Stephen J. Getting

Endogenous lipid- and peptide-derived anti-inflammatory pathways generated with glucocorticoid and aspirin treatment activate the lipoxin A4 receptor.

Aspirin (ASA) and dexamethasone (DEX) are widely used anti-inflammatory agents yet their mechanism(s) for blocking polymorphonuclear neutrophil (PMN) accumulation at sites of inflammation remains unclear. Here, we report that inhibition of PMN infiltration by ASA and DEX is a property shared by aspirin-triggered lipoxins (ATL) and the glucocorticoid-induced annexin 1 (ANXA1)-derived peptides that are both generated in vivo and act at the lipoxin A4 receptor (ALXR/FPRL1) to halt PMN diapedesis. These structurally diverse ligands specifically interact directly with recombinant human ALXR demonstrated by specific radioligand binding and function as well as immunoprecipitation of PMN receptors. In addition, the combination of both ATL and ANXA1-derived peptides limited PMN infiltration and reduced production of inflammatory mediators (that is, prostaglandins and chemokines) in vivo. Together, these results indicate functional redundancies in endogenous lipid and peptide anti-inflammatory circuits that are spatially and temporally separate, where both ATL and specific ANXA1-derived peptides act in concert at ALXR to downregulate PMN recruitment to inflammatory loci.

Aberrant inflammation and resistance to glucocorticoids in Annexin 1-/- Mouse

The 37‐kDa protein annexin 1 (Anx‐1; lipocortin 1) has been implicated in the regulation of phagocytosis, cell signaling, and proliferation and is postulated to be a mediator of glucocorticoid action in inflammation and in the control of anterior pituitary hormone release. Here, we report that mice lacking the Anx‐1 gene exhibit a complex phenotype that includes an altered expression of other annexins as well as of COX‐2 and cPLA2. In carrageenin‐ or zymosan‐induced inflammation, Anx‐1−/− mice exhibit an exaggerated response to the stimuli characterized by an increase in leukocyte emigration and IL‐1β generation and a partial or complete resistance to the antiinflammatory effects of glucocorticoids. Anx‐1−/− polymorphonuclear leucocytes exhibited increased spontaneous migratory behavior in vivo whereas in vitro, leukocytes from Anx‐1−/− mice had reduced cell surface CD 11b (MAC‐1) but enhanced CD62L (L‐selectin) expression and Anx‐1−/− macrophages exhibited anomalies in phagocytosis. There are also gender differences in activated leukocyte behavior in the Anx‐1−/−mice that are not seen in the wild‐type animals, suggesting an interaction between sex hormones and inflammation in Anx‐1−/− animals.

Anti-inflammatory effects of a novel, potent inhibitor of poly (ADP-ribose) polymerase

Abstract.Objective and design: Oxygen- and nitrogen-derived free radicals and oxidants play an important role in the pathogenesis of various forms of inflammation. Recent work emphasizes the importance of oxidant-induced DNA strand breakage and activation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP) in the pathogenesis of various inflammatory diseases. We have recently demonstrated the efficacy of PJ34, a novel, potent phenanthridinone derivative PARP inhibitor, in rodent models of diabetic vascular dysfunction and stroke. Here we tested the efficacy of PARP inhibition in various models of local inflammation in rodents.¶Materials and methods: PJ34 (at doses of 0.03-30 mg/kg) was tested in rats and mice subjected to standard models of inflammation, with relevant parameters of inflammation measured using standard methods.¶Results: PJ34 treatment (s.c, i.p. and i.v.) dose-dependently suppressed neutrophil infiltration and nitric oxide (but not KC and IL-1β) production in peritonitis. In a model of systemic endotoxemia, PJ34 pretreatment significantly reduced plasma levels of TNF-α, IL-1β and nitrite/nitrate (breakdown products of nitric oxide) production. PJ34 treatment (oral gavage) induced a significant suppression of the inflammatory response in dextran sulfate colitis, multiple low dose streptozotocin diabetes and cyclophosphamide-accelerated autoimmune diabetes in the non-obese diabetic mice, and reduced the degree of mononuclear cell infiltration into the iris in an endotoxin-induced uveitis model. Delaying the start of PJ34 administration in the colitis model conferred significant protective effects, while in the arthritis model the post-treatment paradigm lacked protective effects.¶Conclusions: PJ34 provides significant, dose-dependent, anti-inflammatory effects in a variety of local inflammation models. Some of its actions are maintained in the post-treatment regimen and/or after discontinuation of treatment. We conclude that PARP inhibition offers a powerful means for reducing the severity of various forms of local inflammatory responses.

