Upregulation of interferon-induced indoleamine 2,3-dioxygenase in human macrophage cultures by lipopolysaccharide, muramyl tripeptide, and interleukin-1

The tryptophan decyclizing enzyme indoleamine 2,3-dioxygenase (IDO) was induced in human monocyte-derived macrophages (MDM) treated with human recombinant interferon-β (IFN-β) or interferon-γ (IFN-γ). Treated cells exhibited dose-dependent increases in IDO when assayed 48 hr after treatment. Cells exposed to IFN-γ were observed to exhibit consistently higher peak levels of IDO when compared with cells incubated in the presence of IFN-β. When IFN-β-treated cells were incubated in the presence of specified amounts of bacterial lipopolysaccharide (LPS) or liposome-encapsulated muramyl tripeptide (MTP), peak IDO activity increased such that enzyme activity was comparable to maximal activity observed with IFN-γ-treated cells. LPS and MTP also upregulated IFN-γ-mediated IDO activity when suboptimal amounts of IFN-γ were used. When macrophages were costimulated with various concentrations of human recombinant interleukin 1α (IL-1α), along with either maximum-stimulating amounts of IFN-β or suboptimal amounts of IFN-γ, IDO activity was upregulated in a manner similar to results obtained using the microbial products as stimuli. While neither IL-1α or IL-1β was detected in culture supernatants from macrophages treated with either LPS or MTP (alone or in combination with IFN), IL-1α was detected in cell lysates of macrophages treated with these upregulators. Although neutralizing antibody to IL-1α abolished the upregulatory effect of exogenous IL-1α, it had no effect on upregulation by LPS or MTP. This suggests that although LPS and MTP may induce production of cell-associated IL-1α, upregulation of IDO activity by these agents is independent of IL-1α production and may be mediated through distinct pathways.     

Suppression of fibroblast proliferation by activated macrophages: involvement of H2O2 and a non-prostaglandin E product of the cyclooxygenase pathway

Macrophages are considered promoters of fibroblast proliferation; however, suppression by activated macrophages may outweigh this effect. Activated murine peritoneal macrophages obtained by in vivo exposure to C. parvum or by in vitro LPS-activation of thioglycollate-induced macrophages, were tested for their effect on normal syngeneic dermal fibroblasts. C. parvum-activated macrophages, but not resident peritoneal macrophages suppressed fibroblast proliferation. Similarly, macrophages activated in vitro by LPS, but not those unexposed to LPS, suppressed fibroblast proliferation. Catalase partially protected fibroblasts from suppression by either activated macrophage population, suggesting involvement of H2O2 in the suppression. The effect of cyclooxygenase inhibitors on the suppression was also tested. Indomethacin, acetylsalicyclic acid, or eicosatetraynoic acid, all partially protected the fibroblasts from macrophage-mediated suppression. Prostaglandins E2, E1, and F2 alpha, added exogenously at concentrations as high as 10(-6) M, failed to suppress the proliferation of the fibroblasts. These findings suggest that a non-prostaglandin product of the cyclooxygenase pathway is involved in macrophage-mediated suppression of fibroblast proliferation.     

Palmitate- and lipopolysaccharide-activated macrophages evoke contrasting insulin responses in muscle cells

