Jean M. Daley

Use of Ly6G-specific monoclonal antibody to deplete neutrophils in mice

The anti‐granulocyte receptor‐1 (Gr‐1) mAb, RB6‐8C5, has been used extensively to deplete neutrophils in mice and to investigate the role of these cells in host defense. RB6‐8C5 binds to Ly6G, which is present on neutrophils, and to Ly6C, which is expressed on neutrophils, dendritic cells, and subpopulations of lymphocytes and monocytes. It is thus likely that in vivo administration of RB6‐8C5 may deplete not only neutrophils but also other Gr‐l+ (Ly6C+) cells. This study describes the use of an Ly6G‐specific mAb, 1A8, as an alternative means to deplete neutrophils. In vivo administration of RB6‐8C5 reduced blood neutrophils and Gr‐1+ monocytes, whereas administration of 1A8 reduced blood neutrophils but not Gr‐1+ monocytes. Plasma TNF‐α in endotoxemia was increased 20‐fold by RB6‐8C5 pretreatment and fourfold by 1A8 pretreatment. In a wound model, pretreatment with either antibody decreased wound neutrophils and macrophages. TNF‐α staining in brefeldin‐treated wound leukocytes was increased by pretreatment with RB6‐8C5, but not 1A8. Neutrophil depletion with 1A8 offers advantages over the use of RB6‐8C5, as it preserves non‐neutrophil Gr‐1+ cells depleted by the anti‐Gr‐1 antibody. The loss of non‐neutrophil Gr‐1+ populations in RB6‐8C5‐treated animals is associated with increased TNF‐α responses, suggesting these cells may function to suppress TNF‐α production.

The phenotype of murine wound macrophages

The phenotype of wound macrophages has not been studied by direct examination of these cells, yet macrophages recruited to sites of injury are described as alternatively activated macrophages, requiring IL‐4 or IL‐13 for phenotypic expression. This study characterized wound macrophage phenotype in the PVA sponge wound model in mice. Eighty‐five percent of wound macrophages isolated 1 day after injury expressed Gr‐1, but only 20% of those isolated at 7 days expressed this antigen. Macrophages from 1‐, 3‐, and 7‐day wounds expressed markers of alternative activation, including mannose receptor, dectin‐1, arginase 1, and Ym1, but did not contain iNOS. Day 1 wound macrophages produced more TNF‐α, more IL‐6, and less TGF‐β than Day 7 wound macrophages. Wound macrophages did not produce IL‐10. The cytokines considered necessary for alternative activation of macrophages, IL‐4 and IL‐13, were not detected in the wound environment and were not produced by wound cells. Wound macrophages did not contain PStat6. Wound fluids inhibited IL‐13‐dependent phosphorylation of Stat6 and contained IL‐13Rα2, a soluble decoy receptor for IL‐13. The phenotype of wound macrophages was not altered in mice lacking IL‐4Rα, which is required for Stat6‐dependent signaling of IL‐4 and IL‐13. Wound macrophages exhibit a complex phenotype, which includes traits associated with alternative and classical activation and changes as the wound matures. The wound macrophage phenotype does not require IL‐4 or IL‐13.

Modulation of Macrophage Phenotype by Soluble Product(s) Released from Neutrophils

The regulation of macrophage phenotype by neutrophils was studied in the s.c. polyvinyl alcohol sponge wound model in mice made neutropenic by anti-Gr-1 Ab, as well as in cell culture. Wounds in neutropenic mice contained 100-fold fewer neutrophils than those in nonneutropenic controls 1 day after sponge implantation. Wound fluids from neutropenic mice contained 68% more TNF-α, 168% more IL-6, and 61% less TGF-β1 than those from controls. Wound fluid IL-10 was not different between the two groups, and IL-4 was not detected. Intracellular TNF-α staining was greater in cells isolated from neutropenic wounds than in those from control wounds. The hypothesis that wound neutrophil products modulate macrophage phenotype was tested in Transwell cocultures of LPS-stimulated J774A.1 macrophages and day 1 wound cells (84% neutrophils/15% macrophages). Overnight cocultures accumulated 60% less TNF-α and IL-6 than cultures of J774A.1 alone. The suppression of cytokine release was mediated by a soluble factor(s), because culture supernatants from wound cells inhibited TNF-α and IL-6 release from LPS-stimulated J774A.1 cells. Culture supernatants from purified wound neutrophils equally suppressed TNF-α release from LPS-stimulated J774A.1 cells. Wound cell supernatants also suppressed TNF-α and superoxide release from murine peritoneal macrophages. The TNF-α inhibitory factor has a molecular mass <3000 Da and is neither PGE2 nor adenosine. The present findings confirm a role for neutrophils in the regulation of innate immune responses through modulation of macrophage phenotype.

