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CXC chemokine CXCL12 tissue expression and circulating levels in peptic ulcer patients with Helicobacter pylori infection
Cytokine 85 (2016) 1–4
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Cytokine journal homepage: www.journals.elsevier.com/cytokine
CXC chemokine CXCL12 tissue expression and circulating levels in peptic ulcer patients with Helicobacter pylori infection Vahid Bagheri a, Gholamhossein Hassanshahi a, Vahid Mirzaee b, Hossein Khorramdelazad a,⇑ a b
Molecular Medicine Research Center, Rafsanjan University of Medical Sciences, Rafsanjan, Iran Department of Internal Medicine, Rafsanjan University of Medical Sciences, Rafsanjan, Iran
a r t i c l e
i n f o
Article history: Received 11 December 2015 Received in revised form 25 May 2016 Accepted 27 May 2016
Keywords: Helicobacter pylori Chemokine CXCL12 Peptic ulcer Angiogenesis
a b s t r a c t Helicobacter pylori (H. pylori) infection is among the most prevalent human infections. CXCL12 is a wellknown CXC chemokine involved in inflammation and play major roles in angiogenesis. There is currently very limited data on the role of CXCL12 in peptic ulcer disease. Hence, we aimed to explore whether CXCL12 is involved in the pathogenesis of peptic ulcer induced by H. pylori. In this study, we enrolled 102 H. pylori-infected patients, including 51 with active ulcer (GA) and 51 with healing ulcer (GH). We also recruited 50 healthy subjects as control, which did not show any sign or symptoms of chronic inflammatory diseases, infection, or immune-related disorders. Endoscopy was performed to determine the stage of the disease. ELISA was used for detection of H. pylori infection and CXCL12 measurement. We also employed western blotting to detect CXCL12 in ulcerative lesions of H. pylori. Demographic data were also collected by questionnaire. Our results demonstrated that CXCL12 serum levels in GA group (151.8 ± 18.31 pg/mL) were significantly higher than those in GH (36.89 ± 6.78 pg/mL) and control groups (33.77 ± 9.12 pg/mL) (P < 0.0001). However, we did not observe a significant difference between GH and control groups. Moreover, overexpression of CXCL12 in gastric lesions of patients in GA group was confirmed by Western blot analysis. According to the result of the present study, it could be concluded that CXCL12 is involved in the pathogenesis and healing of H. pylori-induced peptic ulcer. CXCL12 serum levels may also be used to distinguish between GA and GH phases of the disease. Ó 2016 Elsevier Ltd. All rights reserved.
1. Introduction Helicobacter pylori infection is found in approximately 50% of the world’s population and provokes chronic inflammation which can further cause peptic ulcer disease (10–15%) and gastric cancer (1–3%) [1]. Recently, H. pylori infection has been found to be a major risk factor for peptic ulcer disease (PUD), including gastric and duodenal ulcers [2]. The etiology and pathogenesis of infections induced by H. pylori have yet to be identified, however, some parameters such as environmental risk factors, bacterial virulence determinants, and host genetic factors play fundamental roles in its pathogenesis. The most important virulence factors in the pathogenesis of H. pylori infection are the cag pathogenicity island (PAI), CagA, and VacA [3]. Chemokine family include small proteins containing conserved cysteines that form two disulfide bonds. More recent data indicate that chemokines are divided into four subfamilies: CXC, CC, (X)C, and CX3C based on the position of N-terminal cysteines. It has been demonstrated that chemokines ⇑ Corresponding author. E-mail address: [email protected] (H. Khorramdelazad). http://dx.doi.org/10.1016/j.cyto.2016.05.025 1043-4666/Ó 2016 Elsevier Ltd. All rights reserved.
play a role in leukocyte migration and attract T lymphocytes, monocytes, and neutrophils to the site of inflammation [4,5]. CD4 and CD8 are important T cells which commonly observed in intestinal inflammation [6]. Inflammatory responses are triggered in the gastric mucosa following infection with H. pylori, which result in production of proinflammatory cytokines. Data show that a number of chemokines such as CXCL10 and CXCL11 participate in gastritis induced by H. pylori [7]. Chemokines contribute to the processes of wound healing owing to induction of inflammatory cells migration in the wound site. The presence of their receptors on resident cells such as endothelial cells shows that chemokines are also involved in the regulation of epithelialization, tissue remodeling, and angiogenesis [8,9]. Chemokine (CXC motif) ligand 12 (CXCL12), also called stromal cell-derived factor-1 (SDF-1), is a CXC chemokine which utilizes CXCR4 and CXCR7 to perform its biological functions. CXCL12/CXCR4 axis plays physiological roles, including hematopoiesis, cardiogenesis, vascular formation, and neurogenesis. On the other hand, it has been implicated in HIV-1 infection, WHIM syndrome (Warts, Hypogammaglobulinemia, Infections, and Myelokathexis), tumor metastasis, and autoimmunity [10–12].
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We studied patients with H. pylori-positive gastric ulcers divided into active (GA) and healing (GH) stages according to endoscopic results [13]. There is already evidence that the CXCL12/ CXCR4 axis is involved in different stages of patients with H. pylori-positive gastritis. Due to limited information regarding the contribution of this chemokine in H. pylori infection, we designed the present study to examine both serum concentrations and tissue expression of CXCL12 in two distinct stages of peptic ulcer (active and healing) in patients infected with H. pylori.
