- Email: [email protected]
St2 pathway on alteration of iron and hematological parameters in acute inflammation
Experimental and Molecular Pathology 99 (2015) 687–692
Contents lists available at ScienceDirect
Experimental and Molecular Pathology journal homepage: www.elsevier.com/locate/yexmp
Effects of Il-33/St2 pathway on alteration of iron and hematological parameters in acute inflammation Marija S. Stankovic a, Vladimir Turuntas b, Silvio R. De Luka a, Sasa Jankovic c, Srdjan Stefanovic c, Nela Puskas d, Ivan Zaletel d, Sanja Milutinović-Smiljanic e, Alexander M. Trbovich a,⁎ a
Department of Pathophysiology, Faculty of Medicine, University of Belgrade, Dr Subotica 8, 11000, Belgrade, Serbia Pediatrics, University hospital Foca, Studentska 5, 73300 Foca, Bosnia and Herzegovina Institute of Meat Hygiene and Technology, Kacanskog 13, 11000, Belgrade, Serbia d Institute of Histology and Embryology “Aleksandar Đ. Kostić”, School of Medicine, University of Belgrade, Dr Subotica 8, 11000, Belgrade, Serbia e General and Oral Histology and Embryology, Faculty of Dentistry, University of Belgrade, Dr Subotica 8, 11000, Belgrade, Serbia b c
a r t i c l e
i n f o
Article history: Received 3 July 2015 and in revised form 3 October 2015 Accepted 9 November 2015 Available online 11 November 2015 Keywords: Acute inflammation IL-33/ST2 axis Iron Hematological parameters
a b s t r a c t Aim: The aim of this study was to examine the role of the IL-33/ST2 pathway in pathogenesis of acute inflammation by investigating its possible role in alteration of iron and hematological parameters in experimental model of acute inflammation. Material and methods: Wild-type and ST2 knockout BALB/c mice were divided into four groups: wild-type control group, ST2−/− control group, wild-type inflammatory group, and ST2−/− inflammatory group. Acute inflammation was induced by intramuscular injection of turpentine oil, while control groups were injected with saline. After 12 h animals were anesthetized, and the treated tissue, blood and spleen were collected. Iron concentration in the treated tissue, hemoglobin blood concentration, mean corpuscular hemoglobin (MCH), hematocrit, erythrocyte, neutrophil and lymphocyte blood count, and erythrocytes percentage in spleen were determined. Results: Iron concentration in the treated tissue was significantly higher in wild-type inflammatory group (WT-I) when compared to both, the wild-type control group (WT-C) and ST2−/− inflammatory group (KO-I). There was no significant difference in iron concentration between ST2−/− control group (KO-C) and the KO-I. MCH had significantly decreased in WT-I when compared to WT-C, while there was no significant difference between KO-C and KO-I. Hemoglobin blood concentration significantly increased in KO-I in comparison to KO-C, while it did not significantly differ between WT-I and KO-I. Erythrocyte count and hematocrit had significantly increased, while the percentage of erythrocytes in spleen decreased in both inflammatory groups when compared to their controls. Neutrophil count significantly decreased in WT-I, when compared to WT-C. Lymphocyte count decreased in both inflammatory groups when compared to their controls. Conclusion: Results of this study indicate that the IL-33/ST2 axis could have a role in the alteration of iron in acute inflammation, namely in an increase of iron concentration at the site of acute inflammation and a decrease of blood mean corpuscular hemoglobin. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Acute inflammation is an early response to cell injury, whose main purpose is to eliminate the cause of injury. However, inflammation can also cause harm to an organism and it is a basis of different diseases. It is important to comprehend different mechanisms of acute inflammation, since improved understanding of pathogenesis of acute inflammation
Abbreviations: WT-C, wild-type control group; KO-C, ST2−/− control group; WT-I, wild-type inflammatory group; KO-I, ST2−/− inflammatory group; MCH, mean corpuscular hemoglobin;. ⁎ Corresponding author. E-mail addresses: [email protected] (M.S. Stankovic), [email protected] (A.M. Trbovich).
http://dx.doi.org/10.1016/j.yexmp.2015.11.016 0014-4800/© 2015 Elsevier Inc. All rights reserved.
may improve therapy, and facilitate prevention of harmful effects and modulation of further course. Some of the key events in acute inflammation include alterations of leukocytes, iron homeostasis and hemoglobin (Mitchell and Cotran, 2003; Sukumaran et al., 2012). Hemoglobin is an iron-containing protein. Most of the iron in organism is, actually, incorporated in hemoglobin found in erythrocytes. Iron balance shifts in acute inflammation, which is characterized by modulation of cytokine profile and acute phase proteins that leads to lower iron concentration in blood and increased iron storage (Sukumaran et al., 2012; D'Anna et al., 2011). Although iron is a necessary element in organism, it can lead to cell damage due to its role in production of free radicals and oxidative stress (Djordjevich et al., 2012). Leukocyte extravasation and alteration of leukocyte blood count also occur in acute inflammation due to release of mediators of inflammation.
688
M.S. Stankovic et al. / Experimental and Molecular Pathology 99 (2015) 687–692
Leukocytes at the site of acute inflammation contribute to the removal of the initial cause of cell injury and of the necrotic cells. Neutrophils are the first line of leukocytes that alter in acute inflammation, while lymphocytes are, at the beginning, slightly changed (Mitchell and Cotran, 2003). IL-33 is a member of IL-1 cytokine family, and it is a specific ligand for the ST2 receptor (Talabot-Ayer et al., 2014; Perbal, 2006). Since 2005, when IL-33 was discovered (Schmitz et al., 2005), a role of IL-33 and ST2 was examined in different diseases and disorders, such as asthma (Bartemes and Kita, 2012), atherogenesis (McLaren et al., 2010), tumors (Liu et al., 2014), and autoimmune uveitis (Barbour et al., 2014). IL-33/ST2 is implicated in Th2 immune response, but it was also shown that IL-33/ST2 affected neutrophil alteration and chemotaxis in infection and rheumatoid arthritis (Le et al., 2012; Verri et al., 2010). To our knowledge, the effects of IL-33/ST2 axis on iron and hemoglobin alteration in acute inflammation have not yet been examined. The aim of this study was to examine the role of the IL-33/ST2 pathway in pathogenesis of acute inflammation by revealing its possible role in alteration of hemoglobin concentration in blood, mean corpuscular hemoglobin and iron concentration at the site of acute inflammation, as well as erythrocyte, neutrophil and lymphocyte blood count in experimental model of acute inflammation. 