Molecular and Cellular Biochemistry 278: 53–57, 2005.
cgSpringer
2005
Intestinal and cardiac inflammatory response shows enhanced endotoxin receptor (CD14) expression in magnesium deficiency Joanna J. Chmielinska,1 M. Isabel Tejero-Taldo,1 I. Tong Mak1 and William B.Weglicki1,2 1
Division of Experimental Medicine, Department of Biochemistry and Molecular Biology; 2 Department of Medicine, The George Washington University Medical Center, Washington, DC, USA Received 24 November 2004; accepted 23 February 2005
Abstract Substance P is elevated in plasma and in other tissues during Mg-deficiency, and was found localised to neuronal C-fibres of cardiac and intestinal tissues, where it could promote neurogenic inflammation. Plasma prostaglandin E2 (PGE2 ), indicative of systemic inflammation, rose significantly (≥4 fold, p < 0.01) after 1 week and remained elevated through week 2 and 3 in rat on the Mg-deficient (MgD) diet. Concomitantly, total blood glutathione decreased by 50%. Immunohistochemical staining for endotoxin (lipopolysaccaride, LPS) receptor, CD14 was prominent in macrophage-type cells in intestinal tissue; more importantly, cardiac tissue revealed both CD11b (monocyte/macrophage surface protein) and CD14 positive cells after 3 weeks in rats on MgD diet. Western blot analysis indicated a significant increase in the endotoxin receptor protein level in the 3 week MgD hearts. Since CD14 is known to be up-regulated in cells exposed to LPS, these observations suggest that prolonged Mg-deficiency results in increased intestinal permeability to bacterial products that induce the endotoxin receptor in cells localized to myocardial and intestinal tissues. These CD14 positive cells may amplify the cardiomyopathic inflammatory process by stimulating TNF-α and other pro-inflammatory cytokines. (Mol Cell Biochem 278: 53–57, 2005) Key words: cardiomyopathic inflammation, endotoxin receptor CD14, lipopolysaccaride (LPS), magnesium deficiency, oxidative stress
Introduction In Mg-deficiency many vital cellular processes are altered. Severe dietary Mg-deficiency leads to diverse pathology in animal models: myocardial necrosis, neuromuscular hyperexcitability, arrhythmia, hyper-irritability, hemorrhagic skin lesions, enhanced atherosclerosis, increased indices of oxidative injury including decreased RBC glutathione [1] and enhanced myocardial susceptibility to ischemia/reperfusion stress [2]. Our studies of the pathologic mechanisms of Mg-
deficiency in rodents, led to the discovery that circulating levels of the pro-inflammatory neuropeptide substance P (SP), were significantly elevated within 3 days of initiating a severe MgD diet [3]. Because the earliest significant rise in plasma SP (day 3) preceded increases in circulating white blood cells and other circulating inflammatory mediators [3], we proposed that this neuropeptide may be inducing many of the later inflammatory/oxidative events which eventually promote cardiomyopathy. Substance P may trigger inflammatory processes and oxidative stress in endothelial cells, mast
Address for offprints: J. Chmielinska, Division of Experimental Medicine, Department of Biochemistry and Molecular Biology, The George Washington University Medical Center, 2300 Eye Street N.W., Washington, DC 20037, USA (E-mail:
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54 cells, macrophages and circulating white blood cells. In vivo treatment with SP receptor blockers also attenuated endogenous activation of neutrophils in MgD rats characterized by enhanced ex vivo superoxide generation in fresh isolates [4]. Of relevant interest, intestinal tissue is a source of SP-rich C-fibres [5]. During Mg-deficiency, the gut may contribute to the early elevation of circulating SP. In a recent study [6], it has been shown that during the acute phase of Mg-deficiency, mucosal inflammation in the intestine occurred, as indicated by neutrophil infiltration. Enhanced free radical generation from the infiltrated neutrophils may contribute to functional abnormalities of inflamed intestine leading to a loss of mucosal barrier function and increased intestinal permeability. As a consequence, endotoxin may be released into the blood stream of the portal circulation, where it would encounter Kupffer cells in the liver and their activation could promote systemic secretion of TNF-α, IL-1, and IL-6 by hepatic Kupffer cells and macrophages present in other tissues [7–9]. LPS can also stimulate secretion of TNF-α directly from adult rat cardiomyocytes through activation of the CD14 receptor [10]. However, hitherto evidence to support such a scenario in MgD is lacking. In this communication, we investigated the effect of severe Mg-deficiency on CD14 (LPS receptor protein) ex- pression in the intestinal and cardiac tissues. The data support the notion that increased endotoxin release from the intestine occurs and may participate in the cardiac inflammatory re- sponses during Mg-deficiency.
