was not inhibited by pretreatment of the tissue with dexamethasone. In
contrast to the lack of response with intact tissue, LPS induced
/
production from minced intestinal tissue. However, unlike the activity
stimulated by CT, LPS-induced activity was inhibited by dexamethasone.
Such inhibition of the LPS effects is typical of a
Ca2+-independent "macrophage
type" NOS (27). No signs of inflammation (e.g., polymorphonuclear,
macrophage, or lymphocytic infiltrates) disruption, or morphological
changes in epithelial cells were noted 8-12 h after CT exposure
(Fig. 7), despite significant fluid and
/
accumulation.
|
|
Antibacterial Activity of NO
In association with superoxide radicals normally found in the intestine (29), NO may combine to form peroxynitrite, a product with potent bactericidal activity (6). SIN-1, a donor of both compounds (NO · +
|
The bactericidal activity of the pure long- and short-acting
NO · donors (DETA-NO and DEA-NO, respectively) was increased in a synergistic fashion by the addition of HX/XO, which releases copious amounts of (Fig.
8B). Moreover, the enhanced
inhibitory activity of the NO · and oxygen radical donors combined was consistent in five strains each of V. cholerae and E. coli
O157:H7, a common intestinal pathogen
(P < 0.03 for each organism with vs.
without an O2 donor; data not
shown).
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DISCUSSION |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We have shown that both severe natural cholera infection and
symptomatic experimental infection with V. cholerae in humans is associated with increased levels
of stable metabolites of NO in both plasma and urine. These elevations
were restricted to patients with diarrhea. Our data also demonstrate
that the increases in
/
were unrelated to the presence of systemic symptoms such as hypotension
or fever. The known restriction of this noninflammatory noninvasive
infection to the superficial mucosa suggested an intestinal source of
NO in subjects with V. cholerae-associated diarrhea.
Indeed, we discovered a mucosal source for NO synthesis in a classic
animal model. Purified CT given intraluminally induced dose-dependent
production of both fluid and
/
in the intestinal lumen, in association with increased NOS activity in
the ileal tissue. Staining of intestinal tissue for NADPH diaphorase, a
marker for NOS activity, confirmed the local upregulation of NOS, a
change confined exclusively to the epithelial cells. Induction of NOS
in intestinal epithelial cells has been described in rats given LPS
systemically (37). In contrast, our results demonstrated that in vivo
exposure of intact bowel mucosa to LPS on the luminal surface does not
induce local production of either fluid secretion or NO, highlighting
the importance of unimpaired epithelial cell integrity. LPS was shown
to elicit
/
production from minced intestinal tissue in vitro but not from intact
mucosa in vivo.
Studies in animals have demonstrated increased intestinal NO production
after local injury and inflammation (10, 21), as have studies in humans
with inflammatory bowel disease (1, 20, 26). In tissues from patients
with active colitis, corticosteroids decreased intestinal NO
production, characteristic of a macrophage type of inducible
Ca2+-independent NOS isoenzyme
(26). Similarly, corticosteroids inhibited the LPS-induced
/
in our disrupted intestinal tissue. In contrast, our data suggest that
CT-induced NO production originates from a
Ca2+-dependent and
dexamethasone-resistant NOS in intact intestinal epithelium in the
absence of inflammation. These characteristics are similar to those of
a constitutive NOS, such as those identified in endothelial cells,
gastrointestinal smooth muscle, and neuronal cells (22, 35), but the
specific designation of the intestinal epithelial enzyme described here
has not yet been established. Although each of these cells may have
contributed to the increases in
/
we observed, none showed the dramatic increase in NADPH diaphorase
staining observed in the epithelial cells.
Our studies are the first to characterize the induction of NOS activity by CT in vivo in epithelial cells, in the absence of obvious local or systemic inflammation. Increased NO production during V. cholerae infection may serve both adaptive and protective functions. NO promotes peristalsis directly through effects on intestinal smooth muscle and indirectly via activation of VIP-secreting neurons (8, 9, 14). This relaxing effect on the bowel may accommodate the dramatic increases in intestinal volume and facilitate clearance of the fluid and bacterial load. Moreover, NO is also an endogenous mediator of relaxation of the vasculature. This powerful vasodilator effect may facilitate the increase in local blood flow required to support the necessary secretion of fluid and electrolytes stimulated by CT (25).
Protective effects of CT-induced NO may include both its bactericidal and cytotoxic effects. Similar to its ability to kill Salmonella (6), NO acts synergistically with oxygen radicals to kill V. cholerae in vitro. Such local bactericidal activity in the intestine may contribute to the host's ability to limit the magnitude of the infection and subsequent CT release to facilitate spontaneous resolution of infection, which occurs in most cases. However, whether the levels and the precise stoichiometric balance of NO and oxygen radicals achieved locally actually result in vibriocidal activity in vivo is unknown. NO also may affect mucosal cell viability. Whereas low levels of NO likely contribute to maintaining cell viability, large amounts of NO can be cytotoxic to the intestinal epithelium (10, 37). Such cytotoxicity may ultimately shorten the duration of diarrhea during acute V. cholerae infection because epithelial cell activation and secretory activity continue relentlessly until the cell dies (7, 11). The effects on both bacterial and epithelial cells in vivo remain speculative at this point.
In summary, our studies show that V. cholerae-associated diarrheal disease activates the NO pathway. This NO is primarily derived from enhancement of a Ca2+-dependent form of NOS produced in intestinal epithelial cells. This local activity is stimulated by CT but not LPS and is not suppressed by dexamethasone. Mucosal production of NO in the intestine during acute diarrheal disease may have adaptive effects by increasing local blood flow and peristalsis to accommodate fluid secretion. Protective effects of NO may include the bactericidal activity of reactive NO metabolites to limit the infectious burden and decreasing epithelial cell viability to promote removal of secreting cells. These data reveal a previously unrecognized aspect of the biology of the host response to this serious diarrheal disease.
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ACKNOWLEDGEMENTS |
---|
We thank Karen Coffee, Audrey Fleming, Dawn Holmes, and Dennis Knapp for technical assistance, John Besser-Weik (Minnesota Department of Health) for E. coli isolates, and Ann Emery for secretarial expertise.
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FOOTNOTES |
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This work was supported by the Veterans Affairs Research Service, National Institutes of Health Grants AI-31373 and DE-42600, and the Virus Research Institute (Cambridge, MA) through a cooperative research and development agreement with the Walter Reed Army Institute of Research.
Address for reprint requests: L. Raij, Veterans Affairs Medical Center, Renal Section (111J), One Veterans Drive, Minneapolis, MN 55417.
Received 15 October 1996; accepted in final form 11 August 1997.
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