2. Material and methods 2.1. Animal model BALB/c and ST2 knockout BALB/c male mice, purchased from the Military Medical Academy in Belgrade, Serbia, were used in this study. All animals were 8–9 weeks old and were allowed to acclimatize for one week prior to experiments. Animal facility provided standard conditions, temperature 22 ± 2 °C, relative humidity 50%, 12 h light/dark cycle, and ad libitum access to fresh water and food pellets. This experiment was approved by the University of Belgrade Faculty of Medicine Ethics Committee. Animals were randomly divided into four groups: WT-C (wild-type control group), KO-C (ST2−/− control group), WT-I (wild-type inflammatory group), and KO-I (ST2 −/− inflammatory group). There was no difference in age, and body mass among the groups. The control group received 10 ml/kg of saline (Hemofarm, Vrsac, Serbia) into the right and the left hind limb muscles, while acute inflammation in WT-I and KO-I was induced by intramuscular injection of turpentine oil (Sigma-Aldrich, Munich, Germany) of the same dosage and procedure (Sultan et al., 2012). After 12 h, there was no difference in body mass among groups, and animals received anesthetics, 100 mg/kg of ketamine hydrochloride (i.p., Ketamidor, Richter Pharma, Wels, Austria) and 5 mg/kg of xylazine (i.p., Rompun, Bayer, Leverkusen, Germany). Treated muscle, blood from the heart and spleen were collected. 2.2. Histopathology Parts of the treated muscles were fixed in 4% formaldehyde (Centrohem, Stara Pazova, Serbia), paraffin-embedded and sliced by microtome (Leica CM1800, Leica Microsystems, Nussloch, Germany). Sections were mounted on slides (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) and stained with hematoxylin (Bio-Optica, Milano, Italy) and eosin (MP Biomedicals LLC, France). Analysis of slides was performed by two independent histopathologists, who used a light microscope Olympus BX41 (Olympus America Inc., Melville, NY, USA). Micrographs were taken by digital camera (Olympus America Inc., Melville, NY, USA). 2.3. Tissue iron concentration Part of the treated muscle, not fixed in formaldehyde, was used for determination of iron concentration. The tissue was treated with nitric
acid (Zorka Pharma, Sabac, Serbia) and 30% hydrogen peroxide (Zorka Pharma, Sabac, Serbia), in ratio 0.5 g of the tissue with 8 ml of nitric acid and 2 ml of 30% hydrogen peroxide, and digested in microwave (ETHOS TC, Milestone S.r.l., Sorisole, Italy) at room temperature of 180 °C for 5 min and then at 180 °C for 10 min. When temperature protocol was completed, the digested tissue was cooled down and transferred into a 50 ml flask (Sigma-Aldrich, St. Louis, Missouri, USA) with deionisated water. The sample was analyzed by the atomic absorption spectrometer “SpectrAA 220” (Varian, Palo Alto, California, USA), respecting Varian Atomic Absorption Spectrometers (AAS) Analytical Methods, and iron concentration was expressed as milligram of iron per kilogram of dried analyzed tissue. 2.4. Hematological parameters Blood collected from the heart was analyzed by a hematological counter (ABX Pentra 80X, Montpellier, France), and blood hemoglobin concentration, mean corpuscular hemoglobin (MCH), hematocrit, erythrocyte, neutrophil and lymphocyte count were determined according to the manufacturer's instructions. Spleen was excided, and after removing connective and fat tissue, part of the spleen was macerated, dispersed through 0.1 mm cell sieve and used for preparation of smears. Spleen smears were stained with May-Grünwald-Giemsa (Carlo Erba, Rodano, Italy) and used for counting 100 nucleated cells per visual field in ten visual fields and determination of erythrocytes' percentage of all counted cells (Djordjevich et al., 2012). 2.5. Statistical analysis Differences among groups were tested by Student's t-test or Mann–Whitney–Wilcoxon test for unpaired groups, depending on distribution of values. Data were presented as mean ± SEM, and p b 0.05 was considered as statistically significant. Statistical analysis was performed by SPSS 15.0 (Chicago, IL, USA). 3. Results Inflammatory infiltration was histopathologically confirmed in both inflammatory groups, WT-I and KO-I, while the examined tissue in WT-C and KO-C was preserved and without inflammatory infiltration (Fig. 1). Iron concentration in the wild-type inflamed tissue (WT-I, 13.781 ± 0.876 mg/kg) was significantly higher than in either control group (WT-C, 10.646 ± 1.007 mg/kg) or inflamed tissue in knockout animals (KO-I, 10.857 ± 0.681 mg/kg). In addition, there was no significant difference in iron concentration in treated tissue between WT-C and KO-C (10.544 ± 0.605 mg/kg) (Fig. 2, Panel A). Mean corpuscular hemoglobin (MCH) significantly decreased in WT-I (17.4 ± 0221 pg) when compared to WT-C (18.143 ± 0.143 pg), while there was no significant difference between KO-C (16.143 ± 0.143 pg) and KO-I (16.222 ± 0.147 pg). MCH was significantly lower in KO-C when compared to WT-C, and in KO-I when compared to WT-I (Fig. 2, Panel B). In comparison to KO-C (107.143 ± 6.801 g/l), total hemoglobin concentration in blood significantly increased in KO-I (134.444 ± 4.747 g/l). Hemoglobin concentration in blood did not significantly differ between WT-I (142 ± 3.59 g/l) and KO-I, while it was significantly lower in KO-C, when compared to WT-C (137.143 ± 1.844 g/l) (Fig. 2, Panel C). Erythrocyte count and hematocrit significantly increased in both inflammatory groups — WT-I (8.19 ± 0.185 × 1012/l; 0.399 ± 0.009, respectively) and KO-I (8.289 ± 0.295 × 1012/l; 0.402 ± 0.014, respectively), when compared to their controls WT-C (7.443 ± 0.1 × 1012/l; 0.361 ± 0.004, respectively) and KO-C (6.643 ± 0.403 × 1012/l; 0.32 ± 0.02, respectively), respectively (Fig. 3, Panel A and Panel B). Percentage of erythrocytes in spleen decreased in WT-I (20.722 ± 1.424%) when compared to
M.S. Stankovic et al. / Experimental and Molecular Pathology 99 (2015) 687–692
689
Fig. 1. Histopathological analysis of the tissue of wild-type control group (Panel A), ST2−/− control group (Panel B), wild-type inflammatory group (Panel C), and ST2−/− inflammatory group (Panel D). Intact tissue devoid of inflammatory infiltration at Panel A and Panel B. Presence of necrotic cells and inflammatory infiltration at Panel C and Panel D.
WT-C (45.04 ± 3.097%), and also in KO-I (20.544 ± 2.412%) in comparison to KO-C (46.23 ± 2.236%) (Fig. 3, Panel C). Neutrophil count significantly decreased in WT-I (0.069 ± 0.021 × 10 9 /l), when compared to WT-C (0.166 ± 0.03 × 10 9 /l) (Fig. 4, Panel A). There was no significant difference in neutrophil count between WT-I and KO-I (0.073 ± 0.019 × 109/l). Neutrophil count had a tendency to decrease in KO-I when compared to KO-C (0.134 ± 0.029 × 109/l) (Fig. 4, Panel A). Lymphocyte count decreased in WT-I (1.272 ± 0.143 × 109/l) and KO-I (1.059 ± 0.131 × 109/l), when compared to WT-C (3.266 ± 0.476 × 109/l) and KO-C (2.807 ± 0.425 × 109/l), respectively. Significant difference was not observed when WT-C was compared to KO-C, or when WT-I was compared to KO-I (Fig. 4, Panel B). 4. Discussion To our knowledge, this is the first study that reports that IL-33/ST2 axis is involved in an increase of iron concentration at the site of acute inflammation, and in a decrease of mean corpuscular hemoglobin (MCH) in acute inflammation. In our research, an experimental model of acute inflammation was induced in male, wild-type and ST2 knockout BALB/c mice. After 12 h, blood, treated tissue and spleen were sampled. We determined iron concentration in the treated tissue, blood hemoglobin concentration, mean corpuscular hemoglobin (MCH), hematocrit, erythrocyte, neutrophil and lymphocyte count, and the percentage of erythrocytes in spleen.
