The tripeptide feG inhibits leukocyte adhesion
© Mathison et al. 2008
Received: 12 September 2007
Accepted: 20 May 2008
Published: 20 May 2008
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© Mathison et al. 2008
Received: 12 September 2007
Accepted: 20 May 2008
Published: 20 May 2008
The tripeptide feG (D-Phe-D-Glu-Gly) is a potent anti-inflammatory peptide that reduces the severity of type I immediate hypersensitivity reactions, and inhibits neutrophil chemotaxis and adhesion to tissues. feG also reduces the expression of β1-integrin on circulating neutrophils, but the counter ligands involved in the anti-adhesive actions of the peptide are not known. In this study the effects of feG on the adhesion of rat peritoneal leukocytes and extravasated neutrophils to several different integrin selective substrates were evaluated.
The adhesion of peritoneal leukocytes and extravasated neutrophils from rats to adhesive proteins coated to 96-well plates was dependent upon magnesium (Mg2+) ion, suggestive of integrin-mediated adhesion. feG inhibited leukocyte adhesion, but only if the cells were stimulated with PAF (10-9M), indicating that feG's actions in vitro require cell activation. In the dose range of 10-10M to 10-12M feG inhibited the adhesion of peritoneal leukocytes to fibrinogen and fibronectin, but not IgG, vitronectin or ICAM-1. feG inhibited the binding of extravasated neutrophils to heparin, IgG, fibronectin and CD16 antibody. Antigen-challenge of sensitized rats reduced the adhesion of peritoneal leukocytes to most substrates and abolished the inhibitory effects of feG. However, pretreating the animals with intraperitoneal feG (100 μg/kg) 18 h before collecting the cells from the antigen-challenged animal restored the inhibition of adhesion by in vitro feG of peritoneal leukocytes and extravasated neutrophils to fibronectin.
The modulation of leukocyte adhesion by feG appears to involve actions on αMβ2 integrin, with a possible interaction with the low affinity FcγRIII receptor (CD16). The modulation of cell adhesion by feG is dual in nature. When administered in vivo, feG prevents inflammation-induced reductions in cell adhesion, as well as restoring its inhibitory effect in vitro. The mechanism by which in vivo treatment with feG exerts these effects remains to be elucidated.
The tripeptide FEG (Phe-Glu-Gly) was isolated from rat submandibular glands as a component of a heptapeptide (SGPT) located at the C-terminal end transcript of the variable coding sequence a1 gene (Vcsa1; also known as a submandibular rat 1 gene; (SMR1)) [1, 2]. The D-isomer of FEG (feG), is active in all species tested to date including mice , sheep , cats , dogs and isolated human neutrophils . The peptide has potent anti-inflammatory action that effectively reduces allergic or type I immediate hypersensitivity reactions, as revealed by attenuated vascular leakage, intestinal motility disturbances, systemic hypotension, bronchoconstriction and hyper-reactivity, and pulmonary inflammation [4, 7–10]. Additionally, these peptides modulate neutrophil function by inhibiting their adhesion, chemotaxis, and production of intracellular superoxide [6, 11–13].
By interfering with leukocyte adhesion and chemotaxis, feG arrests the movement of cells into the extravascular space and prevents their migration to the site of inflammation [7, 14], thereby reducing the severity of the inflammation. Some of the anti-adhesive actions of feG stem from the peptide modifying the expression of integrins and the binding properties of the integrin-associated IgG receptor – CD16 (FcγRIII) [6, 10, 12]. The integrins, heterodimeric cell surface receptors involved in diverse biological responses from embryonic development, thrombosis, and immune and inflammatory responses, are essential players in the adhesion, extravasation and migration of leukocytes [15, 16].
The objective of this study was to further characterize the specificity of feG's inhibitory action on leukocyte adhesion by examining adhesion to substrates that show selectivity for the integrins expressed on neutrophils. These include: highly expressed β2-integrins, αLβ2 (CD11a/CD18) and αMβ2 (CD11b/CD18), and others that are poorly expressed, such as αXβ2 (CD11c/CD18), α2β1 (CD49b/CD29), α4β1 (CD49d/CD29), α5β1(CD49e/CD29), and αVβ3 (CD51/CD61) [17–19]. α4β1 is of interest because its expression is up-regulated on activated neutrophils [12, 17–19].
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 [6, 7, 10, 20]. 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 , and in several studies pre-treatment with feG 18 h before leukocyte isolation has demonstrated attenuated inflammatory responses to endotoxin and allergen [12, 14].
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 [22, 23]. 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 Ca2+-PMN buffer.
Extravasated neutrophils were collected by placing, under halothane anaesthesia, a small sponge soaked in 0.5% carrageenan subcutaneously into the intrascapular region . 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 [25–27]. 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; ), fibronectin (2.5 μg/ml; ), rat serum IgG (10 μg/ml; ), or vitronectin (350 ng/ml; ). 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 × 105 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 , 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.
The presence of an allergic response in the sensitized rats was established by monitoring differential cell counts in blood . 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.
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).
In keeping with other studies using human neutrophils [33, 34], we found that the adhesion of rat leukocytes required the presence of Mg2+ ion in the incubating buffer, indicating that leukocyte adhesion is mediated by an integrin possessing a metal ion-dependent adhesion site (MIDAS). This Mg2+/Mn2+ binding site is located in the I domain of seven integrin α-subunits (α1, α2, αL, αM, αX, αD, αV and αE) . The requirement of cell stimulation with PAF (Figure 1) for feG to inhibit adhesion reflects previous results showing that leukocytes activation was essential for feG to inhibit cell adhesion to atrial tissue , binding of CD16 antibody to neutrophils  and superoxide production . Although the molecular basis for this activation requirement for an effect of feG is not known functional activation by pro-inflammatory mediators with resulting changes in integrin affinity is a common feature of integrin-mediated actions [36, 37].
