Cardiovascular Research

Cardiovascular Research 2002 54(1):95-104; doi:10.1016/S0008-6363(02)00228-6
© 2002 by European Society of Cardiology

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Copyright © 2002, European Society of Cardiology

Induction of iNOS expression in skeletal muscle by IL-1β and NFB activation: an in vitro and in vivo study

Volker Adams^*, Britta Nehrhoff, Ulrike Späte, Axel Linke, Paul C Schulze, Angelika Baur, Stephan Gielen, Rainer Hambrecht and Gerhard Schuler

University Leipzig–Heart Center Leipzig, Clinic of Cardiology, Leipzig, Germany

^* Corresponding author. Universität Leipzig, Herzzentrum GmbH, Klinik für Innere Medizin/Kardiologie, Strümpellstrasse 39, D-04289 Leipzig, Germany. Tel.: +49-341-865-1620; fax: +49-341-865-1461 adav{at}medizin.uni-leipzig.de

Received 25 September 2001; accepted 18 December 2001

	Abstract

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 
Objective: The intracellular pathway and the regulation of inducible nitric oxide synthase (iNOS) expression in skeletal muscle is incompletely understood. In vitro studies, using different cell types, suggest that inflammatory cytokines are potential triggers to induce iNOS expression. Methods: To analyze intracellular pathways leading to iNOS induction, rat skeletal myoblasts were incubated with inflammatory cytokines and assessed for iNOS expression by Western blot and Griess reaction. To confirm the in vitro findings, local cytokine levels were determined in skeletal muscle biopsies of patients with chronic heart failure (CHF) and correlated with iNOS expression. Results: Nitrite accumulation in the myoblast culture supernatant or iNOS protein in the cell pellet was significantly increased after incubation with IL-1β in combination with -IFN. Priming experiments revealed that -IFN elevated the expression of IL-1β receptor mRNA, whereby IL-1β was able to induce iNOS expression. The cytokine-mediated iNOS induction was significantly reduced by blocking ERK1/ERK2 activation and completely abolished by the inhibition of NFB. In skeletal muscle biopsies of CHF patients the local content of IL-1β was significantly increased as compared to healthy controls. Furthermore, a linear correlation between IL-1β content and iNOS expression in the skeletal muscle was detected. Conclusions: These data demonstrate that IL-1β, together with the priming effect of -IFN, induces iNOS expression in skeletal muscle via activation of ERK1/ERK2 and NFB.

KEYWORDS Heart failure; Nitric oxide; Cytokines; Signal transduction

	1. Introduction

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 
Conditions with increased levels of inflammatory cytokines such as septic shock [1] and chronic heart failure (CHF) [2] have been shown to be associated with depressed myocardial contractility. It has been suggested that cardiac dysfunction can be attributed to excessive nitric oxide (NO) production resulting from cytokine-induced expression of inducible nitric oxide synthase (iNOS) [3–6]. Recently, we and others have demonstrated an increased expression of iNOS in skeletal muscle biopsies of CHF patients, which was inversely correlated with exercise capacity [7–9]. These results implied that iNOS expression may also contribute to the reduced contractile performance of peripheral skeletal muscle in CHF.

Genomic analysis of the iNOS promoter [10,11], as well as cell culture experiments in different cell types [12–17], suggested that inflammatory cytokines may be necessary for the induction of iNOS expression. The circulating levels of inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-), interleukin-1β (IL-1β) and interleukin-6 (IL-6), have been reported to be increased in patients with CHF [18–21]. Once bound to their specific receptor on the target cell, an internal signaling cascade is initiated, finally leading to the activation of transcription factors and the induction of iNOS.

In cardiac myocytes the activation of ERK1/ERK2 appears to be necessary for the induction of iNOS by IL-1β and -interferon ( -IFN) [22]. In other cell types, e.g., bovine chondrocytes, the IL-1β induction of iNOS expression is mediated via p38 mitogen-activated protein kinase (p38MAPK) [23]. The same pathway could be identified in mouse astrocytes [24].

So far the regulation of iNOS induction in skeletal muscle of patients with CHF, and the intracellular signal transduction leading to iNOS expression remains largely unknown. Therefore, the aim of this study was to analyze, in an in vitro system, which cytokines and intracellular signaling pathways are involved in the regulation of iNOS induction in the skeletal muscle. To confirm the in vitro findings, the local cytokine levels, together with iNOS expression, were assessed immunohistochemically in skeletal muscle biopsies of patients with CHF.

