Interleukin-8 Is Produced in Neoplastic and Infectious Diseases of the Human Central Nervous System1

[CANCER RESEARCH 52, 4297-4305, August 15, 1992] Interleukin-8 Is Produced in Neoplastic and Infectious Diseases of the Human Central Nervous System1

Erwin Van Meir,2 Miroslav Ceska, Fritz Effenberger, Alfred Walz, Eric Grouzmann, Isabelle Desbaillets, Karl Frei, Adriano Fontana, and Nicolas de Tribolet

Neurosurgery Service, University Hospital (CHUV), 1011 Lausanne, Switzerland [E. V. M., E. G., I. D., N. d. T.J; Theodor Kocher Institut, UniversitÃ¤tBern, 3000 Bern, Switzerland [A. W.]; Sandoz Forschungsinstitut, Vienna, Austria [M. C., F. E.]; Section of Clinical Immunology, University Hospital, 8044 Zurich, Switzerland [K. F., A. F.]

ABSTRACT leukotriene B4 are well known as neutrophil chemoattractants that have little effect on blood lymphocytes (5, 6). However, The presence of Â¡nterleukin-8 (IL-8), a leukocyte chemotactic factor, very little is known about lymphocyte-specific attractants (7,8). was examined in primary and metastatic central nervous system tumors We have chosen, therefore, to examine and compare glioblas and in nonneoplastic acute meningoencephalitides. In vitro: (a) 11 of 12 glioblastoma cell lines constitutively expressed toma, a CNS tumor, with nonneoplastic acute viral or bacterial IL-8 mRNA; (b) 5 of 6 of these cell lines secreted IL-8 protein as meningoencephalitides for the presence of a candidate chemo detected by enzyme-linked immunosorbent assay and a glucosaminidase tactic factor with potential to govern leukocyte accumulations. release bioassay; and (e) II.-I/i or tumor necrosis factor was able to Chemotactic factors may originate either in response to a augment both IL-8 mRNA steady state levels and protein secretion of all cellular immune reaction to a tumor and/or from tumor cells cell lines tested except IN-319. themselves. Chemotactic factors are released by sensitized lym IL-8 was also found in vivo, (a) IL-8 poly A mRNA was detected in phocytes when stimulated by specific antigens (9, 10). Mono- 2 of 2 low grade astrocytomas, 1 of 2 anaplastic astrocytomas, and 6 of 6 glioblastomas. (b) IL-8 protein was present in the cyst fluid of 1 of 4 nuclear infiltrates of human glioblastomas consist predomi nantly of T-lymphocytes and macrophages as well as low grade astrocytomas, 1 anaplastic astrocytoma, 2 of 2 glioblastomas, 1 oligodendroglioma grade III, and one central nervous system cervical B-lymphocytes and natural killer cells (1-3). In addition, hu carcinoma metastasis, (c) The cerebrospinal fluid of 3 of 4 metastatic man glioblastoma cell lines have been shown to release various lymphomas, 2 of 16 glioblastomas, 1 of 2 low grade astrocytomas, but cytokines in vitro such as an interferon-/3-like activity (11), IL-1 none of 3 anaplastic astrocytomas and none of 9 meningiomas contained (12, 13), granulocyte CSF, and granulocyte-macrophage CSF IL-8. The presence of IL-8 was not restricted to central nervous system (14, 15), TGF-/32 (16), TNF (13, 17), MCP-1 (18-21 ), and IL-6 tumors as 2 of 2 bacterial meningitis and 5 of 5 acute viral meningitis (22). Although some of these cytokines such as IL-1 and patients contained considerable IL-8 levels in the cerebrospinal fluid. (d) Immunohistochemical analysis showed IL-8 immunoreactivity in MCP-1 have been reported to have chemotactic activities for perivascular tumor cells in 11 of 15 glioblastoma sections. neutrophils (23) or monocytes (18, 19), no lymphocyte chemo These data suggest that IL-8 secretion could be a key factor involved tactic factor has been identified as the mediator of the impor in the determination of the lymphoid infiltrates observed in brain tumors tant T-lymphocyte infiltrates observed in glioblastoma. Re and the development of cerebrospinal fluid pleocytosis in meningoen cently, IL-8 has been shown to be a chemoattractant for cephalitides. T-lymphocytes as well as neutrophils and basophils but not monocytes (8, 24, 25). Therefore, we decided to evaluate the INTRODUCTION role of this inflammatory cytokine as a candidate factor to explain the specific T-lymphocyte infiltrates observed in CNS A feature common to certain neoplastic and nonneoplastic human CNS3 diseases is the presence of a cellular inflammatory tumors such as glioblastoma as compared to those observed in nonneoplastic meningoencephalitides. response. Primary CNS tumors are infiltrated by mononuclear Inflammatory stimuli such as IL-1 and TNF have been shown lymphoid populations (1-3), while nonneoplastic acute bacte to induce the synthesis of IL-8 mRNA in human endothelial rial meningoencephalitides are characterized primarily by poly- cells (26, 27), T-lymphocytes (28), alveolar macrophages (29), morphonuclear lymphoid infiltrates (4). Neither the mediators dermal fibroblasts (30, 31), keratinocytes (32), retinal pigment nor the mechanism for the development of these specific cell epithelial cells (33), synovial cells (34), neutrophils (35), as well infiltrates have, as yet, been identified. However, cell specificity as 2 hepatoma cell lines (36), lung giant cell carcinoma line of the inflammatory infiltrates may be established by specific LU65C (37), pulmonary epithelial cell line A549 (38), bladder chemoattractants defining the particular classes of leukocytes. For example, N-formylated peptides such as yV-formyl-Met- carcinoma cell line 5637 (39), and astrocytoma cell line Leu-Phe or polypeptides such as complement-derived C5a or U373MG (20). Here we demonstrate the inducible IL-8 mRNA expression and the secretion of IL-8 protein by 13 glioblastoma cell lines. Received 12/11/91; accepted 5/29/92. IL-8 mRNA was also present in frozen astrocytoma and glio The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accord blastoma tumors but not in meningioma. IL-8 was detected in ance with 18 U.S.C. Section 1734 solely to indicate this fact. vivo by ELISA in cyst fluid and CSF of patients with primary 1This work was supported by Grants 3.595.087 (N. d. T.) and 31.28402.90 (A. F.) of the Swiss National Science Foundation. and metastatic CNS tumors as well as in the CSF of patients 2 To whom requests for reprints should be addressed, at Ludwig Institute for with inflammatory diseases such as viral or bacterial meningitis. Cancer Research, 9500 Oilman Drive. San Diego, CA 92093-0660. 3 The abbreviations used are: CNS, central nervous system; CSF, cerebrospinal Immunohistochemical analysis of glioblastoma suggests that fluid; cDNA, complementary DNA; ELISA, enzyme-linked immunosorbent assay; IL-8 is produced by the tumor cells themselves. These data GFAP, glial fibrillary acidic protein; rhu, recombinant human; IL, interleukin; suggest that in vivo IL-8 production might be an important mAb, monoclonal antibody; PGE2, prostaglandin Â£2;TGF, transforming growth factor; PBS, phosphate-buffered saline; MCP-1, monocyte chemoattractant pro- factor in determining the leukocyte infiltrates found in some tein-1; TNF, tumor necrosis factor-Â«;NAP, neutrophil-activating protein. CNS tumors or in meningoencephalitis. 4297

