The protocols for all animal experiments were approved by the University of Calgary Health Sciences Animal Care Committee, which conforms to the guidelines of the Canadian Council on Animal Care. Male Sprague-Dawley rats (Charles River Canada, Saint-Constant, QC), of an initial weight or 160–175 g, were housed under controlled lighting conditions (lights on from 7:00 H to 19:00 H), and provided with food and water ad libitum. Previous studies have established that feG does not affect leukocyte function in normal animals or cells, but its effects are revealed upon imposition of an inflammatory stimulus 671020. Thus, several groups of animals were used that included: 1) normal, unsensitized rats; 2) unsensitized rats treated with 100 μg/kg feG 18 h before harvesting the cells; 3) ovalbumin (OA)-sensitized rats challenged with antigen 18 h before harvesting cells; and 4) ovalbumin-sensitized rats challenged with antigen and treated with 100 μg/kg feG 18 h before harvesting cells. feG has a half-life of approximately 12 h 21, and in several studies pre-treatment with feG 18 h before leukocyte isolation has demonstrated attenuated inflammatory responses to endotoxin and allergen 1214.

Rats were sensitized with an intraperitoneal injection of 1 mg OA and 50 ng pertussis toxin (Sigma Chemical, St. Louis, Mo.) as adjuvant: a sensitization method generating elevated IgE titres 2223. The animals were used 28 to 35 days post-sensitization. Rats received oral antigen by gastric lavage with 100 mg/kg of OA in 0.9% saline, whereas unchallenged sensitized animals received a neutral antigen, bovine serum albumin (BSA).

Leukocytes were obtained from three sources: blood, the peritoneal cavity or a carrageenan-soaked, implanted sponge. Underhalothane anaesthesia 9–10 mL of blood was collected by cardiac puncture into a 12 mL syringe, containing 1 ml of 3.8% Na citrate, an anticoagulant. The blood (10–12 mL) was diluted with polymorphonuclear leukocyte (PMN) buffer without calcium to 50 mL in a polypropylene centrifuge tube, and centrifuged at 400 g for 15 min at 4°C. The PMN buffer was of the following composition: 138 mM NaCl, 2.7 mM KCl, 3.2 mM Na₂HPO₄.12H₂O, 5.5 mM glucose. The white blood cells were removed from the surface of the pellet with a plastic Pasteur pipette, and contaminating red blood cells were lysed with 4 volumes of 0.15 M NH₄Cl for 10 min at room temperature. The volume of the polypropylene centrifuge tube was completed to 50 mL with PMN buffer without calcium, and after a second spin at 400 g for 10 min at 4°C, the supernatant was discarded. The pellet was washed with calcium free PMN buffer and centrifuged again at 400 g for 10 min at 20°C. The supernatant was discarded and the cells resuspended in 1 mL of PMN buffer containing calcium (1.2 mM CaCl₂), magnesium (1.5 mM MgCl₂).

Peritoneal cells were obtained by injecting 10 ml of 0.9% saline into the peritoneum, and after massaging, a laparotomy was performed and the saline aspirated with a plastic Pasteur pipette. The cells were washed twice in calcium free PMN buffer as described for the blood cells before resuspending them in Ca^2+-PMN buffer.

Extravasated neutrophils were collected by placing, under halothane anaesthesia, a small sponge soaked in 0.5% carrageenan subcutaneously into the intrascapular region 24. To implant the sponge a 2–3 cm incision was made dorsally, between the shoulder blades, and connective tissue was cleared from the exposed area. The skin was then closed with sutures of 3-0 Dexon thread. Eighteen hours later the sponge was removed and the fluid was squeezed from it into 5 mLs of PMN buffer. Following centrifugation at 400 g for 10 min, the exudate was decanted and the remaining cells were suspended in Ca²⁺-PMN buffer. Total leukocyte counts were determined with a Hylite hemocytometer (Hauser Scientific, Boulder, CO) using Trypan Blue exclusion as a marker of cell viability.

