Both MC1 and MC3 Receptors Provide Protection From Cerebral Ischemia-Reperfusion–Induced Neutrophil RecruitmentSignificance
Objective—Neutrophil recruitment is a key process in the pathogenesis of stroke, and may provide a valuable therapeutic target. Targeting the melanocortin (MC) receptors has previously shown to inhibit leukocyte recruitment in peripheral inflammation, however, it is not known whether treatments are effective in the unique cerebral microvascular environment. Here, we provide novel research highlighting the effects of the MC peptides on cerebral neutrophil recruitment, demonstrating important yet discrete roles for both MC1 and MC3.
Approach and Results—Using intravital microscopy, in 2 distinct murine models of cerebral ischemia-reperfusion (I/R) injury, we have investigated MC control for neutrophil recruitment. After global I/R, pharmacological treatments suppressed pathological neutrophil recruitment. MC1 selective treatment rapidly inhibited neutrophil recruitment while a nonselective MC agonist provided protection even when coadministered with an MC3/4 antagonist, suggesting the importance of early MC1 signaling. However, by 2-hour reperfusion, MC1-mediated effects were reduced, and MC3 anti-inflammatory circuits predominated. Mice bearing a nonfunctional MC1 displayed a transient exacerbation of neutrophil recruitment after global I/R, which diminished by 2 hours. However importantly, enhanced inflammatory responses in both MC1 mutant and MC3−/− mice resulted in increased infarct size and poor functional outcome after focal I/R. Furthermore, we used an in vitro model of leukocyte recruitment to demonstrate these anti-inflammatory actions are also effective in human cells.
Conclusions—These studies reveal for the first time MC control for neutrophil recruitment in the unique pathophysiological context of cerebral I/R, while also demonstrating the potential therapeutic value of targeting multiple MCs in developing effective therapeutics.
Inflammation plays a central role in cerebral ischemia-reperfusion (I/R) injury. Infiltrating neutrophils contribute to a highly neurotoxic milieu as illustrated by the reduced infarct size and improved functional outcome in models of cerebral I/R after depletion of circulating neutrophils.1 Anti-inflammatory strategies focused on inhibiting neutrophil recruitment by blocking adhesion molecules have, however thus far, proven ineffective in clinical trials.1 The most probable limiting factor to such therapies is that the inflammatory response is a robust system, propagated by diverse pathways, and as such, cannot be effectively subdued by neutralizing a single component. Thus, harnessing endogenous mechanisms for the resolution of inflammation, which impact multiple elements of the inflammatory response, may provide a fruitful strategy.
Five G-protein coupled melanocortin receptors (MC1–5) and the endogenous agonists, adrenocorticotrophic hormone, α, β, and γ melanocyte-stimulating hormones (MSHs) make up the MC receptor system.2 During the past 15 years, research by our team has been pivotal in helping to unravel the biological effects of peptides within this system demonstrating robust anti-inflammatory actions in many inflammatory situations, including gouty, rheumatoid, osteoarthritis, as well as in cardiovascular and I/R models.3–5 These actions are proposed to be mediated primarily through inhibition of NF-κB. Furthermore, leukocytes are both a target for and a source of MCs suggesting that the MC receptor system may provide a self-limiting anti-inflammatory loop, serving to promote inflammatory resolution.2 Such pleiotropic anti-inflammatory actions make these receptors a promising therapeutic candidate to address aberrant inflammation in stroke.6
Of the 5 MCs identified, anti-inflammatory actions have been attributed primarily to MC1 and MC3.2 These receptors have both been shown to be expressed at varying levels in the brain and also on endothelial cells and immune cells (MC1 expression on neutrophils; monocytes and macrophages; dendritic cells; natural killer cells and B lymphocytes. MC3 expression on monocytes; macrophages and B lymphocytes).2 However, the exact anti-inflammatory role of MC subtypes remains unclear, and may vary with the pathophysiological environment.
In this study, we use 2 distinct murine models of cerebral I/R to evaluate the dynamic recruitment of neutrophils in the cerebral microcirculation. Using both pharmacological and genetic approaches, we have demonstrated potent inhibitory actions of the MCs on cerebral leukocyte trafficking, and gained an insight into the relative importance of MC subtypes in mediating these effects.
