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The Journal of Nutrition Vol. 128 No. 2 February 1998,
pp. 166-174
Institut National de la Santé et de la Recherche Médicale et Institut Féderatif de Recherches Multidisciplinaires sur les Peptides, 76233 Bois-Guillaume Cedex, France and * Groupe de Biochimie et de Physiopathologie Digestive et Nutritionnelle, Faculté de Médecine, 76800 Saint Etienne Rouvray Cedex, France
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ABSTRACT |
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Abstract
Introduction
Methods
Results
Discussion
References
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The acute-phase protein (APP) response is regulated by cytokines such as interleukin-6 (IL-6), interleukin-1 (IL-1) and tumor necrosis factor (TNF), but may also be influenced by malnutrition. The aims of this study were as follows: 1) to determine in rats the effect of a protein-deficient diet on IL-6 mRNA expression in intestine, liver and peripheral blood mononuclear cells (PBMC), and on 
-1 acid glycoprotein (AGP) and -2 macroglobulin (A2M) serum levels and hepatic mRNA expression; 2) to compare, in protein-deficient rats, the IL-6 and APP responses after a turpentine (TO)- or a lipopolysaccharide (LPS)-induced inflammation; and 3) to determine the effect of a protein malnutrition on IL-6 mRNA expression in rat PBMC treated ex vivo with LPS. Interleukin-6 mRNA was present in intestine and PBMC but not in the liver of malnourished rats, and was absent in any tissue or cells of controls. A2M was present in the serum from malnourished rats but not after refeeding. AGP mRNA expression was not influenced by protein malnutrition. In malnourished rats, IL-6 serum level peaked later than in controls after TO and LPS treatment. In malnourished TO-treated rats, A2M mRNA increased earlier than in controls and remained detectable later than in controls. AGP mRNA expression after TO was not influenced by protein malnutrition. In PBMC of malnourished rats, LPS-induced IL-6 mRNA expression occurred earlier and lasted longer than in controls. Our results indicate that protein malnutrition by itself induces IL-6 and A2M expression, and that it modulates the APP response to inflammation.
In response to tissue injury or acute inflammation, many plasma proteins synthesized by the liver exhibit quantitative changes that become apparent at a variable time after onset of the acute-phase response. Proteins with a transient increase in synthesis and plasma concentration are called positive acute-phase proteins (APP),4 whereas proteins whose synthesis decreases are referred to as negative APP (Koj 1985 Nutritional status may affect the acute-phase protein response to TO-induced inflammation because IL-6 and A2M responses are delayed and reduced in protein-deficient rats (Jennings and Elia 1994 To achieve better documentation of the effect of protein malnutrition on the syntheses of APP and cytokines, the aims of this study were as follows: 1) to determine in rats the effect of a protein-deficient diet on IL-6 mRNA expression in intestine, peripheral blood mononuclear cells (PBMC) and liver, and on AGP and A2M serum levels as well as on hepatic A2M mRNA expression; 2) to compare, in protein-deficient rats, the AP response to a systemic induction of inflammation with LPS to that induced by a localized abscess obtained with TO treatment in terms of IL-6, AGP and A2M serum levels; and 3) to study, ex vivo, the effects of LPS on IL-6 synthesis by PBMC obtained from rats fed a protein-deficient diet.
Chemicals.
Lipopolysaccharide (LPS) (from Escherichia coli, serotype 055:B5) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma Chemical (Saint Louis, MO). TO was purchased from Cooperative Pharmaceutics (Paris, France). Recombinant human IL-6 (rhIL-6) was purchased from Boehringer-Mannheim (Meylan, France).
Experimental protocols.
Six-week-old male Sprague-Dawley rats (~300 g) (Charles River, Cleón, France) were housed with a 12-h light:dark cycle and received a 23% protein diet (23 g casein/100 g synthetic diet; UAR, Epinay sur Orge, France) and water. After an acclimation period of at least 7 d, rats were housed in individual metabolic cages and given free access to the above control diet or an isocaloric protein-poor (0% casein) diet over 14 d. The composition of the diets is summarized in Table 1. Weight, oral intakes and urinary losses were monitored daily. After 14 d of either diet treatment, an acute-phase reaction was induced by LPS or TO injection. LPS was dissolved in sterile 9 g/L NaCl and injected intravenously via the saphenous vein (600 µg/kg body weight) (Geisterfer et al. 1993
Assays
IL-6 activity.