Hemoglobin consists of globin and heme, and heme is composed of porphyrin and iron. Maintaining iron homeostasis is complex, aiming for a fine balance between iron in tissues and iron in hemoglobin (Sukumaran et al., 2012). Iron balance has a special place in acute inflammation, since iron is involved in oxidative stress, which may cause tissue and cell damage (Lagan et al., 2008). Certain cytokines and acute-phase proteins are included in regulation of iron concentration in acute inflammation, such as IL-6, IL-10, hepcidin, ferritin, transferrin etc. (Sukumaran et al., 2012; Lagan et al., 2008; Malik et al., 2011). We here showed for the first time that IL-33/ST2 axis was involved in an increase of iron concentration at the site of acute inflammation, since in acute inflammation iron concentration increased in wild-type mice, while iron concentration remained the same in ST2−/− mice in acute inflammation at 12 h. Moreover, tissue iron concentration was significantly higher in wild-type animals than in ST2 deficient animals in acute inflammation. On the other hand, mean corpuscular hemoglobin (MCH) decreased in wild-type mice in acute inflammation, but it did not significantly change in ST2 deficient mice in acute inflammation. In inflammation, due to release of inflammatory cytokines and acute-phase proteins, iron is retained in the tissue (D'Anna et al., 2011; Darshan et al., 2010). Furthermore, inflammatory cells are migrating in the affected areas and deposit iron (Andersen et al., 2014), and all these changes may result in increased iron concentration in the inflamed tissue (Van Snick et al., 1974; Andersen et al., 2014). Increase of iron concentration in the inflamed tissue was reported in
690
M.S. Stankovic et al. / Experimental and Molecular Pathology 99 (2015) 687–692
Fig. 2. Concentration of iron in the treated tissue, mean corpuscular hemoglobin and concentration of hemoglobin in blood in acute inflammation in wild-type and ST2−/− mice. Panel A. Iron in the treated tissue was significantly higher in wild-type inflammatory group (WT-I) when compared to wild-type control group (WT-C) and ST2 −/− inflammatory group (KO-I) (p˂ 0,05; Student's t-test). WT-C (n = 8), ST2 −/− control group (KO-C) (n = 9), WT-I (n = 8), KO-I (n = 9). Panel B. Mean corpuscular hemoglobin (MCH) significantly decreased in WT-I when compared to WT-C (p˂ 0,05). No significant difference between KO-C and KO-I. MCH was significantly lower in KO-C and KO-I, when compared to WT-C and WT-I, respectively. Panel C. Concentration of hemoglobin in blood was significantly lower in KO-C when compared to WT-C and KO-I (p˂ 0,01). [Panel B and Panel C. Data were analyzed by Mann–Whitney–Wilcoxon test. WT-C (n = 7), KO-C (n = 7), WT-I (n = 10), KO-I (n = 9)]. * compared to WT-C; ‡ compared to KO-C; § compared to WT-I.
Fig. 3. Erythrocyte count, hematocrit and percentage of erythrocytes in spleen in acute inflammation in wild-type and ST2−/− mice. Panel A. Erythrocyte count was significantly higher in wild-type inflammatory group (WT-I) and ST2−/− inflammatory group (KO-I), when compared to wild-type control group (WT-C) and ST2−/− control group (KO-C), respectively (p˂ 0,01). Panel B. Hematocrit was significantly higher in WT-I and KO-I, when compared to WT-C and KO-C, respectively (p˂ 0,01). [Panel A and Panel B. Statistical analysis by Mann–Whitney–Wilcoxon test. WT-C (n = 7), KO-C (n = 7), WT-I (n = 10), KO-I (n = 9)]. Panel C. Percentage of erythrocytes in spleen decreased in WT-I when compared to WT-C, and in KO-I in comparison to KO-C (p˂ 0,01). Data were analyzed by Student's t-test. WT-C (n = 10), KO-C (n = 10), WT-I (n = 9), KO-I (n = 9). * compared to WT-C; ‡ compared to KO-C.
different inflammatory disorders, i.e. in synovial tissue in rheumatoid arthritis (Hider and Singh, 2012), in bowel inflammatory disorders and inflamed brain tissue (Andersen et al., 2014) etc. Our results of
M.S. Stankovic et al. / Experimental and Molecular Pathology 99 (2015) 687–692
691
still to be explained. In addition, we reported that erythrocyte count and hematocrit increased, whereas the percentage of erythrocyte in spleen decreased in both inflammatory groups, when compared to their controls. It was shown that erythrocyte count is increasing in stress and after catecholamine release as a result of spleen contraction (Thornton et al., 2001; Lai et al., 2006). Since acute inflammation is a stressful condition, our results were in accordance with literature data. Total hemoglobin concentration in blood in wild-type animals did not significantly change after inducing acute inflammation, while blood hemoglobin concentration increased in ST2 knockout mice. The results of total hemoglobin concentration could be a consequence of the erythrocyte count increase in both inflammatory groups; however, MCH decreased only in wild-type animals after inducing inflammatory model, while MCH remained the same in ST2 deficient mice after inducing acute inflammation. Neutrophil count in blood significantly decreased in wild-type mice in acute inflammation, while there was tendency of neutrophil count decrease in ST2 deficient mice in acute inflammation. However, there was no significant difference in neutrophil count between wild-type and ST2 deficient animals in acute inflammation. Lymphocyte count decreased in both inflammatory groups. Since lymphocytes are predominant leukocyte lineage in rodents (Russell and Bernstein, 1966) we expected that lymphocyte count would change. Although it was shown that IL-33 had a role in chemotaxis of neutrophils in Candida albicans infection (Le et al., 2012), in our study IL-33/ST2 pathway had not caused any differences in neutrophil and lymphocyte count. Perhaps, if the experimental model of acute inflammation lasted longer, the effects of IL-33/ST2 axis on alteration of neutrophils and lymphocytes number in blood would also be detected. In inflammation, blood leukocytes count might decrease due to their massive emigration to the site of injury, where they are needed as a defense mechanism (Gaspard and Twite, 2005). In that respect, we confirmed mixed inflammatory infiltration in the inflamed tissue of both inflammatory groups.
Fig. 4. Neutrophil and lymphocytes count in acute inflammation in wild-type and ST2 −/− mice. Panel A. Neutrophil count significantly decreased in WT-I when compared to WT-C (p˂ 0,05). The same tandency was observed in knockout animals but it was without statistical significance. Panel B. Lymphocyte count significantly decreased in WT-I and KO-I when compared to WT-C (p˂ 0,01) and KO-C (p˂ 0,01), respectively. Statistical analysis by Mann–Whitney–Wilcoxon test. WT-C (n = 7), KO-C (n = 7), WT-I (n = 10), KO-I (n = 9). * compared to WT-C; ‡ compared to KO-C.
