- Email: [email protected]
Suppressive effects of lysozyme on polyphosphate-mediated vascular inflammatory responses
Biochemical and Biophysical Research Communications 474 (2016) 715e721
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Suppressive effects of lysozyme on polyphosphate-mediated vascular inflammatory responses Jiwoo Chung a, 1, Sae-Kwang Ku b, 1, Suyeon Lee a, Jong-Sup Bae a, * a
College of Pharmacy, CMRI, Research Institute of Pharmaceutical Sciences, BK21 Plus KNU Multi-Omics Based Creative Drug Research Team, Kyungpook National University, Daegu 41566, Republic of Korea b Department of Anatomy and Histology, College of Korean Medicine, Daegu Haany University, Gyeongsan 38610, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history: Received 1 May 2016 Accepted 4 May 2016 Available online 4 May 2016
Lysozyme, found in relatively high concentration in blood, saliva, tears, and milk, protects us from the ever-present danger of bacterial infection. Previous studies have reported proinflammatory responses of endothelial cells to the release of polyphosphate(PolyP). In this study, we examined the antiinflammatory responses and mechanisms of lysozyme and its effects on PolyP-induced septic activities in human umbilical vein endothelial cells (HUVECs) and mice. The survival rates, septic biomarker levels, behavior of human neutrophils, and vascular permeability were determined in PolyP-activated HUVECs and mice. Lysozyme suppressed the PolyP-mediated vascular barrier permeability, upregulation of inflammatory biomarkers, adhesion/migration of leukocytes, and activation and/or production of nuclear factor-kB, tumor necrosis factor-a, and interleukin-6. Furthermore, lysozyme demonstrated protective effects on PolyP-mediated lethal death and the levels of the related septic biomarkers. Therefore, these results indicated the therapeutic potential of lysozyme on various systemic inflammatory diseases, such as sepsis or septic shock. © 2016 Elsevier Inc. All rights reserved.
Keywords: Lysozyme Polyphosphate Inflammation Barrier integrity
1. Introduction Inorganic polyphosphate (PolyP), which is a linear polymer that is made up of several orthophosphate residues that are linked by adenosine triphosphate-like phosphoanhydride bonds [1], is present in all bacterial and animal cells [2]. Recent studies that were mostly conducted on microorganisms have reported a number of diverse biological functions of PolyP, including inflammation, apoptosis, proliferation, and blood coagulation, in mammalian systems [3e6]. Our recent studies indicated proinflammatory activities of PolyP, such as mediating vascular hyperpermeability, increasing the adhesion and migration of leukocytes, and upregulating the expression of cell adhesion molecules (CAMs), including vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and e-selectin [6e8]. Lysozymes, which thwart bacterial growth and are found in
* Corresponding author. College of Pharmacy, Kyungpook National University, 80 Daehak-ro, Buk-gu, Daegu 41566, Republic of Korea. E-mail address: [email protected] (J.-S. Bae). 1 First two authors contributed equally to this work. http://dx.doi.org/10.1016/j.bbrc.2016.05.016 0006-291X/© 2016 Elsevier Inc. All rights reserved.
relatively high concentration in blood, saliva, tears, and milk, are 1,4-b-N-acetylmuramidases that cleave the glycosidic bond between the C-1 of N-acetylmuramic acid (NAM) and the C-4 of Nacetylglucosamine (NAG) in bacterial peptidoglycans [9]. Lysozyme is a small protein that protects us from the ever-present danger of bacterial infection by attacking the cell walls of bacteria [10,11]. Bacteria build a tough cell wall composed of carbohydrate chains interlocked by short peptide strands, which braces their delicate membrane against the cell’s high osmotic pressure [10,11]. Lysozyme breaks these carbohydrate chains, destroying the structural integrity of the cell wall [10,11], leading to rupturing of the bacteria under their own internal pressure. Because it is commonly found in places where microorganisms are most likely to enter the body, lysozyme is one of the powerful first-line defenses against bacterial infection [10,11]. However, to the best of our knowledge, the effects of lysozyme on PolyP-mediated inflammatory responses have not yet been studied. Thus, noting that lysozyme has a pleiotropic role in bacterial defense, and that PolyP induces a variety of inflammatory responses, we hypothesized that lysozyme might possess anti-PolyP activity. Therefore, in the current study, we investigated the effect of lysozyme on PolyP-mediated inflammatory responses in human endothelial cells and in mice.
716
J. Chung et al. / Biochemical and Biophysical Research Communications 474 (2016) 715e721
2. Methods
2.7. Cell-cell adhesion assay
2.1. Reagents
The adhesion of purified human neutrophils to the HUVECs was tested by fluorescent labeling, as previously described [22]. Briefly, after the neutrophils were labeled with fluorescein, confluent HUVECs were activated with PolyP65 (50 mM, 4 h) and then incubated with without lysozyme (6 h). The percentage of adherent neutrophils was calculated as previously described [22].
Fetal bovine serum and Vybrant® DiD were purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA). Lysozyme from chicken egg white (L7651), PolyP65, Evans blue, crystal violet, 2mercaptoethanol, catalase-polyethylene glycol, and antibiotics (penicillin G and streptomycin) were purchased from SigmaAldrich Co. LLC (St. Louis, MO, USA). 2.2. Animals and husbandry Male mice (C57BL/6 strain; average weight, 27 g; 6e7 weeks old) were obtained from Orient Bio Co., Ltd. (Sungnam, Republic of Korea) and used after a 12-day acclimatization period. The mice were maintained as described previously [12,13]. All of the animals were handled in accordance with the Guidelines for the Care and Use of Laboratory Animals that was issued by Kyungpook National University (IRB No. KNU 2016-54). 2.3. Cell culture Primary human umbilical vein endothelial cells (HUVECs) were purchased from Cambrex Corporation (Charles City, IA, USA) and maintained as previously described [12e16]. The neutrophils were freshly isolated from whole blood (15 mL) that was obtained by the venipuncture of five healthy volunteers and maintained as previously described [17,18].
2.8. In vitro migration assay The migration of purified human neutrophils to the HUVECs was evaluated as previously described [23]. Lysozyme-treated (6 h) confluent HUVECs were activated with PolyP65 (50 mM, 4 h). Purified human neutrophils were then applied to the upper chamber, and the migration index was measured as previously described [23]. 2.9. In vivo permeability and leukocyte migration assay The mice were treated with PolyP65 (6.5 mg/mouse, intravenous administration) or 0.5% dimethyl sulfoxide (DMSO), which was used as a control. The mice were then intravenously administered lysozyme (0.29e2.86 mg/mouse), and 1% Evans blue dye solution in normal saline was injected after 4 h. The vascular permeability and leukocyte migration were determined as previously described [24,25]. 2.10. ELISA for nuclear factor (NF)-kB, extracellular signal-regulated kinase (ERK)1/2, interleukin (IL)-6, and tumor necrosis factor (TNF)-
2.4. Permeability assay in vitro
a
In order to examine any changes in vascular permeability in response to increasing concentrations of lysozyme, the flux of Evans blue-bound albumin was measured, as described previously [19]. Briefly, confluent HUVEC monolayers were treated with increasing concentrations of lysozyme for 6 h and then activated with PolyP65 (50 mM) for 4 h.
Commercially available ELISA kits were used to determine the levels of expression of total and phosphoeNFekB p65 (# 7174 and # 7173, Cell Signaling Technology, Inc.) and total/phospho ERK1/2 (R&D Systems, Inc., Minneapolis, MN, USA) in the nuclear lysates of HUVECs and the levels of IL-6 and TNF-a (R&D Systems, Inc.) in the cell culture supernatants of the HUVECs.
2.5. Enzyme-linked immunosorbent assay (ELISA) of phosphorylated (phospho) p-38
2.11. PolyP-induced lethal model
A commercially available ELISA kit (Cell Signaling Technology, Inc., Danvers, MA, USA) was used to measure the expression levels of phospho p-38. 2.5.1. Immunofluorescence staining Confluent HUVECs on glass coverslips that were coated with 0.05% Poly-L-Lysine were maintained for 2 days. The HUVECs were then activated with PolyP (50 mM, 4 h) with or without lysozyme (50 or 100 nM, 6 h). The cytoskeletal staining was assessed as previously described [20].