Factors secreted by macrophages contribute to whole body insulin resistance, acting in part on adipose tissue. Muscle is the major tissue for glucose disposal, but how macrophage-derived factors impact skeletal muscle glucose uptake is unknown, or whether the macrophage environment influences this response. We hypothesized that conditioned medium from macrophages pretreated with palmitate or LPS would directly affect insulin action and glucose uptake in muscle cells. L6-GLUT4myc myoblasts were exposed to conditioned medium from RAW 264.7 macrophages pretreated with palmitate or LPS. Conditioned medium from palmitate-treated RAW 264.7 macrophages inhibited myoblast insulin-stimulated glucose uptake, GLUT4 translocation, and Akt phosphorylation while activating JNK p38 MAPK, decreasing IkappaBalpha, and elevating inflammation markers. Surprisingly, and opposite to its effects on adipose cells, conditioned medium from LPS-treated macrophages stimulated myoblast insulin-stimulated glucose uptake, GLUT4 translocation, and Akt phosphorylation without affecting stress kinases or inflammation indexes. This medium had markedly elevated IL-10 levels, and IL-10, alone, potentiated insulin action in myoblasts and partly reversed the insulin resistance imparted by medium from palmitate-treated macrophages. IL-10 neutralizing antibodies blunted the positive influence of LPS macrophage-conditioned medium. We conclude that myoblasts and adipocytes respond differently to cytokines. Furthermore, depending on their environment, macrophages negatively or positively influence muscle cells. Macrophages exposed to palmitate produce a mixture of proinflammatory cytokines that reduce insulin action in muscle cells; conversely, LPS-activated macrophages increase insulin action, likely via IL-10. Macrophages may be an integral element in glucose homeostasis in vivo, relaying effects of circulating factors to skeletal muscle.     

Characterization of heterokaryons between skeletal myoblasts and somatic cells formed by fusion with HVJ (Sendai virus); effects on myogenic differentiation

In skeletal myogenic differentiation, myoblasts fuse with myogenic cells spontaneously, but do not fuse with non-myogenic cells either in vivo or in vitro, suggesting that the fusion of myoblasts with non-myogenic cells is unsuitable for differentiation. To understand the inevitability of the fusion among myoblasts, we prepared heterokaryons in crosses between quail myoblasts transformed with a temperature-sensitive mutant of Rous sarcoma virus (QM-RSV cells) and rodent non-myogenic cells, such as tumor cells, fibroblasts, or neurogenic cells by HVJ (Sendai virus) and examined how myogenic differentiation was influenced in the prepared heterokaryons, focusing on myogenin expression and myofibril formation as markers of differentiation. When presumptive QM-RSV cells were fused with non-myogenic cells by HVJ and induced to differentiate, both myogenin expression and myofibril formation were suppressed. When myotubes of QM-RSV cells that had already expressed myogenin and formed myofibrils were fused with non-myogenic cells, both myogenin and myofibrils disappeared. Especially, fibrous structures of myofibrils were significantly lost and dots or aggregations of F-actin were formed within 24 hr after formation of heterokaryons. However, the fusion of presumptive or differentiated QM-RSV cells with rodent myoblasts did not disturb myogenin expression or myofibril formation. These results suggest that mutual fusion of myoblasts is indispensable for normal myogenic differentiation irrespective of the species, and that some factors inhibiting myogenic differentiation exist in the cytoplasm of non-myogenic cells, but not in myoblasts.     

Gangliosides block the inhibition of macrophage Fc-dependent phagocytosis by lipopolysaccharide

The mechanism whereby bacterial lipopolysaccharide (LPS) exerts its biologic effects on mammalian cells is unknown. Plasma membrane gangliosides bind bacterial toxins and have been implicated in modulating the effects of a variety of immunoregulatory substances. We investigated the possibility that gangliosides can inhibit the effect of lipopolysaccharide on Fc-dependent phagocytosis by murine peritoneal macrophages. Protein-free lipopolysaccharide preparations significantly inhibited Fc-mediated phagocytosis (less than 71% of control) at concentrations of 100 ng/ml or greater after 90 min of incubation. The inhibitory effect of LPS (1 micrograms/ml) was blocked when macrophages were incubated with mono-, di-, or trisialogangliosides (25-50 micrograms/ml). Neither asialoganglioside nor sialic acid alone were capable of blocking the effect of LPS. When chromatographed separately on a Sepharose 4B column, LPS and trisialoganglioside had different elution profiles. LPS and trisialoganglioside coeluted, however, when premixed at 37 degrees C for 60 min and then applied to the column. Therefore, abrogation of the effect of LPS on Fc-dependent phagocytosis may occur as a consequence of direct interaction between LPS and gangliosides. These data suggest that gangliosides may modulate the response of macrophages to bacterial lipopolysaccharide.     