MATERIALS AND METHODS 473, 475, 515, and 523), and 5 meningiomas (patients M-391, 421, 433,448, and 523) were used. The 500-base pair EcoRI fragment of the Preparation of Supernatants from Glioma Cell Lines. The l S per IL-8 gene coding region of plasmid pUC19hIL-8 was used as a probe manent glioblastoma cell lines used in this study and their culture for IL-8 (41), the 450-base pair EcoRl/Pstl fragment of the IL-la gene conditions have been described previously (22). For production of su- coding region as probe for IL-1Â«,and the ft/I 1100-base pair fragment pernatants, 5 x IO5 cells of each glioblastoma cell line were plated in of plasmid pAL41 as probe for ÃŸ-actin(22). 6-well plates (Costar). After 24 h, the subconfluent cultures were Immunohistochemical Staining. Biopsy samples obtained from var washed 3 times and replaced with 2 ml serum-free RPMI 1640 (Se- ious CNS tumors and 3 normal adult brains (autopsy or lobectomy) romed). In some experiments, rhu-IL-10 (0.1 to 100 units/ml; Gen- were stored at -70Â°C. Serial sections of 7- to 8-Mm thickness were zyme, Cambridge, MA), rhu-TNF (0.1 to 100 units/ml; Glaxo), rhu- processed for immunohistochemistry as described previously (22). The IL-4 (IO3 units/ml; Genzyme), rhu-TGF-/32 (0.1 to 10 ng/ml; R&D antibodies used were an anti-IL-8 hybridoma supernatant 46E5 (42), a Systems, Minneapolis, MN), and PGE2 ( 1O^8to 10~6 M; Sigma Chem 1:800 dilution of anti-GFAP mAb N358 (Amersham), a 1:20 dilution ical Co., St. Louis, MO) containing bovine serum albumin (Sigma) as a of pan-T-lymphocyte anti-Leu-4 (CD3) mAb (Beckton Dickinson), 2 carrier protein were added at this step. The Supernatants were collected antibodies commonly used to detect macrophages [a 1:20 dilution of 24 h later, centrifuged at 1000 x g, and stored at -20Â°C until being anti-Leu-M3 (CD14) mAb (Beckton Dickinson) and a 1:20 dilution of tested. Supernatants were also prepared from a culture of pure astro- anti-Leu-Ml (CD15) mAb (Beckton Dickinson)], and supernatant of cytes from a human fetal brain (W323-HF 9.12:10.1 ) and from a mixed P3X63Ag8, a 7l-producing myeloma cell line as a negative control. culture containing 60% astrocytes and 40% microglial cells (W360- The following 15 glioblastomas were analyzed: 403, 467,473, 475, 479, HAM 10.31). 515, 523, 546, 549, 621, 628, 637, 658, 669, and 696. Harvesting Cerebrospinal Fluid and Glioma Cyst Fluid. CSF sam Radiolabeling of IL-8. Four Mghuman recombinant IL-8 in 50 n\ of ples tested for IL-8 activity were obtained from 16 patients with suprat- 0.1 M phosphate buffer, pH 7.5, were added to 400 nC\ of [125I]Na entorial glioblastoma, 2 patients with benign astrocytoma, 3 patients (Amersham IMS 30) for 15 min at room temperature in a tube con with anaplastic astrocytoma, 4 patients with brain metastatic lympho- taining 2.5 Mg lodogen (Pierce) (43). The reaction was stopped by mas, 9 patients with meningioma, 4 patients with nonhistologically adding 50 M!of a 120mM KI, 5 m\i ascorbic acid solution in0.9%NaCl. confirmed CNS tumors, 2 patients with bacterial meningitis, 5 patients The mixture was fractionated using a 50-cm Sephadex G15 column and with viral meningitis, 3 patients with multiple sclerosis, and 18 patients PBS containing 1% bovine serum albumin as eluent. The yield of la with unrelated diseases such as herniated lumbar disc syndrome or beling was evaluated to be 50% and the specific activity 50 pCi/pg. tension headache. In the latter patients, inflammatory or tumoral dis Biological activity of the labeled and cold IL-8 was evaluated in an eases of the CNS were excluded. CSF was collected by a lumbar punc elastase release assay (44). The biological activity of the iodinated IL-8 ture before operation at myelography. Glioma cyst fluids were obtained was estimated to be 15% of the unlabeled IL-8. Radioreceptor Assay of IL-8 on Glioblastoma Cell Lines. Binding peroperatively from 2 patients with glioblastoma, 4 patients with be experiments were performed in parallel l h at 37Â°Corfor 2 h at 4Â°Cto nign astrocytoma, 1 patient with anaplastic astrocytoma, 1 patient with avoid internalization (45) using RPMI containing 10 mg/ml bovine oligodendroglioma grade III, and 1 patient with brain metastasis of a serum albumin and 10 mMA'-(2-hydroxyethyl)piperazine-A''-(2-ethane- cervical carcinoma. The CSF and cyst fluids were centrifuged, filtered through Millex GV 0.22 MHI(Millipore), and stored at -70Â°Cuntil the sulfonic acid) as binding buffer and 90,000 cpm of I25l-labeled IL-8 assay. (1.18 ng/tube). Nonspecific binding was obtained with 1000-fold excess Quantification of IL-8 by ELISA. Mouse anti-NAP-l/lL-8 mAb of unlabeled IL-8. Human blood granulocytes were used as positive were used for coating microtiter plate wells (for 16 h at 4Â°C).After 4 control. The 13 following glioblastoma cell lines were analyzed: LN- washes (PBS, pH 7.5 containing 0.05% Tween-20), rhu-NAP-l/IL-8 229, LN-319, LN-340, LN-382, LN-427, LN-428, LN-308, LN-Z308, LN-443, LN-444, LN-464, U-118, and U-251. (kindly provided by Sandoz, Basel, Switzerland) at concentrations rang Quantification of IL-4 by ELISA. IL-4 was measured with the com ing from 0.02 to 10 ng/ml, or CSF (diluted at 1:2, 1:4, 1:10, or 1:100) was added to precoated plates and incubated for 2 h at 37Â°C.After 4 mercial Intertest-4 ELISA kit (Genzyme) according to the manufactur er's instructions. The Supernatants of 9 glioblastoma cell lines pre- washes, goat anti-NAP-l/IL-8 alkaline phosphatase conjugate was added, and the plates were incubated for an additional 2 h at 37Â°C.After treated with rhu-IL-1 (0.1 to 100 units/ml) or untreated were tested at the incubation with /7-nitrophenyl phosphate, the enzymatic reaction 1:1 dilution. The detection limit at this dilution was 0.18 ng/ml. The following cell lines were tested: LN-18, LN-215, LN-229, U-251, LN- was terminated by the addition of 2 N NaOH. Optical reading was Z308, LN-319, LN-443, LN-444, and LN-464. performed at 405 nm. The sensitivity of the assay is 3 pg/ml in assay buffer. Bioassay for IL-8. The IL-8-like activity was measured by the release RESULTS of A'-acetyl-/5-glucosaminidase from azurophil granules, released from cytochalasin B-treated human neutrophils (40). In brief, samples were Twelve permanent glioblastoma cell lines were tested by diluted 3- to 30-fold with PBS containing 2.5 mg/ml bovine serum Northern blotting for the presence of IL-8 mRN A using an IL-8 albumin (final volume 150 M!)and incubated for 15 min at 37Â°Cwith cDNA probe (Fig. 1). Variable amounts of constitutive IL-8 100 M!of cytochalasin B-pretreated (5 Mg/ml, 5 min at 37Â°C)human mRNA were detected in 11 of 12 cell lines tested, in one skin neutrophils (IO7 cells/ml). After centrifugation, 50 M' of the cell-free fibroblast culture taken from the scalp (Fbl 445), and in the supernatant were transferred to a new microtiter plate and incubated histiocytic lymphoma line U937 (data not shown). No mRNA with 50 n\ of 10 mm 4-methylumbelliferyl-2-acetamido-2-deoxy-/3-D- was detected in cell line LN-319, whereas strong constitutive glucopyranoside (Sigma) in 0.1 Msodium citrate buffer for l h at 37Â°C. expression was obtained with cell lines LN-215, U-251, LN- The reaction was then stopped by addition of 100 M' of 0.4 M glycine 308, and LN-443. All cell lines tested had low to high IL-8 buffer. All samples were tested in the presence and absence of neutro mRNA levels after induction with IL-1/3 or TNF (Fig. I, A and phils to control for background levels of enzymes. To obtain absolute IL-8 concentrations, corrected fluorescence values were fitted on a stan B). The enhancement ranged between 2 and 200 times as esti dard curve determined in the same assay with 72-amino acid rhu-IL-8. mated by scanning the autoradiograph and standardizing to the RNA Isolation and Hybridization. Poly A+ selected mRJ^JAwas iso /3-actin mRNA levels. IL-1/3 could also induce a weak expres sion of the IL-8 mRNA in cell line LN-319. The induction or lated from cell lines or solid tumors and used for Northern blotting experiments with random labeled probes as described previously (22). enhancement of the IL-8 mRNA by IL-1/3 or TNF was maximal Solid tumors from 8 low grade astrocytomas (patients AII-460, 466, at 4 h and decreased slowly between 24 and 48 h (Fig. l C). The 471, 472, 483, 501, 528, and 552), 2 anaplastic astrocytomas (patients stimulation was already detected with 0.1 unit/ml IL-1/3 (data AIII-485 and 496), 8 glioblastomas (patients G-403, 435, 467, 468, not shown) and 1 unit/ml TNF. The optimal concentrations 4298

