Changes in total white blood cell counts were only seen at 3 h after antigen challenge (Figure 1A). A significant increase was observed in rats receiving only antigen (7.8 ± 0.5 × 10⁶ cells/mL), relative to sensitized control rats (5.6 ± 0.4 × 10⁶ cells/mL). This increase was prevented by the treatment with feG (6.0 ± 0.55 × 10⁶ cells/mL). The rats treated with dexamethasone experienced a sharp drop in their total white blood cell counts (3.2 ± 0.4 × 10⁶ cells/mL) at 3 h after antigen challenge. At this time a significant increase in circulating neutrophils was seen in the OA-challenged animals (Figure 1B). This increase in circulating neutrophils was prevented by treatment with feG and dexamethasone, although at 24 h neutrophil number increased more than five fold from 6.0 ± 0.5 (×10⁵) cells/mL to 33.5 ± 5.2 (×10⁵) cells/mL in the dexamethasone, but not feG, treated rats. No changes were seen in the number of circulating lymphocytes at any of the time points sampled (not shown).

Intragastric challenge with antigen did not modify hematocrit, relative to unchallenged sensitized rats (40.4 ± 0.8%), at any of the time points tested. However, rats treated with feG experienced a small decrease in hematocrit at 0.5 h after antigen challenge (39.1 ± 0.5%). In contrast, dexamethasone treated rats had significantly higher hematocrit values, than the other 3 groups of animals, at 0.5 h (45.6 ± 0.4%) and 24 h (45.5 ± 1.8%), but not at 3 h (40.2 ± 0.6%) post-challenge.

The effect of intestinal anaphylaxis and the treatment with feG on bone marrow counts was examined at 3 h after antigen challenge. In control-sensitized rats the bone marrow was 13.6 ± 1.2 (×10⁶ cells), and this was not significantly altered by either antigen alone 11.0 ± 1.4 (×10⁶ cells) or in feG-treated challenged rats 11.1 ± 1.0 (×10⁶ cells).

The acute (30 min) changes in vascular permeability induced by antigen in the pinna, jejunum and duodenum was prevented by feG, but not by dexamethasone, when these compounds were given 20 min before antigen challenge (Table 1). On the other hand dexamethasone prevented the changes in vascular permeability provoked by antigen when given 12 h before initiation of the allergic reaction, whereas this prior treatment with feG was ineffective. Antigen challenge alone or in drug treated rats did not affect vascular permeability in the heart or the trachea (not shown).

Leukocyte infiltration into the heart, measured with the MPO assay, increased ~75% in OA challenged animals (0.69 ± 0.09 units/mg tissue) relative to controls (0.40 ± 0.05 units/mg tissue) at 24 h after antigen challenge (Figure 2). Treatment with feG or dexamethasone at the time of antigen challenge prevented this accumulation of neutrophils in the myocardium. Interestingly, at 3 h after antigen challenge the MPO content of the heart decreased, relative to control animals, in both feG and dexamethasone treated rats. Only at 0.5 h were significant changes in tissue MPO content of the lung seen, with feG treated rats exhibiting a decrease from 1.01 ± 0.08 units/mg tissue to 0.79 ± 0.08 units/mg tissue. On the other hand, at 24 h after antigen challenge dexamethasone treatment resulted in a significant increase in MPO activity to 1.55 ± 0.03 units/mg of lung tissue.

To investigate a relationship between the effects of feG on CD11b and CD49d expression, these antigens were measured on the surface of peritoneal neutrophils isolated from sensitized rats 24 h after exposure to antigen and treatment with feG or the saline vehicle. The inhibitory effect of feG, seen on basal expression of CD11b was not observed in rats that had received an antigen challenge 24 h previously (Figure 3). CD49d was not modified by sensitization or antigen challenge, but feG treatment significantly reduced the expression of this adhesion molecule in OA-exposed animals to values that were 50% of those measured in rats receiving antigen only.

