Immunohistochemical Evaluation of CXCL12/CXCR4 and Adiponectin in deep Infiltrating Endometriosis

Research Article

Austin J Reprod Med Infertil. 2015; 2(4): 1023.

Immunohistochemical Evaluation of CXCL12/CXCR4 and Adiponectin in deep Infiltrating Endometriosis

Kim JY¹*, Kang G¹, Kim HJ¹, Park K¹ and Kim KS²

¹Department of Pathology, Inje University Sanggye Paik Hospital, Republic of Korea

²East-West Bone & Joint Disease Research Institute, Kyung Hee University Hospital, Republic of Korea

*Corresponding author: Kim JY, Department of pathology, Inje University Sanggye Paik Hospital, 1342 Dongil-ro, Nowon-gu, Seoul, Republic of Korea

Received: July 06, 2015; Accepted: August 05, 2015; Published: August 07, 2015

Abstract

Chemokine CXCL12 and its receptor CXCR4 are involved in tumor cell migration, invasion and metastasis. Adiponectin is produced by adipose tissue, and exerts anti-inflammatory and anti-angiogenic effects. The aim of this study was to compare the expression of CXCL12, CXCR4 and adiponectin by immunohistochemical staining in intestinal endometriosis (IE, 6 cases) and abdominal wall endometriosis (AE, 12 cases) tissue, to ovarian endometriosis (OE, 12 cases). In addition, the relationship between adiponectin and the CXCL12/CXCR4 axis in these tissues was examined. In IE, CXCL12 expression was detected in 83.3%, and 16.7% of glandular and stromal cells, respectively, but CXCR4 expression was only found in stromal cells (33.3%). In AE, CXCL12 expression was found in 75.0% of glandular and 33.3% of stromal cells, and CXCR4 was found in 50% and 75% of glandular and stromal cells, respectively. There was no difference inCXCL12/CXCR4 expression among three endometriosis groups. The expression of adiponectin isoforms did vary, but there were no differences among the groups. The high molecular weight adiponectin isoform (HMW) was found in both glandular and stromal cells in all three groups. Positive staining for the low molecular weight (LMW) isoform was seen only in stromal cells, and the intermediate molecular weight (IMW) isoform was not found in either glandular or stromal cells. No significant relationship between CXCL12, CXCR4, and adiponectin was found in IE and AE. The CXCL12/CXCR4 axis and adiponectin were not found to be dominant in deep infiltrating endometriosis.

Keywords: Infiltrating endometriosis; CXCL12; CXCR4; Adiponectin

Abbreviations

IE: Intestinal Endometriosis; AE: Abdominal Wall Endometriosis; OE: Ovarian Endometriosis; LMW: Low Molecular Weight; IMW: Intermediate Molecular Weight; HMW: High Molecular Weight

Introduction

Endometriosis is defined by the presence of endometrial tissue outside of uterine cavity. Deep infiltrating endometriosis that penetrates the muscles of pelvic organs, may cause pelvic pain and induce disability and infertility [1]. Immunological factors such as chemokines enhance the implantation of endometrial cells and the progression of disease [2,3], and are elevated in the peritoneal fluid of female patient with endometriosis [4]. Endometriosis in extraovarian sites, especially in the intestine (IE) or in the abdominal wall (AE), has infiltrative properties similar to neoplasms, which results in diagnostic difficulties and unnecessary operations.

Chemokines are 8-10kDa cytokines that regulate a variety of immune responses, and are classified into four groups: CXC, CC, C and CX3C. Chemokine CXCL12, also known as stromal cell derived factor-1 (SDF-1), and its receptor CXCR4, are important for the initiation and progression of primary and metastatic cancers, including breast cancer [5-7]. They have also been associated with angiogenesis and cancer cell invasion [8,9]. CXCR4was known to be expressed in normal endometrium [10] and in endometriosis cells [11], but the expression of CXCL12 has not been investigated extensively in normal endometrium or endometriosis tissue. Whether the activation of CXCL12/CXCR4 is associated with deep infiltrating endometriosis remains unclear.

In addition to its function as a lipid storage organ, adipose tissue is known to have endocrine functions. Adiponectin is produced by adipose tissue, and can suppress some of the metabolic arrangements that result in type 2 diabetes, obesity, atherosclerosis and non-alcoholic fatty liver disease [12-14]. In addition to anti-inflammatory and antiangiogenic properties, adiponectin can also stimulate the expression of vascular endothelial growth factor (VEGF), matrix metalloproteinase (MMP)-1, and MMP-13, and is involved in synovitis and joint destruction in RA [15]. In the ovaries and endometrium, adiponectin affects periovulatory remodeling of the ovarian follicle, steroid synthesis/secretion, energy supply, and the inflammatory response of endometrial cells [16]. Adiponectin concentrations in serum and peritoneal fluid are lower in endometriosis patients compared to nonendometriosis patients [17]. Therefore, adiponectin may be an antiendometriosis factor, but its role in endometriosis is still unclear.

