Balduino Mastrofrancesco

Distinct arginase isoforms expressed in primary and transformed macrophages : regulation by oxygen tension

Experiments were performed to identify arginase isoforms expressed in primary and transformed rodent macrophages and to determine the molecular mechanisms for the previously observed increase in arginase activity in macrophages cultured in hypoxia or anoxia. Results demonstrate the following: 1) mRNA and protein for hepatic-type AI arginase are expressed in primary cultures of rat and mouse peritoneal macrophages and are enhanced seven- and ninefold, respectively, by lipopolysaccharide (LPS). 2) mRNA for extrahepatic-type AII arginase is constitutively expressed in mouse, but not rat, peritoneal macrophages and is detected in RAW264.7 cells after LPS treatment; neither J774A.1 nor P388D1 cells contain arginase mRNA. 3) AI arginase mRNA, arginase activity in cell lysates, andl-arginine flux through arginase in intact cells are all increased in rat wound-derived and mouse peritoneal macrophages by hypoxic or anoxic culture; AII arginase mRNA is, in contrast, suppressed >50% by O2deprivation. 4) Expression of thel-arginine transporter mCAT-2 is increased greater than twofold by reduced O2 culture. These results demonstrate substantial variability in arginase isoform expression among primary and transformed rodent macrophages. They also identify AI and AII arginase and the mCAT-2 l-arginine transporter as O2-regulated genes.

Molecular and Metabolic Evidence for the Restricted Expression of Inducible Nitric Oxide Synthase in Healing Wounds

Tissue injury initiates a temporally ordered sequence of local cellular and metabolic responses presumably necessary for successful repair. Previous investigations demonstrated that metabolic evidence for nitric oxide synthase (NOS) activity is detectable in wounds only during the initial 48 to 72 hours of the repair process. Present results identify the cell types contributing inducible NOS (iNOS) to experimental wounds in rats. iNOS antigen was expressed in most macrophages present in wounds 6 to 24 hours after injury, and these cells exhibited NAPDH diaphorase and NOS activity. Polymorphonuclear leukocytes contained little iNOS antigen and no NADPH diaphorase activity and were minimally able to convert L-arginine to L-citrulline. The frequency of iNOS-positive macrophages declined on days 3 and 5 after wounding. By day 10, most macrophages in the wound were negative for iNOS. These cells, however, acquired iNOS antigen and activity in culture. Wound fluids, but not normal rat serum, suppressed the induction of iNOS during culture. Findings indicate that the expression of iNOS in healing wounds is restricted to macrophages present during the early phases of repair and that components of wound fluid suppress the induction of iNOS in macrophages in late wounds. Polymorphonuclear leukocytes contribute little iNOS activity to the healing wound.

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.

Prostaglandin E2 Suppresses Lipopolysaccharide-Stimulated IFN-β Production

Macrophages activate the production of cytokines and chemokines in response to LPS through signaling cascades downstream from TLR4. Lipid mediators such as PGE2, which are produced during inflammatory responses, have been shown to suppress MyD88-dependent gene expression upon TLR4 activation in macrophages. The study reported here investigated the effect of PGE2 on TLR3- and TLR4-dependent, MyD88-independent gene expression in murine J774A.1 macrophages, as well as the molecular mechanism underlying such an effect. We demonstrate that PGE2 strongly suppresses LPS-induced IFN-β production at the mRNA and protein levels. Poly (I:C)-induced IFN-β and LPS-induced CCL5 production were also suppressed by PGE2. The inhibitory effect of PGE2 on LPS-induced IFN-β expression is mediated through PGE2 receptor subtypes EP2 and EP4, and mimicked by the cAMP analog 8-Br-cAMP as well as by the adenylyl cyclase activator forskolin. The downstream effector molecule responsible for the cAMP-induced suppressive effect is exchange protein directly activated by cAMP (Epac) but not protein kinase A. Moreover, data demonstrate that Epac-mediated signaling proceeds through PI3K, Akt, and GSK3β. In contrast, PGE2 inhibits LPS-induced TNF-α production in these cells through a distinct pathway requiring protein kinase A activity and independent of Epac/PI3K/Akt. In vivo, administration of a cyclooxygenase inhibitor before LPS injection resulted in enhanced serum IFN-β concentration in mice. Collectively, data demonstrate that PGE2 is a negative regulator for IFN-β production in activated macrophages and during endotoxemia.