Materials and Methods
Materials and Methods are available in the online-only Data Supplement.
α-MSH Abrogates Neutrophil Recruitment After Cerebral I/R
To investigate leukocyte recruitment in the cerebral microvasculature, global ischemia was induced, followed by 40-minute reperfusion or 2-hour reperfusion and leukocyte to endothelial cell interactions in pial vessels were assessed by intravital microscopy (Figure 1). Sham surgery, produced little to no leukocyte recruitment, however, I/R caused significant leukocyte rolling (191.0±31.49 cells/mm2 per minute) and adherence (282.3±49.4 cells/mm2 per minute), with 2-hour reperfusion resulting in a further significant increase in adhesion to 555.2±85.3 cells/mm2 per minute (Figure 1C; Movies I and II in the online-only Data Supplement). Correlating with the observed effects on leukocyte recruitment was an enhanced serum soluble E-selectin by 2-hour reperfusion, as detected by ELISA, and a trend toward an increase in the number of intracellular adhesion molecule-1 (ICAM-1)– and vascular cellular adhesion molecule-1 (VCAM-1)–positive vessels detected in the brain. No effect was observed with respect to soluble P-selectin (Figure II in the online-only Data Supplement). α-MSH (10 µg IP), a nonselective MC agonist, given at the start of reperfusion strongly inhibited leukocyte recruitment at 40 minutes, reducing rolling by 80% and adhesion by 68%, to levels comparable with sham animals. Furthermore, these protective effects remained highly significant even after 2 hours of reperfusion (Movie III in the online-only Data Supplement). In line with the reduced leukocyte recruitment at 2 hours, α-MSH was also found to reduce levels of soluble E-selectin and a modest reduction in vessel ICAM-1and VCAM-1 expression.
Finally, to ascertain the role of neutrophils, some mice were depleted of neutrophils before I/R. After I/R, neutropenic mice displayed significant reductions of leukocyte rolling (81%) and adhesion (76%), consistent with the majority or all cells observed being neutrophils (Figure I in the online-only Data Supplement).
Effect of α-MSH on NF-κB–Related Cytokine and mRNA Expression
The effect of cerebral I/R on serum cytokines was investigated using multicytokine analysis (Figure IIIA–IIIC in the online-only Data Supplement). Expression of interleukin (IL)-12p70, interferon (IFN)-γ, and MCP-1 (monocyte chemoattractant protein) remained below the reliable detection range across all treatments (Data not shown). I/R induced a significant increase in serum tumor necrosis factor (TNF)-α by 2 hours, which was abolished by α-MSH treatment. The anti-inflammatory cytokine IL-10 showed a trend toward an increase at 2-hour reperfusion (to 34.7 pg/mL), but significantly increased (88.7 pg/mL) after α-MSH treatment. Levels of the pleotropic cytokine IL-6 remained unchanged after I/R, however, treatment with α-MSH was found to result in a significant upregulation of IL-6 by 2 hours. Considering IL-6 signaling via STAT3 has been shown to reduce neutrophil recruitment,7 we also investigated levels of tyrosine 705 phosphorylated STAT3 in leukocyte nuclear fractions by Western blot, finding a slight enhancement of STAT3 in α-MSH–treated animals at 2 hours (Figure IV in the online-only Data Supplement).
To investigate whether this influence over serum cytokines could be because of NF-κB inhibition, mRNA levels of the NF-κB regulatory protein IκB (which closely corresponds to NF-κB activation8) were assessed in both blood and brain using quantitative reverse transcription polymerase chain reaction (Figure IVA and IVB in the online-only Data Supplement). IκB levels were not significantly elevated 40 minutes after I/R, however by 2 hours, IκB was significantly increased in the blood, and this was suppressed by α-MSH treatment. Suppression of NF-κB activation in leukocytes at 2-hour reperfusion was further confirmed by Western blot analysis of serine 536 phophorylated NF-kB p65 in leukocyte nuclear fractions.
