,
* Departments of Pediatrics,
Microbiology and Immunology, and
Pathology, Dalhousie University, and the Dalhousie Inflammation Group, Halifax, Nova Scotia, Canada
Correspondence: Andrew Stadnyk, Ph.D., Infection and Immunology Research Laboratories, IWK Grace Health Centre, 5850 University Avenue, Halifax, Nova Scotia, B3J 3G9 Canada. E-mail: Andrew.Stadnyk{at}Dal.Ca
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Key Words: Trichinella spiralis • interleukin-1 • macrophage inflammatory peptide-2
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It is noteworthy that IEC are reported as a source of cytokines in various gastrointestinal inflammatory conditions, including infections, in which neutrophil infiltrates are implicated in exacerbating the underlying disease [7 ]. It is widely appreciated that neutrophils may damage host tissues through the secretion of various inflammatory mediators but the specific effects of neutrophils on the epithelium are poorly understood. In a rabbit ligated intestinal loop model of Shigella flexneri infection, low numbers of neutrophils infiltrated the gut after early infection via Peyer’s patches. This was followed by a generalized disseminated bacterial infection through the enterocytes, which was prevented if the rabbits were first treated with anti-CD18 antibodies [8 ]. Thus neutrophils presumably contributed to the IEC susceptibility to this infection and exacerbation of the inflammation. That neutrophils may directly damage the epithelium was well demonstrated with the use of a Transwell model in which human neutrophils migrating in response to a chemotactic gradient wounded a monolayer of T84 colon carcinoma cells [9 , 10 ]. The neutrophil-inflicted wound may be mechanistically similar to the injury by T. spiralis, and this has led us to hypothesize that neutrophils potentially contribute to the IEC cytokine induction during this helminth infection.
Neutrophil migration into the T. spiralis-infected rodent intestine has been incompletely examined. Using a dose of 7,500 worms in rats, Smith and Castro [11 ] reported significantly increased myeloperoxidase in gut samples beginning on day 2 but did not distinguish between the lamina propria and epithelium, nor between neutrophil and eosinophil peroxidase. In a second study, Castro et al. [12 ] reported that the peroxidase-positive cells were confined to the lamina propria between days 7 and 10, the earliest time points studied. Others have provided evidence that during infection leukocytes cross the epithelium and were detected in the lumen [13 ]. In this report we expand on these data, and in addressing the hypothesis, we show that neutrophil migration parallels the cytokine expression by IEC and that cytokine levels are diminished when neutrophils are prevented from reaching the epithelium during infection. We interpret our findings to suggest that a loop of leukocyte recruitment through the stimulation of further cytokine production by the epithelium may occur, and support a model wherein regulation of neutrophil infiltration may help control inflammation.
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Neutrophil isolation and labeling
Neutrophils were isolated using hydroxy ethyl starch (Hespan,
DuPont-Merck Pharmaceuticals, Wilmington, DE) transfusion exchange and
Percoll density gradient separation as reported elsewhere
[14
, 15
]. Briefly, donor male Lewis rats,
with adjuvant arthritis to induce neutrophilia [14
],
were exchange transfused via the femoral vein using 6% hydroxy ethyl
starch/saline with blood collected into acid-citrate-dextrose (ACD,
Formula A, Fenwal-Travenol, Malton, Ontario) anticoagulant.
Leukocyte-rich plasma was collected from whole blood after red cell
sedimentation (1 g) at room temperature. The leukocyte-rich
plasma was centrifuged, and the cell pellet was resuspended in
Ca2+ Mg2+-free Tyrode’s solution containing
5% platelet-poor plasma (PPP) with 3 mg/mL pyrogen-free human serum
albumin (HSA; Connaught Laboratories, Toronto, Ontario). The cells were
then layered on a discontinuous Percoll (Pharmacia, Baie dUrfe, Quebec)
density gradient made with the same medium (63%/74%) and centrifuged
at 400 g for 30 min at room temperature. Neutrophils were
collected from the 63%/74% interface and were consistently >95%
pure.