increased iron concentration in the inflamed tissue in wild-type mice corroborate these findings. As noted above, the importance of iron in the tissue is that it may cause additional tissue and cell damage (Lagan et al., 2008), which may contribute to a more severe course of inflammation. On the other hand, hemoglobin synthesis is dependent on iron availability (Gaspard and Twite, 2005). Hemoglobin synthesis starts at early erythroblast stage, and continues even during final maturation of reticulocytes to erythrocytes (Gaspard and Twite, 2005). In inflammation, due to increased iron storage in the tissue (Darshan et al., 2010), decreased release of iron from macrophages and decreased iron absorption through gastrointestinal tract (Roy, 2010), less iron is available for hemoglobin synthesis (Nemeth et al., 2004). Our results of decreased mean corpuscular hemoglobin (MCH) in wild-type inflammatory group are in accordance with these findings. However, in our experiment, an increase of iron concentration in the inflamed tissue, and a decrease of MCH detected in wild-type group, did not occur in ST2 knock out mice. This is the first time that the involvement of IL-33/ST2 pathway in iron alteration in acute inflammation is evidenced. IL-33/ST2 pathway may have a direct effect on iron homeostasis and its redistribution and/or it may affect cytokines or some acute-phase proteins that have a role in iron storage and balancing during acute inflammation. We also observed that blood hemoglobin and MCH were lower in ST2 deficient control animals, when compared to wild-type control animals. These differences are
5. Conclusion To our knowledge, the results of this study indicate for the first time, that IL-33/ST2 axis could have a role in alteration of iron in acute inflammation, namely in an increase of iron concentration at the site of injury and a decrease of mean corpuscular hemoglobin in blood. Acknowledgments This research was funded by the Ministry of Education, Science and Technological Development (RS, grant III 41013). References Andersen, H.H., Johnsen, K.B., Moos, T., 2014. Iron deposits in the chronically inflamed central nervous system and contributes to neurodegeneration. Cell. Mol. Life Sci. 71, 1607–1622. Barbour, M., Allan, D., Xu, H., Pei, C., Chen, M., Niedbala, W., Fukada, S.Y., Besnard, A.G., Alves-Filho, J.C., Tong, X., Forrester, J.V., Liew, F.Y., Jiang, H.R., 2014. IL-33 attenuates the development of experimental autoimmune uveitis. Eur. J. Immunol. 44, 3320–3329. Bartemes, K.R., Kita, H., 2012. Dynamic role of epithelium-derived cytokines in asthma. Clin. Immunol. 143, 222–235. D'Anna, M.C., Giorgi, G., Roque, M.E., 2011. Immunohistochemical studies on duodenum, spleen and liver in mice: distribution of ferroportin and prohepcidin in an inflammation model. Int. J. Morphol. 29, 747–753. Darshan, D., Frazer, D.M., Wilkins, S.J., Anderson, G.J., 2010. Severe iron deficiency blunts the response of the iron regulatory gene Hamp and pro-inflammatory cytokines to lipopolysaccharide. Haematologica 95, 1660–1667. Djordjevich, D.M., De Luka, S.R., Milovanovich, I.D., Janković, S., Stefanović, S., Vesković-Moračanin, S., Cirković, S., Ilić, A.Ž., Ristić-Djurović, J.L., Trbovich, A.M., 2012. Hematological parameters' changes in mice subchronically exposed to static magnetic fields of different orientations. Ecotoxicol. Environ. Saf. 81, 98–105. Gaspard, K.J., Twite, K., 2005. Hematopoietic Function. In: Porth, C.M. (Ed.), Pathophysiology: Concept of Altered Health States. Lippincott Williams & Wilkins, Philadelphia, pp. 277–337.
692
M.S. Stankovic et al. / Experimental and Molecular Pathology 99 (2015) 687–692
Hider, R.C., Singh, S., 2012. Iron chelators of clinical significance. In: Silver, J. (Ed.), Chemistry of Iron, Springer Science & Business Media, New York, ch. 8. Lagan, A.L., Melley, D.D., Evans, T.W., Quinlan, G.J., 2008. Pathogenesis of the systemic inflammatory syndrome and acute lung injury: role of iron mobilization and decompartmentalization. Am. J. Physiol. Lung Cell. Mol. Physiol. 294, L161–L174. Lai, J.C., Kakuta, I., Mok, H.O., Rummer, J.L., Randall, D., 2006. Effects of moderate and substantial hypoxia on erythropoietin levels in rainbow trout kidney and spleen. J. Exp. Biol. 209, 2734–2738. Le, H.T., Tran, V.G., Kim, W., Kim, J., Cho, H.R., Kwon, B., 2012. IL-33 priming regulates multiple steps of the neutrophil-mediated anti-Candida albicans response by modulating TLR and dectin-1 signals. J. Immunol. 189, 287–295. Liu, J., Shen, J.X., Hu, J.L., Huang, W.H., Zhang, G.J., 2014. Significance of interleukin-33 and its related cytokines in patients with breast cancers. Front. Immunol. 5, 141. Malik, I.A., Naz, N., Sheikh, N., Khan, S., Moriconi, F., Blaschke, M., Ramadori, G., 2011. Comparison of changes in gene expression of transferrin receptor-1 and other iron-regulatory proteins in rat liver and brain during acute-phase response. Cell Tissue Res. 344, 299–312. McLaren, J.E., Michael, D.R., Salter, R.C., Ashlin, T.G., Calder, C.J., Miller, A.M., Liew, F.Y., Ramji, D.P., 2010. IL-33 reduces macrophage foam cell formation. J. Immunol. 185, 1222–1229. Mitchell, R., Cotran, R., 2003. Acute and Chronic Inflammation. In: Cotran, R., Robbins, S.L., Kumar, V. (Eds.), Robbins Basic Pathology. W.B. Saunders, Philadelphia, pp. 33–34. Nemeth, E., Rivera, S., Gabayan, V., Keller, C., Taudorf, S., Pedersen, B.K., Ganz, T., 2004. IL-6 mediates hypoferremia of inflammation by inducing the synthesis of the iron regulatory hormone hepcidin. J. Clin. Invest. 113, 1271–1276.
Perbal, B., 2006. New insight into CCN3 interactions–nuclear CCN3 : fact or fantasy? Cell Commun. Signal. 4, 6. Roy, C.N., 2010. Anemia of inflammation. Hematol. Am. Soc. Hematol. Educ. Program. 276–280. Russell, E.S., Bernstein, S.E., 1966. Blood and Blood Formation. In: Green, E.L. (Ed.), Biology of the Laboratory Mouse. Dover publications Inc., New York ch. 17. Schmitz, J., Owyang, A., Oldham, E., Song, Y., Murphy, E., McClanahan, T.K., Zurawski, G., Moshrefi, M., Qin, J., Li, X., Gorman, D.M., Bazan, J.F., Kastelein, R.A., 2005. IL-33, an interleukin-1-like cytokine that signals via the IL-1 receptor-related protein ST2 and induces T helper type 2-associated cytokines. Immunity 23, 479–490. Sukumaran, A., Venkatraman, A., Jacob, M., 2012. Inflammation-induced effects on iron-related proteins in splenic macrophages and the liver in mice. Blood Cells Mol. Dis. 49, 11–19. Sultan, S., Pascucci, M., Ahmad, S., Malik, I.A., Bianchi, A., Ramadori, P., Ahmad, G., Ramadori, G., 2012. LIPOCALIN-2 is a major acute-phase protein in a rat and mouse model of sterile abscess. Shock 37, 191–196. Talabot-Ayer, D., Martin, P., Seemayer, C.A., Vigne, S., Lamacchia, C., Finckh, A., Saiji, E., Gabay, C., Palmer, G., 2014. Immune-mediated experimental arthritis in IL-33 deficient mice. Cytokine 69, 68–74. Thornton, S.J., Spielman, D.M., Pelc, N.J., Block, W.F., Crocker, D.E., Costa, D.P., LeBoeuf, B.J., Hochachka, P.W., 2001. Effects of forced diving on the spleen and hepatic sinus in northern elephant seal pups. Proc. Natl. Acad. Sci. U. S. A. 98, 9413–9418. Van Snick, J.L., Masson, P.L., Heremans, J.F., 1974. The involvement of lactoferrin in the hyposideremia of acute inflammation. J. Exp. Med. 140, 1068–1084. Verri Jr., W.A., Souto, F.O., Vieira, S.M., Almeida, S.C., Fukada, S.Y., Xu, D., Alves-Filho, J.C., Cunha, T.M., Guerrero, A.T., Mattos-Guimaraes, R.B., Oliveira, F.R., Teixeira, M.M., Silva, J.S., McInnes, I.B., Ferreira, S.H., Louzada-Junior, P., Liew, F.Y., Cunha, F.Q., 2010. IL-33 induces neutrophil migration in rheumatoid arthritis and is a target of anti-TNF therapy. Ann. Rheum. Dis. 69, 1697–1703.
