IFN-gamma inhibits the production of latent transforming growth factor-beta1 by mouse inflammatory macrophages

Transforming growth factor (TGF)-beta is a multifunctional cytokine, which in mammals exists in three isoforms (TGF-beta1, 2 and 3). It is synthesized by a variety of cells including macrophages, and exerts potent immunoregulatory effects such as the inhibition of Th1 development and the suppression or reversal of IFN-gamma-induced macrophage activation. In this study we analyzed the effect of IFN-gamma on the production of TGF-beta1 by thioglycolate-elicited mouse peritoneal macrophages under serum-free conditions. Untreated macrophages released TGF-beta1 in its latent form, which became detectable in a capture ELISA specific for active TGF-beta1 after acid activation of the culture supernatants. Treatment with IFN-gamma reduced the amount of latent TGF-beta1 in the culture supernatants in a dose-dependent fashion. The effect of IFN-gamma was confirmed by a newly developed Western blot system for the detection of mouse TGF-beta1 protein. IFN-gamma only weakly (16-24 %) reduced the levels TGF-beta1 mRNA at early and late time points of stimulation, and no evidence was obtained that IFN-gamma suppresses the secretion of latent TGF-beta1. Thus, inhibition of TGF-beta1 production by IFN-gamma is most likely due to decreased synthesis and/or stability of the TGF-beta1 protein, and might be important for the generation of fully activated macrophages and a Th1 response.     

Role of urokinase in the activation of macrophage-associated TGF-beta in silica-induced lung fibrosis

Since tumor growth factor beta (TGF-beta) and its receptor are ubiquitously expressed and because latent TGF-beta cannot bind to the cell surface receptor, the ability of a cell to activate latent TGF-beta upon secretion represents an important regulatory mechanism of TGF-beta action. In vivo, the protease plasmin is considered to be one of the main enzymes operative in the proteolytic cleavage of the latency-associated peptide moiety from TGF-beta, which converts it into the biologically active form. The TGF-beta response was characterized in alveolar macrophages during pulmonary inflammation (d 3) and fibrosis (d 120) induced by a single intratracheal instillation of silica particles (5 mg/mouse). To appreciate the role of urokinase-type plasminogen activator (uPA) in the activation of TGF-beta, the production of total, active and latent TGF-beta by explanted alveolar macrophages was compared in uPA-deficient (uPA-/-) mice and their normal counterparts (uPA+/+). At d 3 and 120 after silica treatment, a significant increase in cell-associated PA activity was found in uPA+/+ mice compared to that of saline controls. As expected, this response was almost totally absent in uPA-/- mice. Alveolar macrophages from uPA+/+ controls were found to release TGF-beta mainly expressed in a biologically active form. In response to silica treatment, inflammatory cells were found to upregulate, especially at the fibrotic stage, their secretion of total and bioactive TGF-beta. No significant difference was found between uPA-/- and uPA+/+ silica-treated animals for the expression of total, active, or latent TGF-beta. Although it has previously been reported that macrophage surface activation of TGF-beta is dependent on both plasmin generation and uPA cell surface receptor, no evidence was found to support this hypothesis in the present study.     

Cellular inflammatory response after spinal cord injury in Sprague-Dawley and Lewis rats

The distribution of microglia, macrophages, T-lymphocytes, and astrocytes was characterized throughout a spinal contusion lesion in Sprague-Dawley and Lewis rats by using immunohistochemistry. The morphology, spatial localization, and activation state of these inflammatory cells were described both qualitatively and quantitatively at 12 hours, 3, 7, 14, and 28 days after injury. By use of OX42 and ED1 antibodies, peak microglial activation was observed within the lesion epicenter of both rat strains between three and seven days post-injury preceding the bulk of monocyte influx and macrophage activation (seven days). Rostral and caudal to the injury site, microglial activation plateaued between two and four weeks post-injury in the dorsal and lateral funiculi as indicated by morphological transformation and the de-novo expression of major histocompatibility class II (MHC II) molecules. Similar to the timing of microglial reactions, T-lymphocytes maximally infiltrated the lesion epicenter between three and seven days post-injury. Reactive astrocytes, while present in the acute lesion, were more prominent at later survival times (7-28 days). These cells were interspersed with activated microglia but appeared to surround and enclose tissue sites occupied by reactive microglia and phagocytic macrophages. Thus, trauma-induced central nervous system (CNS) inflammation, regardless of strain, occurs rapidly at the site of injury and involves the activation of resident and recruited immune cells. In regions rostral or caudal to the epicenter, prolonged activation of inflammatory cells occurs preferentially in white matter and primarily consists of activated microglia and astrocytes. Differences were observed in the magnitude and duration of macrophage activation between Sprague-Dawley (SD) and Lewis (LEW) rats throughout the lesion. Increased expression of complement type 3 receptors (OX42) and macrophage-activation antigens (ED1) persisted for longer times in LEW rats while expression of MHC class II molecules was attenuated in LEW compared to SD rats at all times examined. Variations in the onset and duration of T-lymphocyte infiltration also were observed between strains with twice as many T-cells present in the lesion epicenter of Lewis rats by 3 days post-injury. These strain-specific findings potentially represent differences in corticosteroid regulation of immunity and may help predict a range of functional neurologic consequences affected by neuroimmune interactions.     

