Increase in complement component C3 is an early response to experimental magnesium deficiency in rats

Life Sciences 73 (2003) 499 – 507 www.elsevier.com/locate/lifescie

Increase in complement component C3 is an early response to experimental magnesium deficiency in rats F.I. Bussie`re a, A. Tridon b, W. Zimowska a, A. Mazur a, Y. Rayssiguier a,* a

Centre de Recherches en Nutrition Humaine d’Auvergne, Unite´ Maladies Me´taboliques et Micronutriments, INRA, Theix, 63122 St-Gene`s-Champanelle, France b Laboratoire d’Immunologie, CHRU Clermont-Ferrand, France Received 10 February 2003; accepted 25 February 2003

Abstract The importance of the inflammatory process in the pathology of experimental Mg-deficiency has been reconsidered but the sequence of events leading to inflammatory response remains unclear. In this study, the effect of Mg-deficiency on complement system by measuring total C3 concentration, mRNA abundance for rat pre-pro complement C3 in liver by RT-PCR, complement haemolytic activity and C3 activation by Western Blot was studied. Weaning male Wistar rats were fed either Mg-deficient or control experimental diets for 2 or 8 days. At 8 days, a characteristic inflammatory response of Mg-deficiency including hyperaemia, leukocytosis and enlarged spleen was accompanied by an increase in the total C3 quantity in plasma. Moreover, at 8 days, RT-PCR analysis indicated higher level of mRNA rat pre-pro complement C3 in liver from Mg-deficient rats compared to control rats. Even if the inflammatory syndrome was not observed in rats after 2 days, total plasma C3 was shown to be significantly increased as compared to total plasma C3 level in control rats. Because of the high variability of complement haemolytic activity values in Wistar rats, weaning male Sprague-Dawley rats were used in a second experiment. At 8 days, the inflammatory response of Sprague-Dawley rats was accompanied by an increase in total C3 quantity and by a higher haemolytic activity. The Western Blot technique failed to display distinct bands resulting from C3 cleavage in plasma from Mg-deficient rats. Since, the complement C3 is a positive acute phase reactant, the elevation of C3 indicates that the modification of inflammatory response is an early event of Mgdeficiency. However, complement activation does not appear to be involved in the acute phase of the deficiency. D 2003 Elsevier Science Inc. All righs reserved. Keywords: Magnesium; Complement; Inflammation

* Corresponding author. Tel.: +33-4-73-62-42-30; fax: +33-4-73-62-46-38. E-mail address: [email protected] (Y. Rayssiguier). 0024-3205/03/$ - see front matter D 2003 Elsevier Science Inc. All righs reserved. doi:10.1016/S0024-3205(03)00291-1

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Introduction Magnesium (Mg), the second most abundant intracellular cation plays an essential role in a wide range of fundamental cellular reactions and many signs, symptoms and disease states are attributed to altered Mg homeostasis (Shils, 1994). In developed countries, the marginal Mg intake may induce a high prevalence of marginal Mg-deficiency (Galan et al., 1997). Mg-deficiency has been extensively studied in the rat where it is readily produced by dietary depletion. Many studies underline the importance of the immuno-inflammatory process in the pathology of Mg-deficiency (Rayssiguier et al., 2001; Weglicki and Phillips, 1992). In 1932, Kruse and coworkers. (Kruse et al., 1932) reported a peripheral vasodilatation with hyperaemia as one of the symptoms of Mg-deficiency in rats. Many investigators have since recognised that these symptoms are related to the activation of mast cells with the release of histamine and other inflammatory mediators (Kraeuter and Schwartz, 1980). Recent studies from our laboratory as well as from others suggest that in Mg-deficiency, phagocytes are metabolically primed and produce reactive oxygen species leading to tissue damage (Mak et al., 1997; Malpuech-Bruge`re et al., 2000). However, the precise mechanism of this characteristic response to Mg-deficiency is still unknown. During normal inflammatory response, the activity of phagocytic cells are closely related to the complement system (Frank and Fries, 1991). Not only does the complement system regulate the phagocytic process but it also plays a major role in inflammation. Mg is required in the initiation of both the classical and alternative pathways of the complement system (McCoy and Kenney, 1992) and preliminary studies suggested that experimental Mg-deficiency may induce functional changes in the complement system (Kawanobe and Sakamoto, 1989). Thus, activation of the complement system could be responsible for early mast cell degranulation and phagocyte activation in Mg-deficient rats. The aim of this study is to assess the effect of Mg-deficiency on early modifications of the complement system by measuring the total complement component C3 quantity by ELISA, the abundance of mRNA for rat prepro complement C3 in liver by RT-PCR, the serum haemolytic activity and some components of C3 cleavage by Western Blot.

