Brain, Vol. 123, No. 2, 374-379,
February 2000
© 2000 Oxford University Press
Synergistic effect of ß-amyloid protein and interferon gamma on nitric oxide production by C2C12 muscle cells
Institute of Neurology, University of Milan, IRCCS Ospedale Maggiore Policlinico, Milan, Italy
Correspondence to:
Pierluigi Baron, MD, Institute of Neurology, University of Milan, IRCCS Ospedale Maggiore Policlinico, Via F. Sforza 35, 20122 Milan, Italy
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Abstract |
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Nitric oxide (NO) is an important mediator of diverse physiological and pathological responses. NO-induced oxidative stress has been proposed in the pathogenesis of muscle tissue damage in inclusion-body myositis (IBM), which is characterized by deposition of ß-amyloid protein (Aß) in vacuolated muscle fibres. To determine whether Aß can induce NO production in skeletal muscle, we stimulated C2C12 mouse skeletal muscle cells in vitro with Aß[1–42] or Aß[25–35] peptides in the presence or absence of interferon gamma (IFN-
). Neither Aß peptides nor IFN- were able to stimulate nitrite (NO2–) production by C2C12 cells when given alone. However, combination of IFN- with either Aß[1–42] or Aß[25–35] resulted in significant NO2– release into cell-free supernatants. Northern blot analysis of RNA obtained from Aß/IFN--stimulated C2C12 cells revealed increased mRNA accumulation of inducible nitric oxide synthase (iNOS). Moreover, ~4% of muscle cells incubated with Aß peptides and IFN- showed ultrastructural features of DNA fragmentation. These findings, taken together, indicate that the association of Aß with IFN- stimulates NO2– production via induction of iNOS gene expression in skeletal muscle cells, with occasional evidence for nuclear changes suggesting apoptotic morphology. These data further support a role for Aß deposition in the pathogenesis of postulated oxidative damage in IBM. nitric oxide; ß-amyloid; muscle cell; IBM; apoptosis
Aß = ß-amyloid protein; ßAPP = ß-amyloid precursor protein; s-IBM = inclusion-body myositis; h-IBM = inclusion-body myopathy; IFN- = interferon gamma; NO = nitric oxide; NOS = nitric oxide synthase; L-NMMA = N
-methyl-L-arginine; DAPI = 4',6'-diamidino-2-phenylindole-dihydrochloride
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Introduction |
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Nitric oxide (NO) is a ubiquitous paracrine substance that is synthesized through the conversion of L-arginine to citrulline by catalytic action of the enzyme nitric oxide synthase (NOS). NOS has three major isoforms: neuronal, inducible and endothelial, each encoded by a different gene (Dawson and Snyder, 1994
; Forstermann et al., 1994; Nathan and Xie, 1994). The inducible NOS (iNOS) is predominantly present in activated macrophages (Stuehr et al., 1991), but it can also be induced in various other cell types, including muscle cells (Williams et al., 1994).
NO has been the subject of numerous recent investigations documenting its role in the function of macrophages, the control of vascular tone by endothelial cells, and neurotrasmission and synaptic plasticity in neurons (Dawson and Dawson, 1994). Even though NO can act as an intracellular second messenger in various cells and tissues, excessive synthesis of NO can induce oxidative stress via combination with superoxide (O2–) to produce highly toxic peroxynitrite (ONOO–) (Blough and Zafiriou, 1985).
Excessive production of NO has been postulated to play an important pathogenic role in various diseases, including Alzheimer's disease and in sporadic and hereditary forms of muscle disease called inclusion-body myositis (s-IBM) and myopathy (h-IBM) (Good et al., 1996; Yang et al., 1996, 1998; Smith et al., 1997). These conditions share pathological abnormalities, because senile plaques in Alzheimer's disease as well as vacuolated muscle fibres in IBM contain a unique combination of proteins, including ß-amyloid protein (Aß), which is a 39- to 42-residue polypeptide derived from the cleavage of a large precursor protein (ßAPP) (Askanas et al., 1992a, b; Gambetti and Perry, 1994).
The mechanisms leading to oxidative stress in both Alzheimer's disease and IBM are still not well defined, but because abnormal accumulation of Aß occurs very early in the disease processes and precedes other abormalities, it is currently believed that Aß deposition is a crucial event in the pathogenesis of either senile plaques in Alzheimer's disease or vacuolated muscle fibres in both s-IBM and h-IBM (Maury, 1995; Yang et al., 1998). Even though Alzheimer's disease brain contains extracellular deposits of Aß, which instead are intracellular in IBM, the presence in pathological muscle fibres of Aß fibrils with the same features as those of senile plaques raises the question of whether Aß deposition may trigger a similar response from different cell types.