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Fig. l. IL-8 mRNA detection in glioblastoma cell lines. Northern blot analysis was performed on 10 Mgof total RNA using an IL-8 cDNA probe or a /3-actin cDNA probe as control. I. glioblastoma cells were cultured for 24 h in medium alone (- columns) or in medium plus S units/ml IL-1/3 (+ columns). B, as in A but stimulation with medium plus TNF (100 units/ml). C, as in A but the cells were cultured for 4, 24, or 48 h with IL-1/3 (5 units/ml). Five Mgof total fibroblast RNA were used as a positive control (Fbl 445). D, as in A but the cells were stimulated by IL-1/3 (1 unit/ml) or TNF (0.1 to 100 units/ml). The blots were autoradiographed 48 h (A), 11 h (B), 1 days (O, and 24 h (D). Kb, Kilobase.

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1992 American Association for Cancer Research. IL-8 IS PRODUCED IN HUMAN CENTRAL NERVOUS SYSTEM DISEASES were 1 unit/ml IL-iÃŸand 10 units/ml TNF for cell line LN-229 (Fig. \D). Interferon-7 had no effect on IL-8 mRNA expression by LN-229 cells at 100 units/ml (data not shown). To test whether the synthesis of IL-8 was also detected at the protein level and whether the increased amount of IL-8 mRNA 28S obtained in response to IL-IÃŸor TNF resulted in a direct in crease in secreted protein, we tested the supernatants of 6 se lected cell lines expressing various levels of IL-8 mRNA for 18S - - IL-8 IL-8 by ELISA (Fig. 2). All of them except LN-319 showed variable constitutive IL-8 levels by ELISA ranging from 3 to 190 ng/ml. Both IL-1/3 (Fig. 2A) and TNF (data not shown) enhanced IL-8 production in a dose-dependent way. Although doubling times of the different cell lines vary between 36 and 48 - IL-1a h, a semiquantitative comparison is possible since equal cell 18S numbers (5 x 10s) were used and subsequent supernatants were harvested at 48 h. The IL-8 levels increased up to more than - ÃŸ-actin 850 ng/ml for cell line LN-215 with a 24-h stimulation with 100 18S units/ml IL-1/3 for 24 h. The enhancement of IL-8 production

Fig. 3. IL-8 mRNA detection in ex vivo tumors. Northern blot analysis was used to test IL-8 and IL-1Â«mRNA expression on variable amounts (0.1 to 5 Mg) A. ELISA poly A+ selected RNA extracted from 2 low grade astrocytoma (AH), 2 anaplastic astrocytoma (AIII), and 6 glioblastoma (G) depending on the available tissue. The 1000 blot was autoradiographed for 2 weeks for IL-8 and 3 weeks for IL-1Â«.The mRNA degradation level and proportional mRNA amount loaded were assessed control":i%10 using a ÃŸ-actincDNA probe. Kb. kilobase. 1U/mlIL-1H U) 800 - IL-1010 U/ml l IL-1\1!""Jâ€ž1l;J|J 100 U/ml (0 was proportional to the amount of IL-1/3 added and correlated 600 - o. with the mRNA levels detected by Northern blots. No IL-8 was U) detected in the supernatants of LN-319 cells treated with IL-lÃŸ C at 1 to 100 units/ml, whereas a small amount (8 ng/ml) was 400 - obtained with a TNF treatment at 100 units/ml (data not shown). IL-la could also enhance IL-8 secretion as tested on o> cell line LN-Z308, but at 10 times higher concentrations than C 200 - r IL-1/3; whereas 0.1 to 10 Â¿ig/mlLPS and 100 units/ml IL-6 had no effect (data not shown). .H To further evaluate whether the IL-8 detected by ELISA is LN-18 LN-215 LN-229 LN-Z308 LN-308 LN-319 LN-428 biologically active, the same supernatants were tested for their cell lines ability to induce the secretion of glucosaminidase by neutrophils (see "Materials and Methods"). All of the supernatants tested were bioactive, and their IL-8-like activity was inducible by IL-1/3 and TNF except for LN-319 (Fig. 2B). Although the Glucosaminidase release assay B. general profile of the data was identical to those obtained by 100 ELISA, the amount of IL-8-like activity detected by bioassay control0 was on average 10 times lower (compare Fig. 2, A and B). This 1U/mlIL-10 difference was not due to interfering activities in the superna 80 - IL-1010 U/ml IL-1il 100 U/ml tants as similar results were obtained with diluted samples (1:5 l\pa and 1:10). To test whether proteases present in the supernatants could be responsible for this result, we produced new superna 60 - tants in the presence of protease inhibitors (leupeptin 4 x 10~4

^\lrJp1JP1 M and aprotinin 3 units/ml), however, no difference was no 40 - ticed. Furthermore, 0.1 to 10 ng/ml IL-4, an IL-8 gene expres sion regulatory factor (46), 0.1 to 10 ng/ml TGF-/32, and 10~8 to 10~6 M PGE2 showed no inhibitory effect (data not shown). 20 - Supernatants from a purified astrocyte culture from human fetal brain and a mixed culture of 60% astrocytes and 40% microglial cells contained 38 and 370 pg/ml IL-8, respectively. -vrÃl 1â€¢Frj^spai|\\ The relevance of the in vitro data to the in vivo situation was LN-18 LN-215 LN-229 LN-Z308 LN-308 LN-319 LN-428 first addressed on a Northern blot with isolated poly A"1"mRNA cell lines from ex vivo frozen tumors (Fig. 3). Two of 2 low grade astro- Fig. 2. IL-8 detection in glioblastoma cell line supernatants. Cell lines were cytomas, 1 of 2 anaplastic astrocytomas, and 6 of 6 glioblasto- cultured for 24 h in medium alone (control) or in medium plus IL-1/3 (1 to 100 mas were faintly to strongly positive on the original autoradio- units/ml). The supernatants were tested in parallel for IL-8 by ELISA (A) and the glucosaminidase release assay (B). LN-Z308 is a subculture of line LN-308 at 15 graph. No signal was obtained with mRNA of a 17-week fetal passages that has been cultured independently in ZÃ¼richsince 1982. brain and 5 meningiomas (data not shown). 4300

