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B Activation and Proinflammatory Cytokine Production During Early Rehabilitation of Protein Malnourished Mice1 ,2
*
Department of Human Nutrition and
Division of Oral Biology, School of Dentistry, Ohio State University, Columbus, OH 43210
3To whom correspondence should be addressed. E-mail: bray.21{at}osu.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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B (NFB) activation and proinflammatory cytokine production in protein malnutrition. We further investigated molecular mechanisms by which protein malnutrition influenced inflammatory responses. CD-1 mice were fed for 3 wk a normal protein (150 g/kg) diet or a protein-deficient (5 g/kg) diet, or for 2 wk a protein-deficient diet followed by 1 wk of N-acetylcysteine (NAC) supplementation. Lipopolysaccharide (LPS) was injected intravenously, and liver was collected at 0, 15 min, 1, 4, 24 and 48 h after LPS administration. Protein malnutrition significantly increased the activation of NFB and transcription levels of its downstream genes interleukin-1ß and tumor necrosis factor-
. Peak NFB activation was inversely associated with GSH levels (r = -0.939, P < 0.0001) but positively correlated with the GSH disulfide/2GSH reduction potential (r = 0.944 P < 0.0001). We noted unusual NFB p50/p50 homodimer translocation that was significantly elevated in tissue from protein malnourished mice, along with decreased peak levels of normal p65/p50 heterodimer translocation. Interestingly, mRNA levels of IB- were not affected by protein malnutrition. However, early supplementation of NAC to protein malnourished mice without replenishing with dietary protein restored GSH levels and reduction potential, and normalized NFB activation and proinflammatory cytokine production. Taken together, these findings provide evidence supporting the role of GSH in NFB activation and inflammatory response in protein malnutrition, and the use of NAC in early rehabilitation of protein malnutrition without a high protein diet.
KEY WORDS: • protein malnutrition • nuclear factor B • glutathione • N-acetylcysteine • proinflammatory cytokine
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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,2
). In developed countries, it occurs mainly in the elderly, the hospitalized and those with chronic diseases (3
,4
). Protein malnutrition reduces antioxidant levels, impairs immune functions and increases sensitivity to opportunistic infections and septic shock (5
–7
).
Among important antioxidants, glutathione (GSH4
; 
-glutamylcysteinylglycine) is the most abundant nonprotein thiol in mammalian cells and plays an important role in the reduction of reactive oxygen species and the detoxification of xenobiotics (8
). Blood GSH was decreased in protein malnourished children, whose incidence of opportunistic infection was increased (9
,10
). Protein malnourished patients were also found to have increased sensitivity to septic shock (5
,7
,11
).
Septic shock is caused by overwhelming bacterial infection, and results in an acute overproduction of proinflammatory cytokines including interleukin-1ß (IL-1ß) and tumor necrosis factor- (TNF-) (12
,13
). Bacterial endotoxin lipopolysaccharide (LPS) plays an important role in the development of septic shock and consequent multiorgan failure of the host (14
). LPS induces translocation of nuclear factor B (NFB), a ubiquitous transcription factor that mediates the overproduction of IL-1ß and TNF- (15
,16
).
LPS-induced NFB translocation can be responsive to oxidative stress and tissue redox environment (17
). A more oxidized redox environment has been associated with increased NFB translocation. In contrast, a more reduced redox status might decrease NFB activation. The GSH disulfide (GSSG)/2GSH reduction potential is considered a comprehensive indicator of tissue redox environment. Antioxidants, in particular GSH, have been shown to decrease GSSG/2GSH reduction potential and NFB activation (18
–20
). N-Acetylcysteine (NAC), a cysteine prodrug that increases GSH levels, was found to decrease NFB activation (17
,21
). In contrast, buthioninesulfoximine, a compound that inhibits GSH synthesis, potentiates LPS-induced NFB activation and tissue damage (22
,23
).
Therefore, we proposed that the increased sensitivity to septic shock in protein malnourished hosts might be caused by increased NFB activation and subsequent overproduction of proinflammatory cytokines. This abnormal activation of NFB might be associated with decreased tissue GSH levels and elevated GSSG/2GSH reduction potentials. Hence, supplementation of NAC might restore GSH levels and reduction potentials, which in turn would reduce NFB translocation and proinflammatory cytokine production.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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LPS (0111:B4 from Escherichia coli) and chemicals needed for GSH and GSSG analysis were obtained from Sigma-Aldrich (St. Louis, MO). AIN-93G (24
) purified rodent powder diet (Dyets, Bethlehem, PA) was formulated to be isocaloric, and contained either 5 g (PM) or 150 g (Normal) of protein per kilogram of diet (Table 1
). The amount of NAC added to the PM diet was calculated so that the supplemented diet (PM + NAC) had the same amount of sulfur amino acids as the normal protein diet.