PolyP65 (6.5 mg/mouse) in DMSO or 0.5% DMSO, which was a control, were intravenously injected into the mice. At 12 h or 50 h after the PolyP65 injection, male C57BL/6 mice were administered lysozyme (1.43 or 2.86 mg/mouse). Animal survival was monitored every 6 h after the PolyP65 injections for 132 h. 2.12. Measurements of organ injury markers The plasma levels of aspartate transaminase, alanine transaminase, blood urea nitrogen, and creatinine were measured with commercial assay kits (Pointe Scientific, Inc., Canton, MI, USA).
2.6. Levels of expression of the protein and mRNA of CAMs
2.13. Statistical analysis
The confluent monolayers of the HUVECs were treated with PolyP65 (50 mM) for 16 h for VCAM-1 and ICAM-1 or 24 h for eselectin and then with without lysozyme (50 or 100 nM, 6 h). A whole-cell ELISA was performed to determine the levels of expression of the ICAM-1, e-selectin, and VCAM-1 proteins on the HUVECs, as previously described [21,22]. For the real-time polymerase chain reaction, RNA was isolated with TRI Reagent (Thermo Fisher Scientific Inc.) according to the manufacturer’s protocol. The real-time polymerase chain reaction was performed as previously described [8].
Each experiment was independently performed at least 3 times, and each value was expressed as the mean ± standard deviations (SD). SPSS for Windows (version 16.0; IBM Corporation, Armonk, NY, USA) was used to evaluate the statistical significance of the differences between the test groups. Statistical relevance was determined with one-way analysis of variance and/or Tukey’s posthoc test. A KaplaneMeier analysis was used to evaluate the survival data on the outcomes of the cecal ligation and punctureinduced sepsis. P values less than 0.05 were considered statistically significant.
J. Chung et al. / Biochemical and Biophysical Research Communications 474 (2016) 715e721
3. Results and discussion 3.1. Inhibitory effects of lysozyme on the PolyP-induced hyperpermeability Vascular permeability was assessed to test the effects of lysozyme on the PolyP-induced disruptions of the vascular barrier as the endothelial barrier integrity is cleaved by PolyP [6,7]. Our previous studies reported the PolyP parameters (50 mM and 4 h) that optimize the disruption of endothelial integrity [6,7]. The cells were activated with PolyP (50 mM) for 4 h and then various concentrations of lysozyme for 6 h. The results showed the inhibitory effects of lysozyme on the PolyP-mediated hyperpermeability, with the optimal dose occurring at concentrations above 20 nM (Fig. 1A). Furthermore, lysozyme alone (100 nM) did not alter the barrier integrity of the HUVECs (Fig. 1A). Next, to confirm in vitro data, lysozyme was intravenously injected into mice with PolyPmediated hyperpermeability. The results showed that PolyP enhanced vascular permeability, and this was suppressed by lysozyme (Fig. 1B). Because the average blood volume is 72 mL/kg [26] and the average weight of the mice that were used in this study was 27 g, the amount of lysozyme (0.29, 0.59, 1.43 or 2.86 mg/mouse) injected was equivalent to 10, 20, 50 or 100 nM in peripheral blood. Vascular inflammatory inducers, such as lipopolysaccharide and high-mobility group box 1 protein, mediate inflammatory responses by activating p38 mitogen-activated protein kinase (MAPK) [27,28]. Therefore, the effects of lysozyme on the PolyPinduced activation of p38 MAPK were determined. The results revealed that lysozyme inhibited the PolyP-induced upregulation of phospho p38 expression (Fig. 1C).
717
Previous studies have indicated the importance of cytoskeletal proteins for maintaining cell integrity and shape [29] and the involvement of vascular integrity in the detachment of cell-cell contact and redistribution of the actin cytoskeleton [30,31]. Thus, the effects of lysozyme on PolyP-mediated actin cytoskeletal arrangement in HUVECs were examined by staining the HUVECs with fluorescein phalloidin-labeled F-actin. Compared to the control HUVECs that displayed an irregular distribution of F-actin, the disruption in the barrier integrity that was induced by treatment with PolyP (50 mM) was demonstrated by the formation of paracellular gaps in the HUVECs, and these were reduced by treatment with lysozyme (100 nM) (Fig. 1D). To exclude the possibility that the barrier-protecting effects of lysozyme were due to the cellular cytotoxicity of lysozyme, cellular viability assays were conducted. Data showed that lysozyme was not cytotoxic in the HUVECs at concentrations up to 500 nM (data not shown). Because the high morbidity and mortality that are seen in patients with serious inflammatory diseases result from the disruption of vascular integrity [32] and because the reagents used to treat a number of inflammatory diseases are designed to inhibit vascular hyperpermeability [33], our results indicated the potential of lysozyme as a therapeutic agent in various vascular inflammatory diseases.
3.2. Effects of lysozyme on the PolyP effects on CAMs expression and neutrophil adhesion and migration Previous studies have confirmed the pivotal roles of CAMs (VCAM-1, ICAM-1, and e-selectin) during cell-cell adhesion in processes of vascular inflammation [34,35]. Thus, inhibiting the
Fig. 1. Effects of lysozyme on Polyphosphate P (PolyP)-induced barrier disruptions in vitro and in vivo. (A) The effects of various concentrations of lysozyme on PolyP-induced (50 mM, 4 h) barrier disruption, which was monitored by the flux of Evans blue-bound albumin across human umbilical vein endothelial cells (HUVECs). (B) The effects of lysozyme on PolyP-induced [3.5 mg/mouse, intravenous (i.v.)] vascular permeability in mice were examined by the flux of Evans blue in the mice (expressed mg/mouse, n ¼ 5). (C) After activation with PolyP (50 mM, 4 h), the HUVECs were treated with different concentrations of lysozyme for 6 h. The effects of each compound on the expression of PolyP-mediated phosphorylated (phospho) p38 (p-p38) were determined with an ELISA. (D) Staining for F-actin. The HUVEC monolayers grown on glass coverslips were stimulated with PolyP (50 mM, 4 h) and then treated with lysozyme for 6 h. F-actin was examined with immunofluorescent staining. The arrows indicate intercellular gaps. The results are expressed as means ± SD of three independent experiments. P indicates vehicle [PBS] only. *p < 0.05 versus PolyP (AeC). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
718
J. Chung et al. / Biochemical and Biophysical Research Communications 474 (2016) 715e721
expression of CAMs is considered a promising therapeutic approach for treating vascular inflammatory diseases. We recently showed that the levels of expression of CAMs are increased by PolyP, thereby stimulating leukocyte behaviors, such as adhesion and migration, toward HUVECs [6,7]. In this study, we determined the effects of lysozyme on the levels of expression of CAMs and on the adhesion and migration of leukocytes toward HUVECs, which were both affected by PolyP. The results showed that lysozyme suppressed the increases in the levels of protein and transcript expression of CAMs (Fig. 2A and 2B). In addition, the enhancement in the expression of CAMs correlated with the increased binding and migration of leukocytes to PolyP-treated HUVECs, and this was inhibited by lysozyme treatment in a concentration-dependent manner (Fig. 2CeE). In order to confirm these results in vivo, we examined the effects of injected lysozyme on PolyP-induced leukocyte migration in mice. PolyP increased the number of migrated leukocytes in the peritoneal cavities of mice, and this was reduced by lysozyme treatment (Fig. 3F). Collectively, the results of this study showed that lysozyme downregulated PolyP-mediated vascular inflammatory responses by inhibiting the augmentation by PolyP of inflammatory signaling pathways, such as the adhesion and migration of leukocytes to inflamed endothelium.
3.3. Effects of lysozyme on the PolyP-stimulated activation of NF-
kB/ERK and the production of IL-6/TNF-a Based on the results of the present study and findings that the activation of NF-kB or ERK and the increased production of TNF-a or IL-6 amplifies vascular inflammatory responses [36e40], we hypothesized that the activation and expression of these proinflammatory molecules were suppressed by lysozyme. To confirm this hypothesis, the levels of activation and expression of these proinflammatory molecules were measured with ELISA in PolyPactivated and lysozyme-treated HUVECs. The results showed that the increased levels of protein expression of TNF-a and IL-6 (Fig. 3A and B) and the increased activation of NF-kB and ERK1/2 (Fig. 3C and D) that were induced by PolyP were reduced by lysozyme. Therefore, these results suggested that lysozyme can control important vascular inflammatory signaling pathways by regulating the molecules involved.