Contents lists available at ScienceDirect
Experimental and Molecular Pathology journal homepage: www.elsevier.com/locate/yexmp
Effects of Il-33/St2 pathway on alteration of iron and hematological parameters in acute inflammation Marija S. Stankovic a, Vladimir Turuntas b, Silvio R. De Luka a, Sasa Jankovic c, Srdjan Stefanovic c, Nela Puskas d, Ivan Zaletel d, Sanja Milutinović-Smiljanic e, Alexander M. Trbovich a,⁎ a
Department of Pathophysiology, Faculty of Medicine, University of Belgrade, Dr Subotica 8, 11000, Belgrade, Serbia Pediatrics, University hospital Foca, Studentska 5, 73300 Foca, Bosnia and Herzegovina Institute of Meat Hygiene and Technology, Kacanskog 13, 11000, Belgrade, Serbia d Institute of Histology and Embryology “Aleksandar Đ. Kostić”, School of Medicine, University of Belgrade, Dr Subotica 8, 11000, Belgrade, Serbia e General and Oral Histology and Embryology, Faculty of Dentistry, University of Belgrade, Dr Subotica 8, 11000, Belgrade, Serbia b c
a r t i c l e
i n f o
Article history: Received 3 July 2015 and in revised form 3 October 2015 Accepted 9 November 2015 Available online 11 November 2015 Keywords: Acute inflammation IL-33/ST2 axis Iron Hematological parameters
a b s t r a c t Aim: The aim of this study was to examine the role of the IL-33/ST2 pathway in pathogenesis of acute inflammation by investigating its possible role in alteration of iron and hematological parameters in experimental model of acute inflammation. Material and methods: Wild-type and ST2 knockout BALB/c mice were divided into four groups: wild-type control group, ST2−/− control group, wild-type inflammatory group, and ST2−/− inflammatory group. Acute inflammation was induced by intramuscular injection of turpentine oil, while control groups were injected with saline. After 12 h animals were anesthetized, and the treated tissue, blood and spleen were collected. Iron concentration in the treated tissue, hemoglobin blood concentration, mean corpuscular hemoglobin (MCH), hematocrit, erythrocyte, neutrophil and lymphocyte blood count, and erythrocytes percentage in spleen were determined. Results: Iron concentration in the treated tissue was significantly higher in wild-type inflammatory group (WT-I) when compared to both, the wild-type control group (WT-C) and ST2−/− inflammatory group (KO-I). There was no significant difference in iron concentration between ST2−/− control group (KO-C) and the KO-I. MCH had significantly decreased in WT-I when compared to WT-C, while there was no significant difference between KO-C and KO-I. Hemoglobin blood concentration significantly increased in KO-I in comparison to KO-C, while it did not significantly differ between WT-I and KO-I. Erythrocyte count and hematocrit had significantly increased, while the percentage of erythrocytes in spleen decreased in both inflammatory groups when compared to their controls. Neutrophil count significantly decreased in WT-I, when compared to WT-C. Lymphocyte count decreased in both inflammatory groups when compared to their controls. Conclusion: Results of this study indicate that the IL-33/ST2 axis could have a role in the alteration of iron in acute inflammation, namely in an increase of iron concentration at the site of acute inflammation and a decrease of blood mean corpuscular hemoglobin. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Acute inflammation is an early response to cell injury, whose main purpose is to eliminate the cause of injury. However, inflammation can also cause harm to an organism and it is a basis of different diseases. It is important to comprehend different mechanisms of acute inflammation, since improved understanding of pathogenesis of acute inflammation
Abbreviations: WT-C, wild-type control group; KO-C, ST2−/− control group; WT-I, wild-type inflammatory group; KO-I, ST2−/− inflammatory group; MCH, mean corpuscular hemoglobin;. ⁎ Corresponding author. E-mail addresses: [email protected] (M.S. Stankovic), [email protected] (A.M. Trbovich).
http://dx.doi.org/10.1016/j.yexmp.2015.11.016 0014-4800/© 2015 Elsevier Inc. All rights reserved.
may improve therapy, and facilitate prevention of harmful effects and modulation of further course. Some of the key events in acute inflammation include alterations of leukocytes, iron homeostasis and hemoglobin (Mitchell and Cotran, 2003; Sukumaran et al., 2012). Hemoglobin is an iron-containing protein. Most of the iron in organism is, actually, incorporated in hemoglobin found in erythrocytes. Iron balance shifts in acute inflammation, which is characterized by modulation of cytokine profile and acute phase proteins that leads to lower iron concentration in blood and increased iron storage (Sukumaran et al., 2012; D'Anna et al., 2011). Although iron is a necessary element in organism, it can lead to cell damage due to its role in production of free radicals and oxidative stress (Djordjevich et al., 2012). Leukocyte extravasation and alteration of leukocyte blood count also occur in acute inflammation due to release of mediators of inflammation.
688
M.S. Stankovic et al. / Experimental and Molecular Pathology 99 (2015) 687–692
Leukocytes at the site of acute inflammation contribute to the removal of the initial cause of cell injury and of the necrotic cells. Neutrophils are the first line of leukocytes that alter in acute inflammation, while lymphocytes are, at the beginning, slightly changed (Mitchell and Cotran, 2003). IL-33 is a member of IL-1 cytokine family, and it is a specific ligand for the ST2 receptor (Talabot-Ayer et al., 2014; Perbal, 2006). Since 2005, when IL-33 was discovered (Schmitz et al., 2005), a role of IL-33 and ST2 was examined in different diseases and disorders, such as asthma (Bartemes and Kita, 2012), atherogenesis (McLaren et al., 2010), tumors (Liu et al., 2014), and autoimmune uveitis (Barbour et al., 2014). IL-33/ST2 is implicated in Th2 immune response, but it was also shown that IL-33/ST2 affected neutrophil alteration and chemotaxis in infection and rheumatoid arthritis (Le et al., 2012; Verri et al., 2010). To our knowledge, the effects of IL-33/ST2 axis on iron and hemoglobin alteration in acute inflammation have not yet been examined. The aim of this study was to examine the role of the IL-33/ST2 pathway in pathogenesis of acute inflammation by revealing its possible role in alteration of hemoglobin concentration in blood, mean corpuscular hemoglobin and iron concentration at the site of acute inflammation, as well as erythrocyte, neutrophil and lymphocyte blood count in experimental model of acute inflammation. 