Clinical implications of expression of interleukin-8 related to myometrial invasion with angiogenesis in uterine endometrial cancers

J. Fujimoto,+, I. Aoki, S. Khatun, H. Toyoki and T. Tamaya

Department of Obstetrics and Gynecology, Gifu University School of Medicine, Gifu, Japan

Received 6 April 2001; revised 6 September 2001; accepted 18 September 2001.


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Background

Angiogenesis is essential for development, growth and advancement of solid tumors. The tumor-associated macrophage has been recognized among inflammatory cells as a candidate for supplying tumor angiogenic factors. Interleukin (IL)-8 is assumed to be a macrophage-derived mediator of angiogenesis. This prompted us to study the clinical implications of macrophage-derived angiogenesis in uterine endometrial cancers.

Patients and methods

Sixty patients underwent curative resection for uterine endometrial cancers. The patient prognosis was analyzed with a 48 month survival rate after curative resection. In tissue of uterine endometrial cancers, the levels of IL-1{alpha}, IL-1ß, tumor necrosis factor-{alpha}, IL-8, basic fibroblast growth factor, vascular endothelial growth factor and platelet-derived endothelial cell growth factor were determined by enzyme immunoassay, and the localization and counts of microvessels and macrophages were determined by immunohistochemistry.

Results

There was a significant correlation between microvessel counts and IL-8 levels and between infiltrated macrophage counts and IL-8 levels in uterine endometrial cancers. Immunohistochemical staining revealed that the localization of IL-8 was similar to that of CD68 for macrophages. IL-8 levels were significantly increased during myometrial invasion from stage Ia to stages Ib through IV.

Conclusions

IL-8 might act as an angiogenic switch in myometrial invasion in stage I uterine endometrial cancers. Furthermore, IL-8 supplied from infiltrated macrophages within and around the tumor might not be a prognostic indicator of advancement, but may be associated with myometrial invasion in uterine endometrial cancers.

Key words: angiogenesis, IL-8, macrophage, myometrial invasion, uterine endometrial cancer


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Angiogenesis is essential for the development, growth and advancement of solid tumors (myometrial invasion, vascular and lymphatic involvement, vaginal invasion, secondary spreading to distant organs, etc., in uterine endometrial cancers) [1]. Various angiogenic factors in endometrial cancers have been studied as follows. Vascular endothelial growth factor (VEGF), especially VEGF isomers VEGF165 and VEGF121, are dominantly expressed in cancer cells and contribute to tumor growth in stage I of well-differentiated uterine endometrial cancers [2]. VEGF levels in uterine endometrial cancers were down-regulated during advancement. Platelet-derived endothelial cell growth factor (PD-ECGF) is dominantly expressed in interstitial cells, and contributes to myometrial invasion and tumor growth in the early stage of uterine endometrial cancers [3]. PD-ECGF levels were also down-regulated during advancement. The down-regulation of VEGF and PD-ECGF might be derived from a lack of hormone dependency developing with advancement. On the other hand, basic fibroblast growth factor (bFGF) is expressed in both cancer and interstitial cells, and not only does its level show good correlation with patient prognosis [4], but it is also recognized as a reliable indicator of prognosis. Furthermore, it is well known that infiltration of inflammatory cells is stimu-lated in malignant tumors, which contributes to angiogenesis. Tumor-associated macrophages have been recognized among inflammatory cells as a candidate for supplying tumor angiogenic factors [5]. Macrophages that have infiltrated tumors of the liver and gastrointestinal tract supply bFGF [6], those found in breast and ovarian cancers supply VEGF [7] and tumor necrosis factor (TNF)-{alpha} [8], and those found in uterine cervical cancers supply IL-8 [9]. Although TNF-{alpha} possesses weak angiogenic activity in itself, the angiogenic potential of TNF-{alpha} appears to be modulated through the induction of the strong angiogenic factors IL-8, VEGF and bFGF, and this pathway is regulated through paracrine and/or autocrine mechanisms [10]. IL-8 is expressed in macrophages and fibroblasts derived from the interstitium [6, 9] and is recognized as a macrophage-derived mediator of angiogenesis [9, 11]. This prompted us to study the clinical implications of macrophage-derived angiogenesis in uterine endometrial cancers.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patients
Consent for the following studies was obtained from all patients and approval was given by the Research Committee for Human Subjects, Gifu University School of Medicine. Sixty patients ranging from 31 to 83 years of age (stage Ia, 15 cases; stage Ib, 15 cases; stage Ic, 15 cases; stage II, seven cases; stage III, five cases; stage IV, three cases; and well-differentiated adenocarcinoma, 38 cases; moderately differentiated adenocarcinoma, 13 cases; poorly differentiated adenocarcinoma, nine cases) underwent resection for uterine endometrial cancers. The patient prognosis was analyzed with a 48 month survival rate after curative resection at the Department of Obstetrics and Gynecology, Gifu University School of Medicine, from June 1994 to April 1998. None of the patients had received any preoperative therapy. A sample of each uterine endometrial cancer was obtained immediately after hysterectomy and was snap-frozen in liquid nitrogen to determine the levels of IL-1{alpha}, IL-1ß, TNF-{alpha}, IL-8, bFGF, VEGF and PD-ECGF, and a sample of neighboring normal tissue was submitted for histopathological study. The clinical stage of uterine endometrial cancers was determined by International Federation of Obstetrics and Gynecology (FIGO) classification [12].