Methods Experimental design The institution’s guide for the care and use of laboratory animals was used. In a first experiment, male weaning Wistar rats (IFFA-CREDO, L’Arbresle, France) weighing 61 F 3 g were randomly divided into Mg-deficient and control groups containing 20 rats each. Half of the animals received the experimental diets for 2 days and the other half received the experimental diets for 8 days. In a second experiment, male weaning Sprague-Dawley rats (IFFA-CREDO, L’Arbresle, France) weighing 55 F 2 g were fed either the Mg-deficient or the control diet (12 rats per group) for 8 days. The rats were housed in wire-bottomed cages in a temperature-controlled room (22 jC) with a 12 h dark (20.00
08.00 h) and 12 h light period. Distilled water and diet were provided ad libitum. The synthetic diets contained (g/kg): 200 casein, 650 sucrose, 50 corn oil, 50 alphacel, 3 D,L-methionine, 2 choline bitartrate, 35 modified AIN-76 mineral mix and 10 AIN-76A vitamin mix (ICN biomedicals, Orsay, France). The MgO was omitted from the mineral mix in the Mg-deficient diet. The Mg

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concentrations of the diets, determined by flame atomic absorption spectrometric analysis were 30 and 960 mg/kg for Mg-deficient and control diets, respectively. Rats were anaesthetised with pentobarbital (40 mg/kg body weight). Blood was collected from the abdominal artery in both EDTA sterile tubes and tubes without any anticoagulant. Plasma was obtained after centrifugation (2000 g, 15 min) and serum was obtained after clot formation and centrifugation (1000 g, 15 min). Plasma EDTA and serum were stored at
80 jC for biochemical analysis. Spleen was removed and weighed. Liver was removed and stored frozen in liquid nitrogen for C3 mRNA analysis. General parameters of inflammation and analytical procedures Flame atomic absorption spectrometry (Perkin Elmer 800) was used to determine serum Mg concentration. Hyperaemia was recorded daily using scores from 0 to 3 (score 0: no hyperaemia, score 1: hyperaemia at the base of the ears, score 2: hyperaemia over half of the ears, score 3: hyperaemia over the entire ears). The number of total white cells was determined by a cell counter (Cobas, Hoffmann, La Roche). Determination of total C3 concentration by ELISA Microtiter plates (96 wells) were coated with goat antiserum to rat complement C3 (1:2000; ICN, CAPPEL, Aurora, USA) for 12 h at 4 jC. After saturation of unspecific sites with gelatin (0.5%) dissolved in phosphate buffer saline (PBS), 100 AL of diluted rat plasma (1:20,000; PBS-Tween-Gelatin) were added to the wells and the plates were incubated for 90 min at 37 jC. After washing, the plates were incubated with 100 AL of horseradish peroxidase conjugated goat anti-rat C3 polyclonal antibody (1:2000; ICN, CAPPEL) for 1 h at 37 jC and washed again. A substrate solution of o-phenylenediamine (SIGMA, St Louis, USA) dissolved in citrate buffer (0.1 mol/L, pH 5) with added hydrogen peroxide has been added to the wells and the color developed in proportion to the amount of C3. After 5 minutes incubation, the color development was stopped by addition of 1N HCl. The absorbance was measured at 492 nm using a microtiter plate reader. A standard curve was obtained using pooled normal rat plasma. Corrected absorbance was calculated by subtracting background (OD without plasma sample) from mean values of samples and standard dilutions. Determination of the abundance of mRNA for rat pre-pro complement C3 by RT-PCR The abundance of mRNA for rat pre-pro-complement C3 was assessed by both qualitative and quantitative RT-PCR. Total cellular RNA from the liver was isolated according to the method of Chomczynski and Sacchi (1987). Total RNA (3 Ag) was converted into first strand cDNA using ReadyTo-Go You-Prime First-Strand Beads Kit (Amersham Pharmacia Biotech Inc, Piscataway, USA) and 0.5 mg/ml oligo (dT)15 Primer (Promega; 1 Al) were incubated at 37 jC for 60 minutes. For qualitative analysis of the abundance of C3 mRNA the PCR was carried out in a total volume of 25 Al. The RT mixture (1 AL) and 25 AM concentration of the forward and reverse specific primers (Isoprim, Toulouse, France) were added to the Ready-To-Go PCR Beads Kit (Amersham Pharmacia Biotech Inc). All PCR reactions were performed individually for each primer pair in a Hybaid OmnE thermocycler (Hybaid Ltd, Teddington Middlesex, UK) that was programmed as follows: an initial 4 minute period for complete denaturation at 94 jC ; a primer-specific number of cycles of 30 s denaturation at 94 jC, 30 s

