The time course of ?2-macroglobulin (?2M) synthesis in rat liver was investigated using immunohistochemistry. Furthermore, correlations between synthesis of ?2M in hepatocytes and interleukin (IL)-6 and cytokine-induced neutrophil chemoattractant-1 (CINC-1), which are considered to contribute to the production of ?2M, were evaluated. The presence of ?2M in the liver was investigated by immunohistochemistry, and serum levels of ?2M, IL-6 and CINC-1 were measured by enzyme-linked immunosorbent assay. ?2M was not detected in the liver before injection of turpentine oil. ?2M was detected throughout the liver at 12 hours after injection of turpentine oil, when high serum levels of IL-6 and CINC-1 were observed. ?2M was distributed mainly around the central vein of the liver at 36 hours. Only small amounts of ?2M were detected in the liver at 48 hours, when peak serum levels of ?2M were observed. Synthesis of ?2M in hepatocytes peaked long before peak ?2M serum levels were seen. In conclusion, ?2M was considered to be synthesized in response to stimulation by IL-6 and CINC-1

Honjo, T

Kuribayashi, T

Momotani, E

Seita, T

Yamamoto, S

Yamazaki, S

English

Journals from University of Tartu

– 1 –sjlas2014, Volume 40, Number 3ISSN 2002-0112Scandinavian Journal of Laboratory Animal ScienceIntroductionAcute-phase proteins are useful infl ammatory mark-ers due to their obvious increases during bacterial infections and aft er surgical treatments as well as their changes in serum levels under disease con-ditions (Ceron et al., 2005; Jinbo et al., 2002; Honjo et al., 2010; Kuribayashi et al., 2003; Lazovic, 2012; Pepys et al., 1983). C-reactive protein (CRP) is a typ-ical acute-phase protein in humans and dogs (Capsi et al., 1984; Castell et al., 1988; Ceron et al., 2005; Du Clos, 2000; Eckersall et al., 1993; 1994; Karadag et al., 2008; Morris et al., 1982, Rahman et al., 2008). On Correlation between synthesis of α2-macroglobulinin hepatocytes and changes in serum cytokine levels in rats after infl ammatory stimulationby Takashi Kuribayashi1, Tetsurou Seitα1, Eiichi Momotani2, Toshio Honjo3, Shunsuke Yamazaki4 & Shizuo Yamamoto11Laboratory of Immunology, School of Life and Environmental Science, Azabu University, Sagamihara, Kanagawa, Japan2Research Team for Paratuberculosis, National Institute of Animal Health, Tsukuba, Ibaraki, Japan3Seminar on Environment, School of Life and Environmental Science, Azabu University, Sagamihara, Kanagawa, Japan4Laboratory of Physiology, Department of Food and Nutrition, Kamakura Women’s University, Kamakura, Kanagawa, JapanCorrespondence: Shizuo YamamotoLaboratory of Immunology, School of Life and Environmental ScienceAzabu University1-17-71 Fuchinobe, Chuo-ku, Sagamihara, Kanagawa 252-5201, JapanTel +81-42-754-7111, Fax +81-42-754-2461, E-mail kuribaya@azabu-u.ac.jpSummaryTh e time course of α2-macroglobulin (α2M) synthesis in rat liver was investigated using im-munohistochemistry. Furthermore, correlations between synthesis of α2M in hepatocytes and interleukin (IL)-6 and cytokine-induced neutrophil chemoattractant-1 (CINC-1), which are considered to contribute to the production of α2M, were evaluated. Th e presence of α2M in the liver was investigated by immunohistochemistry, and serum levels of α2M, IL-6 and CINC-1 were measured by enzyme-linked immunosorbent assay. α2M was not detected in the liver before injection of turpentine oil. α2M was detected throughout the liver at 12 hours aft er in-jection of turpentine oil, when high serum levels of IL-6 and CINC-1 were observed. α2M was distributed mainly around the central vein of the liver at 36 hours. Only small amounts of α2M were detected in the liver at 48 hours, when peak serum levels of α2M were observed. Synthesis of α2M in hepatocytes peaked long before peak α2M serum levels were seen. In conclusion, α2M was considered to be synthesized in response to stimulation by IL-6 and CINC-1.the other hand, α2-macroglobulin (α2M) and α1-acid glycoprotein (AAG) are typical acute-phase proteins in rats (Honjo et al., 2010; Honjo et al., 2010; Inoue et al., 2001; Jinbo et al., 2001; Jinbo et al., 2002). Serum levels of α2M and AAG increase in rats aft er injection of turpentine oil, surgical treatment or inoculation with Staphylococcus aureus (Honjo et al., 2010). How-ever, α2M reacts more sensitively than AAG in rats during acute infl ammation. Th us, α2M is considered to be more useful than AAG as an acute-phase pro-tein in rats.