2. Materials and methods 2.1. Subjects Current cross-sectional study was carried out on H. pyloriinfected patients with peptic ulcers in the active stage, patients infected with H. pylori in the healing stage, and healthy controls. The occurrence of disease in both groups was determined by clinical and para-clinical findings such as positive result of H. pylori antigens by ELISA. An expert internist diagnosed the event of the active and healing stages based on endoscopy results. Healthy controls were selected among blood donor volunteers who had no evidence of infection, gastrointestinal diseases, and immune-related disorders. All members of control group were tested for H. pylori antigens and had negative IgG and IgM results. In addition, they were thoroughly examined by an experienced internist. This study was approved by the Ethics Committee at Rafsanjan University of Medical Sciences and written informed consent was obtained from patients and controls before enrollment in the study. The demographic data were also collected by questionnaire. Following specimen collection, samples were transferred to the Molecular Medicine Research Center laboratories. The tissues were stored at 80 °C, while serum isolated from blood samples was stored at 20 °C for further use. 2.2. Determination of H. pylori-specific antibodies The serum levels of anti-H. Pylori antibody (IgG) were measured by using a commercial ELISA kit (Equipar, Italy). According to the manufacturer’s instructions, the value of 5 Uarb/ml was used to differentiate positive samples from negative samples. 2.3. Detection of serum CXCL12 The serum levels of CXCL12 were measured by ELISA (R&D systems, UK) in patients and healthy controls immediately after blood collection. Ninety-six-well EIA/RIA plates were coated with 100 lL per well of the diluted mouse anti-human CXCL12/SDF-1 capture antibody and were then blocked by adding 300 lL of reagent diluent (1% bovine serum albumin in PBS) to each well. Samples (100 lL) were diluted in diluent reagent and incubated for 2 h at room temperature. Biotinylated goat anti-human CXCL12/SDF-1 detection antibody, diluted in diluent reagent, was added (100 lL) to each well and incubated for 2 h at room temperature. The working dilution of Streptavidin-HRP was added (100 lL) to each well and incubated for 20 min at room temperature. Substrate solution was added (100 lL) to each well and incubated for 20 min at room temperature. Stop solution was added (50 lL) to each well and the optical density of each well was determined immediately, using a microplate reader set to 450 nm. A standard curve was generated for each set of samples assayed from seven points of a twofold dilution series. Each standard or sample was assayed in duplicate. The sensitivity of kit was 2 pg/ml and inter and intraassay assessments of reliability of the kit were conducted.
2.4. Western blotting Ulcerative tissues were obtained by endoscopic biopsy and subjected to lysis. Samples were used for Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Equal amounts of protein lysate were loaded and resolved by a 10% SDS-PAGE and then transferred to a nitrocellulose membrane. After blocking with 5% skim milk in PBS for 1 h at room temperature, the nitrocellulose membrane was incubated with anti-human primary antibody against CXCL12 (R&D System, Minneapolis, MN, USA) overnight at 4 °C in PBS/Tween containing 3% (w/v) skim milk. Subsequently, the membrane was washed three times with 5% skim milk in PBS and then incubated with anti-mouse immunoglobulin G (IgG) horseradish peroxidase-conjugated secondary antibody (Amersham Life Science, Amersham, UK) (diluted 1:1000) was used for further detection of CXCL12. The amount of CXCL12 in each sample was visualized using enhanced chemiluminescence reagents (Amersham Life Science, UK), followed by autoradiography. b-actin was used as a loading control. 2.5. Statistical analysis Data were expressed as mean ± SEM. Statistical analysis was conducted using SPSS version 18 (SPSS, Inc, Chicago, IL, USA). One-way ANOVA was used to evaluate differences among groups. A P value of less than 0.05 was considered statistically significant. 3. Results 3.1. Patient demographics In this study, we enrolled 102 patients suffering from peptic ulcer induced by H. pylori, along with 50 healthy controls. H. pylori infection was in active form in 51 patients, while 51 patients had recovered from the disease and were in the healing phase. Statistical analysis of the demographic data of the present study showed that the average age of participants was 37.9 ± 10.48, 38.72 ± 12.62, and 36.38 ± 11.99 years in control, GA and GH group, respectively (Table 1). 3.2. Serum levels of CXCL12 Our results demonstrated that the mean circulating levels of CXCL12 levels were 151.8 ± 18.31 pg/mL, 36.89 ± 6.78 pg/mL, and 33.77 ± 9.12 pg/mL in patient with active peptic ulcer (GA), healing peptic ulcer (GH), and control group, respectively (Fig. 1). Statistical analysis of data showed that there was a significant difference between the two groups of patients. In the GA group, CXCL12 levels were found to be substantially higher than those in the GH and control groups (P < 0.0001). In contrast, we did not observe a significant difference between the GH and control groups. 3.3. Expression of CXCL12 in gastric ulcer tissues In addition to serum concentration, the protein levels of CXCL12 was examined by Western blotting of tissue extracts. As clearly shown in Fig. 2, CXCL2 protein was highly upregulated in GA group compared with GH and control groups. However, we observed very Table 1 Patient demographics in the GA, GH, and control groups. Clinical status
N
Age, years (mean ± SEM)
Gender (male/female)
Healthy controls Active stage (GA) Healing stage (GH)
50 51 51
37.90 ± 10.48 38.72 ± 12.62 36.38 ± 11.99
28/22 25/26 23/28
V. Bagheri et al. / Cytokine 85 (2016) 1–4
Fig. 1. Circulating levels of CXCL12 in the GA and GH stages of H. pylori-associated peptic ulcer. ⁄Significant difference among the three groups.