Disruption of Interleukin-1 Signaling Improves the Quality of Wound Healing

In this study, we investigated the role of interleukin (IL)-1 signaling in wound healing. IL-1 receptor type I (IL-1R) knockout (KO) mice showed reduced fibrosis in both cutaneous and deep tissue wounds, which was accompanied by a reduction in inflammatory cellular infiltration in cutaneous but not in deep tissue wounds. There were no differences in either total collagenolytic activity or in the expression of selected matrix metalloproteinases or tissue inhibitors of metalloproteinases between the wound fluids from wild-type or IL-1R KO mice. However, wound fluids from IL-1R KO mice contained lower levels of IL-6 compared with wild-type controls. In addition, the infusion of IL-6 into wounds in IL-1R KO mice did not increase fibrosis. Skin wounds in IL-1R KO animals had lower levels of collagen and improved restoration of normal skin architecture compared with skin wounds in wild-type mice. However, neither the tensile strength of incisional skin wounds nor the rate of closure of excisional wounds differed between IL-1R KO and wild-type animals. The reduced fibrotic response in wounds from IL-1R KO mice could be reproduced by the administration of an IL-1R antagonist. These findings suggest that pharmacological interference with IL-1 signaling could have therapeutic value in the prevention of hypertrophic scarring and in the treatment of fibrotic diseases.

The Monocyte to Macrophage Transition in the Murine Sterile Wound

The origin of wound repair macrophages is incompletely defined and was examined here in sterile wounds using the subcutaneous polyvinyl alcohol sponge implantation model in mice. Phenotypic analysis identified F4/80+Ly6ChiCD64+MerTK– monocytes and F4/80+Ly6ClowCD64+MerTK+ macrophages in the wound. Circulating monocytes were the precursors of inflammatory Ly6Chi wound monocytes. Ly6ClowMerTK+ macrophages appeared later, expressed CD206, CD11c, and MHC class II, produced cytokines consistent with repair function, and lacked a gene expression profile compatible with mesenchymal transition or fibroblastic transdifferentiation. Data also demonstrated that Ly6Chi wound cells were precursors of Ly6Clow macrophages, although monocytes did not undergo rapid maturation but rather persisted in the wound as Ly6ChiMerTK– cells. MerTK-deficient mice were examined to determine whether MerTK-dependent signals from apoptotic cells regulated the maturation of wound macrophages. MerTK-deficient mice had day 14 cell compositions that resembled more immature wounds, with a smaller proportion of F4/80+ cells and higher frequencies of Ly6G+ neutrophils and Ly6Chi monocytes. The cytokine profile and number of apoptotic cells in day 14 wounds of MerTK-deficient mice was unaffected despite the alterations in cell composition. Overall, these studies identified a differentiation pathway in response to sterile inflammation in which monocytes recruited from the circulation acquire proinflammatory function, persist in the wound, and mature into repair macrophages.

Macrophage arginase regulation by CCAAT/enhancer-binding protein beta.

Arginase activity is expressed by macrophages in healing wounds and other sites of inflammation and has been shown to modulate the synthesis of nitric oxide, polyamines, and collagen. The role of CCAAT/enhancer-binding protein β (C/EBPβ) in the regulation of macrophage arginase by different agonists was investigated using C/EBPβ−/− and +/+ macrophage cell lines. 8-Bromo-cyclic adenosine monophosphate (8-Br-cAMP, 0.5 mM), recombinant murine interleukin 4 (rmIL-4, 20 U/mL), Escherichia coli lipopolysaccharide (100 ng/mL), and hypoxia (1% O2) induced arginase activity in C/EBPβ+/+ macrophages, where enzyme activity correlated with arginase I protein. Only rmIL-4 increased arginase activity in C/EBPβ−/− cells. Arginase II protein was expressed constitutively in wild-type and C/EBPβ−/− cell lines and was unaltered by 8-Br-cAMP or rmIL-4. rmIL-4-stimulated immortalized C/EBPβ−/− macrophages demonstrated higher nuclear signal transducer and activator of transcription-6 (STAT6) and phospho-STAT6 content than their +/+ counterparts. Validating the biological relevance of findings with the cell lines, additional experiments examined wound fluids and peritoneal macrophages from C/EBPβ−/− mice and demonstrated that both contained less arginase activity than those from wild-type controls. Wounds in C/EBPβ−/− animals showed signs of delayed maturation, as manifested by the persistence of neutrophils in the inflammatory infiltrate. Peritoneal macrophages from C/EBPβ+/+ animals responded to 8-Br-cAMP and rmIL-4 with increased arginase activity, whereas those from C/EBPβ−/− mice did not respond to cAMP. Results demonstrate a key mechanistic role for C/EBPβ in the modulation of macrophage arginase I expression in vivo and in vitro.