Bacterial Colonization and the Expression of Inducible Nitric Oxide Synthase in Murine Wounds

The expression of inducible nitric oxide synthase (iNOS) in two different murine wound models was investigated. Animals were subjected to either full-thickness linear skin incision with subcutaneous implantation of sterile polyvinyl alcohol sponges, or to 1.5 x 1.5-cm dorsal skin excision. Reverse transcriptase-polymerase chain reaction detected iNOS mRNA in all cell samples retrieved from the sponges. Immunoblotting of lysates of inflammatory cells harvested from the sponges failed to detect iNOS protein, and immunohistochemistry of the incisional wound was mildly positive. Inflammatory cells of excisional wounds stained strongly positive for iNOS. Cutaneous wounds were found to be colonized with Staphylococcus aureus. The detection of iNOS in cells from sponges inoculated in vivo with heat-killed bacteria and the reduction of immunohistochemical signal for iNOS in excisional wounds of animals treated with antibiotics support a role of bacteria in the induction of iNOS in wounds. The expression of iNOS in excisional wounds requires interferon-gamma and functional lymphocytes because interferon-gamma knockout and SCID-Beige mice exhibited attenuated iNOS staining in excisional wounds. The expression of iNOS in the inflammatory cells of murine wounds is a response to bacterial colonization and not part of the normal repair process elicited by sterile tissue injury.

Modulatory activities of wound fluid on fibroblast proliferation and collagen synthesis.

Several different cell types play a role in the regulatory mechanisms involved in wound healing. A rat wound model was used to evaluate temporal changes in the cellular infiltrate, histology, and effects of wound fluid (WF) on fibroblast growth and collagen synthesis in vitro. Polyvinyl alcohol sponges were implanted in male Sprague/Dawley rats and harvested after 1, 3, 5, 7, 10, and 15 days. Rat wound fibroblasts were cultured in different media with 10 or 20% WF pooled from five or more rats from each time interval, and then pulsed with [3H]thymidine. Days 1 through 5 WF stimulated proliferation, whereas Days 10 and 15 WF inhibited proliferation. Stimulatory activity was found in the greater than 300 kDa molecular weight fraction; inhibitory activity was in the less than 10 kDa molecular weight fraction. Ten percent WF from both Day 1 and Day 15 sponges exerted a stimulatory effect in incubated fibroblasts on collagen production, measured as protein-bound [3H]hydroxyproline. Fibroblast proliferation and collagen synthesis appeared to be independently regulated functions. Fibroblasts were stimulated by the wound environment to proliferate for about 1 week after injury, at which point further growth was inhibited, while collagen production was maintained.

Evidence for aerobic glycolysis in λ-carrageenan-wounded skeletal muscle

Abstract Classically, increased lactate production in wounded tissue is ascribed to anaerobic glycolysis although its oxygen consumption has been found to be similar to normal tissue. This apparent inconsistency was studied in a standardized isolated perfused wound model. Male Sprague-Dawley rats were wounded (group W) with intramuscular injections of λ-carrageenan and fed ad lib.; not wounded and pair fed to the decreased food intake of the wounded animals (group PFC); or not wounded and fed ad lib. (group ALC). After 5 days, the hindlimbs of animals from each group were either perfused using a standard perfusate with added [U-14C]glucose or [1-14C]pyruvate or assayed for the tissue content of lactate and pyruvate. In addition, the effect of a 30% hemorrhage on the tissue lactate and pyruvate concentration was examined. Wounding increased glucose uptake and lactate production by 100 and 96%, respectively, above that seen in ALC animals. Oxygen consumption was unchanged by wounding (5.74, 5.14, and 5.83 μmole/min/100 g in W, PFC, and ALC, respectively). Glucose and pyruvate oxidation were also unaltered among the groups. Hemorrhage resulted in a comparable increase in lactate and pyruvate in tissue from wounded and pair-fed control animals (above those concentrations found in tissue harvested without preexisting hemorrhage). As a consequence, the same relationship in L/P ratio was maintained after hemorrhage. Taken together, these results confirm the presence of aerobic glycolysis in wounded tissue (unchanged oxygen consumption, glucose, and pyruvate oxidation). In addition, pyruvate dehydrogenase activity in the wound was apparently the same as that found in muscle from pair-fed control animals.