Fig. 2. Western blot analysis of CXCL12 in tissues from patients with different stages of H. pylori-associated peptic ulcer. b-actin was used as a loading control.
weak expression of CXCL12 in GH and control groups. These results are consistent with observations obtained from ELISA. 4. Discussion H. pylori and non-steroidal anti-inflammatory drugs (NSAIDs) are the main causes of peptic ulcer disease [14]. For instance, it has been reported that H. pylori infection is more prevalent among south Iranian patients with peptic ulcer disease [15]. There is evidence that H. pylori eradication programs can reduce the rate of gastric atrophy and peptic ulcer disease [16]. Due to recruitment of immune cells to the site of inflammation, CXCL12 is considered to have a proinflammatory role in autoimmune diseases, including rheumatoid arthritis (RA) and nephritis [17]. In the intestinal immune system, CXCL12 is expressed by intestinal epithelial cells and upregulated in inflammatory bowel disease, suggesting an inflammatory role in the gut [18]. The findings of the present study revealed that after being infected with H. pylori and during active peptic ulcer disease, increased CXCL12 production is initiated in ulcerative lesions. Expression of chemokines (CXC and CC) have been widely evaluated in mucosal biopsies during H. pylori infection. These findings have demonstrated that CXC chemokines (Gro-alpha (CXCL-1), IL-8 (CXCL-8), and ENA-78 (CXCL5)) and CC chemokines (MCP-1 (CCL-2) and RANTES (CCL5)) expression is markedly increased in patients with H. pylori infection [19–23]. Production of Gro-alpha and IL-8, which act as neutrophilrecruiting chemokines, is induced through H. pylori lipopolysaccharides (LPS) in human monocytes, suggesting recruitment of neutrophils to inflamed gastric mucosa and ulcer formation [24]. Additionally, endothelial and gastric epithelial cells produce Groalpha and IL-8 after infection with H. pylori [25,26]. Other in vivo studies have indicated that CXC chemokines are associated with H. pylori-induced gastric inflammation in comparison with CC
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chemokines [27,28]. Interestingly, elevated levels of Gro-alpha and IL-8 have been observed in mucosal tissues of patients with peptic ulcer, but not in patients with gastritis or normal mucosa [29]. Taking together, one possible mechanism which may explain enhanced CXCL12 level in both serum and ulcerative lesions of patients in the GA stage could be host inflammatory responses developed during peptic ulcer. On the other hand, a large number of studies have shown that CXCL12 can play a major role in angiogenesis. CXCL12 is also able to upregulate VEGF-A and this lead to attraction of pro-angiogenic VEGFR2+ and CXCR4+ cells [30,31]. This angiogenic activity of CXCL12 has been shown in different cancers. For instance, CXCR4+ monocytes are recruited by CXCL12, which induce angiogenesis by releasing angiogenic factors such as angiopoietin and VEGF-A [32]. Several reports indicate that CXCL12/CXCR4 axis could have a role not only in vascularization of the adult human gastrointestinal tract but also in the repair process of inflamed human intestinal mucosa [33]. Production of CXCL12 achieved to its peak level during the active stage and declined in patients with healing stage of disease. To the best of our knowledge, this study is the first report of serum concentrations and protein expression of CXCL12 in patients with H. pylori-positive peptic ulcer disease. The results are consistent with an earlier research by Akimoto and co-workers on mRNA expression of several angiogenic factors in gastric ulcers. They demonstrated that CXCL12/CXCR4 axis have a significant role in gastric ulcer healing. Increased expression of CXCL12 in the GA group and CXCR4 in the GH group led to the suggestion that CXCR4 is involved in late angiogenesis [34]. The results of the present investigation may re-emphasize an involvement of CXCL12 in H. pylori infection. Increased circulating levels and tissue expression of CXCL12 may be related to the crucial roles played by this chemokine in angiogenesis phenomenon. The current data show that CXCL12/CXCR4 chemokine signaling pathway is responsible for wound healing by recruitment and migration of epidermal stem cells (ESCs) and bone marrow-derived stromal stem cells (BMSCs) to wound sites [35,36]. For example, expression of CXCL12 is elevated during the early phases of acute myocardial infarction and skeletal repair [37]. It is therefore proposed that increased expression of CXCL12 may facilitate gastric ulcer healing and tissue regeneration in the early stages of peptic ulcer. These effects have been confirmed in a mouse model of diabetes. It was found that treatment of diabetic wounds with a plasmid expressing CXCL12 can accelerate wound healing process [38]. It was previously believed that CXCL12 was the only ligand for CXCR4, while macrophage migration inhibitory factor (MIF) and extracellular ubiquitin (eUb) have been recently recognized. MIF is a cytokine with chemokine-like functions such as chemotactic activity and exerts its effects by binding to the CXCR4 and CXCR2 chemokine