Inhibition of neutrophil and monocyte recruitment by endogenous and exogenous lipocortin 1

The role played by endogenous lipocortin 1 in the anti‐migratory action exerted by dexamethasone (Dex) on monocyte recruitment in an in vivo model of acute inflammation was investigated by use of several neutralizing polyclonal antibodies raised against lipocortin 1 or a lipocortin 1‐derived N‐terminus peptide (peptide Ac2‐26). The efficacy of peptide Ac2‐26 in inhibiting monocyte and polymorphonuclear leucocyte (PMN) recruitment was also tested. Intraperitoneal (i.p.) injection of zymosan A (1 mg) produced a time‐dependent cell accumulation into mouse peritoneal cavities which followed a typical profile of acute inflammation: PMN influx was maximal at 4 h post‐zymosan (between 15 and 20×106 cells per mouse), and this was followed by an accumulation of monocytes which peaked at the 24 h time‐point (between 10 and 15×106 cells per mouse). Dex administration to mice reduced zymosan‐induced 4 h PMN infiltration and 24 h monocyte accumulation with similar efficacy: approximately 50% of inhibition of recruitment of both cell types was achieved at the dose of 30 μg per mouse (∼1 mg kg−1, subcutaneously (s.c.)). Maximal inhibitions of 64% and 67% on PMN and monocyte recruitment, respectively, were measured after a dose of 100 μg per mouse (∼3 mg kg−1, s.c.). Dex (30 μg s.c.) inhibited monocyte (53%) and PMN (69%) accumulation in response to zymosan application in mice which had been treated with a non‐immune sheep serum (50 μl s.c.). In contrast, the steroid was no longer active in reducing cell accumulation in mice which had been passively immunized against full length human recombinant lipocortin 1 (serum LCS3), or against lipocortin 1 N‐terminus peptide. Treatment of mice with vinblastine (1 mg kg−1, intravenously (i.v.)) produced a remarkable leucopenia as assessed 24 h after administration. This was accompanied by a 60% reduction in 4 h‐PMN influx, and by a 27% reduction in 24 h‐monocyte accumulation, measured after zymosan administration. The inhibitory effect of Dex on monocyte recruitment was not significantly modified in vinblastine‐treated mice, with 36% and 57% of inhibition calculated at the dose of 30 μg Dex, and 70% and 60% of inhibition at 100 μg Dex, in vehicle‐ and vinblastine‐treated mice, respectively. Treatment of mice with peptide Ac2‐26 dose‐dependently attenuated PMN influx at 4 h post‐zymosan with a significant effect at 100 μg per mouse (45% of inhibition, n=9, P<0.05) and a maximal effect of 61% inhibition at the highest dose tested of 200 μg s.c. (n=14, P<0.05). No effect of peptide Ac2‐26 (200 μg s.c.) was seen on zymosan‐induced 24 h monocyte recruitment. In contrast, administration of 200 μg peptide Ac2‐26 every 6 h was effective in reducing the number of monocytes harvested from the inflamed peritoneal cavities at 24 h post‐zymosan: 9.40±0.58×106 monocytes per mouse (n=13) and 5.74±0.34 monocytes per mouse (n=14) in vehicle‐ and peptide Ac2‐26‐treated mice, respectively (P<0.05). Finally, peptide Ac2‐26 produced a concentration‐dependent inhibition of the rate of phagocytosis of mouse resident peritoneal macrophages as measured by flow cytometry, with a maximal reduction of 34% at the highest concentration tested of 100 μg ml−1 (n=8 experiments performed in duplicate; P<0.05). In conclusion, this study suggests that in vivo monocyte recruitment during acute inflammation is, at least in part, under the negative modulatory control of endogenous lipocortin 1 (as seen after administration of Dex by using the specific antisera) and exogenous lipocortin 1 mimetics (as observed with peptide Ac2‐26). In addition to the neutrophil, we can now propose that the monocyte also can be a target for the in vivo anti‐inflammatory action of lipocortin 1.