2. Material and methods 2.1. Animal model BALB/c and ST2 knockout BALB/c male mice, purchased from the Military Medical Academy in Belgrade, Serbia, were used in this study. All animals were 8–9 weeks old and were allowed to acclimatize for one week prior to experiments. Animal facility provided standard conditions, temperature 22 ± 2 °C, relative humidity 50%, 12 h light/dark cycle, and ad libitum access to fresh water and food pellets. This experiment was approved by the University of Belgrade Faculty of Medicine Ethics Committee. Animals were randomly divided into four groups: WT-C (wild-type control group), KO-C (ST2−/− control group), WT-I (wild-type inflammatory group), and KO-I (ST2 −/− inflammatory group). There was no difference in age, and body mass among the groups. The control group received 10 ml/kg of saline (Hemofarm, Vrsac, Serbia) into the right and the left hind limb muscles, while acute inflammation in WT-I and KO-I was induced by intramuscular injection of turpentine oil (Sigma-Aldrich, Munich, Germany) of the same dosage and procedure (Sultan et al., 2012). After 12 h, there was no difference in body mass among groups, and animals received anesthetics, 100 mg/kg of ketamine hydrochloride (i.p., Ketamidor, Richter Pharma, Wels, Austria) and 5 mg/kg of xylazine (i.p., Rompun, Bayer, Leverkusen, Germany). Treated muscle, blood from the heart and spleen were collected. 2.2. Histopathology Parts of the treated muscles were fixed in 4% formaldehyde (Centrohem, Stara Pazova, Serbia), paraffin-embedded and sliced by microtome (Leica CM1800, Leica Microsystems, Nussloch, Germany). Sections were mounted on slides (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) and stained with hematoxylin (Bio-Optica, Milano, Italy) and eosin (MP Biomedicals LLC, France). Analysis of slides was performed by two independent histopathologists, who used a light microscope Olympus BX41 (Olympus America Inc., Melville, NY, USA). Micrographs were taken by digital camera (Olympus America Inc., Melville, NY, USA). 2.3. Tissue iron concentration Part of the treated muscle, not fixed in formaldehyde, was used for determination of iron concentration. The tissue was treated with nitric
acid (Zorka Pharma, Sabac, Serbia) and 30% hydrogen peroxide (Zorka Pharma, Sabac, Serbia), in ratio 0.5 g of the tissue with 8 ml of nitric acid and 2 ml of 30% hydrogen peroxide, and digested in microwave (ETHOS TC, Milestone S.r.l., Sorisole, Italy) at room temperature of 180 °C for 5 min and then at 180 °C for 10 min. When temperature protocol was completed, the digested tissue was cooled down and transferred into a 50 ml flask (Sigma-Aldrich, St. Louis, Missouri, USA) with deionisated water. The sample was analyzed by the atomic absorption spectrometer “SpectrAA 220” (Varian, Palo Alto, California, USA), respecting Varian Atomic Absorption Spectrometers (AAS) Analytical Methods, and iron concentration was expressed as milligram of iron per kilogram of dried analyzed tissue. 2.4. Hematological parameters Blood collected from the heart was analyzed by a hematological counter (ABX Pentra 80X, Montpellier, France), and blood hemoglobin concentration, mean corpuscular hemoglobin (MCH), hematocrit, erythrocyte, neutrophil and lymphocyte count were determined according to the manufacturer's instructions. Spleen was excided, and after removing connective and fat tissue, part of the spleen was macerated, dispersed through 0.1 mm cell sieve and used for preparation of smears. Spleen smears were stained with May-Grünwald-Giemsa (Carlo Erba, Rodano, Italy) and used for counting 100 nucleated cells per visual field in ten visual fields and determination of erythrocytes' percentage of all counted cells (Djordjevich et al., 2012). 2.5. Statistical analysis Differences among groups were tested by Student's t-test or Mann–Whitney–Wilcoxon test for unpaired groups, depending on distribution of values. Data were presented as mean ± SEM, and p b 0.05 was considered as statistically significant. Statistical analysis was performed by SPSS 15.0 (Chicago, IL, USA). 3. Results Inflammatory infiltration was histopathologically confirmed in both inflammatory groups, WT-I and KO-I, while the examined tissue in WT-C and KO-C was preserved and without inflammatory infiltration (Fig. 1). Iron concentration in the wild-type inflamed tissue (WT-I, 13.781 ± 0.876 mg/kg) was significantly higher than in either control group (WT-C, 10.646 ± 1.007 mg/kg) or inflamed tissue in knockout animals (KO-I, 10.857 ± 0.681 mg/kg). In addition, there was no significant difference in iron concentration in treated tissue between WT-C and KO-C (10.544 ± 0.605 mg/kg) (Fig. 2, Panel A). Mean corpuscular hemoglobin (MCH) significantly decreased in WT-I (17.4 ± 0221 pg) when compared to WT-C (18.143 ± 0.143 pg), while there was no significant difference between KO-C (16.143 ± 0.143 pg) and KO-I (16.222 ± 0.147 pg). MCH was significantly lower in KO-C when compared to WT-C, and in KO-I when compared to WT-I (Fig. 2, Panel B). In comparison to KO-C (107.143 ± 6.801 g/l), total hemoglobin concentration in blood significantly increased in KO-I (134.444 ± 4.747 g/l). Hemoglobin concentration in blood did not significantly differ between WT-I (142 ± 3.59 g/l) and KO-I, while it was significantly lower in KO-C, when compared to WT-C (137.143 ± 1.844 g/l) (Fig. 2, Panel C). Erythrocyte count and hematocrit significantly increased in both inflammatory groups — WT-I (8.19 ± 0.185 × 1012/l; 0.399 ± 0.009, respectively) and KO-I (8.289 ± 0.295 × 1012/l; 0.402 ± 0.014, respectively), when compared to their controls WT-C (7.443 ± 0.1 × 1012/l; 0.361 ± 0.004, respectively) and KO-C (6.643 ± 0.403 × 1012/l; 0.32 ± 0.02, respectively), respectively (Fig. 3, Panel A and Panel B). Percentage of erythrocytes in spleen decreased in WT-I (20.722 ± 1.424%) when compared to
M.S. Stankovic et al. / Experimental and Molecular Pathology 99 (2015) 687–692
689
Fig. 1. Histopathological analysis of the tissue of wild-type control group (Panel A), ST2−/− control group (Panel B), wild-type inflammatory group (Panel C), and ST2−/− inflammatory group (Panel D). Intact tissue devoid of inflammatory infiltration at Panel A and Panel B. Presence of necrotic cells and inflammatory infiltration at Panel C and Panel D.
WT-C (45.04 ± 3.097%), and also in KO-I (20.544 ± 2.412%) in comparison to KO-C (46.23 ± 2.236%) (Fig. 3, Panel C). Neutrophil count significantly decreased in WT-I (0.069 ± 0.021 × 10 9 /l), when compared to WT-C (0.166 ± 0.03 × 10 9 /l) (Fig. 4, Panel A). There was no significant difference in neutrophil count between WT-I and KO-I (0.073 ± 0.019 × 109/l). Neutrophil count had a tendency to decrease in KO-I when compared to KO-C (0.134 ± 0.029 × 109/l) (Fig. 4, Panel A). Lymphocyte count decreased in WT-I (1.272 ± 0.143 × 109/l) and KO-I (1.059 ± 0.131 × 109/l), when compared to WT-C (3.266 ± 0.476 × 109/l) and KO-C (2.807 ± 0.425 × 109/l), respectively. Significant difference was not observed when WT-C was compared to KO-C, or when WT-I was compared to KO-I (Fig. 4, Panel B). 4. Discussion To our knowledge, this is the first study that reports that IL-33/ST2 axis is involved in an increase of iron concentration at the site of acute inflammation, and in a decrease of mean corpuscular hemoglobin (MCH) in acute inflammation. In our research, an experimental model of acute inflammation was induced in male, wild-type and ST2 knockout BALB/c mice. After 12 h, blood, treated tissue and spleen were sampled. We determined iron concentration in the treated tissue, blood hemoglobin concentration, mean corpuscular hemoglobin (MCH), hematocrit, erythrocyte, neutrophil and lymphocyte count, and the percentage of erythrocytes in spleen.