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Male CD-1 mice (4–5 wk old) were obtained from Harlan (Indianapolis, IN) and singly housed in a temperature- and humidity-controlled environment (12-h light/dark cycle) with free access to tap water and diet. Mice were acclimated for 3 d before initiation of dietary treatments. All animal protocols were reviewed and approved by the Ohio State University Institutional Laboratory Animal Care and Use Committee.
Experimental design.
Mice were fed for 3 wk a 5 g/kg protein diet (PM), a 150 g/kg protein diet (Normal), or a 5 g/kg protein diet for 2 wk followed by 1 wk supplementation of NAC (PM + NAC). At the end of dietary treatment, LPS was injected intravenously via the tail vein. Mice were killed by CO2 asphyxiation. Liver was collected at 0, 15 min, 1, 4, 24 and 48 h post-LPS injection. Saline was also injected intravenously to serve as vehicle control where liver was collected 1 h after injection. The purpose of adding the saline group was to ensure that any effect observed after LPS injection was due to LPS, not to the intravenous injection itself.
NFB nuclear translocation.
Crude nuclear extracts were prepared from the liver as described by Deryckere and Gannon (25
). An electrophoretic mobility shift assay (EMSA) was used to determine the transcriptional activity of NFB by assaying the extent of binding of nuclear extracts to NFB consensus sequences as described by Gupta et al. (26
). The intensity of the binding was quantified using densitometry. In the supershift assay, EMSA was performed as described above except that the binding reaction contained 3 µg of polyclonal antibodies to either p50 or p65, the two main subunits of NFB.
GSSG/2GSH reduction potential.
The tissue reduction potential, based on the GSSG/2GSH couple, was calculated from the Nernst equation: Ehc = -240 - (59.1/2) log ([GSH]2/[GSSG]) mV at 25°C, pH 7.0. It is assumed that measured levels of reduction potential relate to the thermodynamic concentrations.
NFB subunit translocation (ELISA).
A Trans-AM NFB ELISA kit was used according to the manufacturer’s manual (Active Motif America, Carlsbad, CA) to determine NFB subunit translocation. The primary antibody was either anti-p65 or anti-p50. A secondary antibody conjugated to horseradish peroxidase provided colorimetric readout, which was quantified by spectrophotometry.
In situ hybridization (fluorescence).
The in situ hybridization protocols were performed as described previously for ribonucleotide (cRNA) probes (27
). First, paraffin-embedded tissue sections were dewaxed, dehydrated, incubated in pepsin, fixed and acetylated. Second, antisense probes were transcribed using Riboprobe System (Promega, Madison, WI) with T7 RN polymerase. Mouse IB- cDNA was generously provided by Dr. Rebecca Taub (University of Pennsylvania, Philadelphia, PA). A 672-bp DNA sequence of murine IL-1ß mRNA was prepared from commercially available insert #963357 in vector pT7T3D-pac cloned in host E. coli (American Type Culture Collection, Manassas, VA). TNF- cDNA was generously provided by Dr. Karl Decker (Albert-Ludwigs-Universität, Freiburg, Germany). To control for the specificity of the probe, sense probes were also generated by transcribing the same plasmid with T3 RNA polymerase. Finally, probes were diluted in the riboprobe hybridization buffer and applied to liver section slides. After overnight incubation at 55°C in a humidified chamber, slides were first washed in SSC of decreasing concentrations (55°C) and then incubated in a blocking buffer for 30 min. Diluted anti-digoxigenin antibody was added onto the slides, followed by anti-mouse antibody, primary streptavidin, biotinyl tyramide solution and finally CY2-streptavidin. The slides were then viewed with a microscope through a fluorescence filter. The mRNA production was quantified by measuring both the intensity (number of pixels) and the number of cells expressing cytokine mRNA using Adobe Photoshop (Adobe Systems, Mountain View, CA).
High-performance liquid chromatography (HPLC).
Determination of GSH and GSSG was achieved by using the method described by Melnyk et al. (28
) with slight modification. Briefly, an HPLC-electrochemical was obtained from Enviromental Sciences Associates, Inc. (Chelmsford, MA), along with two Shimadzu solvent delivery systems, a reverse phase C18 column (5 µm; 4.6 x 150 mm; mobile crystalline material, Tokyo, Japan) and an autosampler. GSH eluted 4 min after sample was autoinjected into the system, whereas GSSG eluted at 10 min. Sample vials were placed in a temperature-controlled (4°C) tray to minimize potential artifacts in the GSH assay. The specificity of GSH and GSSG peak and the reliability of the assay were determined by adding GSH and GSSG standards of known concentrations to the sample.