Effect of MC1 and MC3 Agonists on Neutrophil Recruitment and Circulating Cytokines
The relative contribution of MC subtypes on I/R-induced neutrophil recruitment was investigated using more selective MC agonists. Activation of MC1 by BMS-470539 provided a potent inhibition of bilateral common carotid artery occlusion (BCCAo)–induced neutrophil rolling and adhesion at 40-minute reperfusion (Figure 2A and 2B), however, became less effective by 2 hours, with only the level of adhesion being significant versus vehicle. However, treatment with [DTRP8]-γ-MSH, which has a high affinity for MC3, only inhibited cell adhesion at 40-minute reperfusion, yet by 2 hours this effect was stronger, with neutrophil rolling also being significantly reduced. Furthermore, [DTRP8]-γ-MSH significantly reduced BCCAo-induced TNF-α release by 2 hours, while also elevating serum levels of the pleiotropic cytokine IL-6 and the anti-inflammatory IL-10 (Figure 2C–2E).
Pharmacological Investigations Using the MC3/4 Antagonist SHU9119
BMS-470539 is almost entirely selective for MC1, while [DTRP8]-γ-MSH may activate other MCs than MC3.9 To further examine the roles of specific MC subtypes, the MC3/4 antagonist SHU9119 was coadministered with either α-MSH or [DTRP8]-γ-MSH (Figure 3C and 3D), revealing that MC3/4 antagonism caused no increase in rolling or adhesion at 40-minute reperfusion versus α-MSH or [DTRP8]-γ-MSH alone. In fact SHU9119 administered alone, or in conjunction with α-MSH, reduced neutrophil rolling. However, by 2 hours, coadministration of SHU9119 blunted the α-MSH–induced reductions in rolling and adhesion, and prevented the protective effects of [DTRP8]-γ-MSH.
MC Receptor Expression
To determine whether the delayed importance of MC3 was because of change in receptor expression antibody-based investigations into receptor expression were undertaken. However, initial analysis of MC1 and MC3 protein expression using Western blotting revealed antibody binding in MC3−/− mice, using both the Sigma Aldrich (M4937) and Acris (AP10124PU-N) MC3 antibodies, despite polymerase chain reaction confirmation of the MC3−/− (Figure V in the online-only Data Supplement). This suggests that both antibodies tested display nonspecific binding to protein at a similar molecular weight to MC3, as has been previously described.10 Therefore, quantitative reverse transcription polymerase chain reaction was used to quantify MC expression at the mRNA level. MC1–5 was detected in both blood and brain, however, BCCAo induced no changes at either 40 minutes or 2 hours after BCCAo (Figure VI in the online-only Data Supplement).
Physiological Effects of MC1 and 3 in Cerebral I/R-Induced Inflammation
We tested whether the physiological effects of receptor deficiency would support our findings from pharmacological treatments. Recessive yellow (e/e) MC1 mutant mice displayed enhanced (nearly 3×) neutrophil rolling at 40 minutes after I/R versus wild-type (WT; Figure 4A) accompanied by elevated serum TNF-α (Figure 4C). In the absence of a functional MC1, α-MSH reduction of rolling was also hampered at this time point, however, e/e mice displayed no derangements in cell adhesion and were able to respond to α-MSH treatment. MC3 null mice showed no significant differences in neutrophil recruitment, or in their ability to respond to α-MSH at 40 minutes. This is consistent with the predominat anti-inflammatory role of MC1 at early reperfusion. In agreement with the diminished role of MC1 observed in pharmacological studies at 2 hours, at this time point the inflammatory phenotype was not maintained, with TNF-α levels and neutrophil rolling and adhesion being comparable to WT (Figure 5).