Hemoglobin consists of globin and heme, and heme is composed of porphyrin and iron. Maintaining iron homeostasis is complex, aiming for a fine balance between iron in tissues and iron in hemoglobin (Sukumaran et al., 2012). Iron balance has a special place in acute inflammation, since iron is involved in oxidative stress, which may cause tissue and cell damage (Lagan et al., 2008). Certain cytokines and acute-phase proteins are included in regulation of iron concentration in acute inflammation, such as IL-6, IL-10, hepcidin, ferritin, transferrin etc. (Sukumaran et al., 2012; Lagan et al., 2008; Malik et al., 2011). We here showed for the first time that IL-33/ST2 axis was involved in an increase of iron concentration at the site of acute inflammation, since in acute inflammation iron concentration increased in wild-type mice, while iron concentration remained the same in ST2−/− mice in acute inflammation at 12 h. Moreover, tissue iron concentration was significantly higher in wild-type animals than in ST2 deficient animals in acute inflammation. On the other hand, mean corpuscular hemoglobin (MCH) decreased in wild-type mice in acute inflammation, but it did not significantly change in ST2 deficient mice in acute inflammation. In inflammation, due to release of inflammatory cytokines and acute-phase proteins, iron is retained in the tissue (D'Anna et al., 2011; Darshan et al., 2010). Furthermore, inflammatory cells are migrating in the affected areas and deposit iron (Andersen et al., 2014), and all these changes may result in increased iron concentration in the inflamed tissue (Van Snick et al., 1974; Andersen et al., 2014). Increase of iron concentration in the inflamed tissue was reported in
690
M.S. Stankovic et al. / Experimental and Molecular Pathology 99 (2015) 687–692
Fig. 2. Concentration of iron in the treated tissue, mean corpuscular hemoglobin and concentration of hemoglobin in blood in acute inflammation in wild-type and ST2−/− mice. Panel A. Iron in the treated tissue was significantly higher in wild-type inflammatory group (WT-I) when compared to wild-type control group (WT-C) and ST2 −/− inflammatory group (KO-I) (p˂ 0,05; Student's t-test). WT-C (n = 8), ST2 −/− control group (KO-C) (n = 9), WT-I (n = 8), KO-I (n = 9). Panel B. Mean corpuscular hemoglobin (MCH) significantly decreased in WT-I when compared to WT-C (p˂ 0,05). No significant difference between KO-C and KO-I. MCH was significantly lower in KO-C and KO-I, when compared to WT-C and WT-I, respectively. Panel C. Concentration of hemoglobin in blood was significantly lower in KO-C when compared to WT-C and KO-I (p˂ 0,01). [Panel B and Panel C. Data were analyzed by Mann–Whitney–Wilcoxon test. WT-C (n = 7), KO-C (n = 7), WT-I (n = 10), KO-I (n = 9)]. * compared to WT-C; ‡ compared to KO-C; § compared to WT-I.
Fig. 3. Erythrocyte count, hematocrit and percentage of erythrocytes in spleen in acute inflammation in wild-type and ST2−/− mice. Panel A. Erythrocyte count was significantly higher in wild-type inflammatory group (WT-I) and ST2−/− inflammatory group (KO-I), when compared to wild-type control group (WT-C) and ST2−/− control group (KO-C), respectively (p˂ 0,01). Panel B. Hematocrit was significantly higher in WT-I and KO-I, when compared to WT-C and KO-C, respectively (p˂ 0,01). [Panel A and Panel B. Statistical analysis by Mann–Whitney–Wilcoxon test. WT-C (n = 7), KO-C (n = 7), WT-I (n = 10), KO-I (n = 9)]. Panel C. Percentage of erythrocytes in spleen decreased in WT-I when compared to WT-C, and in KO-I in comparison to KO-C (p˂ 0,01). Data were analyzed by Student's t-test. WT-C (n = 10), KO-C (n = 10), WT-I (n = 9), KO-I (n = 9). * compared to WT-C; ‡ compared to KO-C.
different inflammatory disorders, i.e. in synovial tissue in rheumatoid arthritis (Hider and Singh, 2012), in bowel inflammatory disorders and inflamed brain tissue (Andersen et al., 2014) etc. Our results of
M.S. Stankovic et al. / Experimental and Molecular Pathology 99 (2015) 687–692
691
still to be explained. In addition, we reported that erythrocyte count and hematocrit increased, whereas the percentage of erythrocyte in spleen decreased in both inflammatory groups, when compared to their controls. It was shown that erythrocyte count is increasing in stress and after catecholamine release as a result of spleen contraction (Thornton et al., 2001; Lai et al., 2006). Since acute inflammation is a stressful condition, our results were in accordance with literature data. Total hemoglobin concentration in blood in wild-type animals did not significantly change after inducing acute inflammation, while blood hemoglobin concentration increased in ST2 knockout mice. The results of total hemoglobin concentration could be a consequence of the erythrocyte count increase in both inflammatory groups; however, MCH decreased only in wild-type animals after inducing inflammatory model, while MCH remained the same in ST2 deficient mice after inducing acute inflammation. Neutrophil count in blood significantly decreased in wild-type mice in acute inflammation, while there was tendency of neutrophil count decrease in ST2 deficient mice in acute inflammation. However, there was no significant difference in neutrophil count between wild-type and ST2 deficient animals in acute inflammation. Lymphocyte count decreased in both inflammatory groups. Since lymphocytes are predominant leukocyte lineage in rodents (Russell and Bernstein, 1966) we expected that lymphocyte count would change. Although it was shown that IL-33 had a role in chemotaxis of neutrophils in Candida albicans infection (Le et al., 2012), in our study IL-33/ST2 pathway had not caused any differences in neutrophil and lymphocyte count. Perhaps, if the experimental model of acute inflammation lasted longer, the effects of IL-33/ST2 axis on alteration of neutrophils and lymphocytes number in blood would also be detected. In inflammation, blood leukocytes count might decrease due to their massive emigration to the site of injury, where they are needed as a defense mechanism (Gaspard and Twite, 2005). In that respect, we confirmed mixed inflammatory infiltration in the inflamed tissue of both inflammatory groups.
Fig. 4. Neutrophil and lymphocytes count in acute inflammation in wild-type and ST2 −/− mice. Panel A. Neutrophil count significantly decreased in WT-I when compared to WT-C (p˂ 0,05). The same tandency was observed in knockout animals but it was without statistical significance. Panel B. Lymphocyte count significantly decreased in WT-I and KO-I when compared to WT-C (p˂ 0,01) and KO-C (p˂ 0,01), respectively. Statistical analysis by Mann–Whitney–Wilcoxon test. WT-C (n = 7), KO-C (n = 7), WT-I (n = 10), KO-I (n = 9). * compared to WT-C; ‡ compared to KO-C.
