1 Department of Integrative Biology, University of Texas at Houston Medical School, Houston 77030; and 2 Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas 77030
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ABSTRACT |
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Rotaviral infection in neonatal animals and
young children leads to acute self-limiting diarrhea, but infected
adults are mainly asymptomatic. Recently, significant in-roads have
been made into our understanding of this disease: both viral infection and virally manufactured nonstructural protein (NSP)4 evoke
intracellular Ca2+ ([Ca2+]i)
mobilization in native and transformed gastrointestinal epithelial cells. In neonatal mouse pup mucosa models,
[Ca2+]i elevation leads to age-dependent
halide ion movement across the plasma membrane, transepithelial
Cl secretion, and, unlike many microbial enterotoxins,
initial cyclic nucleotide independence to secretory diarrhea.
Similarities between rotavirus infection and NSP4 function suggest that
NSP4 is responsible for these enterotoxigenic effects. NSP4-mediated
[Ca2+]i mobilization may further facilitate
diarrhea by signaling through other Ca2+-sensitive cellular
processes (cation channels, ion and solute transporters) to potentiate
fluid secretion while curtailing fluid absorption. Apart from these
direct actions in the mucosa at the onset of diarrhea, innate
host-mediated defense mechanisms, triggered by either or both viral
replication and NSP4-induced [Ca 2+]i
mobilization, sustain the diarrheal response. This secondary component
appears to involve the enteric nervous system and may be cyclic
nucleotide dependent. Both phases of diarrhea occur in the absence of
significant inflammation. Thus age-dependent rotaviral disease
represents an excellent experimental paradigm for understanding a
noninflammatory diarrhea.
ion transport; virus-induced diarrhea
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INTRODUCTION |
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ROTAVIRAL DIARRHEAL ILLNESS is one of the most common infectious diseases in preschool children. Approximately 3 million children worldwide die of diarrhea annually, of which 600,000-800,000 deaths are attributed to rotavirus (RV). Most of these deaths occur in developing countries (the Indian subcontinent, sub-Saharan Africa, and some areas of Central and South America), where a child's risk of mortality after infection is estimated to be 1 in 200 or greater. The first licensed vaccine significantly prevented severe disease [1 million children vaccinated, 69-91%] but was voluntarily withdrawn from the United States, and thence the world market, because of a significantly increased relative risk of intussusception after the first or second dose of the vaccine (22). The continuing need for new therapeutic approaches for RV disease prevention and treatment highlights the great necessity for better understanding of the pathobiology underlying this disease. This themes article focuses on new and interesting developments in this area. The reader is also referred to several reviews relevant to rotavirus infection (5, 28). Limitations on the number of citations allowed in this article format have necessarily meant that many important papers have not been credited.
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PATHOPHYSIOLOGY OF ROTAVIRAL GASTROENTERITIS |
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Individuals from all age groups are susceptible to RV infection, but diarrhea is predominantly induced in young children, in whom infections are the most frequent source of acute, self-limiting diarrheal disease at the ages of 6 months to 1-2 years. Thus a clear age dependence exists. RV is relatively unique in this respect, because many bacterial toxigenic diarrheas cause severe clinical disease in children but exhibit no age dependence or predominantly affect older age groups (i.e., the highest incidences of most Vibrio cholera and Escherichia coli toxigenic diarrheas occur in adolescent/adult populations; Ref. 9). Besides diarrhea, other clinical features of RV disease include anorexia, depression, dehydration, and vomiting. In developing nations, death attributed to severe dehydration secondary to gastrointestinal fluid loss is often exacerbated by the poor nutritional status of these children and high incidences of concomitant infection with other gastrointestinal pathogens (5, 9).
RV has been shown to chronically infect immunocompromised children within the 6- to 18-month age group and older children and adults. Under these circumstances, sporadic longer-term diarrheal disease is recorded and has been associated with a prolonged period of viral replication and shedding. Normally, both viral replication and shedding resolve within 5-12 days of the onset of infection and gastrointestinal inflammation is low and infrequent or absent. RV-induced diarrhea in older populations can be explained by age-dependent decreases in immune status and innate defense. This diarrhea is correspondingly more complex than that recorded in infants (9). Our understanding in this area is accordingly very limited. What do we know concerning the mucosal and cellular mechanisms underlying noninflammatory RV diarrhea in children? Recent research from a number of different areas has expanded our knowledge.
