Autonomic regulation of anti-inflammatory activities from salivary glands.

The cervical sympathetic nerves which innervate the medial basal hypothalamus-hypophyseal complex, primary and secondary lymph organs, and numerous glands, such as the pineal, thyroid, parathyroid and salivary glands form a relevant neuroimmunoendocrine structure that is involved in the regulation of systemic homeostasis. The superior cervical ganglia and the submandibular glands form a 'neuroendocrine axis' called the cervical sympathetic trunk submandibular gland (CST-SMG) axis. The identification of this axis usurps the traditional view of salivary glands as accessory digestive structures and reinforces the view that they are important sources of systemically active immunoregulatory and anti-inflammatory factors whose release is intimately controlled by the autonomic nervous system, and in particular the sympathetic branch. An end component of the CST-SMG axis is the synthesis, processing and release of submandibular rat-1 protein (SMR1), a prohormone, that generates several different peptides, one from near its N-terminus called sialorphin and another from its C-terminus called - submandibular gland peptide-T (SGP-T). SGP-T formed the template for tripeptide fragment (FEG) and its metabolically stable D-isomeric peptide feG, which are potent inhibitors of allergy and asthma (IgE-mediated allergic reactions) and several non-IgE-mediated inflammations. The translation from rat genetics and proteomics to humans has yielded structural and functional correlates that hopefully will lead to the development of new medications and therapeutic approaches for difficult to treat disorders. Although the CST-SMG axis has barely been explored in humans recognition of the importance of this axis could facilitate an understanding and improved management of periodontal disease, and other diseases with a more systemic and nervous system basis such as asthma, autoimmunity, graft-versus-host disease and even Parkinson's disease.

Salivary gland derived peptides as a new class of anti-inflammatory agents: review of preclinical pharmacology of C-terminal peptides of SMR1 protein.

The limitations of steroidal and non steroidal anti-inflammatory drugs have prompted investigation into other biologically based therapeutics, and identification of immune selective anti-inflammatory agents of salivary origin. The traditional view of salivary glands as accessory digestive structures is changing as their importance as sources of systemically active immunoregulatory and anti-inflammatory factors is recognized. Salivary gland involvement in maintenance of whole body homeostasis is regulated by the nervous system and thus constitutes a "neuroendocrine axis". The potent anti-inflammatory activities, both in vivo and in vitro, of the tripeptide Phe-Glu-Gly (FEG) are reviewed. FEG is a carboxyl terminal peptide of the prohormone SMR1 identified in the rat submandibular salivary gland, The D-isomeric form (feG) mimics the activity of its L-isomer FEG. Macropharmacologically, feG attenuates the cardiovascular and inflammatory effects of endotoxemia and anaphylaxis, by inhibition of hypotension, leukocyte migration, vascular leak, and disruption of pulmonary function and intestinal motility. Mechanistically, feG affects activated inflammatory cells, especially neutrophils, by regulating integrins and inhibiting intracellular production of reactive oxygen species. Pharmacodynamically, feG is active at low doses (100 µg/kg) and has a long (9-12 hour) biological half life. As a therapeutic agent, feG shows promise in diseases characterized by over exuberant inflammatory responses such as systemic inflammatory response syndrome and other acute inflammatory diseases. Arthritis, sepsis, acute pancreatitis, asthma, acute respiratory inflammation, inflammatory bowel disease, and equine laminitis are potential targets for this promising therapeutic peptide. The term "Immune Selective Anti-Inflammatory Derivatives" (ImSAIDs) is proposed for salivary-derived peptides to distinguish this class of agents from corticosteroids and nonsteroidal anti-inflammatory drugs.

The tripeptide feG inhibits leukocyte adhesion.

BACKGROUND: 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 beta1-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. RESULTS: 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 mug/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. CONCLUSION: The modulation of leukocyte adhesion by feG appears to involve actions on alphaMbeta2 integrin, with a possible interaction with the low affinity FcgammaRIII 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 analog feG ameliorates severity of acute pancreatitis in a caerulein mouse model.