As further proof for the production of IL-8 in vivo, the pres surrounding necrosis were also positive (data not shown). Ad ence of IL-8 in CSF and cyst fluid of patients was measured jacent sections were positive for GFAP, suggesting that the (Fig. 4). Eighteen CSF from patients with unrelated diseases tumor cells produce IL-8 in vivo (Fig. 5D). No reactivity with such as herniated lumbar disc syndrome or tension headache neoplastic vascular endothelial cells or T-lymphocytes was were used as controls. CSF of patients with infectious CNS found. Gliosis surrounding metastatic CNS lymphomas was diseases consisting of 2 bacterial meningitis and 5 viral menin also positive, therefore reactive astrocytes may also produce gitis were also tested. Two of 16 glioblastomas (569 and 20 IL-8. The presence of macrophages was assessed with anti- pg/ml), 1 of 2 benign astrocytomas (110 pg/ml), 0 of 3 anaplas- CD 14 and anti-CD 15 mAbs. In some glioblastomas, 10 to 50% tic astrocytomas, 3 of 4 metastatic lymphomas (138, 66 and 54 of the cells were positive with anti-CD 14 as well as the gliosis pg/ml), 0 of 9 meningiomas, and 2 of 4 other nonhistologically surrounding metastatic CNS lymphomas (data not shown). The confirmed CNS tumors (271 and 108 pg/ml) were positive for expression pattern of the CD 14 epitope correlated with the IL-8 by ELISA. Two of 18 control CSF (50 and 30 pg/ml), 2 of regions expressing IL-8. In contrast, only a few cells surround 2 bacterial meningitis (24,340 and 289 pg/ml), and 5 of 5 viral ing vessels were positive with anti-CD 15 (Fig. 5, B and F). meningitis (4411, 1857, 1184, 549 and 250 pg/ml) were posi These results suggest that infiltrating macrophages may also tive for IL-8. The CSF of 3 patients with multiple sclerosis were participate in the IL-8 secretion or that the tumor cells and negative. The tumor cyst fluids of 2 glioblastomas (7787 and reactive astrocytes expressing IL-8 are a subpopulation also 612 pg/ml), 1 of 4 astrocytomas grade I or II (72 pg/ml), 1 expressing the CD 14 antigen. No IL-8 staining was detected on astrocytoma grade III (860 pg/ml), 1 oligodendroglioma grade 3 normal adult brain sections examined (data not shown). III (885 pg/ml), and 1 metastasis of a cervical carcinoma (1239 To determine whether IL-8 secretion could have a direct pg/ml) were also positive for IL-8. biological effect on the tumor cells, we checked for the presence The presence of IL-8 poly A+ mRNA in 9 of 10 ex vivo of IL-8 receptors on 13 glioblastoma cell lines with 125I-labeled astrocytoma and glioblastoma specimens analyzed and of IL-8 IL-8. Freshly isolated human blood granulocytes were used as protein in 4 of 7 cyst fluids of gliomas suggests that cells within positive control. The binding experiments were performed at the tumor tissue release IL-8 in vivo. Some candidate cells are 4Â°Cto avoid internalization and subsequent degradation of the the tumor cells themselves, reactive astrocytes, endothelial ligand-receptor complex (45). Granulocytes exhibited many cells, T- and B-lymphocytes, and macrophage or microglial binding sites with 92.5% specific binding, whereas no specific cells. To address this question, immunohistochemical analyses binding was obtained for the 13 glioblastoma cell lines tested on frozen tissue sections were performed. Adjacent serial sec (data not shown). tions were studied by utilizing anti-IL-8 mAb 46E5 (42), anti- GFAP serum, 2 antibodies commonly used to detect macroph ages [anti-Leu-Ml (CD 15) mAb and anti-Leu-M3 (CD 14) DISCUSSION mAb], a pan-T-lymphocyte antibody [anti-Leu-4 (CD3) mAb], The results presented here indicate that in vivo IL-8 secretion and supernatant of P3X63Ag8 as a negative control (Fig. 5). In occurs in both primary and metastatic brain tumors (but not in the glioblastoma sections of 11 of 15 patients studied, numer leptomeningeal tumors such as meningiomas) and in nonneo- ous dispersed strongly positive regions were observed. Some of plastic meningoencephalitides. We suggest that IL-8 may be an the positive cells had a foamy cytoplasm and were mostly lo calized around tumor vessels where macrophages and T-lym- important candidate factor determining the leukocyte infiltra tions linked to these CNS diseases. phocytes were present (Fig. 5, A and E). Pseudopalisading areas Brain Tumor Data. For brain tumors, our study shows that 11 of 12 glioblastoma cell lines expressed IL-8 mRNA without 100000. any stimulation. ELISA and bioassay studies showed detectable sclerosisviral IL-8 in the supernatants of 5 of 6 of the same cell lines. IL-1/3 meningitisbacterial meningitisbenign and TNF were able to enhance IL-8 mRNA steady state levels astrocytomaanaplastic and IL-8 secretion in these cell lines. LN-319 was the only cell 10000- Ã€0n astrocyloma line that did not secrete IL-8 constitutively. Despite the induc glioblastomametÃ¡stasismeningiomation of IL-8 mRNA by IL-1 ÃŸandTNF, only TNF could induce IL-8 production in LN-319, suggesting possible posttranscrip- â€¢¿a+ f 1000. AA oligodendroglioma III tional control mechanisms. These results are in agreement with A0 neurinomaCNS previous studies; inflammatory cytokines regulate IL-8 mRNA SVA tumorXXX steady state levels and IL-8 secretion in astrocytoma cell line U373MG (20) and in other cell types (26, 28-34, 36-38). 100. â€¢¿Ã€â€¢ Interestingly, the IL-8 levels measured by ELISA in the su pernatants of all the glioblastoma cell lines tested were about 10 times higher than the IL-8-like activity obtained with the bio- 15 ooTAAAA assay even in the presence of protease inhibitors. This differ XXX XXXX*X ence is likely the result of comparing the 77-amino acid form of AAAAAAAAA IL-8 produced by glioblastoma cell lines4 with the 72-amino AAAAAAAAM*Dâ€¢AAAâ€¢*TocontrolmultipleXXX* acid rhuIL-8 (our standard), which is approximatively 10-fold Tumor Tumor Meningitis Control more bioactive in vitro (31, 47, 48). cyst fluid CSF CSF CSF Fig. 4. IL-8 detection in CSF and cyst fluid of patients with neoplastic and infectious diseases of the CNS. Nine tumor cyst fluids. 39 tumor CSF, 7 viral or bacterial meningitis, 18 control CSF from patients with noninflammatory dis 4 M. Tada, Y. Sawamura. S. Sakuma, H. Abe, K. Suzuki, Y. Yamakawa, E. Van eases such as herniated lumbar disc syndrome or tension headache, and 3 control Meir, and N. de Tribolet. Human astroglial cells produce the 77-amino acid form patients with multiple sclerosis. of interleukin-8 (IL-877), submitted for publication. 4301

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Fig. 5. Identification of IL-8 producing cells on ex vivoglioblastoma tumor specimens. Glioblastoma sections from patient G-696 stained with anti-IL-8 mAh 46ES (A), ami I,cu-Ml (CD15) mAb (A), anti-CD3 mAb (Q, or anti-GFAP serum (D). Glioblastoma section from patient G-403 stained with anti-IL-8 mAb 46E5 (E) or anti-Leu-Ml (CD15) mAb (F). Magnifications: A to D. x 250; E and F, x 400.