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VIROLOGICAL FINDINGS |
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Considerable effort has been expended in identifying viral proteins causally linked to diarrhea. Studies have clearly shown that, although the onset of watery diarrhea follows detection of virus particles in the stool and intestinal contents (usually within 12 h after infection), viral shedding may continue days to weeks after symptomatic recovery, depending on immunocompetence in both humans and animal models (6). Thus, although viral infectivity is important for disease initiation, there is a poor correlation thereafter. The search for causative viral genes and proteins through genetic reassortment studies of this double-stranded RNA virus (containing 11 genes) has identified a number of virulence factors: genes that encode structural proteins (VP3, VP4, VP6, and VP7) and genes that encode nonstructural proteins [NSP1, NSP2, and NSP4; reviewed in Ref. 8]. VP4 and VP7 are capsid proteins found on the outer proteinaceous layer of the virus. VP4 is important for viral adsorption and penetration into epithelial cells, and VP7 may also play a role in these functions. VP3 and VP6 encode proteins required for RNA transcription and correct viral structure. Little is understood concerning the functions of most of the nonstructural proteins; they may facilitate viral replication and thus increase the efficiency of virus formation. However, NSP4 is the first described viral enterotoxin. NSP4 has uniquely been shown to promote Ca2+-mediated enterotoxigenic effects causally linked to diarrhea. Although there is little evidence for the direct roles of any other RV proteins in mediating enterotoxigenic diarrhea, their requirement in the replication process for efficient viral production suggests that they may indirectly influence late stages of diarrhea when the buildup of cellular viral proteins appears to trigger nonimmune innate host-defense mechanisms that sustain and potentiate the mucosal enterotoxigenic effects of NSP4.
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USE OF ANIMAL MODELS |
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Because of the impossibility of studying RV disease in children or surgically excised human neonatal tissues for ethical reasons, most of our current understanding of this multifactorial disease comes from studies in animal models. Both large and small mammals exhibit RV infection and pathogenesis (reviewed in Ref. 6). Disease severity and location within the gastrointestinal tract vary among animal species, inoculum used (viral strain and dose), immune status, age on infection, and host intestinal physiology. Age-dependent RV disease is a major agricultural concern for large-animal (calves, foals, lambs, and piglets) breeding facilities. However, studies in these models are prohibitively expensive. As a result, the best-characterized model is presently the mouse, with investigations in both rabbit and rat models also being conducted (5). Naive mice challenged with low doses of murine RV become infected (homologous infection) and exhibit age-dependent diarrheal symptoms. Furthermore, infection spreads to other naive mice with equal incidence in both pup and adult populations in terms of the intensity and duration of virus shedding. Similar phenomena are recorded for all other homologous and heterologous (e.g., nonmurine virus infection in mice) virus-mammalian host-specific interactions. Viral clearance is linked to the development of virus-specific intestinal IgA, similar to human infection (12). Furthermore, age-dependent resistance to RV infection appears to be mediated by acquired immunity, and a similar mechanism is thought to be operative as children age.
The mucosal site of rotavirus interaction in animal models, as in humans, is usually limited to mature enterocytes at the tips of the villi in the small intestine. However, segmental variability (duodenum > jejunum > ileum) can exist between animal species and between homologous and heterologous RV infection. It is not known whether these tropisms reflect the restricted location of a specific receptor for cellular viral entry or whether differentiated enterocytes express other factors required for efficient infection and replication. The identification of a cellular receptor for human RV, and its homologues in animal models, will clarify these issues.
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PHYSIOLOGICAL FINDINGS |
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A number of reviews have outlined hypotheses explaining age-dependent RV diarrhea in young children and animals (5, 8). Watery diarrhea may be caused by 1) changes in small intestinal surface area, leading to a reduction in net fluid absorption at a time when the colonic absorptive reserve may not be fully developed, 2) changes in mucosal osmotic permeability secondary to mucosal destruction, and 3) changes in fluid and electrolyte secretion. All may contribute at different times to diarrheal production.
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WHAT CELLULAR MECHANISMS DEFINE RV DIARRHEA? |
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There are both cellular and clinical definitions of secretory
diarrhea. The former is based on observations made in vitro and states
that epithelial cells exhibit complex but coordinated interactions
among plasma membrane channels, pumps, and cotransporters to cause
electrogenic exit of Cl across the apical plasma
membrane. The accompanying paracellular movement of Na+ and
H2O leads to accumulation of fluid within the lumen of the gastrointestinal tract and, subsequently, diarrhea. Clinically, secretory diarrhea is diagnosed when measurements of fecal fluid Na+, K+, and accompanying anion concentrations
account for isosmotic balance with plasma. This contrasts with osmotic
diarrheas, when nonabsorbed solutes within the lumen prevent fluid
absorption and an osmotic gap occurs. During RV infection in mice, net
water transport is associated with luminal anion and accompanying
cation concentrations that do not exhibit an osmotic gap and, in fact, can exceed plasma levels (30). This is consistent with the
presence of a mucosal cell-mediated secretory diarrhea.
The complex etiology of RV diarrhea can be viewed from either of two perspectives: 1) that RV diarrhea is initiated by viral interaction (signaling) within the host to ensure transmission and propagation and thus represents a means of virus spread and survival or 2) that RV diarrhea is a consequence of host mucosal defense and thus represents the activation of endogenous mechanisms by which this microorganism is removed from the intestinal environment. These perspectives highlight two cellular mechanisms active during RV diarrhea and provide insights into the expected efficacy of existing therapeutic approaches for the treatment of both early- and late-stage diarrheal disease.