Serum IL-6 activity was assayed by measuring the IL-6-dependent proliferation of the B9 murine plasmacytoma cell line, as described (Aarden et al. 1987 Immunoelectrophoresis.
The amounts of serum A2M and AGP were determined by rocket immunoelectrophoresis as described (Laurell 1966 Nitrogen balance.
After 14 d of either diet treatment, urine was collected before LPS or TO treatment at d 0, and at 1, 3 and 7 d after TO or LPS injection. Urine was subsequently stored at Isolation and handling of PBMC.
Blood was collected from the inferior vena cava in the presence of anticoagulant and incubated ex-vivo with LPS (10 mg/L) for 45 min at 37°C at 800 × g for 15 min; then, PBMC were isolated by ficoll density gradient centrifugation and were incubated for an additional 1, 3, 6, 9 or 24 h. Finally, cells were harversted and frozen at mRNA preparation, Northern-blot analysis and RT-PCR
mRNA extraction.
Rat tissues or cells were homogenized at room temperature in 4 mol/L guanidinium thiocyanate, 0.1 mol/L 2-mercaptoethanol and 20 g/L N-laurylsarcosine. Total RNA were obtained by centrifugation at 120,000 × g for 16 h at 20°C through a 5.7 mol/L CsCl layer (Chirgwin et al. 1979 Northern-blot analysis.
Total liver RNA (20 µg), was pooled from five rats as described (Verma et al. 1992 Reverse transcription.
Reverse transcription (RT) was conducted with the use of 1 µg of total RNA in a total volume of 20 µL reaction buffer (Tris-HCl, KCl, 2.5 mmol/L MgCl2) containing 8 pmol/L hexanucleotide random primer pd(N6) (Pharmacia, Orsay, France), 1 µg prokaryotic chloramphenicol acetyl transferase (CAT) RNA as an exogenous internal standard (Jean et al. 1996 Polymerase chain reaction.
The sets of primers (Bioprobe, Montreuil-ss-Bois, France) used are summarized in Table 2. Each oligonucleotide was localized on different exons of the gene under study. The guanine + cytosine content of each set of primers was selected to achieve a high melting temperature (Tm). Polymerase chain reaction (PCR) was conducted in a final volume of 50 µL with 10 µL of the RT mixture, 2.5 units of Taq polymerase (Promega), 200 µmol/L of each dNTP, 0.2-0.4 mCi (7.4-14.8 Mbq) [
Quantitative analysis of amplified products.
Six microliters of each RT-PCR sample was resolved on a 7.5% polyacrylamide gel. The gels were exposed to Kodak film. The levels of amplified product were measured by quantitative scanning densitometry of autoradiograms. They were normalized to constant amounts of glyceraldehyde-3 phosphate dehydrogenase (GAPDH) or actin mRNA as an endogenous standard and/or prokaryote CAT RNA as an exogenous standard as described (Dukas et al. 1993
Statistical analysis.
Results are presented as means and SD. For serum and liver assays, values were obtained from five rats at each time point in each group. Differences between diet groups were tested by using ANOVA followed by the Fisher's Protected Least Significant Difference test when appropriate. In each group, changes over time were evaluated using ANOVA for repeated measures. P < 0.05 was considered the critical limit.
Variations of food intake, body weight, nitrogen balance and serum albumin level.
After 14 d of protein malnutrition alone, the body weight at d 0 was significantly lower in protein-malnourished rats than in controls (P < 0.001) (Fig. 2A). After TO or LPS injection at d 0, the body weight of malnourished rats decreased further compared with d 0. Injection of TO and LPS had similar effects on food intake and body weight. After induction of inflammation with TO or LPS in control rats, weight did not fluctuate again until d 7 (Fig. 2A) and remained significantly higher than in malnourished rats (P < 0.001). These weight changes were accounted for by a marked decrease in food intake during the 2 d after inflammation in both malnourished and control rats compared with the average daily food intake during the 14 d before inflammation: 13.6 ± 6.3 vs. 23.2 ± 4.6 g/d and 16.7 ± 2.8 vs. 25.1 ± 2.9 g/d (P < 0.01) in the malnourished and control rats, respectively. After d 4 of inflammation, food intake returned to the pre-inflammation level: 26.8 ± 4.6 and 28 ± 3.1 g/d, respectively.