receptors. On the other hand, Ub is a small protein found in eukaryotic cells that is involved in protein degradation. In addition to its intracellular activities, Ub exists in plasma as a natural protein [39]. Several studies have confirmed that MIF is able to induce angiogenesis, raising the possibility that MIF could mediate its angiogenic properties through CXCR4 [40]. It is also interesting to note that production of MIF by gastric epithelial cells is induced in response to the presence of H. pylori [41]. Similarly, Ub/CXCR4 axis has been shown to induce the process of angiogenesis, including endothelial cell migration and capillary network formation [42]. According to the present and previous study [34], expression of CXCL12 is significantly reduced during ulcer healing (GH) in comparison with active ulcer (GA), whereas expression of CXCR4 is significantly increased during GH stage. Based on the above observations, we therefore hypothesize that other CXCR4 ligands (MIF and eUb) may activate CXCR4 to trigger the angiogenic effects and facilitate tissue repair during the healing stage of gastric ulcers. Moreover, our results propose that circulating levels of
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CXCL12 may be a useful marker to differentiate between GA and GH stages of peptic ulcer. In conclusion, the finding of the present study may reemphasize the fact that elevated CXCL12 in patients with peptic ulcer could play a critical part in the healing of peptic ulcer. Due to a dual role in recruitment of inflammatory or stem cells, further studies are needed to clarify the involvement of CXCL12 in inflammation or healing process during H. pylori- associated peptic ulcer. We suggest evaluation of the role of MIF and eUb in the different stages of gastric ulcers. It will be interesting for future research to examine expression of CXCR7, another CXCL12 receptor, in peptic ulcer stages. Finally, our results suggest that the topical application of CXCL12 gene (lipofection, plasmid, or viral vector) in animal models of peptic ulcer may improve stem cells migration and enhance ulcer healing. Conflict of interest None of authors declared conflict of interest. Acknowledgements The authors are grateful to the clinical staff of the endoscopy unit of Ali-ebn-Abitaleb Hospital of Rafsanjan for invaluable help. This project was financially supported by the Rafsanjan University of Medical Sciences. References [1] F. Avilés-Jiménez, A. Reyes-Leon, E. Nieto-Patlán, L.M. Hansen, J. Burgueño, I.P. Ramos, et al., In vivo expression of Helicobacter pylori virulence genes in patients with gastritis, ulcer, and gastric cancer, Infect. Immun. 80 (2012) 594–601. [2] B. Schöttker, M.A. Adamu, M.N. Weck, H. Brenner, Helicobacter pylori infection is strongly associated with gastric and duodenal ulcers in a large prospective study, Clin. Gastroenterology Hepatology 10 (487–93) (2012) e1. [3] D.N. Sgouras, T.T.H. Trang, Y. Yamaoka, Pathogenesis of Helicobacter pylori infection, Helicobacter 20 (2015) 8–16. [4] M. Baggiolini, Chemokines and leukocyte traffic, Nature 392 (1998) 565–568. [5] A. Zlotnik, O. Yoshie, The chemokine superfamily revisited, Immunity 36 (2012) 705–716. [6] R.M. Nandre, A.A. Chaudhari, K. Matsuda, J.H. Lee, Immunogenicity of a Salmonella Enteritidis mutant as vaccine candidate and its protective efficacy against salmonellosis in chickens, Vet. Immunol. Immunopathol. 144 (2011) 299–311. [7] H.-F. Tsai, P.-N. Hsu, Interplay between Helicobacter pylori and immune cells in immune pathogenesis of gastric inflammation and mucosal pathology, Cell. Mol. Immunol. 7 (2010) 255–259. [8] R. Gillitzer, M. Goebeler, Chemokines in cutaneous wound healing, J. Leukoc. Biol. 69 (2001) 513–521. [9] S. Barrientos, O. Stojadinovic, M.S. Golinko, H. Brem, M. Tomic-Canic, Growth factors and cytokines in wound healing, Wound Repair Regeneration 16 (2008) 585–601. [10] T. Nagasawa, CXC chemokine ligand 12 (CXCL12) and its receptor CXCR4, J. Mol. Med. 92 (2014) 433–439. [11] J.A. Wojcechowskyj, J.Y. Lee, S.H. Seeholzer, R.W. Doms, Quantitative phosphoproteomics of CXCL12 (SDF-1) signaling, PLoS One 6 (2011) e24918. [12] X. Sun, G. Cheng, M. Hao, J. Zheng, X. Zhou, J. Zhang, et al., CXCL12/CXCR4/ CXCR7 chemokine axis and cancer progression, Cancer Metastasis Rev. 29 (2010) 709–722. [13] K. Kohda, K. Tanaka, Y. Aiba, M. Yasuda, T. Miwa, Y. Koga, Role of apoptosis induced by Helicobacter pylori infection in the development of duodenal ulcer, Gut 44 (1999) 456–462. [14] B. Bashinskaya, B.V. Nahed, N. Redjal, K.T. Kahle, B.P. Walcott, Trends in peptic ulcer disease and the identification of Helicobacter Pylori as a causative organism: population-based estimates from the US nationwide inpatient sample, J. Global Infect. Dis. 3 (2011) 366–370. [15] M.R. Hashemi, M. Rahnavardi, B. Bikdeli, H pylori infection among 1000 southern Iranian dyspeptic patients, World J. Gastroenterology: WJG 12 (2006) 5479–5482. [16] Y.-C. Lee, T.H.-H. Chen, H.-M. Chiu, C.-T. Shun, H. Chiang, T.-Y. Liu, et al., The benefit of mass eradication of Helicobacter pylori infection: a communitybased study of gastric cancer prevention, Gut 62 (2013) 676–682.
