Oxidant-induced vascular endothelial growth factor expression in human keratinocytes and cutaneous wound healing

Neutrophils and macrophages, recruited to the wound site, release reactive oxygen species by respiratory burst. It is commonly understood that oxidants serve mainly to kill bacteria and prevent wound infection. We tested the hypothesis that oxidants generated at the wound site promote dermal wound repair. We observed that H(2)O(2) potently induces vascular endothelial growth factor (VEGF) expression in human keratinocytes. Deletion mutant studies with a VEGF promoter construct revealed that a GC-rich sequence from bp -194 to -50 of the VEGF promoter is responsible for the H(2)O(2) response. It was established that at microm concentrations oxidant induces VEGF expression and that oxidant-induced VEGF expression is independent of hypoxia-inducible factor (HIF)-1 and dependent on Sp1 activation. To test the effect of NADPH oxidase-generated reactive oxygen species on wound healing in vivo, Rac1 gene transfer was performed to dermal excisional wounds left to heal by secondary intention. Rac1 gene transfer accelerated wound contraction and closure. Rac1 overexpression was associated with higher VEGF expression both in vivo as well in human keratinocytes. Interestingly, Rac1 gene therapy was associated with a more well defined hyperproliferative epithelial region, higher cell density, enhanced deposition of connective tissue, and improved histological architecture. Overall, the histological data indicated that Rac1 might be an important stimulator of various aspects of the repair process, eventually enhancing the wound-healing process as a whole. Taken together, the results of this study indicate that wound healing is subject to redox control.     

Suppression of phorbol myristate acetate-triggering of macrophage H2O2 release by sarcoma 180 originating factor

A high molecular weight proteinaceous factor in the cell extract of sarcoma 180 (S-180) was found to inhibit phorbol myristate acetate (PMA)-triggering of macrophage H2O2 release. This factor (S-180 factor) was stable at 56 C for 1 hr and resistant to ultraviolet-irradiation. The S-180 factor inhibited the specific binding of PMA to macrophages and this was accompanied by a parallel reduction of PMA-triggered H2O2 release. S-180 factor preferentially depressed macrophage H2O2 release in response to phorbol diesters including PMA, 4 beta-phorbol 12 13 beta,13 alpha-diacetate, 4 beta-phorbol 12 beta,13 alpha-didecanoate, 4 beta-phorbol 12 beta,13 alpha-dibenzoate, and 4-omicron-methyl-PMA rather than the H2O2 release triggered by wheat germ agglutinin or by phagocytosis of latex particles. The S-180 factor failed to affect the PMA-elicited macrophage cell spreading and macrophage phagocytic activity against latex beads with or without PMA-mediated stimulation. A similar inhibitory factor was found in the extracts of some other murine tumor cells (Ehrlich carcinoma and thymic leukemia) and normal cells (liver, spleen, and peritoneal exudate cells).     

Gene transfer of CuZn superoxide dismutase enhances the synthesis of vascular endothelial growth factor

Nitric oxide (NO) and reactive oxygen species (ROS) are emerging as important regulators of angiogenesis. NO enhances VEGF synthesis in several cell types and is required for execution of VEGF angiogenic effect in endothelial cells. Similarly, hydrogen peroxide induces VEGF synthesis and recent studies indicate the involvement of ROS in signaling downstream of VEGF stimulation. VEGF synthesis can not only be enhanced by gene transfer of VEGF but also by overexpression of NO synthase genes. Here, we examined the possibility of augmentation of VEGF production by gene transfer of copper/zinc superoxide dismutase (CuZnSOD, SOD1). Overexpression of human SOD1 in mouse NIH 3T3 fibroblasts increased SOD activity, enhanced intracellular generation of H2O2 and significantly stimulated VEGF production as determined by increase in VEGF promoter activity, VEGF mRNA expression and VEGF protein synthesis. The stimulatory effect on VEGF synthesis induced by SOD1 gene transfer was reverted by overexpression of human catalase. The effect of H2O2 produced by engineered cells is mediated by activation of hypoxia-inducible factor response element (HRE) as well as Sp1 recognition site of VEGF promoter. This data suggest the feasibility of stimulation of angiogenesis by overexpression of SOD1.     