The expression of CXCL12/CXCR4 and adiponectin were evaluated in intestinal, abdominal wall, and ovarian endometriosis by immunohistochemical staining, and the relationship between the three factors was analyzed to determine the nature of deep infiltrating endometriosis.

Materials and Methods

Samples from patients who had been surgically treated for IE and AE between 2001 and 2013 (6 cases of IE, and 12 cases of AE) at the Sanggye Paik Hospital, Seoul, Korea, were collected, and compared to resected ovarian endometriosis (12 samples). All specimens were fixed with 10% buffered formalin, and the paraffin-embedded sections were stained with hematoxylin and eosin. Mean age was 37.0±9.7, 33.6±4.6, and 34.8±9.9years in IE, AE, and OE groups, respectively.

Hybridoma production

A monoclonal antibody against adiponectin was produced by hybridoma production and monoclonal antibody purification as follow [15]. Two BALB/c mice were immunized subcutaneously with 100 μL of complete Freund’s adjuvant (DIFCO Laboratories, Detroit, MI) containing 100 μg of recombinant human adiponectin expressed in E. coli (Prospec, Rehobot, Israel). After 2 weeks, the mice were injected with incomplete Freund’s adjuvant as before. The mice were boosted with only antigen, adiponectin (50 μg) i.v. 2 weeks later. Two days after the last boost, the sera were tested for reactivity to recombinant adiponectin by ELISA. The splenic lymphocytes were fused to FO myeloma cells (ATCC) then plated in 96-well plates in DMEM supplemented with 20% FBS (Invitrogen) and HAT component (Sigma-Aldrich). The culture supernatants were tested for reactivity to recombinant human adiponectin by Western blot and ELISA. Monoclonal antibodies were purified from culture supernatants of the screened clones by Protein G-Sepharose column chromatography (GenScript, NJ, USA) according to the manufacturer’s protocol. Antibodies to the three adiponectin isoforms were generated; high (HMW), intermediate (IMW) and low (LMW) molecular weight.

Immunohistochemistry

Sections (4 μm) of the endometriosis tissue were cut and stained by an automated system (Vision Biosystem Ltd, Mount Waverly, Australia) with CXCL12 (Santa Cruz, CA, USA), CXCR4 (Santa Cruz, CA, USA), and three adiponectin isoforms (10mg/ml). Antigen was retrieved with epitope retrieval solution 1 or 2 (Leika Microsystem, Newcastle, UK). Slides were incubated with antibody at room temperature for 20 minutes then with a biotinylated secondary antibody for 8 minutes. The resulting complexes were detected with avidin-peroxidase-conjugated polymer. Color was developed using 3,3’-diaminobenzidine (DAB; ScyTek, Logan, UT, USA). Mayer’s hematoxylin was used as a counterstain. Positive and negative control stains were used in each run. Nuclear and/or cytoplasmic staining for CXCR4 and adiponectin and cell membrane, nuclear and/or cytoplasmic staining for CXCL12 were considered as positive. Expression scoring was defined as follows; o, no positive cells; 1, <10% positive cells; 2, 10-25% positive cells; 3, 26-50% positive cells; 4, >50% positive cells. Scores of more than 2 were considered positive.

Results

CXCL12 expression was found in 83.3% (5/6 cases) and 16.7% (1/6 cases) of glandular and stromal cells in IE, respectively (Table 1, Figure 1). In AE, 75.0% (9/12 cases) of glandular and 33.3% (4/12 cases) of stromal cells were positive for CXCL12. For OE tissue, 50% (6/12 cases) of glandular and stromal cells were positive for CXCL12. CXCR4 expression was found in 33.3% of IE stromal cells (2/6cases), but not in glandular cells. CXCR4 expression was noted in 50% (6/12 cases) and 75% (9/12 cases) of glandular and stromal in AE, respectively, and in 8.3% (1/12 case) and 25% (3/12 cases) of glandular and stromal in OE. Co-expression of CXCL12/CXCR4 was noted in 66.7% of AE tissue (8/12 cases). There was no difference in expression pattern among the three groups; glandular cells expressed slightly more CXCL12, while stromal cells expressed slightly more CXCR4. CXCL12 protein expression did not correlate with CXCR4 expression in IE or AE.