– 2 –sjlasα2M is produced in the liver (Du Clos, 2000; Heinrich et al., 1990), and interleukin (IL)-6 and cytokine-in-duced neutrophil chemoattractant-1 (CINC-1) are thought to contribute to its production (Baumann et al., 1989; Ling et al., 2004; Nijsten et al., 1987; Richard et al., 2008; Sheikh et al., 2006). Changes in serum α2M levels have been widely investigated and the kinetics of serum α2M have been clarifi ed (Honjo et al., 2006; Honjo et al., 2010; Jinbo et al., 2001; Jinbo et al., 2002; Kuribayashi et al., 2011; Kuribayashi et al., 2012). However, the time course of α2M synthesis in rat liver has not been investigated using immunohis-tochemistry. Furthermore, correlations between α2M synthesis and changes in serum levels of α2M, IL-6 and CINC-1 have not been clarifi ed. In the present study, the time course of α2M synthesis in rat liver aft er acute infl ammatory stimulation was investigat-ed by immunohistochemistry. Moreover, correlations between α2M synthesis in the liver and changes in serum levels of α2M, IL-6 and CINC-1 were evalu-ated.MethodsAnimalsTwenty-four male Sprague-Dawley rats (age, 9 weeks) were purchased from Charles River Laborato-ries Japan (Yokohama, Kanagawa, Japan). Rats were kept in cages at a temperature of 23 ± 2°C, and a rela-tive humidity of 55% ± 10%, on a 12/12 dark (18:00-6:00)/light (6:00-18:00) cycle with the air exchanged 12 times or more per hour. Rats were fed MF (Orien-tal 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 Exper-imentation (Japanese Association for Laboratory Animal Science, JALAS, 1987). Th e present animal experiment was approved by the Institutional Animal Care and Use Committee of Azabu University.Animal experimental designTurpentine oil (Wako Pure Chemical Industries, Co., Ltd., Osaka, Japan) was intramuscularly injected at 0.4 ml/rat. Rats were sacrifi ced under anesthesia with an intravenous injection of pentobarbital (Kyoritsu Seiyaku Corporation, Tokyo, Japan) before treatment and at 6, 12, 18, 24, 36, 48 or 72 hours aft er injection of turpentine oil. Th ree rats were sacrifi ced at each time point. Unfortunately, liver samples could not be collected from the same rat at each time point, as pricking with the biopsy needle would constitute infl ammatory stimulation and would infl uence the kinetics of α2M. Th us, whole livers were collected at each time point from diff erent rats. Blood was col-lected from the aorta when the liver was removed. Sera were obtained by centrifugation (1,600 × g, 15 minutes), and were stored at -80°C until measure-ment of α2M, IL-6 and CINC-1.ImmunohistochemistryLiver samples were fi xed in 10% formalin and embed-ded in paraffi  n. Sections (3 mm) were cut, incubat-ed with Blocking One (Nacalai Tesque, Inc., Kyoto, Japan) diluted with 0.01 M phosphate buff ered saline for 15 minutes. Next, mouse anti-α2M monoclonal antibody (Inoue et al., 2001) was applied for 1 hour. Sections were stained using the peroxidase method with Histofi ne Simple Stain Rat MAX-PO(M) (Nichirei Biosciences Inc., Tokyo, Japan) and Simple Stain DAB solution (Nichirei Biosciences Inc.).Measurement of α2M, IL-6 and CINC-1Serum levels of α2M were measured by enzyme-linked immunosorbent assay (ELISA) according to the procedure of Honjo et al. (2010). Serum levels of IL-6 and CINC-1 were measured by ELISA using commercial kits. Commercial kits for IL-6 and CINC-1 were purchased from BioSource Interna-tional, Inc. (Camarillo, CA, USA) and Panapharm Laboratories Co., Ltd. (Kumamoto, Japan), respec-tively. Serum levels at each point are given as means ± standard deviation.ResultsTh e presence of α2M in rat liver aft er acute infl amma-tory stimulation is shown in Figure 1. Similar obser-vations were obtained from other rats at each time point. Changes in serum levels of α2M are shown in Figure 2, while changes in serum levels of IL-6 and CINC-1 are shown in Figures 3 and 4, respectively.α2M was not detected in the liver before treatment, but α2M was detected particularly in perilobular hepatocytes at 6 hours aft er injection of turpentine oil. Th e presence of α2M was observed in hepat-ocytes throughout the liver at 12 hours, and was observed mainly around the central veins in the liver at 24 hours. Small amounts of α2M were observed throughout the liver at 48 hours, and α2M was no longer observed in the liver at 72 hours aft er injec-tion.2014, Volume 40, Number 3– 3 –sjlas2014, Volume 40, Number 30.01.02.03.00 24 48 72Levels of 2M (mg/ml)Time after injection (hrs)Figure 1. Immunohistochemical detection of α2-macroglobulin in liver obtained from rats injected with turpentine oil (0.4 ml/rat). A: pre-treatment; B: 6 h post-injection; C; 12 h post-injection; D: 24 h post-injection; E: 48 h post-injection; F: 72 h post-injectionTh e peak serum level of α2M was 2.2±0.48 mg/ml at 48 hours aft er tur-pentine injection, aft er which serum levels decreased. Th e peak levels of IL-6 and CINC-1 were 235.6±122.3 pg/ml at 18 hours and 4.6±2.1 ng/ml