McCormick Department of Pediatric Gastroenterology, Massachusetts General Hospital, and Department of Microbiology and Molecular Genetics, Harvard Medical School, Charlestown, MA, USA In
Trang 1Bacterial-induced hepoxilin A3 secretion as
a pro-inflammatory mediator
Beth A McCormick
Department of Pediatric Gastroenterology, Massachusetts General Hospital, and Department of Microbiology and Molecular Genetics, Harvard Medical School, Charlestown, MA, USA
Introduction
Recent studies suggest that 8S⁄
R-hydroxy-11,12-epoxyeicosa-5Z,9E,14Z-trienoic acid (hepoxilin A3;
HXA3) plays a central role in the directed migration of
neutrophils across mucosal surfaces infected with
pathogenic bacteria This review will discuss recent
advances made in understanding the complex
molecu-lar events that orchestrate the directional movement of
neutrophils across the mucosal surface during bacterial
infection of the intestinal tract and lung and will, in
particular, emphasize the important role played by the
eicosanoid HXA3
during bacterial infection and is a potent neutrophil chemoattractant
Bacterial pathogens continually confront epithelial bar-riers of the body, such as those of the gastrointestinal, respiratory and reproductive tracts Although mucosal surfaces are generally impermeable to most foreign entities, many microorganisms have developed sophis-ticated strategies to breach or alter this barrier In gen-eral, microbial pathogens have evolved the capacity to engage their host cells in very complex interactions commonly involving the exchange of biochemical
Keywords
arachidonic acid; chemotaxis; eicosanoid;
hepoxolin A3; inflammation; intestine; lung;
neutrophils; Pseudomonas aeruginosa;
Salmonella typhimurium
Correspondence
B A McCormick, Department of Pediatric
Gastroenterology, Massachusetts General
Hospital, Harvard Medical School, CNY 114
16th Street (114–3503), Charlestown,
MA 02129, USA
Fax: +1 617 7264172
Tel: +1 617 7264168
E-mail: mccormic@helix.mgh.harvard.edu
(Received 13 Oct 2006, accepted 23 May
2007)
doi:10.1111/j.1742-4658.2007.05911.x
Bacterial infections at epithelial surfaces, such as those that line the gut and the lung, stimulate the migration of neutrophils through the co-ordi-nated actions of chemoattractants secreted from pathogen-stimulated epi-thelial cells One such factor involved in attracting polymorphonuclear leukocytes across the epithelium and into the lumen has until recently remained elusive In 2004, we identified the eicosanoid, hepoxilin A3, to be selectively secreted from the apical surface of human intestinal or lung epi-thelial cells stimulated with Salmonella enterica serotype Typhimurium or Pseudomonas aeruginosa, respectively In this role, the function of hepoxilin
A3 is to guide neutrophils, via the establishment of a gradient, across the epithelial tight junction complex Interestingly, interruption of the synthetic pathway of hepoxilin A3 blocks the apical release of hepoxilin A3 in vitro and the transmigration of neutrophils induced by S typhimurium both in
in vitro and in vivo models of inflammation Such results have led to the discovery of a completely novel pathway that is not only critical for responses to bacterial pathogens but also has broad implications for inflammatory responses affecting mucosal surfaces in general Thus, the objective of this review was to highlight the recent findings that implicate hepoxilin A3as a key regulator of mucosal inflammation
Abbreviations
AA, arachidonic acid; HpETE, hydroperoxy-eicosatetraenoic acid; HXA 3 , 8S ⁄ R-hydroxy-11,12-epoxyeicosa-5Z,9E,14Z-trienoic acid
(hepoxilin A3); IL-8, interleukin-8; LOX, lipoxygenase; PKC, protein kinase C; PLA2, phospholipase A2.