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annealing at 58 jC, and 60 s primer extension at 72 jC ; a 8 min period at 72 jC, for final extension. For the PCR step, a specific primer was used: rat pre-pro-complement C3 (Misumi et al., 1990) of product size 500 bp, 22 cycles, annealing temperature 58 jC, forward 5V- TGA CTG GCT TTA TTC CAG ACA C -3V, reverse 5V- ATC TGA GCC TGA CTT GAT GAC-3V. 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. As a control, a housekeeping gene GADPH was used. The PCR product (12 Al) was resolved on 1% agarose gel containing ethidium bromide (0.75 mg/ml) and visualised under UV light. For quantitative analysis of C3 mRNA abundance PCR was performed in a fluorescence temperature cycler (LightCycler; Roche Diagnostics GmbH, Mannheinm, Germany). Amplification was performed in a 20 Al final reaction volume containing LC-FastStart DNA Master SYBR Green I (FastStart Taq DNA, reaction buffer, dNTP mix with dUTP instead of dTTP, SYBR Green I dye; 2 Al), 3 mM MgCl2 (1.6 Al), 0.5 AM of each specific primers (Isoprim, Toulouse, France; 2 Al), H2O sterile PCR grade (Roche; 7.4 Al), and the cDNA template (1:100; 5 Al). For the PCR step, the following specific primers were used: rat C3, product size 184 bp, Forward 5V-AAGAACACCCTCATCATCTACC-3Vand Reverse 5V-GCATTCCATCGTCCTTCTCC-3Vand for or normalization a rat GAPDH: product size 179, Forward 5V-ACCCCTTCATTGACCTCAAC-3V and Reverse 5V-ATACTCAGCACCAGCATCAC-3V was used. The amplification program included the initial denaturation step at 95 jC for 600 s and 45 cycles of denaturation at 95 jC for 12 s, annealing at 59 jC for 5 s, and extension at 72 jC for 10 s. The temperature transition rate was 20 jC/s. Fluorescence was measured at the end of each cycle. After amplification, a melting curve was acquired by heating the product at 20 jC/s to 95 jC, cooling it at 20 jC/s to 69 jC, holding it at 69 jC for 5 s, and slowly heating it at 0.1 jC/s to 95 j. Melting curves were used to determine the specificity of the PCR. This analysis was performed in triplicate on pooled RNA samples from each studied group. Serum haemolytic activity The haemolytic activity (CH50) of serum from Mg-deficient and control rats was measured according to Mayer’s method (Kabat and Mayer, 1961). Briefly, rat serum (0.12 mL) was added to sheep erythrocytes (Sanofi, Diagnostics Pasteur; 4 mL) sensitised with anti-sheep antibody raised in rabbit (Sanofi, Pasteur) and suspended in a calcium-Mg buffer (Mayer buffer). Evolution of erythrocyte lysis was performed by recording the kinetics of the OD at 650 nm. The lag time to lyse 50% of the erythrocyte suspension was measured and expressed in seconds. Previous studies showing a high variability of serial total haemolytic activity determinations on Wistar male rats (Arroyave et al., 1977) compelled us to perform serum haemolytic activity on Sprague-Dawley rats. Determination of C3 cleavage by Western Blot The C3 cleavage was determined by a blot technique described by Tridon et al. (1992). Briefly, plasma was diluted to 1:5 in PBS and Paragon gel electrophoresis was performed (Beckmann). The gel was transferred to a nitrocellulose membrane and unspecific sites were saturated with 5% non fat dry milk. After washing with distilled water, membranes were incubated for 1 h with horseradish peroxidaseconjugated goat anti-rat C3 polyclonal antibody (1:500; ICN, CAPPEL). Then, the substrate (diaminobenzidine and hydrogen peroxide) was added to the membrane. The reaction was stopped using 1 N