Physiological Role in Focal Stroke Model
Stroke is highly variable in its severity and magnitude. The global model of cerebral I/R, represents human stroke conditions caused by atherosclerotic degeneration of the common carotid arteries and respiratory or cardiac arrest. To investigate whether the MC anti-inflammatory effects, we were observing were specific to global I/R, we also undertook investigations using a focal stroke model (ie, the middle cerebral artery occlusion model). We chose this model because of the fact that focal ischemia accounts for ≈80% of ischemic stroke. Figure 6 demonstrates that at 24 hours post ischemia while e/e mice showed elevated adhesion, MC3−/− displayed a more severe inflammatory phenotype with significantly enhanced rolling and adhesion. Importantly, the increased neutrophil recruitment observed, translated into elevated infarct volume and poor functional outcome (detected through neurological scoring) in animals with deficits in either receptor, suggesting both receptors to be potential pharmacological targets.
In Vitro Investigations of MC Effects on Neutrophil Functioning
The MC receptor system displays many disparities between humans and rodents and many MC agonists and antagonists have different selectivity in MCs from different species.9,11 To assess the effectiveness of MC treatments on human cells, we used the neutrophil flow chamber model and chemotaxis assay to investigate neutrophil inflammatory function (Figure VII in the online-only Data Supplement). In the flow chamber, treating neutrophils with just 10 µg/mL of α-MSH (a comparable dose to in vivo studies) resulted in significant reductions in capture (54% reduction), adherence (68%), and transmigration (67%) versus saline (Figure VIIA in the online-only Data Supplement). Treating human umbilical vein endothelial cells (HUVECs) with α-MSH (≤100 µg/mL), however, failed to induce significant reductions in neutrophil recruitment (data not shown), suggesting neutrophils to be the effector cells of MC actions in this context. In the chemotaxis assay, pretreatment with 10 µg/mL of α-MSH or [DTRP8]-γ-MSH failed to inhibit neutrophil migration toward fMLP, however, BMS-470539 significantly reduced this response, suppressing the number of migrated cells by ≈75% (Figure VIIB in the online-only Data Supplement), suggesting MC1 to play a specific role in inhibiting neutrophil chemotaxis.
This study provides, for the first time, evidence that MC treatments can effectively inhibit neutrophil recruitment in the unique microenvironment of the cerebral microvasculature. Using both selective ligands and MC mutant mice, we have gained an insight into MCs involved in modulating neutrophil recruitment in 2 separate models of cerebral I/R, finding both MC1 and MC3 to display important inhibitory roles. In addition, MC treatment was effective in modulating human neutrophil inflammatory functioning, and may represent a novel treatment to stem poststroke inflammation.
Both ischemic models used (BCCAo and middle cerebral artery occlusion) here resulted in a pronounced increase in neutrophil rolling and adhesion compared with sham, as has previously been observed.12,13 Nonselective treatment with α-MSH caused an abrogation of BCCAo-induced neutrophil rolling and adhesion, congruent with a trend toward a reduction in ICAM-1 and VCAM-1 expression in the cerebral vascular. Considering these effects were not significant, other adhesion molecules and integrin activation state may also play a role in the MC influence for leukocyte adhesion. Although anti–ICAM-1 antibody Enlimomab failed clinical trials for stroke (possibly because of inflammatory side effects to the mouse monoclonal antibody) strategies inhibiting CAMs have shown great promise preclinically. Soluble P- and E-selectin are early markers of endothelial activation. Although levels of soluble P-selectin were found only to produce a trend toward an increase after BCCAo, which was unchanged by treatment, soluble E-selectin was increased by 2 hours post ischemia. Elevated levels of soluble E-selectin have been demonstrated in human stroke and during sepsis.14 In vitro studies have also shown that endothelial cells stimulated with IL-1β, TNF-α, endotoxin or serum deprivation, and TNF-α release E-selectin into the culture supernatant.15,16 α-MSH treatment significantly reduced BCCAo-induced soluble E-selectin, probably a reflection of reduced endothelial cell activation, consistent with the reduced levels of IL-1β and TNF-α observed.