Vestigial respiratory burst activity in wound macrophages

Macrophages from experimental wounds in rats were tested for their capacity to generate reactive oxygen intermediates. Measurements of superoxide and H2O2release, [Formula: see text]-dependent lucigenin chemiluminescence, oxygen consumption, hexose monophosphate shunt flux, and NADPH oxidase activity in cell lysates indicated, at best, the presence of a vestigial respiratory burst response in these cells. The inability of wound cells to release[Formula: see text] was not rekindled by priming with endotoxin or interferon-γ in vivo or in vitro. NADPH oxidase activity in a cell-free system demonstrated that wound macrophage membranes, but not their cytosols, were capable of sustaining maximal rates of [Formula: see text] production when mixed with their corresponding counterparts from human neutrophils. Immune detection experiments showed wound macrophages to be particularly deficient in the cytosolic component of the NADPH oxidase p47- phox. Addition of recombinant p47- phox to the human neutrophil-cell membrane/wound macrophage cytosol cell-free oxidase assay, however, failed to support[Formula: see text] production. Present findings indicate an unexpected deficit of wound macrophages in their capacity to generate reactive oxygen intermediates.Macrophages from experimental wounds in rats were tested for their capacity to generate reactive oxygen intermediates. Measurements of superoxide and H2O2 release, O-2-dependent lucigenin chemiluminescence, oxygen consumption, hexose monophosphate shunt flux, and NADPH oxidase activity in cell lysates indicated, at best, the presence of a vestigial respiratory burst response in these cells. The inability of wound cells to release O-2 was not rekindled by priming with endotoxin or interferon-gamma in vivo or in vitro. NADPH oxidase activity in a cell-free system demonstrated that wound macrophage membranes, but not their cytosols, were capable of sustaining maximal rates of O-2 production when mixed with their corresponding counterparts from human neutrophils. Immune detection experiments showed wound macrophages to be particularly deficient in the cytosolic component of the NADPH oxidase p47-phox. Addition of recombinant p47-phox to the human neutrophil-cell membrane/wound macrophage cytosol cell-free oxidase assay, however, failed to support O-2 production. Present findings indicate an unexpected deficit of wound macrophages in their capacity to generate reactive oxygen intermediates.

The temporal characteristics of the metabolic and endocrine response to injury.

The neuroendocrine and substrate responses immediately after injury have been extensively investigated in man and animals. The purpose of the present study was to examine simultaneously, the temporal, metabolic and endocrine consequences of a single uniform injury induced by the injection of lambda-carrageenan into the hindlimbs of male Sprague-Dawley rats and to compare this response to that observed in semistarved pair-fed control animals. Immediately after injury there was a decrease in the plasma hematocrit, increase in tissue water and peripheral vasoconstriction that suggested hypovolemia. This was followed by a restoration of the blood volume by 1 day as reflected in hemodilution. Alterations in insulin, glucagon, ACTH, corticosterone, epinephrine, norepinephrine, and dopamine in wounded animals occurred during the first 5 days. However, similar changes were observed in pair-fed control animals from days 1 to 5. These findings implied that the early endocrine response observed from 0 to 24 hours after injury arises, primarily as a result of hypovolemia, whereas the response observed from 1 to 5 days appeared to be the result of semistarvation. In contrast to the endocrine alterations observed, alterations in the plasma concentrations of lactate, acetoacetate and beta-hydroxybutyrate persisted for up to 15 days. The presence of these substrate alterations in the absence of hormonal stimuli suggest that nonendocrine mechanisms exist to induce these alterations. The possibility is raised that these substrate alterations may be, at least in part, the result of the inflammatory infiltrate.

A macrophage-mediated factor that increases the high energy phosphate content of skeletal muscle

A marked cellular infiltrate accompanies wounding. The phagocytic and bacteriocidal activities of this infiltrate require increased substrate and O2 consumption. This rapid utilization of available oxygen and substrates could jeopardize an already compromised resident cellular component of a wound. Recent studies have demonstrated macrophage-mediated cell stimulatory agents which induce proliferation of nonlymphoid mesenchymal cells. This study was designed to examine macrophage-resident tissue interactions and their possible significance in wounded tissue. A reconstituted wound system was designed which combines the major components of a lambda-carrageenan skeletal muscle wound (muscle + macrophages). The extensor digitorum longi (EDL) of male Fisher rats were incubated in a standardized fashion. The groups of EDL were muscle incubated alone, muscle with the addition to the incubate of activated peritoneal macrophages or muscle with the addition of a conditioned supernatant from the incubation of activated or nonactivated peritoneal macrophages. Muscle ATP and CP content were noted to be increased 46 and 22%, respectively, when macrophages and skeletal muscle were coincubated. Macrophage-conditioned media from activated or nonactivated macrophages increased the ATP and CP muscle content 44 and 37%, respectively. Preliminary characterization of this high energy phosphate (HEP) promoting factor demonstrates it to be heat and cold stable and less than 10,000 Da. Therefore, a macrophage-mediated transferable factor is capable of increasing the HEP content of skeletal muscle in an in vitro system. This may have important consequences in maintaining host cell integrity following injury.