Statistical analysis.
Data were reported as mean ± SEM. Homogeneity of the error variance of the data were achieved after data were logarithm-transformed. Six one-way ANOVA on diet (one for each time point) were conducted to determine whether diet had a significant effect at each time point. When a significant effect was found, Tukey’s HSD all pair-wise comparisons were performed. In addition, three one-way ANOVA of time (one for each dietary group: PM, Normal, PM + NAC) were conducted to determine whether time had an effect within each dietary group. When a significant effect was found, Tukey’s HSD all pair-wise comparisons were performed. Thus, a total of nine one-way ANOVA were conducted for each variable. To control for the experiment-wise error, the level for each one-way ANOVA was set at 0.005 so that the overall experiment-wise error was 0.005 x 9 = 0.045, which is <0.05. Thus, differences in each ANOVA were considered significant if P < 0.005.
Pearson’s correlations were determined between NFB activation and GSH levels, and between NFB activation and GSSG/2GSH reduction potentials. All statistical analyses were performed using SAS software (SAS Institute, Cary, NC).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Feeding mice the PM diet for 3 wk decreased their body weight by 
16%, whereas 1-wk dietary supplementation of NAC did not affect the decrease (Fig. 1
). Food intake did not differ among the three dietary groups (data not shown).
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B nuclear translocation.
None of the variables measured differed between the saline group and the pre-LPS group (data not shown). Data from the pre-LPS group were then used as the controls (0 min). In protein malnourished mice, NFB nuclear translocation in liver was observed 15 min after LPS injection. The intensity peaked at 1 h and was still detected at 4 h (Figs. 2A
and 3
). In contrast, in mice fed the normal protein diet, NFB translocation did not occur until 1 h post-LPS and was not detectable at 4 h. In addition, NFB activation in mice fed the PM diet was significantly stronger than that in controls (Fig. 3)
. Notably, dietary supplementation of NAC to protein malnourished mice effectively inhibited the increase in NFB nuclear translocation, restoring it to the level in controls (Figs. 2
B and 3
).
|
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The basal GSH level was substantially lower in mice fed the PM diet compared with control mice (none Table 2
). In normal mice, hepatic GSH was doubled 1 h post-LPS treatment and remained elevated. In protein malnourished mice, however, GSH was not increased until 24 h after LPS injection. Hepatic GSSG in mice fed the PM diet was slightly increased at 4 h post-LPS (P = 0.01) treatment and significantly elevated at 24 h (Table 2)
. This is in contrast to controls, in which GSSG was unaffected by LPS injection.
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B activation and GSH levels and reduction potentials.
LPS-induced NFB activation in liver peaked at 1 h post-LPS treatment for both mice fed the PM diet and control mice (Figs. 2
A and 3
). However, high basal GSH levels in normal mice were associated with a lower intensity of NFB translocation, whereas low GSH levels in mice fed the PM diet were correlated with higher NFB activation (r = -0.939, P < 0.0001) (Fig. 4A
). NAC supplementation to protein malnourished mice restored both GSH levels and intensity of NFB translocation.
|
B activation (r = 0.944, P < 0.0001) (Fig. 4
B). Thus, NFB activation in liver was inversely correlated with basal GSH levels and positively associated with the GSSG/2GSH reduction potential.
NFB subunit translocation.
An EMSA supershift assay indicated that p50 was the major NFB subunit translocated to the nucleus in mice fed the PM diet 15 min after LPS administration (Fig. 2
B, lanes 2 and 3). An ELISA further confirmed that the translocation of the NFB p50 subunit in mice fed the PM diet began at 15 min, peaked at 1 h and returned to basal levels at 48 h (Table 3
). NFB p50 translocation at 1 h in mice fed the PM diet was approximately four times as intense as that in controls, where the translocation was unaffected by LPS injection. Interestingly, whereas protein malnutrition increased p50 translocation, it decreased peak p65 translocation compared with controls (Table 3)
.
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B-none mRNA production.
In mice fed the PM diet, mRNA production of IB- in liver started to increase at 15 min, peaked at 1 h and decreased to baseline levels at 4 h (Table 4
). Similar patterns and magnitudes of the response were found in normal and NAC-supplemented mice.
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mRNA production.