Effects of a protein-deficient diet alone or after a localized abscess on serum levels and hepatic mRNA amounts of A2M and AGP.
After 14 d of protein-deficient diet, rats fed that diet had A2M present in serum, whereas it was not present in controls (see d 0, Fig. 3A); in contrast, the serum level of AGP was not affected by protein deprivation (Fig. 3B, d 0). After TO injection, the A2M serum level showed a maximum at 48 h in control rats; in contrast, in malnourished rats, A2M levels were not significantly different than controls from d 1 through 3, but the A2M level was higher than in controls at d 7 (168 h) (P < 0.05, Fig. 3A). In both groups, the serum AGP level was maximal at d 2, with no significant difference between groups. However, a greater AGP level was still observed at d 7 in the rats fed the protein-deficient diet vs. controls (P < 0.05, Fig. 3B).
Effects of a protein-deficient diet alone or after an LPS-induced systemic inflammation on serum levels of A2M and AGP.
As indicated in Figure 5, the maximum 48-h increase of serum A2M and AGP levels in well- and malnourished rats was less marked after LPS- than after TO-induced inflammation (P < 0.001). After LPS injection, the increase in serum level for A2M did not differ between groups until after d 1. Thereafter, the increase of A2M was significantly greater in malnourished rats from 48 to 168 h (Fig. 5A). In contrast, there was no difference in serum AGP between groups at any time (Fig. 5B).
Effects of refeeding after a period of protein-deficient diet on A2M serum levels.
After refeeding, previously malnourished rats returned to their basal body weight between day +4 and +7 and continued to gain weight until the end of the experiment (Fig. 6). In contrast, the serum A2M level was highest at d 0 (after the period of protein-deficient diet intake) and lowest at d +14 after reintroduction of the normal protein diet.
Effects of a protein-deficient diet alone on IL-6 mRNA expression in liver, intestine and PBMC.
In the absence of inflammation, IL-6 mRNA was absent in any organ or cell of well-nourished rats as analyzed by RT-PCR (Fig. 7, 23% lanes). In contrast, in malnourished rats (0% lanes), IL-6 mRNA was present in intestine and PBMC, but absent in the liver and in mucosal scrapings of intestine.
Effects of a protein-deficient diet on IL-6 serum levels after LPS- vs. TO-induced inflammation.
In the absence of inflammation, no serum IL-6 activity could be measured in either controls or malnourished rats (data not shown). The maximal responses of serum IL-6 activity at chosen times were not significantly different between the well- and malnourished rats, during both LPS- and TO-induced inflammation (Fig. 8). However, there was a delay in the kinetics of IL-6 synthesis in the malnourished compared with well-nourished rats. Indeed, in malnourished rats, serum IL-6 level peaked at 9 and 24 h in LPS- and TO-treated rats, respectively, in contrast with 6 and 9 h, respectively, in controls (P < 0.001, Fig. 8).
Effects of ex vivo LPS stimulation on IL-6 synthesis by PBMC.
PBMC from protein-deficient and control rats were stimulated ex vivo by LPS for the periods of time indicated in Figure 9. Interleukin-6 mRNA was maximal at 3 h and none was present at 24 h in cells from well-nourished rats, whereas it was maximal at 6 h and still present at 24 h in cells from malnourished rats.
Severe protein malnutrition influences the ability of the body to face an infectious or inflammatory challenge. Some experimental and clinical studies have suggested that malnutrition may affect cytokine and acute-phase response (Carlson et al. 1994 The authors thank G. H. Fey for the gift of cDNA for A2M, M. Dehoux for the gift of B9 cells, D. Biou for the gift of anti-AGP antiserum, F. Gauthier for the gift of anti-A2M antiserum, R. Vranckx for the gift of cDNA for 18S and V. Coquin for her skillful assistance.
INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References
, Lebreton et al. 1979). In all mammalian species, the regulation of expression of the APP genes is mediated mainly by inflammatory cytokines that include mainly interleukin-6 (IL-6), interleukin-1 (IL-1) and tumor necrosis factor (TNF). The effects of cytokines on the pattern of liver gene expressions during the inflammatory process enable us to distinguish the following two APP classes: class 1 in humans includes haptoglobin, C-reactive protein (CRP), serum amyloid A (SAA), -1 acid glycoprotein (AGP) and hemopexin. It is regulated mainly by IL-1 or combinations of IL-1 + IL-6 and glucocorticoids. Class 2 includes fibrinogen, -1 antichymotrypsin and -1 antitrypsin and is regulated by IL-6 and glucocorticoids exclusively (Koj 1985, Kushner and Mackiewicz 1993). Similarly, in rats, the gene for AGP (class 1) is induced by IL-6, IL-1 and glucocorticoids, whereas the gene for -2 macroglobulin (A2M) (class 2) responds to IL-6 or leukemia inhibitory factor and glucocorticoids (Gabay et al. 1996, Hocke et al. 1993, Koj 1985). Two types of injury are commonly used for eliciting an acute inflammatory reaction in animals. The first is a localized inflammation induced by the formation of a sterile abscess after subcutaneous injection of turpentine oil (TO). The second is a systemic inflammation produced by intravenous injection of lipopolysaccharide (LPS) (Geisterfer et al. 1993, Scotté et al. 1996).
). In contrast, protein-depleted rats exhibited higher basal levels of A2M before the challenge with TO, whereas serum IL6 levels were not affected (Jennings et al. 1992a and 1992b). Recently, experiments in mice with disruption of the IL-6 gene showed that the induction of acute-phase proteins is dramatically reduced after TO-induced localized inflammation, whereas the protein response is only slightly modified after LPS-induced systemic inflammation (Fattori et al. 1994). These results indicate that IL-6 is an important mediator of the APP response after tissue damage but not after systemic inflammation. Thus the acute-phase response may differ according to the inducing agent employed. However, the influence of malnutrition on the APP response to LPS in rats has not been investigated. This question is of clinical importance, because malnutrition is a frequent complication during inflammatory diseases such as enterocolitis, e.g., Crohn's disease. Indeed, it has been suggested that malnutrition and nutritional support may affect AGP levels in Crohn's disease patients (Carlson et al. 1994), as well as CRP levels in malnourished children (Doherty et al. 1993). However, the influence of malnutrition on APP response in humans remains largely unknown.
MATERIALS AND METHODS
Abstract
Introduction
Methods
Results
Discussion
References
). TO was injected at a single subcutaneous site in the back (5 mL/kg) (Geisterfer et al. 1993). In an additionnal set of rats, the 14-d period of protein-restricted diet was followed by a 14-d refeeding period with the normal 23% casein diet. Rats were killed while under ether anesthesia. Blood was collected via the vena cava and centrifuged at 1000 × g for 15 min; the serum was stored at 
20°C. The liver was rinsed with sterile 9 g/L NaCl, removed and quickly frozen in liquid nitrogen. The intestines were removed, thoroughly rinsed with sterile 9 g/L NaCl and either scraped with a glass blade or left unscraped before freezing in liquid nitrogen. All experiments were performed according to the guiding principles in the care and use of animals of the French Ministry of Agriculture.
View this table:
Table 1.
Composition of control and protein-deficient diets1
, Helle et al. 1988, Mosmann 1983). Briefly, IL-6 activities were determined spectrophotometrically by assessing the 72-h proliferation of cell line B9 by using serial dilution of plasma and MTT. The cells were incubated at 35°C for an additional 4-h period. The supernatants were removed and 100 µL isopropanol-HCl was added to cells that were incubated in the dark at room temperature overnight. The absorbance was read at 550 nm. A standard curve with 2 × 105 units/L of rhIL-6 was prepared by plotting absorbancy values vs. log concentration of the standard. Values were determined by comparison with the standard curve. A unit of activity is defined as the dilution that gives half-maximal B9 cell growth in 72 h and corresponds to ~2 ng/L rhIL-6.
). Albumin and AGP were expressed in grams per liter with purified rat albumin and AGP as a standard. In the absence of standard, A2M was expressed as a percentage of the maximal response obtained at 48 h post-TO injection. The level of detection was 0.1% for serum A2M and 2.5 mg/L for serum AGP and serum albumin.
20°C before nitrogen assay with the use of the Antek pyrochemiluminescence technique (Antek Instruments, Houston, TX) as described (Ward et al. 1980). An approximation of the nitrogen balance was made by calculating the difference between input and urinary excretion.
20°C for mRNA extraction.