3.4. Vascular protective effects of lysozyme in the PolyP-induced lethality model Finally, we hypothesized that lysozyme would prevent PolyP-
Fig. 2. Effects of lysozyme on the PolyP-induced expression of cellular adhesion molecules (CAMs), cell adhesion and migration of neutrophils. (A) The PolyP-mediated (50 mM) levels of expression of vascular cell adhesion molecule-1 (VCAM-1; white bar), intercellular adhesion molecule-1 (ICAM-1; gray bar), and E-selectin (black bar) proteins in HUVECs were analyzed with whole-cell ELISA after treating the monolayers with lysozyme. (B) The PolyP-mediated (50 mM) levels of expression of VCAM-1 (white bar), ICAM-1 (gray bar), and E-selectin (black bar) transcription (mRNA) in HUVECs were analyzed with whole-cell ELISA after treating the monolayers with lysozyme. (C) The PolyPmediated (50 mM) adherence of neutrophils to HUVECs was analyzed after treating the cells with lysozyme. (D) Representative photomicrographs of neutrophil adhesion to HUVECs. (E) The PolyP-mediated (50 mM) migration of neutrophils through HUVEC monolayers was analyzed after treating the cells with lysozyme. (F) Effects of lysozyme on PolyP-induced (3.5 mg/mouse, i.v.) leukocyte migration in mice (expressed as 106, n ¼ 5). The results are expressed as the means ± SD of three independent experiments. *p < 0.05 versus PolyP.
J. Chung et al. / Biochemical and Biophysical Research Communications 474 (2016) 715e721
719
Fig. 3. Effects of lysozyme on the PolyP-stimulated activation of nuclear factor (NF)-kB/extracellular signal-regulated kinase (ERK) 1/2 and production of interleukin (IL-6)/ tumor necrosis factor (TNF)-a. The PolyP-mediated (50 mM) production of TNF-a (A) and IL-6 (B) in HUVECs was analyzed after the treatment of the cells with the indicated concentrations of lysozyme for 6 h. (C) The PolyP-mediated (50 mM) activation of phosphoeNFekB p65 (white bar) and total NF-kB p65 (black bar) in the HUVECs was analyzed after the treatment of the cells with lysozyme for 6 h. (D) The PolyP-mediated (50 mM) activation of phospho-ERK1/2 (white bar) and total ERK1/2 (black bar) in the HUVECs was analyzed after the treatment of the cells with lysozyme for 6 h *p < 0.05 vs. PolyP only.
mediated lethality in mice. To confirm this, mice were administered with lysozyme after PolyP injections. The results showed that treatment with a single dose of lysozyme (1.43 or 2.86 mg/mouse, 12 h after the PolyP injection) did not prevent PolyP-induced death
(data not shown). Thus, lysozyme was administered twice (once at 12 h and then 50 h after the PolyP injection), which resulted in an increase in the survival rate from 0 to 50% in the Kaplan-Meier survival analysis (Fig. 4A, p < 0.0001). Because the liver and
Fig. 4. Effects of lysozyme on PolyP-induced lethality and organ damage markers. (A) Male C57BL/6 mice (n ¼ 20) were intravenously administered lysozyme 12 h and 50 h after PolyP (3.5 mg/mouse, i.v.) injections. Animal survival was monitored every 6 h after the PolyP injections for 132 h. The PolyP-injected mice (C) and control sham mice (B) were administered PBS (n ¼ 20). A Kaplan-Meier survival analysis was used to determine the overall survival rates versus the PolyP-treated mice. The plasma levels of the hepatic injury markers aspartate transaminase (AST) and alanine transaminase (ALT) (B), renal injury markers creatinine (C) and bun urea nitrogen (BUN) (D), and tissue injury marker lactate dehydrogenase (LDH) (E) were measured (n ¼ 5) 72 h after the PolyP injections. D indicates vehicle (0.5% DMSO) only. *p < 0.05 vs. PolyP alone.
720
J. Chung et al. / Biochemical and Biophysical Research Communications 474 (2016) 715e721
kidney are major target organs of systemic inflammatory diseases and multiple organ failure is caused by systemic inflammatory diseases, such as sepsis and septic shock [41], we examined the plasma levels of tissue damage markers. As shown in Fig. 4BeE, lysozyme reduced the polyP-induced increases in the plasma levels of alanine transaminase and aspartate transaminase (markers of hepatic injury, Fig. 4B) and creatinine and blood urea nitrogen (markers of renal injury, Fig. 4C and D). In addition, the levels of lactate dehydrogenase, which is a marker of tissue injury, were reduced by lysozyme in PolyP-injected mice (Fig. 4E). The molecular mechanisms underlying the anti-inflammatory effects of lysozyme on PolyP-mediated septic responses may be mediated by the suppression of PolyP-mediated hyperpermeability (Fig. 1A and 1B) through the inhibition of the activation of p38 (Fig. 1C). Furthermore, the inhibitory mechanisms of lysozyme on the interaction of leukocytes with endothelial cells are mediated by the inhibition of the expression of CAMs, such as VCAM, ICAM, and e-selectin (Fig. 2). The underlying mechanisms of these antiinflammatory effects of lysozyme involve the downregulation of the production of inflammatory cytokines (TNF-a and IL-6, Fig. 3A and 4) and the activation of inflammatory transcriptional factors (NF-kB and ERK1/2, Fig. 3C and D). Finally, the results of this study showed the protective activities of lysozyme on the vascular barrier disruptions induced by PolyP in both human endothelial cells and mice. Therefore, these results suggested that lysozyme is a potential candidate in the treatment of severe vascular inflammatory diseases. Conflict of interest statement The authors declare no conflicts of interest. Acknowledgments This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (NRF2012R1A5A2A42671316 and 2014R1A2A1A11049526).
[10] [11]
[12]
[13]
[14] [15]
[16] [17]
[18]
[19] [20]
[21]
[22] [23]
[24]
[25]
[26]
Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2016.05.016.