Previous studies have shown that a variety of Aß peptides, in the presence of interferon gamma (IFN-), can activate microglia with production of free radicals potentially involved in neurodegeneration associated with Alzheimer's disease (Goodwin et al., 1995; Meda et al., 1995, 1996). Drawing from these reports, in the present study we investigated whether Aß[25–35] and Aß[1–42] peptides, together with IFN-, can also trigger NO2– production from cultured muscle cells as they do for microglia in vitro. We have approached this issue using C2C12 cells, which are an extensively studied skeletal myoblast cell line derived from the hindlimb of normal mice (Yaffe and Saxel, 1977). Our data demonstrate that Aß plus IFN- trigger NO2– production by skeletal muscle cells via induction of iNOS gene expression. We also provide evidence for occasional apoptotic morphology in Aß/IFN--stimulated C2C12 myogenic cells. These data further support a role for Aß deposition in the pathogenesis of postulated oxidative damage in IBM.
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Material and methods |
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Material
Aß[1–42] and Aß[25–35] peptides (Bachem, Hannover, Germany) were resuspended in sterile H2O, at a concentration of 1 mg/ml, and kept at –20°C. Recombinant murine IFN-
and N-methyl-L-arginine (L-NMMA) were purchased from Sigma Chemical Company (St Louis, Mo., USA). IFN- was resuspended in PBS (phosphate buffered saline) supplemented with 1% foetal bovine serum (Gibco-BRL, Bethesda, Md., USA), while L-NMMA was suspended in sterile H2O.
Muscle cell cultures
C2C12 murine cell line was provided by the European Collection of Animal Cell Cultures (ECACC). Cells were plated in 96-well plates at a concentration of 1 x 104/100 µl/well or in six-well plates at a concentration of 1 x 106/ml/well, and cultured in DMEM (Dulbecco's modified Eagle's medium) with 5% glucose (Gibco-BRL), supplemented with 10% heat-inactivated foetal bovine serum, 100 U/ml penicillin and 100 mg/ml streptomycin (Sigma). After incubation with culture stimuli for the indicated times, cell-free supernatants were collected and stored at –70°C for subsequent assays. Total RNA was extracted from adherent cells cultured in six-well plates.
Nitrite assay
Nitrite (NO2–) is a stable end-product used extensively as an indicator of NO production by cultured cells. In our experimental conditions, NO2– accumulation was assayed by the Griess reaction, according to the method described previously (Rockett et al., 1992). Briefly, cell-free supernatants were mixed with equal amounts of Griess reagent (p-aminobenzene sulfonilamide 1%, naphtylethylenediamide 0.1% in phosphoric acid 2.5%) in 96-well plates. Samples were incubated at room temperature for 10 min, and subsequently absorbance was read at 540 nm using a microplate reader. NO2– concentrations were calculated in accordance with a sodium nitrite standard curve.
RNA extraction and Northern blot analysis
C2C12 muscle cells were plated in six-well plates at a concentration of 1 x 106/ml/well and then incubated with stimuli. At the indicated times, total RNA was extracted and analysed as described previously (Meda et al., 1996). The cDNA fragments encoding mouse macrophage iNOS (Lyons et al., 1992) were labelled with 32P using a Ready-To-Go labelling kit (Pharmacia, Uppsala, Sweden) before hybridization of nylon filters and autoradiography. Blots were subsequently rehybridized with a human G3PDH (glyceraldehyde-3-phosphate dehydrogenase) cDNA probe (Clontech Laboratories Inc., Palo Alto, Calif., USA) as an internal control.
Statistical analysis
Data are expressed as means ± standard deviations. Statistical evaluation was performed by repeated measures ANOVA (analysis of variance) followed by Dunnet's test for specific comparisons. Statistical significance was set at P < 0.05.
Nuclear morphology
Nuclear morphology was examined by staining C2C12 cells with 2 mg/ml DAPI (4',6'-diamidino-2-phenylindole-dihydrochloride) (Boehringer Mannheim, GmbH, Germany). To estimate the number of cells that fulfilled the morphological (DAPI) criteria of apoptosis, at least 300 nuclei were counted from three coverslips and results were expressed as the ratio of fragmented nuclei to total nuclei x 100.