Acute pancreatitis (AP) is associated with significant morbidity and mortality; however, there is no specific treatment for this disease. A novel salivary tripeptide analog, feG, reduces inflammation in several different animal models of inflammation. The aims of this study were to determine whether feG reduced the severity of AP and modifies the expression of pancreatic ICAM-1 mRNA during AP in a mouse model. AP was induced in mice by hourly (x12) intraperitoneal injections of caerulein. A single dose of feG (100 microg/kg) was coadministered with caerulein either at time 0 h (prophylactic) or 3 h after AP induction (therapeutic). Plasma amylase and pancreatic MPO activities and pancreatic ICAM-1 mRNA expression (by RT-PCR) were measured. Pancreatic sections were histologically assessed for abnormal acinar cells and interstitial space. AP induction produced a sevenfold increase in plasma amylase, a tenfold increase in pancreatic MPO activity, and a threefold increase in interstitial space, and 90% of the acinar cells were abnormal. Prophylactic treatment with feG reduced the AP-induced plasma amylase activity by 45%, pancreatic MPO by 80%, the proportion of abnormal acinar cells by 30%, and interstitial space by 40%. Therapeutic treatment with feG significantly reduced the AP-induced abnormal acinar cells by 10% and the interstitial space by 20%. Pancreatic ICAM-1 mRNA expression was upregulated in AP and was reduced by 50% with prophylactic and therapeutic treatment with feG. We conclude that feG ameliorates experimental AP acting at least in part by modulating ICAM-1 expression in the pancreas.

Vcsa1 gene peptides for the treatment of inflammatory and allergic reactions.

The recently emerged Vcsa1 gene is one member of the variable coding sequence (VCS) multigene family of Rattus norvegicus. This gene encodes the precursor prohormone SMR1 (submandibular rat-1), which on enzymatic processing gives rise to several 5 to 11 amino acid peptides that modulate a variety of physiological functions. The analgesic pentapeptide sialorphin and anti-inflammatory heptapeptide submandibular gland peptide-T (TDIFEGG) are the most intensively studied. Although the Vcsa1 gene and its protein product are unique to rats, TDIFEGG or a derivative acts on all species examined to date, including human cells, in functions related to allergic reactions and inflammation. In this review, the patent and academic literature on SMR1 and its natural peptides and their derivatives are reviewed for consideration of biological targets and relevance to the development of novel therapeutic agents. The VCS gene family is discussed and we speculate on possible human homologs of these potent anti-inflammatory rat-derived peptides. The biologically active peptide products of SMR1 are considered and the mechanism of action and structure-activity relationships of the anti-inflammatory submandibular gland peptide-T and its derivatives are discussed.

Effects of a novel tripeptide on neurological outcomes after spinal cord injury.

A biologically active tripeptide [phenylalanine glutamate glycine (feG)] derived from the submandibular gland has anti-inflammatory actions. We have shown that intravenous treatment with feG after spinal cord injury decreases the intraspinal infiltration of leukocytes and associated oxidative damage within 72 h after injury. The present study assessed effects of this treatment on chronic neurological outcomes after clip-compression spinal cord injury at the 12th thoracic segment. Locomotor scores of feG-treated rats were significantly higher than those of controls at 7 weeks after spinal cord injury. Treated rats had significantly less hind paw mechanical allodynia than controls at this time. In conclusion, the anti-inflammatory and anti-oxidative actions of feG treatment correlate with improved neurological outcomes after spinal cord injury.

The tripeptide feG regulates the production of intracellular reactive oxygen species by neutrophils.

BACKGROUND: The D-isomeric form of the tripeptide FEG (feG) is a potent anti-inflammatory agent that suppresses type I hypersensitivity (IgE-mediated allergic) reactions in several animal species. One of feG's primary actions is to inhibit leukocyte activation resulting in loss of their adhesive and migratory properties. Since activation of neutrophils is often associated with an increase in respiratory burst with the generation of reactive oxygen species (ROS), we examined the effect of feG on the respiratory burst in neutrophils of antigen-sensitized rats. A role for protein kinase C (PKC) in the actions of feG was evaluated by using selective isoform inhibitors for PKC. RESULTS: At 18 h after antigen (ovalbumin) challenge of sensitized Sprague-Dawley rats a pronounced neutrophilia occurred; a response that was reduced in animals treated with feG (100 microg/kg). With antigen-challenged animals the protein kinase C (PKC) activator, PMA, significantly increased intracellular ROS of circulating neutrophils, as determined by flow cytometry using the fluorescent probe dihydrorhodamine-123. This increase was prevented by treatment with feG at the time of antigen challenge. The inhibitor of PKCdelta, rottlerin, which effectively prevented intracellular ROS production by circulating neutrophils of animals receiving a naïve antigen, failed to inhibit PMA-stimulated ROS production if the animals were challenged with antigen. feG treatment, however, re-established the inhibitory effects of the PKCdelta inhibitor on intracellular ROS production. The extracellular release of superoxide anion, evaluated by measuring the oxidative reduction of cytochrome C, was neither modified by antigen challenge nor feG treatment. However, hispidin, an inhibitor of PKCbeta, inhibited the release of superoxide anion from circulating leukocytes in all groups of animals. feG prevented the increased expression of the beta1-integrin CD49d on the circulating neutrophils elicited by antigen challenge. CONCLUSION: feG reduces the capacity of circulating neutrophils to generate intracellular ROS consequent to an allergic reaction by preventing the deregulation of PKCdelta. This action of feG may be related to the reduction in antigen-induced up-regulation of CD49d expression on circulating neutrophils.