Resident macrophages activated by lipopolysaccharide suppress muscle tension and initiate inflammatory response in the gastrointestinal muscle layer

A great number of macrophages is found to be evenly distributed in the muscle layer of the gastrointestinal tract. We investigated their effects on smooth muscle contraction and the initiation of immune reactions such as inflammatory responses. Macrophages were demonstrated by the uptake of FITC-dextran and their ultrastructural features were elucidated by electron microscopy. Muscle layers of rats’ ileums were incubated with lipopolysaccharide (LPS) for 4–8 h and the force of smooth muscle contraction was measured. The induction effect of inducible nitric oxide synthase (iNOS) on macrophages was then checked by immunohistochemistry. The expression of major histocompatibility complex (MHC) class II was also examined. Macrophages in the muscle layer were confirmed as resident macrophages and were different from a population of dendritic cells. After incubation with LPS, macrophages began to express iNOS and produced NO, and it reduced smooth muscle contraction. iNOS-immunopositive cells increased in a time-dependent manner. Macrophages also began to express MHC class II. The total number of macrophages did not alter after incubation. Results indicate that resident macrophages in the muscle layer induced iNOS as an inflammatory reaction, affected smooth muscle contraction, and initiated immune response in the smooth muscle layer of the gastrointestinal tract, when activated by LPS. Accepted: 24 November 1999     

Inhibition of prostaglandin E2-stimulated cAMP accumulation by lipopolysaccharide in murine peritoneal macrophages.

Treatment of murine peritoneal macrophages with 100 nM prostaglandin E2 (PGE2) produced a rapid biphasic increase in intracellular cAMP that was maximal at 1 min and sustained through 20 min. Pretreatment of macrophages with 100 ng/ml of lipopolysaccharide (LPS) for 60 min prior to PGE2 decreased the magnitude of cAMP elevation by 50%, accelerated the decrease of cAMP to basal levels, and abolished the sustained phase of cAMP elevation. The effect of LPS was concentration-dependent, with maximal effect at 10 ng/ml in cells incubated in the presence of 5% fetal calf serum and at 1 microgram/ml in the absence of fetal calf serum. LPS also inhibited cAMP accumulation in cells treated with 100 microM forskolin, but the decrease was about half that seen in cells treated with PGE2. LPS concentrations that inhibited cAMP accumulation produced a 30% increase in soluble low Km cAMP phosphodiesterase activity while having no effect on particulate phosphodiesterase activity. The nonspecific phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine, as well as the more specific inhibitors rolipram and Ro-20-1724 were effective in inhibiting soluble phosphodiesterase activity in vitro, producing synergistic elevation of cAMP in PGE2-treated cells, and blocking the ability of LPS to inhibit accumulation of cAMP. Separation of the phosphodiesterase isoforms in the soluble fraction by DEAE chromatography indicated that LPS activated a low Km cAMP phosphodiesterase. The enzyme(s) present in this peak could be activated 6-fold by cGMP and were potently inhibited by low micromolar concentrations of Ro-20-1724 and rolipram. Using both membranes from LPS-treated cells and membranes incubated with LPS, no decrease in adenylylcyclase activity could be attributed to LPS. Although effects of LPS on the rate of synthesis of cAMP cannot be excluded, the present evidence is most consistent with a role for phosphodiesterase activation in the inhibitory effects of LPS on cAMP accumulation in murine peritoneal macrophages.     