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Table 1 Indices of inflammation of rats fed either the control or the Mg-deficient diety Days

Dietary groups

Serum Mg (mmol/L)

Relative spleen weight (g/100 g bwt)

Blood leukocytes (106 cells/mL)

Hyperemia (A.U.z)

Plasma C3 (A.U.z)

2

Control Mg-deficient Control Mg-deficient

1.05 F 0.04 0.67 F 0.05*** 0.94 F 0.04 0.33 F 0.03***

0.55 F 0.03 0.51 F 0.03 0.54 F 0.03 0.67 F 0.05*

10.2 F 1.5 10.2 F 1.3 7.1 F 0.4 20.0 F 3.6**

2.9 F 0.03

0.96 F 0.05 1.10 F 0.04* 1.10 F 0.05 1.88 F 0.05***

8

Values represent the mean F SEM for 10 rats per group. Mean values were significantly different from those for the control group: *P < 0.05; **P < 0.01; ***P < 0.001. y Wistar rats were fed either the Mg-deficient diet or the control diet for 2 days or 8 days. z A.U.: Arbitrary Units.

HCl. Rat serum with or without activation by sheep erythrocytes sensitised with anti-sheep antibody raised in rabbit were used as controls. Statistical analysis Statistical analysis were conducted using an InStat package (InStat, Graph Pad, San Diego, USA). Results were expressed as means with standard errors. The statistical significance of differences between means was undertaken using the Student’s t test. When error variance was found to be heterogeneous, the statistical significance between groups was undertaken using the Mann-Whitney test. The differences were considered to be statistically significant when the P value was less than 0.05.

Results In accordance with previous observations, after 8 days on the experimental diet, Mg-deficient Wistar rats presented growth retardation (100 F 3 vs. 114 F 2 g for controls, P < 0.01), low serum Mg levels and symptoms of inflammation including hyperaemia, increased number of blood leukocytes and enlarged spleen. At this stage of the deficiency, total C3 was found to be significantly higher in the plasma from Mg-deficient rats than that of control rats (Table 1). As shown in Fig. 1, RT-PCR analysis indicates a higher level of mRNA rat pre-pro complement C3 in the liver from Mg-deficient rats

Fig. 1. Hepatic pre-pro complement C3 abundance in control and Mg-deficient rats assessed by RT-PCR. Wistar rats were fed either the control or the Mg-deficient diet for 8 days. M wt: molecular weight standard.