Twenty-four hours after the administration of oral antigen a decreased number of cells expressed the CD18 marker in the smooth muscle of the jejunum (Table 2). This reduction was partially reversed in animals receiving feG. Two morphological distinct leukocytes that stained for ED9 (LPS receptor) could be identified. One type was round and displayed the typical nucleus structure of neutrophils, and the other type had the stellate shape of interstitial macrophages. The number of macrophage-like leukocytes binding ED9 antibody was not affected by antigen challenge, whereas there was a 70% decrease in the number of ED9⁺ neutrophils. This decrease was significantly reduced by feG.

All experiments described in this study were approved by The University of Calgary, Animal Care Committee in accordance with the guidelines of the Canadian Council on Animal Care. Male Hooded-Lister rats, weighing 150–170 g, were raised at the Life and Environmental Sciences Animal Resource Centre, The University of Calgary. The rats were housed under controlled lighting conditions (lights on from 7:00 H to 19:00 H), and provided with food and water ad libitum. All surgical procedures were performed with the animal under halothane anesthesia.

The rats were sensitized by intraperitoneal injection of 10 μg of the antigen chicken ovalbumin (OA) (Grade V, Sigma Chemical Co., St. Louis, MO) and 10 mg of aluminum hydroxide (Al(OH)₃) adjuvant in saline. This sensitization protocol results in an IgE-dependent anaphylaxis 47, and blood anti-OA IgE antibody titer was measured by passive cutaneous anaphylaxis 10 days after sensitization 3 using a small aliquot of blood (500 μl) obtained by cardiac puncture. The serum was then diluted serially (1:8 to 1:64) and 300 μl of each sample was injected into the shaved back of Sprague-Dawley rats. After 48 h, the rats were challenged with an intracardiac injection of a solution containing 2.5 mg of Evan's blue and 5 mg of OA in a total volume of 2.5 mL. Evan's blue extravasation was determined at the injection site, and a serum anti-OA antibody titer of 1:64 was required for an animal to be included in the experiments.

Four different groups of sensitized rats received one of the following treatments: (1) control animals received 0.9% saline only; (2) OA-challenged rats received 100 mg/kg of OA in 0.9% saline; (3) feG (Core Laboratories, Queen's University, Kingston, ON) treated rats received 100 μg/kg of the peptide and antigen; and (4) dexamathasone treated rats received 4 mg/kg of Azium (Schering, Point Claire, QC) intraperitoneally.

Under halothane anesthesia whole blood (200 μl) was drawn by cardiac puncture at 0.5, 3 and 24 h after antigen challenge. Total leukocyte counts were determined using a Unopette microcollection system (Becton Dickinson, Franklin Lakes, NJ) and Hylite hemocytometer (Hauser Scientific, Boulder, CO). Another sample was used to prepare blood smears for microscopic leukocyte differentiation using a Giemsa stain. The hematocrit was determined with a microcapillary centrifuge.

The animals were killed with an overdose of sodium pentobarbital (MTC Pharmaceuticals, Cambridge, ON). The right femur bone marrow was eluted with 10 mL of phosphate buffered saline (PBS) and leukocyte counts were determined using a hemocytometer. The PBS had the following composition (mM): NaCl (137), KCl (2.7), Na₂HPO₄ (8.1), KH₂PO₄ (1.47), CaCl₂ (1.19,) MgCl₂ (0.54), glucose (7.5), pH (7.4).

The rats were anesthetized with halothane and injected with Evans Blue (20 mg/kg; Sigma) in 0.9% saline via cardiac puncture, and 10 min later they were orally challenged with OA and the drugs feG and dexamethasone were administered. Two other groups of rats were treated with feG or dexamethasone 12 hours before the OA challenge. Thirty minutes later, and under halothane anesthesia, the rats were perfused with 1 L/kg of PBS through the left ventricle. Pieces of heart, trachea, pinna, jejunum, and ileum tissue were excised, weighed, and placed in 1 mL of formamide (Sigma) for 72 h at 20°C. The optical density of the formamide was then read at 620 nm with a Beckman DU-50 spectrophotometer (Beckman Coulter Canada Inc., Mississauga, ON) and the amount of Evan's Blue present was calculated from a standard curve.