Role of porin of Shigella dysenteriae type 1 in modulation of lipopolysaccharide mediated nitric oxide and interleukin-1 release by murine peritoneal macrophages

The ability of Shigella dysenteriae type 1 porin to induce the release of nitric oxide (NO) and interleukin-1 (IL-1) from peritoneal macrophages of mouse and to regulate lipopolysaccharide (LPS) and gamma interferon (IFN-gamma) mediated release of the two proinflammatory mediators was investigated. Porin released nitrite when added to macrophage cultures. A maximum of 3.2-fold nitrite release by macrophages was observed with 100 ng ml(-1) of porin. The nitrite release of LPS was enhanced significantly by lower concentrations of porin, whereas the effect of IFN-gamma was enhanced by porin at higher concentrations. Polysaccharide (PS) moiety of LPS stimulated the nitrite release of elicited macrophages by 1.6-fold compared to untreated control. It also enhanced the stimulatory effect of 1 and 10 ng ml(-1) of porin by 1.3-fold. Lipid A (LPA) moiety of LPS did not release nitrite, nor did it increase the porin mediated nitrite production. Porin treated 24 h old macrophage culture supernatants were applied for ConA activated thymocyte proliferation as a measure for determination of IL-1 release. Sixty percent depletion of thymocyte proliferation was observed when the porin treated macrophage supernatants were absorbed with anti-IL-1 antibody. A maximum of 5.5-fold increase of thymocyte proliferation over control was found with 1 and 10 ng ml(-1) of porin. One or 10 ng ml(-1) of porin and LPS augmented the thymocyte growth, 1.5-fold beyond that obtained by porin and 1.8-/1. 7-fold more than that obtained by LPS, alone. Similarly, porin and IFN-gamma co-stimulated the cell growth also. PS enhanced the thymocyte proliferation by 5-fold. It also enhanced the thymocyte growth by co-stimulating 1.4-fold the effect observed by 1 or 10 ng ml(-1) of porin alone. LPA could not participate in the cell proliferating activity nor did it enhance the stimulatory effect of porin. Therefore, both nitrite release and thymocyte proliferation by LPS could be substituted by PS only. The tight association of the two bacterial outer membrane components, porin and LPS, could be a necessary co-signal for boosting the release of the two proinflammatory mediators, namely NO and IL-1, which may be associated with the inflammatory response of the colon during Shigella invasion.     

Effect of cytokines and lipopolysaccharide on CD14 antigen expression in human monocytes and macrophages.

The 52 kD myeloid membrane glycoprotein CD14 represents the receptor for complexes of lipopolysaccharide (LPS) and LPS binding protein (LBP); it is involved in LPS induced tumor necrosis factor-alpha production. Expression of CD14 increases in monocytes differentiating into macrophages, and it is reduced by rIFNg in monocytes in vitro. In the present study CD14 membrane antigen expression was investigated in cultures of human mononuclear leucocytes (PBL), in elutriated, purified monocytes, and in blood monocyte derived Teflon cultured macrophages. Cells were incubated for 15 or 45 h with rIL-1, rIL-2, rIL-3, rIL-5, rIL-6, rTNFa, rGM-CSF, rM-CSF, rTGFb1, rIFNa, lipopolysaccharide (LPS), and, as a control, rIFNg. The monoclonal antibodies Leu-M3 and MEM 18 were used for labelling of CD14 antigen by indirect immunofluorescence and FACS analysis of scatter gated monocytes or macrophages. IFNg concentrations were determined in PBL culture supernatants by ELISA. rIFNa and rIL-2 reduced CD14 in 15 and 45 h PBL cultures, an effect mediated by endogenous IFNg, since it was abolished by simultaneous addition of an anti-IFNg antibody. rIFNa and rIL-2 were ineffective in purified monocytes or macrophages. rIL-4 strongly reduced CD14 in PBL and purified monocytes after 45 h, whereas in macrophages the decrease was weak, although measurable after 15 h. The other cytokines investigated did not change CD14 antigen expression. Cycloheximide alone reduced CD14, but when added in combination with rIFNg the effect on CD14 downregulation was more pronounced. The effect of rIFNg on CD14 in PBL cultures was dose-dependently inhibited by rIL-4 and this inhibition is probably due to an IL-4 mediated blockade of IFNg secretion. LPS at a low dose increased CD14, at a high dose it produced a variable decrease of CD14 in PBL, which was probably due to LPS induced IFNg secretion. LPS strongly enhanced CD14 in 45 h cultures of purified monocytes. The results, showing that CD14 antigen expression is upregulated by LPS and downregulated by rIFNg and rIL-4, suggest that the LPS-LBP receptor is involved in the feedback response of IFNg and IL-4 to LPS stimulation.     