Trang 2signals, the net result of which is often the triggering
of a host pro-inflammatory response At the forefront
of this inflammatory response is the infiltration of
neu-trophils to the site of bacterial insult Neuneu-trophils
rep-resent a class of crucial white cells needed to defend
the host from such pathogenic injury, and thus the
accumulation of neutrophils at inflamed sites
repre-sents a characteristic feature of the innate host
response However, the mechanisms by which
neu-trophils eradicate offending bacteria are nonspecific
and can lead to tissue damage, which, if excessive,
con-tributes to the pathology of the disease
To reach inflamed sites, neutrophils traverse various
barriers, including the endothelium, basement
mem-brane (intestine)⁄ interstitium (lung) and epithelium, in
response to localized inflammatory mediators Overall,
such directed migration of neutrophils involves the
integrated actions of cytokines, adhesion molecules
with specificity for specific ligands, as well as highly
timed and compartmentalized secretion of various
neu-trophil-specific chemokines Research from my
laborat-ory has begun to disclose the molecular and cellular
events underlying the directed infiltration of neutrophils
across epithelial mucosal surfaces during states of
bac-terial infection This work has led to the current
para-digm that intestinal epithelial cells respond to luminal
pathogens, such as Salmonella typhimurium, by
releas-ing distinctive pro-inflammatory neutrophil
chemo-attractants that sequentially orchestrate neutrophil
movement across the intestinal epithelium [1–4] More
specifically, S typhimurium–intestinal epithelial cell
interactions induce the epithelial release of the potent
neutrophil chemokine, interleukin-8 (IL-8) Such
baso-lateral IL-8 release imprints subepithelial matrices with
long-lived haptotactic gradients that serve to guide
neu-trophils through the lamina propria to a subepithelial
position [2] However, basolateral IL-8 release is
insuffi-cient to induce the migration of neutrophils across the
intestinal epithelium, suggesting that the production of
other inflammatory mediators, whose release would
probably be polarized apically, are important for the
execution of this step in the inflammatory pathway
[1,2] In support of this contention, Kucharzik et al
recently developed a double transgenic mouse model
with the ability to induce human IL-8 expression
restricted to the intestinal epithelium [5] The results
from this transgenic model showed that although acute
induction of IL-8 in the intestinal epithelium is
suffi-cient to trigger neutrophil recruitment to the lamina
propria, additional signals are required for neutrophil
transepithelial migration and mucosal tissue injury
Owing to the restrictive actions of the intestinal
epi-thelial tight junctions present at the neck of adjacent
epithelial cells, a distinct apical chemotactic factor would be required for the continued migration of neu-trophils across the epithelial tight junction We recently discovered that neutrophil transit through the epithelial monolayer to the luminal surface is directed by the ap-ically released eicosanoid, HXA3[4] HXA3is a hyroxy epoxide derivative formed from 12S-hydroperoxyei-cosa-5Z,8Z,10E,14Z-tetraenoic acid (12S-HpETE), the primary product of arachidonic acid (AA) formed by 12S-lipoxygenase (12S-LOX) Hepoxilins are documen-ted to possess a wide range of biological activities, with the A3 form having been shown to potentiate glucose-dependent insulin secretion [6], open S-type K+ chan-nels in Aplysia [7], modulate synaptic neurotransmission
in rat hippocampus [8], increase vascular permeability in rat skin [9] and induce chemotaxis of neutrophils at con-centrations as low as 30–40 nm [10] Our findings repre-sent the first demonstration that HXA3can be secreted from epithelial cells, and that such secretion is regulated
by conditions that contribute to inflammation [4]
HXA3 directly stimulates neutrophils via a pertussis toxin-sensitive receptor and elicits a Ca2+ signal [11] While these features are shared by most other chemo-kines, analysis of HXA3-elicited neutrophil activation reveals that, unlike other lipid- or peptide-based chemo-attractants, HXA3, even at saturating concentrations, elicits chemotactic activity in the absence of stimula-tion of superoxide producstimula-tion and⁄ or release of pri-mary and⁄ or secondary granules [3] Thus, HXA3 appears to function as a ‘pure’ neutrophil chemo-attractant Induction of polarized movement by neu-trophils across the tight junction in response to HXA3
