INTRODUCTION

Acute-phase proteins are useful as inflammatory markers [1–5]. α2-Macroglobulin (α2M) is a typical acute-phase protein in rats [6–8]. α2M reacted more sensitively than did α1-acid glycoprotein (AAG) after inflammatory stimulation in rats [9, 10]. The authors previously reported α2M kinetics in rats. On the other hand, C-reactive protein (CRP) is a typical acute-phase protein in dogs [1, 4, 10, 11]. The serum peak concentration of CRP in 1-month-old dogs after injection of turpentine oil was lower than that in 3- or 18-month-old dogs [12]. However, AAG did have a significantly different level among the three groups of dogs [12]. Therefore, characteristic acute-phase protein levels change with age. To our knowledge, differences in synthesis of α2M by juvenile vs. mature rats have not been investigated previously. The aim of this study was to clarify whether such differences exist. Furthermore, levels of interleukin (IL)-6 and cytokine-induced neutrophil chemoattractant (CINC)-1, considered to contribute to the synthesis of α2M [10, 13, 14], were assessed.

MATERIALS AND METHODS

Animals

A total of 15 Sprague–Dawley rats (3, 7, and 11 weeks of age) were purchased from Charles River Laboratories Japan (Yokohama, Kanagawa, Japan). The body weights of adult male rats ranged from 200 g for 7-week-old rats to 400 g at 11 weeks of age [15]. Rats aged 7 and 11 weeks were considered “mature” rats in this study, and rats weaned 3 weeks after delivery [16] were considered “juvenile” rats. Rats were kept in isolation at a temperature of 23 ± 2°C and relative humidity of 55 ± 10 % on a 12/12 dark (20:00–8:00)/light (8:00–20:00) cycle, and the air was exchanged 12 or more times per hour. Rats were fed MF (Oriental Yeast Co., Ltd., Tokyo, Japan) and were allowed free access to water. All experiments conformed to Japanese regulations concerning animal care and use, as described in the Guidelines for Animal Experimentation (Japanese Association for Laboratory Animal Science, JALAS, 1987). The present animal experiment was approved by the Institutional Animal Care and Use Committee of Azabu University.

Animal Experimental Design

Turpentine oil (Wako Pure Chemical Industries, Co., Ltd., Osaka, Japan) was intramuscularly injected at 0.2 ml/kg body weight. Turpentine oil has been used to induce inflammation in many previous studies, reliably and with little individual variation [26–28] and, therefore, was chosen to induce inflammation in this study. Blood was collected by ventricular puncture before turpentine oil injection and 6, 12, 24, 48, 72, and 96 h after injection under anesthesia with pentobarbital (Kyoritsu Seiyaku Corporation, Tokyo, Japan).

Measurements of α2M, IL-6, and CINC-1

The serum concentrations of α2M were measured by enzyme-linked immunosorbent assay (ELISA) of samples collected before treatment and 24, 48, 72, and 96 h after turpentine oil injection by the method described by Honjo et al. [7]. The commercial ELISA kits for measurement of serum concentrations of IL-6 and CINC-1 in preinjection and 6, 12, and 24 h postinjection samples were purchased from Invitrogen Corporation (CA, USA) and Panapharm Laboratories Co., Ltd. (Kumamoto, Japan), respectively.

Statistical Analysis

The peak serum concentrations (C max) of α2M, IL-6, and CINC-1 of individual rats were used for analysis. The area under the concentration vs. time curve (AUC) was calculated by the traperizol method. The serum concentrations of α2M, IL-6, and CINC-1 were analyzed using the Student's t test. Differences in p values <0.05 were considered significant.

RESULTS

Changes of serum concentrations of α2M, IL-6, and CINC-1 are shown in Figs. 1, 2, and 3, respectively. The C max of α2M was observed 48 h after turpentine oil injection. The C max of IL-6 was observed 12 h after turpentine oil injection. The serum concentrations of α2M in 3- and 7-week-old rats at 48, 72, and 96 h after turpentine oil injection were significantly lower than that in 11-week-old rats. The serum concentrations of IL-6 and CINC-1 in 3-week-old rats 6 h after turpentine oil injection were significantly higher than those in 7- and 11-week-old rats.

Fig. 1
figure 1

Changes in serum concentrations of α2-macroglobulin (α2M) in 3-, 7-, and 11-week-old rats after injection of turpentine oil. Mean ± standard deviation (n = 5). *Value differs significantly from that of 11-week-old rats (p < 0.05). **Value differs significantly from that of 7-week-old rats (p < 0.05).

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Fig. 2
figure 2

Changes in serum concentrations of interleukin (IL)-6 in 3-, 7-, and 11-week-old rats after injection of turpentine oil. Mean ± standard deviation (n = 5). *Value differs significantly from that of 3-week-old rats (p < 0.05). **Value differs significantly from that of 11-week-old rats (p < 0.05).