Materials and methods Animal model Male Spraque–Dawley rats (approx. 7 weeks old, weight 150–155 g) were housed under 12-h light/dark cycle, with free access to double-distilled water and food. Animals were randomly separated into two dietary groups: one received rat chow containing 100% of the RDA recommended Mg, and the second group was provided with severely deficient diet containing 9% Mg. After maintaining the animals on the diets for 1, 2, or 3 weeks they were sacrificed; 5 ml of blood was collected from inferior vena cava, the hearts, parts of small intestine, and livers were rapidly excised and rinsed in saline. Each specimen was split. Parts of tissue were snapfrozen in liquid nitrogen and the other parts quickly embedded in OCT Compound (Tissue-Tek, Sakura, USA), frozen in methyl-butane cooled in dry-ice and stored at −70 ◦ C until used. Immunochemistry Tissue sections were studied by indirect, immunofluorescence technique to localize the target proteins in the tissues.
Incubation with primary antibodies rabbit anti-rat CD14 (1:500, Santa Cruz Biotechnology Inc., Santa Cruz, CA) and mouse anti-rat ICAM-1 (1:50, Serotec Ltd, Oxford, UK) was conducted overnight at 4 ◦ C in humidity chamber. After washes, samples were exposed to the secondary antibodies (goat anti-rabbit IgG conjugated with FITC and goat antimouse IgG conjugated with Texas Red, both from Molecular Probes Inc., Eugene, OR). Samples were examined under a fluorescence microscope (Olympus BX60) and multiple microphotographs were taken with a digital camera (Evolution Colour MP; Media Cybernetics, Silver Spring, MD). CD11b antigen was visualized using mouse anti-rat CD11b (1:500, Chemicon International, Temecula, CA) and Vectastain Elite ABC kit immunoperox- idase system (Vector Laboratories Inc.; Burlingame, CA). Negative controls were included in all assays where the primary antibodies were omitted. Western blot analyses of ventricular tissue Snap-frozen heart tissue was homogenized at 4 ◦ C 1:5 (v/v) in RIPA buffer. Samples containing 50 µg of proteins were separated by SDS-PAGE electrophoresis, and transferred to a nitrocellulose membrane as previously described [Tejero-Taldo et al., 2002]. Membranes were probed overnight with rabbit anti-CD14 antibodies (1:200, Santa Cruz Biotechnology Inc., Santa Cruz, CA), followed by incubation with horseradish peroxidase conjugated donkey anti-rabbit IgG (1:20,000; Amersham Bio-sciences Inc., Piscataway, NJ). Membranes were developed using enhanced chemoluminescence (ECL plus, Amersham Biosciences Inc., Piscataway, NJ), and exposed to X-ray film. Band densitometry and analyses were performed using a Personal Densitometer SI (Molecular Dynamics) and computerized analysis system (Image Quant v 5.2, Molecular Dynamics, 1999). PGE2 and RBC glutathione Prostaglandin E2 (PGE2 ) levels were determined using ELISA KIT from R&D Systems (Minneapolis, MN). The assay is based on the competitive binding technique in which PGE2 present in the sample competes with a fixed amount of alkaline phosphatase-labeled PGE2 for sites on a mouse monoclonal antibody coating the wells on the microplate. Total RBC glutathione was determined by the enzymatic ‘recyclic’ method as described previously [1]. Statistics The data were reported as mean ± S.E.M. Statistical significance between treatment groups was evaluated using ANOVA and all pair wise comparisons were analysed by Bonferroni Multiple Comparison. Test using Graph PAD software. The
55 differences were considered statistically significant at the value of p < 0.05.
Results and discussion The primary objective of this study was to determine if CD14 might be up-regulated during Mg-deficiency in selected tissues. Immunohistochemical staining of the intestine sections for CD14 was prominent in MgD animals in comparison with Mg-sufficient (MgS) controls at 3 weeks on the diet (Fig. 1), suggesting exposure to LPS. The same intestinal sections were stained for ICAM-1 and showed increases at 3 weeks in MgD animals (Fig. 1) In prior studies myocardial ICAM1 expression displayed a progressive increase during Mgdeficiency [12].
The heart tissue sections (ventricles) showed positive staining for CD14 in MgD animals at 3 weeks on MgD diet in comparison with MgS rats; the MgD ventricles also stained positive for CD11b at 3 weeks (Fig. 2). Wetern blotting analyses of homogenized hearts showed significantly higher levels of CD14 protein (187.1 ± 29.49%; p < 0.01; n = 7) in MgD rats than in MgS (100%) controls at 3 weeks; at 1 week, the differences between dietary groups were not significant for CD14 (Fig. 2). The CD11b protein levels rose significantly at 3 weeks in MgD animals (186.7 ± 32.54%; p < 0.01; n = 4) in comparison with MgS (100%) controls and there was no significant difference in CD11b expression between dietary groups at week one (Fig. 2). Also CD11b levels increased significantly from week 1 at 140.2 ± 18.85% to week 3 at 187.7 ± 32.54%; p < 0.05 in comparison with MgS groups (Fig. 2).
