Cellular Immunology 290 (2014) 164–168
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Cellular Immunology journal homepage: www.elsevier.com/locate/ycimm
Prolactin increases tumor necrosis factor alpha expression in peripheral CD14 monocytes of patients with rheumatoid arthritis Chun Tang a, Yun Li b, Xiaojun Lin a, Jinghua Ye a, Weinian Li a, Zhixiang He a, Fangfei Li a, Xiaoyan Cai a,⇑ a b
Department of Rheumatology, Guangzhou First People’s Hospital, Guangzhou Medical University, 1 Panfu Road, Guangzhou, China Department of Cardiothoracic Surgery, The Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, China
a r t i c l e
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Article history: Received 2 March 2014 Accepted 17 June 2014 Available online 24 June 2014 Keywords: Rheumatoid arthritis Tumor necrosis factor-a Prolactin Mitogen activated protein Methylation specific PCR
a b s t r a c t Tumor necrosis factor (TNF)-a is one of the major proinflammatory mediators of rheumatic arthritis (RA); the regulatory factors for TNF-a release is not fully understood. This study aims to investigate the role of prolactin receptor (PRLR) activation in regulating the expression and release of TNF-a from CD14+ monocytes. The results showed that the expression of PRLR was detectable in CD14+ monocytes of healthy subjects, which was markedly increased in RA patients. Exposure to PRL in the culture increased the expression and release of TNF-a from CD14+ monocytes, which was abolished by the PRLR gene silencing or blocking the mitogen activated protein (MAPK) pathway. We conclude that exposure to PRL increases TNF-a release from CD14+ monocytes of RA patients, which can be abolished by PRLR gene silencing or treating with MAPK inhibitor. Ó 2014 Elsevier Inc. All rights reserved.
1. Introduction Rheumatoid arthritis (RA) is a chronic immune inflammatory disease in the body; it mainly affects the joints. The inflammation can be destructive, cause severe pain and functional disability [1]. The pathogenesis of RA is not fully understood yet. The therapeutic effect of RA is not satisfactory currently [2]. Tumor necrosis factor (TNF)-a is one of the major proinflammatory mediators of RA [3]. Under the physiological circumstance, TNF-a plays a critical role in the body defensive system by activating the cytocidal activities in phagocytes [4]. TNF-a can be produced by monocytes, macrophages, and T cells upon being stimulated by pathogens or microbial products; it may play a key role in bone metabolism and is important in inflammatory bone diseases such as the TNF-a-induced osteoclast recruitment is probably central to the pathogenesis of RA [5]. However, the regulatory factors of TNF-a release are not fully elucidated yet. CD14+ cells include several cell types, such as monocytes, macrophages and dendritic monocytes, which are one of the major sources of TNF-a in the body. TNF-a is one of the proinflammatory cytokines involving in a number of inflammatory diseases, such as inflammatory bowel disease [6] and rheumatoid arthritis [7]. Published data indicate that prolactin (PRL) can modulate monocyte
⇑ Corresponding author. Address: 1 Panfu Road, Guangzhou, Guangdong 510180, China. Fax: +86 13503080062. E-mail address: [email protected] (X. Cai). http://dx.doi.org/10.1016/j.cellimm.2014.06.005 0008-8749/Ó 2014 Elsevier Inc. All rights reserved.
activities [8]. PRL is mainly produced by the pituitary gland; it plays a critical role in lactation. Some investigators indicate that PRL is associated with RA [9]. It is suggested that PRL plays a role in dermatitis [10]; and represents a prognostic marker for obese children and a predictive factor for progression to metabolic syndrome [11]. PRL is the ligand of PRL receptor (PRLR). The interaction of PRL and PRLR modulates the target cell activities. Whether PRL modulate the TNF-a release from CD14+ monocytes has not been reported. In this study, we observed that CD14+ monocytes expressed PRLR. Exposure to PRL up regulated the release of TNF-a from CD14+ monocytes, which was much more in RA patients than healthy control subjects.