Purified neutrophils were washed twice in Tyrode’s solution and labeled with 2 µCi 111In-oxine/107 cells (Amersham, Oakville, Ontario) for 10 min at room temperature. Labeled cells were washed twice in Tyrode’s-5% PPP-HSA solution and resuspended in Tyrode’s containing 10% PPP. Recipient animals were lightly anesthetized using halothane (Benson Medical Industries, Markham, Ontario) and 5 x 106 neutrophils were injected intravenously and allowed to circulate for 18 h.
Collection of samples and measurement of neutrophil migration
Recipient rats were killed with a CO2 overdose at
18, 50, or 72 h, or 7 days post-infection. Whole blood (1 mL) was
collected in heparin for gamma counting. The proximal, middle, and
distal thirds of the small intestine and lumen washout of each segment,
and the mesenteric lymph nodes were recovered for 111In
content determination. The lumen washouts were centrifuged and both the
supernatant and pellet (cell)-associated counts were determined. Values
are expressed as cpm/g/106 cpm injected. In addition, a
0.5-cm piece of the proximal end of each intestinal segment was
collected and preserved in buffered formalin for histological staining
with hematoxylin and eosin. A second piece was snap-frozen in liquid
nitrogen, and cryostat sections stained for peroxidase using
diaminobenzedine reactivity.
Neutrophil depletion and adhesion molecule blockade
Infected rats were treated to deplete circulating neutrophils
using 2- or 3-mL intravenous or intraperitoneal injections of the
monoclonal antibody, RP-3 [16
] (kindly provided by Dr.
F. Sendo, Yamagata University School of Medicine, Yamagata, Japan)
every 12 h for either 24 or 48 h before killing. In
experiments intended to block neutrophil migration, infected rats were
treated intravenously with 3 mg (at time of infection) or 2 mg (at
24 h) WT3 (anti-CD18) monoclonal antibody (kindly provided by Dr.
M. Miyasaka, Osaka, Japan), and all rats were killed at 48 h of
infection.
Isolation and enrichment for IEC and intraepithelial lymphocytes
(IEL)
IEC and IEL isolation and purification were performed as
described previously [17
]. Briefly, animals were killed
and the small intestine removed and divided into quarters or thirds.
The segments were flushed with phosphate-buffered saline (PBS),
everted, then inflated by first ligating one end with surgical silk,
injecting PBS, then ligating the opposite end. The segments were placed
in PBS containing 2 mM dithiothreitol (DTT; Life Technologies,
Burlington, Ontario) and vortexed on high for 10 s to remove mucus
and debris from the epithelial surface. The segments were transferred
to 30 mL complete RPMI medium (cRPMI, RPMI containing 5%
heat-inactivated fetal calf serum, 2 mM L-glutamine, 10 mM
HEPES, 50 U/mL penicillin, 50 µg/mL streptomycin, all from Life
Technologies) and vortexed on high for 15-s bursts, repeated four
times, to slough the epithelium. The resulting cell suspension was
passed through two layers of cheesecloth, then Percoll was added to a
final concentration of 30%, and the cells finally collected by
centrifugation at 550 g for 15 min at room temperature. The
cell pellet was resuspended in 45% Percoll, which was then layered
over 75% Percoll. The gradient was centrifuged at 550 g for
30 min. Epithelial cells were collected at the top of the 45% layer
and IEL at the 45%/75% interface. A sample of the Percoll-enriched
cell fractions was used to make cytocentrifuge preparations and the
remainder were lysed for RNA extraction and reverse
transcriptase-polymerase chain reaction (RT-PCR) analysis.
RNA extraction and relative RT-PCR
RNA was extracted using Trizol reagent (Life Technologies)
following the manufacturer’s instructions. RT and PCR were performed
as described previously [3
]. Briefly, 1 µg of total
cellular RNA from each sample was reverse transcribed using Maloney
murine leukemia virus reverse transcriptase (Life Technologies) with
0.01 mM dNTPs (Pharmacia, Nutley, NJ) and 1 µg random hexamers
(Pharmacia). The cDNA was diluted 1:10 for ß-actin measurements,
otherwise an equal volume was used as template for all the cytokine
determinations. PCR mixes contained (in final concentrations) 50 mM
KCl, 20 mM Tris-HCl, pH 8.4, 2.5 mM MgCl2, 0.1 µg/mL
bovine serum albumin (BSA), 0.2 mM dNTPs, and 2.5 pmol of each primer.