Treatment also reduced serum TNF-α and IL-1β. IL-1β, in particular, plays a pivotal role in propagating inflammatory responses and is an established pathological factor in cerebrovascular disease, with significant preclinical and clinical evidence demonstrating blockade of IL-1β signaling to be beneficial in stroke.17 MSH treatment also simultaneously enhanced anti-inflammatory IL-10 by 2-hour reperfusion. The ability of MCs to suppress proinflammatory responses while enhancing anti-inflammatory signals suggests that these receptors form an endogenous proresolving system. In humans, α-MSH concentrations increases in myocardial infarction and infection2 and higher MC levels correlate with better outcome in patients wirh stroke.18 Thus, given the results from this study the MC receptor system may prove a valuable therapeutic target for the treatment of stroke.
Although others have demonstrated a contribution of either MC1 or MC3 in providing anti-inflammatory protection in different systemic inflammatory models, we have, for the first time, identified protective roles for both receptors in the cerebral-microvasculature. In particular, we have identified that MC1-mediated effects predominate in early protection, while MC3 actions are more delayed and induce proresolving factors. This study reveals an additional layer of complexity in MC inflammatory modulation, emphasizing the importance of drug treatment directed at both receptors.
Pharmacological investigations in the BCCAo model revealed the MC1 agonist BMS-470539, significantly inhibited early neutrophil rolling and adhesion, while activation of MC3 using [D-TRP8]-γ-MSH only reduced adhesion. However, by 2 hours after BCCAo, despite a robust pharmacodynamic half-life of ≈8 hours,19 BMS-470539 lost its inhibitory actions on rolling and the reduction of adhesion was diminished slightly. However at 2 hours, [D-TRP8]-γ-MSH anti-inflammatory actions were enhanced. Thus suggesting MC3-mediated effects become more prominent at later time points. SHU9119 (MC3/4 antagonist) was used to further explore the role of MC subtypes. By 40-minute reperfusion, SHU9119 enhanced, rather than inhibited, α-MSH and [D-TRP8]-γ-MSH effects. Furthermore, administered alone, SHU9119 reduced neutrophil adhesion at 40 minutes, possibly because of its seldom reported agonist actions at MC1/5,11 further supporting the predominance of MC1-mediated effects at early stages of reperfusion. By 2-hour reperfusion, SHU9119 lost these anti-inflammatory effects, and cotreatments with SHU9119 inhibited the protective effects of α-MSH and [D-TRP8]-γ-MSH, illustrating role for MC3 in mediating delayed effects on neutrophil adhesion.
Despite the apparent change in MC engagement, no change in MC RNA expression was detected after BCCAo. Whether this is also reflected in protein expression is difficult to discern given the lack of specificity of the antibodies. Even if the surface expression of MC1 or MC3 is increased after stroke, α-MSH and [D-TRP8]-γ-MSH have short half-lives and, therefore, are most probably exerting their effects before such upregulation occurs. The shift in receptor importance may instead reflect activation of distinct mechanisms of action or effector cells. Neutrophils express MC1, while MC3 expression is limited to endothelial cells and macrophages/monocytes. Rapid inhibitory actions exerted by MC1 may be through direct interactions with neutrophils, while the more prolonged MC3 actions are likely via actions on other effector cells. Inhibition of NF-κB is a key element of the protective MC actions.2 Given NF-κB DNA binding occurs only after 30 minutes after TNF-α stimulation, followed by gene transcription 30 minutes later,8 NF-κB inhibition is unlikely to mediate the rapid effects on neutrophil recruitment at 40 minutes. Indeed, IκB transcript levels were not significantly increased at 40 minutes, yet by 2-hours blood IκB elevated 25-fold and nuclear levels of phosphorlyated NF-κB protein were enhanced, which was inhibited by α-MSH. Given our finding of the delayed effect of MC3-targeted treatment on neutrophil this later effect by α-MSH on IκB may be mediated predominantly via MC3 signaling.
MC treatments inhibited neutrophil recruitment at 40 minutes after BCCAo, a timeframe incompatible with transcriptional changes. Such a phenomenon has previously been observed in other models; in zymosan-stimulated macrophages AP214 reduced IL-1β and TNF-α release with no effect on mRNA levels,20 and α-MSH can act via MC1 to shed cell surface CD14 on macrophages21 and IL-8 receptors on neutrophils22 independently of mRNA changes. Although the understanding of NF-κB–independent MC effects is still in its infancy, these mechanisms may fit well with the early efficacy of MC1 treatments in suppressing neutrophil recruitment.