Involvement of the Receptor for Formylated Peptides in the in Vivo Anti-Migratory Actions of Annexin 1 and its Mimetics

An innovative avenue for anti-inflammatory therapy is inhibition of neutrophil extravasation by potentiating the action of endogenous anti-inflammatory mediators. The glucocorticoid-inducible protein annexin 1 and derived peptides are effective in inhibiting neutrophil extravasation. Here we tested the hypothesis that an interaction with the receptor for formylated peptide (FPR), so far reported only in vitro, could be the mechanism for this in vivo action. In a model of mouse peritonitis, FPR antagonists abrogated the anti-migratory effects of peptides Ac2-26 and Ac2-12, with a partial reduction in annexin 1 effects. A similar result was obtained in FPR (knock-out) KO mice. Binding of annexin 1 to circulating leukocytes was reduced (>50%) in FPR KO mice. In vitro, annexin binding to peritoneal macrophages was also markedly reduced in FPR KO mice. Finally, evidence of direct annexin 1 binding to murine FPR was obtained with HEK-293 cells transfected with the receptor. Overall, these results indicate a functional role for FPR in the anti-migratory effect of annexin 1 and derived peptides.

The central role of myostatin in skeletal muscle and whole body homeostasis

Myostatin is a powerful negative regulator of skeletal muscle mass in mammalian species. It plays a key role in skeletal muscle homeostasis and has now been well described since its discovery. Myostatin is capable of inducing muscle atrophy via its inhibition of myoblast proliferation, increasing ubiquitin‐proteasomal activity and downregulating activity of the IGF–Akt pathway. These well‐recognized effects are seen in multiple atrophy causing situations, including injury, diseases such as cachexia, disuse and space flight, demonstrating the importance of the myostatin signalling mechanism. Based on this central role, significant work has been pursued to inhibit myostatins actions in vivo. Importantly, several new studies have uncovered roles for myostatin distinct from skeletal muscle size. Myostatin has been suggested to play a role in cardiomyocyte homeostasis, glucose metabolism and adipocyte proliferation, all of which are examined in detail below. Based on these effects, myostatin inhibition has potential to be widely utilized in many Western diseases such as chronic obstructive pulmonary disease, type II diabetes and obesity. However, if myostatin inhibitors are to successfully translate from bench‐top to bedside in the near future, awareness must be raised on these non‐traditional effects of myostatin away from skeletal muscle. Indeed, further research into these novel areas is required.

Redundancy of a functional melanocortin 1 receptor in the anti-inflammatory actions of melanocortin peptides: studies in the recessive yellow (e/e) mouse suggest an important role for melanocortin 3 receptor.

The issue of which melanocortin receptor (MC-R) is responsible for the anti-inflammatory effects of melanocortin peptides is still a matter of debate. Here we have addressed this aspect using a dual pharmacological and genetic approach, taking advantage of the recent characterization of more selective agonists/antagonists at MC1 and MC3-R as well as of the existence of a naturally defective MC1-R mouse strain, the recessive yellow (e/e) mouse. RT-PCR and ultrastructural analyses showed the presence of MC3-R mRNA and protein in peritoneal macrophages (Mφ) collected from recessive yellow (e/e) mice and wild-type mice. This receptor was functional as Mφ incubation (30 min) with melanocortin peptides led to accumulation of cAMP, an effect abrogated by the MC3/4-R antagonist SHU9119, but not by the selective MC4-R antagonist HS024. In vitro Mφ activation, determined as release of the CXC chemokine KC and IL-1β, was inhibited by the more selective MC3-R agonist γ2-melanocyte stimulating hormone but not by the selective MC1-R agonist MS05. Systemic treatment of mice with a panel of melanocortin peptides inhibited IL-1β release and PMN accumulation elicited by urate crystals in the murine peritoneal cavity. MS05 failed to inhibit any of the inflammatory parameters either in wild-type or recessive yellow (e/e) mice. SHU9119 prevented the inhibitory actions of γ2-melanocyte stimulating hormone both in vitro and in vivo while HS024 was inactive in vivo. In conclusion, agonism at MC3-R expressed on peritoneal Mφ leads to inhibition of experimental nonimmune peritonitis in both wild-type and recessive yellow (e/e) mice.