increased iron concentration in the inflamed tissue in wild-type mice corroborate these findings. As noted above, the importance of iron in the tissue is that it may cause additional tissue and cell damage (Lagan et al., 2008), which may contribute to a more severe course of inflammation. On the other hand, hemoglobin synthesis is dependent on iron availability (Gaspard and Twite, 2005). Hemoglobin synthesis starts at early erythroblast stage, and continues even during final maturation of reticulocytes to erythrocytes (Gaspard and Twite, 2005). In inflammation, due to increased iron storage in the tissue (Darshan et al., 2010), decreased release of iron from macrophages and decreased iron absorption through gastrointestinal tract (Roy, 2010), less iron is available for hemoglobin synthesis (Nemeth et al., 2004). Our results of decreased mean corpuscular hemoglobin (MCH) in wild-type inflammatory group are in accordance with these findings. However, in our experiment, an increase of iron concentration in the inflamed tissue, and a decrease of MCH detected in wild-type group, did not occur in ST2 knock out mice. This is the first time that the involvement of IL-33/ST2 pathway in iron alteration in acute inflammation is evidenced. IL-33/ST2 pathway may have a direct effect on iron homeostasis and its redistribution and/or it may affect cytokines or some acute-phase proteins that have a role in iron storage and balancing during acute inflammation. We also observed that blood hemoglobin and MCH were lower in ST2 deficient control animals, when compared to wild-type control animals. These differences are
5. Conclusion To our knowledge, the results of this study indicate for the first time, that IL-33/ST2 axis could have a role in alteration of iron in acute inflammation, namely in an increase of iron concentration at the site of injury and a decrease of mean corpuscular hemoglobin in blood. Acknowledgments This research was funded by the Ministry of Education, Science and Technological Development (RS, grant III 41013). References Andersen, H.H., Johnsen, K.B., Moos, T., 2014. Iron deposits in the chronically inflamed central nervous system and contributes to neurodegeneration. Cell. Mol. Life Sci. 71, 1607–1622. Barbour, M., Allan, D., Xu, H., Pei, C., Chen, M., Niedbala, W., Fukada, S.Y., Besnard, A.G., Alves-Filho, J.C., Tong, X., Forrester, J.V., Liew, F.Y., Jiang, H.R., 2014. IL-33 attenuates the development of experimental autoimmune uveitis. Eur. J. Immunol. 44, 3320–3329. Bartemes, K.R., Kita, H., 2012. Dynamic role of epithelium-derived cytokines in asthma. Clin. Immunol. 143, 222–235. D'Anna, M.C., Giorgi, G., Roque, M.E., 2011. Immunohistochemical studies on duodenum, spleen and liver in mice: distribution of ferroportin and prohepcidin in an inflammation model. Int. J. Morphol. 29, 747–753. Darshan, D., Frazer, D.M., Wilkins, S.J., Anderson, G.J., 2010. Severe iron deficiency blunts the response of the iron regulatory gene Hamp and pro-inflammatory cytokines to lipopolysaccharide. Haematologica 95, 1660–1667. Djordjevich, D.M., De Luka, S.R., Milovanovich, I.D., Janković, S., Stefanović, S., Vesković-Moračanin, S., Cirković, S., Ilić, A.Ž., Ristić-Djurović, J.L., Trbovich, A.M., 2012. Hematological parameters' changes in mice subchronically exposed to static magnetic fields of different orientations. Ecotoxicol. Environ. Saf. 81, 98–105. Gaspard, K.J., Twite, K., 2005. Hematopoietic Function. In: Porth, C.M. (Ed.), Pathophysiology: Concept of Altered Health States. Lippincott Williams & Wilkins, Philadelphia, pp. 277–337.
692
M.S. Stankovic et al. / Experimental and Molecular Pathology 99 (2015) 687–692
Hider, R.C., Singh, S., 2012. Iron chelators of clinical significance. In: Silver, J. (Ed.), Chemistry of Iron, Springer Science & Business Media, New York, ch. 8. Lagan, A.L., Melley, D.D., Evans, T.W., Quinlan, G.J., 2008. Pathogenesis of the systemic inflammatory syndrome and acute lung injury: role of iron mobilization and decompartmentalization. Am. J. Physiol. Lung Cell. Mol. Physiol. 294, L161–L174. Lai, J.C., Kakuta, I., Mok, H.O., Rummer, J.L., Randall, D., 2006. Effects of moderate and substantial hypoxia on erythropoietin levels in rainbow trout kidney and spleen. J. Exp. Biol. 209, 2734–2738. Le, H.T., Tran, V.G., Kim, W., Kim, J., Cho, H.R., Kwon, B., 2012. IL-33 priming regulates multiple steps of the neutrophil-mediated anti-Candida albicans response by modulating TLR and dectin-1 signals. J. Immunol. 189, 287–295. Liu, J., Shen, J.X., Hu, J.L., Huang, W.H., Zhang, G.J., 2014. Significance of interleukin-33 and its related cytokines in patients with breast cancers. Front. Immunol. 5, 141. Malik, I.A., Naz, N., Sheikh, N., Khan, S., Moriconi, F., Blaschke, M., Ramadori, G., 2011. Comparison of changes in gene expression of transferrin receptor-1 and other iron-regulatory proteins in rat liver and brain during acute-phase response. Cell Tissue Res. 344, 299–312. McLaren, J.E., Michael, D.R., Salter, R.C., Ashlin, T.G., Calder, C.J., Miller, A.M., Liew, F.Y., Ramji, D.P., 2010. IL-33 reduces macrophage foam cell formation. J. Immunol. 185, 1222–1229. Mitchell, R., Cotran, R., 2003. Acute and Chronic Inflammation. In: Cotran, R., Robbins, S.L., Kumar, V. (Eds.), Robbins Basic Pathology. W.B. Saunders, Philadelphia, pp. 33–34. Nemeth, E., Rivera, S., Gabayan, V., Keller, C., Taudorf, S., Pedersen, B.K., Ganz, T., 2004. IL-6 mediates hypoferremia of inflammation by inducing the synthesis of the iron regulatory hormone hepcidin. J. Clin. Invest. 113, 1271–1276.
Perbal, B., 2006. New insight into CCN3 interactions–nuclear CCN3 : fact or fantasy? Cell Commun. Signal. 4, 6. Roy, C.N., 2010. Anemia of inflammation. Hematol. Am. Soc. Hematol. Educ. Program. 276–280. Russell, E.S., Bernstein, S.E., 1966. Blood and Blood Formation. In: Green, E.L. (Ed.), Biology of the Laboratory Mouse. Dover publications Inc., New York ch. 17. Schmitz, J., Owyang, A., Oldham, E., Song, Y., Murphy, E., McClanahan, T.K., Zurawski, G., Moshrefi, M., Qin, J., Li, X., Gorman, D.M., Bazan, J.F., Kastelein, R.A., 2005. IL-33, an interleukin-1-like cytokine that signals via the IL-1 receptor-related protein ST2 and induces T helper type 2-associated cytokines. Immunity 23, 479–490. Sukumaran, A., Venkatraman, A., Jacob, M., 2012. Inflammation-induced effects on iron-related proteins in splenic macrophages and the liver in mice. Blood Cells Mol. Dis. 49, 11–19. Sultan, S., Pascucci, M., Ahmad, S., Malik, I.A., Bianchi, A., Ramadori, P., Ahmad, G., Ramadori, G., 2012. LIPOCALIN-2 is a major acute-phase protein in a rat and mouse model of sterile abscess. Shock 37, 191–196. Talabot-Ayer, D., Martin, P., Seemayer, C.A., Vigne, S., Lamacchia, C., Finckh, A., Saiji, E., Gabay, C., Palmer, G., 2014. Immune-mediated experimental arthritis in IL-33 deficient mice. Cytokine 69, 68–74. Thornton, S.J., Spielman, D.M., Pelc, N.J., Block, W.F., Crocker, D.E., Costa, D.P., LeBoeuf, B.J., Hochachka, P.W., 2001. Effects of forced diving on the spleen and hepatic sinus in northern elephant seal pups. Proc. Natl. Acad. Sci. U. S. A. 98, 9413–9418. Van Snick, J.L., Masson, P.L., Heremans, J.F., 1974. The involvement of lactoferrin in the hyposideremia of acute inflammation. J. Exp. Med. 140, 1068–1084. Verri Jr., W.A., Souto, F.O., Vieira, S.M., Almeida, S.C., Fukada, S.Y., Xu, D., Alves-Filho, J.C., Cunha, T.M., Guerrero, A.T., Mattos-Guimaraes, R.B., Oliveira, F.R., Teixeira, M.M., Silva, J.S., McInnes, I.B., Ferreira, S.H., Louzada-Junior, P., Liew, F.Y., Cunha, F.Q., 2010. IL-33 induces neutrophil migration in rheumatoid arthritis and is a target of anti-TNF therapy. Ann. Rheum. Dis. 69, 1697–1703.