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Table 2 Effect of dietary Mg on complement activity and total plasma C3 levelsy Dietary groups

Serum Mg (mmol/L)

Complement activity (seconds)yy

Plasma C3 (A.U.z)

Control Mg-deficient

1.00 F 0.02 0.33 F 0.01***

44.8 F 3.6 28.8 F 3.6**

1.33 F 0.07 2.35 F 0.16***

Values represent the mean F SEM for 12 rats per groups. Mean values were significantly different from those for the control group: **P < 0.01; ***P < 0.001. y Sprague Dawley rats were fed either the Mg-deficient diet or the control diet for 8 days. yy Lag-time to lyse 50% erythrocyte suspension. z A.U.: Arbitrary Units.

compared to control rats. The quantitative RT-PCR analysis confirmed these results showing 1.8 fold greater C3mRNA values for Mg-deficient rats than in control animals. The precocity of inflammation involved in Mg-deficiency was also evaluated. After 2 days on the experimental diet, rats fed Mgdeficient diet showed low serum Mg level. The body weight of the control and the Mg-deficient rats were comparable (73 F 1 vs. 76 F 1 g) and clinical signs of inflammation were not observed (Table 1). However, at this stage of the deficiency, total C3 was significantly higher in plasma from Mg-deficient rats compared to control rats. Using a Western Blot technique, C3 from control and Mg-deficient rats was not cleaved (data not shown). Sprague-Dawley Mg-deficient rats developed the same classical symptoms of inflammation after 8 days on the experimental diet as the Mg-deficient Wistar rats showing low serum Mg levels, hyperaemia (2.7 F 0.22 A.U.), growth retardation (101 F 1 vs. 114 F 2 g for controls; P < 0.001), an increased number of blood leukocytes (24.5 F 2.6 vs. 5.5 F 0.8  106 cells/mL for controls; P < 0.001) and enlarged spleens (1.01 F 0.07 vs. 0.54 F 0.03 g / 100 g b. wt for controls; P < 0.001). Similarly to Mg-deficient Wistar rats, total C3 was found to be significantly higher in plasma from Mg-deficient Sprague-Dawley rats than in controls. Indeed, in this experiment, the lag time for 50% of erythrocyte lysis was shorter for serum from Mg-deficient than for serum from control rats and haemolytic activity of serum from Mg-deficient rats was significantly higher than haemolytic activity of serum from control animals (Table 2). Using the Western Blot technique, C3 from control and Mg-deficient SpragueDawley rats was not cleaved (Fig. 2) whereas activated serum showed two distinct bands. A slight C3 activation also occurred spontaneously in normal serum as a result of blood coagulation during serum preparation.

Fig. 2. Determination of C3 activation by Western Blot. C3 cleavage was assessed on plasma from control and Mg-deficient Sprague Dawley rats after 8 days on the experimental diet. Rat serum without (1) or with (2) activation by sheep erythrocytes sensitised with anti-sheep antibody raised in rabbit were used as controls. A slight C3 activation occurs spontaneously in normal serum as a result of blood coagulation during serum preparation.