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DIRECT VIRUS-MEDIATED DIARRHEA: CELLULAR EFFECTS OF ROTAVIRUS NSP4 ENTEROTOXIN |
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Rotaviral infection has been shown by a number of groups
to cause sustained intracellular Ca2+
([Ca2+]i) mobilization in gastrointestinal
cell lines (28), and the pleiotropic consequences of
elevated [Ca2+]i have led to the generation
of several new ideas (Fig. 1).
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The first of these relates to the fact that rotavirus infection and elevated [Ca2+]i in cultured, nonpolarized epithelial cells leads to cytolysis. It has therefore been hypothesized that similar events occur in vivo, leading to a loss of small intestinal surface mucosa and reduced fluid absorptive area. This cellular destruction scenario would explain the dramatic villus flattening observed in pig and calf models. However, such dramatic histological changes would also be expected to be associated with activation of significant immune responses, similar to those reported for mucosal-damaging bacterial toxins elaborated during dysentery and invasive diarrheal syndromes. This would result in a large inflammatory component to diarrhea. However, this does not occur in children and in many animal models. In mice, rotavirus infection is not associated significantly with mucosal inflammation; only mild mononuclear infiltration of the lamina propria is seen (4, 8, 12), and histopathological changes are limited to cytoplasmic vacuolization in a percentage of small intestinal enterocytes at the tips of the villi. Pigs, rabbits, calves, and lambs exhibit more pronounced histological changes that are seen at the site of viral replication 24-72 h after infection. The conclusion that mucosal destruction is unlikely to play a primary role in the propagation of diarrhea in animals is based on the findings that 1) diarrheal onset occurs during subclinical levels of infectious load, before alterations in cytopathology, 2) prophylactic treatment with cytoprotective growth factors inhibits histologic changes in pig models while failing to affect diarrhea (reviewed in Ref. 8), and 3) polarized monolayers of cultured epithelial cells, in contrast to their nonpolarized counterparts, fail to exhibit cytolysis when infected with RV (15).
The novel discovery of a rotavirus-produced enterotoxin was made by
serendipity when individual RV genes were expressed in cells (Ref.
8; Fig. 2A). The
nonstructural protein NSP4 was identified as the only gene product
capable of eliciting [Ca2+]i mobilization,
thus mimicking RV infection. Furthermore, when mice were injected with
a 22-amino acid peptide synthesized from the COOH terminus of this
175-amino acid protein, diarrhea was recorded within 4 h. The
full-length protein similarly produced diarrhea, whereas peptides from
regions outside of amino acids 96-135 did not. Diarrhea mimicked
that recorded for virulent, homologous virus infection, exhibiting a
similar severity but less prolonged time course. Furthermore,
immunization of pregnant dams with physiologically active
NSP4114-135 peptide conferred resistance against
homologous RV-elicited diarrhea to pups, confirming a major role of
this peptide in the pathogenic process. When
NSP4114-135 was added to pup small intestinal mucosal
sheets mounted in Ussing chambers, a Ca2+-dependent
component to transepithelial Cl secretion was recorded.
However, whereas this current was macroscopically similar to carbachol
responses in pup mucosa, it was lost in adult mucosa. A unique age
dependency similar to the whole animal diarrheal response was therefore
demonstrated for NSP4 and not for any other RV protein.
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More recently, in vitro studies have shown that after viral replication in cells, a 7-kDa peptide of NSP4 containing a physiologically active COOH-terminal domain (amino acids 112-175) is released into the medium of virus-infected cells via a nonclassic, Golgi-independent cellular secretory pathway (33). This suggests that extracellular NSP4 (eNSP4) is available for subsequent enterotoxigenic effects on the surrounding mucosa and provides a physiological basis for immunoprotection after NSP4 immunization. As indicated by recombinant protein studies, this endogenously produced eNSP4 peptide binds to an as yet unidentified apical membrane receptor to mobilize [Ca2+]i through phospholipase C (PLC) signaling and to preferentially stimulate [Ca2+]i-sensitive halide influx into neonatal, but not adult, intestinal enterocytes and colonocytes. The fact that [Ca2+]i mobilization occurs regardless of mucosal age but differs from classic PLC agonists in its pertussis toxin sensitivity (unpublished observation) and age-dependent secretory activity supports the hypothesis that NSP4's enterotoxigenic actions occur distal to the Ca2+ rise, at the level of the plasma membrane anion movement. NSP4 is proposed to elicit anion secretion through an interaction with luminal, age-dependent Ca2+-sensitive anion channels.
Secondary effects on basolateral Ca2+-sensitive
K+ conductances, which could facilitate transcellular
Cl secretion via cellular hyperpolarization and the
upregulation of basolateral secondary active Cl
uptake
(20), together provide a cellular basis for the
age-dependent transcellular secretion recorded during RV diarrhea.
Evidence supporting this hypothesis comes from studies made in cystic
fibrosis (CF) transmembrane conductance regulator (CFTR)-deficient mice (21). In CFTR-deficient CF mouse pups, NSP4 continues to
evoke both age-dependent diarrhea and age-dependent
[Ca2+]i-sensitive changes in enterocyte and
colonocyte plasma membrane halide influx. In contrast, the classic
[Ca2+]i-mobilizing secretagogue carbachol and
the cAMP-mobilizing diterpene forskolin fail to provoke similar
phenomena. Thus RV-induced secretory diarrhea is not mediated by CFTR,
and the molecular identity of the responsible channel remains to be determined.