Immunohistochemistry
Sections (4 µm) of formalin-fixed paraffin-embedded tissue samples from uterine endometrial cancers were cut with a microtome and dried overnight at 37°C on a silanized-slide (Dako; Carpinteria, CA, USA). Samples were deparaffinized in xylene at room temperature for 80 min and washed with a graded ethanol/water mixture and then with distilled water. Immuno-histochemical staining for factor VIII-related antigen, which is synthesized by vascular endothelial cells, is specific for the endothelial cells of blood vessels [13] and is useful for detecting tumor angiogenesis [14]. Samples for the immunohistochemical analysis of the IL-8 and CD68 antigens were soaked in a citrate buffer, and then microwaved at 100°C for 10 min, and those for factor VIII-related antigen were treated with 0.3 µg/ml trypsin in phosphate buffer at room temperature for 20 min. The protocol for the DAKO LSAB2 kit, peroxidase (Dako) was followed for each sample. In the described procedures, rabbit anti-human IL-8 (Biosource; Camarillo, CA, USA), mouse anti-human macrophage CD68 (Dako), and rabbit anti-factor VIII-related antigen (Zymed; San Fran-cisco, CA, USA) were used at dilutions of 1:50, 1:50, and 1:2, respectively, as the first antibodies. The addition of the first antibody, either rabbit anti-human IL-8, mouse anti-human macrophage CD68, or rabbit anti-factor VIII-related antigen, was omitted to act as a negative control for IL-8, CD68 or factor VIII-related antigen, respectively.

Vessels and macrophages were counted in the five areas of highest density at 200x magnification (using a combination of 20x objective and 10x ocular, 0.785 mm2 per field). Microvessel and macrophage counts were expressed as the mean numbers of vessels and macrophages in these areas, respectively [15, 16]. Microvessel density and macrophage infiltration were evaluated by the counting of microvessels and macrophages, respectively.

Enzyme immunoassay for determination of IL-1{alpha}, IL-1ß, TNF-{alpha}, IL-8, bFGF, VEGF and PD-ECGF antigens
All steps were carried out at 4°C. Tissues of uterine endometrial cancers (wet weight: 10–20 mg) were homogenized in HG buffer (5 mM Tris–HCl, pH 7.4, 5 mM NaCl, 1 mM CaCl2, 2 mM ethyleneglycol-bis-[ß-amino-ethyl ether]-N,N,N',N'-tetraacetic acid, 1 mM MgCl2, 2 mM dithiothreitol, 25 µg/ml aprotinin, and 25 µg/ml leupeptin) with a Polytron homogenizer (Kinematics; Luzern, Switzerland). This suspension was centrifuged in a microfuge at 10 000 g for 3 min to obtain the supernatant. The protein concentration of samples was measured by the method of Bradford [17] to standardize IL-1{alpha}, IL-1ß, TNF-{alpha}, IL-8, bFGF, VEGF and PD-ECGF antigen levels.