Effect of hydrocortisone and bacterial lipopolysaccharide on colony-stimulating activity production from mouse marrow adherent cells, spleen cells and peritoneal macrophages in vitro

The effect of hydrocortisone (HC) on colony-stimulating activity (CSA) production from mouse bone marrow adherent cells, spleen cells and peritoneal macrophages with or without bacterial lipopolysaccharide (LPS) stimulation was studied. CSA in the supernatant from bone marrow adherent cells incubated with HC was found to be five times higher than CSA from cultures without LPS stimulation. In contrast, the CSA production by spleen cells and peritoneal macrophages were significantly suppressed by HC in both LPS-stimulated and non-stimulated cultures. These studies suggest that the effect of HC on CSA production was quite different depending on the target cells.     

Regulation by PGE2 of the production of oxygen intermediates by LPS-activated macrophages

The regulation by prostaglandin E2 (PGE2) of production of oxygen radicals by bacterial lipopolysaccharide-(LPS) activated macrophages was studied in vitro. A 48-hr incubation of murine thioglycollate-elicited macrophages with LPS (0.1 micrograms/ml) resulted in an enhanced ability of these cells to produce oxygen radicals when challenged with phorbol myristate acetate (PMA). Macrophages incubated for 48 hr without LPS did not produce measurable amounts of oxygen radicals when exposed to this triggering stimulus. Thus, PMA-triggered production of oxygen radicals was the result of macrophage activation by LPS. The PMA-triggered production of oxygen radicals by the LPS-activated macrophages was inhibited when PGE2 (10(-5) to 10(-9) M) was present during the incubation with LPS. Inhibition by PGE2 occurred during the early stages of macrophage activation, since the addition of PGE2 24 hr after LPS no longer inhibited the production of oxygen radicals by the macrophages. This inhibitory effect of PGE2 on the LPS-induced activation of macrophages could be reproduced by cyclic-adenosine-monophosphate (cAMP) agonists, such as isoproterenol and cholera toxin as well as by the cAMP analog dibutyryl-cAMP, suggesting a cAMP-mediated mechanism for the inhibitory effect of PGE2 on macrophage activation by LPS. Previous reports have implicated prostaglandins as mediators of destructive processes associated with chronic inflammation. Our findings suggest that PGE2 may, on the other hand, reduce tissue damage in a chronic inflammatory site by inhibiting the production of oxygen radicals by macrophages activated in the sera.     