[27]
[28]
References [1] K.D. Kumble, A. Kornberg, Inorganic polyphosphate in mammalian cells and tissues, J. Biol. Chem. 270 (1995) 5818e5822. [2] I.S. Kulaev, V.M. Vagabov, Polyphosphate metabolism in micro-organisms, Adv. Microb. Physiol. 24 (1983) 83e171. [3] T. Shiba, D. Nishimura, Y. Kawazoe, Y. Onodera, K. Tsutsumi, R. Nakamura, M. Ohshiro, Modulation of mitogenic activity of fibroblast growth factors by inorganic polyphosphate, J. Biol. Chem. 278 (2003) 26788e26792. [4] L. Hernandez-Ruiz, I. Gonzalez-Garcia, C. Castro, J.A. Brieva, F.A. Ruiz, Inorganic polyphosphate and specific induction of apoptosis in human plasma cells, Haematologica 91 (2006) 1180e1186. [5] F. Muller, N.J. Mutch, W.A. Schenk, S.A. Smith, L. Esterl, H.M. Spronk, S. Schmidbauer, W.A. Gahl, J.H. Morrissey, T. Renne, Platelet polyphosphates are proinflammatory and procoagulant mediators in vivo, Cell 139 (2009) 1143e1156. [6] J.S. Bae, W. Lee, A.R. Rezaie, Polyphosphate elicits pro-inflammatory responses that are counteracted by activated protein C in both cellular and animal models, J. Thromb. Haemost. 10 (2012) 1145e1151. [7] S.K. Ku, J.S. Bae, Concentration dependent anti-inflammatory effects thrombin on polyphosphate-mediated inflammatory responses in vitro and in vivo, Inflamm. Res. 62 (2013) 609e616. [8] I.C. Lee, J.S. Bae, Anti-inflammatory effects of vicenin-2 and scolymoside on polyphosphate-mediated vascular inflammatory responses, Inflamm. Res. 65 (2016) 203e212. [9] J.A. Kelly, A.R. Sielecki, B.D. Sykes, M.N. James, D.C. Phillips, X-ray
[29]
[30]
[31]
[32] [33] [34] [35]
[36]
[37]
crystallography of the binding of the bacterial cell wall trisaccharide NAMNAG-NAM to lysozyme, Nature 282 (1979) 875e878. R. Clingolani, Bioinspired Approaches for Human-centric Technologies, Springer, New York, 2014. D. Nakimbugwe, B. Masschalck, D. Deckers, L. Callewaert, A. Aertsen, C.W. Michiels, Cell wall substrate specificity of six different lysozymes and lysozyme inhibitory activity of bacterial extracts, FEMS Microbiol. Lett. 259 (2006) 41e46. J.S. Bae, W. Lee, J.O. Nam, J.E. Kim, S.W. Kim, I.S. Kim, Transforming growth factor beta-induced protein promotes severe vascular inflammatory responses, Am. J. Respir. Crit. Care Med. 189 (2014) 779e786. S.K. Ku, E.K. Yoon, H.G. Lee, M.S. Han, T. Lee, J.S. Bae, Inhibitory effects of lysozyme on endothelial protein C receptor shedding in vitro and in vivo, BMB Rep. 48 (2015) 624e629. E.K. Yoon, S.K. Ku, W. Lee, S. Kwak, H. Kang, B. Jung, J.S. Bae, Antitcoagulant and antiplatelet activities of scolymoside, BMB Rep. 48 (2015) 577e582. S.K. Ku, W. Lee, M. Kang, J.S. Bae, Antithrombotic activities of aspalathin and nothofagin via inhibiting platelet aggregation and FIIa/FXa, Arch. Pharm. Res. 38 (2015) 1080e1089. W. Lee, S.K. Ku, J.S. Bae, Antiplatelet, anticoagulant, and profibrinolytic activities of baicalin, Arch. Pharm. Res. 38 (2015) 893e903. R. Hofbauer, D. Moser, H. Salfinger, M. Frass, S. Kapiotis, Sufentanil inhibits migration of human leukocytes through human endothelial cell monolayers, Anesth. Analg. 87 (1998) 1181e1185. J.S. Bae, A.R. Rezaie, Thrombin inhibits HMGB1-mediated proinflammatory signaling responses when endothelial protein C receptor is occupied by its natural ligand, BMB Rep. 46 (2013) 544e549. J.S. Bae, A.R. Rezaie, Activated protein C inhibits high mobility group box 1 signaling in endothelial cells, Blood 118 (2011) 3952e3959. W. Lee, S.K. Ku, D.H. Na, J.S. Bae, Anti-inflammatory effects of lysozyme against HMGB1 in human endothelial cells and in mice, Inflammation 38 (2015) 1911e1924. W. Che, N. Lerner-Marmarosh, Q. Huang, M. Osawa, S. Ohta, M. Yoshizumi, M. Glassman, J.D. Lee, C. Yan, B.C. Berk, J. Abe, Insulin-like growth factor-1 enhances inflammatory responses in endothelial cells: role of Gab1 and MEKK3 in TNF-alpha-induced c-Jun and NF-kappaB activation and adhesion molecule expression, Circ. Res. 90 (2002) 1222e1230. J.W. Bae, J.S. Bae, Barrier protective effects of lycopene in human endothelial cells, Inflamm. Res. 60 (2011) 751e758. T.H. Kim, S.K. Ku, I.C. Lee, J.S. Bae, Anti-inflammatory functions of purpurogallin in LPS-activated human endothelial cells, BMB Rep. 45 (2012) 200e205. J.S. Bae, W. Lee, A.R. Rezaie, Polyphosphate elicits proinflammatory responses that are counteracted by activated protein C in both cellular and animal models, J. Thromb. Haemost. 10 (2012) 1145e1151. J.D. Lee, J.E. Huh, G. Jeon, H.R. Yang, H.S. Woo, D.Y. Choi, D.S. Park, Flavonolrich RVHxR from Rhus verniciflua Stokes and its major compound fisetin inhibits inflammation-related cytokines and angiogenic factor in rheumatoid arthritic fibroblast-like synovial cells and in vivo models, Int. Immunopharmacol. 9 (2009) 268e276. K.H. Diehl, R. Hull, D. Morton, R. Pfister, Y. Rabemampianina, D. Smith, J.M. Vidal, C. van de Vorstenbosch, A good practice guide to the administration of substances and removal of blood, including routes and volumes, J. Appl. Toxicol. 21 (2001) 15e23. K.L. Walton, L. Holt, R.B. Sartor, Lipopolysaccharide activates innate immune responses in murine intestinal myofibroblasts through multiple signaling pathways, Am. J. Physiol. Gastrointest. Liver Physiol. 296 (2009) G601eG611. J.S. Bae, Role of high mobility group box 1 in inflammatory disease: focus on sepsis, Arch. Pharm. Res. 35 (2012) 1511e1523. H.J. Schnittler, S.W. Schneider, H. Raifer, F. Luo, P. Dieterich, I. Just, K. Aktories, Role of actin filaments in endothelial cell-cell adhesion and membrane stability under fluid shear stress, Pflugers Arch. 442 (2001) 675e687. J. Friedl, M. Puhlmann, D.L. Bartlett, S.K. Libutti, E.N. Turner, M.F. Gnant, H.R. Alexander, Induction of permeability across endothelial cell monolayers by tumor necrosis factor (TNF) occurs via a tissue factor-dependent mechanism: relationship between the procoagulant and permeability effects of TNF, Blood 100 (2002) 1334e1339. I. Petrache, A. Birukova, S.I. Ramirez, J.G. Garcia, A.D. Verin, The role of the microtubules in tumor necrosis factor-alpha-induced endothelial cell permeability, Am. J. Respir. Cell Mol. Biol. 28 (2003) 574e581. Y.A. Komarova, D. Mehta, A.B. Malik, Dual regulation of endothelial junctional permeability, Sci. STKE 2007 (2007) re8. S.M. Dudek, J.G. Garcia, Cytoskeletal regulation of pulmonary vascular permeability, J. Appl. Physiol. 91 (2001) 1487e1500. C.J. Frijns, L.J. Kappelle, Inflammatory cell adhesion molecules in ischemic cerebrovascular disease, Stroke 33 (2002) 2115e2122. H. Ulbrich, E.E. Eriksson, L. Lindbom, Leukocyte and endothelial cell adhesion molecules as targets for therapeutic interventions in inflammatory disease, Trends Pharmacol. Sci. 24 (2003) 640e647. K. Javaid, A. Rahman, K.N. Anwar, R.S. Frey, R.D. Minshall, A.B. Malik, Tumor necrosis factor-alpha induces early-onset endothelial adhesivity by protein kinase Czeta-dependent activation of intercellular adhesion molecule-1, Circ. Res. 92 (2003) 1089e1097. J.M. Lockyer, J.S. Colladay, W.L. Alperin-Lea, T. Hammond, A.J. Buda, Inhibition of nuclear factor-kappaB-mediated adhesion molecule expression in human
J. Chung et al. / Biochemical and Biophysical Research Communications 474 (2016) 715e721 endothelial cells, Circ. Res. 82 (1998) 314e320. [38] H. Yamagami, S. Yamagami, T. Inoki, S. Amano, K. Miyata, The effects of proinflammatory cytokines on cytokine-chemokine gene expression profiles in the human corneal endothelium, Investig. Ophthalmol. Vis. Sci. 44 (2003) 514e520. [39] N. Marui, M.K. Offermann, R. Swerlick, C. Kunsch, C.A. Rosen, M. Ahmad, R.W. Alexander, R.M. Medford, Vascular cell adhesion molecule-1 (VCAM-1)
721
gene transcription and expression are regulated through an antioxidantsensitive mechanism in human vascular endothelial cells, J. Clin. Investig. 92 (1993) 1866e1874. [40] B.A. Rose, T. Force, Y. Wang, Mitogen-activated protein kinase signaling in the heart: angels versus demons in a heart-breaking tale, Physiol. Rev. 90 (2010) 1507e1546. [41] M.E. Astiz, E.C. Rackow, Septic shock, Lancet 351 (1998) 1501e1505.