	2. Methods

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 
2.1 In vitro experiments
2.1.1 Cell culture
L6 rat skeletal myoblast cells (generous gift of Dr C. Sers, Institute of Pathology, Charité Berlin) were cultured as recently described [7]. Primary rat skeletal myoblasts were isolated from rat hindlimbs according to the method recently described [25]. Cells, 60–80% confluent, were incubated with inflammatory cytokines like IL-1β (50 ng/ml), TNF- (20 ng/ml) or IFN- (100 U/ml) (Sigma, Deissenhofen, Germany) for up to 24 h. For studying the signal transduction pathway, the cells were preincubated with specific inhibitors for 1 h before the addition of IL-1β, TNF- or IFN-. The following inhibitors were used: SB203580 (Calbiochem, La Jolla, CA, USA), a highly specific, cell-permeable inhibitor of p38MAPK with no significant inhibition of p42/44MAPK [26]; PD98059 (Calbiochem), a specific blocker for the activation of p42/44MAPK, also known as ERK1/2 [27], or pyrrolidine dithiocarbamoate (PDTC, Sigma), an anti-oxidant often used to inhibit the activation of NFB [28,29].

For priming experiments, cells were incubated for 8 h with the priming cytokine as described in the literature [3]. After this priming period the medium was changed and the stimulatory cytokine was added for an additional 24 h to the cell culture medium.

2.1.2 Assay for iNOS induction
Synthesis and release of NO was quantified by assaying the cell culture supernatant for nitrite by Griess reaction [30], whereas the supernatant of untreated cells was used a blank.

The induction of the iNOS protein expression was investigated in the cell pellet by Western blot analysis [8], whereas the expression of iNOS mRNA was evaluated by semiquantitative reverse transcription–polymerase chain reaction (RT–PCR) using specific primers (NOS-R3, 5'-GCGGAGCGATGGGAAGCATG-3' and NOS-R4, 5'-CCCGAGCTCCTGGAACCACT-3')

2.1.3 Detection of IL-1 receptor
The expression of IL-1 receptor subtype 1 was evaluated by quantitative RT–PCR using the following primers: IL1βR1-U, 5'-AGATGGAAGGACCTATGATG-3' and IL1βR1-L, 5'-TGCAGCATCTGACGACAGGA-3' The expression was quantified by densitometry and expressed as a ratio over GAPDH.

2.1.4 Nuclear extract preparation and electrophoretic mobility shift assay
The cells were detached from the plate by phosphate-buffered saline (PBS)/EDTA and collected by centrifugation. After washing once with PBS the cell pellet was resuspended in 1 ml ice-cold buffer A (HEPES 10 mmol/l, KCl 10 mmol/l, EDTA 0.1 mmol/l, EGTA 1 mmol/l, dithiothreitol 1 mmol/l, phenylmethylsulfonyl fluoride 1 mmol/l, Aprotinine 10 µg/ml, Pepstatine 10 µg/ml, Leupeptine 10 µg/ml) and incubated for 15 min at 4 °C. Subsequently Nonidet P-40 was added to 0.5% and the suspension was centrifuged at 3000 g for 10 min. The supernatant was aspirated and the cell pellet was resuspended in buffer B (HEPES 20 mmol/l, NaCl 400 mmol/l, EDTA 0.1 mmol/l, EGTA 1 mmol/l, dithiothreitol 1 mmol/l, phenylmethylsulfonyl fluoride 1 mmol/l, Aprotinine 10 µg/ml, Pepstatine 10 µg/ml, Leupeptine 10 µg/ml). After 15 min incubation on ice and subsequent centrifugation (10 min at 8000 g at 4 °C) the supernatant containing the nuclear proteins was used in the electrophoretic mobility shift assay (EMSA).

The EMSA was performed using an electrophoretic mobility shift assay kit (Promega GmbH, Mannheim, Germany) and an NFB-specific oligonucleotide 5'-AGTTGAGGGGACTTTCCCAGGC-3' [31].

2.2 In vivo experiments
2.2.1 Patients
Thirty-six male patients 70 years with CHF (New York Heart Association functional class II to III) as a result of dilated cardiomyopathy (n=32) or ischemic heart disease (n=4) and 10 sedentary normal individuals were studied. Inclusion and exclusion criteria were the same as recently published [7].

The protocol for this study was approved by the Ethics Committee of the University of Leipzig, and written informed consent was obtained from all subjects prior to enrolment in the study.