This unique aspect of NSP4-mediated [Ca2+]i signaling (i.e., a role as the first described viral enterotoxin) may represent only part of the overall picture. Dramatic intracellular NSP4 production recorded 24 h after RV infection, as evidenced by in situ hybridization studies in intestinal villi isolated from virus-infected mice (2), and the pathophysiological consequences of sustained [Ca2+]i mobilization during this period may underlie many cellular responses recorded after RV infection. When coupled with virus-mediated repression of endogenous (host cell) protein synthesis, [Ca2+]i mobilization is expected to affect the functioning of a variety of host cell Ca2+-sensitive processes, enzymes, and transporters. Thus inhibition of Ca2+- and G protein-sensitive intracellular vesicular transport and protein folding (reviewed in Ref. 1) may explain decreases in apical hydrolase polarization indices recorded during RV infection (15) and changes in absorptive sugar transport (reviewed in Ref. 14). Recent evidence additionally suggests that NSP4 may directly inhibit the functioning of the cellular Na+-dependent glucose transporter SGLT-1 (14). Extracellular and/or intracellular NSP4 may further contribute to diarrheal pathogenesis by altering the dynamics of intracellular actin distribution and intracellular contacts, as well as effecting changes in paracellular permeability (reviewed in Ref. 5). All of the above phenomena are evident after viral replication.
Another related and potentially important new finding has been the publication of the crystal structure for NSP495-137 (3). Modeling predicts that this portion of the full-length NSP4 molecule, encompassing the enterotoxigenic peptide sequence (NSP4114-135), may form a homotetrameric pore. This could potentially span the cellular endoplasmic reticulum (ER) membrane and act as a Ca 2+ channel. No direct evidence currently supports this hypothesis. However, 1) the interior hydrophilic surface of the predicted pore contains a metal (Ca2+)-binding region (3) and 2) previous studies (reviewed in Ref. 8) have shown that endogenously expressed NSP4, although failing to alter plasma membrane divalent cation permeability, can induce non-PLC-dependent changes in ER membrane Ca2+ release when expressed within cells. This potentially important secondary mode of NSP4-mediated [Ca2+]i mobilization, again requiring intracellular NSP4 synthesis, may both potentiate the enterotoxigenic [Ca2+]i signal of extracellular NSP4 or itself initiate disease at later times after infection.
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SECONDARY HOST-MEDIATED DIARRHEA: VILLUS ISCHEMIA AND INVOLVEMENT OF THE ENTERIC NERVOUS SYSTEM |
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RV infection in mouse pups has been correlated with changes in
vascular circulation and reversible cellular ischemia in the villus tip during the periods of highest viral replication [48-72 h after infection (24); Fig. 2B]. After this
period, incipient hyperemia is recorded for another 24-48 h before
a second phase of ischemia leading to the initiation of villus
damage. The significance of these detailed ultrastructural studies has
until recently been difficult to ascertain. The original authors
suggested that release of a vasoactive substance was likely to underlie
the initial ischemic response, leading to functional changes
to, but not loss of, mucosal enterocyte integrity. Whether similar
phenomena are widespread in other RV-infected animal models has not
been clearly researched. However, other microbial toxins, most notably
cholera toxin, have been shown to cause parallel and often exaggerated
structural changes in human small intestinal villi, the extent of which
determines the severity of clinical illness (18).
Furthermore, the duration of both cholera and RV diarrhea illness is
reduced after oral aspirin (nonsteroidal anti-inflammatory) therapy,
and elevated levels of prostaglandins (notably PGE2 and
PGF2) have been recorded in the plasma and stool of
RV-infected children (32) and cholera-infected adults
(reviewed in Ref. 17). These facts point toward the
existence of similar prostaglandin-dependent cellular mechanisms
responsible for ultrastructural changes in both instances. In cholera,
this phenomenon is linked to activation of submucosal nervous reflexes
that form part of the enteric nervous system (ENS).
Histological staining in rodents and other animal models has clearly
demonstrated that vasoactive intestinal polypeptide (VIP) is
ubiquitously expressed throughout neuronal cells of the myenteric plexus, establishing it as a major secretagogue released by a number of
local ENS intramural reflexes. In cholera, good experimental evidence
demonstrates that mucosal enterochromaffin cells release 5-hydroxytryptamine (5-HT). Interactions with mucosal
5-HT2 receptors and local neuronal 5-HT3 or
5-HT4 receptors (species specific) elicit neuronal VIP
release. In addition, mucosal VIP effects and the luminal overflow of
mucosally derived PGE2 (reviewed in Refs. 17
and 25) result in both protein kinase C and phosphoinositol hydrolysis-dependent increases in fluid (Cl or
HCO
Alternatively, RV-induced ischemia could evoke acute changes in cellular nitric oxide (NO) production (reviewed in Ref. 27), with corresponding vascular effects leading to mucosal cell PGE2 production and cGMP-dependent anion secretion via a local nonadrenergic, noncholinergic branch of the ENS. Mucosal cGMP effects differentiate the pathologies of E. coli. heat labile enterotoxin (LT) and heat stable enterotoxin A (STa) from the effects of cholera toxin-stimulated 5-HT production, even though all are dependent on ENS-neuronal reflex pathways (10).