Electron microscopy
C2C12 muscle cells, after incubation with stimuli for the indicated times, were fixed in glutaraldehyde in PBS and post-fixed in 1% osmium tetroxide in the same buffer. They were then dehydrated and embedded in epoxy resin as described previously (Moggio et al., 1989). Thin sections were stained with uranyl acetate and lead citrate and examined with Zeiss EM 109 electron microscope.
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Results |
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To determine whether Aß can induce NO2– production by skeletal muscle in vitro, C2C12 cells were stimulated with Aß[1–42] or Aß[25–35]. These peptides at concentrations up to 100 µg/ml, as well as IFN-
100 U/ml, used alone, did not trigger statistically significant NO2– release during 72 h stimulation. However, when Aß[25–35] or Aß[1–42] was combined with IFN-, accumulation of NO2– was detected after 48 h in cell free supernatants (16 ± 2- and 12 ± 2.8-fold increase over control for Aß[25–35] and Aß[1–42], respectively; P < 0.05, n = 5) (Fig. 1). Dose–response studies demonstrated that the amount of NO2– released into the culture medium was related to increasing concentrations of Aß[1–42] or Aß[25–35] (Fig. 2). In additional experiments, release of NO2– triggered by Aß peptides plus IFN- was inhibited by 88% using 100 µM L-NMMA, a specific inhibitor of iNOS activity (not shown). Specificity of the effect of Aß[25–35] was demonstrated by using a scrambled analogue, which failed to induce NO2– production over that triggered by IFN- alone (Fig. 3). Stimulation of C2C12 myoblasts and differentiated myotubes with Aß peptides and IFN- resulted in statistically indistinguishable NO2– levels in cell-free supernatants, demonstrating that the stage of differentiation did not affect the ability of C2C12 cells to produce NO2– (Fig. 4).
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To investigate whether the production of NO2– triggered by Aß peptides in the presence of IFN-
reflected induction of iNOS gene expression, Northern blot analysis was performed on C2C12 total RNA, using a probe complementary to the mouse macrophage iNOS coding sequence. In resting conditions as well as after incubation with individual IFN- or Aß peptides, the mRNA expression for iNOS in C2C12 cells was absent. Steady-state mRNA levels of iNOS were clearly increased after stimulation of the cells with Aß[1–42]or Aß[25–35] at the concentration of 50 µg/ml, in the presence of IFN- (100 U/ml) (Fig. 5).
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Finally, to detect the presence of apoptotic morphology of C2C12 cells upon incubation with Aß and IFN-
, muscle cultures were examined by DAPI staining and electron microscopy. As shown in Fig. 6, ~4% of muscle cells showed morphological evidence of apoptosis after exposure to Aß plus IFN- for 48 h.
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Discussion |
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TopAbstractIntroductionMaterial and methodsResultsDiscussionReferences |
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In this study we demonstrate that Aß[25–35] and Aß[1–42] peptides, in the presence of IFN-
, are able to induce the production of NO2– by cultured murine C2C12 cells. Similar NO2– levels were obtained in cell-free supernatants from C2C12 myoblasts and differentiated myotubes, showing that the stage of differentiation did not affect the ability of C2C12 cells to release NO2– in response to Aß peptides plus IFN-. The effect of Aß and IFN- on NO2– production was regulated at the level of mRNA accumulation, as indicated by increased expression of iNOS mRNA in Aß/IFN--stimulated C2C12 myogenic cells, and it was obtained with both Aß[25–35] and the full-length Aß[1–42]. Moreover, L-NMMA, a specific inhibitor of iNOS activity, almost completely suppressed NO2– production triggered by Aß peptides plus IFN-. Specificity of the effects of Aß[25–35] was demonstrated using a peptide with the same amino acid composition as Aß[25–35], but with a scrambled sequence, which did not influence NO2– accumulation by C2C12 cells treated with IFN-.
Production of NO2– by C2C12 skeletal muscle cells stimulated with Aß[25–35] or Aß[1–42], in the presence of IFN-, although ~30% lower, was in the same range of concentrations of those observed in microglial cells under similar experimental conditions (Goodwin et al., 1995; Meda et al., 1995; Smith et al., 1997). However, while IFN- alone has been shown to be a stimulus for NO2– accumulation in microglia, it was incapable of inducing NO2– release in C2C12 cells by itself, even though it appeared to be a necessary component for the biological effect of Aß peptides. The possible explanations for the essential role of IFN- in NO2– release and induction of iNOS mRNA in C2C12 muscle cells include upregulation of an as yet undiscovered receptor for Aß protein, activation of intracellular signalling pathways, or co-induction of necessary transcription factors.