2.2.2 Skeletal muscle biopsy
Percutaneous needle biopsies were obtained from the middle part of the m. vastus lateralis under local anesthesia, as described in detail by Bergström et al. [32]. The biopsies were fixed with 4% buffered formaldehyde and subsequently paraffin embedded or frozen immediately in liquid nitrogen.

2.2.3 Immunohistochemical detection of cytokine expression
The local expression of IL-1β, TNF- and -IFN was visualized and quantified using specific polyclonal antibodies; IL-1β (Upstate Biotechnology, Lake Placid, NY, USA), TNF- (Sigma, St. Louis, MO, USA) and -IFN (Serotec, Oxford, UK) following a protocol recently described [8].

For consecutive duplicate biopsy samples, the variation regarding the percentage of IL-1β, TNF-, IFN--positive stained tissue area has been shown to be in the order of less than 10%.

2.2.4 Detection of iNOS by immunohistochemistry
The expression of iNOS was visualized and quantified using an iNOS-specific polyclonal antibody (Transduction Laboratories, Lexington, KY, USA) and biotinylated tyramid as recently described in detail [8].

2.2.5 Statistical analysis
Values are given as mean±S.E.M. for all variables. Single comparisons were performed by a non-parametric test (Mann–Whitney U-test) and a P-value of 0.05 was considered statistically significant.

	3. Results

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 
3.1 In vitro experiments
3.1.1 Induction of iNOS in L6 myoblasts by different cytokines
To analyze the inducibility of iNOS expression in skeletal muscle by different inflammatory cytokines, rat skeletal myoblasts were incubated for 24 h with either IL-1β, TNF-, IFN- or with possible combinations of these cytokines. The analysis of the cell culture supernatant by Griess reaction and of the cell pellet by Western blot analysis (Fig. 1A, B) showed, that a single cytokine was unable to induce iNOS expression. The combination of IL-1β and IFN- was capable to stimulate iNOS expression, whereas the combinations of IL-1β and TNF- or IFN- and TNF- failed to initiate iNOS expression. The induction pattern was identical for the L6 cell line or the primary rat skeletal muscle myoblast. The addition of TNF- to the combination of IL-1β and IFN- further increased the iNOS induction significantly by 50–70% (L6: 73.0±6.04 vs. 124.8±6.2 nmol nitrite/mg cell protein, P<0.05; primary myoblasts: 335.0±10.5 vs. 515.6±19.9 nmol nitrite/mg cell protein, P<0.05). The rise of nitrite in the cell culture supernatant was always accompanied by an increased cellular iNOS protein synthesis (Fig. 1A, B).

View larger version (15K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Cytokine-induced iNOS expression and nitrite accumulation in L6 (A) and primary rat skeletal myoblast cells (B). After stimulation of cells with various cytokines or cytokine combinations; IL-1β (50 ng/ml), -IFN (100 U/ml), TNF- (20 ng/ml), nitrite accumulation in culture supernatant was measured by the Griess reaction and iNOS protein expression in the cell pellet was detected by Western blot. The positive Western blot signal with a molecular weight of 130 kDa is marked with an arrow. Values for nitrite production are expressed as mean±S.E.M. for four independent experiments. *P<0.05 vs. control, #P<0.05 vs. IL1β/IFN-.

 
3.1.2 Time-course for iNOS induction
Based on the above observation, that L6 cells and primary cardiomyocytes showed the same stimulation pattern for iNOS induction, all the following experiments were performed only with L6 cells. iNOS mRNA expression occurred 1 h after IL-1β/IFN- or IL-1β/IFN-/TNF- stimulation (Fig. 2B). Nitrite accumulation (Fig. 2A) in the culture medium, as well as iNOS protein expression (Fig. 2C), lagged behind iNOS mRNA expression, but then increased up to 24 h, the last time-point measured.

View larger version (43K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Time-course of cytokine-mediated iNOS expression in L6 cells. The nitrite accumulation in culture supernatant (A), RNA expression (B) or iNOS protein expression as detected by Western blot (C) after stimulation with IL-1β (50 ng/ml)/-IFN (100 U/ml) or with IL-1β (50 ng/ml)/-IFN (100 U/ml)/TNF- (20 ng/ml). Values for nitrite production are expressed as mean±S.E.M. for four independent experiments. *P<0.05 vs. unstimulated; P<0.05 vs. 24 h stimulated with IL-1β/-IFN.

