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(Journal of Leukocyte Biology. 2000;68:821-827.)
© 2000 by Society for Leukocyte Biology

Neutrophil migration stimulates rat intestinal epithelial cell cytokine expression during helminth infection

Andrew W. Stadnyk*, Cheryl D. Dollard* and Andrew C. Issekutz*,{dagger},{ddagger}

* Departments of Pediatrics,
{dagger} Microbiology and Immunology, and
{ddagger} 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


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We are interested in understanding the role of epithelial cells during inflammation, and we previously reported that rat small intestinal epithelial cells express interleukin-1ß (IL-1ß) during infection by Trichinella spiralis. We now report that the epithelium also produces the potent neutrophil chemotactic factor, macrophage inflammatory protein-2 (MIP-2), and an IL-1 antagonist: the type II IL-1 receptor. Consequently we investigated the pattern of neutrophil infiltration into the infected intestine, which closely paralleled the epithelial cytokine expression. Speculating that neutrophil infiltration may provoke epithelial cytokine expression, neutrophil migration into the infected gut was reduced by depleting circulating cells through the use of a specific antibody, or by preventing migration through the use of a function-blocking anti-CD18 monoclonal antibody. Either treatment reduced the number of neutrophils recoverable from the small intestinal epithelium and was paralleled by reduced mRNA levels for epithelial cytokines. These results demonstrate that neutrophil infiltration of the small intestinal epithelium contributes to the stimulation of epithelial cell cytokines.

Key Words: Trichinella spiralis • interleukin-1 • macrophage inflammatory peptide-2


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Helminth infections of rodents have become popular models for investigations into the pathology and immunity mediated by T helper lymphocyte subsets in the gastrointestinal tract, yet the early events of the intestinal inflammatory response to these pathogens remains poorly understood. The mechanisms underlying inflammation resulting from Trichinella spiralis are particularly intriguing because this nematode is an intracellular parasite of the proximal small intestinal epithelium [1 , 2 ]. We discovered that intestinal epithelial cells (IEC) respond to the infection with early and transient interleukin-1ß (IL-1ß) production [3 ]. Human IEC have since been reported to produce IL-1ß during infection by Entamoeba histolytica in a model of human fetal intestinal transplant into SCID mice [4 ]. However, the specific mechanism by which the infection induces IEC cytokines in either in vivo model is not understood. The possibility that T. spiralis directly induces IEC cytokines has drawn attention since a breakthrough showing infection of human colon carcinoma lines in vitro [5 ]. Li et al. [6 ] reported that T. spiralis infection of HT29 cells led to IL-1ß and chemokine production by the epithelial cells.

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.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and infection
T. spiralis was maintained in female C3H/HEN mice (Charles River, St Constant, Quebec); infectious larvae were collected from infected mouse carcasses by digestion in pepsin-HCl for 2 h at 37°C with aeration and subsequent filtration through cheesecloth, as reported in detail elsewhere [3 ]. Male Lewis rats (220–224 g, Harlan Sprague Dawley, Indianapolis, IN) were infected with 5,000 T. spiralis larvae suspended in 0.2% agar through a feeding tube (0.5 mL final volume). All work was undertaken in compliance with the guidelines of the Canadian Council on Animal Care.