at 12 hours aft er turpentine oil injec-tion, respectively.DiscussionTh e time course of α2M synthesis in rat liver was investigated using immu-nohistochemistry. Th e pattern of changes in serum α2M was similar to previous data obtained in individual rats aft er infl ammatory stimulation, with peak serum levels observed at 48 hours aft er turpentine oil injection (Jinbo et al., 2001; Jinbo et al., 2002). Synthesis of CRP in rab-bits has been evaluated using an immunoenzymatic technique (Macintyre et al., 1982), and CRP was not detected in hepatocytes of rabbits not injected with turpentine oil. Similarly, in the present study, α2M was not detected in the liver prior to turpentine oil injection, despite small amounts of α2M being pres-Figure 2. Changes in α2-macroglobulin (α2M) in rat serum aft er injection of turpentine oil (0.4 ml/rat). Data represent means ± SD (n=3).ent serum prior to infl ammatory stimulation. α2M was observed particularly in perilobular areas of the liver at 6 hours aft er turpentine oil injection, and subsequently increasing numbers of hepatocytes pro-duced α2M around the central veins of liver. Th ese synthesis patterns of α2M in rats were similar to those of CRP in rabbits. α2M was detected through-out the liver of rats at 12 hours, when mean serum levels of IL-6 and CINC-1 showed high levels. How-ever, α2M levels in serum were very low (0.09 mg/ml)at 12 hours. On the other hand, α2M was weakly detected in hepatocytes at 48 hours, when peak serum levels of α2M were observed. Jinbo et al. investigated serum changes of α2M and several cytokines in rats aft er injection of turpentine oil and found that only IL-6 and CINC-1 increased prior to elevation of α2M (Jinbo et al., 2002). We believe that IL-6 and CINC-1 contribute to synthe-sis of α2M in rats (Honjo et al., 2006; Honjo et al., 2010; Jinbo et al., 2002; Ling et al., 2004; Richard et – 4 –sjlasal., 1991; Sheikh et al., 2006; Kuribayashi et al., 2011). Correlations between synthesis of α2M in the liver and changes in IL-6 and CINC-1 in serum were thus evaluated in this study. Circulating blood enters the liver lobules from interlobular arteries and veins through the sinusoidal capillaries and fl ows into the central vein of each lobule. Honjo et al. (2010) reported that serum levels of α2M were elevated aft er injection of a mixture of IL-6- and CINC-1-rich fractions separated from rat sera aft er acute infl am-matory stimulation; however, α2M was not elevated aft er individual injection of IL-6- or CINC-1-rich fraction. Peak synthesis of α2M in hepatocytes cor-responded to peak serum levels of IL-6 and CINC-1. Th is suggests that both IL-6 and CINC-1 are involved in signaling hepatocytes to synthesize α2M.AcknowledgementsTh is research was partially supported by Th e Promo-tion and Mutual Aid Corporation for Private Schools of Japan, and a Grant-in-Aid for Matching Fund Sub-sidy for Private Universities.ReferencesBaumann H, KR Prowse & S Marinkovic: Stimulation of hepatic acute phase response by cytokines an glucocorti-coids. Ann NY Acad Med Sci, 1989, 557, 180-195.Capsi D, ML Baltz, F Snel, E Gruys, D Niv, RM Batt, EA Munn, N Buttress & MB Pepys: Isolation and Character-ization of C-reactive protein from the dog. 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Lab Anim, 2011, 45, 215-218.Kuribayashi T, T Seita, T Honjo, S Yamazaki, E Momotani & S Yamamoto; Impairment of α2-macroglobulin synthesis in experimental hepatopathic rats treated with turpentine oil, Exp Anim, 2012, 61, 125-130.Lazovic B: Correlation of CRP and serum levels fi briino-gen with severity of disease in chronic obstructive pulmo-nary disease patients. Med Arh, 2012, 66, 159-60.Ling PP, RJ Smith, S Kie, P Boyce & BR Bistrian: Eff ects of protein malnutrition on IL-6-mediated signaling in the liver and the systemic acute-phase response in rats. Am J Physiol Regul Integr Comp Physiol, 2004, 287, 801-808.Macintyre SS, D Schultz & I Kushner: Biosynthesis of C-re-active protein. Ann NY Acad Med Sci, 1982, 389, 76-87.Morris SG, D Gomez & KR Prasad: C-reactive protein in liver cancer surgery. Eur J Surg Oncol, 2008, 34, 727-729.Nijsten MWN, ER DeGroot, HJ TenDuis, JH Klaren, CE Hack & LA Arden: Serum levels of interleukin-6 and acute phase respose. Lancet, 1987, 330, 921. 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Correlation between synthesis of ?2-macroglobulin in hepatocytes and changes in serum cytokine levels in rats after infl ammatory stimulation

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Correlation between synthesis of ?2-macroglobulin in hepatocytes and changes in serum cytokine levels in rats after infl ammatory stimulation

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