Department of Obstetrics and Gynecology, Gifu University School of Medicine, Gifu, Japan
Received 6 April 2001; revised 6 September 2001; accepted 18 September 2001.
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Abstract |
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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, IL-1ß, tumor necrosis factor-
, 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
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Introduction |
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Materials and methods |
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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, IL-1ß, TNF-
, IL-8, bFGF, VEGF and PD-ECGF antigens
All steps were carried out at 4°C. Tissues of uterine endometrial cancers (wet weight: 1020 mg) were homogenized in HG buffer (5 mM TrisHCl, 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, IL-1ß, TNF-
, IL-8, bFGF, VEGF and PD-ECGF antigen levels.
IL-1, IL-1ß, TNF-
, IL-8, bFGF and VEGF antigen levels in the samples were determined by a sandwich enzyme immunoassay using a Human IL-1
Quantikine (R&D System; Minneapolis, MN, USA), a Human IL-1ß Quantikine (R&D System), a Human TNF-
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
, IL-1ß, TNF-
, IL-8, bFGF, VEGF and PD-ECGF were standardized with the corresponding cellular protein concentrations.
Statistics
Survival curves were calculated using the KaplanMeier method and analyzed by the log-rank test. IL-1, IL-1ß, TNF-
, 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 Students t-test. Differences were considered significant for values of P <0.05.
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Results |
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Discussion |
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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, IL-1ß, TNF-
, bFGF, VEGF and PD-ECGF did not. Therefore, there appears to be no distinct cytokine network among IL-1
, IL-1ß, TNF-
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.
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Acknowledgements |
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Footnotes |
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References |
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