Activation of mouse peritoneal macrophages in vitro and in vivo by interferon-gamma

To determine the role of IFN-gamma in the activation of resident mouse peritoneal macrophages, crude macrophage-activating lymphokines were incubated with a monoclonal anti-murine IFN-gamma antibody. This treatment abolished the capacity of mitogen-induced lymphokines to enhance either H2O2 release or activity against the intracellular protozoa Toxoplasma gondii and Leishmania donovani. All macrophage-activating factor detected by these assays was also removed by passing the lymphokines over a Sepharose column to which the monoclonal anti-IFN-gamma antibody had been coupled. Therefore, pure murine rIFN-gamma was tested both in vitro and in vivo as a single activating agent. After 48 hr of pretreatment in vitro with 0.01 to 1 antiviral U/ml, macrophage H2O2-releasing capacity was enhanced an average of 6.4-fold; half-maximal stimulation was induced by 0.03 U/ml. Resident macrophages infected with T. gondii half-maximally inhibited parasite replication after 24 hr of preincubation with 0.14 U/ml of rIFN-gamma, and near complete inhibition was achieved by pretreatment with 100 U/ml. Half-maximal leishmanicidal activity was induced by 0.08 U/ml of rIFN-gamma, and 67 to 75% of intracellular L. donovani amastigotes were killed after macrophages were preincubated with 10 to 100 U/ml. Eighteen hours after parenteral injection of rIFN-gamma, peritoneal macrophages displayed a dose-dependent enhancement of H2O2-releasing capacity and antiprotozoal activity. Half-maximal enhancement required 85 to 250 U or rIFN-gamma given i.p. Peritoneal macrophages were also activated by rIFN-gamma injected i.v. and intramuscularly. These results suggest that, in the mouse model, IFN-gamma is likely to be a primary factor within mitogen-induced lymphokines responsible for activating macrophage oxidative metabolism and antiprotozoal activity, and indicate that rIFN-gamma is a potent activator of these effector functions both in vitro and in vivo. These findings provide a rationale for evaluating rIFN-gamma in the treatment of systemic intracellular infections, and indicate that murine models are appropriate for such studies.     

Macrophage colony-stimulating factor (M-CSF) enhances the capacity of murine macrophages to secrete oxygen reduction products

The capacity of macrophage colony-stimulating factor (M-CSF) to enhance respiratory burst activity in peritoneal macrophages was measured. Macrophages incubated for 48 hr or more with concentrated L cell-conditioned medium as a source of M-CSF released two to three times as much O2- in response to PMA as did unexposed macrophages. Stimulation was noted at concentrations of colony-stimulating activity from 0.1 to 2000 U/ml and was maximal at 10 to 100 U/ml. Purified, endotoxin-free CSF enhanced secretion to a similar degree as unpurified L cell-conditioned medium. Release of O2- by M-CSF macrophages occurred over 60 min and was triggered by opsonized zymosan as well as PMA. H2O2 release was also enhanced in macrophages exposed to both unpurified and purified M-CSF. These data indicate that M-CSF enhances the capacity of mature macrophages to release oxygen reduction products, and they are consistent with reports that CSF can stimulate the release of other secretory products.     

Macrophage activation by Tetrahymena pyriformis. I. Active subcellular fractions of Tetrahymena

Experiments were carried out to determine what subcellular fractions of Tetrahymena pyriformis could, after inoculation into mice, activate macrophages to kill Toxoplasma gondii in vitro. Peritoneal macrophages from mice inoculated intraperitoneally with cilia, pellicles, mitochondria, and microsomes exhibited strong toxoplasmacidal activity and had an enhanced capacity to release hydrogen peroxide (H2O2) by stimulation of a membrane-active agent as compared with resident macrophages. In contrast, macrophages from mice inoculated with macronuclei and postmicrosomal supernatant showed no toxoplasmacidal activity and a low level of H2O2 release. Similar dose response was observed on the active subcellular fractions with regard to the degree of macrophage activation. Treatment of the active subcellular fractions with heating and trypsin markedly reduced their activity.     