Natural and synthetic agonists of the melanocortin receptor type 3 possess anti‐inflammatory properties

The effects of the natural and synthetic ligands for the melanocortin receptor type 3 (MC3‐R) have been evaluated in a murine model of experimental gout. Systemic treatment of mice with γ2‐melanocyte‐stimulating hormone (γ2‐MSH) and the synthetic agonist MTII inhibited accumulation of KC, interleukin‐1 beta (IL‐1β), and PMN elicited by urate crystals in the peritoneal cavity. In vitro, macrophage (Mø) activation, determined as release of KC and IL‐1β, was inhibited by γ2‐MSH and MTII. The mixed MC3/4‐R antagonist SHU9119 prevented the inhibitory actions of γ2‐MSH and MTII in vitro and in vivo, whereas the selective MC4‐R antagonist HS024 was without effect. Western blotting also showed the presence of MC3‐R protein on murine peritoneal Mø. Furthermore, agonism at the MC3‐R evoked accumulation of cAMP within the Mø, which was inhibited by SHU9119. Thus, naturally occurring melanocortins, as well as the synthetic long‐acting compound MTII, activate MC3‐R on peritoneal Mø to inhibit the experimental inflammatory response.

Melanocortin 3 receptors control crystal-induced inflammation

In this study we have characterized the anti‐inflammatory profile of a selective melanocortin type 3 receptor (MC3‐R) ligand [D‐Trp8]‐γ‐MSH, validating in vitro results with analyses in mice deficient for this receptor subtype. In wild‐type (WT) macrophages, [D‐Trp8]‐γ‐MSH activated MC3‐R (as tested by accumulation of cyclic AMP) and inhibited (~50%) the release of interleukin (IL)‐1 and the chemokine KC (CXCL1), but was ineffective in cells taken from MC3‐R null mice. In vivo, administration of 3–30 μg [D‐Trp8]‐γMSH significantly inhibited leukocyte influx and cytokine production in a model of crystal‐induced peritonitis, and these effects were absent in MC3‐R null mice or blocked by coadministration of an MC3‐R antagonist. Finally, in a model of gouty arthritis, direct injection of urate crystals into the rat joint provoked a marked inflammatory reaction that was significantly inhibited (~70%) by systemic or local administration of [D‐Trp8]‐γ‐MSH. In conclusion, using an integrated transgenic and pharmacological approach, we provide strong proof of concept for the development of selective MC3‐R agonists as novel anti‐inflammatory therapeutics.—Getting, S. J., Lam, C. W., Chen, A. S., Grieco, P., Perretti, M. Melanocortin 3 receptors control crystal‐induced inflammation. FASEB J. 20, 2234–2241 (2006)

Saliva: physiology and diagnostic potential in health and disease

Saliva has been described as the mirror of the body. In a world of soaring healthcare costs and an environment where rapid diagnosis may be critical to a positive patient outcome, saliva is emerging as a viable alternative to blood sampling. In this review, we discuss the composition and various physiological roles of saliva in the oral cavity, including soft tissue protection, antimicrobial activities, and oral tissue repair. We then explore saliva as a diagnostic marker of local oral disease and focus particularly on oral cancers. The cancer theme continues when we focus on systemic disease diagnosis from salivary biomarkers. Communicable disease is the focus of the next section where we review the literature relating to the direct and indirect detection of pathogenic infections from human saliva. Finally, we discuss hormones involved in appetite regulation and whether saliva is a viable alternative to blood in order to monitor hormones that are involved in satiety.