IL-1{alpha}, IL-1ß, TNF-{alpha}, IL-8, bFGF and VEGF antigen levels in the samples were determined by a sandwich enzyme immunoassay using a Human IL-1{alpha} Quantikine (R&D System; Minneapolis, MN, USA), a Human IL-1ß Quantikine (R&D System), a Human TNF-{alpha} Quantikine (R&D System), a Human IL-8 Quantikine (R&D System), a Human bFGF Quantikine (R&D System) and a Human VEGF Assay kit (Immuno Biological Laboratories; Gunma, Japan), respectively, and PD-ECGF antigen levels were determined by the method of Nishida et al. [18]. The levels of IL-1{alpha}, IL-1ß, TNF-{alpha}, IL-8, bFGF, VEGF and PD-ECGF were standardized with the corresponding cellular protein concentrations.

Statistics
Survival curves were calculated using the Kaplan–Meier method and analyzed by the log-rank test. IL-1{alpha}, IL-1ß, TNF-{alpha}, IL-8, bFGF, VEGF and PD-ECGF levels were measured from three different parts of the same tissue in triplicate. Statistical analysis was performed with Student’s t-test. Differences were considered significant for values of P <0.05.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
There was a significant correlation between microvessel counts (MVC) and IL-8 (P <0.01 as shown in Figure 1), bFGF (P <0.01, data not shown), VEGF (P <0.01, data not shown) and PD-ECGF levels (P <0.01, data not shown) in uterine endometrial cancers, but not between MVC and IL-1{alpha}, IL-1ß or TNF-{alpha}. There was a significant correlation between infiltrated macrophage counts (IMC) and IL-8 levels in uterine endometrial cancers as shown in Figure 2 (P <0.01), but not between IMC and IL-1{alpha}, IL-1ß, TNF-{alpha}, bFGF, VEGF or PD-ECGF (data not shown).



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Figure 1. Correlation between microvessel count and IL-8 level in uterine endometrial cancer.

 


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Figure 2. Correlation between infiltrated macrophage count and IL-8 level in uterine endometrial cancer.

 
Immunohistochemical staining for IL-8 and CD68 in representative cases of stage Ia, Ib and III uterine endometrial cancers are shown in Figure 3. In stage Ia uterine endometrial cancers, IL-8 was sparsely distributed in the interstitium. In stage Ib, IL-8 was specifically expressed in the area of myometrial invasion of the cancer cells. In stage III, the cancer cells completely invaded the entire uterine wall; cancer and stromal cells in the whole layer were occupied, and IL-8 was diffusely expressed in the interstitium. CD68 was localized in a manner similar to IL-8 in all cases.



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Figure 3. Immunohistochemical staining for IL-8 and CD68 in uterine endometrial cancers (original magnification x100). Cases of stages Ia, Ib and III adenocarcinomas of the uterine endometrium. Rabbit anti-human IL-8 (Biosource; Camarillo, CA, USA) and mouse anti-human macrophage CD68 (Dako; Carpinteria, CA, USA) were each used at a dilution of 1:50 as the first antibody. Dark brown staining represents a positive signal for the antigens IL-8 and CD68.

 
There was no specificity related to histopathological types or IL-8 levels (data not shown). Furthermore, there was no significant correlation between IL-8 levels and 48 month survival rate (data not shown). However, there was a significant difference (P <0.05) in IL-8 levels between stage Ia and stages Ib to IV uterine endometrial cancers, as shown in Figure 4. IL-8 in all cases of stage Ia uterine endometrial cancers was <1000 pg/mg protein. Curiously, IL-8 in four out of 15 cases of stages Ib, Ic, and II to IV uterine endometrial cancer was present at levels <1000 pg/mg protein.