AN ANALYSIS OF MYOGENESIS BY THE USE OF FLUORESCENT ANTIMYOSIN

Antibodies against myosin of adult chicken skeletal muscle were labelled with fluorescein and used as staining reagents to analyze the development of trunk myoblasts in the chick embryo. Myoblasts from the brachial myotomes were studied in three ways: (a) Specimens were fixed, sectioned, and stained with iron-hematoxylin. (b) Living myoblasts, and myoblasts prepared by glycerol extraction, were teased and examined by phase contrast microscopy. (c) Embryo trunks were treated with fluorescent antimyosin or with a control solution of fluorescent normal globulin, and were examined by fluorescence and phase contrast microscopy. Both glycerol-extracted and fixed materials were used. Cross-striated myofibrils appeared first in stage 16 to 17 embryos in the series studied by antimyosin staining and fluorescence microscopy. Striated myofibrils appeared first in stage 18 to 19 embryos, in the series stained by iron-hematoxylin, and at stage 22 to 23, in the series studied by glycerol extraction and phase contrast microscopy. In each series, myofibrils without apparent cross-striations were detected shortly before cross-striations were observed. Specific staining by antimyosin occurred only in differentiating myoblasts. Within the myoblasts antimyosin staining was confined to the A bands of the slender myofibrils. The following observations suggest that the first delicate striated structure to appear in the early 3 day myoblast was remarkably mature: (1) The sarcomere pattern both in length and in internal detail, was similar to that of adult muscle. (2) The distribution of myosin, as revealed by antimyosin staining, was the same in the embryonic as in the mature myofibril. (3) Glycerol-extracted myoblasts contracted vigorously on exposure to ATP. The changes in sarcomere band pattern were indistinguishable from those occurring during contraction of adult muscle induced by ATP. (4) ATP contraction was blocked by prior antimyosin staining in embryonic myoblasts as in mature muscle. It is suggested that the early myofibril grows laterally as a thin sheet associated with the sarcolemma, and that growth in length occurs in the growth tips of the elongating myoblast.     

Heterochromatin protein (HP)1γ is not only in the nucleus but also in the cytoplasm interacting with actin in both cell compartments

Confocal and electron microscopy images, and WB analysis of cellular fractions revealed that HP1γ is in the nucleus but also in the cytoplasm of C2C12 myoblasts, myotubes, skeletal and cardiac muscles, N2a, HeLa and HEK293T cells. Signal specificity was tested with different antibodies and by HP1γ knockdown. Leptomycin B treatment of myoblasts increased nuclear HP1γ, suggesting that its nuclear export is Crm-1-dependent. HP1γ exhibited a filamentous pattern of staining partially co-localizing with actin in the cytoplasm of myotubes and myofibrils. Immunoelectron microscopic analysis showed high-density immunogold particles that correspond to HP1γ localized to the Z-disk and A-band of the sarcomere of skeletal muscle. HP1γ partially co-localized with actin in C2C12 myotubes and murine myofibrils. Importantly, actin co-immunoprecipitated with HP1γ in the nuclear and cytosolic fractions of myoblasts. Actin co-immunoprecipitated with HP1γ in myoblasts incubated in the absence or presence of the actin depolymerizing agent cytochalasin D, suggesting that HP1γ may interact with G-and F-actin. In the cytoplasm, HP1γ was associated to the perinuclear actin cap that controls nuclear shape and position. In the nucleus, re-ChIP assays showed that HP1γ-actin associates to the promoter and transcribed regions of the house keeping gene GAPDH, suggesting that HP1γ may function as a scaffold protein for the recruitment of actin to control gene expression. When HP1γ was knocked-down, myoblasts were unable to differentiate or originated thin myotubes. In summary, HP1γ is present in the nucleus and the cytoplasm interacting with actin, a protein complex that may exert different functions depending on its subcellular localization.     

Effect of fast neutrons on the process of muscle cell differentiation

A study was made of the effect of fast neutron on differentiation of myoblasts in vitro. The results obtained indicate that irradiation inhibits the differentiation of myoblasts in culture. Irradiation with fast neutrons (1.5 and 2.0 Gy) inhibits the uninucleate myoblast fusion thus preventing the formation of multinuclear tissue structures with myofibrils, myosymplasts.     

Lipopolysaccharide and proinflammatory cytokines stimulate interleukin-6 expression in C2C12 myoblasts: role of the Jun NH2-terminal kinase