is presumed to be achieved through its actions as a
Ca2+ signaling molecule, and our earlier report of intracellular Ca2+ events following HXA3 administra-tion to isolated human neutrophils are consistent with this hypothesis [3] Most recent studies indicate that the Ca2+signaling induced by HXA3appears to occur through the activation of an intracellular receptor [12]
A previous elegant study by Mills et al showed that HXA3induces a reorganization of Ca2+within human neutrophils from the endoplasmic reticulum into mito-chondria [13] In fact, HXA3has been shown to inhibit subsequent Ca2+ signaling events in cells where Ca2+ signaling is normally induced by fMet-Leu-Phe, plate-let-activating factor and leukotriene B4 [14] Further-more, the binding of HXA3 to a receptor in human neutrophils shows clear specificity for this eicosanoid compared with other compounds [15] in a manner sim-ilar to our previously reported observations [4]
Trang 3Functional consequences of HXA3
release: the role of AA metabolism
Intestinal inflammation
The biological capacity of 12-LOX and its enzymatic
products, such as HXA3, is underappreciated
com-pared with the well-documented functional roles of
5-LOX and 15-LOX products, such as leukotriene B4
and lipoxins, respectively Nevertheless, key pieces of
work have not only demonstrated the formation of
hepoxilins through the 12-LOX pathway [10], but have
also uncovered the intriguing observation that
epithe-lial 12-LOX can be regulated at sites of mucosal
inflammation Shannon et al noticed that in the
healthy colonic mucosal epithelium, cells do not
express 12-LOX, whereas in tissue from patients with
inflammatory bowel disease, the colonic tissue is not
only actively involved with the disease, but also
expres-ses 12-LOX in mucosal epithelial cells and displays an
increase in 12-LOX enzymatic activity [16] This study
was the first to demonstrate that 12-LOX participates
in colonic epithelial function It also provides the first
in situ evidence for a selective increase in epithelial
12-LOX in inflammatory disease Additionally, our
recent findings further demonstrate that inhibition of
the 12-LOX pathway, which is required for the
synthe-sis of HXA3, dramatically reduces neutrophil-mediated
tissue trauma associated with enteric infection [4]
Although these collective observations establish the
12-LOX pathway as yet another avenue for AA
meta-bolism involved in the events underlying inflammation,
such observations further underscore an emerging
con-cept suggesting that modulation of the 12-LOX
path-way during intestinal inflammation may be unique to
polarized epithelia and involved in host defense In the
particular case of HXA3, its stimulated production and
release from the apical surface of infected intestinal
epithelial cells provides an unprecedented pathway of
regulated actions by a chemoattractant and, in
addi-tion, identifies a new scheme in innate immune
responses crucial for mediating neutrophil movement
through epithelial surfaces Thus, while the epithelium
probably evolved to generate significant levels of
HXA3 in response to colonization by pathogens, it is
certainly possible that HXA3 generation is
dysregulat-ed under conditions such as inflammatory bowel
dis-ease because 12-LOX activity is induced at active sites
of this disease
Because HXA3 may play an important step
underly-ing the pathophysiology of inflammatory diseases, such
as inflammatory bowel disease, the investigation of
human 12-LOX genes at mucosal surfaces, and their
involvement with HXA3 production, becomes an important area of study As this is an area of research truly at its embryonic stage, at present, one can only speculate as to the 12-LOX gene(s) responsible for the synthesis of HXA3 at mucosal surfaces There are at least four 12-LOX isoforms expressed in human tissue [17,18] These include platelet-type 12-LOX (p12-LOX), epidermal-type 12-LOX (e12-(p12-LOX), 12R-LOX, and 12⁄ 15-LOX (human 15-LOX-1) Of these four, only three appear to be functional 12-LOXs [18]; although the human e12-lox transcript is expressed in skin and hair follicles, it has been reported to be a pseudogene, which lacks function [18], making it a less likely candidate for the synthesis of HXA3 Platelet-type 12-LOX is expressed in multiple tissues aside from platelets, and can also be regulated at the transcriptional level [17,18] The enzymatic expression