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Suppressive effects of lysozyme on polyphosphate-mediated vascular inflammatory responses Jiwoo Chung a, 1, Sae-Kwang Ku b, 1, Suyeon Lee a, Jong-Sup Bae a, * a
College of Pharmacy, CMRI, Research Institute of Pharmaceutical Sciences, BK21 Plus KNU Multi-Omics Based Creative Drug Research Team, Kyungpook National University, Daegu 41566, Republic of Korea b Department of Anatomy and Histology, College of Korean Medicine, Daegu Haany University, Gyeongsan 38610, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history: Received 1 May 2016 Accepted 4 May 2016 Available online 4 May 2016
Lysozyme, found in relatively high concentration in blood, saliva, tears, and milk, protects us from the ever-present danger of bacterial infection. Previous studies have reported proinflammatory responses of endothelial cells to the release of polyphosphate(PolyP). In this study, we examined the antiinflammatory responses and mechanisms of lysozyme and its effects on PolyP-induced septic activities in human umbilical vein endothelial cells (HUVECs) and mice. The survival rates, septic biomarker levels, behavior of human neutrophils, and vascular permeability were determined in PolyP-activated HUVECs and mice. Lysozyme suppressed the PolyP-mediated vascular barrier permeability, upregulation of inflammatory biomarkers, adhesion/migration of leukocytes, and activation and/or production of nuclear factor-kB, tumor necrosis factor-a, and interleukin-6. Furthermore, lysozyme demonstrated protective effects on PolyP-mediated lethal death and the levels of the related septic biomarkers. Therefore, these results indicated the therapeutic potential of lysozyme on various systemic inflammatory diseases, such as sepsis or septic shock. © 2016 Elsevier Inc. All rights reserved.
Keywords: Lysozyme Polyphosphate Inflammation Barrier integrity
1. Introduction Inorganic polyphosphate (PolyP), which is a linear polymer that is made up of several orthophosphate residues that are linked by adenosine triphosphate-like phosphoanhydride bonds [1], is present in all bacterial and animal cells [2]. Recent studies that were mostly conducted on microorganisms have reported a number of diverse biological functions of PolyP, including inflammation, apoptosis, proliferation, and blood coagulation, in mammalian systems [3e6]. Our recent studies indicated proinflammatory activities of PolyP, such as mediating vascular hyperpermeability, increasing the adhesion and migration of leukocytes, and upregulating the expression of cell adhesion molecules (CAMs), including vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and e-selectin [6e8]. Lysozymes, which thwart bacterial growth and are found in
* Corresponding author. College of Pharmacy, Kyungpook National University, 80 Daehak-ro, Buk-gu, Daegu 41566, Republic of Korea. E-mail address: [email protected] (J.-S. Bae). 1 First two authors contributed equally to this work. http://dx.doi.org/10.1016/j.bbrc.2016.05.016 0006-291X/© 2016 Elsevier Inc. All rights reserved.
relatively high concentration in blood, saliva, tears, and milk, are 1,4-b-N-acetylmuramidases that cleave the glycosidic bond between the C-1 of N-acetylmuramic acid (NAM) and the C-4 of Nacetylglucosamine (NAG) in bacterial peptidoglycans [9]. Lysozyme is a small protein that protects us from the ever-present danger of bacterial infection by attacking the cell walls of bacteria [10,11]. Bacteria build a tough cell wall composed of carbohydrate chains interlocked by short peptide strands, which braces their delicate membrane against the cell’s high osmotic pressure [10,11]. Lysozyme breaks these carbohydrate chains, destroying the structural integrity of the cell wall [10,11], leading to rupturing of the bacteria under their own internal pressure. Because it is commonly found in places where microorganisms are most likely to enter the body, lysozyme is one of the powerful first-line defenses against bacterial infection [10,11]. However, to the best of our knowledge, the effects of lysozyme on PolyP-mediated inflammatory responses have not yet been studied. Thus, noting that lysozyme has a pleiotropic role in bacterial defense, and that PolyP induces a variety of inflammatory responses, we hypothesized that lysozyme might possess anti-PolyP activity. Therefore, in the current study, we investigated the effect of lysozyme on PolyP-mediated inflammatory responses in human endothelial cells and in mice.
716
J. Chung et al. / Biochemical and Biophysical Research Communications 474 (2016) 715e721
2. Methods
2.7. Cell-cell adhesion assay
2.1. Reagents
The adhesion of purified human neutrophils to the HUVECs was tested by fluorescent labeling, as previously described [22]. Briefly, after the neutrophils were labeled with fluorescein, confluent HUVECs were activated with PolyP65 (50 mM, 4 h) and then incubated with without lysozyme (6 h). The percentage of adherent neutrophils was calculated as previously described [22].
Fetal bovine serum and Vybrant® DiD were purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA). Lysozyme from chicken egg white (L7651), PolyP65, Evans blue, crystal violet, 2mercaptoethanol, catalase-polyethylene glycol, and antibiotics (penicillin G and streptomycin) were purchased from SigmaAldrich Co. LLC (St. Louis, MO, USA). 2.2. Animals and husbandry Male mice (C57BL/6 strain; average weight, 27 g; 6e7 weeks old) were obtained from Orient Bio Co., Ltd. (Sungnam, Republic of Korea) and used after a 12-day acclimatization period. The mice were maintained as described previously [12,13]. All of the animals were handled in accordance with the Guidelines for the Care and Use of Laboratory Animals that was issued by Kyungpook National University (IRB No. KNU 2016-54). 2.3. Cell culture Primary human umbilical vein endothelial cells (HUVECs) were purchased from Cambrex Corporation (Charles City, IA, USA) and maintained as previously described [12e16]. The neutrophils were freshly isolated from whole blood (15 mL) that was obtained by the venipuncture of five healthy volunteers and maintained as previously described [17,18].
2.8. In vitro migration assay The migration of purified human neutrophils to the HUVECs was evaluated as previously described [23]. Lysozyme-treated (6 h) confluent HUVECs were activated with PolyP65 (50 mM, 4 h). Purified human neutrophils were then applied to the upper chamber, and the migration index was measured as previously described [23]. 2.9. In vivo permeability and leukocyte migration assay The mice were treated with PolyP65 (6.5 mg/mouse, intravenous administration) or 0.5% dimethyl sulfoxide (DMSO), which was used as a control. The mice were then intravenously administered lysozyme (0.29e2.86 mg/mouse), and 1% Evans blue dye solution in normal saline was injected after 4 h. The vascular permeability and leukocyte migration were determined as previously described [24,25]. 2.10. ELISA for nuclear factor (NF)-kB, extracellular signal-regulated kinase (ERK)1/2, interleukin (IL)-6, and tumor necrosis factor (TNF)-
2.4. Permeability assay in vitro
a
In order to examine any changes in vascular permeability in response to increasing concentrations of lysozyme, the flux of Evans blue-bound albumin was measured, as described previously [19]. Briefly, confluent HUVEC monolayers were treated with increasing concentrations of lysozyme for 6 h and then activated with PolyP65 (50 mM) for 4 h.
Commercially available ELISA kits were used to determine the levels of expression of total and phosphoeNFekB p65 (# 7174 and # 7173, Cell Signaling Technology, Inc.) and total/phospho ERK1/2 (R&D Systems, Inc., Minneapolis, MN, USA) in the nuclear lysates of HUVECs and the levels of IL-6 and TNF-a (R&D Systems, Inc.) in the cell culture supernatants of the HUVECs.
2.5. Enzyme-linked immunosorbent assay (ELISA) of phosphorylated (phospho) p-38
2.11. PolyP-induced lethal model
A commercially available ELISA kit (Cell Signaling Technology, Inc., Danvers, MA, USA) was used to measure the expression levels of phospho p-38. 2.5.1. Immunofluorescence staining Confluent HUVECs on glass coverslips that were coated with 0.05% Poly-L-Lysine were maintained for 2 days. The HUVECs were then activated with PolyP (50 mM, 4 h) with or without lysozyme (50 or 100 nM, 6 h). The cytoskeletal staining was assessed as previously described [20].