Leukocyte adhesion was performed using modifications of a crystal violet assay 252627. The wells of 96-cell polystyrene Nunclon plates (Nalge Nunc International, Naperville, IL) were coated with various substrate molecules, using literature cited amounts – fibrinogen (500 ng/ml; 28), fibronectin (2.5 μg/ml; 29), rat serum IgG (10 μg/ml; 30), or vitronectin (350 ng/ml; 31). The plates were allowed to dry at room temperature overnight, then washed twice with 0.9% saline, dried again and then stored at 4°C until use within 2–3 weeks. Leukocytes(2.5 × 10 ⁵ in 200 μl of PMN buffer) were distributed into the protein-coated wells and different final concentrations (10^-10M to 10^-12M) of the peptide feG were added (10 μL) to separate wells. The cells were allowed to adhere for 45 min at 37°C. The wells were then washed 3 times in 200 μl of PMN buffer, fixed with 10% formalin for 10 min, before adding crystal-violet (crystal-violet 7·5 g/l, NaCl 2·5 g/l, formaldehyde 1·57%, methanol 50%) for an additional 5 min. The cells were washed 3 times with distilled water, solubilized with 10% sodium dodecyl sulphate (SDS), and the plates were read at 540 nm (Multiskan Ascent, Thermo Scientific, Waltham, MA). After subtraction of non-specific colorimetric readings to obtain absolute binding, the percent inhibition of leukocyte adhesion by feG was calculated relative to the wells containing only PAF (10^-9M).

The adhesion of neutrophils to various antibodies to various cell adhesion molecules was evaluated using anti-rat CD11b (clone OX-42; isotype – IgG2a; BD-Pharmingen, San Diego, CA); mouse anti-rat CD18 (clone WT.3; isotype – IgG1; AbD Serotec, Cedarlane Laboratories Ltd; Hornby ON); mouse anti-Rat CD32 (Clone: D34-485; isotype – IgG1; RDI Research Diagnostics Inc., Concord MA); hamster anti-rat CD62L (Clone: HRL1; isotype – IgG2a; BD-Pharmingen) and anti-human CD16 (clone LNK16; isotype – IgG1; Advanced ImmunoChemical Inc, Long Beach, CA). 0.1 μg of antibody was added to each well of a 96-well plate 32, and the adhesion study performed as described above.

feG was synthesized by American Peptide Co., Sunnyvale, CA. Platelet activating factor PAF(C₁₆) (1-Hexadecyl-2-acetyl-sn-glycero-3-phosphocholine), obtained from Sigma-Aldrich, St. Louis was dissolved in 100% ethanol at a concentration of 10^-2M and stored at -20°C in 5 μl aliquots, and diluted 10⁷ fold for use at a final concentration of 10^-9M. Rat tail collagen, IgG from rat sera, vitronectin from human plasma were purchased from Sigma-Aldrich. Fibrinogen (plasminogen-depleted from human plasma) was obtained from Calbiochem, San Diego, CA. Fibronectin (human) BD Biosciences, San Jose, CA Recombinant human soluble ICAM-1 from Bender MedSystems Inc. Burlingame, CA. Heparin was from Organon Canada Ltd. Toronto, ON.

The results are presented as the mean ± SEM. The statistical functions used are associated with Excel (Microsoft Office XP, Redmond, WA). Comparisons between treatment groups were made with one-way analysis of variance (ANOVA), and if warranted differences between two groups were evaluated using the unpaired Student's t-test. Statistical values reaching probabilities of p < 0.05 were considered significant.

Adhesion of circulating and peritoneal leukocytes, as well as extravasated neutrophils, to fibrinogen and fibronectin increased significantly when magnesium ion (Mg²⁺) was present in the buffer. The adhesion of blood leukocytes, predominately monocytes/lymphocytes, was at least 50% less than that of peritoneal cells (macrophages and neutrophils) and extravasated neutrophils. Due to this low adhesion of blood leukocytes the effects of feG on adhesion were evaluated using peritoneal leukocytes and extravasated neutrophils.

In a previous study stimulation with PAF (10^-9M) was required to observe an inhibitory effect of feG both on rat leukocyte adhesion to atrial tissue 20, and inhibition of CD16 antibody binding to human neutrophils 6. This requirement for PAF also was observed for feG (10^-11M) inhibition of adhesion of peritoneal leukocytes from unsensitized animals to fibrinogen and fibronectin (Figure 1A). Similar results were seen with extravasated neutrophils adhesion to fibronectin (Figure 1B), although feG did not inhibit the adhesion of these cells to fibrinogen (Figure 1B) or IgG (not shown). Extravasated neutrophils did not bind to collagen. For all subsequent experiments PAF was included in the adhesion assay.

The presence of an allergic response in the sensitized rats was established by monitoring differential cell counts in blood 12. Antigen challenge of sensitized animals caused a circulating neutrophilia (48.7 ± 4.4% of circulating white blood cells) that was ~2.5 times greater than that of unsensitized animals (19.2 ± 2.9%). Treatment with feG did not alter white blood cell counts in unsensitized animals, but effectively prevented the neutrophilia occurring in sensitized animals (28.9 ± 3.4%).