Identification of a binding site for the anti-inflammatory tripeptide feG.

The mechanism of action of feG, an anti-inflammatory peptide, was explored using data mining, molecular modeling, and enzymatic techniques. The molecular coordinates of protein kinase A (PKA) were used to create six virtual isoforms of protein kinase C (PKCalpha, betaI, betaII, delta, iota, and zeta). With in silico techniques a binding site for feG was identified on PKCbetaI that correlated significantly with a biological activity, the inhibition of intestinal anaphylaxis. Since feG selectively increased the binding of a PKCbetaI antibody, it is proposed that this peptide inhibits the reassociation of the hydrophobic tail of PKCbetaI with its binding site and prevents the enzyme from assuming an inactive conformation.

Modulation of neutrophil function by the tripeptide feG.

BACKGROUND: Neutrophils are critical in the defense against potentially harmful microorganisms, but their excessive and inappropriate activation can contribute significantly to tissue damage and a worsening pathology. Through the release of endocrine factors submandibular glands contribute to achieving a balance in neutrophil function by modulating the state of activation and migratory potential of circulating neutrophils. A putative hormonal candidate for these effects on neutrophils was identified as a heptapeptide named submandibular gland peptide T (SGP-T; sequence = TDIFEGG). Since the tripeptide FEG, derived from SGP-T, and its D-amino acid analogue feG had similar inhibitory effects on inflammatory reactions, we investigated the effects of feG on human and rat neutrophil function. RESULTS: With human neutrophils feG had no discernible effect on oxidative burst or phagocytosis, but in picomolar amounts it reduced PAF-induced neutrophil movement and adhesion, and the binding of CD11b by 34% and that of CD16b close to control values. In the rat feG (10-11M) reduced the binding of CD11b and CD16 antibodies to PAF-stimulated circulating neutrophils by 35% and 43%, respectively, and at 100 micrograms/kilograms intraperitoneally feG reduced neutrophil in vivo migration by 40%. With ovalbumin-sensitized rats that were challenged with antigen, feG inhibited binding of antibodies against CD16b but not CD11b, on peritoneal leukocytes. CONCLUSIONS: The inhibitory effect of feG on neutrophil movement may be mediated by alterations in the co-stimulatory molecules CD11b and CD16.

Role of submandibular salivary glands in LPS-induced lung inflammation in rats.

OBJECTIVE: Literature data suggest that rodent salivary glands can exert a neuroimmunomodulatory influence on distant inflammatory events. The release of regulatory factors by salivary glands appears to be influenced by time-dependent factors. In this paper we examined this possibility directly by studying the role of submandibular salivary glands in the temporal profile of lypopolysaccharide (LPS)-induced lung inflammation in rats. METHODS: The submandibular glands were removed (SMGx) or not (sham) and, 4 days later, the animals received an intravenous LPS injection (Salmonella abortus equi, 1 mg/kg). Cells in peripheral blood and in bronchoalveolar and bone marrow lavages were quantified after 90 min, 1, 3 and 5 days. Tumor necrosis factor (TNF) activity and corticosterone concentrations in serum were also determined. Baseline values were determined in a group of naïve rats. RESULTS: One day after the LPS injection, neutrophil counts in lungs and blood in both animal groups were elevated, but the SMGx rats presented a significantly lower response in comparison to the sham-operated controls. Five days after LPS treatment, however, SMGx rats had higher neutrophil counts in the lungs than did sham animals, but numbers of blood neutrophils were equal. Ninety minutes after LPS injection, a peak of serum TNF activity was detected in both groups compared with naïve levels. At this time point, TNF activity was about 135% higher in the serum of the SMGx group than in controls. Corticosterone levels of sham-operated controls rose only on the 5th day after LPS, whereas SMGx rats had significant peaks of corticosterone both on the 1st and the 5th day, but not on the 3rd day. CONCLUSION: Our data indicate that submandibular glands have a dual effect on inflammatory pulmonary response by differentially modulating the profile of lung neutrophil influx.