Direct evidence for ENS involvement in late-phase RV-induced diarrhea in mice has been published (16). Treatment of mice with drugs that affect ENS function significantly inhibited RV-mediated net fluid transport in organ bath experiments and altered transmucosal potentials toward values consistent with anion secretion and away from cation absorption. These studies, conducted 48-60 h after infection, were performed in mice exhibiting classic macroscopic signs of reactive hyperemia: tissue edema, vasodilation, and ischemia. Analysis of the data demonstrated that 66% of RV-induced net fluid secretion was mediated by this ENS secretomotor reflex arc because of its sensitivity to TTX (neuronal cation channel inhibitor), lidocaine (local anesthetic), or mesalamine (ganglionic inhibitor). Diarrhea was also inhibited in mice injected intraperitoneally with lidocaine, demonstrating consistency of the effects at the whole animal level.
One clinical consequence of the activation of such a mechanism would be
the potentiation but not initiation of diarrhea evoked by NSP4
enterotoxin. In this respect, clinical studies with the enkephalinase
inhibitor racedotril (acetorphan) in hospitalized RV-infected children
have shown that this drug reduces diarrhea duration from an average of
48 to 24 h after admission (29). Racedotril, a
useful agent for the treatment of a variety of acute and chronic
infectious and inflammatory diarrheas in all age groups, prevents the
breakdown of endogenous enkephalins within the gastrointestinal mucosa,
notably those that interact with antisecretory (cAMP lowering) -receptors expressed on mucosal epithelial cells. This prevents elevations in cAMP levels and, hence, cyclic nucleotide-dependent anion
secretion. These studies provide strong clinical evidence for the
activation of this innate defense mechanism triggered by reactive
hyperemia after subcytotoxic mucosal ischemia. Any subsequent
inflammatory burden caused by ischemia-induced mucosal damage,
the amplifying effects of local or species-specific proinflammatory cytokine release, or immunodeficiency would then serve to magnify this
host-mediated diarrheal component.
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CORRELATIONS AND DIFFERENCES BETWEEN DIARRHEAL EFFECTS OF VIRAL NSP4 AND BACTERIALLY DERIVED TOXINS |
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Many infectious agents and all bacterial enterotoxins cause
diarrhea by affecting cellular ion secretion. The classic example is
the direct mucosal action of cholera toxin, which increases cAMP
levels, leading to the opening of the apical plasma membrane cAMP-responsive anion channel to cause fluid secretion. This
conductance has been studied extensively (reviewed in Ref.
20) and is a property of CFTR. In CF patients, this
channel either does not function very well or does not function at all.
This results in a lack of fluid secretion within epithelial organs
including the epithelial linings of the gut and airways. Heat-stable
enterotoxins produced from both E. coli and Yersinia
enterocolitica use a parallel pathway to cause secretory diarrhea
via elevated cGMP (reviewed in Ref. 23). On the other
hand, enteroadherent bacteria such as Salmonella typhimurium
produce a soluble mediator, flagellin, that potently stimulates an
inflammatory diarrhea through nuclear factor (NF)-B dependent
interleukin (IL)-8 production with ultimately the luminal recruitment
of polymorphonuclear leukocytes (PMN; Ref. 13). PMN are a
source of luminal 5'-AMP that, on conversion to adenosine, leads to
receptor activation and CFTR Cl
channel opening.
Therefore, a common theme for both enterotoxigenic and enteroadherent
bacteria is direct or indirect activation of the cellular CFTR
Cl
channel through elevation in cellular cyclic
nucleotide levels. Furthermore, these pathways are influenced by the
ENS. Noninflammatory and inflammatory conditions cause the release of
multiple peptides from the mucosal epithelium and/or luminal PMN, which
potentiate this response by stimulating either the cholinergic
interneurons or the secretomotor VIPergic afferent nerves within the
myenteric plexus to produce the same net hypersecretory effect
(reviewed in Ref. 10).
The primary cellular mechanism used by the NSP4 enterotoxin appears to
be different from all other enterotoxins. Loss of function of the
cAMP-activated CFTR Cl channel protects individuals with
CF and transgenic mice from the diarrheal effects of both V. cholera (CTX) and E. coli (STa and LT) toxins and,
importantly, ENS-VIPergic nerve activation. However, this genotype
fails to protect CF children and mice from age-dependent RV diarrhea.
Thus age-dependent diarrheal onset induced by RV and NSP4, unlike the
non-age-dependent diarrhea recorded after toxigenic increases in
cellular cyclic nucleotide levels, does not appear to be mediated by
activation of the cellular CFTR Cl
channel. Therapeutic
approaches aimed at preventing ENS involvement during RV disease would
therefore be expected to affect the second phase of the disease process
but not to affect the primary diarrhea initiated by NSP4.