Stimulation of macrophage H2O2 release by resident thymocytes: effect of a soluble factor distinct from interferon-gamma

Activated T cells are known to stimulate macrophage oxidative metabolism and antimicrobial activity through release of interferon-gamma (IFN-gamma). In contrast, the role of nonactivated T cells in regulating macrophage effector functions is less well defined. We have previously reported that a low molecular weight soluble factor derived from resident (nonactivated) thymocytes enhances macrophage receptor-mediated phagocytosis. In the present study, we examined the capacity of resident murine thymocytes to stimulate the respiratory burst and microbicidal activity of peritoneal macrophages. Macrophages cultured for 1-2 days with cell-free thymocyte supernatant (TS) released two to three times more H2O2 in response to PMA or opsonized zymosan than did control macrophages. The H2O2-stimulating factor in TS was distinguished from IFN-gamma by its heat stability (100 degrees C, 20 min), approximate MW of 2400 Da (gel filtration high-pressure liquid chromatography), and absence of interferon activity in both antiviral and enzyme-linked immunosorbent assays. TS-treated macrophages, however, did not exhibit a greater capacity to kill or inhibit the intracellular growth of Toxoplasma gondii, indicating that the thymocyte factor did not fully activate macrophage microbicidal mechanisms. These data suggest that thymocytes can increase the respiratory burst capacity of macrophages in the absence of antigen-specific immune responses.     

Transcription of the vascular endothelial growth factor gene in macrophages is regulated by liver X receptors

Macrophages are an important source of angiogenic activity in wound healing, cancer, and chronic inflammation. Vascular endothelial growth factor (VEGF), a cytokine produced by macrophages, is a primary inducer of angiogenesis and neovascularization in these contexts. VEGF expression by macrophages is known to be stimulated by low oxygen tension as well as by inflammatory signals. In this study, we provide evidence that Vegfa gene expression is also regulated by activation of liver X receptors (LXRs). VEGF mRNA was induced in response to synthetic LXR agonists in murine and human primary macrophages as well as in murine adipose tissue in vivo. The effects of LXR ligands on VEGF expression were independent of hypoxia-inducible factor HIF-1alpha activation and did not require the previously characterized hypoxia response element in the VEGF promoter. Rather, LXR/retinoid X receptor heterodimers bound directly to a conserved hormone response element (LXRE) in the promoter of the murine and human Vegfa genes. Both LXRalpha and LXRbeta transactivated the VEGF promoter in transient transfection assays. Finally, we show that induction of VEGF expression by inflammatory stimuli was independent of LXRs, because these effects were preserved in LXR null macrophages. These observations identify VEGF as an LXR target gene and point to a previously unrecognized role for LXRs in vascular biology.     

Ganglioside expression in macrophages from endotoxin responder and nonresponder mice

Peritoneal macrophage ganglioside patterns and ganglioside sialic acid content were compared for two congenic strains of mice having differing responses to bacterial lipopolysaccharide. Resident macrophage ganglioside patterns from C3H/HeJ mice (endotoxin hyporesponsive) and C3H/HeN mice (endotoxin responsive) were similar. Macrophages elicited with phenol-extracted or butanol-extracted endotoxin showed distinctly more complex ganglioside patterns in C3H/HeN mice. C3H/HeJ macrophages showed distinct, but less complex changes when elicited with butanol-extracted endotoxin. As expected, there were minimal alterations induced by phenol-extracted endotoxin in the C3H/HeJ patterns. When injected with whole killed E. coli, both strains of mice exhibited complex ganglioside patterns; however, there were relative differences in the quantities of multiple gangliosides. Differences in ganglioside patterns were mirrored in the relative ratios of N-acetyl- to N-glycolylneuraminic acid. When macrophages were activated by administration of either endotoxin preparation, macrophage gangliosides from C3H/HeN mice always contained a higher proportion of N-acetylneuraminic acid compared with C3H/HeJ macrophage gangliosides. Oxidative metabolism of the macrophage populations was assessed by PMA-induced H2O2 release. This indicated that endotoxin activation produced an increase in PMA-induced H2O2 release as well as a shift of sialic acid class from the N-glycolyl type to the N-acetyl type. However, no direct correlation could be made between ganglioside composition, sialic acid content, and macrophage function. These data indicate that both ganglioside composition and sialic acid composition of macrophages are profoundly altered with endotoxin activation. The data further indicate that under conditions which C3H/HeJ mice respond to Gram-negative bacteria, their macrophage ganglioside patterns still differ from normal mice.     