Expression of TGF-beta 1, PDGF and IGF-1 mRNA in lung of bleomycin-A5-induced pulmonary fibrosis in rats

OBJECTIVE: To investigate the influence of alveolar macrophages (AMs), fibroblasts and interstitial cells on development of lung fibrosis, and the interactions among TGF-beta 1 PDGF and IGF-1 and these cytokines-effects on lung fibrosis. MATERIAL AND METHODS: Expressions of TGF-beta 1, PDGF and IGF-1 mRNA in the lung cells and lung tissues in different stages of Bleomycin-A5-induced pulmonary fibrosis in rats were studied through Northern hybridization. RESULTS: The expressions of TGF-beta 1 and PDGF mRNA reached their peaks in AMs of pulmonary fibrosis in rats on the 7th day after Bleomycin-A5 instillation. It was similar with that in the lung tissues. IGF-1 mRNA remained relatively stable in AMs during the course. PDGF and IGF-1 mRNA increased gradually in fibroblasts, and reached the highest expressions in the interstitial cells. There was almost no TGF-beta 1 mRNA expression in all groups of fibroblasts. CONCLUSIONS: AMs are the main sources of TGF-beta 1 and PDGF in the lung tissues with fibrosis induced by Bleomycin-A5 AMs are activated in the first weekend and secrete TGF-beta 1 and PDGF to promote fibroblasts proliferation and fibrosis. As fibrosis developed, fibroblasts have established PDGF and IGF-1 autocrine and these three cytokines paracrine nets combined with the interstitial cells to promote lung fibrosis.     

Protein translocation functions of Escherichia coli SecY: in vitro characterization of cold-sensitive secY mutants

Activated macrophages are among the major constituents of the periapical granuloma. Their state of activation may persist for long periods after the local irritant is removed and may delay resolution and repair of the lesion. The effect of activated macrophages on fibroblast growth was studied in vitro. Circular fibroblast colonies were formed using a drop containing 7.5 x 10(5) murine dermal fibroblasts and allowed to grow for 7 days. When peritoneal exudate macrophages were added (0.5-3.0 x 10(6) cells/dish) and activated in vitro by LPS (1 microgram/ml), the fibroblast colony's growth was suppressed. LPS alone, at the concentration used, had no effect on the fibroblast growth. Hydrocortisone (> or = 10(-7) M) totally reversed the suppression, when added either simultaneously with or 6, 24, or 48 h after the LPS. The efficacy of late hydrocortisone treatment suggests that its effect was through prevention of the expression of the LPS activation of the macrophages. These findings may provide a possible clue to a pharmacological modulation of the healing processes that occur in the periapical lesion once its infective source had been eliminated.     

In vitro activation of mouse macrophages by rat lymphocyte mediators

Macrophage-activating factors (MAF)3 were released by presensitized rat lymphocytes stimulated in vitro with the appropriate antigens. Different supernatants of presensitized rat lymphocytes specifically stimulated in vitro with several different mouse, dog, and rat tumor or normal cells were capable of rendering normal rat and mouse macrophages nonspecifically cytotoxic in vitro to their respective syngeneic tumor cells. The release of active mediators by rat lymphocytes sensitized in vivo was dependent upon immunologically specific recognition of an antigen in vitro. When rat lymphocytes were incubated in vitro with antigens unrelated to the in vivo sensitizing antigens, no release of MAF occurred. Once rat MAF was released, it activated both syngeneic (rat) and xenogeneic (mouse) macrophages to kill tumor cells in vitro. These activated marcophages destroyed all syngeneic tumor targets. Such cytotoxicity was obtained even when the cells used to elicit release of MAF were totally unrelated to the target tumor cells. The data thus demonstrated that MAF can cross strain and even species specificities and can activate macrophages to kill tumors in a nonspecific manner. The cytotoxicity mediated by in vitro activated mouse macrophages decreased with time once the macrophages were removed from MAF; and by 7 days postactivation, the macrophages were not cytotoxic. However, when incubated again with MAF, significant reactivation was observed. This suggested that activation of macrophages in vivo may be a continuous process of lymphocyte-macrophage interaction.     