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IS NSP4 ENTEROTOXIN UNIQUE IN ITS MUCOSAL INTERACTIONS? |
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The direct mucosal interaction of E. coli-derived
heat-stable enterotoxin B (STb) may be an exception to the concept that NSP4's mucosal interactions are unique. Cellular extracts of
STb-elaborating E. coli have been shown to elicit cyclic
nucleotide-independent fluid secretion and to increase short-circuit
current across pig small intestinal mucosa mounted in Ussing chambers
(reviewed in Refs. 7, 26). Measurement of
electrolyte content of intestinal segments further suggests that STb
stimulates HCO
In mouse intestinal loop assays, purified STb toxin has been confirmed to cause second-phase ENS-mediated diarrhea that is inhibited by aspirin and indomethacin without altering cAMP or cGMP levels; it also causes luminal PGE2 release and vascular dilation. Furthermore, the quantity of fluid secreted has been correlated with PGE2 generation and mucosal cell levels of arachidonic and phosphatidic acid metabolism. More recently, both mucosal PGE2 release and 5-HT generation by intestinal enterochromaffin cells have been shown to underlie this response, which in rodents is partially sensitive to the 5-HT antagonist ketanserin and to drugs that inhibit ENS activity such as lidocaine, tetrodotoxin, and atropine. At the cellular level, STb, like NSP4, has also been shown to cause the mobilization of [Ca2+]i in mucosal cell lines through a pertussis toxin-sensitive G protein-dependent mechanism. Thus a number of similarities between NSP4 and STb exist at the level of primary and secondary phases of the disease process.
However, unlike NSP4, STb does not exhibit a pronounced age dependence, and, in fact, disease is nearly always seen in adult animals and is rarely recorded in humans (reviewed in Ref. 7). These results suggest that, whereas NSP4 requires a cellular specificity expressed in neonatal mucosa, STb conversely utilizes a receptor specifically expressed in adult mucosa. Apart from this dramatic difference, which determines the susceptibility of the host to diarrhea onset, the Ca2+-mobilizing properties of both toxins through changes in mucosal cell PLC and phospholipase A2 activity and subsequent mucosal PGE2 production may, in concert with local ENS reflexes, promote the second phase of diarrhea. If NSP4-induced [Ca2+]i mobilization is limited to cells of the intestinal villus and crypt and not found in neuronal plexus cells, an enteroendocrine cell axis for NSP4-ENS interaction should be found.
Finally, sustained [Ca2+]i mobilization in
cells by a wide variety of agonists leads to cellular stress and a
NF-B-driven cAMP- and PLC-independent diarrhea with both
similarities and differences to that evoked by NSP4. This pathway
involves the upregulation of mucosal galanin-1 receptors, typically
over a time course of 3 days, leading to a Ca2+- and
pertussis toxin-sensitive fluid
(Cl
/HCO
secretion is unknown, both enteric nerve- and
inflammatory cell-derived galanin sustain this phenomena. Presently, it
is unclear whether this represents a pathway common to or separate from
that characterized by ischemia-derived reactive hyperemia.
Although this Ca2+-mediated, CFTR-independent diarrhea
resembles that elicited by RV infection and NSP4 inoculation, salient
differences include its extended time of onset (days vs. hours for
NSP4), the lack of both PLC and age dependence, and the presence of
significant mucosal PMN recruitment and/or inflammation. Finally, NSP4
bears no structural resemblance to galanin and would not be expected to
activate the galanin-1 receptor. However, the question remains as to
whether NSP4 could possibly be circumventing the cellular requirement
for this receptor by acting at an intracellular site, for instance, at
the level of a common pertussis toxin sensitivity to G protein
signaling. In conclusion, although this ENS-dependent mechanism is
unlikely to be active during the onset of diarrhea mediated by RV in
noninflamed mucosa, an intriguing question remains as to whether NSP4
has evolved to utilize intracellular signaling aspects of the galanin-1
receptor response to promote a second-phase diarrheal disease.
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FUTURE DIRECTIONS |
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There are still many unanswered questions with regard to the
cellular basis of the multifaceted RV-induced disease that leads to
age-dependent diarrhea. When defining RV secretory diarrhea, current
studies have emphasized early events stimulating age-dependent plasmalemma anion movement at the level of the mucosal cell before more
complex secondary phenomena arise. A number of salient questions at
this stage of the disease process remain unanswered. At the single-cell
level these questions remain: 1) What is the biophysical nature of the NSP4 enterotoxin-stimulated, age- and
Ca2+-dependent cellular halide secretion? 1)
Does non-CFTR-dependent Ca2+-activated
HCO transport contribute to
the secretory diarrheal response? 3) What is the importance
of the nontypical pertussis toxin sensitivity to NSP4-mediated
[Ca2+]i mobilization, particularly with
regard to cellular PLC isoform requirement? 4) What cell
type(s) are affected (exocrine, goblet, endocrine)? Even less is known
at the whole tissue level and the development of the second-stage
diarrheal response. For instance, does a specific cell type transfer
NSP4 mucosal signaling effects into the ENS through release of a
mucosal cell neuroactive substance, or is this effect more general,
reflecting ischemia-derived reactive hyperemia and an innate
mucosal defense mechanism? Finally, although RV disease is not
characterized by inflammation, how inflammatory conditions within the
mucosa affect RV diarrhea remains largely unanswered. The close
parallels arising from recent investigations into bacterial microbial
interactions with the mucosa and enterotoxigenic signaling will provide
excellent blueprints for further understanding the pathogenesis of RV-
and NSP4-mediated diarrhea.