IL-8 was detected by ELISA in tumor cyst fluids of 1 of 4 low plastic astrocytomas, and 6 of 6 glioblastoma ex vivo tumor grade astrocytomas, 1 anaplastic astrocytoma, and 2 of 2 glio- specimens contained IL-8 poly A+ mRNA. blastomas. The cyst fluids of one oligodendroglioma grade III Different cell types such as T- and B-lymphocytes, mono- and one CNS cervical carcinoma metastasis were also positive. cytes, and macrophages, fibroblasts, microglial cells, astrocytes, In addition, IL-8 was also present in the CSF of 3 of 4 meta- and endothelial cells or the tumor cells themselves are potential static lymphomas, in 2 of 16 glioblastomas, in 1 of 2 low grade candidates for the production of IL-8 in vivo. Some of these astrocytomas, but not in 3 anaplastic astrocytomas and 9 men- cells are known to release IL-8 in vitro (26-29, 31). Immuno- ingiomas analyzed. Other cytokines such as IL-1, IL-6, TGF-/3, cytochemical analysis of glioblastoma sections showed that in but not TNF have been reported in the cyst fluid or CSF of 11 of 15 patients, between 30 to 80% of the cells were positive brain tumors (15, 22). for IL-8 staining in regions expressing GFAP, suggesting that The presence of IL-8 in vivo in gliomas was further supported the tumor cells themselves are able to produce IL-8 in vivo. by the finding that 2 of 2 low grade astrocytomas, 1 of 2 ana- Although endothelial cells have been shown to secrete the IL-8 4302

77-amino acid form (26, 27), neoplastia vascular endothelium difficult to establish whether IL-8 production in vivo in both was not stained; neither were T-lymphocytes and sections of diseases may be a common feature having different origins (tu normal brain. The positively staining cells were usually focused mor cells producing constitutively IL-1 and IL-8 as a result of around vessels in regions containing macrophages and T-lym transformation and nonneoplastic cells producing IL-1 and phocytes as shown by staining with anti-CD 15 and anti-CD3 IL-8 in response to viral or bacterial infection) or a more gen mAbs. Therefore, we cannot exclude that in addition to tumor eral phenomenon linked to the common production of inflam cells, macrophages may participate directly in the IL-8 secre matory cytokines such as IL-1 by macrophages, microglial cells, tion; or that inflammatory cytokines such as IL-1 or TNF, or astrocytes in response to the presence of bacterial li- produced by macrophages and microglial cells, could indirectly popolysaccharides and viral or tumor antigen presentation. elicit IL-8 secretion by the tumor cells in vivo, as occurs in vitro. What Could Be the Consequence of IL-8 Secretion for Both Reactive astrocytes present in the gliosis surrounding meta- Diseases in Vivo? First, does IL-8 act as an autocrine growth static CNS lymphomas were also positive, and primary cultures factor for glioblastoma cells? To test whether IL-8 could have a of astrocytes secreted IL-8, suggesting that when activated, nor direct effect on glioblastoma cells through an IL-8 receptor- mal astrocytes are also able to secrete IL-8. mediated mechanism, we decided to check for the presence of What might be the mechanism of IL-8 production by the IL-8 receptors by binding of 125I-labeled rhIL-8 on 13 glioblas neoplastic cells? Our in vitro data show that glioblastoma cells toma cell lines. No binding was observed, suggesting by this are able to secrete IL-8 constitutively, therefore the first possi technique that IL-8 has no direct effect on glioblastoma cells. bility is that in vivo they also produce IL-8 without induction. Second, does IL-8 play an important role for the leukocyte The strong IL-8 induction capacities of IL-1 and TNF in vitro chemotaxis in CNS tumors or meningitis? These diseases show suggest, however, that these factors may also play an autocrine differences in the localization (tumor or CSF) and the leukocyte or paracrine role in vivo. Although it is not a general charac content of their infiltrates. CNS brain tumors have a predom teristic, some astrocytoma and glioblastoma cell lines are able inance of mononuclear infiltrates within the tumor consisting to release TNF and IL-1 in vitro (12, 13, 49). The same heter mainly of T-lymphocytes and macrophages, whereas almost no ogeneity is observed in vivo: we have been unable to detect IL-lev leukocytes were found in the CSF (1-3). The leukocyte infil mRNA in the tumors positive for IL-8 mRNA, and neither trates in the CSF of bacterial meningoencephalitis patients con IL-lt* nor IL-1/3 was detected by radioimmunoassay in the CSF sist of more than 90% polymorphonuclear leukocytes, whereas viral meningoencephalitis patients' CSF contain mostly lym of our glioma patients (49). Nevertheless, IL-la and -ÃŸwere recently found in the cyst fluid but not CSF of 3 of 7 glioblas phocytes (4). IL-8 has been shown to be chemotactic in vitro for toma patients (15); IL-1/3 mRNA was detected in 3 fresh astro- human neutrophils predominantly, as well as for T-lympho cytomas (50), and IL-la immunostaining was reported on glio cytes and basophils (8, 24, 25). In our study, IL-8 was found in blastoma sections (49). These studies suggest that tumor cells, the CSF of all 7 meningitis patients tested at low to high con infiltrating macrophages, microglial cells, or reactive astrocytes centrations (up to 24,340 pg/ml). IL-8 was also present in the that all secrete IL-1 in vitro (12, 20, 51) could in some cases tumor cyst fluid at low to moderate concentrations (70 to 7000 produce IL-1 in vivo and so induce IL-8 secretion. pg/ml) in 6 of 9 CNS tumors, but only in 8 of 35 CSF. Tada et a/.5 have also found a neutrophil chemotactic activity in 2 of 2 Nonneoplastic Meningoencephalitides Data. All 7 CSF of inflammatory diseases such as viral and bacterial meningitis regional fluid and 2 of 4 CSF of operated glioma patients with were positive for IL-8 (up to 24,340 pg/ml in one case). The out the presence of neutrophils in vivo. Although IL-8 is present presence of other inflammatory cytokines such as TNF (52- in these body fluids, we only rarely detected the presence of 54), IL-1 (54, 55), and IL-6 (15, 54, 56) in the CSF of menin granulocytes by immunohistochemistry in the tumor sections analyzed in this study (data not shown). T-lymphocyte infil gitis has already been reported. The cellular origin of the cy trates without the presence of neutrophils after rhIL-8 injection tokines produced in vivo has not yet been identified. The initiation of meningitis occurs primarily in the arachnoid layer have been observed previously in the guinea pig lung and the ear of the leptomeninges and can subsequently spread to the pia skin of rats (24, 61). mater and the brain. Therefore, the activated endothelial cells Several factors may explain these different leukocyte con of the blood vessels and scattered macrophages as well as the tents, (a) The neutrophil response could be transient due to down-regulating functions that may impinge its further accu arachnoid cap cells and fibrocytes present in these layers are all mulation. Possible inhibitory factors include IL-4 (46), PGE2, candidates for the initial cytokine production. These cytokines and TGF-/32 (15). However, we have shown by ELISA that IL-4 could then be released either directly in the blood circulation or is not secreted by 9 glioblastoma cell lines in vitro and that IL-4, in the CSF through the subarachnoid space and initiate CSF pleocytosis (57-59). Intrathecal injection of IL-1 and TNF or PGE2, and TGF-/32 do not affect the IL-1 stimulated IL-8 lipopolysaccharide causes meningitis and leukocytosis in the secretion and bioactivity in vitro (data not shown), (b) The CSF of rabbits (60), and IL-6 and IL-8 production is induced by concentration of IL-8 could be below a threshold level necessary IL-1 and TNF in macrophages and glial cells in vitro (22, 29). to elicit a response. Injection of rIL-8 into the ear skin of rats at Therefore, part of the action of TNF and IL-1 in meningitis 1 ng/ml induced a selective T-lymphocyte infiltration, whereas pathogenesis might be mediated by their induction of IL-8 and neutrophil recruitment necessitated 100-fold higher doses (24). IL-6 in the inflamed regions of the CNS. Therefore, the low IL-8 concentrations (100 pg/ml to 1 ng/ml) Although it is tempting to parallel the IL-8 production in found in the cyst fluid or CSF of the patients studied could meningitis and CNS neoplasia, the involvment of IL-1 and possibly elicit only a T-lymphocyte infiltrate response. This is, TNF is better established in the first case than in the second, in however, unlikely since similar concentrations were observed in which the tumor cells may constitutively secrete IL-8 without IL-1 or TNF induction. The finding of IL-1 but not TNF in in 5 M. Tada. Y. Sawamura. S. Sakuma. K. Suzuki. H. Ohta, T. Aida, and H. Abe. Cellular and cytokine responses in the human central nervous system to intracranial vivo samples of glioblastoma suggests, however, that at least administration of tumor necrosis factor-a for the treatment of malignant gliomas, IL-1 may play a role in IL-8 secretion. It will, however, be submitted for publication. 4303

