Patient data
Control brains without neuropathological alterations were obtained from an established normal tissue brain bank of the Institute of Brain Research of the University of Tuebingen as published previously [20]. Autopsy data of these cases are listed in Table 1. We investigated 50 primary brain tumor specimens, consisting of 3 protoplasmatic astrocytomas (WHO grade II), 21 fibrillary astrocytomas (WHO grade II), 7 anaplastic astrocytomas (WHO grade III), and 19 glioblastoma multiforme (WHO grade IV; Table 2). The ages of the patients (19 females and 31 males) ranged 20–77 years. All tumor specimens were obtained before either radiotherapy or chemotherapy. Histological grading of the human tumor specimens was performed according to the revised WHO classification guidelines [15]. Tumors were resected at the Department of Neurosurgery at Tuebingen.
Table 2 Data of patients with different astrocytomas
Animal model
The rat C6 glioblastoma cell line was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in RPMI 1640 medium with Glutamax II (Gibco, BRL, Paisely, UK) containing 10% fetal calf serum (Gibco BRL) and 1.2% penicillin/streptomycin (Fluka, Buchs, Switzerland) at 37°C and 5% CO2. C6 glioma cells were implanted as follows: 15 male Sprague-Dawley rats (220–280 g, Charles River, Sulzfeld, Germany) were anesthetized by intraperitoneal injection of Ketanest (Ketaminehydrochloride, Parke Davis, USA; 100 mg/kg) and Rompun (Xylazine-hydrochloride, Bayer, Germany; 10 mg/kg). In order to avoid xerophthalmia during anesthesia, the eyes were covered with Oculotect Gel (Retinolpalmitate, CIBA Vision, Novartis, Germany). After reaching an adequate depth of anesthesia, the skin of the head was shaved, cleaned, and disinfected with 75% ethanol and incised (midline incision) to expose the skull. After drilling a 2-mm hole 3 mm to the right and 3 mm posterior to bregma, the rat was fixed in a stereotactic apparatus (ASI Instruments, Germany). The needle of an Hamilton syringe (900 series; Hamilton Company, Reno, NV, USA) was inserted 4 mm from the pial surface vertically into the brain. Five μl of C6 glioblastoma cell suspension (2×105 cells/μl) were injected. Thereafter the needle was slowly withdrawn and the skin was restored with a suture (4/0, FS-2, Ethicon; Johnson & Johnson, Brussels, Belgium). After two weeks the rats were sacrificed and perfused intracardially with fixative (4% formalin in 0.1 M phosphatebuffered saline, pH 7.5). Four normal rats (without suffering sham injection) were used as controls. All rats were kept under controlled conditions of temperature and light, with food and water available ad libitum. The animals were cared for in accordance with published International Health Guidelines under a protocol approved by the local Institutional Animal Care and the Administration District Official Committee.
Immunohistochemistry
Tissue samples were fixed in buffered 4% formalin (pH 7.4) and paraffin-embedded by routine methods. After dewaxing, sections (3 μm) were immersed in 0.01 M citrate buffer and irradiated for 15 min in a microwave oven set at 600 W to enhance the fraction of accessible epitopes. Endogenous peroxidase was blocked with 1% H2O2 in methanol (15 min). Non-specific protein binding was inhibited by incubation with normal porcine serum (Biochrom, Berlin, Germany) for 15 min. Tissue sections were incubated with purified mouse anti-IL-16 monoclonal antibody (BMA, Augst, Switzerland; diluted at 1:100 in TBS-BSA) overnight at 4°C. Specific antibody binding was detected by incubating the sections with a secondary biotinylated rabbit anti-mouse IgG F(ab)2 antibody fragment (1:400; Dako, Hamburg, Germany) for 30 min, followed by incubation with a peroxidase-conjugated streptavidin-biotin-complex (Dako). Labeled antigens were visualized by application of 3,3′-diaminobenzidine (DAB; Fluka, Neu-Ulm, Germany) as a chromogen. All sections were counterstained with Mayer's Hemalaun.
Negative controls consisted of sections incubated in the absence of the primary antibody. The specificity of monoclonal IL-16 antibody was analyzed by isotype control and by preincubation of the monoclonal antibody with an irrelevant cytokine, EMAP II (endothelial-monocyte activation polypeptide II) [22]. As a positive control for IL-16 immunoreactivity we used rat and human spleen sections, as macrophages are abundantly found in this organ.
Double-labeling experiments
Double-labeling experiments were performed on human tissues of GBM and rat C6 glioma. Antibodies used for GBM included monoclonal antibody against CD68 (clone KP-1, diluted 1:100 in TBS-BSA, Dako) for microglia/macrophage identification. Activated microglia/macrophages were detected with antibodies directed against HLA-DR, -DP, and -DQ (MHC class II, clone CR3/43, 1:50, Dako). Lymphocytic subsets were typed by monoclonal antibodies against CD4 (T-helper lymphocytes, clone 1F6, 1:10, Dako) and CD3 (pan-T-cell marker, clone PS1, 1:100, Novocastra, Dossenheim, Germany) and were applied overnight to tissue sections at 4°C. For rat C6 glioma, sections were incubated with monoclonal antibodies against ED1 (1:100; clone ED1, BMA, Augst, Switzerland) to demonstrate activated microglia/macrophages. Briefly, sections were dewaxed, irradiated in a microwave oven for antigen retrieval, and incubated with porcine serum as described above. Visualization was achieved by adding biotinylated secondary rabbit anti-mouse IgG diluted 1:400 for 30 min and alkaline phosphatase-conjugated ABC complex/AP (Dako, Kenmark) (1:400 in TBS-BSA) for 30 min. The sections were developed with Fast Blue BB salt (Fluka, Buchs, Switzerland), yielding a blue reaction product. To avoid cross-reactivity, the probes were irradiated again in a microwave oven. After administration of porcine serum for 15 min, the sections were labeled with IL-16 antibody as already described. Bound antibodies were visualized with DAB (brown).
Evaluation of human astrocytomas and rat C6 gliomas
IL-16+ cells with the typical morphology of activated microglial cells and with a clearly visible nucleus and all counterstained cells with a visible nucleus were counted in 10 high-power fields (×200 magnification with an eyepiece grid representing 0.25 mm2) in 10 independent areas of the tumor tissue for each section. The ratio of positively labeled and counterstained nuclei was calculated in each brain tumor specimen and the control brain section to obtain the percentage distribution of labeled TAMs. IL-16+ cells in perivascular spaces (cells nestled against the outer vessel wall and cells in perivascular spaces) were counted per 10 vessels and considered to be positive if a minimum of two IL-16+ cells were present.
Statistical analysis
In order to stabilize variances, percentages were subjected to an arcsine transformation before a one way analysis of variance (Anova) was performed. Differences of means were compared by a Tukey's honest significant difference post-hoc test. Data are presented as means and their 95% confidence intervals (95%CI) after back-transformation. The percentages of IL-16+ perivascular cells were calculated as means of labeled perivascular cells (MLPVC). Data of the density of IL-16+ parenchymal cells were calculated as means of labeled parenchymal cells (MLPC).