PolyP65 (6.5 mg/mouse) in DMSO or 0.5% DMSO, which was a control, were intravenously injected into the mice. At 12 h or 50 h after the PolyP65 injection, male C57BL/6 mice were administered lysozyme (1.43 or 2.86 mg/mouse). Animal survival was monitored every 6 h after the PolyP65 injections for 132 h. 2.12. Measurements of organ injury markers The plasma levels of aspartate transaminase, alanine transaminase, blood urea nitrogen, and creatinine were measured with commercial assay kits (Pointe Scientific, Inc., Canton, MI, USA).
2.6. Levels of expression of the protein and mRNA of CAMs
2.13. Statistical analysis
The confluent monolayers of the HUVECs were treated with PolyP65 (50 mM) for 16 h for VCAM-1 and ICAM-1 or 24 h for eselectin and then with without lysozyme (50 or 100 nM, 6 h). A whole-cell ELISA was performed to determine the levels of expression of the ICAM-1, e-selectin, and VCAM-1 proteins on the HUVECs, as previously described [21,22]. For the real-time polymerase chain reaction, RNA was isolated with TRI Reagent (Thermo Fisher Scientific Inc.) according to the manufacturer’s protocol. The real-time polymerase chain reaction was performed as previously described [8].
Each experiment was independently performed at least 3 times, and each value was expressed as the mean ± standard deviations (SD). SPSS for Windows (version 16.0; IBM Corporation, Armonk, NY, USA) was used to evaluate the statistical significance of the differences between the test groups. Statistical relevance was determined with one-way analysis of variance and/or Tukey’s posthoc test. A KaplaneMeier analysis was used to evaluate the survival data on the outcomes of the cecal ligation and punctureinduced sepsis. P values less than 0.05 were considered statistically significant.
J. Chung et al. / Biochemical and Biophysical Research Communications 474 (2016) 715e721
3. Results and discussion 3.1. Inhibitory effects of lysozyme on the PolyP-induced hyperpermeability Vascular permeability was assessed to test the effects of lysozyme on the PolyP-induced disruptions of the vascular barrier as the endothelial barrier integrity is cleaved by PolyP [6,7]. Our previous studies reported the PolyP parameters (50 mM and 4 h) that optimize the disruption of endothelial integrity [6,7]. The cells were activated with PolyP (50 mM) for 4 h and then various concentrations of lysozyme for 6 h. The results showed the inhibitory effects of lysozyme on the PolyP-mediated hyperpermeability, with the optimal dose occurring at concentrations above 20 nM (Fig. 1A). Furthermore, lysozyme alone (100 nM) did not alter the barrier integrity of the HUVECs (Fig. 1A). Next, to confirm in vitro data, lysozyme was intravenously injected into mice with PolyPmediated hyperpermeability. The results showed that PolyP enhanced vascular permeability, and this was suppressed by lysozyme (Fig. 1B). Because the average blood volume is 72 mL/kg [26] and the average weight of the mice that were used in this study was 27 g, the amount of lysozyme (0.29, 0.59, 1.43 or 2.86 mg/mouse) injected was equivalent to 10, 20, 50 or 100 nM in peripheral blood. Vascular inflammatory inducers, such as lipopolysaccharide and high-mobility group box 1 protein, mediate inflammatory responses by activating p38 mitogen-activated protein kinase (MAPK) [27,28]. Therefore, the effects of lysozyme on the PolyPinduced activation of p38 MAPK were determined. The results revealed that lysozyme inhibited the PolyP-induced upregulation of phospho p38 expression (Fig. 1C).
717
Previous studies have indicated the importance of cytoskeletal proteins for maintaining cell integrity and shape [29] and the involvement of vascular integrity in the detachment of cell-cell contact and redistribution of the actin cytoskeleton [30,31]. Thus, the effects of lysozyme on PolyP-mediated actin cytoskeletal arrangement in HUVECs were examined by staining the HUVECs with fluorescein phalloidin-labeled F-actin. Compared to the control HUVECs that displayed an irregular distribution of F-actin, the disruption in the barrier integrity that was induced by treatment with PolyP (50 mM) was demonstrated by the formation of paracellular gaps in the HUVECs, and these were reduced by treatment with lysozyme (100 nM) (Fig. 1D). To exclude the possibility that the barrier-protecting effects of lysozyme were due to the cellular cytotoxicity of lysozyme, cellular viability assays were conducted. Data showed that lysozyme was not cytotoxic in the HUVECs at concentrations up to 500 nM (data not shown). Because the high morbidity and mortality that are seen in patients with serious inflammatory diseases result from the disruption of vascular integrity [32] and because the reagents used to treat a number of inflammatory diseases are designed to inhibit vascular hyperpermeability [33], our results indicated the potential of lysozyme as a therapeutic agent in various vascular inflammatory diseases.
3.2. Effects of lysozyme on the PolyP effects on CAMs expression and neutrophil adhesion and migration Previous studies have confirmed the pivotal roles of CAMs (VCAM-1, ICAM-1, and e-selectin) during cell-cell adhesion in processes of vascular inflammation [34,35]. Thus, inhibiting the
Fig. 1. Effects of lysozyme on Polyphosphate P (PolyP)-induced barrier disruptions in vitro and in vivo. (A) The effects of various concentrations of lysozyme on PolyP-induced (50 mM, 4 h) barrier disruption, which was monitored by the flux of Evans blue-bound albumin across human umbilical vein endothelial cells (HUVECs). (B) The effects of lysozyme on PolyP-induced [3.5 mg/mouse, intravenous (i.v.)] vascular permeability in mice were examined by the flux of Evans blue in the mice (expressed mg/mouse, n ¼ 5). (C) After activation with PolyP (50 mM, 4 h), the HUVECs were treated with different concentrations of lysozyme for 6 h. The effects of each compound on the expression of PolyP-mediated phosphorylated (phospho) p38 (p-p38) were determined with an ELISA. (D) Staining for F-actin. The HUVEC monolayers grown on glass coverslips were stimulated with PolyP (50 mM, 4 h) and then treated with lysozyme for 6 h. F-actin was examined with immunofluorescent staining. The arrows indicate intercellular gaps. The results are expressed as means ± SD of three independent experiments. P indicates vehicle [PBS] only. *p < 0.05 versus PolyP (AeC). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
718
J. Chung et al. / Biochemical and Biophysical Research Communications 474 (2016) 715e721
expression of CAMs is considered a promising therapeutic approach for treating vascular inflammatory diseases. We recently showed that the levels of expression of CAMs are increased by PolyP, thereby stimulating leukocyte behaviors, such as adhesion and migration, toward HUVECs [6,7]. In this study, we determined the effects of lysozyme on the levels of expression of CAMs and on the adhesion and migration of leukocytes toward HUVECs, which were both affected by PolyP. The results showed that lysozyme suppressed the increases in the levels of protein and transcript expression of CAMs (Fig. 2A and 2B). In addition, the enhancement in the expression of CAMs correlated with the increased binding and migration of leukocytes to PolyP-treated HUVECs, and this was inhibited by lysozyme treatment in a concentration-dependent manner (Fig. 2CeE). In order to confirm these results in vivo, we examined the effects of injected lysozyme on PolyP-induced leukocyte migration in mice. PolyP increased the number of migrated leukocytes in the peritoneal cavities of mice, and this was reduced by lysozyme treatment (Fig. 3F). Collectively, the results of this study showed that lysozyme downregulated PolyP-mediated vascular inflammatory responses by inhibiting the augmentation by PolyP of inflammatory signaling pathways, such as the adhesion and migration of leukocytes to inflamed endothelium.
3.3. Effects of lysozyme on the PolyP-stimulated activation of NF-
kB/ERK and the production of IL-6/TNF-a Based on the results of the present study and findings that the activation of NF-kB or ERK and the increased production of TNF-a or IL-6 amplifies vascular inflammatory responses [36e40], we hypothesized that the activation and expression of these proinflammatory molecules were suppressed by lysozyme. To confirm this hypothesis, the levels of activation and expression of these proinflammatory molecules were measured with ELISA in PolyPactivated and lysozyme-treated HUVECs. The results showed that the increased levels of protein expression of TNF-a and IL-6 (Fig. 3A and B) and the increased activation of NF-kB and ERK1/2 (Fig. 3C and D) that were induced by PolyP were reduced by lysozyme. Therefore, these results suggested that lysozyme can control important vascular inflammatory signaling pathways by regulating the molecules involved.