2. Materials and methods 2.1. Reagents The antibodies of PRLR, p-38, p-p38, TNF-a, and shRNA of PRLR were purchased from Santa Cruz Biotech (Shanghai, China). The ELISA kit of TNF-a and protein of prolactin were purchased from R&D Systems (Shanghai, China). The reagents for qRT-PCR and Western blot were purchased from Invitrogen (Shanghai, China). The DNA purification kit was purchased from Promega (Beijing, China). The EpiXploreTM Methyl Detection kit was purchased from Clontech (Beijing, China). The CD14+ monocyte isolation kits were purchased from Miltenyi Biotec (Beijing, China). The MAPK inhibi-
C. Tang et al. / Cellular Immunology 290 (2014) 164–168
tor, PD98059, was purchased from Sigma Aldrich (Shanghai, China). The endotoxin levels in all reagents were detected using the Limulus assay (Limulus amebocyte lysate QCL 1000, Bio Whittaker, Walkersville, MD, USA). The reagents used in this study contained
Contents lists available at ScienceDirect
Cellular Immunology journal homepage: www.elsevier.com/locate/ycimm
Prolactin increases tumor necrosis factor alpha expression in peripheral CD14 monocytes of patients with rheumatoid arthritis Chun Tang a, Yun Li b, Xiaojun Lin a, Jinghua Ye a, Weinian Li a, Zhixiang He a, Fangfei Li a, Xiaoyan Cai a,⇑ a b
Department of Rheumatology, Guangzhou First People’s Hospital, Guangzhou Medical University, 1 Panfu Road, Guangzhou, China Department of Cardiothoracic Surgery, The Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, China
a r t i c l e
i n f o
Article history: Received 2 March 2014 Accepted 17 June 2014 Available online 24 June 2014 Keywords: Rheumatoid arthritis Tumor necrosis factor-a Prolactin Mitogen activated protein Methylation specific PCR
a b s t r a c t Tumor necrosis factor (TNF)-a is one of the major proinflammatory mediators of rheumatic arthritis (RA); the regulatory factors for TNF-a release is not fully understood. This study aims to investigate the role of prolactin receptor (PRLR) activation in regulating the expression and release of TNF-a from CD14+ monocytes. The results showed that the expression of PRLR was detectable in CD14+ monocytes of healthy subjects, which was markedly increased in RA patients. Exposure to PRL in the culture increased the expression and release of TNF-a from CD14+ monocytes, which was abolished by the PRLR gene silencing or blocking the mitogen activated protein (MAPK) pathway. We conclude that exposure to PRL increases TNF-a release from CD14+ monocytes of RA patients, which can be abolished by PRLR gene silencing or treating with MAPK inhibitor. Ó 2014 Elsevier Inc. All rights reserved.
1. Introduction Rheumatoid arthritis (RA) is a chronic immune inflammatory disease in the body; it mainly affects the joints. The inflammation can be destructive, cause severe pain and functional disability [1]. The pathogenesis of RA is not fully understood yet. The therapeutic effect of RA is not satisfactory currently [2]. Tumor necrosis factor (TNF)-a is one of the major proinflammatory mediators of RA [3]. Under the physiological circumstance, TNF-a plays a critical role in the body defensive system by activating the cytocidal activities in phagocytes [4]. TNF-a can be produced by monocytes, macrophages, and T cells upon being stimulated by pathogens or microbial products; it may play a key role in bone metabolism and is important in inflammatory bone diseases such as the TNF-a-induced osteoclast recruitment is probably central to the pathogenesis of RA [5]. However, the regulatory factors of TNF-a release are not fully elucidated yet. CD14+ cells include several cell types, such as monocytes, macrophages and dendritic monocytes, which are one of the major sources of TNF-a in the body. TNF-a is one of the proinflammatory cytokines involving in a number of inflammatory diseases, such as inflammatory bowel disease [6] and rheumatoid arthritis [7]. Published data indicate that prolactin (PRL) can modulate monocyte
⇑ Corresponding author. Address: 1 Panfu Road, Guangzhou, Guangdong 510180, China. Fax: +86 13503080062. E-mail address: [email protected] (X. Cai). http://dx.doi.org/10.1016/j.cellimm.2014.06.005 0008-8749/Ó 2014 Elsevier Inc. All rights reserved.
activities [8]. PRL is mainly produced by the pituitary gland; it plays a critical role in lactation. Some investigators indicate that PRL is associated with RA [9]. It is suggested that PRL plays a role in dermatitis [10]; and represents a prognostic marker for obese children and a predictive factor for progression to metabolic syndrome [11]. PRL is the ligand of PRL receptor (PRLR). The interaction of PRL and PRLR modulates the target cell activities. Whether PRL modulate the TNF-a release from CD14+ monocytes has not been reported. In this study, we observed that CD14+ monocytes expressed PRLR. Exposure to PRL up regulated the release of TNF-a from CD14+ monocytes, which was much more in RA patients than healthy control subjects.
2. Materials and methods 2.1. Reagents The antibodies of PRLR, p-38, p-p38, TNF-a, and shRNA of PRLR were purchased from Santa Cruz Biotech (Shanghai, China). The ELISA kit of TNF-a and protein of prolactin were purchased from R&D Systems (Shanghai, China). The reagents for qRT-PCR and Western blot were purchased from Invitrogen (Shanghai, China). The DNA purification kit was purchased from Promega (Beijing, China). The EpiXploreTM Methyl Detection kit was purchased from Clontech (Beijing, China). The CD14+ monocyte isolation kits were purchased from Miltenyi Biotec (Beijing, China). The MAPK inhibi-
C. Tang et al. / Cellular Immunology 290 (2014) 164–168
tor, PD98059, was purchased from Sigma Aldrich (Shanghai, China). The endotoxin levels in all reagents were detected using the Limulus assay (Limulus amebocyte lysate QCL 1000, Bio Whittaker, Walkersville, MD, USA). The reagents used in this study contained