Fig. 1. Small intestine tissue sections stained for CD14 (upper panels; green) and ICAM-1 (middle panels; red) in Mg-sufficient and Mg-deficient rats placed on their respective diets for 3 weeks. Lower panels exhibit partial co-localization of CD14 and ICAM-1 (yellow-orange). Magnification 20×.
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Fig. 2. Western blot analysis and immunohistochemical staining assessed CD14 and CD11b protein expression and tissue localization in the heart of rats at 1 and 3 weeks on MgD diet. Band density of the MgD group (n = 7) was compared to a time matched 100% MgS group (n = 4). Upper panel presents significantly higher expression of CD14 in heart of MgD animals over MgS after 3 weeks ( p < 0.01) in Western Blots. Increased protein expression was verified by positive immunofluorescence staining for CD14 (green). Magnification 20×. No significant differences were observed between diet groups after 1 week. Lower panel presents significantly higher Cd11b protein expression in the heart of the MgD group compared with the MgS group after 3 weeks ( p < 0.01) in Western Blots. In addition, CD11b protein expression was significantly elevated in the heart after 3 weeks on MgD diet compared with the 1 week deficient animals ( p < 0.05). Positive immunohistochemical staining (brown) with antibody against CD11b and secondary antibody conjugated with HRP was observed in the heart of the MgD group at 1 and in abundance at 3 weeks on Mg-deficient diet. Magnification 20×.
Western blot analysis of homogenized liver tissue revealed significant increase of CD14 at 1 week (222.4%±36.58; p < 0.01) in MgD rats in comparison with MgS (100%) animals, and by 3 weeks, there was no significant difference in CD14 expression between Mg-deficient and Mg-sufficient animals; this suggests an early response to endotoxin exposure through the portal circulation. Since Mg-deficiency may cause a systemic cascade of inflammatory events we measured plasma levels of PGE2 , which were significantly higher in rats on MgD diet in comparison with rats on MgS diet after only 1 week (861 ± 188 pg/ml vs. 253 ± 44 pg/ml; p < 0.05) and remained elevated at week 2 (1210 ± 162 pg/ml vs. 317 ± 106 pg/ml; p < 0.01) and at week 3 (930 ± 122 pg/ml vs. 33.6 ± 44 pg/ml; p < 0.01). As an index of oxidative stress, Mg-deficiency also resulted in a significant loss of RBC glutathione from 6.11 ± 0.3 umol/gm Hb at time 0 to 3.09 ± 0.22 ( p < 0.01) at the end of week 3. This study focused on histochemical and biochemical changes in cardiac and intestinal CD14 expression in an MgD animal model. It has been proposed that hypomagne-
saemia allows for calcium influx at the neuronal N-methyl-Daspartate (NMDA) glutamate receptor level by reducing its Mg-blockade and allowing activation of neuronal tissue. This is followed by the release of substance P and CGRP from sensory motor neurons, which triggers (through NK-1 specific receptors on a variety of cell types) a cascade of inflammatory and oxidative stress events leading to cardiovascular lesion formation [12]. Also myocardial functional recovery (aortic output, peak systolic pressure and developed pressure) after postischemic stress is substantially depressed in animals on MgD-diet in comparison with controls [11, 13]. It has been reported that during intestinal inflammation the concentration of SP is increased due to down-regulation of neutral endopeptidase resulting in reduced SP degradation [14]; the elevated SP levels may enhance functional abnormalities and the inflammatory process. SP significantly increases sensitivity of macrophages to LPS stimulation causing enhancement of proinflammatory cytokine secretion compared to LPS alone [15]. Responses of monocytes, macrophages, and neutrophils to bacterial LPS require CD14 receptor [16]. Normally the CD14 receptor is absent from intestinal mucosa (no m-RNA
57 for CD14 on mucosal macrophages); this explains the lack of inflammation in normal intestine despite the constant presence of bacterial products [17]. The elevated levels of CD14 detected in our MgD animals may represent part of the inflammatory response subsequent to altered in- testinal contractile function and increased permeability [18]. Systemic exposure to circulating LPS is an important stimu- lus enhancing LPS receptor levels in macrophage-like cells found in hepatic and cardiac tissues of MgD rats. It is also known, that endotoxin induces systemic elevation of TNF-α, IL-1, and IL-6 [7, 9]; all three cytokines were found to be elevated in our rat model [19]. In conclusion, our data indicate that Mg-deficiency results in increased intestinal permeability to bacterial products. We also propose that enhanced production of TNF-α and other pro-inflammatory cytokines due to LPS stimulation may contribute to cardiac lesion formation observed in our rat model during prolonged severe Mg- deficiency [3, 19]. Whether our observations are also applicable for chronic, marginal magnesium deficiency, a condition commonly encountered in man, remains to be investigated.
Acknowledgments This study was supported by NIH RO1 Grant HL-62282 and NIH RO1 Grant HL-65718. We thank Jonathon Hall, MSc. MPH. for his help in formatting illustrations included in the manuscript.
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