of 12R-LOX forms 12R-hydroperoxy-eicosatetraenoic acid (12R-HpETE) from AA with high specificity However, human 12R-LOX has very limited tissue dis-tribution and, to date, only normal and psoriatic human skin and tonsils have been found to express the enzyme and convert exogenous AA to 12R-HETE [19] Lastly, 15-LOX-1 produces primarily 15-HpETE, but can also produce 12-HpETE [20,21] Consequently, this enzyme has been referred to as 12⁄ 15-LOX and displays high homology (86.3%) at the protein level to bovine leukocyte type 12-LOX [22] Although the pro-duction of 12-HpETE is a side reaction of 15-LOX-1,
it represents an intriguing candidate for the involve-ment in HXA3production considering its expression in both intestinal and airway epithelial cells [23]
release
Identification of a factor such as HXA3, which is responsible for the transmigration of neutrophils across the mucosal barrier for entry into the intestinal lumen, has addressed an important question of epithe-lial pathobiology Studies exploring the mechanism underlying the release of HXA3 during infection with
S typhimurium revealed the involvement of the
S typhimurium type III secreted effector protein, SipA [24] The Salmonella effector protein, SipA, promotes
a lipid signal transduction cascade that recruits an ADP-ribosylation factor 6 guanine nucleotide exchange factor (such as ARNO) to the apical plasma mem-brane ARNO facilitates ADP-ribosylation factor 6 activation at the apical membrane, which in turn stimulates phospholipase D recruitment to and activity
at this site The phospholipase D product, phosphati-dic acid, is metabolized by a phosphohydrolase into
Trang 4diacylglycerol, which recruits cytosolic protein
kin-ase C (PKC)-alpha to the apical membrane Through
a process that is less understood, activated PKC-alpha
phosphorylates downstream targets that are
respon-sible for the production and apical release of HXA3,
which drives transepithelial neutrophil movement [25]
(Fig 1)
Although immune cells recruited in response to
S typhimurium, especially neutrophils, are thought to
be responsible for the clinical manifestations of this
infection, they probably play an important role in host
defense because nonimmunocompromised hosts
gener-ally clear this infection without medicinal intervention
(beyond hydration) Given that apically directed
migration of neutrophils is, by itself, thought to
contribute to epithelial cell dysfunction in a host of
mucosal diseases (i.e cystic fibrosis and chronic
obstructive pulmonary disease of the lung, cirrhosis of
the skin, and urinary tract infections) [26], it is
conceivable that HXA3 is produced by epithelial cells
at other mucosal surfaces Indeed, we have shown that lung epithelial cells produce HXA3 in response to Pseudomonas aeruginosa infection and HXA3, in turn, appears to mediate neutrophils transmigration across airway epithelial cells [27]
Lung inflammation The inflammatory response mounted against bacterial pathogens infecting the mucosal surface of the lung is highly complex and multifaceted Like the intestine, one of the destructive consequences of an over-aggres-sive inflammatory response is the accumulation of activated neutrophils in the airway lumen that can damage lung tissue Mounting evidence reveals that epithelial cells lining the luminal cavity, which separate the lumenal contents from the underlying tissue, are key players in orchestrating innate immune responses
Fig 1 Model of bacterial-induced signaling leading to the release of S ⁄ R-hydroxy-11,12-epoxyeicosa-5Z,9E,14Z-trienoic acid (hepoxilin A 3 ; HXA3) Interaction of bacterial pathogens of the intestine (S typhimurium) and lung (P aeruginosa) leads to the activation of a unique lipid signal transduction cascade resulting in the up-regulation and activation of phosphorylated protein kinase C (pPKC), the signaling kinase required for HXA 3 production Whether pPKC directly or indirectly leads to the activation of phospholipase A 2 (PLA 2 ) has yet to be deter-mined, but this enzyme is responsible for the membrane release of arachidonic acid (AA), the precursor of HXA3 Once liberated within the cytosol, AA is available as a substrate for 12-lipoxygenase (12-LOX), the enzyme responsible for the synthesis of HXA3 Through a mechan-ism yet to be determined, HXA 3 is then released apically where it forms a concentration gradient through the epithelial cell tight junction, resulting in the directed movement of neutrophils across the epithelial barrier PMN, polymorphonuclear leukocyte.