The tripeptide feG was found to inhibit leukocyte adhesion to several integrin-selective substrates, although the identity of the specific integrin was not conclusively identified. The inhibition of adhesion of peritoneal leukocytes to fibrinogen is indicative of modification of αMβ2 integrin-mediated adhesion. Leukocytes can adhere to fibrinogen by using αMβ2, αXβ2 and αVβ3 integrins [15, 38]. However, since feG did not modify adhesion to vitronectin, an αVβ3 integrin selective substrate , nor adhesion to collagen, which interacts preferentially with αXβ2 (CD11c), α1β1, α2β1, α10β1 and α11β1[40, 41] and αXβ2 is generally, with some exceptions [42, 43], poorly expressed on neutrophils, feG's probably alters αM-mediated adhesion. An apparent anomalous observation is that feG did not modify adhesion to ICAM-1 which also interacts with αMβ2 . However, the binding sites on αMβ2 for ICAM-1 and fibrinogen are distinct [44, 45], and other αMβ2 integrin inhibitors block adhesion to fibrinogen but not to ICAM-1 .
An apparent exception to the selectivity of feG interfering with αMβ2-mediated adhesion is the inhibition by this peptide of leukocyte and neutrophil adhesion to fibronectin, which is generally considered to adhere to four integrins (α3β1, α4β1, α5β1, αVβ3) using the RGD (arginine-glycine-aspartic acid) motif . However, several studies have shown that αMβ2 integrin binds to fibronectin via coordinate interactions with β1 integrins [47, 48]. This interaction may involve initial engagement of β1 integrins on neutrophils with a resulting cross-talk signal leading to activation of αMβ2-mediated adhesion . Thus, feG probably interacts or modifies a restricted subset of binding sites on the versatile and promiscuous αMβ2 integrin . Cross-talk between α4β1 and αVβ3 also occurs , and may account for the trend towards inhibitory actions of ex vivo feG on extravasated neutrophils binding to vitronectin (Figure 4E).
A previously proposed role for FcγRIII in the actions of feG [6, 50] is supported by the observation that the peptide inhibited the adhesion of extravasated neutrophils to a CD16 antibody (Figure 5), and IgG (Figure 3B). αMβ2 is known to cooperate with FcγRIII for the internalization of IgG-coated particles  and the generation of a respiratory burst . In these cells, a physical proximity and association exists between CD16 and αMβ2 integrin [53, 54]. The absence of an effect of feG on peritoneal leukocyte adhesion to IgG may reflect transient binding observed for human circulating neutrophils .
It is not known whether feG, which has its origins in the salivary glands of rats [2, 55], acts as hormonal regulator of integrin-mediated adhesive interactions, or reflects a binding motif on an integrin or an integrin ligand. Most adhesive interactions between ligands and their substrates involve "a substrate recognition sequences" . A FEG-like motif does not exist in IgG, fibrinogen or fibronectin, thus feG is probably not acting as "substrate recognition motif" to prevent integrin-substrate interactions. A FEG-like motif (FEA at F302-A304) is found on the α7 tail of αM integrin, and this sequence deserves attention as contributing to αM integrin-mediated adhesion. Exogenous FEG may be acting as a mimic of this α7 tail motif. Although a FEG sequence is found on laminins, tenascin C and versican, it is not known if this motif in these proteins serves as a recognition site for adhesive events, which are generally considered to be mediated by the RGD motif for laminin interactions with several integrin heterodimers (α1β1, α2β1, α3β1, α6β1, α7β1 and α6β4) , as is the case for tenascin interactions with α5β1. Moreover, αMβ2 integrins do not play a major role in the adhesion of leukocytes to these extracellular matrix molecules .
The low adhesion of mixed blood leukocytes from rats to the fibrinogen and fibronectin precluded their study, and probably reflects the high proportion of lymphocytes/monocytes in rat blood, which only exhibit significant adhesion when stimulated with cytokines , or to more complex substrates such as heart tissue or cultured epithelial and endothelial cells [20, 58, 59]. The reduced adhesion of peritoneal leukocytes from antigen-challenged rats relative to unsensitized rats (Figure 2) may reflect a state of unresponsiveness or phenotypic modification of the cells resulting from the activation of the immediate hypersensitivity reaction. A similar loss of response by neutrophils is seen in other pathologies, such as portal hypertension, sepsis and severe injury [60–62]. Pretreatment with feG (18 h before cell collection) prevents the development of reduced adhesion in antigen-challenged animals, as is also seen with the increased production of intracellular superoxide by circulating neutrophils of antigen-challenged sensitized rats . The reduced adhesion of peritoneal leukocytes was not due to lower expression of CD11b, since the surface expression of this integrin was increased by antigen challenge, and feG pre-treatment prevented this increase (unpublished observations). In what appears to be a paradox, antigen challenge had the opposite effect on extravasated neutrophils, enhancing their adhesion to fibronectin and vitronectin (Figure 3), indicating that feG may have differential actions depending upon the source of the cells, and possibly cross-talk interactions between integrins discussed above. The basis of these differences and interactions should become clear once the mechanism of action of feG is elucidated.
The tripeptide feG, an anti-inflammatory peptide, may inhibit leukocyte adhesion by interfering with αMβ2 integrin-mediated adhesion. Several facets to feG's actions exist: an acute ex vivo inhibitory effect; and when the peptide is administered in vivo, a prevention of loss of peritoneal leukocyte binding in antigen-challenged animals with restoration of ex vivo inhibition.
submandibular gland peptide-T (Thr-Asp-Ile-Phe-Glu-Gly-Gly)
This study was supported by Allergen NCE Inc.
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