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1992 American Association for Cancer Research. IL-8 IS PRODUCED IN HUMAN CENTRAL NERVOUS SYSTEM DISEASES the CSF of one of our patients with bacterial meningitis where phils (66). Therefore, the possible concomitant release of other neutrophil pleocytosis was present, (c) Tumor-secreting factors members of these 2 families and their concentrations in the may inhibit patients' neutrophil migration abilities. Some cell CNS will influence and determine the final makeup of leuko ular immune responses are impaired in patients with primary cytes in the tumor tissue. intracranial tumors (62). However, no granulocytopenia exists in brain tumor patients (62) and no impairement of their neu ACKNOWLEDGMENTS trophil immune responsiveness has been reported so far. In addition, granulocyte-macrophage CSF, initially characterized We wish to thank Dr. J. Antel, Dr. E. Christophers, Dr. T. Gauthier, as a neutrophil migration inhibitory factor (63), is produced by Dr. I. Lindley, D. Piani, Dr. K. Matsushima, Dr. K. Schwechheimer, glioblastoma cells in vitro but not in vivo (15). (d) The neoplas- Dr. M. Sticherling, Dr. M. Tada, Dr. K. Williams, and Sandoz for the tic vascular endothelium of the tumors was not stained for IL-8, generous gifts of cytokines, cDNA probes, cell culture supernatants, CSF, antibodies, helpful advice, and unpublished data. We would also suggesting either a lack of inducing factors such as TNF or a like to thank A-C. Diserens, I. Guignard, and M-F. Hamou for excel difference in function as compared with normal endothelium lent technical assistance, Dr. T. Gauthier for expert computer assis (27). Endothelial cells play an important role in neutrophil tance, and Dr. J-P. Deruaz and Dr. R. Janzer for the neuropathological diapedesis: TNF, IL-1/3, and lipopolysaccharide induce endo- diagnosis of the tumors. We are grateful to Dr. W. Cavenee, Dr. T. thelial cell-derived IL-8 secretion that in turn generates a Mikkelsen, Dr. S. Rempel, and Dr. Y. Sawamura for critically reading chemotactic gradient and allows neutrophil-endothelium inter the manuscript. action by regulating adhesion molecules such as LECAM-1 and /32 integrins (27). (e) The primary IL-8 form produced may by REFERENCES itself be unable to elicit neutrophil migration in the human 1. von Hanwehr, R. I., Hofman, F. M., Taylor, C. R., and Apuzzo, M. L. CNS. An attractive hypothesis would be that the 77-amino acid Mononuclear lymphoid populations infiltrating the microenvironment of pri form of IL-8 produced by glioblastoma cells4 and the IL-8 mary CNS tumors. Characterization of cell subsets with monoclonal anti bodies. J. Neurosurg., 60: 1138-1147, 1984. molecular forms produced in meningoencephalitides would 2. Kuppner, M. C., Hamou, M. F., and de Tribolet, N. Immunohistological and need an additional proteolytic cleavage to become chemoattrac- functional analyses of lymphoid infiltrates in human glioblastomas. Cancer Res., 48: 6926-6932, 1988. tant in vivo for neutrophils. The 72-amino acid form of IL-8 is 3. Wood, G. W., and Morantz, R. A. Immunohistologic evaluation of the lym- about 10-fold more chemotactic in vitro than the 77- and 79- phoreticular infiltrate of human central nervous system tumors. J. Nati. amino acid forms of IL-8 (31,47). TNF that is produced in vivo Cancer Inst.. 62: 485-491, 1979. 4. Oehminger, M. Cerebrospinal Fluid Cytology. Stuttgart: Thieme, 1976. in bacterial meningitis (where neutrophil infiltrates are pre 5. Snyderman, R., and Goetzl, E. J. Molecular and cellular mechanisms of dominant) but not in viral meningitis (52) or glioblastoma (15) leukocyte chemotaxis. Science (Washington DC), 213: 830-837, 1981. 6. Wilkinson, P. C., and Haston, W. S. Chemotaxis: an overview. Methods could be indirectly involved in this mechanism. TNF could Enzymol., 762:3-16, 1988. induce the opening of endothelial junctions leading to an influx 7. Wilkinson, P. C, Haston, W. S., and Shields, J. M. Some determinants of the of blood components containing proteases able to cleave imma locomotory behaviour of phagocytes and lymphocytes in vitro. Clin. Exp. Immunol., 50: 461-473, 1982. ture IL-8. TNF also regulates the coagulant/anticoagulant ac 8. Leonard, E. J., Skeel, A., Yoshimura, T., Noer, K., Kutvirt, S., and Van Epps, tivity at the endothelial cell-blood interface, resulting in pro D. Leukocyte specificity and binding of human neutrophil attractant/ activation protein-1. J. Immunol.. 144: 1323-1330. 1990. duction of a proteolytic activity (47). Two clinical studies show 9. Ward, P. A., Remold, H. G.. and David, J. R. The production by antigen- that a neutrophil response can be obtained when TNF or IL-2 stimulated lymphocytes of a leukotactic factor distinct from migration inhib is injected in the CNS: first, Tada et a/.5 have shown that TNF itory factor. Cell Immunol.. /: 162-174. 1970. 10. Snyderman, R., Altman, L. C., Hausman, M. S., and Mergenhagen, S. E. injected intracerebrally in human glioma patients could elicit Human mononuclear leukocyte chemotaxis: a quantitative assay for humoral leukocyte bursts both in regional fluid and CSF. These events and cellular chemotactic factors. J. Immunol.. 108: 857-860. 1972. correlated with the appearance of a neutrophil chemotactic ac 11. Larsson, L, Landstrom, L. E., Larner. E., Lundgren, E., Miorner, H., and Strannegard, L. Interferon production in glia and glioma cell lines. Infect. tivity that copurified with IL-8 on high performance liquid Immun., 22: 786-789, 1978. chromatography.5 Second, intraventricular injection of IL-2 in 12. Lee, J. C., Simon, P. L., and Young, P. R. Constitutive and PMA-induced interleukin-1 production by the human astrocytoma cell line T24. Cell Im patients with leptomeningeal carcinomatosis induced a tran munol., IIS: 298-311, 1989. sient rapid influx of neutrophilic leukocytes, followed by a pro 13. Velasco, S., Tarlow, M., Olsen, K., Shay, J. W., McCracken, G. H., and Nisen, P. D. Temperature-dependent modulation of lipopolysaccharide-in- longed presence of lymphocytes correlating with the appear duced interleukin-1/j and tumor necrosis factor alpha expression in cultured ance of the IL-8 inducers IL-1 and TNF (64). Clearly the human astroglial cells by dexamethasone and indomethacin. J. Clin. Invest., potential of IL-8 as a leukocyte chemoattractant in the human 87: 1674-1680, 1991. CNS will only be established with IL-8 injections into the CNS. 14. Tweardy, D. J., Molt, P. L., and Glazer, E. W. Monokine modulation of human astroglial cell production of granulocyte colony-stimulating factor This will only be possible if correlations between good progno and granulocyte-macrophage colony-stimulating factor. I. Effects of IL-1 sis and IL-8 levels can be established. In the meantime, injec alpha and IL-beta. J. Immunol., 144: 2233-2241, 1990. 15. Frei, K., Malipieiro, U., Piani, D., Van Meir, E., de Tribolet, N., and Fon tion of IL-8 in the CSF or the brain of an animal model will tana, A. GM-CSF production by glioblastoma cells: despite the presence of allow one to address the above-discussed hypotheses. inducing signals GM-CSF is not expressed in vivo. i. Immunol.. 1480:3140- Finally, the fact that IL-8 belongs to a superfamily of cytok- 3146, 1992. 16. Bodmer, S., Strommer, K., Frei, K., Siepi, C., de Tribolet, N., Heid, I., and ines sharing 4 half-cystines (the IL-8 and MCP-1 families) (21) Fontana, A. Immunosuppression and transforming growth factor-beta in opens a new chapter in the understanding of the leukocyte glioblastoma. Preferential production of transforming growth factor-beta 2. chemotaxis in CNS diseases. Macrophage inflammatory pro J. Immunol., 143: 3222-3229, 1989. 17. Bethea, J. R., Gillespie, G. Y., Chung, J. Y., and Benveniste, E. N. Tumor tein-1 and -2 have been shown to exert important inflammatory necrosis factor production and receptor expression by a human malignant responses in the CNS in a rabbit model (60, 65). MCP-1 is glioma cell line. D54-MG. J. Neuroimmunol., 30: 1-13, 1990. 18. Matsushima, K., and Oppenheim, J. Interleukin-8 and MCAF: novel inflam chemoattractant for monocytes but not neutrophils and is also matory cytokines inducible by IL-1 and TNF. Cytokine, /: 2-13, 1989. induced by IL-1 and TNF in human astrocytomas and glioblas- 19. Robinson, E. A., Yoshimura, T., Leonard, E. J., Tanaka, S., Griffin, P. R., tomas in vitro (18-21). Of further particular interest will be Shabanowitz. J., Hunt, D. F., and Appella, E. Complete amino acid sequence of a human monocyte chemoattractant, a putative mediator of cellular im RANTES, which attracts both T-lymphocytes of the memory mune reactions. Proc. Nati. Acad. Sci. USA, 86: 1850-1854, 1989. type and (at 200-fold higher doses) monocytes but not neutro 20. Kasahara. T., Mukaida. N., Yamashita, K., Yagisawa, H., Akahoshi, T., and 4304