Changes, reported below, in cell adhesion with sensitized animals were not due to differential cell numbers in the peritoneal lavage fluid or in the carrageenan-soaked sponge, since peritoneal lavage fluid contained 11 to 12% neutrophils and 35 to 43% macrophages and was the same in the 4 animal groups studied. The carrageenan-soaked sponge cells were >99% neutrophils in all animal groups.

When cells were collected from sensitized animals that were not challenged with antigen, the adhesion of peritoneal leukocytes to any of the substrates was not significantly different from that seen with unsensitized animals (not shown). However, when peritoneal leukocytes were collected from antigen-challenged animals, lower adhesion to heparin, fibrinogen, fibronectin and vitronectin (Figures 2A, C, D and 2E) but not to IgG (Figure 2B) was observed. Treatment with feG (100 μg/kg) at the time of antigen challenge reversed this antigen-induced reduction in adhesion to these substrates except for heparin. The adhesion of extravasated neutrophils to IgG and fibrinogen was not affected by antigen challenge (Figures 3B and 2C), although adhesion to heparin of the extravasated cells was reduced (Figure 3A) and adhesion to fibronectin was increased. With unsensitized animals the extravasated neutrophils did not adhere to vitronectin (Figure 3E), although antigen challenge of the sensitized animals resulted in significant adhesion to vitronectin.

Figure 4 shows dose response relationships (10^-10M to 10^-12M; 612) for the inhibitory effect of feG on PAF-stimulated peritoneal leukocytes from unsensitized rats that were not pretreated with feG (Figure 4A), and those that received intraperitoneal feG (100 μg/kg) 18 hr before isolating the cells (Figure 4B). With cells from animals that were not pretreated with feG (Figure 4A), the ex vivo addition of feG to the assay wells at 10^-10M and 10^-11M inhibited leukocyte adhesion to fibrinogen by 24.2 ± 3.7% and 14.3 ± 4.4%, respectively, and to fibronectin by 17.3 ± 8.4% and 16.0 ± 5.3%, respectively. Peritoneal leukocytes from unsensitized animals avidly bound to ICAM-1 (OD of 2.36 ± 0.08/10⁶ cells), although feG did not affect the adhesion to this substrate (not shown).

In contrast, with feG pretreatment (Figure 4B), the inhibition of adhesion of peritoneal leukocytes to fibronectin and fibrinogen increased significantly to an average of 32.0 ± 7.5% and 31.7 ± 6.1%, respectively, for the three concentrations of feG. A sensitization of the leukocytes to the inhibitory effect of ex vivo feG occurred as the significant inhibition of adhesion seen with 10^-12M peptide was absent if the animals were not pretreated with feG.

The inhibitory effects of ex vivo feG on peritoneal leukocyte adhesion to fibrinogen and fibronectin were abolished when sensitized animals were challenged with antigen (Figure 2). However, the in vivo pretreatment with feG re-established the inhibitory effect of feG on adhesion to fibronectin, but not fibrinogen (Figure 2C and 2D).

With extravasated neutrophils from unsensitized animals ex vivo feG only inhibited adhesion to heparin (Figure 3A), and with antigen-challenged animals inhibition of adhesion of these cells to IgG occurred (Figure 3B). Pretreatment with feG enabled an inhibitory effect of ex vivo feG on extravasated neutrophil adhesion to IgG in unsensitized rats (Figure 3B) and fibronectin with sensitized rats (Figure 3D).

Since feG inhibits the binding of CD11b and CD16b antibody to human neutrophils 6, the effects of feG on the adhesion of extravasated neutrophils to integrin antibodies and CD16b were evaluated. feG did not modify neutrophil adhesion to CD18b (β2 integrin), CD62L (L-selectin) or CD32 (FcγRII; intermediate affinity IgG receptor) (not shown). feG modestly, but dose-dependently, inhibited the adhesion of neutrophils to human CD16 (FcγRIII; intermediate affinity IgG receptor) antibody, and at the highest dose tested (10^-9M) inhibited neutrophil adhesion to CD11b antibody by 24 % (Figure 5). The inhibition of adherence to CD11b and CD16 antibodies by feG is not due to non-specific binding effects as anti-CD62L was the same isotype as anti-CD11b (IgG2a), and anti-CD18 and anti-CD32 were the same isotype (IgG1) as CD16.