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Contents lists available at ScienceDirect
Cytokine journal homepage: www.journals.elsevier.com/cytokine
CXC chemokine CXCL12 tissue expression and circulating levels in peptic ulcer patients with Helicobacter pylori infection Vahid Bagheri a, Gholamhossein Hassanshahi a, Vahid Mirzaee b, Hossein Khorramdelazad a,⇑ a b
Molecular Medicine Research Center, Rafsanjan University of Medical Sciences, Rafsanjan, Iran Department of Internal Medicine, Rafsanjan University of Medical Sciences, Rafsanjan, Iran
a r t i c l e
i n f o
Article history: Received 11 December 2015 Received in revised form 25 May 2016 Accepted 27 May 2016
Keywords: Helicobacter pylori Chemokine CXCL12 Peptic ulcer Angiogenesis
a b s t r a c t Helicobacter pylori (H. pylori) infection is among the most prevalent human infections. CXCL12 is a wellknown CXC chemokine involved in inflammation and play major roles in angiogenesis. There is currently very limited data on the role of CXCL12 in peptic ulcer disease. Hence, we aimed to explore whether CXCL12 is involved in the pathogenesis of peptic ulcer induced by H. pylori. In this study, we enrolled 102 H. pylori-infected patients, including 51 with active ulcer (GA) and 51 with healing ulcer (GH). We also recruited 50 healthy subjects as control, which did not show any sign or symptoms of chronic inflammatory diseases, infection, or immune-related disorders. Endoscopy was performed to determine the stage of the disease. ELISA was used for detection of H. pylori infection and CXCL12 measurement. We also employed western blotting to detect CXCL12 in ulcerative lesions of H. pylori. Demographic data were also collected by questionnaire. Our results demonstrated that CXCL12 serum levels in GA group (151.8 ± 18.31 pg/mL) were significantly higher than those in GH (36.89 ± 6.78 pg/mL) and control groups (33.77 ± 9.12 pg/mL) (P < 0.0001). However, we did not observe a significant difference between GH and control groups. Moreover, overexpression of CXCL12 in gastric lesions of patients in GA group was confirmed by Western blot analysis. According to the result of the present study, it could be concluded that CXCL12 is involved in the pathogenesis and healing of H. pylori-induced peptic ulcer. CXCL12 serum levels may also be used to distinguish between GA and GH phases of the disease. Ó 2016 Elsevier Ltd. All rights reserved.
1. Introduction Helicobacter pylori infection is found in approximately 50% of the world’s population and provokes chronic inflammation which can further cause peptic ulcer disease (10–15%) and gastric cancer (1–3%) [1]. Recently, H. pylori infection has been found to be a major risk factor for peptic ulcer disease (PUD), including gastric and duodenal ulcers [2]. The etiology and pathogenesis of infections induced by H. pylori have yet to be identified, however, some parameters such as environmental risk factors, bacterial virulence determinants, and host genetic factors play fundamental roles in its pathogenesis. The most important virulence factors in the pathogenesis of H. pylori infection are the cag pathogenicity island (PAI), CagA, and VacA [3]. Chemokine family include small proteins containing conserved cysteines that form two disulfide bonds. More recent data indicate that chemokines are divided into four subfamilies: CXC, CC, (X)C, and CX3C based on the position of N-terminal cysteines. It has been demonstrated that chemokines ⇑ Corresponding author. E-mail address: [email protected] (H. Khorramdelazad). http://dx.doi.org/10.1016/j.cyto.2016.05.025 1043-4666/Ó 2016 Elsevier Ltd. All rights reserved.
play a role in leukocyte migration and attract T lymphocytes, monocytes, and neutrophils to the site of inflammation [4,5]. CD4 and CD8 are important T cells which commonly observed in intestinal inflammation [6]. Inflammatory responses are triggered in the gastric mucosa following infection with H. pylori, which result in production of proinflammatory cytokines. Data show that a number of chemokines such as CXCL10 and CXCL11 participate in gastritis induced by H. pylori [7]. Chemokines contribute to the processes of wound healing owing to induction of inflammatory cells migration in the wound site. The presence of their receptors on resident cells such as endothelial cells shows that chemokines are also involved in the regulation of epithelialization, tissue remodeling, and angiogenesis [8,9]. Chemokine (CXC motif) ligand 12 (CXCL12), also called stromal cell-derived factor-1 (SDF-1), is a CXC chemokine which utilizes CXCR4 and CXCR7 to perform its biological functions. CXCL12/CXCR4 axis plays physiological roles, including hematopoiesis, cardiogenesis, vascular formation, and neurogenesis. On the other hand, it has been implicated in HIV-1 infection, WHIM syndrome (Warts, Hypogammaglobulinemia, Infections, and Myelokathexis), tumor metastasis, and autoimmunity [10–12].
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We studied patients with H. pylori-positive gastric ulcers divided into active (GA) and healing (GH) stages according to endoscopic results [13]. There is already evidence that the CXCL12/ CXCR4 axis is involved in different stages of patients with H. pylori-positive gastritis. Due to limited information regarding the contribution of this chemokine in H. pylori infection, we designed the present study to examine both serum concentrations and tissue expression of CXCL12 in two distinct stages of peptic ulcer (active and healing) in patients infected with H. pylori.