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Figure 4. Levels of IL-8 in uterine endometrial cancer classified according to clinical stages. Clinical stages of uterine endometrial cancer are according to International Federation of Obstetrics and Gynecology (FIGO) criteria. Each level is the mean of nine determinations.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Infiltrated macrophages in tumors are stimulated, which leads to the induction of angiogenic potential; in fact, macrophage infiltration has been reported to correlate with microvessel density in breast and uterine cervical cancers [5, 9]. In the present study, there was a significant correlation between IMC and MVC (P <0.05) in uterine endometrial cancers (data not shown). Therefore, we studied which angiogenic factors derived from tumor-associated macrophages promote tumor angiogenesis in uterine endometrial cancers. Generally, VEGF, PD-ECGF, bFGF, TNF-{alpha} and IL-8 have been recognized as tumor-associated macrophage-derived angiogenic factors [611].

In the present study, positive correlations between MVC and VEGF, PD-ECGF, bFGF and IL-8 demonstrate that they might work as angiogenic factors. In uterine endometrial cancers the VEGF isomers VEGF165 and VEGF121, that are expressed in cancer cells, contribute to tumor growth in the early stages [2]. Platelet-derived endothelial cell growth factor expressed in interstitial cells contributes to myometrial invasion in the early stages of uterine endometrial cancers [3]. On the other hand, bFGF expression is up-regulated during advancement, and acts as an adequate indicator of angiogenic potential related to advancement of uterine endometrial cancers. In other angiogenesis-dependent diseases, IL-8 has been reported to contribute to growth related to angiogenesis, in bronchogenic carcinoma [19], glioblastoma [20], melanoma [21], ovarian carcinoma [22, 23] and uterine cervical cancers [9].

There was a significant correlation between infiltrated macrophage counts and IL-8 levels in uterine endometrial cancers. Although PD-ECGF and bFGF seem to be partly provided by tumor-associated macrophages, only IL-8 showed a positive correlation with macrophage infiltration whereas IL-1{alpha}, IL-1ß, TNF-{alpha}, bFGF, VEGF and PD-ECGF did not. Therefore, there appears to be no distinct cytokine network among IL-1{alpha}, IL-1ß, TNF-{alpha} and IL-8 in tumor angiogenesis derived from infiltrated macrophages. Furthermore, immunohistochemical staining revealed that the localization of IL-8 was similar to that of CD68 for macrophages. These results demonstrate that IL-8 may be dominantly supplied by tumor-associated macrophages.

In addition, since IL-8 was dominantly localized in the area of myometrial invasion in stage Ib endometrial cancers, IL-8 levels might be increased during the myometrial invasion from stage Ia to stages Ib through IV. Therefore, although IL-8 dominantly supplied from infiltrated macrophages within and around the tumor might not be a prognostic indicator, it may act as an angiogenic switch in myometrial invasion in stage I uterine endometrial cancers.


    Acknowledgements
 
This study was supported in part by funds from the following Ministry of Health and Welfare programs of the Japanese Government: Grant for Scientific Research Expenses for Health and Welfare Programs, Foundation for Promotion of Cancer Research, and Grant-in-Aid for the Second Term Comprehensive 10-year Strategy for Cancer Control. The authors wish to thank Mr John Cole for proofreading the English of this manuscript.


    Footnotes
 
+ Correspondence to: Dr J. Fujimoto, Department of Obstetrics and Gynecology, Gifu University School of Medicine, 40 Tsukasa-machi, Gifu 500-8705, Japan. Tel: +81-58-267-2631; Fax: +81-58-265-9006; E-mail: jf@cc.gifu-u.ac.jp Back


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 Discussion
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