Differential expression and regulation of macrophage inflammatory protein (MIP)-1alpha and MIP-2 genes by alveolar and peritoneal macrophages in LPS-hyporesponsive C3H/HeJ mice

A point mutation in Toll-like receptor 4 (Tlr4) gene in C3H/HeJ mice underlies a defect in LPS-induced cytokine production by peritoneal macrophages (PMphi;). Whether the C-C and the C-X-C chemokines are induced differently by LPS between alveolar macrophages (AMphi;) and PMphi; in this mice remains unclear. Thus, we examined the expression and regulation of macrophage inflammatory protein-1alpha (MIP-1alpha) and macrophage inflammatory protein-2 (MIP-2) in C3H/HeJ macrophages. These results showed that the accumulation of MIP-1alpha and MIP-2 mRNA increased dose dependently in response to LPS. PMphi; responded to LPS to produce significantly higher levels of both chemokine mRNA and protein than AMphi;. In addition, both macrophages produced much more MIP-2 than MIP-1alpha by the same doses of LPS stimulation. Moreover, the chemokine production by C3H/HeN macrophages was significantly higher than that of the C3H/HeJ macrophages. IFN-gamma suppressed the LPS-induced MIP-1alpha release but enhanced the LPS-induced MIP-2 secretion in both macrophages. These results show that the chemokine production was induced and regulated differentially in AMphi; and PMphi;.     

15d-PGJ2 inhibits oxidized LDL-induced macrophage proliferation by inhibition of GM-CSF production via inactivation of NF-kappaB

Macrophage-derived foam cells play an important role in atherosclerotic lesions. Oxidized low-density lipoprotein (Ox-LDL) induces macrophage proliferation via production of GM-CSF in vitro. This study investigated the effects of 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), a natural ligand for peroxisome proliferator-activated receptor gamma, on macrophage proliferation. Mouse peritoneal macrophages and RAW264.7 cells were used for proliferation study and reporter gene assay, respectively. Twenty microgram per milliliter of Ox-LDL induced [3H]thymidine incorporation in mouse peritoneal macrophages, and 15d-PGJ(2) inhibited Ox-LDL-induced [3H]thymidine incorporation in a dose-dependent manner. Ox-LDL increased GM-CSF release and GM-CSF mRNA expression, and activated GM-CSF gene promoter, all of which were prevented by 15d-PGJ(2) or 2-cyclopenten-1-one, a cyclopentenone ring of 15d-PGJ(2). The suppression of GM-CSF promoter activity by 15d-PGJ(2) and 2-cyclopenten-1-one was mediated through reduction of NF-kappaB binding to GM-CSF promoter. These results suggest that 15d-PGJ(2) inhibits Ox-LDL-induced macrophage proliferation through suppression of GM-CSF production via NF-kappaB inactivation.     

Deregulated unfolded protein response in chronic wounds of diabetic ob/ob mice: A potential connection to inflammatory and angiogenic disorders in diabetes-impaired wound healing