Trang 5Given the complexity of the route that neutrophils
must travel to reach the airway lumen (i.e through the
endothelium), the basement membrane, the interstitial
space, the epithelial basement membrane and the
epi-thelial layer, it is likely that multiple neutrophil
chemo-attractants participate at discrete steps during this
recruitment process In a scenario similar to the
intes-tine, during bacterial infection of the lung, such as
P aeruginosa infection, IL-8 probably plays a major
role in recruiting neutrophils from the bloodstream to
the epithelium, whereas the production and apical
secretion of the eicosanoid HXA3 is PKC dependent
and necessary for guiding neutrophils across the
infec-ted epithelium [27]
Current work from my laboratory is attempting to
define the mechanism(s) underlying HXA3 production
and neutrophil transepithelial migration in response to
infection with P aeruginosa One probable means to
increase the production of HXA3 is to increase the
availability of its precursor, AA The liberation of AA
from phospholipid membranes is presumed to be the
rate-limiting step for the generation of eicosanoids,
with the idea being that the greater amount of free AA
to serve as substrate for the 12-lipoxygenase enzyme,
the greater potential to produce 12-HpETE and
HXA3 The major mechanism to generate free AA for
subsequent conversion to eicosanoids (via
lipoxygenas-es and cyclooxygenaslipoxygenas-es) is by the action of
phospho-lipase A2 (PLA2) [28] PLA2 represents a family of at
least 19 distinct proteins, which have been grouped
into three subfamilies The sPLA2 subfamily contains
small (14–19 kDa) enzymes that are secreted by cells
and act on the lumenal surface of cell membranes to
liberate AA [29] Members of the cPLA2 family are
distinguished by a dependency on calcium and are
acti-vated by phosphorylation In addition, members of
this group are capable of shifting from the cytosol to
the perinuclear membrane where they interact with
phospholipids, resulting in the liberation of AA [29]
The third group is iPLA2, which resides in the cytosol
but its activation is independent of calcium [29]
Although isoforms of iPLA2 have generally been
believed to participate in phospholipid remodeling,
recent studies have also documented the involvement
of iPLA2 in mediating AA under certain circumstances
[30] Indeed, our recent studies have shown that PLA2
activity is required for P aeruginosa-induced
neutro-phil transepithelial migration [31] In addition, upon
infection, lung epithelial cells phosphorylate cPLA2
and release significantly more AA from membrane
stores [31] Based on these observations we have
hypo-thesized that increased PLA2 activity, which mediates
AA release, is obligatory for the production of HXA3,
which in turn is required for orchestrating neutrophil movement across lung epithelial monolayers (Fig 1)
It is worth noting, however, that is it controversial as
to whether the activation of PLA2 occurs by PKC Regardless, determination of the particular phospholi-pase A2 responsible for orchestrating neutrophil trans-epithelial migration may lead to targeted therapies designed to dampen inflammation in the lung
Summary
Thus far it has been shown that pathogenic bacterial interactions with either intestinal or airway epithelial cells results in signal transduction cascades, which lead
to the production and secretion of HXA3, most prob-ably through the action of the 12-LOX enzymatic pathway Therefore, this pathway may represent a con-served innate immune mechanism for detection and eradiation of pathogens interfacing with the host mucosal surface Of potential clinical significance, signaling pathways leading to HXA3 secretion may provide an important new therapeutic target for the treatment of acute and chronic diseases of intestinal, lung and perhaps other mucosal surfaces
Acknowledgements
I am indebted to the members of the McCormick Laboratory, past and present, who have contributed to this work A special thank you is reserved for Dr Randall J Mrsny This work was supported by the National Institutes of Health (DK56754)
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