2. Materials and methods 2.1. Subjects Current cross-sectional study was carried out on H. pyloriinfected patients with peptic ulcers in the active stage, patients infected with H. pylori in the healing stage, and healthy controls. The occurrence of disease in both groups was determined by clinical and para-clinical findings such as positive result of H. pylori antigens by ELISA. An expert internist diagnosed the event of the active and healing stages based on endoscopy results. Healthy controls were selected among blood donor volunteers who had no evidence of infection, gastrointestinal diseases, and immune-related disorders. All members of control group were tested for H. pylori antigens and had negative IgG and IgM results. In addition, they were thoroughly examined by an experienced internist. This study was approved by the Ethics Committee at Rafsanjan University of Medical Sciences and written informed consent was obtained from patients and controls before enrollment in the study. The demographic data were also collected by questionnaire. Following specimen collection, samples were transferred to the Molecular Medicine Research Center laboratories. The tissues were stored at 80 °C, while serum isolated from blood samples was stored at 20 °C for further use. 2.2. Determination of H. pylori-specific antibodies The serum levels of anti-H. Pylori antibody (IgG) were measured by using a commercial ELISA kit (Equipar, Italy). According to the manufacturer’s instructions, the value of 5 Uarb/ml was used to differentiate positive samples from negative samples. 2.3. Detection of serum CXCL12 The serum levels of CXCL12 were measured by ELISA (R&D systems, UK) in patients and healthy controls immediately after blood collection. Ninety-six-well EIA/RIA plates were coated with 100 lL per well of the diluted mouse anti-human CXCL12/SDF-1 capture antibody and were then blocked by adding 300 lL of reagent diluent (1% bovine serum albumin in PBS) to each well. Samples (100 lL) were diluted in diluent reagent and incubated for 2 h at room temperature. Biotinylated goat anti-human CXCL12/SDF-1 detection antibody, diluted in diluent reagent, was added (100 lL) to each well and incubated for 2 h at room temperature. The working dilution of Streptavidin-HRP was added (100 lL) to each well and incubated for 20 min at room temperature. Substrate solution was added (100 lL) to each well and incubated for 20 min at room temperature. Stop solution was added (50 lL) to each well and the optical density of each well was determined immediately, using a microplate reader set to 450 nm. A standard curve was generated for each set of samples assayed from seven points of a twofold dilution series. Each standard or sample was assayed in duplicate. The sensitivity of kit was 2 pg/ml and inter and intraassay assessments of reliability of the kit were conducted.
2.4. Western blotting Ulcerative tissues were obtained by endoscopic biopsy and subjected to lysis. Samples were used for Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Equal amounts of protein lysate were loaded and resolved by a 10% SDS-PAGE and then transferred to a nitrocellulose membrane. After blocking with 5% skim milk in PBS for 1 h at room temperature, the nitrocellulose membrane was incubated with anti-human primary antibody against CXCL12 (R&D System, Minneapolis, MN, USA) overnight at 4 °C in PBS/Tween containing 3% (w/v) skim milk. Subsequently, the membrane was washed three times with 5% skim milk in PBS and then incubated with anti-mouse immunoglobulin G (IgG) horseradish peroxidase-conjugated secondary antibody (Amersham Life Science, Amersham, UK) (diluted 1:1000) was used for further detection of CXCL12. The amount of CXCL12 in each sample was visualized using enhanced chemiluminescence reagents (Amersham Life Science, UK), followed by autoradiography. b-actin was used as a loading control. 2.5. Statistical analysis Data were expressed as mean ± SEM. Statistical analysis was conducted using SPSS version 18 (SPSS, Inc, Chicago, IL, USA). One-way ANOVA was used to evaluate differences among groups. A P value of less than 0.05 was considered statistically significant. 3. Results 3.1. Patient demographics In this study, we enrolled 102 patients suffering from peptic ulcer induced by H. pylori, along with 50 healthy controls. H. pylori infection was in active form in 51 patients, while 51 patients had recovered from the disease and were in the healing phase. Statistical analysis of the demographic data of the present study showed that the average age of participants was 37.9 ± 10.48, 38.72 ± 12.62, and 36.38 ± 11.99 years in control, GA and GH group, respectively (Table 1). 3.2. Serum levels of CXCL12 Our results demonstrated that the mean circulating levels of CXCL12 levels were 151.8 ± 18.31 pg/mL, 36.89 ± 6.78 pg/mL, and 33.77 ± 9.12 pg/mL in patient with active peptic ulcer (GA), healing peptic ulcer (GH), and control group, respectively (Fig. 1). Statistical analysis of data showed that there was a significant difference between the two groups of patients. In the GA group, CXCL12 levels were found to be substantially higher than those in the GH and control groups (P < 0.0001). In contrast, we did not observe a significant difference between the GH and control groups. 3.3. Expression of CXCL12 in gastric ulcer tissues In addition to serum concentration, the protein levels of CXCL12 was examined by Western blotting of tissue extracts. As clearly shown in Fig. 2, CXCL2 protein was highly upregulated in GA group compared with GH and control groups. However, we observed very Table 1 Patient demographics in the GA, GH, and control groups. Clinical status
N
Age, years (mean ± SEM)
Gender (male/female)
Healthy controls Active stage (GA) Healing stage (GH)
50 51 51
37.90 ± 10.48 38.72 ± 12.62 36.38 ± 11.99
28/22 25/26 23/28
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Fig. 1. Circulating levels of CXCL12 in the GA and GH stages of H. pylori-associated peptic ulcer. ⁄Significant difference among the three groups.