IL-6 is a major inflammatory cytokine that plays a central role in coordinating the acute-phase response to trauma, injury, and infection in vivo. Although IL-6 is synthesized predominantly by macrophages and lymphocytes, skeletal muscle is a newly recognized source of this cytokine. IL-6 from muscle spills into the circulation, and blood-borne IL-6 can be elevated >100-fold due to exercise and injury. The purpose of the present study was to determine whether inflammatory stimuli, such as LPS, TNF-alpha, and IL-1beta, could increase IL-6 expression in skeletal muscle and C2C12 myoblasts. Second, we investigated the role of mitogen-activated protein (MAP) kinases, and the Jun NH2-terminal kinase (JNK) in particular, as a mediator of this response. Intraperitoneal injection of LPS in mice increased the circulating concentration of IL-6 from undetectable levels to 4 ng/ml. LPS also increased IL-6 mRNA 100-fold in mouse fast-twitch skeletal muscle. Addition of LPS, IL-1beta, or TNF-alpha directly to C2C12 myoblasts increased IL-6 protein (6- to 8-fold) and IL-6 mRNA (5- to 10-fold). The response to all three stimuli was completely blocked by the JNK inhibitor SP-600125 but not as effectively by other MAP kinase inhibitors. SP-600125 blocked LPS-stimulated IL-6 synthesis dose dependently at both the RNA and protein level. SP-600125 was as effective as the synthetic glucocorticoid dexamethasone at inhibiting IL-6 expression. SP-600125 inhibited IL-6 synthesis when added to cells up to 60 min after LPS stimulation, but its inhibitory effect waned with time. LPS stimulated IL-6 mRNA in both myoblasts and myotubes, but myoblasts showed a proportionally greater LPS-induced increase in IL-6 protein expression compared with myotubes. SP-600125 and the proteasomal inhibitor MG-132 blocked LPS-induced degradation of IkappaB-alpha/epsilon and LPS-stimulated expression of IkappaB-alpha mRNA. Yet, only SP-600125 and not MG-132 blocked LPS-induced IL-6 mRNA expression. This suggests that IL-6 gene expression is a downstream target of JNK in C2C12 myoblasts.     

Electron-microscopic study of the rat masseter muscle following injection of lidocaine

The ultrastructure of rat masseter muscle was examined at 15 min, 1 and 6 h, and 1 and 2 days following a single injection of 2% lidocaine. Lesions developed within 15 min. The plasma membrane was disrupted and invaginated. The nuclei were pyknotic and the mitochondria appeared swollen. The myofibrils separated and became disoriented. By 1 and 6 h, these changes were severe. By 1 day, the macrophages appeared in damaged myofibers. The presence of a few presumptive myoblasts signaled the onset of regeneration. By 2 days, presumptive myoblasts formed within the basement membrane. The basal lamina proved most resistant to injury. Regeneration of masseter muscle following the damage produced by lidocaine appeared discontinuous in nature. The singly nucleated presumptive myoblasts seemed to arise within the lesions.     

Granulocyte colony-stimulating factor treatment protects rodents against lipopolysaccharide-induced toxicity via suppression of systemic tumor necrosis factor-alpha.

Pretreatment with recombinant human granulocyte CSF (G-CSF) protected mice in two different models of septic shock. Intravenous injection of 250 micrograms/kg G-CSF to mice prevented lethality induced by 5 mg/kg LPS. Injection of 50 micrograms/kg G-CSF protected galactosamine-sensitized mice against LPS-induced hepatitis. In either case, this protection was accompanied by a suppression of LPS-induced serum TNF activity. In contrast, when galactosamine-sensitized mice were pretreated with 50 micrograms/kg murine recombinant granulocyte/macrophage CSF instead of G-CSF and subsequently challenged with LPS, serum TNF activity was significantly enhanced and mortality was increased. The suppressive effect of G-CSF on LPS-induced TNF production was also demonstrated in rats. In vivo, no TNF was detectable in the blood of LPS-treated rats, which had been pretreated with G-CSF. Ex vivo, alveolar macrophages, bone marrow macrophages, Kupffer cells, or peritoneal macrophages prepared from G-CSF-treated rats produced significantly less TNF upon stimulation with LPS than corresponding populations from control rats. However, when these macrophage populations were incubated with G-CSF in vitro, LPS-induced TNF production was unaffected. These data suggest that the G-CSF-mediated suppression of TNF production is not a direct effect of G-CSF on macrophages. To examine whether, independent of the protection against LPS, G-CSF treatment still activated neutrophils, it was demonstrated that granulocytes from G-CSF-treated rats were primed for PMA-induced oxidative burst and for ionophore/arachidonic acid-stimulated lipoxygenase product formation. The experiments of this study support the notion that G-CSF is a negative feedback signal for macrophage-derived TNF-alpha production during Gram-negative sepsis.     