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ACKNOWLEDGEMENTS |
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We thank the following for their helpful critique of this article: Dr. Johnny Peterson, Department of Microbiology and Immunology, University of Texas Medical Branch at Galveston; Dr. Norman Weisbrodt, Department of Integrative Biology, University of Texas at Houston; Dr. Robert Bridges, Department of Cell Biology and Physiology, University of Pittsburgh; and Drs. Max Ciarlet and Margaret Conner, Department of Molecular Virology and Microbiology, Baylor College of Medicine.
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FOOTNOTES |
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Research on RV in the authors' laboratories is supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-30144 and DK-56338.
Address for reprint requests and other correspondence: A. P. Morris, Dept. of Integrative Biology and Internal Medicine-Gastroenterology, Medical School, Univ. of Texas Health Science Center at Houston, 6431 Fannin, Houston, TX 77030 (E-mail: Andrew.P.Morris{at}uth.tmc.edu).
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REFERENCES |
---|
1.
Ashby, MC,
and
Tepikin AV.
ER calcium and the functions of intracellular organelles.
Semin Cell Dev Biol
12:
11-17,
2001[ISI][Medline].
2.
Boshuizen, JA,
Reimerink J,
Rossen JW,
Koopmans M,
Buller H,
Dekkar J,
and
Einerhand AW.
Rotavirus infection induces a shut-off of endogenous gene expression in mouse small intestine (Abstract).
Gastroenterology
118:
A2373,
2000.
3.
Bowman, GD,
Nodelman IM,
Levy O,
Lin SL,
Tian P,
Zamb TJ,
Udem SA,
Venkataraghavan B,
and
Schutt CE.
Crystal structure of the oligomerization domain of NSP4 from rotavirus reveals a core metal-binding site.
J Mol Biol
304:
861-871,
2000[ISI][Medline].
4.
Casola, A,
Estes MK,
Crawford SE,
Ogra PL,
Ernst PB,
Garofalo RP,
and
Crowe SE.
Rotavirus infection of cultured intestinal epithelial cells induces secretion of CXC and CC chemokines.
Gastroenterology
114:
947-955,
1998[ISI][Medline].
5.
Ciarlet, M,
and
Estes MK.
Rotaviruses and calicivirus infections of the gastrointestinal tract.
Curr Opin Gastroenterol
17:
10-16,
2001[ISI].
6.
Conner, ME,
and
Ramig RF.
Viral enteric diseases.
In: Viral Pathogenesis, edited by Nathanson N.. New York: Lippincott-Raven, 1997, p. 713-743.
7.
Dubreuil, JD.
Escherichia coli STb enterotoxin.
Microbiology
143:
1783-1795,
1997
8.
Estes, MK,
and
Morris AP.
A viral enterotoxin. A new mechanism of virus-induced pathogenesis.
Adv Exp Med Biol
473:
73-82,
1999[ISI][Medline].
9.
Farthing, MJ.
Diarrhoea: a significant worldwide problem.
Int J Antimicrob Agents
14:
65-69,
2000[ISI][Medline].
10.
Farthing, MJ.
Enterotoxins and the enteric nervous systema fatal attraction.
Int J Med Microbiol
290:
491-496,
2000[ISI][Medline].
11.
Fernandez-Moreno, MD,
Arilla E,
and
Prieto JC.
Effect of intestinal ischaemia on intestinal VIP levels and VIP interaction with intestinal epithelial cells from rat.
Comp Biochem Physiol A Physiol
93:
463-466,
1989[ISI].
12.
Franco, MA,
and
Greenberg HB.
Immunity to homologous rotavirus infection in adult mice.
Trends Microbiol
8:
50-52,
2000[ISI][Medline].
13.
Gewirtz, AT,
Rao AS,
Simon PO, Jr,
Merlin D,
Carnes D,
Madara JL,
and
Neish AS.
Salmonella typhimurium induces epithelial IL-8 expression via Ca(2+)- mediated activation of the NF-kappaB pathway.
J Clin Invest
105:
79-92,
2000
14.
Halaihel, N,
Lievin V,
Alvarado F,
and
Vasseur M.
Rotavirus infection impairs intestinal brush-border membrane Na+-solute cotransport activities in young rabbits.
Am J Physiol Gastrointest Liver Physiol
279:
G587-G596,
2000
15.