3.4. Vascular protective effects of lysozyme in the PolyP-induced lethality model Finally, we hypothesized that lysozyme would prevent PolyP-
Fig. 2. Effects of lysozyme on the PolyP-induced expression of cellular adhesion molecules (CAMs), cell adhesion and migration of neutrophils. (A) The PolyP-mediated (50 mM) levels of expression of vascular cell adhesion molecule-1 (VCAM-1; white bar), intercellular adhesion molecule-1 (ICAM-1; gray bar), and E-selectin (black bar) proteins in HUVECs were analyzed with whole-cell ELISA after treating the monolayers with lysozyme. (B) The PolyP-mediated (50 mM) levels of expression of VCAM-1 (white bar), ICAM-1 (gray bar), and E-selectin (black bar) transcription (mRNA) in HUVECs were analyzed with whole-cell ELISA after treating the monolayers with lysozyme. (C) The PolyPmediated (50 mM) adherence of neutrophils to HUVECs was analyzed after treating the cells with lysozyme. (D) Representative photomicrographs of neutrophil adhesion to HUVECs. (E) The PolyP-mediated (50 mM) migration of neutrophils through HUVEC monolayers was analyzed after treating the cells with lysozyme. (F) Effects of lysozyme on PolyP-induced (3.5 mg/mouse, i.v.) leukocyte migration in mice (expressed as 106, n ¼ 5). The results are expressed as the means ± SD of three independent experiments. *p < 0.05 versus PolyP.
J. Chung et al. / Biochemical and Biophysical Research Communications 474 (2016) 715e721
719
Fig. 3. Effects of lysozyme on the PolyP-stimulated activation of nuclear factor (NF)-kB/extracellular signal-regulated kinase (ERK) 1/2 and production of interleukin (IL-6)/ tumor necrosis factor (TNF)-a. The PolyP-mediated (50 mM) production of TNF-a (A) and IL-6 (B) in HUVECs was analyzed after the treatment of the cells with the indicated concentrations of lysozyme for 6 h. (C) The PolyP-mediated (50 mM) activation of phosphoeNFekB p65 (white bar) and total NF-kB p65 (black bar) in the HUVECs was analyzed after the treatment of the cells with lysozyme for 6 h. (D) The PolyP-mediated (50 mM) activation of phospho-ERK1/2 (white bar) and total ERK1/2 (black bar) in the HUVECs was analyzed after the treatment of the cells with lysozyme for 6 h *p < 0.05 vs. PolyP only.
mediated lethality in mice. To confirm this, mice were administered with lysozyme after PolyP injections. The results showed that treatment with a single dose of lysozyme (1.43 or 2.86 mg/mouse, 12 h after the PolyP injection) did not prevent PolyP-induced death
(data not shown). Thus, lysozyme was administered twice (once at 12 h and then 50 h after the PolyP injection), which resulted in an increase in the survival rate from 0 to 50% in the Kaplan-Meier survival analysis (Fig. 4A, p < 0.0001). Because the liver and
Fig. 4. Effects of lysozyme on PolyP-induced lethality and organ damage markers. (A) Male C57BL/6 mice (n ¼ 20) were intravenously administered lysozyme 12 h and 50 h after PolyP (3.5 mg/mouse, i.v.) injections. Animal survival was monitored every 6 h after the PolyP injections for 132 h. The PolyP-injected mice (C) and control sham mice (B) were administered PBS (n ¼ 20). A Kaplan-Meier survival analysis was used to determine the overall survival rates versus the PolyP-treated mice. The plasma levels of the hepatic injury markers aspartate transaminase (AST) and alanine transaminase (ALT) (B), renal injury markers creatinine (C) and bun urea nitrogen (BUN) (D), and tissue injury marker lactate dehydrogenase (LDH) (E) were measured (n ¼ 5) 72 h after the PolyP injections. D indicates vehicle (0.5% DMSO) only. *p < 0.05 vs. PolyP alone.
720
J. Chung et al. / Biochemical and Biophysical Research Communications 474 (2016) 715e721
kidney are major target organs of systemic inflammatory diseases and multiple organ failure is caused by systemic inflammatory diseases, such as sepsis and septic shock [41], we examined the plasma levels of tissue damage markers. As shown in Fig. 4BeE, lysozyme reduced the polyP-induced increases in the plasma levels of alanine transaminase and aspartate transaminase (markers of hepatic injury, Fig. 4B) and creatinine and blood urea nitrogen (markers of renal injury, Fig. 4C and D). In addition, the levels of lactate dehydrogenase, which is a marker of tissue injury, were reduced by lysozyme in PolyP-injected mice (Fig. 4E). The molecular mechanisms underlying the anti-inflammatory effects of lysozyme on PolyP-mediated septic responses may be mediated by the suppression of PolyP-mediated hyperpermeability (Fig. 1A and 1B) through the inhibition of the activation of p38 (Fig. 1C). Furthermore, the inhibitory mechanisms of lysozyme on the interaction of leukocytes with endothelial cells are mediated by the inhibition of the expression of CAMs, such as VCAM, ICAM, and e-selectin (Fig. 2). The underlying mechanisms of these antiinflammatory effects of lysozyme involve the downregulation of the production of inflammatory cytokines (TNF-a and IL-6, Fig. 3A and 4) and the activation of inflammatory transcriptional factors (NF-kB and ERK1/2, Fig. 3C and D). Finally, the results of this study showed the protective activities of lysozyme on the vascular barrier disruptions induced by PolyP in both human endothelial cells and mice. Therefore, these results suggested that lysozyme is a potential candidate in the treatment of severe vascular inflammatory diseases. Conflict of interest statement The authors declare no conflicts of interest. Acknowledgments This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (NRF2012R1A5A2A42671316 and 2014R1A2A1A11049526).
[10] [11]
[12]
[13]
[14] [15]
[16] [17]
[18]
[19] [20]
[21]
[22] [23]
[24]
[25]
[26]
Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2016.05.016.