). The RNA pellets were dissolved in 5 g/L SDS and 0.01 mol/L Tris-HCl (pH 7.5) and precipitated twice with ethanol. The RNA pellets were diluted in 100 µL of diethylpyrocarbonate-treated water. The purity and yield of total RNA were determined spectrophotometrically. The 260/280 ratio considered acceptable for the purity of RNA preparation was 1.8-2.0. RNA integrity was checked by agarose gel electrophoresis.
), electrophoresed in 10 g/L agarose gel containing formaldehyde and transferred to nylon membrane Hybond-N+ (Amersham, Les Ulis, France) by capillary action. Each cDNA probe was labeled with 0.2-0.4 mCi (7.4-14.8 MBq) of [-32P]dCTP (3000 Ci/mmol) (1.1 × 105 GBq/mmol) (Isotopchim, Ganagobie-Peyruis, France) by using a standard random-primed reaction. The membranes were hybridized with a probe at 2 × 109 cpm/L in QuickHyb solution (Stratagene, La Jolla, CA) at 42°C for 6 h and washed twice in 2X SSC/1g/L SDS at room temperature for 15 min and once in 0.1X SSC/0.1% SDS at 60°C for 45 min. The membranes were then exposed to an X-Omat AR film (Eastman Kodak, Rochester, NY) with intensifying screens at 80°C. The membranes were subsequently stripped and rehybridized with a [32-P]-labeled fragment of the mouse 18S cDNA. Ribosomal 18S RNA was used as standard. The autoradiograms were analyzed by quantitative scanning densitometry and the amounts of mRNA under study were normalized with the ribosomal 18S RNA signal as standard.
), 10 mmol/L of each dNTP (Pharmacia), 100 units RNasin (Promega, Charbonnières, France), 50 units murine Moloney leukemia virus (MMLV) reverse transcriptase (Promega). First-strand cDNAs were obtained after 1 h at 37°C.
-33P]dATP (Isotopchim), 100 pmol/L of each 5
and 3 specific primer sequence, in a buffer consisting of 50 mmol/L KCl, 10 mmol/L Tris-HCl, pH 8.3, and 2.5 mmol/L MgCl2. The samples were first denatured at 94°C for 3 min; amplification was performed with denaturation at 94°C for 45 s, annealing at the appropriate Tm for 45 s and extension at 72°C for 60 s, for 20-30 cycles as appropriate. The final cycle was followed by a 10-min extension step at 72°C. The absence of contaminants was routinely checked by RT-PCR of negative control samples, in which the RNA samples were replaced with sterile water or MMLV RT was omitted.
View this table:
Table 2.
Oligonucleotide primers
, Jean et al. 1996). The number of PCR cycles in each system was chosen within the linear phase to use this assay as a relative measure of gene expression (Fig. 1).
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Fig 1.
Determination of the amplification cycle numbers. For evaluating -2 macroglobulin (A2M) mRNA levels, glyceraldehyde-3 phosphate dehydrogenase (GAPDH) and chloramphenicol acetyl transferase (CAT) (data not shown) standards were used, and the number of polymerase chain reaction (PCR) cycles was chosen within the linear phase. The levels of amplified product were measured by quantitative scanning densitometry of autoradiograms.
RESULTS
Abstract
Introduction
Methods
Results
Discussion
References
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Fig 2.
Effects of a protein-deficient diet on the body weight (panel A), nitrogen balance (panel B) and serum albumin concentration (panel C) of rats after induction of inflammation. Rats received either a 23% or a 0% casein diet 14 d before receiving an injection of turpentine oil (TO) (5 mL/kg) or lipopolysaccharide (LPS) (600 µg/kg) at d 0. Data are means ± SD, n = 5. Differences between diet groups at a time: *P < 0.05, **P < 0.01, ***P < 0.001. Changes over time: panel A: a, P < 0.05 vs. d 14; panel B: a, P < 0.05 vs. d 0 (23%); panel C: a, P < 0.05 vs. d 0 (23%); b, P < 0.05 vs. d 0 (0%).
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Fig 3.