Matsushima, K. IL-1 and TNF-a induction of IL-8 and monoeyte chemot- lycoluril. Biochem. Biophys. Res. Commun., 80: 849-857, 1978. actic and activating factor (MCAF) mRNA expression in a human astrocy- 44. Dewald, B., and Baggiolini, M. Evaluation of PAF antagonists using human toma cell line. Immunology, 74: 60-67, 1991. neutrophils in a microtiter plate assay. Biochem. Pharmacol., 36:2505-2510, 21. Leonard, E. J., and Yoshimura, T. Human monoeyte chemoattractant pro- 1987. tein-I (MCP-1). Immunol. Today, //: 97-101, 1990. 45. Samanta, A. K., Oppenheim, J. J., and Matsushima, K. Interleukin 8 (mono 22. Van Meir, E., Sawamura, Y., Diserens. A. C.. Hamou, M. F.. and de Tribolet, cyte-derived neutrophil chemotactic factor) dynamically regulates its own N. Human glioblastoma cells release interleukin 6 in vivoand in vitro. Cancer receptor expression on human neutrophils. J. Biol. Chem.. 265: 183-189, Res., 50:6683-6688, 1990. 1990. 23. Kampschmidt, R. F., and Pulliam, L. A. Effect of human monoeyte pyrogen 46. Standiford, T. J., Strieter, R. M., Chensue, S. W., Westwick, J., Kasahara, on plasma iron, plasma zinc, and blood neutrophils in rabbits and rats. Proc. K., and Kunkel, S. L. II.4 inhibits the expression of IL-8 from stimulated Soc. Exp. Biol. Med., 158: 32-35, 1978. human monocytes. J. Immunol., 145: 1435-1439, 1990. 24. Larsen, C. G., Anderson, A. O., Appella, E.. Oppenheim, J. J., and Mat- 47. Hebert, C. A., Luscinskas, F. W., Kiely, J. M., Luis, E. A., Darbonne, W. C., sushima, K. The neutrophil-activating protein (NAP-1) is also chemotactic Bennett, G. L., Liu, C. C., Obin, M. S., Gimbrone, M. A., Jr., and Baker, J. for T lymphocytes. Science (Washington DC), 243: 1464-1466, 1989. B. Endothelial and leukocyte forms of IL-8. Conversion by thrombin and 25. Bacon. K. B., and Camp, R. D. Interleukin (ILj-8-induced in vitro human interactions with neutrophils. J. Immunol., 145: 3033-3040, 1990. lymphocyte migration is inhibited by cholera and pertussis toxins and inhib- 48. Nourshargh, S., Perkins, J. A., Showell, H. J., Matsushima, K., Williams, T. itors of protein kinase C. Biochem. Biophys. Res. Commun., 169: 1099- J., and Collins, P. D. A comparative study of the neutrophil stimulatory 1104. 1990. activity in vitro and pro-inflammatory properties in vivoof 72 amino acid and 26. Stricter, R. M., Kunkel, S. L., Showell, H. J., Remick, D. G., Phan, S. H., 77 amino acid IL-8. J. Immunol., 148: 106-111, 1992. Ward, P. A., and Marks, R. M. Endothelial celi gene expression of a neu- 49. Gauthier, T., Hamou, M. F., Monod, L., (Â¡allay.P., Carrel, S., and de trophil chemotactic factor by TNF-a/pAu, LPS, and IL-1 bÃªta. Science Tribolet, N. Expression and release of interleukin-1-a/pAa and beta by human (Washington DC), 243: 1467-1469, 1989. glioblastoma cells in vitro and in vivo. Acta Neurochir., in press, 1992. 27. Huber. A. R., Kunkel. S. L.. Todd, R. F.. Ill, and Weiss, S. J. Regulation of 50. Lichtor, T., Dohrmann, G. J., and Gurney, M. E. Cytokine gene expression transendothelial neutrophil migration by endogenous interleukin-8. Science by human gliomas. Neurosurgery. 26: 788-793. 1990. (Washington DC), 254: 99-102, 1991. 51. Fontana, A., Kristensen, F., Dubs, R., and Weber, E. Production of prostag- 28. Gregory, H., Young, J., Schroder, J. M., Mrowietz, U., and Christophers, E. landin E and an interleukin-1 like factor by cultured astrocytes and C6 glioma Structure determination of a human lymphocyte derived neutrophil activat cells. J. Immunol., 129: 2413-2419, 1982. ing peptide (LYNAP). Biochem. Biophys. Res. Commun., /5/: 883-890, 52. Leist, T. P., Frei, K., Kam-Hansen, S., Zinkernagel, R. M., and Fontana, A. 1988. Tumor necrosis factor alpha in cerebrospinal fluid during bacterial, but not 29. Stricter, R. M.. Chensue, S. W., Basha, M. A., Standiford, T. J., Lynch, J. P., viral, meningitis. Evaluation in murine model infections and in patients. J. Baggiolini, M., and Kunkel, S. L. Human alveolar macrophage gene expres Exp. Med., 167: 1743-1748, 1988. sion of interleukin-8 by tumor necrosis factor-alpha, lipopolysaccharide, and 53. Mustafa, M. M., Ramilo, O., Saez-Llorens, X., Olsen, K. D., Magness, R. R., interleukin-1 beta. Am. J. Respir. Cell Mol. Biol., 2: 321-326, 1990. and McCracken, G. H., Jr. Cerebrospinal fluid prostaglandins, interleukin 1 30. Stricter, R. M., Phan, S. H., Showell, H. J.. Remick. D. G., Lynch, J. P., beta, and tumor necrosis factor in bacterial meningitis. Clinical and labora Genord, M., Raiford, C., Eskandari, M.. Marks, R. M., and Kunkel. S. L. tory correlations in placebo-treated and dexamethasone-treated patients. Am. Monokine-induced neutrophil chemotactic factor gene expression in human J. Dis. Child, 144: 883-887, 1990. fibroblasts. J. Biol. Chem., 264: 10621-10626, 1989. 54. Waage, A., Halstensen, A., Shalaby, R., Brandtzaeg, P., Kierulf, P., and 31. Schroder, J. M., Sticherling, M., Henneicke, H. H., Preissner, W. C., and Espevik, T. Local production of tumor necrosis factor alpha, interleukin 1, Christophers, E. IL-1 alpha or tumor necrosis factor-a/pAa stimulate release and interleukin 6 in meningococcal meningitis. Relation to the inflammatory of three NAP-l/IL-8-related neutrophil chemotactic proteins in human der response. J. Exp. Med., 170: 1859-1867, 1989. mal fibroblasts. J. Immunol.. 144: 2223-2232, 1990. 55. Ramilo, O., Mustafa, M. N.. Porter, J., Saez-Llorens, X., Mensola, J., Olsen, 32. Larsen, C. G., Anderson, A. O., Oppenheim, J. J., and Matsushima, K. K. D., Luby, J. P., Beutler, B., and McCracken, G. H., Jr. Detection of Production of interleukin-8 by human dermal fibroblasts and keratinocytes in interleukin-Ib but not tumor necrosis factor-a in cerebrospinal fluid of chil response to interleukin-1 or tumour necrosis factor. Immunology, 68:31-36, dren with aseptic meningitis. Am. J. Dis. Child, 144: 349-352, 1990. 