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.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Cytokine mRNA expression in IEC
We previously reported that IL-1ß mRNA became detectable in IEC by 48 h of a 2,000-dose T. spiralis infection of rats [3 ]. In complementary in vitro studies intended to address the mechanisms leading to cytokine expression, we discovered that detaching IEC (the IEC-18 rat cell line) induced IL-1ß expression, as well as the type II IL-1 receptor (IL-1RII) [18 ]. We consequently re-examined mRNA from freshly isolated T. spiralis-infected (5,000 dose) epithelial cells for IL-1RII, and because IL-1 is a potent stimuli for chemokine expression, macrophage inflammatory peptide-2 (MIP-2, a C-X-C family member and neutrophil chemoattractant), and monocyte/macrophage chemoattractant peptide-1 (MCP-1, a C-C family member). Figure 1 shows concurrent expression of IL-1RII, IL-1ß, and MIP-2 mRNA expression in the epithelial cell (leukocyte-depleted) fraction from the top of the 45% Percoll gradient. The greatest levels of mRNA were found in the proximal-most quarter of the small intestine, the preferred site of infection by this helminth. Each time point represents mRNA isolated from one infected rat; however, repeats of similar experiments using a dose of 5,000 worms consistently showed elevated mRNA for the cytokines in the small intestinal epithelium by day 2, which remained detectable on day 3. MCP-1 mRNA was not detected at any time point examined, although we readily detect this molecule in IL-1-stimulated IEC-18 cells [19 ]. Our discovery of the chemokine MIP-2 in the proximal intestine is compatible with the recruitment of neutrophils, and thus we chose to examine the migration kinetics by dividing the intestine into thirds.



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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.

 
Pattern of neutrophil infiltration
Neutrophils are not typically found in the small intestine of this strain of rat but a few eosinophils are (Fig. 2 A-C ). On the other hand, the presence of neutrophils in the infected gut is readily observed in hematoxylin and eosin-stained histological sections of 2-day infected rat small intestine (Fig. 2D 2E 2F) . Although neutrophils are present throughout the villi and crypts, the higher-power magnification (Fig. 2E) shows that many are concentrated in some villus tips. By day 4 the crypt hyperplasia and villus atrophy, a characteristic response to helminth infection, is clear, and there is hemorrhaging in the villus tips. Red blood cells can be seen between the epithelial cells at the villus tip (Fig. 2H) . Also in Figure 2 , the goblet cell hyperplasia is evident by day 8, yet neutrophil numbers identified with hematoxylin and eosin staining are reduced, and there is an apparent increase in eosinophils. The neutrophils in the villus tips of the 2-day-infected rat do not stain positive for peroxidase but rather there is a diffuse staining, suggesting that the cells have degranulated within the lamina propria (Fig. 2F) . Peroxidase staining otherwise does reveal positively stained cells scattered throughout the lamina propria, some of which appear at higher magnifications to be eosinophils. The number of discrete peroxidase-positive cells is increased by day 8 of the infection (Fig. 2L) but most of these cells appear to be eosinophils, considering the shape of their nucleus.



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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.

 
Using an 18-h period of radiolabeled cell migration, we more closely mapped the time course kinetics and magnitude of neutrophil infiltration into the small intestinal tissue (Fig. 3A ) and lumen (Fig. 3B) of T. spiralis-infected rats. Statistically significant numbers of neutrophils were detected in both the lumen and tissue by day 2 of infection but not before 18 h. The number of neutrophils migrating into the intestine was reduced to near baseline levels by day 7 of the infection. Greater than 90% of the counts recovered from the infected rat lumen were associated with the pelleted material after centrifugation and thus are most likely cell-associated. The higher number of neutrophils found distal in the lumen, in contrast to the high number of tissue-bound neutrophils in the proximal third, is likely a result of cells in the lumen washing downstream during the 18 h of incubation. We were unable to detect 111In in the enriched IEL preparations, although neutrophils were readily visible in cytocentrifuge preparations (for example, see Fig. 4A ).



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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.