The results of our study suggest that NO production triggered by Aß and IFN- might have manifold consequences for the pathophysiology of skeletal muscle and is important on several accounts. NO has been shown to inhibit muscle contractility through a cGMP dependent mechanism (Kobzik et al., 1994) and to decrease basal glucose transport into skeletal muscle (Balon and Nadler, 1994). Moreover, NO can combine with O2– to produce highly toxic ONOO–, which in turn may induce aberrant protein nitration (Yang et al., 1998) and damage to the muscle cell surface. NO, because of its lipophilic properties, can then readily diffuse into the extracellular milieu to react with surrounding structures and cells (Brown, 1995). Finally, NO can mediate toxic effects of cytokines, playing a pathophysiological role during the acute and chronic phases of inflammatory reactions (Williams et al., 1994).
The significance of Aß deposits in IBM represents a subject of debate. The intracellular location of IBM-associated Aß favours a protein synthesized within the cell. However, whether it represents an altered protein derived from a normal gene product or an isoform of a normal protein resulting from a gene mutation is presently unknown (Mendell et al., 1991). Regardless of the source of Aß in IBM, the potential importance of Aß deposition and oxidative damage in human muscle pathology is supported by several findings. First, abnormal accumulation of Aß in IBM often precedes other abnormalities, including congophilia and vacuolization (Askanas et al., 1992b; Sarkozi et al., 1994). Secondly, direct transfer of the ßAPP gene into cultured normal muscle cells is sufficient to induce several of the typical morphological aspects of IBM (Askanas et al., 1996, 1997). Thirdly, iNOS immunoreactivity is detectable in vacuolated muscle fibres and co-localizes with Aß deposition in both s-IBM and h-IBM (Yang et al., 1998). Therefore, our data suggest that the synergistic effect of Aß and IFN- on NO production from C2C12 muscle cells in culture might be at least partly responsible for the increase of iNOS in vacuolated fibres of IBM. However, since Aß acted synergistically with IFN-, a question raised by our study is the pathophysiological role of IFN- in vivo in s-IBM and h-IBM. Because sIBM is characterized by varying degrees of inflammation (Karpati and Carpenter, 1993; Mikol and Engel, 1994), the presence of CD8+ cytotoxic T cells and macrophages might be a potential source of IFN-. The lack of lymphocytic inflammation in h-IBM (Askanas and Engel, 1993) makes more intringuing the role of IFN- in this condition. Future studies will address whether other molecules besides IFN-, for example products of muscle catabolism, may cooperate with Aß in mediating its biological effects in h-IBM.
We also provide evidence that incubation of C2C12 muscle cultures with Aß and IFN- induces ultrastructural nuclear abnormalities such as irregular clusters of condensed chromatin, suggesting the presence of apoptotic morphology. However, since these features were only occasionally detectable in our culture conditions, we do not have sufficient data to support the idea that apoptosis may be a relevant mode of muscle cell death related to NO production driven by Aß and IFN- stimulation. This notion is also in agreement with the lack of typical apoptotic changes in IBM, even though some muscle fibres have been found to express the oncoprotein Bcl-2 (Figarella-Branger et al., 1988), which is an inhibitor of apoptosis and whose expression has been considered to be an attempt to escape programmed cell death (Hockenbery et al., 1990).
In conclusion, our study demonstrates that the combination of Aß peptides plus IFN- induces production of NO2– and accumulation of iNOS mRNA from myogenic cells in vitro. These findings suggest that the mechanism leading to the increased accumulation of iNOS in IBM might be triggered by the synergistic effects of Aß and IFN-. This study also provides a useful in vitro experimental system to further investigate the potential role of oxidative stress driven by Aß deposition in the pathogenesis of muscle tissue damage in IBM.
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Acknowledgments |
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This work was supported by Associazione Amici del Centro Dino Ferrari, and by a grant from IRCCS Ospedale Maggiore Policlinico (Progetto a Concorso Ricerca Corrente anno 1993), Milan, Italy.
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Received January 7, 1999. Revised May 26, 1999. Second revision on September 13, 1999. Accepted September 13, 1999.
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