Exosomes derived from inflammatory myoblasts promote M1 polarization and break the balance of myoblast proliferation/differentiation

BACKGROUNDAcute muscle injuries are one of the most common injuries in sports. Severely injured muscles are prone to re-injury due to fibrotic scar formation caused by prolonged inflammation. How to regulate inflammation and suppress fibrosis is the focus of promoting muscle healing. Recent studies have found that myoblasts and macrophages play important roles in the inflammatory phase following muscle injury; however, the crosstalk between these two types of cells in the inflammatory environment, particularly the exosome-related mechanisms, had not been well studied. AIMTo evaluate the effects of exosomes from inflammatory C2C12 myoblasts (IF-C2C12-Exos) on macrophage polarization and myoblast proliferation/differentiation.METHODSA model of inflammation was established in vitro by lipopolysaccharide stimulation of myoblasts. C2C12-Exos were isolated and purified from the supernatant of myoblasts by gradient centrifugation. Multiple methods were used to identify the exosomes. Gradient concentrations of IF-C2C12-Exos were added to normal macrophages and myoblasts. PKH67 fluorescence tracing was used to identify the interaction between exosomes and cells. Microscopic morphology, Giemsa stain, and immunofluorescence were carried out for histological analysis. Additionally, ELISA assays, flow cytometry, and western blot were conducted to analyze molecular changes. Moreover, myogenic proliferation was assessed by the BrdU test, scratch assay, and CCK-8 assay.RESULTSWe found that the PKH-67-marked C2C12-Exos can be endocytosed by both macrophages and myoblasts. IF-C2C12-Exos induced M1 macrophage polarization and suppressed the M2 phenotype in vitro. In addition, these exosomes also stimulated the inflammatory reactions of macrophages. Furthermore, we demonstrated that IF-C2C12-Exos disrupted the balance of myoblast proliferation/differentiation, leading to enhanced proliferation and suppressed fibrogenic/myogenic differentiation.CONCLUSIONIF-C2C12-Exos can induce M1 polarization, resulting in a sustained and aggravated inflammatory environment that impairs myoblast differentiation, and leads to enhanced myogenic proliferation. These results demonstrate why prolonged inflammation occurs after acute muscle injury and provide a new target for the regulation of muscle regeneration.     

Lipopolysaccharide-induced cell cycle arrest in macrophages occurs independently of nitric oxide synthase II induction

Lipopolysaccharide (LPS, a Gram-negative bacterium cell wall component) is a potent macrophage activator that inhibits macrophage proliferation and stimulates production of nitric oxide (NO) via NO synthase II (NOSII). We investigated whether NO mediates the LPS-stimulated cell cycle arrest in mouse bone marrow-derived macrophages (BMM). The addition of the NO donor DETA NONOate (200 microM) inhibited BMM proliferation by approx. 80%. However, despite NO being an antimitogen, LPS was as potent at inhibiting proliferation in BMM derived from NOSII-/- mice as from wild-type mice. Consistent with these findings, LPS-induced cell cycle arrest in normal BMM was not reversed by the addition of the NOSII inhibitor S-methylisothiourea. Moreover, in both normal and NOSII-/- BMM, LPS inhibited the expression of cyclin D1, a protein that is essential for proliferation in many cell types. Despite inhibiting proliferation DETA NONOate had no effect on cyclin D1 expression. Our data indicate that while both LPS and NO inhibit BMM proliferation, LPS inhibition of BMM proliferation can occur independently of NOSII induction.