Differences in cytokine regulation between MC1-targeted activation and MC3-targeted/nonspecific activation further support the hypothesis that MC1 and MC3 act via distinct mechanisms. Both α-MSH and [D-TRP8]-γ-MSH significantly reduced BCCAo-induced TNF-α while enhancing anti-inflammatory IL-10. TNF-α has multiple roles in stroke pathology and early increases in blood TNF have been shown to correlate with stroke severity in humans23 while the anti-inflammatory cytokine IL-10 has been shown to be protective in several models of cerebral I/R.24,25 BMS-470539 treatments, however, had no effect on these NF-κB–controlled cytokines further supporting a distinct mechanism of action for the MC1-mediated early inhibition of neutrophil recruitment, while MC3 may be more prominent in initiating proresolving effects. Conflicting reports have been made as the effect of MC treatments on IL-6 levels, however, in the current investigations both α-MSH and [D-TRP8]-γ-MSH increased this pleiotropic cytokine at 2 hours, despite a decrease in NF-κB levels. As such the observed IL-6 release may be as a result of elevated release of presynthesized IL-6. α-MSH has previously been shown to influence IL-6 levels via MC3,26 thus, explaining why IL-6 induction was not observed in BMS-470539–treated animals. Although IL-6 is an endogenous pyrogen with chemotactic activity, IL-6 knockout mice show no protection after experimental stroke.27 Indeed IL-6 signaling via STAT3, has been shown to limit the inflammatory recruitment of neutrophils,7 given that IL-10 may also activate STAT3 and levels were increased after α-MSH treatment such findings seem to be consistent with the present results. Furthermore, it has been demonstrated that NF-κB signaling may induce expression of IL-6 and IL-10 in cells with a high level of STAT3 phosphorylation.28 In line with such observations, we found nuclear extracts from α-MSH-treated BCCAo animals to show a trend toward an elevation of phosphorylated STAT-3 protein.
Our investigations using MC mutant mice, in 2 independent models of stroke, further support a temporal difference in MC1 and MC3 actions. Recessive yellow e/e mice displayed a transiently enhanced neutrophil rolling and elevated TNF-α at the onset of the BCCAo-induced inflammatory response, while MC3−/− mice showed no inflammatory phenotype at this early time point. It is possible that compensatory upregulation of other MCs may mask the anti-inflammatory role of this receptor in MC3−/−. Montero-Melendez et al20 have previously shown that while in macrophages isolated from WT and e/e zymosan challenge induced no change in MC1, MC3, or MC5 expression, in MC3−/− inflammatory challenge led to marked gene activation for MC1 and MC5, perhaps indicating compensatory regulation of other MC in the absence of MC3. However, in the focal stroke model, at 24-hours MC3−/− animals displayed enhanced rolling and adhesion further supporting a delayed role of this receptor, while e/e animals only showed elevated adhesion compared with WT, perhaps as a result enhanced rolling before the observation period. Previous reports and unpublished data from our laboratory have shown no significant differences in the basal circulating levels of leukocytes between WT, e/e, and MC3−/−.3 As such, the observed enhanced leukocyte recruitment is unlikely to be because of an initially high leukocyte count in these animals. As observed in BCCAo animals both receptors seem to be of importance with MC3 anti-inflammatory circuits predominating at later time points.
Previously, Leoni et al3 demonstrated that the MC3−/− mouse displays higher levels of leukocyte adhesion and emigration in the mesenteric microcirculation after I/R, while the response of the e/e mouse was not significantly different from WT. Yet in the same model, the MC1 agonist BMS-470539 was effective in reducing leukocyte recruitment (an effect which was absent in e/e mice).4 These results led the authors to postulate that the receptors show different physiological roles but that both may be harnessed pharmacologically. The present findings demonstrate a physiological role for both receptors in the cerebral microcirculation. Indeed, the observed of temporally distinct roles of these 2 receptors may help to explain such apparently conflicting findings across studies investigating the anti-inflammatory actions of these receptors. Crucially, our study demonstrates that the absence of signaling from either receptor resulted in enhanced infarct size and worse functional outcome compared with WT, further illustrating that both receptors are important pharmacological targets.