 
3.1.3 Priming of L6 cells
Incubation of L6 cells with either IL-1β or -IFN alone failed to stimulate nitrite production; however, if the cells were first primed by an 8-h exposure to -IFN, subsequent exposure to IL-1β led to a significant increase in nitrite accumulation (Table 1). This effect was not observed if the cells were primed with IL-1β and subsequently stimulated with -IFN. To evaluate if this priming effect of -IFN was due to an increase in IL-1-receptor expression quantitative RT–PCR was performed (Fig. 3A). As shown in Fig. 3B the expression of the IL-1β receptor subtype 1 is significantly increased in cells pretreated for 8 h with -IFN as compared to untreated or IL-1β primed cells.

View this table: [in this window] [in a new window]   Table 1 Priming of L6 cells by cytokines

 

View larger version (45K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 L6 cells were incubated with either IL-1β (50 ng/ml) or -IFN (100 U/ml) for 8 h and the expression of IL-1β receptor was quantified by RT–PCR. (A) Representative agarose gel stained with ethidium bromide of PCR (IL-1βR and GAPDH). (B) Quantitative analysis is expressed as mean±S.E.M. for four independent experiments. *P<0.05 vs. untreated cells.

 
3.1.4 Inhibition of NO synthesis by specific inhibitors of the signaling cascade
To further elucidate the signaling pathway, by which IL-1β/-IFN-mediated iNOS transcription is achieved, L6 cells were incubated with IL-1β (50 ng/ml) and IFN- (100 U/ml) in the presence or absence of SB203580 (10 µM), PD98059 (50 µM) or PDTC (100 µM) and the generation of nitrite in the culture medium was measured. As shown in Fig. 4A the presence of SB203580 reduces the concentration of nitrite in the supernatant only by 19±11%, whereas the inhibitor of ERK1/ERK2 (PD98059) inhibits the concentration of nitrite in the cell culture medium by 68±13%. The combination of both inhibitors did not further decrease the nitrite concentration (69±16%). Also different concentrations of the inhibitors did not change the degree of cytokine-induced nitrite production (Fig. 4B, C). If PDTC, an inhibitor of NFB activation, was added to the cell culture, a dose-dependent inhibition of nitrite production was observed (Fig. 4D). The activation of NFB by the cytokine mixture IL-1β/-IFN was further confirmed by EMSA. As shown in Fig. 5, a significant shift of the labeled oligonucleotide was observed in cytokine treated cells (lanes 3 and 4) as compared to untreated cells (lane 2). The specificity of the shift was proven by competition experiments with unlabeled oligonucleotide containing the specific NFB sequence (lane 5).

View larger version (23K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 L6 cells were preincubated for 1 h with various inhibitors (A) and concentrations (B–D) of different signaling cascade molecules before a combination of IL-1β (50 ng/ml) and -IFN (100 U/ml) (cyt) was added to the medium. The nitrite accumulation after 24 h was determined in the medium by Griess reaction. The amount of nitrite generated by cytokines without the addition of an inhibitor was set as 100%. Values for nitrite production are expressed as mean±S.E.M. for four independent experiments. *P<0.001 vs. cytokine, #P<0.05 vs. cytokine.

 

View larger version (96K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 The effect of IL-1β/-IFN incubation of the NFB DNA binding activity. Nuclear extracts isolated from unstimulated and stimulated cells were incubated with digoxigenin-labeled NFB-specific oligonucleotide and separated on a polyacrylamide gel. After transfer to a nitrocellulose membrane, digoxigenin was detected by specific antibodies and enhanced chemiluminescence. Lane 1: only labeled oligonucleotide without nuclear extract; lane 2: extract from unstimulated cells; lane 3, 4: extract from cytokine-stimulated cells; lane 5: extract from stimulated cells plus an access of unlabeled NFB oligonucleotide. The result shown is a representative of four independent experiments.

 
3.2 In vivo experiments
3.2.1 Detection of inflammatory cytokines in skeletal muscle biopsies of patients with CHF
To evaluate a possible contribution of different cytokines to the in vivo regulation of iNOS expression, the expression of iNOS, IL-1β, TNF- and -IFN was measured immunohistochemically in 36 skeletal muscle biopsies of patients with CHF and of 10 healthy, age-matched controls. The patient characteristics are presented in Table 2. Left ventricular ejection fraction (22.4±1.8 vs. 72.7±1.5%; P<0.001) and maximal oxygen uptake (17.6±0.8 vs. 27.2±0.3 ml/kg/min, P<0.001) were significantly impaired, and a lower body weight (83.5±2.4 vs. 94.2±3.7 kg; P=0.04) was detected as compared to the healthy control subjects.