 
Neutrophil depletion and migration blocking studies
Having established that significant numbers of neutrophils migrated into the proximal small intestine, including the lumen, on day 2 of the infection, we decided to impede neutrophil infiltration of the epithelium in infected rats then examine the IEC cytokine expression. To prevent neutrophils from reaching the epithelium we first chose to deplete cells from the blood, up to the 48-h time point. The specific anti-rat neutrophil antibody RP-3 [16 ] was administered every 12 h for either 24 or 48 h before killing infected rats and harvesting the epithelium from the first third of the intestine. The RP-3 injections reduced the circulating neutrophils to less than 10% of all blood leukocytes, despite a rise from approximately 20 to 40% neutrophils by day 2 of the infection. As mentioned above, neutrophils were present in the IEL preparations; Table 1 reports the number of neutrophils as a percent of all epithelial leukocytes found in the control-infected and RP-3-treated, infected rats. Figure 4 shows the cytocentrifugation preparation of an RP-3 experiment (experiment 2 in Table 1 ) and demonstrates that increased doses of RP-3 effectively reduced the numbers of neutrophils contaminating the epithelial leukocyte preparation. The stepwise titration in numbers of neutrophils reaching the epithelium was paralleled with a decline in the epithelial cytokine mRNA signal, most strikingly with IL-1ß, and to a lesser extent with IL-1RII, whereas MIP-2 showed a reduction in only one of four experiments (Fig. 5A ). A second approach to preventing neutrophil migration out of the blood into the infected intestine was used to affirm that the reduced cytokine mRNA levels by the epithelium was not unique to the RP-3 antibody. Various studies have shown that blocking CD18 is an effective strategy to prevent neutrophil migration into the inflamed intestine [8 , 20 ]. Accordingly, groups of rats were injected with a monoclonal antibody to rat CD18 (WT3) for either 24 or 24 and 48 h before killing and examining the epithelial cell fraction for cytokine mRNA. Saturating levels of anti-CD18 were achieved as determined by staining fresh rat leukocytes for flow cytometry using dilutions of the WT3-treated rat serum (not shown). Table 1 shows that the anti-CD18 treatments effectively reduced the numbers of neutrophils recovered from the epithelium, although the blockage was not absolute. Figure 5C , derived from the pooled results from the four experiments in Table 1 , shows that blocking infiltration systematically reduces the cytokine mRNA signal for IL-1ß (by roughly 50-fold in 48-h-treated animals, using the titration curve in Fig. 5B ) and to a lesser extent, IL-1RII. The pooled data illustrate that MIP-2 mRNA levels do not change with the neutrophil blockade.


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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.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Infection of rats with intestinal helminth parasites elicits an inflammatory response that includes villus atrophy, crypt hyperplasia, goblet cell and smooth muscle cell hyperplasia, leukocytic infiltrations, and the hallmark increase in mast cell numbers and circulating IgE levels. Considering that many of these events also occur in other intestinal inflammatory diseases, we use the infection to model the sequence of events leading to intestinal inflammation. This particular pathogen directly infects the small intestinal epithelium, which we reported responded with IL-1ß expression [3 ], and now with MIP-2 and the IL-1 antagonist, IL-1RII. Although we have not localized the epithelial cell source of the cytokines relative to the worms, the possibility that the worm infection directly stimulates IEC cytokine expression (including IL-1ß and chemokines) was recently demonstrated in vitro, using transformed human colon carcinoma cell lines [6 ]. In addition, we have shown that the small intestinal rat cell line, IEC-18, transcribes and secretes IL-1ß when detached, in a model of anoikis or detachment-induced cell death by apoptosis [18 ]. Thus the worms may be inducing cytokine expression by stimulating anoikis or a related route to apoptosis in the IEC. This paradigm of infection linked to apoptosis leading to cytokine production resembles that reported in macrophages [21 ] and for human IEC infected by bacteria, except that unlike the infection by T. spiralis, human IEC do not seem to respond to bacterial infection with IL-1ß [22 ].

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.


   ACKNOWLEDGEMENTS
 
This research was funded by a grant from Crohn’s and Colitis Foundation of Canada to A. W. S., and the Medical Research Council of Canada to A. C. I. A. W. S. is the recipient of an IWK Grace Research Investigatorship Award. We wish to thank Carol Jordan and Derek Rowter for help with neutrophil isolation and labeling, and Drs. T. Issekutz and C. Waterhouse for insightful discussions.

Received August 21, 1999; revised July 3, 2000; accepted July 5, 2000.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Wright, K. A. (1979) Trichinella spiralis: An intracellular parasite in the intestinal phase J. Parasitol. 65,441-445[Medline]
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