1989. 56. Gallo. P., Frei, K., Rordorf, C., Lazdins, J., Tavolato, B., and Fontana, A. 33. Einer, V. M., Stricter, R. M., Einer. S. G., Baggiolini, M., Lindley, I., and Human immunodeficiency virus type 1 (HIV-1) infection of the central ner Kunkel, S. L. Neutrophil chemotactic factor (IL-8) gene expression by cy- vous system: an evaluation of cytokines in cerebrospinal fluid. J. Neuroim- tokine-treated retinal pigment epithelial cells. Am. J. Pathol., 136: 745-750, munol., 23: 109-116, 1989. 1990. 57. Righi, M., Mori, L., De-Libero, G., Sironi, M., Biondi, A., Mantovani, A., 34. Brennan, F. M., Zachariae, C. O., Chantry, D., Larsen, C. G., Turner, M., Donini, S. D., and Ricciardi-Castagnoli, P. Monokine production by micro- Maini, R. N., Matsushima, K., and Feldmann, M. Detection of interleukin 8 glial cell clones. Eur. J. Immunol., 19: 1443-1448, 1989. biological activity in synovial fluids from patients with rheumatoid arthritis 58. Selmaj, K. J., Farooq, M., Norton, W. T., Raine, C. S., and Brosnan, C. F. and production of interleukin 8 mRNA by isolated synovial cells. Eur. J. Proliferation of astrocytes in vitro in response to cytokines. A primary role for Immunol., 20: 2141-2144, 1990. tumor necrosis factor. J. Immunol., 144: 129-135, 1990. 35. Bazzoni, F., Cassatella, M. A., Rossi, F., Ceska, M., Dewald, B., and Bag- 59. Chung, I. Y., and Benveniste, E. N. Tumor necrosis factor-alpha production giolini, M. Phagocytosing neutrophils produce and release high amounts of by astrocytes. Induction by lipopolysaccharide, IFN-gammo, and IL-lbeta. J. the neutrophil-activating peptide 1/interleukin 8. J. Exp. Med., 173: 771- Immunol., 144: 2999-3007, 1990. 774, 1991. 60. Ramilo, O., Saez-Llorens, X., Mensola, J., Jafari, H., Olsen, K. D., Hansen, 36. Thornton, A. J., Stricter, R. M., Lindley, I., Baggiolini, M.. and Kunkel, S. E., Yoshinaga, M., Ohkawara, S., Nariuchi, H., and McCracken, G. H. L. Cytokine-induced gene expression of a neutrophil chemotactic factor/IL-8 Tumor necrosis factor-a/cachectin and interleukin Ib initiate meningea! in in human hepatocytes. J. Immunol., 144: 2609-2613, 1990. flammation. J. Exp. Med., 772:497-507. 1990. 37. Hotta, K., Hayashi, K., Ishikawa, J., Tagawa, M., Hashimoto, K., Mizuno, 61. Burrows, L. J., Piper, P. J., Lindley, I. D., and Westwick, J. Intraperitoneal S., and Suzuki, K. Coding region structure of interleukin-8 gene of human injection of human recombinant neutrophil-activating factor/interleukin 8 lung giant cell carcinoma LU65C cells that produce LUCT/interleukin-8: (hrNAF/IL-8) produces a T cell and eosinophil infiltrate in the guinea pig homogeneity in interleukin-8 genes. Immunol. Lett., 24: 165-169, 1990. lung. Effect of PAF agonist WEB2086. Ann. NY Acad. Sci., Â«29:422-424, 38. Standiford, T. J., Kunkel, S. L., Basha, M. A., Chensue. S. W., Lynch, J. P., 1989. Toews, G. B., Westwick, J., and Strieter, R. M. Interleukin-8 gene expression 62. Brooks, W. H., Netsky, M. G., Normansell, D. E., and Horwitz, D. A. by a pulmonary epithelial cell line. A model for cytokine networks in the lung. Depressed cell-mediated immunity in patients with primary intracranial tu J. Clin. Invest., 86: 1945-1953, 1990. mors. J. Exp. Med., 136: 1631-1647, 1972. 39. Kaashoek, J. G., Mout, R., Falkenburg. J. H., Willemze, R.. Fibbe, W. E., 63. Weisbarth, R. H.. Chan, G., Spotter, L., and Golde, D. W. Further purifica and Landegent. J. E. Cytokine production by the bladder carcinoma cell line tion of neutrophil migration inhibitory factor from T lymphocytes (NIF-T): 5637: rapid analysis of mRNA expression levels using a cDNA-PCR proce evidence that NIF-T and leukocyte inhibitory factor (LIF) are immunologi- dure. Lymphokine Cytokine Res., 10: 231-235, 1991. cally distinct. Clin. Immunol. Immunopathol., 32: 269-274, 1984. 40. Baggiolini, M., and Dewald, B. Cellular models for the detection and evalu- 64. List, J., Moser, R. P., Steuer, M., Loudon, W. G., Blacklock, J. B., and ation of drugs that modulate human phagocyte activity. Experientia, 44: Grimm, E. A. Cytokine responses to intraventricular injection of interleukin 841-848. 1988. 2 into patients with leptomeningeal carcinomatosis: rapid induction of tumor 41. Matsushima, K., Morishita, K., Yoshimura, T., Lavu, S., Kobayashi, Y., Lew, necrosis factor a, interleukin Ib, interleukin 6. 7-interferon, and soluble W., Appella, E., Kung, H. F., Leonard, E. J., and Oppenheim, J. J. Molecular interleukin 2 receptor (M, 55,000 protein). Cancer Res.. 52: 1123-1128, cloning of a human monocyte-derived neutrophil chemotactic factor 1992. (MDNCF) and the induction of MDNCF mRNA by interleukin 1 and tumor 65. Saukkonen, K., Sande, S., Cioffe, C., Wolpe, S., Sherry, B., Cerami, A., and necrosis factor. J. Exp. Med., 167: 1883-1893, 1988. Tuomanen, E. The role of cytokines in the generation of inflammation and 42. Sticherling, M., Schroder, J. M., and Christophers, E. Production and char tissue damage in experimental gram-positive meningitis. J. Exp. Med., 777: acterization of monoclonal antibodies against the novel neutrophil activating 439-448, 1990. peptide NAP/IL-8. J. Immunol., 143: 1628-1634, 1989. 66. Schall, T. J., Bacon, K., Toy, K. J., and Goeddel, D. V. Selective attraction 43. Fraker, P. J., and Speck, J. C., Jr. Protein and cell membrane iodinations of monocytes and T lymphocytes of the memory phenotype by cytokine with a sparingly soluble chloroamide, l,3,4,6-tetrachloro-3a,6a-diphrenylg- RANTES. Nature (Lond.), 347: 669-671, 1990. 4305

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1992 American Association for Cancer Research. Interleukin-8 Is Produced in Neoplastic and Infectious Diseases of the Human Central Nervous System

Erwin Van Meir, Miroslav Ceska, Fritz Effenberger, et al.

Cancer Res 1992;52:4297-4305.

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