View this table: [in this window] [in a new window]   Table 2 Clinical parameters of study population

 
Analyzing the expression of inflammatory cytokines in skeletal muscle biopsies of CHF patients, a significantly higher concentration of IL-1β (1.05±0.06 vs. 0.73±0.06 positive stained tissue area; P=0.03) was detected in myocytes as compared to healthy controls, whereas the expression of TNF- (1.33±0.16 vs. 1.27±0.13 positive stained tissue area; P=N.S.) and -IFN (1.78±0.25 vs. 1.73±0.26 positive stained tissue area; P=N.S.) was not significant between both groups (Fig. 6A–C).

View larger version (37K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 6 The local concentration of IL-1β (A), -IFN (B) and TNF- (C) was determined by quantitative immunohistochemistry in paraffin-embedded skeletal muscle biopsies of CHF patients (CHF) and in healthy controls (control). The quantity of detected cytokine is expressed as % positive stained tissue area. The linear correlation between the local concentration of IL-1β in skeletal muscle biopsies of patients with CHF and the expression of iNOS in the skeletal muscle is depicted (D).

 
3.2.2 Correlation between iNOS and cytokine expression in skeletal muscle biopsies
To investigate the role of the cytokine concentration in skeletal muscle for the regulation of iNOS expression, linear regression analyses were performed. There was a close linear correlation between the local IL-1β levels and iNOS protein expression (r=0.70, P<0.0001) (Fig. 6D). TNF- and -IFN however, were not significantly correlated with iNOS expression.

	4. Discussion

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 
The following key messages emerge from these in vitro and in vivo experiments:

(1) In vitro, in skeletal muscle myoblasts the expression of iNOS is activated by IL-1β in combination with -IFN and further increased by the addition of TNF-.

(2) -IFN primes L6 myoblasts by increasing the expression of IL-1β receptor for IL-1β to stimulate iNOS expression.

(3) Intracellular, the activation of ERK1/ERK2 is the major pathway involved in iNOS induction. The activation of NFB is an essential step in this activation process.

(4) In vivo, the concentration of IL-1β is significantly increased in the skeletal muscle of CHF patients, and demonstrated a close linear correlation to iNOS expression.

Taken together, these findings extend our knowledge of cytokine-mediated induction of iNOS in vitro and in vivo in the skeletal muscle of patients with CHF.

4.1 Cytokine-mediated iNOS induction
The analysis of several different cell lines revealed that, depending on the cell type, different inflammatory mediators are necessary to induce the expression of iNOS [14,16]. The molecular basis for the involvement of cytokines for the induction of iNOS transcription is based on the localization of cytokine responsive elements in the promoter region of the gene [10,11]. Transfection studies, using a luciferase reporter construct, showed that several cytokine responsive elements are located –16 kb upstream of the iNOS transcriptional start site [11]. These include -IFN responsive elements and NFB binding consensus sequences. In L6 cells, a rat skeletal muscle cell line, and primary rat skeletal myoblasts, only IL-1β/-IFN was the minimal cytokine combination which was able to induce iNOS transcription and translation. This result is in line with an investigation performed by Okuda et al., who also found that iNOS is induced in L6 cells by IL-1β/-IFN [33]. In C2C12 cells, a mouse skeletal muscle myocyte cell line, the combination of TNF-/-IFN or IL-1β/-IFN leads to an activation of iNOS transcription [17]. In L6 cells and isolated myoblasts, as already demonstrated for C2C12 cells [17], the addition of TNF- to the combination of IL-1β/-IFN further increased iNOS transcription and translation. Taking all results together it is obvious that -IFN is a necessary component for cytokine-induced iNOS expression.

What is the function of -IFN for the cytokine-induced iNOS transcription? At least in L6- and C2C12 skeletal muscle cells it seems, as demonstrated by priming experiments, that -IFN primes the cells for the iNOS-inducing action of either IL-1β (L6 cells) or TNF- (C2C12 cells) [17]. It appears that in L6 cells this priming effect is due to an increased expression of IL-1β receptor (IL-1βR), an effect that has also been reported in a human fibroblast cell line [34].

It is therefore conceivable that in skeletal myocytes and possibly in other cells, -IFN enhances the expression of the respective cytokine receptor, which stimulates the cell to initiate iNOS transcription.