[27]
[28]
References [1] K.D. Kumble, A. Kornberg, Inorganic polyphosphate in mammalian cells and tissues, J. Biol. Chem. 270 (1995) 5818e5822. [2] I.S. Kulaev, V.M. Vagabov, Polyphosphate metabolism in micro-organisms, Adv. Microb. Physiol. 24 (1983) 83e171. [3] T. Shiba, D. Nishimura, Y. Kawazoe, Y. Onodera, K. Tsutsumi, R. Nakamura, M. Ohshiro, Modulation of mitogenic activity of fibroblast growth factors by inorganic polyphosphate, J. Biol. Chem. 278 (2003) 26788e26792. [4] L. Hernandez-Ruiz, I. Gonzalez-Garcia, C. Castro, J.A. Brieva, F.A. Ruiz, Inorganic polyphosphate and specific induction of apoptosis in human plasma cells, Haematologica 91 (2006) 1180e1186. [5] F. Muller, N.J. Mutch, W.A. Schenk, S.A. Smith, L. Esterl, H.M. Spronk, S. Schmidbauer, W.A. Gahl, J.H. Morrissey, T. Renne, Platelet polyphosphates are proinflammatory and procoagulant mediators in vivo, Cell 139 (2009) 1143e1156. [6] J.S. Bae, W. Lee, A.R. Rezaie, Polyphosphate elicits pro-inflammatory responses that are counteracted by activated protein C in both cellular and animal models, J. Thromb. Haemost. 10 (2012) 1145e1151. [7] S.K. Ku, J.S. Bae, Concentration dependent anti-inflammatory effects thrombin on polyphosphate-mediated inflammatory responses in vitro and in vivo, Inflamm. Res. 62 (2013) 609e616. [8] I.C. Lee, J.S. Bae, Anti-inflammatory effects of vicenin-2 and scolymoside on polyphosphate-mediated vascular inflammatory responses, Inflamm. Res. 65 (2016) 203e212. [9] J.A. Kelly, A.R. Sielecki, B.D. Sykes, M.N. James, D.C. Phillips, X-ray
[29]
[30]
[31]
[32] [33] [34] [35]
[36]
[37]
crystallography of the binding of the bacterial cell wall trisaccharide NAMNAG-NAM to lysozyme, Nature 282 (1979) 875e878. R. Clingolani, Bioinspired Approaches for Human-centric Technologies, Springer, New York, 2014. D. Nakimbugwe, B. Masschalck, D. Deckers, L. Callewaert, A. Aertsen, C.W. Michiels, Cell wall substrate specificity of six different lysozymes and lysozyme inhibitory activity of bacterial extracts, FEMS Microbiol. Lett. 259 (2006) 41e46. J.S. Bae, W. Lee, J.O. Nam, J.E. Kim, S.W. Kim, I.S. Kim, Transforming growth factor beta-induced protein promotes severe vascular inflammatory responses, Am. J. Respir. Crit. Care Med. 189 (2014) 779e786. S.K. Ku, E.K. Yoon, H.G. Lee, M.S. Han, T. Lee, J.S. Bae, Inhibitory effects of lysozyme on endothelial protein C receptor shedding in vitro and in vivo, BMB Rep. 48 (2015) 624e629. E.K. Yoon, S.K. Ku, W. Lee, S. Kwak, H. Kang, B. Jung, J.S. Bae, Antitcoagulant and antiplatelet activities of scolymoside, BMB Rep. 48 (2015) 577e582. S.K. Ku, W. Lee, M. Kang, J.S. Bae, Antithrombotic activities of aspalathin and nothofagin via inhibiting platelet aggregation and FIIa/FXa, Arch. Pharm. Res. 38 (2015) 1080e1089. W. Lee, S.K. Ku, J.S. Bae, Antiplatelet, anticoagulant, and profibrinolytic activities of baicalin, Arch. Pharm. Res. 38 (2015) 893e903. R. Hofbauer, D. Moser, H. Salfinger, M. Frass, S. Kapiotis, Sufentanil inhibits migration of human leukocytes through human endothelial cell monolayers, Anesth. Analg. 87 (1998) 1181e1185. J.S. Bae, A.R. Rezaie, Thrombin inhibits HMGB1-mediated proinflammatory signaling responses when endothelial protein C receptor is occupied by its natural ligand, BMB Rep. 46 (2013) 544e549. J.S. Bae, A.R. Rezaie, Activated protein C inhibits high mobility group box 1 signaling in endothelial cells, Blood 118 (2011) 3952e3959. W. Lee, S.K. Ku, D.H. Na, J.S. Bae, Anti-inflammatory effects of lysozyme against HMGB1 in human endothelial cells and in mice, Inflammation 38 (2015) 1911e1924. W. Che, N. Lerner-Marmarosh, Q. Huang, M. Osawa, S. Ohta, M. Yoshizumi, M. Glassman, J.D. Lee, C. Yan, B.C. Berk, J. Abe, Insulin-like growth factor-1 enhances inflammatory responses in endothelial cells: role of Gab1 and MEKK3 in TNF-alpha-induced c-Jun and NF-kappaB activation and adhesion molecule expression, Circ. Res. 90 (2002) 1222e1230. J.W. Bae, J.S. Bae, Barrier protective effects of lycopene in human endothelial cells, Inflamm. Res. 60 (2011) 751e758. T.H. Kim, S.K. Ku, I.C. Lee, J.S. Bae, Anti-inflammatory functions of purpurogallin in LPS-activated human endothelial cells, BMB Rep. 45 (2012) 200e205. J.S. Bae, W. Lee, A.R. Rezaie, Polyphosphate elicits proinflammatory responses that are counteracted by activated protein C in both cellular and animal models, J. Thromb. Haemost. 10 (2012) 1145e1151. J.D. Lee, J.E. Huh, G. Jeon, H.R. Yang, H.S. Woo, D.Y. Choi, D.S. Park, Flavonolrich RVHxR from Rhus verniciflua Stokes and its major compound fisetin inhibits inflammation-related cytokines and angiogenic factor in rheumatoid arthritic fibroblast-like synovial cells and in vivo models, Int. Immunopharmacol. 9 (2009) 268e276. K.H. Diehl, R. Hull, D. Morton, R. Pfister, Y. Rabemampianina, D. Smith, J.M. Vidal, C. van de Vorstenbosch, A good practice guide to the administration of substances and removal of blood, including routes and volumes, J. Appl. Toxicol. 21 (2001) 15e23. K.L. Walton, L. Holt, R.B. Sartor, Lipopolysaccharide activates innate immune responses in murine intestinal myofibroblasts through multiple signaling pathways, Am. J. Physiol. Gastrointest. Liver Physiol. 296 (2009) G601eG611. J.S. Bae, Role of high mobility group box 1 in inflammatory disease: focus on sepsis, Arch. Pharm. Res. 35 (2012) 1511e1523. H.J. Schnittler, S.W. Schneider, H. Raifer, F. Luo, P. Dieterich, I. Just, K. Aktories, Role of actin filaments in endothelial cell-cell adhesion and membrane stability under fluid shear stress, Pflugers Arch. 442 (2001) 675e687. J. Friedl, M. Puhlmann, D.L. Bartlett, S.K. Libutti, E.N. Turner, M.F. Gnant, H.R. Alexander, Induction of permeability across endothelial cell monolayers by tumor necrosis factor (TNF) occurs via a tissue factor-dependent mechanism: relationship between the procoagulant and permeability effects of TNF, Blood 100 (2002) 1334e1339. I. Petrache, A. Birukova, S.I. Ramirez, J.G. Garcia, A.D. Verin, The role of the microtubules in tumor necrosis factor-alpha-induced endothelial cell permeability, Am. J. Respir. Cell Mol. Biol. 28 (2003) 574e581. Y.A. Komarova, D. Mehta, A.B. Malik, Dual regulation of endothelial junctional permeability, Sci. STKE 2007 (2007) re8. S.M. Dudek, J.G. Garcia, Cytoskeletal regulation of pulmonary vascular permeability, J. Appl. Physiol. 91 (2001) 1487e1500. C.J. Frijns, L.J. Kappelle, Inflammatory cell adhesion molecules in ischemic cerebrovascular disease, Stroke 33 (2002) 2115e2122. H. Ulbrich, E.E. Eriksson, L. Lindbom, Leukocyte and endothelial cell adhesion molecules as targets for therapeutic interventions in inflammatory disease, Trends Pharmacol. Sci. 24 (2003) 640e647. K. Javaid, A. Rahman, K.N. Anwar, R.S. Frey, R.D. Minshall, A.B. Malik, Tumor necrosis factor-alpha induces early-onset endothelial adhesivity by protein kinase Czeta-dependent activation of intercellular adhesion molecule-1, Circ. Res. 92 (2003) 1089e1097. J.M. Lockyer, J.S. Colladay, W.L. Alperin-Lea, T. Hammond, A.J. Buda, Inhibition of nuclear factor-kappaB-mediated adhesion molecule expression in human
J. Chung et al. / Biochemical and Biophysical Research Communications 474 (2016) 715e721 endothelial cells, Circ. Res. 82 (1998) 314e320. [38] H. Yamagami, S. Yamagami, T. Inoki, S. Amano, K. Miyata, The effects of proinflammatory cytokines on cytokine-chemokine gene expression profiles in the human corneal endothelium, Investig. Ophthalmol. Vis. Sci. 44 (2003) 514e520. [39] N. Marui, M.K. Offermann, R. Swerlick, C. Kunsch, C.A. Rosen, M. Ahmad, R.W. Alexander, R.M. Medford, Vascular cell adhesion molecule-1 (VCAM-1)
721
gene transcription and expression are regulated through an antioxidantsensitive mechanism in human vascular endothelial cells, J. Clin. Investig. 92 (1993) 1866e1874. [40] B.A. Rose, T. Force, Y. Wang, Mitogen-activated protein kinase signaling in the heart: angels versus demons in a heart-breaking tale, Physiol. Rev. 90 (2010) 1507e1546. [41] M.E. Astiz, E.C. Rackow, Septic shock, Lancet 351 (1998) 1501e1505.