Our studies have further demonstrated that melancortin peptides may affect neutrophil inflammatory functioning in human cells. As the MC receptor system displays several disparities in organization and function between humans and rodents. Such as differences in potency and selectivity of MC ligands, and receptor expression between species, we also investigated MC roles in human neutrophil functioning. The facts that α-MSH treatment was found to suppress neutrophil recruitment to HUVECs via actions on neutrophils, and that MC1, but not MC3, is expressed on neutrophils supports the concept of an MC1-specific effect. These effects were rapid, occurring within 30 minutes after treatments, again a timeframe incompatible with transcriptional mechanisms. MC1-specific direct effects on neutrophils were further illustrated by the ability of MC1-targeted treatments to significantly reduce neutrophil chemotaxis (performed in the absence of other cell types). Further investigation, however, will need to be made to establish the effector cell type of the delayed, but potent, effects of MC3 on neutrophil recruitment.
Taken together, novel experimental data presented here highlight a role for use of MC-based treatments as potential therapeutics for stroke, and potentially other neurovascular diseases. This work also demonstrates important roles for both MC1 and MC3, showing MC1 effects to provide rapid inhibition of leukocyte recruitment via mechanisms independent of NF-κB regulation, while MC3 actions seem more robust at later time points. Given the complexity of MC regulation along with the variable nature of stroke, strategies targeting multiple MCs in a non(or perhaps partially) selective manner may be more fruitful in providing robust protection, rather than targeting 1 MC alone. Indeed, it may be telling that currently the most promising MC compounds (NDP-α-MSH and AP214) are both nonselective MC agonists. Further investigations into later time points of treatment and in animals with comorbidities will help to reveal the full potential of this promising therapeutic target for stroke.
P.M. Holloway performed, designed, and analyzed experiments and wrote the article. P.F. Durrenberger performed experiments, D. Cooper performed some flow chamber experiments, M. Perretti provided scientific input and provided the e/e and MC3−/− animals, S.J. Getting and F.N.E. Gavins designed and analyzed the experiments and wrote the article. We also thank Monika Dowejko (University of Westminster, United Kingdom) for genotyping the MC3−/− mouse, Dr Lucy Norling (William Harvey Research Institute, United Kingdom) for her help with the flow chamber model and Jonette Green (LSUHSC-S) for help with the immunofluorescent staining. Marjan Trutschl and Urska Cvek provided input on the statistical analysis and techniques.
Sources of Funding
This study was funded by The British Heart Foundation (studentship FS/09/020/27184). Drs Trutschl and Cvek work reported in this publication was supported by the National Institute Of General Medical Sciences of the National Institutes of Health under Award no. P30GM110703.
The online-only Data Supplement is available with this article at http://atvb.ahajournals.org/lookup/suppl/doi:10.1161/ATVBAHA.115.305348/-/DC1.
- Nonstandard Abbreviations and Acronyms
- bilateral common carotid artery occlusion
- MC1 mutant recessive yellow e/e
- melanocortin receptor 3 null
- melanocyte-stimulating hormone
- Received January 19, 2015.
- Accepted June 11, 2015.
- © 2015 American Heart Association, Inc.
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Stroke is a leading cause of mortality and morbidity worldwide. Following the initial ischemic brain damage an ensuing inflammatory response may exacerbate injury. Neutrophils are key arbitrators of this damaging response and inhibiting neutrophil recruitment in cerebral ischemia-reperfusion injury may provide therapeutic benefit. We have previously demonstrated that melanocortin treatments can reduce leukocyte recruitment in peripheral tissues, but these actions have yet to be demonstrated in the unique microcirculation of the brain. Here in, we show that melanocortins reduce post ischemic leukocyte recruitment in the brain, and that these effects are mediated by the distinct actions of both melanocortin receptors 1 and 3. Therefore, targeting these receptors provides a novel therapeutic strategy for treating stroke, and other cerebrovascular diseases.