Primer sequences are published elsewhere [17
]. PCR was
carried out in a Biotherm Biooven III Thermocycler (Bio/Can Scientific)
under the following cycling conditions: 93°C 30 s, 60°C
30 s, 72°C 15 s for 30 or 35 cycles, followed by a 5-min
extension at 72°C. Products were visualized on 1.5% agarose gels
containing 5 µg/mL ethidium bromide and photographed using Polaroid
667 film. For densitometric quantification and comparison between
treatment groups, the Polaroid image was digitized at 600 dpi, and the
pixel density of a fixed area enclosing each amplicon was determined
using UN-SCAN-IT software (Silk Sciences, Orem, UT). The data are
represented by the cytokine amplicon pixel density divided by the
ß-actin amplicon pixel density for the same treatment group.
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Figure 1. Early time course expression of epithelial cytokine mRNAs, shown for
the different quarters of the small intestine (numbers above lanes,
reading proximal to distal). Total cellular RNA was extracted from the
Percoll-enriched IEC fraction from the indicated small intestinal
segment of rats infected with 5,000 T. spiralis. The
epithelial cell preparations were typically greater than 93% pure (7%
contaminated with leukocytes, including neutrophils); however, in this
experiment the 36 h second segment and 48 h first segment
were 89 and 85% pure, respectively. The first segment at 72 h was
highly clumped and we determined to include 3–5% neutrophils.
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Figure 2. Hematoxylin and eosin-stained (A, B, D, E, G, H, J, K), and
peroxidase-stained (C, F, I, L) sections of T.
spiralis-infected rat small intestine showing the presence of
neutrophils (e.g., arrow). (A, C, D, F, G, I, J, L) original
magnification x10; (B, E, H, K) original magnification x400. A, B, C
are normal rat intestine; D, E, F are day 2-infected; G, H, I are day
4-infected; J, K, L are day 8-infected.
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Figure 3. Time course kinetics of 111In-labeled neutrophil
infiltration into the small intestinal tissue (A) and lumen (B) of
T. spiralis-infected rats. Substantially greater numbers of
neutrophils are retarded in the tissue than accumulate in the lumen, as
represented by higher counts. Figure is a composite of multiple
experiments to illustrate the temporal pattern of migration;
uninfected, n = 7; 18 h, n = 3;
50 h, n = 4; 72 h, n = 4; 7
days, n = 2; error bars are SEM. The
Kruskal-Wallis nonparametric test followed by post hoc
Dunn’s multiple comparisons test was used to measure the significance
of the 18- and 50-h neutrophil accumulations. A statistically
significant (P < 0.05) number of neutrophils
infiltrated the first segment tissue by 50 h but the first segment
lumen could not be tested as the standard deviation of the uninfected
group was 0.
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Figure 4. Neutrophil numbers in the Percoll-enriched IEL fraction from the small
intestinal epithelium after treatment of infected rats with RP-3 (Table 1
, Experiment no. 2). (A) fraction from infected non-antibody-treated
rats. (B) fraction from rats treated for 24 h before killing (at
48 h of infection). (C) fraction from rats treated for 48 h
before killing.
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Table 1. Neutrophils as a Percent of all Intraepithelial Leukocytes in the
Infected Rat Small Intestinal Epithelium After Depletion with RP-3 or
CD18 Blockade
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Figure 5. (A) RT-PCR analysis of IEC cytokine mRNA levels in RP-3-treated
T. spiralis-infected rats. (B) densitometric interpretation
of IL-1ß amplicons from serially diluted first-strand cDNAs prepared
from an RP-3-treated rat. The linear part of the amplicon pixel density
versus cDNA concentration relationship shows that a 50-fold change in
message concentration is detectable. (C) densitometric representation
of RT-PCR data pooled from the four experiments in Table 1
. The pixel
density of a fixed area around each PCR amplicon for each cytokine is
divided by the pixel density of ß-actin for the same mRNA source.