Effect of enkephalins and endorphins on cytotoxic activity of natural killer cells and macrophages/monocytes in mice

The effect of [Met5]enkephalin, [Leu5]enkephalin, proenkephalin, dynorphin-(1-17) or beta-endorphin on the cytotoxic (51Cr release assay) activity of natural killer cells and macrophages/monocytes was studied in mice. It was found that a single i.p. injection of [Met5]enkephalin, [Leu5]enkephalin, beta-endorphin, dynorphin or proenkephalin as well as repeated treatment with both enkephalins increased natural killer cell activity. In vitro only [Met5]enkephalin stimulated natural killer cells. Opioid peptides did not affect the cytotoxic activity of macrophages/monocytes. In addition to functional alterations, both enkephalins and beta-endorphin increased the percentage of cells with natural killer phenotype. The results of this study suggest that the increase in natural killer cytotoxicity after opioid peptides injected once or for 14 days may result from an increased number of natural killer cells in the spleen.     

Effects of transforming growth factor beta on corneal epithelial and stromal cell function in a rat wound healing model after excimer laser keratectomy

BACKGROUND: Transforming growth factor beta (TGF-beta) regulates extracellular matrix deposition, cell proliferation, and migration, and is expressed in cornea. TGF-beta is thought to be involved in the corneal wound healing process. METHODS: The central corneal area (3 mm in diameter) of Lewis rats was ablated using PTK mode excimer laser and the wound healing process was observed at 12 and 24 h and 2, 5, 10, and 30 days after treatment. The expression of TGF-beta 1, -beta 2 and -beta 3, TGF-beta type I and type II receptors, alpha 3, alpha 5, beta 4 integrin subunits, laminin and fibronectin was studied immunohistochemically. Antibody neutralizing TGF-beta 1, -beta 2 and -beta 3 was administered intraperitoneally, 50 micrograms daily, for 5 days after the laser treatment to investigate the effects of TGF-beta function blockade. RESULTS: At the leading edge of the regenerating epithelium, no TGF-beta type I and type II receptors and beta 4 integrin subunits were expressed after 24 h. Regenerating epithelium covered the ablated area after 2 days. An abnormal fibrotic layer was formed in the subepithelial area. This layer contained round-shaped cells in the stroma in the early stage (2-5 days after laser ablation) and spindle-shaped fibroblast-like keratocytes after 10 days. Laminin and fibronectin expression increased in the fibrotic layer. The increased stromal cells expressed TGF-beta isoforms and TGF-beta receptors. Neutralizing TGF-beta inhibited the stromal cell increase in the laser ablated area after 5 days. CONCLUSION: TGF-beta may be involved in epithelial cell migration and stromal cell reaction during the corneal wound healing process after excimer laser ablation in rat models.     

Identification of an inhibitor targeting macrophage production of monocyte chemoattractant protein-1 as TGF-beta 1

Monocyte chemoattractant protein-1 (MCP-1) is expressed in a diverse range of cells in response to various pathologic stimuli, whereas little is known about endogenous inhibitors of MCP-1 expression. I sought negative regulators of MCP-1 in culture medium conditioned by several cell lines and found that glomerular mesangial cells exclusively secrete a factor that inhibits expression of MCP-1 by activated macrophages. Treatment of J774.2 macrophages with conditioned medium from mesangial cells blunted the induction of MCP-1 by LPS. Even after the induction, subsequent treatment of macrophages with the conditioned medium down-regulated the MCP-1 expression. Medium conditioned by normal rat glomeruli contained a similar inhibitory activity that was enhanced in regenerating glomeruli, where mesangial cells are activated. The activity of the conditioned medium was not diminished, but enhanced by heat treatment, which was consistent with the unique property of TGF-beta family of molecules. Indeed, the mesangial cell-derived medium contained active TGF-beta 1. An anti-TGF-beta 1 neutralizing Ab abolished the inhibitory effect exerted by the mesangial cell medium, and externally added TGF-beta 1 suppressed the MCP-1 expression by macrophages at both mRNA and protein levels. The inhibitory effect of TGF-beta 1 on MCP-1 was observed in other macrophage cell lines, RAW264.7 and NR8383, and peritoneal macrophages, but not in fibroblastic cell line NRK49F. Treatment of J774.2 macrophages with TGF-beta 1 inhibited LPS induction of c-jun that was found to be crucial for the MCP-1 expression. These data demonstrate that TGF-beta 1 functions as an inhibitor of MCP-1 expression in macrophages possibly via down-regulation of c-Jun/activator protein-1.     