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Fig. 3
figure 3

Changes in serum concentrations of cytokine-induced neutrophil chemoattractant-1 (CINC-1) in 3-, 7-, and 11-week-old rats after injection of turpentine oil. Mean ± standard deviation (n = 5). *Value differs significantly from that of 3-week-old rats (p < 0.05). **Value differs significantly from that of 7- and 11-week-old rats (p < 0.05).

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The C max and AUC of α2M, IL-6, and CINC-1 are shown in Table 1. The AUC of α2M significantly increased in an age-associated manner but that of IL-6 did not differ significantly among the three age groups. The AUC of CINC-1 in 3-week-old rats was significantly lower than that in 7- and 11-week-old rats. The C max was calculated from individual rat data. The C max of α2M and IL-6 increased in an age-associated manner. Significant differences in C max of α2M and IL-6 were observed among 3-, 7-, and 11-week-old rats. The C max of CINC-1 in 3-week-old rats was significantly lower than that in 7- and 11-week-old rats; however, no significant difference was observed between 7- and 11-week-old rats.

Table 1 Kinetic Parameters of α2-Macroglobulin, Interlekin-6, and Cytokine-Induced Neutrophil Chemoattractant-1 in 3-, 7-, and 11-Week-Old Rats
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DISCUSSION

The synthesis of α2M by juvenile and mature rats subjected to the same inflammatory stimulation was compared. The C max values for α2M in the three rat groups were observed 48 h after turpentine oil injection, and serum concentrations of α2M decreased. The kinetics of α2M were not different among the three groups. The AUC is an essential parameter for comparing the amount of absorption of candidate drug substances from the small intestine [17, 18]. Also, comparisons of AUC are considered to be more appropriate than comparison of serum concentrations at each time point for ascertaining differences in synthesis of α2M. The AUC of α2M increased with age and significant differences were observed among the three groups of rats. The synthesis of α2M in juvenile rats was significantly lower than that of mature rats in spite of the identical inflammatory stimulation.

Jinbo et al. investigated the serum concentrations of IL-1b, IL-2, IL-4, IL-6, CINC-1, IL-10, and IFN-γ in rats stimulated by injection of turpentine oil [14]. Only the serum concentrations of IL-6 and CINC-1 increased prior to α2M. Other cytokines did not change. Furthermore, Honjo et al. administered IL-6 or CINC-1 separated by gel chromatography from rat serum rich in α2M to rats [13]. The serum levels of α2M increased after injection of a solution including both IL-6 and CINC-1 [13]. IL-6 and CINC-1 were presumed to have contributed to the synthesis of α2M in the livers of these rats [19–24]. Only the concentration of CINC-1 in 3-week-old rats was significantly lower than in 7- and 11-week-old rats. Furthermore, in the current study, the AUC of IL-6 was not significantly different among the three groups of rats. We noted that the synthesis of IL-6 and CINC-1 did not differ among the three age groups. That the synthesis of albumin increased in an age-dependent manner has been reported [25]. Rat liver microsomal protein increased and the age-associated hepatocyte binuclear index increased [26–28]. Furthermore, liver protein synthesis in adult rats increased compared to that in young rats [29], and so the capacity of rat liver for synthesis of protein is thought to increase with age. The AUC of α2M in 11-week-old rats was significantly higher than in 7-week-old rats, even though profiles for IL-6 and CINC-1 were similar. These results suggest that the synthesis of α2M is increased with age due to differences in hepatic function at various ages.

A significant difference in the concentration of α 2M was not seen among the three groups at only 6 h after turpentine oil injection. The reason for this phenomenon may be that the concentrations of both IL-6 and CINC-1 in 3-week-old rats were significantly higher than in 7- and 11-week-old rats. However, Serushago et al. investigated IFN-γ levels in the media of mononuclear cell cultures from newborn and adult rats [30]. The levels of IFN-γ in cultures of mononuclear cells from newborns reached plateau levels on the third day of culture, whereas levels of IFN-γ from adult cell cultures had still increased after 5 days of culture [30]. On the other hand, levels of proinflammatory cytokines, including IL-6, in the extremely premature infant were higher than that of adult patients with Bacillus cereus sepsis [31]. High levels of proinflammatory cytokines means there was a significant response of macrophages/monocytes against B. cereus [31]. We presumed that the functions of phagocytes were more active in juvenile rats than in mature rats. However, the levels of IL-6 and CINC-1 increased earlier in 3- than in 7- and 11-week-old rats. The reason for this phenomenon has not been clarified by the present study. Further studies are needed to understand why IL-6 and CINC-1 in 3-week-old rats increased more quickly than in 7- and 11-week-old rats.

In conclusion, a greater amount of α2M is synthesized by mature than by juvenile rats, in spite of similar inflammatory stimulation. However, the synthesis of IL-6 and CINC-1 did not differ in an age-dependent manner. We think that this result could be attributable to differences in the synthesis of α2M in the liver. On the other hand, IL-6 and CINC-1 in 3-week-old rats increased more quickly than in 7- and 11-week-old rats. Further investigations will be needed to determine why these cytokines increase in juvenile rats.