ß-actin levels do not change significantly from the RP-3 treatments
during the infection. The data are the means ± standard
deviation, n = 4.
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In addition to the IL-1ß, we report the coincidental expression of the IL-1RII and MIP-2 mRNA by the T. spiralis-infected epithelium. Expression of all three genes first became detectable by 36 h in the most proximal quarter of the small intestine, making it unlikely that the IL-1 acted as the stimuli for the chemokine or IL-1RII. The IL-1RII is expressed coincidentally with IL-1ß in the IEC-18 detachment model as well [18 ], a pattern that further suggests that IL-1 is not the stimulus for IL-1RII but that a common stimulus leads to expression of both molecules. Indeed, McGee et al. [23 ] had previously reported that IL-1 was not a stimulus for IL-1RII expression using the rat IEC-6 line. The IL-1RII is a potent IL-1 antagonist when anchored as a transmembrane protein [24 ] and when solubilized by shedding [25 ]. Experiments using mice transgenic for keratinocyte overexpression of the IL-1RII concluded that the primary blocking activities of this antagonist were local to the site of expression [26 ]. In contrast to the keratinocyte model, the precise role of the IL-1RII during intestinal inflammation is unknown, although we are exploring whether IL-1ß (and IL-1RII) affects the fate of cells after detachment.
Of the C-X-C and C-C chemokines measured, MIP-2 was clearly detected in the infected rat epithelium, whereas MCP-1 was not, despite the fact that IL-1 was present and is a potent stimulus for both chemokines [19 ]. The lack of early MCP-1 changes is compatible with another report showing that day 13 was the earliest time that MCP-1 became detectable in the serum of T. spiralis-infected mice [27 ]. The reasons for the lack of epithelial MCP-1 expression despite IL-1 in vivo are not clear but may be explained by a relative overabundance of the IL-1RII protein, which could prevent IL-1 from acting in an autocrine or juxtacrine manner. If this is the case then the MIP-2 expression would be a direct consequence of the same stimulus leading to IL-1ß expression, which we partly attribute to the worms (discussed above) and partly to the infiltrating neutrophils. It is known that detaching a human bronchial epithelial cell line leads to IL-8 production [28 ], but whether detachment induces CC chemokines has not been reported for epithelial cells.
Congruent with expression of MIP-2, we showed here that substantial numbers of neutrophils infiltrate the rat small intestine by day 2 of infection, and moreover, that a considerable number continue to migrate through the epithelium. This early period of neutrophil infiltration is compatible with the report by Smith and Castro [11 ] who detected increased myeloperoxidase in gut samples beginning on day 2. It is noteworthy that despite finding label in the lumen, greater numbers of cells remained in the tissue even after an 18-h migration period (compare the ordinate scale of Fig. 2A and 2B ). This pattern of neutrophil retention in the tissue may be due to a strong chemotactic gradient ending on the basolateral surface of the epithelium, especially because the pathogen is not lumen-dwelling. A dramatic increase in epithelial permeability (which has not been tested on day 2 of T. spiralis infection) would presumably allow paracellular transport of luminal bacterial products, such as N-formyl-methionyl-leucyl-phenylalanine, which might recruit greater numbers of neutrophils into the lumen, but this does not seem to be the case. In fact, little is known about the signals that draw neutrophils across the epithelium in vitro or in vivo. McCormick and co-workers [29 ] have described a novel chemoattractant secreted apically by S. typhimurium-infected T84 cells. It is quite possible that this molecule or related chemoattractants are secreted apically during this helminth infection, but none are presently identified. In murine studies, apical secretion of MIP-2 by urinary tract epithelial cells was shown to be critical for the recruitment of neutrophils into the lumen after infection with E. coli 1177 [30 ]. The possibility that MIP-2 is secreted into the intestinal lumen in the infected rat small intestine may be explored when antibodies become available for detection of the rat molecule.