Fig. 2. Western blot analysis of CXCL12 in tissues from patients with different stages of H. pylori-associated peptic ulcer. b-actin was used as a loading control.
weak expression of CXCL12 in GH and control groups. These results are consistent with observations obtained from ELISA. 4. Discussion H. pylori and non-steroidal anti-inflammatory drugs (NSAIDs) are the main causes of peptic ulcer disease [14]. For instance, it has been reported that H. pylori infection is more prevalent among south Iranian patients with peptic ulcer disease [15]. There is evidence that H. pylori eradication programs can reduce the rate of gastric atrophy and peptic ulcer disease [16]. Due to recruitment of immune cells to the site of inflammation, CXCL12 is considered to have a proinflammatory role in autoimmune diseases, including rheumatoid arthritis (RA) and nephritis [17]. In the intestinal immune system, CXCL12 is expressed by intestinal epithelial cells and upregulated in inflammatory bowel disease, suggesting an inflammatory role in the gut [18]. The findings of the present study revealed that after being infected with H. pylori and during active peptic ulcer disease, increased CXCL12 production is initiated in ulcerative lesions. Expression of chemokines (CXC and CC) have been widely evaluated in mucosal biopsies during H. pylori infection. These findings have demonstrated that CXC chemokines (Gro-alpha (CXCL-1), IL-8 (CXCL-8), and ENA-78 (CXCL5)) and CC chemokines (MCP-1 (CCL-2) and RANTES (CCL5)) expression is markedly increased in patients with H. pylori infection [19–23]. Production of Gro-alpha and IL-8, which act as neutrophilrecruiting chemokines, is induced through H. pylori lipopolysaccharides (LPS) in human monocytes, suggesting recruitment of neutrophils to inflamed gastric mucosa and ulcer formation [24]. Additionally, endothelial and gastric epithelial cells produce Groalpha and IL-8 after infection with H. pylori [25,26]. Other in vivo studies have indicated that CXC chemokines are associated with H. pylori-induced gastric inflammation in comparison with CC
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chemokines [27,28]. Interestingly, elevated levels of Gro-alpha and IL-8 have been observed in mucosal tissues of patients with peptic ulcer, but not in patients with gastritis or normal mucosa [29]. Taking together, one possible mechanism which may explain enhanced CXCL12 level in both serum and ulcerative lesions of patients in the GA stage could be host inflammatory responses developed during peptic ulcer. On the other hand, a large number of studies have shown that CXCL12 can play a major role in angiogenesis. CXCL12 is also able to upregulate VEGF-A and this lead to attraction of pro-angiogenic VEGFR2+ and CXCR4+ cells [30,31]. This angiogenic activity of CXCL12 has been shown in different cancers. For instance, CXCR4+ monocytes are recruited by CXCL12, which induce angiogenesis by releasing angiogenic factors such as angiopoietin and VEGF-A [32]. Several reports indicate that CXCL12/CXCR4 axis could have a role not only in vascularization of the adult human gastrointestinal tract but also in the repair process of inflamed human intestinal mucosa [33]. Production of CXCL12 achieved to its peak level during the active stage and declined in patients with healing stage of disease. To the best of our knowledge, this study is the first report of serum concentrations and protein expression of CXCL12 in patients with H. pylori-positive peptic ulcer disease. The results are consistent with an earlier research by Akimoto and co-workers on mRNA expression of several angiogenic factors in gastric ulcers. They demonstrated that CXCL12/CXCR4 axis have a significant role in gastric ulcer healing. Increased expression of CXCL12 in the GA group and CXCR4 in the GH group led to the suggestion that CXCR4 is involved in late angiogenesis [34]. The results of the present investigation may re-emphasize an involvement of CXCL12 in H. pylori infection. Increased circulating levels and tissue expression of CXCL12 may be related to the crucial roles played by this chemokine in angiogenesis phenomenon. The current data show that CXCL12/CXCR4 chemokine signaling pathway is responsible for wound healing by recruitment and migration of epidermal stem cells (ESCs) and bone marrow-derived stromal stem cells (BMSCs) to wound sites [35,36]. For example, expression of CXCL12 is elevated during the early phases of acute myocardial infarction and skeletal repair [37]. It is therefore proposed that increased expression of CXCL12 may facilitate gastric ulcer healing and tissue regeneration in the early stages of peptic ulcer. These effects have been confirmed in a mouse model of diabetes. It was found that treatment of diabetic wounds with a plasmid expressing CXCL12 can accelerate wound healing process [38]. It was previously believed that CXCL12 was the only ligand for CXCR4, while macrophage migration inhibitory factor (MIF) and extracellular ubiquitin (eUb) have been recently recognized. MIF is a cytokine with chemokine-like functions such as chemotactic activity and exerts its effects by binding to the CXCR4 and CXCR2 chemokine receptors. On the other