Hyperphosphorylation of RyRs Underlies Triggered Activity in Transgenic Rabbit Model of LQT2 Syndrome

Rationale: Loss-of-function mutations in human ether go-go (HERG) potassium channels underlie long QT syndrome type 2 (LQT2) and are associated with fatal ventricular tachyarrhythmia. Previously, most studies focused on plasma membrane–related pathways involved in arrhythmogenesis in long QT syndrome, whereas proarrhythmic changes in intracellular Ca2+ handling remained unexplored. Objective: We investigated the remodeling of Ca2+ homeostasis in ventricular cardiomyocytes derived from transgenic rabbit model of LQT2 to determine whether these changes contribute to triggered activity in the form of early after depolarizations (EADs). Methods and Results: Confocal Ca2+ imaging revealed decrease in amplitude of Ca2+ transients and sarcoplasmic reticulum Ca2+ content in LQT2 myocytes. Experiments using sarcoplasmic reticulum–entrapped Ca2+ indicator demonstrated enhanced ryanodine receptor (RyR)–mediated sarcoplasmic reticulum Ca2+ leak in LQT2 cells. Western blot analyses showed increased phosphorylation of RyR in LQT2 myocytes versus controls. Coimmunoprecipitation experiments demonstrated loss of protein phosphatases type 1 and type 2 from the RyR complex. Stimulation of LQT2 cells with &bgr;-adrenergic agonist isoproterenol resulted in prolongation of the plateau of action potentials accompanied by aberrant Ca2+ releases and EADs, which were abolished by inhibition of Ca2+/calmodulin-dependent protein kinase type 2. Computer simulations showed that late aberrant Ca2+ releases caused by RyR hyperactivity promote EADs and underlie the enhanced triggered activity through increased forward mode of Na+/Ca2+ exchanger type 1. Conclusions: Hyperactive, hyperphosphorylated RyRs because of reduced local phosphatase activity enhance triggered activity in LQT2 syndrome. EADs are promoted by aberrant RyR-mediated Ca2+ releases that are present despite a reduction of sarcoplasmic reticulum content. Those releases increase forward mode Na+/Ca2+ exchanger type 1, thereby slowing repolarization and enabling L-type Ca2+ current reactivation.

Toll‐like receptor 4 signaling regulates the acute local inflammatory response to injury and the fibrosis/neovascularization of sterile wounds

The role of Toll‐like receptor 4 (TLR4) in the regulation of inflammation and fibrosis in sterile wounds was investigated in TLR4 signal‐deficient (C3H/HeJ or TLR4−/−) and control mice using the subcutaneously implanted polyvinyl alcohol sponge wound model. Total and differential wound cell counts 1, 3, and 7 days after injury did not differ between C3H/HeJ and C3H/HeOuJ animals. Blood monocytes from both strains expressed CCR2 equally. Day one wounds in C3H/HeJ mice contained fewer Gr‐1high wound macrophages, CCL3, and CCL5, and more CCL17 than those in controls. The accumulation of CCL2, CX3CL1, tumor necrosis factor‐α, interleukin (IL)‐6, IL‐10, IL‐12, and interferon‐γ in wound fluids was not TLR4 dependent. Wound macrophages from C3H/HeJ and C3H/HeOuJ mice expressed CCR4 and CCR5, but not CCR1 or CCR3. Wound macrophage recruitment was not altered in CCR5−/− mice or in C3H/HeOuJ animals injected with neutralizing anti‐CCL3 and anti‐CCL5 antibodies. Neutralization of the CCR4 ligand CCL17 in C3H/HeJ mice did not alter wound macrophage populations. There was a twofold increase in collagen content and number of neovessels in 21‐day‐old wounds in C3H/HeJ vs. C3H/HeOuJ mice. There were no differences between strains in the number of myofibroblasts in the wounds 7 or 21 days postwounding. The increased fibrosis and angiogenesis in wounds from /HeJ mice correlated with higher concentrations of transforming growth factor‐β and fibroblast growth factor 2 in wound fluids from these animals. Wound fluids did not contain detectable lipopolysaccharide and did not induce IκBα degradation in J774.A1 macrophages. Results support a role for endogenous ligands of TLR4 in the regulation of inflammation and repair in sterile wounds.

A novel method of standardized myocardial infarction in aged rabbits

The incidence of both myocardial infarction (MI) and sudden cardiac death increases with age. Here, we describe the development of a minimally invasive large animal model of MI that can be applied to young or aged animals. We demonstrate that rabbit coronary anatomy is highly variable, more so than described in previous literature. In this work, we categorize the coronary pattern of 37 young rabbits and 64 aged rabbits. Aged rabbits had a higher degree of branching from the left main coronary artery. Standardizing the model across age cohorts required a new approach, targeting an area of myocardium rather than a specific vessel. Here, we present a method for achieving a reproducible infarct size, one that yielded a consistent scar encompassing ~30% of the apical left ventricular free wall. The models consistency allowed for more valid comparisons of MI sequelae between age cohorts.NEW & NOTEWORTHY This study describes the coronary angiographic imaging of young and aged rabbits. We developed and improved a novel minimally invasive approach for coil embolization that targets a specific area of myocardium and yielded a consistent scar encompassing ~30% of the left ventricular free wall of young and aged rabbit hearts.