As a specific enzymatic marker of neutrophil infiltration into the tissue, myeloperoxidase (MPO) activity was determined in myocardial tissue and in lungs according previously described procedures 48. Tissues (100–200 mg), excised from anesthetized rats were homogenized (Polytron homogenizer; Daigger, Vemon Hills, IL) in 0.5% hexadecyltrimethyl ammonium bromide (Sigma) dissolved in 50 mM potassium phosphate buffer at pH 6.0 for 1 min. Homogenates were then centrifuged at 4°C for 15 min at 1000 g. The supernatant was collected and reacted with 0.167 mg/mL o-dianisidine dihydrochloride (Sigma) and 0.005% H₂O₂ in 50 mM phosphate buffer at pH 6.0. The change in absorbance was measured spectrophotometrically at 460 nm. One unit of MPO activity is defined as the quantity of enzyme degrading 1 μmol of peroxide/min at 25°C.

Eighteen hours after allergen-challenged or saline treatment mid-jejunal tissues were removed and whole mounted with pins and fixed in paraformaldehyde for 20 min at ~150% of resting tissue length and ~250% of resting tissue diameter. After fixing, the tissues were stripped of their mucosa, and histochemistry was performed on the muscularis 38. The intestinal tissues were incubated for 24 h at 4°C with either ED9 or CD18 primary antibody (Cedarlane, Hornby, ON) at a 1:100 dilution) followed by three 5-min washes in PBS. The tissues were then incubated with a labeled secondary antibody (goat anti-mouse coupled to biotin) at 4°C overnight. After washing 3 times for 5 min with PBS buffer, avidin coupled horseradish peroxidase was added for 30 min at room temperature. Following further washes diaminobenzadine (DAB – Peroxidase Substrate Kit; Vector Laboratories, Burlingame, CA) was added until desired staining was attained. The intestinal tissues were then dry mounted on chrom alum-coated slides, cover slipped and viewed at ×200 magnification. Counting was performed blind and the number of labeled cells determined in four random fields of each specimen.

Blood was collected by cardiac puncture into a 10 mL syringe from rats anesthetized with halothane. The blood was diluted with approximately 30 mL of PMN buffer (PBS buffer without calcium) in a 50 mL polypropylene centrifuge tube, and spun at 300 g for 15 min at 20°C. After the buffer was carefully decanted from the pellet, the red blood cells were lysed (0.15 M NH₄Cl) for 3 min at room temperature. After a second spin at 200 g for 10 min at 20°C, the supernatant was discarded and the pellet washed with PMN buffer and spun again 200 g for 10 min at 20°C. The supernatant was then discarded and the cells resuspended in 1 mL of PMN buffer, and total cell number determined using Trypan Blue exclusion on a hemocytometer. Cell differentials were determined on cell smears stained with May-Grünwald-Giemsa stain. Lymphocytes were 68.7% of the circulating white blood cells, neutrophils accounted for 20.4% and monocytes were 11.0%.

To isolate peritoneal leukocytes 10 mL of 0.9% sterile saline was injected intraperitoneally into halothane anesthetized rats and the abdomen massaged gently. After a clean laparotomy the peritoneal fluid (7 to 10 mL) was recovered and placed into a 50 mL polypropylene tube and diluted to 30 mL with PMN buffer. The cells were then prepared as described for blood leukocytes. Of the peritoneal cells 21.5% were neutrophils and 46.5% were macrophages.

Results were expressed as mean ± SEM and n is the number of animals. Statistical significance of differences between means was determined at p < 0.05, using, where appropriate, one-way analysis of variance (ANOVA) with the Student t-test applied for comparison of differences between two means.