Engagement of cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) induces transforming growth factor beta (TGF-beta) production by murine CD4(+) T cells

Evidence indicates that cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) may negatively regulate T cell activation, but the basis for the inhibitory effect remains unknown. We report here that cross-linking of CTLA-4 induces transforming growth factor beta (TGF-beta) production by murine CD4(+) T cells. CD4(+) T helper type 1 (Th1), Th2, and Th0 clones all secrete TGF-beta after antibody cross-linking of CTLA-4, indicating that induction of TGF-beta by CTLA-4 signaling represents a ubiquitous feature of murine CD4(+) T cells. Stimulation of the CD3-T cell antigen receptor complex does not independently induce TGF-beta, but is required for optimal CTLA-4-mediated TGF-beta production. The consequences of cross-linking of CTLA-4, together with CD3 and CD28, include inhibition of T cell proliferation and interleukin (IL)-2 secretion, as well as suppression of both interferon gamma (Th1) and IL-4 (Th2). Moreover, addition of anti-TGF-beta partially reverses this T cell suppression. When CTLA-4 was cross-linked in T cell populations from TGF-beta1 gene-deleted (TGF-beta1(-/-)) mice, the T cell responses were only suppressed 38% compared with 95% in wild-type mice. Our data demonstrate that engagement of CTLA-4 leads to CD4(+) T cell production of TGF-beta, which, in part, contributes to the downregulation of T cell activation. CTLA-4, through TGF-beta, may serve as a counterbalance for CD28 costimulation of IL-2 and CD4(+) T cell activation.     

Marrow-derived activated macrophages are required during the effector phase of experimental autoimmune uveoretinitis in rats

PURPOSE: Experimental autoimmune uveoretinitis (EAU), an established model for human endogenous (autoimmune) posterior uveitis, is a CD4+ T cell-mediated disease inducible in Lewis rats by intradermal inoculation with retinal antigens. Immunohistochemical studies have previously documented the lymphocyte profiles during various stages of the disease process. The purpose of the present study was to investigate the role of macrophages in EAU. METHODS: EAU was induced in Lewis rats, and the effect of macrophage depletion, using the drug dichlorodimethylene diphosphonate (Cl2MDP) encapsulated in liposomes and administered intravenously, was assessed based on the clinical and histological profile of the disease. RESULTS: The results have shown that in control animals macrophages occur early, feature prominently throughout the course of the disease and display considerable heterogeneity: marrow-derived ED1+ cells and ED3+ cells are the major infiltrating cells, with many cells also expressing ED7 and ED8. In contrast, few cells expressed the ED2 antigen during EAU, even though ED2+ "resident" macrophages occur in the normal choroid. Macrophage depletion, using intravenously injected dichloromethylene diphosphonate (Cl2MDP) enclosed in liposomes, caused a delay in the onset and a reduction in the severity of EAU when administered during the "effector" stage of the disease, i.e. 9-11 days after inoculation with retinal antigen. The delay in disease onset was greater when liposomes were mannosylated and was accompanied by a reduction in the overall inflammatory cell infiltrate into the eye and reduced tissue damage. In addition, there was a reduction in the level of expression of MHC Class II antigen and CR3 (ED7) antigen, a marker of macrophage activation, in Cl2MDP-treated animals compared to controls. CONCLUSION: These results suggest that blood-borne, activated macrophages are major effectors of tissue damage during EAU.     