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Discussion The complement system is a non specific immune defence system involved in the elimination of invading foreign cells and the initiation of inflammation. The complement system includes proteolytic enzymes, enzyme inhibitors and receptors which are mainly synthesised in liver, but also in peripheral monocytes and to a lesser extent in other extra-hepatic tissues. The complement components must be activated before they can carry out their functions. The third complement component, C3, participates as a major player in the proteolytic reactions of the complement cascade : the antibody dependent classical complement pathway, the antibody independent alternative pathway and the lectin complement pathways (Frank and Fries, 1991). Results of the present experiments indicate that inflammatory response in Mg-deficiency is accompanied by an increase in the concentration of C3 in plasma. This study confirms the occurrence of an inflammatory response in Mg-deficient Wistar rats after 8 days on an experimental diet (Malpuech-Bruge`re et al., 2000). Decreased serum Mg indicates that the rats fed Mg-deficient diet were Mg-deficient. Dietary Mg-deficiency gives rise, after a few days, to a characteristic anaphylactoı¨d reaction, the first visible symptom being a peripheral vasodilatation (Bois et al., 1963; Nishio et al., 1988). The inflammatory response is associated with a marked increase in the neutrophil function (Bussie`re et al., 2002; Rayssiguier et al., 2001). Previous observations point to the spontaneous and non-infectious induction of the inflammatory process in this experimental model (Malpuech-Bruge`re et al., 1998a). The greater spleen size in Mg-deficient rats is believed to be due to infiltration of the spleen with polymorphonuclear leukocytes and macrophages (Malpuech-Bruge`re et al., 1998a). Moreover, elevation of the plasma concentration of interleukin (IL)-6, a known mediator of the acute phase response, has been observed in the same experimental model (Malpuech-Bruge`re et al., 2000; Bussie`re et al., 2002). The third complement component C3 is a positive acute phase reactant and typical of some other positive acute phase reactants, C3 synthesis is induced in response to IL-1, IL-6 and tumour necrosis factor (Frank and Fries, 1991). Thus, the increase in C3 in the plasma level is probably related to the elevated levels of pro-inflammatory cytokines. As shown by RT-PCR, this increase is related to higher mRNA level in liver from Mg-deficient rats as compared to control rats. Furthermore, other results obtained in our laboratory using cDNA array showed a two fold induction in the expression level of the gene encoding for C3 (non published data). Even if the inflammatory syndrome including hyperaemia and leukocytosis was not observed in Wistar rats after 2 days on the experimental diet, total C3 quantity was shown to be significantly elevated suggesting that cytokines may induce C3 synthesis as early as day 2 following Mg deprivation. These results agree with previous studies showing that the response of Mg-deficient rats to lipopolysaccharide was associated with higher production of TNFa than in control rats and that this alteration occurred as early as 48 h following the first Mg-depleted meal. Thus, the release of inflammatory cytokines could be an early pathophysiological event of Mg-deficiency. The lysis of antibody coated erythrocytes has long been used as a means of estimating the complement activity of serum. In Wistar rats, a high variability in complement haemolytic activity values (long and short dura tion of haemolysis) due to a frequent deficiency in C4 in Wistar rats (Arroyave et al., 1977) was observed. Sprague-Dawley rats were therefore used to measure this parameter. After 8 days on the Mg-deficient diet, Sprague-Dawley rats developed the same inflammatory reaction as Wistar rats. In Sprague Dawley Mg-deficient rats, the increase in CH50 activity was observed in agreement with the increase in plasma level in the complement component C3. A Western Blot technique was used to assess the effect of dietary Mg on the complement system activation. This

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qualitative method is sensitive enough to detect a C3 cleavage in sera in which the complement system is activated by the coagulation process. However, this method failed to display distinct bands resulting from C3 cleavage in plasma from Mg-deficient rats. Thus, an increase in the C3 plasma level and in the complement haemolytic activity are not accompanied by the complement system activation as shown in other systemic inflammatory syndromes (Frank and Fries, 1991). Finally, the results suggest that complement activation is not responsible for the early mast cell degranulation and phagocyte activation in this experimental model. The underlying mechanism for inflammation in Mg-deficiency is still unknown. However, feeding the animals with the Mg-deficient diet results in a rapid decline in plasma Mg. Inflammatory response is dependent on cytosolic calcium elevation (Romeo et al., 1975). Since Mg frequently acts as a natural calcium antagonist, the possible role of an increased intracellular free calcium concentration on inflammatory response has been recently suggested (Malpuech-Bruge`re et al., 1998b; Bussie`re et al., 2002).

Conclusion The present experiment indicates that inflammation is an early event occurring during Mg-deficiency. The inflammatory response is accompanied by an early increase of complement component C3 but complement activation does not appear to be responsible for the acute phase response following Mgdeficiency.

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