Type-2 diabetes mellitus (T2D) represents an important metabolic disorder, firmly connected to obesity and low level of chronic inflammation caused by deregulation of fat metabolism. The convergence of chronic inflammatory signals and nutrient overloading at the endoplasmic reticulum (ER) leads to activation of ER-specific stress responses, the unfolded protein response (UPR). As obesity and T2D are often associated with impaired wound healing, we investigated the role of UPR in the pathologic of diabetic-impaired cutaneuos wound healing. We determined the expression patterns of the three UPR branches during normal and diabetes-impaired skin repair. In healthy and diabetic mice, injury led to a strong induction of BiP (BiP/Grp78), C/EBP homologous protein (CHOP) and splicing of X-box-binding protein (XBP)1. Diabetic-impaired wounds showed gross and sustained induction of UPR associated with increased expression of the pro-inflammatory chemokine macrophage inflammatory protein (MIP)2 as compared to normal healing wounds. In vitro, treatment of RAW264.7 macrophages with tunicamycin, and subsequently stimulation with lipopolysaccharide (LPS) and interferon (IFN)-γ enhances MIP2 mRNA und protein expression compared to proinflammatory stimulation alone. However, LPS/IFNγ induced vascular endothelial growth factor (VEGF) production was blunted by tunicamycin induced-ER stress.     

Magnesium-deficient medium enhances NO production in alveolar macrophages isolated from rats

Magnesium deficiency has been shown to increase nitric oxide (NO) levels in plasma and to aggravate endotoxin lethality. The present study was performed to examine the effects of magnesium (Mg(2+))-deficient culture medium, with and without endotoxin (LPS), on NO release and inducible NOS (iNOS) mRNA levels in alveolar macrophages isolated from rats. Decreasing the Mg(2+) concentration in the culture medium from 0.39 mM (normal-Mg(2+) medium) to 0.021 mM (Mg(2+)-deficient medium) increased NO release from alveolar macrophages for 2 h. However, LPS stimulation in Mg(2+)-deficient medium had little effect on NO release. The increased NO release in Mg(2+)-deficient medium was suppressed completely by L-NAME and aminoguanidine. Dexamethasone, pyrrolidine dithiocarbamate and curcumin strongly inhibited NO release. Verapamil, U73122, TMB-8 and W-7 had no significant effect on NO release induced by Mg(2+) deficiency. Preculture of macrophages with Mg(2+)-deficient medium for 22 h markedly increased NO release and iNOS mRNA levels for a further 2 h; these increments were suppressed completely by curcumin. These results suggest that Mg(2+) deficiency enhances NO production via iNOS by alveolar macrophages. In this experimental condition, we can not suggest that NO production from alveolar macrophage plays an essential role in the pathogenesis of enhanced endotoxin lethality in Mg-deficient rats.     

Characteristics of macrophage activation by gamma interferon for tumor cytotoxicity in peritoneal macrophages and macrophage cell line J774.1

We investigated the characteristics of macrophage-mediated tumor cytotoxicity (MTC) against Meth A target, H2O2 generation and release of effector molecule(s) for MTC, by comparing with those of peritoneal macrophages (PMP) and macrophage cell line J774.1 during stimulation with recombinant gamma interferon (IFN-gamma). In PMP, MTC was demonstrated when they were stimulated with IFN-gamma for 12 hr (short-term stimulation) and was abrogated when they were stimulated for 48 hr (long-term stimulation). Enhanced H2O2 generation was observed in PMP activated by long-term stimulation followed by triggering with PMA, but not observed by triggering with Meth A cells. By contrast, whereas non-treated J774.1 cells have already attained a definite level of MTC, a higher MTC level was demonstrated both by short- and long-term stimulations. Conversely, J774.1 cells were unable to generate H2O2 at any stage of IFN-gamma stimulation followed by triggering both with PMA or Meth A cells. The time course for stimulation of PMP by IFN-gamma for release of cytotoxic factor (CF) corresponded to that for MTC by PMP, and activities of the CF released from both activated PMP and J774.1 cells also closely corresponded to those of MTC by both cells. The serological and physicochemical characteristics of CF released from both activated PMP and J774.1 cells were determined to be closely related to those of tumor necrosis factor (TNF). These results indicate that in contrast to PMP, the J774.1 cell line is free from suppression stage for MTC and CF release during stimulation with IFN-gamma. The results suggest that TNF-like CF plays a crucial role for MTC against Meth A target, and that H2O2 is irrelevant for MTC against Meth A.