Lymphocyte and macrophage responses after vaccinia virus infections

Using a semimicromethod with washed whole blood, in vitro lymphocyte responses of rabbits to intradermal infection with vaccinia virus was studied. Peritoneal exudate macrophages were infected with vaccinia in vitro to determine the time of appearance of activated macrophages. After primary infection, an increase in spontaneous incorporation of thymidine by blood cultures was found as early as 2 days postinfection. This effect was at a maximum at 7 to 10 days, with counts up to 100-fold higher than before infection. Incubation of these cultures with concanavalin A showed a marked decrease in stimulation index as compared with normals. Although only a transient stimulation with vaccinia was found during the acute infection, stimulation indexes of 2 to 3 were obtained during convalescence. Macrophages from rabbits early after infection supported vaccinia replication, whereas those at day 6 or later resisted infection. Macrophage resistance persisted for 2 to 3 weeks. The response of lymphocytes from rabbits reinfected with vaccinia after 15 weeks differed, with a small increase in spontaneous thymidine uptake, a smaller depression in concanavalin A stimulation, and a greater specific response to vaccinia. Macrophage activation occurred earlier and persisted for a longer time after secondary infection.     

7-(N-substituted)amino-2,3-polymethylene benzofurane derivatives with tracheal relaxant activity

BACKGROUND: Macrophages play an important role in several ocular diseases. Because macrophages localized in ocular tissues may be derived from blood monocytes, the effect of vitreous [containing transforming growth factor-beta 2 (TGF-beta 2) and hyaluronic acid] on blood monocytes, maturating in the tissue to macrophages, was determined. METHODS: Human monocytes were cultured with and without vitreous in RPMI 1640 medium containing human AB serum. As a parameter of activation the release of interleukin-6 was measured by the B9 bioassay; as an indication of maturation, the content of acid phosphatase and the increase in cell size were assessed. RESULTS: Monocytes in vitreous-containing medium grew more slowly than did control monocytes. Monocytes cultured in 10% vitreous released 51% less, and in 20% vitreous 73% less, interleukin-6 than control monocytes. Vitreous at 20% significantly (P = 0.0075) reduced the amount of acid phosphatase by 80% over a 4-day culture period. This reduction was partially eliminated with neutralizing antibodies to TGF-beta (P = 0.0014). Furthermore, human recombinant TGF-beta 2 increased the activity of acid phosphatase in monocytes at 1.25 ng/ml and reduced it (P < 0.0001) at higher concentrations (5-10 ng/ml). Hyaluronic acid showed an effect additive to that of TGF-beta in further diminishing the amount of acid phosphatase (P = 0.026). CONCLUSION: Vitreous exerts a regulatory effect on monocyte activation and maturation by its content of TGF-beta and possibly hyaluronic acid and may, thus, modify the inflammatory or immune response in the eye.     

Migration inhibitory factor induces killing of Leishmania major by macrophages: dependence on reactive nitrogen intermediates and endogenous TNF-alpha

Macrophage migration inhibitory factor (MIF) is a product of activated T cells, anterior pituitary cells, and macrophages. MIF plays an important role in LPS-induced shock and delayed-type hypersensitivity. Furthermore, MIF exhibits a proinflammatory spectrum of action, promoting TNF-alpha production by macrophages, and counter-regulates glucocorticoid suppression of cytokine production. Here, we report that purified recombinant MIF activates murine macrophages to kill Leishmania major, with maximal effects at concentrations above 1 microg/ml. This MIF-mediated activation is specific, since it can be blocked completely by anti-MIF mAb. The MIF-mediated activation is dependent on TNF-alpha produced endogenously by macrophages, because the administration of anti-TNF-alpha antiserum markedly reduced the MIF effect. No MIF-mediated activation was observed in macrophages derived from TNF receptor p55 knockout mice, thus demonstrating the requirement of the smaller TNF receptor molecule for autocrine TNF-alpha signaling. A highly specific inhibitor of the inducible nitric oxide synthase (iNOS), L-N6-(1-iminoethyl)lysine, dihydrochloride, also inhibited the action of MIF, suggesting an important role for iNOS in the antiparasitic properties of MIF. In line with this, no MIF-mediated activation was detected analyzing macrophages derived from iNOS-deficient mice. The effect of MIF was blocked completely by the macrophage-deactivating cytokines IL-10, IL-13, and TGF-beta. Finally, the expression of MIF mRNA and protein was up-regulated in lymph nodes of mice during the first week after infection with L. major. MIF therefore represents a cytokine involved not only in the recruitment of proinflammatory cells during infection but also in the complex regulation of the antimicrobial activity of these cells.