Effects of a protein-deficient diet on -2 macroglobulin (A2M) (panel A) and -1 acid glycoprotein (AGP) (panel B) serum levels after a turpentine oil (TO)-induced inflammation in rats. Rats received either (23%) or (0%) casein diet for 14 d before injection of TO (5 mL/kg) at time zero. In the absence of standard, A2M was expressed as a percentage of the maximal response obtained at 48 h post-TO injection. The level of detection for serum A2M was 0.1% and for serum AGP was 2.5 mg/L. Data are means ± SD, n = 5. Differences between diet groups: *P < 0.05. Changes over time in each diet group: a, P < 0.05 vs. h 0; b, P < 0.05 vs. h 9; c, P < 0.05 vs. h 24; d, P < 0.05 vs. h 48; e, P < 0.05 vs. h 72.
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Fig 4.
Effects of a protein-deficient diet on -2 macroglobulin (A2M) and -1 acid glycoprotein (AGP) mRNA levels following a turpentine oil (TO)-induced inflammation in rats. Rats received either a 23% or a 0% casein diet for 14 d before injection of TO (5 mL/kg) at time zero. The autoradiograms were analyzed by quantitative scanning densitometry, and the amounts of mRNA under study were normalized with the ribosomal 18S rRNA signal.
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Fig 5.
Effects of a protein-deficient diet on -2 macroglobulin (A2M) (panel A) and -1 acid glycoprotein (AGP) (panel B) serum concentrations after a lipopolysaccharide (LPS)-induced systemic inflammation compared with turpentine oil (TO)-induced localized inflammation in rats. Rats received either a 23% or a 0% casein diet for 14 d before injection of LPS (600 µg/kg) at time zero. In the absence of standard, A2M was expressed as a percentage of the maximal response obtained at 48 h post-TO injection. The level of detection for serum A2M was 0.1% and for serum AGP was 2.5 mg/L. Data are given as means ± SD, n = 5. Differences between diet groups: *P < 0.05, **P < 0.001. Changes over time in each diet group: a, P< 0.05 vs. h 0; b, P < 0.05 vs. h 9; c, P < 0.05 vs. h 24; d, P < 0.05 vs. h 48; e, P < 0.05 vs. h 72.
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Fig 6.
Effects of refeeding rats a normal diet (23% casein diet) after a period of protein-deficient diet (0% casein diet) on body weight and -2 macroblobulin (A2M) serum concentration. Rats were given a protein-deficient diet for 14 d. At d 0, rats received a normal protein diet. In the absence of standard, A2M was expressed as a percentage of the maximal response obtained at 48 h post-turpentine oil (TO) injection. The level of detection for serum A2M was 0.1%.
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Fig 7.
Effects of a protein-deficient diet alone on interleukin-6 (IL-6) mRNA expression in liver (L), peripheral blood mononuclear cells (M), intestine (I) and scraped intestine (sI). The levels of amplified product were measured by quantitative scanning densitometry of autoradiograms. Control (C) is IL-6 mRNA obtained from PBMC treated for 1 h with LPS (see Materials and Methods). Data represent three separate experiments performed with tissues or cells pooled from 5 rats.
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Fig 8.
Effects of a protein-deficient diet on serum interleukin-6 (IL-6) activities after turpentine oil (TO)-induced compared with lipopolysaccharide (LPS)-induced inflammation in rats. To evaluate serum IL-6 levels, total blood, obtained from rats receiving either a 23% casein diet or a 0% casein diet for 14 d before injection of LPS (600 µg/kg) and TO (5 mL/kg) at d 0, was harvested and serum IL-6 activities were analyzed at the indicated times. Data are means ± SD, n = 5. Differences between diet groups: *P < 0.05. Changes over time: a, P < 0.05 vs. h 6; b, P < 0.05 vs. h 9; c, P < 0.05 vs. h 24.
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Fig 9.
Effects of a protein-deficient diet on the expression of mRNA for interleukin-6 (IL-6) in rat peripheral blood mononuclear cells (PBMC) after ex vivo lipopolysaccharide (LPS) stimulation. PBMC were obtained from total blood collected in the inferior vena cava, incubated ex-vivo with LPS (10 mg/L) for 45 min at 37°C, isolated by ficoll density gradient centrifugation, and incubated for the indicated times. The levels of IL-6 amplified product were measured by quantitative scanning densitometry of autoradiograms. Data presented are representative of three separate experiments performed with pooled PBMC from 5 rats.
DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References
, Doherty et al. 1993, Jennings et al. 1992b). Liver A2M synthesis is reduced in rats fed a protein-deficient diet during a localized inflammation (Jennings et al. 1992b). Because IL-6 is the predominant proinflammatory cytokine in the control of hepatic APP production (Gabay et al. 1996, Koj 1985, Kushner and Mackiewicz 1993), we investigated whether synthesis of A2M and IL-6 could be altered after a severe protein deficiency. This study has shown that A2M mRNA was expressed in the liver and that its protein was present in the serum of malnourished rats. In addition, we have shown that IL-6 mRNA was present in PBMC and intestine of these rats, but not in control rats. However, no serum IL-6 activity was found in the systemic circulation of either group. Small amounts of IL-6 may be necessary to induce a maximal acute-phase response (Hocke et al. 1993, Koj 1985). Thus, it is likely that minute amounts of IL-6 may have been produced both from the circulating PBMC and/or in the splanchnic region of animals given a protein-deficient diet. Gut-derived IL-6 may have reached the liver via the portal vein and consequently contributed to the increase in A2M synthesis in the liver that was observed after a severe protein-deficient diet. The source of IL-6 production in intestine could be epithelial cells (Isaacs et al. 1992) or equally, the mononuclear cells of the lamina propria (Stevens et al. 1992). In this study, the fact that IL-6 expression was observed in the whole intestine but not in the mucosal scraping suggests that the site of IL-6 production was mainly the mononuclear cells of the lamina propria. However, some contribution of epithelial cells and of mononuclear cells of mesenteric lymph nodes cannot be completely ruled out.
), weak modifications in AGP synthesis may be more difficult to detect.
). Surprisingly, studies have shown that the maximal response of AGP, among other APP, was overexpressed after TO- compared with LPS-induced inflammation, despite similar IL-6 activity levels in LPS- and TO-treated rats (Geisterfer et al. 1993). Similarly, our results (Fig. 5) demonstrate that A2M belongs to this group of overexpressed proteins and that its overexpression was not abolished by protein depletion. To date, this differential expression of APP remains unexplained.
). Infiltrating leucocytes have also been identified as sources of IL-6 in inflammatory diseases (Stevens et al. 1992). In other studies, IL-6 was detected by immunofluorescence in enterocytes of patients with inflammatory bowel disease as well as in rats after hemorrhagic shock (Jones et al. 1993, Shirota et al. 1990, Tamion et al. 1997). In our study, IL-6 mRNA was present in the whole intestine, but not in the mucosal scraping; it suggests that the main site for IL-6 synthesis is located in the lamina propia and is probably accounted for by mononuclear cells, in accordance with our findings in PBMC. As a consequence of prolonged protein malnutrition, disruption of the gut mucosal barrier may have allowed small amounts of endotoxin or intestinal bacteria to reach the subepithelial region and elicit IL-6 synthesis in submucosal mononuclear cells. However, the mechanisms and significance of bacterial translocation remain controversial (Schlichting et al. 1995).
, Keenan et al. 1982). Indeed, IL-1, obtained from blood monocytes of patients suffering from a wasting malnutrition, induced an attenuated fever in adult rabbits (Hoffman-Goetz et al. 1981). In severely malnourished children, it has been reported that the response of both CRP and SAA to diphteria-pertussis-tetanus vaccine was impaired; accordingly, after LPS stimulation, the in vitro production of proinflammatory cytokines such as TNF- and IL-6 in whole blood from these children was reduced (Doherty et al. 1994) . Finally, it has been reported that the responses of serum IL-1 and granulocyte-macrophage-stimulating factor to acute-phase infection of kwashiorkor patients were defective, and that nutritional-repletion therapies restored the production of these cytokines (Aslan et al. 1996).
ACKNOWLEDGMENTS
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FOOTNOTES |
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-2 macroglobulin; AGP, -1 acid glycoprotein; APP, acute-phase protein; CAT, chloramphenicol acetyl transferase; CRP, C-reactive protein; GAPDH, glyceraldehyde-3 phosphate dehydrogenase; IL, interleukin; LPS, lipopolysaccharide; MMLV murine Moloney leukemia virus; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PBMC, peripheral blood mononuclear cells; PCR, polymerase chain reaction; RT, reverse transcription; SAA, serum amyloid A; TNF, tumor necrosis factor; TO, turpentine oil.
Manuscript received 31 October 1996. Initial reviews completed 22 January 1997. Revision accepted 29 September 1997.
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LITERATURE CITED |
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Abstract
Introduction
Methods
Results
Discussion
References
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