The production of IL-1ß mRNA in mice fed the PM diet was increased significantly at 15 min post-LPS, peaked at 1 h and decreased to basal levels at 4 h (Fig. 5
and Table 5
). A similar trend was found for TNF- in mice fed the PM diet (Table 5)
. Notably, peak mRNA levels of both cytokines in mice fed the PM diet were significantly higher than those in controls. NAC supplementation inhibited the increase in both cytokines, restoring the levels to controls. In addition, TNF- mRNA synthesis in mice fed the PM diet showed a biphasic pattern; i.e., it peaked at 1 h, decreased at 4 h and was increased again at 24 h (Table 5)
.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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B activation. Notably, peak NFB activation was inversely correlated with basal GSH levels and positively correlated with GSSG/2GSH reduction potential, a key indicator of redox status. In addition, dietary supplementation of NAC to protein malnourished mice effectively restored GSH concentrations and redox status and reduced NFB activation. These findings confirmed our hypothesis that the increased sensitivity to septic shock in protein malnourished hosts might be due to increased NFB activation. Although protein malnutrition was previously found to decrease NFB activation in LPS-treated macrophages (29
), how protein malnutrition would modify LPS-induced NFB activation in vivo was first shown in this study.
The strong associations found in this study between GSH levels/redox status and NFB activation further illustrated the role of GSH and redox status in NFB activation. The study also demonstrated that NAC supplementation to protein malnourished mice normalized the overproduction of IL-1ß and TNF-, two proinflammatory cytokines that play an important role in the induction of septic shock. Therefore, this study provided evidence that NAC supplementation could be used in the early rehabilitation of protein malnourished patients to decrease the acute overproduction of deleterious cytokines after endotoxin exposure. This could be especially meaningful because a protein-rich diet that increases GSH levels in animal studies is not recommended for the early rehabilitation of protein malnutrition, because it might impose metabolic stress due to the body’s adaptation to the catabolic protein malnutrition state (30
,31
). In addition, NAC could be beneficial for those protein malnourished patients who have increased exposure to oxidative stress due to the necessary drug and oxygen therapies.
Another interesting finding of this study was the selective activation of p50/p50 homodimers in mice fed the PM diet after LPS injection. Traditionally, translocation of the NFB p65/p50 heterodimer was thought to be stimulus-inducible, whereas p50/p50 homodimer translocation was categorized as constitutive (32
). However, more recent research has demonstrated selective activation of p50/p50 homodimers (33
,34
), which was associated with binding affinity of the inhibitory unit IB- to NFB dimers (35
,36
). In particular, IB- was found to have a much higher affinity to p65/p50 heterodimers than to p50/p50 homodimers (35
,36
). As a result, whereas a stimulus-induced partial reduction of IB- allowed p50/p50 homodimers to be translocated from the cytoplasm to the nucleus, the remaining IB- would seem to be enough to keep p65/p50 heterodimers in the cytoplasm. In our study, LPS-induced IB- mRNA synthesis was not affected by protein malnutrition. Therefore, the up-regulation of p50/p50 homodimers in protein malnutrition could be due to decreased IB- protein synthesis and/or increased IB- protein degradation. It is also noteworthy that in this study p65 subunit translocation was decreased in protein malnourished mice compared with controls, suggesting that translocation of p65/p50 heterodimers in mice fed the PM diet were decreased, because this heterodimer is the most abundant of the Rel/NFB dimers (37
). However, it might be possible that the p65/p50 heterodimers were normally translocated whereas the p65 homodimers were decreased.
The p50/p50 homodimers were believed to have no transactivation domains (34
,38
). However, in our study up-regulation of p50/p50 homodimers in mice fed the PM diet was associated with increased IL-1ß and TNF- synthesis, whereas inhibition of this homodimer translocation by NAC decreased mRNA synthesis of both cytokines. This is supported by previous findings that p50/p50 homodimers could be transcriptionally active in a target gene-specific manner (39
). Indeed, p50/p50 homodimers might have differential regulatory effects on downstream genes because the selective up-regulation of p50/p50 homodimers in protein malnourished mice increased mRNA production of IL-1ß and TNF-, but not IB-.
Taken together, these findings provided evidence supporting a role for GSH in NFB activation and proinflammatory cytokine production, and the use of NAC in early rehabilitation of protein malnutrition without a high protein diet.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Supported by National Institutes of Health Grant RO1 NS 38315 (to T.M.B.).
4 Abbreviations used: EMSA, electrophoretic mobility shift assay; GSH, glutathione; GSSG, GSH disulfide; HPLC, high-performance liquid chromatography; NAC, N-acetylcysteine; NFB, nuclear factor B; LPS, lipopolysaccharide; IL-1ß, interleukin 1ß; TNF-, tumor necrosis factor .
Manuscript received 23 June 2002. Initial review completed 15 July 2002. Revision accepted 8 August 2002.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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