hand, Ub is a small protein found in eukaryotic cells that is involved in protein degradation. In addition to its intracellular activities, Ub exists in plasma as a natural protein [39]. Several studies have confirmed that MIF is able to induce angiogenesis, raising the possibility that MIF could mediate its angiogenic properties through CXCR4 [40]. It is also interesting to note that production of MIF by gastric epithelial cells is induced in response to the presence of H. pylori [41]. Similarly, Ub/CXCR4 axis has been shown to induce the process of angiogenesis, including endothelial cell migration and capillary network formation [42]. According to the present and previous study [34], expression of CXCL12 is significantly reduced during ulcer healing (GH) in comparison with active ulcer (GA), whereas expression of CXCR4 is significantly increased during GH stage. Based on the above observations, we therefore hypothesize that other CXCR4 ligands (MIF and eUb) may activate CXCR4 to trigger the angiogenic effects and facilitate tissue repair during the healing stage of gastric ulcers. Moreover, our results propose that circulating levels of
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CXCL12 may be a useful marker to differentiate between GA and GH stages of peptic ulcer. In conclusion, the finding of the present study may reemphasize the fact that elevated CXCL12 in patients with peptic ulcer could play a critical part in the healing of peptic ulcer. Due to a dual role in recruitment of inflammatory or stem cells, further studies are needed to clarify the involvement of CXCL12 in inflammation or healing process during H. pylori- associated peptic ulcer. We suggest evaluation of the role of MIF and eUb in the different stages of gastric ulcers. It will be interesting for future research to examine expression of CXCR7, another CXCL12 receptor, in peptic ulcer stages. Finally, our results suggest that the topical application of CXCL12 gene (lipofection, plasmid, or viral vector) in animal models of peptic ulcer may improve stem cells migration and enhance ulcer healing. Conflict of interest None of authors declared conflict of interest. Acknowledgements The authors are grateful to the clinical staff of the endoscopy unit of Ali-ebn-Abitaleb Hospital of Rafsanjan for invaluable help. This project was financially supported by the Rafsanjan University of Medical Sciences. References [1] F. Avilés-Jiménez, A. Reyes-Leon, E. Nieto-Patlán, L.M. Hansen, J. Burgueño, I.P. Ramos, et al., In vivo expression of Helicobacter pylori virulence genes in patients with gastritis, ulcer, and gastric cancer, Infect. Immun. 80 (2012) 594–601. [2] B. Schöttker, M.A. Adamu, M.N. Weck, H. Brenner, Helicobacter pylori infection is strongly associated with gastric and duodenal ulcers in a large prospective study, Clin. Gastroenterology Hepatology 10 (487–93) (2012) e1. [3] D.N. Sgouras, T.T.H. Trang, Y. Yamaoka, Pathogenesis of Helicobacter pylori infection, Helicobacter 20 (2015) 8–16. [4] M. Baggiolini, Chemokines and leukocyte traffic, Nature 392 (1998) 565–568. [5] A. Zlotnik, O. Yoshie, The chemokine superfamily revisited, Immunity 36 (2012) 705–716. [6] R.M. Nandre, A.A. Chaudhari, K. Matsuda, J.H. Lee, Immunogenicity of a Salmonella Enteritidis mutant as vaccine candidate and its protective efficacy against salmonellosis in chickens, Vet. Immunol. Immunopathol. 144 (2011) 299–311. [7] H.-F. Tsai, P.-N. Hsu, Interplay between Helicobacter pylori and immune cells in immune pathogenesis of gastric inflammation and mucosal pathology, Cell. Mol. Immunol. 7 (2010) 255–259. [8] R. Gillitzer, M. Goebeler, Chemokines in cutaneous wound healing, J. Leukoc. Biol. 69 (2001) 513–521. [9] S. Barrientos, O. Stojadinovic, M.S. Golinko, H. Brem, M. Tomic-Canic, Growth factors and cytokines in wound healing, Wound Repair Regeneration 16 (2008) 585–601. [10] T. Nagasawa, CXC chemokine ligand 12 (CXCL12) and its receptor CXCR4, J. Mol. Med. 92 (2014) 433–439. [11] J.A. Wojcechowskyj, J.Y. Lee, S.H. Seeholzer, R.W. Doms, Quantitative phosphoproteomics of CXCL12 (SDF-1) signaling, PLoS One 6 (2011) e24918. [12] X. Sun, G. Cheng, M. Hao, J. Zheng, X. Zhou, J. Zhang, et al., CXCL12/CXCR4/ CXCR7 chemokine axis and cancer progression, Cancer Metastasis Rev. 29 (2010) 709–722. [13] K. Kohda, K. Tanaka, Y. Aiba, M. Yasuda, T. Miwa, Y. Koga, Role of apoptosis induced by Helicobacter pylori infection in the development of duodenal ulcer, Gut 44 (1999) 456–462. [14] B. Bashinskaya, B.V. Nahed, N. Redjal, K.T. Kahle, B.P. Walcott, Trends in peptic ulcer disease and the identification of Helicobacter Pylori as a causative organism: population-based estimates from the US nationwide inpatient sample, J. Global Infect. Dis. 3 (2011) 366–370. [15] M.R. Hashemi, M. Rahnavardi, B. Bikdeli, H pylori infection among 1000 southern Iranian dyspeptic patients, World J. Gastroenterology: WJG 12 (2006) 5479–5482. [16] Y.-C. Lee, T.H.-H. Chen, H.-M. Chiu, C.-T. Shun, H. Chiang, T.-Y. Liu, et al., The benefit of mass eradication of Helicobacter pylori infection: a communitybased study of gastric cancer prevention, Gut 62 (2013) 676–682.
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