In addition to the direct effect of the worms to elicit IEC cytokines in vivo, neutrophil migration into the gut also seems to stimulate further cytokine production. Although the specific mechanism of neutrophil-induced cytokines was not deduced here, two non-mutually exclusive models may explain it. First, neutrophils may become activated in the tissue and degranulate, and the various granule constituents may damage the basement membrane and IEC directly. Secondly, neutrophils may cross the intestinal epithelium and in the process, damage the epithelium. Related to this second model, cells in the lumen may continue to influence the IEC by secreting inflammatory mediators, as depicted in experiments by Madara and co-workers. They showed, using an in vitro Transwell system, that 5’-adenosine monophosphate from post-migration neutrophils acted back on a T84 monolayer to induce Cl- secretion [31 ]. We are particularly interested in the nature of the IEC disruption resulting from neutrophil transepithelial migration. Nusrat and co-workers [10 ] showed that on human T84 cell monolayers, ß1 integrins normally undetectable from the apical aspect became detectable after neutrophil transepithelial migration, presumably because the neutrophils disassociated the integrin/ligand interactions. The neutrophil inflicted "wounds" manifested as erosions with a ring of ß1 integrins detectable on the epithelial cells at the erosion perimeter. It was not clear from the study whether the T84 ß1 integrins were initially engaged in cell-to-substrate or possibly cell-to-cell adhesions, but by disrupting epithelial ß1 integrin neutrophils may emote "detachment" leading to cytokine expression. We are currently conducting experiments to test this hypothesis, also using the T84 Transwell system.
If neutrophil infiltration leads to enhanced IEC cytokine expression by any of the mechanisms considered above, then reducing the numbers of neutrophils should have a detectable consequence on the epithelium. We blocked neutrophil infiltration through the use of two approaches, specific depletion and adhesion molecule blockade. Considering the antigen specificities of the antibodies chosen for this study, there is no obvious reason to expect that either antibody had direct effects on the worm viability. We first depleted circulating neutrophils using the specific monoclonal antibody, RP-3. The specificity of this antibody for neutrophils was partly confirmed, by the founders, by depleting neutrophils in T. spiralis-infected rats at a time when eosinophil numbers were increasing in the blood (day 18) [16 , 32 ]. Our experience (which was similar to that of the investigators who derived the clone), was that the depletion was not absolute, and thus different doses were used to vary the number of neutrophils that ultimately reached the epithelium (Table 1) . The titration of neutrophils with increased dosages of the antibody was paralleled by a downward titration of the epithelial cell cytokine mRNA signal, most strikingly with IL-1ß. It is unlikely that the reduced signal is a result of reduced contamination of the epithelial fraction by neutrophils expressing IL-1ß because neutrophils are rare among the epithelial cells after Percoll enrichment of the enterocytes. As an alternative to depletion we used the single-most effective adhesion molecule blockade reported to prevent migration of neutrophils into the inflamed gut, anti-CD18 antibody treatment. This regime of adhesion molecule blockade did not result in the depletion of neutrophils from the blood but did reduce the numbers of neutrophils found in the epithelium. Again, the blockade was not complete, pointing to the intriguing possibility that neutrophils use CD18-independent adhesion molecules to infiltrate the gut and epithelium during (this) helminth infection. Anti-CD18 treatment likely also blocks the migration of other leukocytes, which would then fail to reach the epithelium, unlike the neutrophil depletion protocol. IL-1ß and IL-1RII titrated downward with reduced numbers of neutrophils due to the anti-CD18 treatment, whereas MIP-2 remained unchanged. This result may be explained by a model whereby the MIP-2 expression is due to the worms directly, and not by the infiltrating neutrophils.
Our two approaches to reducing the numbers of neutrophils that reach the epithelium, despite this dose of infection, support the model whereby neutrophil infiltration stimulates epithelial cell cytokine production. Our results would predict that reduction of neutrophil emigration during inflammatory bowel disease may have a hitherto unappreciated beneficial effect through a reduction in epithelial cell inflammatory cytokines.
Received August 21, 1999; revised July 3, 2000; accepted July 5, 2000.
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differentially stimulate MCP-1 and eotaxin production by intestinal epithelial cells J. Interferon Cytokine Res. 20,299-308[Medline]This article has been cited by other articles:
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