4.2 Intracellular signaling pathway involved in cytokine-mediated iNOS induction
In the L6 rat skeletal muscle cell line Okuda and co-worker could show that the addition of a tyrosine kinase inhibitor like genistein or herbimycin completely abolished the IL-1β/-IFN-mediated iNOS expression [33]. The intracellular pathway further downstream of tyrosine kinase activation leading to cytokine-mediated iNOS induction in skeletal muscle cells is not well defined. In several cell culture systems including macrophages, skin-derived dendritic cells, glomerular mesangial cells, cardiac myocytes, oligodendrocytes or vascular smooth muscle cells the importance of p38MAPK, p42/44MAPK (ERK1/ERK2) and NFB for cytokine- or LPS-mediated iNOS induction has been established [22,35–38]. The significant reduction of nitrite accumulation in the cell culture supernatant after IL-1β/-IFN stimulation by the specific ERK1/ERK2 inhibitor PD98059 and the resistance of iNOS induction to SB203580 implies that in skeletal muscle the activation of ERK1/ERK2 via MEK1/MEK2 is involved in cytokine-mediated iNOS induction. The observation that no concentration-dependent inhibition of nitrite synthesis by PD98059 was observed (Fig. 4C) with concentrations as low as 5 µM may be due to the fact that with concentrations higher than the IC₅₀ (2 µM for PD98059 in cell culture experiments) the p42/44MAPK pathway is already completely inhibited. The activation of p38MAPK seems to be negligible in the process of iNOS induction. Further downstream of the MAPK pathway activation, the activation of NFB is an essential step for the initiation of iNOS transcription and translation.

Taken together, one may conclude that in skeletal muscle myocytes the cytokine-mediated iNOS expression is transduced via tyrosine kinase, followed by the activation of ERK1/ERK2, finally leading to the activation of the transcription factor NFB.

4.3 The role of cytokines for iNOS induction in vivo
In patients with CHF, an increased expression of iNOS in heart and skeletal muscle myocytes [7–9], as well as an increased serum concentration of inflammatory cytokines, has been reported [19]. The pathophysiological role of increased cytokine levels with respect to the regulation of iNOS expression in skeletal muscle is not fully understood. As demonstrated by the linear correlation between the local IL-1β concentration and the expression of iNOS in skeletal muscle biopsies of patients with CHF and the localization of both parameters in myocytes, it seems that also in vivo IL-1β in the presence of -IFN may regulate iNOS expression. The lack of a correlation between iNOS expression and local -IFN may be explained by the fact that -IFN only primes the cells for the stimulatory function of IL-1β, as proven in the cell culture experiments. Therefore one may assume that -IFN, already in low concentrations, induces the expression of IL-1β receptor whereby IL-1β as iNOS-inducing cytokine is now able to stimulate iNOS expression

As pointed out by Satoh and coworkers the level of plasma TNF- does not predict the TNF- concentration in the tissue [39]. Therefore, to investigate the role of inflammatory cytokines for the induction of iNOS expression in skeletal muscle it is necessary to determine the local concentration of inflammatory cytokines in the skeletal muscle, and compare this concentration with the concentration of iNOS.

4.4 Limitation of study
The use of an immortalized rat skeletal muscle myoblast cell line raises the question about the application of the in vitro results to the situation in a differentiated human skeletal muscle. Comparing the L6 cell line, at least to primary rat skeletal myoblasts with regard to the cytokines able to induce iNOS expression, no difference was observed, whereas the quantitative response in nitrite synthesis was different (Fig. 1). To transfer the data obtained in this study to the situation in human skeletal muscle, further experiments using isolated and cultured human skeletal myoblasts/myocytes are necessary. Nevertheless, the linear correlation between the local content of IL-1β and iNOS expression in human muscle biopsies support the relevance of IL-1β for the regulation of iNOS expression in skeletal muscle.

At least the influence of peroxynitrite (ONOO^–), a reaction product of NO and superoxide (O₂^–), on muscle contractility, has been reported for skeletal muscle, diaphragm and myocardium [40–44]. Therefore it is conceivable that the cytokine-induced iNOS induction in skeletal muscle may contribute to exercise intolerance observed in patients with CHF.

In conclusion, the data presented in this report together with reports in the literature, indicate that in skeletal muscle, inflammatory cytokines together with the priming effect of -IFN induces iNOS expression. Activation of ERK1/ERK2 is at least in part responsible, whereas the activation of the transcription factor NFB is essential for the induction of iNOS expression.

Time for primary review 16 days.

	Acknowledgements

 
We would like to thank Silke Krabbes and Angela Kricke for excellent technical assistance.

	References

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 

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