Jourdan, N,
Brunet JP,
Sapin C,
Blais A,
Cotte-Laffitte J,
Forestier F,
Quero AM,
Trugnan G,
and
Servin AL.
Rotavirus infection reduces sucrase-isomaltase expression in human intestinal epithelial cells by perturbing protein targeting and organization of microvillar cytoskeleton.
J Virol
72:
7228-7236,
1998
16.
Lundgren, O,
Peregrin AT,
Persson K,
Kordasti S,
Uhnoo I,
and
Svensson L.
Role of the enteric nervous system in the fluid and electrolyte secretion of rotavirus diarrhea.
Science
287:
491-495,
2000
17.
Marquet, F,
Pansu D,
and
Descroix-Vagne M.
Distant intestinal stimulation by cholera toxin in rat in vivo.
Eur J Pharmacol
374:
103-111,
1999[ISI][Medline].
18.
Mathan, MM,
Chandy G,
and
Mathan VI.
Ultrastructural changes in the upper small intestinal mucosa in patients with cholera.
Gastroenterology
109:
422-430,
1995[ISI][Medline].
19.
Matkowskyj, KA,
Danilkovich A,
Marrero J,
Savkovic SD,
Hecht G,
and
Benya RV.
Galanin-1 receptor up-regulation mediates the excess colonic fluid production caused by infection with enteric pathogens.
Nat Med
6:
1048-1051,
2000[ISI][Medline].
20.
Morris, AP.
The regulation of epithelial cell cAMP- and calcium-dependent chloride Channels.
Adv Pharmacol
46:
209-251,
1999[Medline].
21.
Morris, AP,
Scott JK,
Ball JM,
Zeng CQ,
O'Neal WK,
and
Estes MK.
NSP4 elicits age-dependent diarrhea and Ca2+-mediated I influx into intestinal crypts of CF mice.
Am J Physiol Gastrointest Liver Physiol
277:
G431-G444,
1999
22.
Murphy, TV,
Gargiullo PM,
Massoudi MS,
Nelson DB,
Jumaan AO,
Okoro CA,
Zanardi LR,
Setia S,
Fair E,
LeBaron CW,
Wharton M,
and
Livingood JR.
Intussusception among infants given an oral rotavirus vaccine.
N Engl J Med
344:
564-572,
2001
23.
Nair, GB,
and
Takeda Y.
The heat-stable enterotoxins.
Microb Pathog
24:
123-131,
1998[ISI][Medline].
24.
Osborne, MP,
Haddon SJ,
Worton KJ,
Spencer AJ,
Starkey WG,
Thornber D,
and
Stephen J.
Rotavirus-induced changes in the microcirculation of intestinal villi of neonatal mice in relation to the induction and persistence of diarrhea.
J Pediatr Gastroenterol Nutr
12:
111-120,
1991[ISI][Medline].
25.
Peregrin, AT,
Ahlman H,
Jodal M,
and
Lundgren O.
Involvement of serotonin and calcium channels in the intestinal fluid secretion evoked by bile salt and cholera toxin.
Br J Pharmacol
127:
887-894,
1999
26.
Peterson, JW,
and
Whipp SC.
Comparison of the mechanisms of action of cholera toxin and the heat-stable enterotoxins of Escherichia coli.
Infect Immun
63:
1452-1461,
1995[Abstract].
27.
Rolfe, VE,
and
Milla PJ.
Nitric oxide stimulates cyclic guanosine monophosphate production and electrogenic secretion in Caco-2 colonocytes.
Clin Sci (Colch)
96:
165-170,
1999[ISI][Medline].
28.
Ruiz, MC,
Cohen J,
and
Michelangeli F.
Role of Ca2+ in the replication and pathogenesis of rotavirus and other viral infections.
Cell Calcium
28:
137-149,
2000[ISI][Medline].
29.
Salazar-Lindo, E,
Santisteban-Ponce J,
Chea-Woo E,
and
Gutierrez M.
Racecadotril in the treatment of acute watery diarrhea in children.
N Engl J Med
343:
463-467,
2000
30.
Starkey, WG,
Collins J,
Candy DC,
Spencer AJ,
Osborne MP,
and
Stephen J.
Transport of water and electrolytes by rotavirus-infected mouse intestine: a time course study.
J Pediatr Gastroenterol Nutr
11:
254-260,
1990[ISI][Medline].
31.
Wagner, R,
Gabbert H,
and
Hohn P.
The mechanism of epithelial shedding after ischemic damage to the small intestinal mucosa. A light and electron microscopic investigation.
Virchows Arch
30:
25-31,
1979.
32.
Yamashiro, Y,
Shimizu T,
Oguchi S,
and
Sato M.
Prostaglandins in the plasma and stool of children with rotavirus gastroenteritis.
J Pediatr Gastroenterol Nutr
9:
322-327,
1989[ISI][Medline].
33.
Zhang, M,
Zeng CQ,
Morris AP,
and
Estes MK.
A functional NSP4 enterotoxin peptide secreted from rotavirus-infected cells.
J Virol
74:
11663-11670,
2000