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Upregulation of interleukin 8 by oxygen-deprived cells in glioblastoma suggests a role in leukocyte activation, chemotaxis, and angiogenesis.

Desbaillets I, Diserens AC, Tribolet N, Hamou MF, Van Meir EG - J. Exp. Med. (1997)

Bottom Line: In glioblastoma, it further localizes to oxygen-deprived cells surrounding necrosis.Hypoxic/anoxic insults on glioblastoma cells in vitro using anaerobic chamber systems or within spheroids developing central necrosis induced an increase in IL-8 messenger RNA (mRNA) and protein expression. mRNA for IL-8-binding chemokine receptors CXCR1, CXCR2, and the Duffy antigen receptor for chemokines (DARC) were found in all astrocytoma grades by reverse transcription/PCR analysis.These results support a model where IL-8 expression is initiated early in astrocytoma development through induction by inflammatory stimuli and later in tumor progression increases due to reduced microenvironmental oxygen pressure.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Tumor Biology and Genetics, Neurosurgery Department, University Hospital (CHUV), Lausanne, Switzerland.

ABSTRACT
Leukocyte infiltration and necrosis are two biological phenomena associated with the development of neovascularization during the malignant progression of human astrocytoma. Here, we demonstrate expression of interleukin (IL)-8, a cytokine with chemotactic and angiogenic properties, and of IL-8-binding receptors in astrocytoma. IL-8 expression is first observed in low grade astrocytoma in perivascular tumor areas expressing inflammatory cytokines. In glioblastoma, it further localizes to oxygen-deprived cells surrounding necrosis. Hypoxic/anoxic insults on glioblastoma cells in vitro using anaerobic chamber systems or within spheroids developing central necrosis induced an increase in IL-8 messenger RNA (mRNA) and protein expression. mRNA for IL-8-binding chemokine receptors CXCR1, CXCR2, and the Duffy antigen receptor for chemokines (DARC) were found in all astrocytoma grades by reverse transcription/PCR analysis. In situ hybridization and immunohistochemistry localized DARC expression on normal brain and tumor microvascular cells and CXCR1 and CXCR2 expression to infiltrating leukocytes. These results support a model where IL-8 expression is initiated early in astrocytoma development through induction by inflammatory stimuli and later in tumor progression increases due to reduced microenvironmental oxygen pressure. Augmented IL-8 would directly and/or indirectly promote angiogenesis by binding to DARC and by inducing leukocyte infiltration and activation by binding to CXCR1 and CXCR2.

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Measurement of IL-8 mRNA and protein levels in glioblastoma cells exposed to anoxia. (A) Time course of IL-8 mRNA levels in  LN-229 glioblastoma cell spheroids by Northern blotting (left) and graphical display of relative mRNA levels (right). 10 μg of total RNA extracted  from 4-, 7-, and 11-d-old spheroids is displayed in each lane. (This normalizes for cell content in spheroids.) (B) Northern blot with RNA extracted from LN-229 glioblastoma cells, incubated for 24 h in an anoxia-generating chamber system. N, normoxia; A, anoxia; IL-1, simultaneous  IL-1β treatment at 10 U/ml for 24 h; TNF, TNF-α treatment at 100 U/ ml for 24 h. (C) Measurement of IL-8 by ELISA (picogram per milligram  of total cellular protein) in conditioned media of LN-229 cells after 24 h  of normoxic (white column) or anoxic (black column) treatment. Standard  deviations (vertical bars) of triplicates were calculated. (D) Analysis of cell  number and viability of glioblastoma cell lines upon anoxic treatment.  LN-229 (left) and LN-Z308 (right) glioblastoma cells were stained with  trypan blue and counted before (hatched columns) and after a 24-h treatment under normoxic (white columns) or anoxic (black columns) conditions.  Standard deviations of triplicates (vertical bars) were calculated. The  amount of cells permeable to trypan blue was insignificant and is not presented in the graph.
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Figure 3: Measurement of IL-8 mRNA and protein levels in glioblastoma cells exposed to anoxia. (A) Time course of IL-8 mRNA levels in LN-229 glioblastoma cell spheroids by Northern blotting (left) and graphical display of relative mRNA levels (right). 10 μg of total RNA extracted from 4-, 7-, and 11-d-old spheroids is displayed in each lane. (This normalizes for cell content in spheroids.) (B) Northern blot with RNA extracted from LN-229 glioblastoma cells, incubated for 24 h in an anoxia-generating chamber system. N, normoxia; A, anoxia; IL-1, simultaneous IL-1β treatment at 10 U/ml for 24 h; TNF, TNF-α treatment at 100 U/ ml for 24 h. (C) Measurement of IL-8 by ELISA (picogram per milligram of total cellular protein) in conditioned media of LN-229 cells after 24 h of normoxic (white column) or anoxic (black column) treatment. Standard deviations (vertical bars) of triplicates were calculated. (D) Analysis of cell number and viability of glioblastoma cell lines upon anoxic treatment. LN-229 (left) and LN-Z308 (right) glioblastoma cells were stained with trypan blue and counted before (hatched columns) and after a 24-h treatment under normoxic (white columns) or anoxic (black columns) conditions. Standard deviations of triplicates (vertical bars) were calculated. The amount of cells permeable to trypan blue was insignificant and is not presented in the graph.

Mentions: To obtain a quantitative estimate of the increase in IL-8 mRNA production as a result of necrosis development over time, we performed Northern blotting on 10 μg total RNA (thus normalizing for differences in cell numbers between young and old spheroids) extracted from 4-, 7-, and 11-d-old spheroids. Total IL-8 mRNA content of spheroids increased significantly from days 4 to 7 by 2-fold, and from days 4 to 11 by 7.5-fold (Fig. 3 A). These results demonstrate that establishment of a necrotic area in glioblastoma spheroids is sufficient to generate an increase in IL-8 mRNA steady state levels in glioblastoma cells, and that this induction is not dependent on nontumoral accessory cells.


Upregulation of interleukin 8 by oxygen-deprived cells in glioblastoma suggests a role in leukocyte activation, chemotaxis, and angiogenesis.

Desbaillets I, Diserens AC, Tribolet N, Hamou MF, Van Meir EG - J. Exp. Med. (1997)

Measurement of IL-8 mRNA and protein levels in glioblastoma cells exposed to anoxia. (A) Time course of IL-8 mRNA levels in  LN-229 glioblastoma cell spheroids by Northern blotting (left) and graphical display of relative mRNA levels (right). 10 μg of total RNA extracted  from 4-, 7-, and 11-d-old spheroids is displayed in each lane. (This normalizes for cell content in spheroids.) (B) Northern blot with RNA extracted from LN-229 glioblastoma cells, incubated for 24 h in an anoxia-generating chamber system. N, normoxia; A, anoxia; IL-1, simultaneous  IL-1β treatment at 10 U/ml for 24 h; TNF, TNF-α treatment at 100 U/ ml for 24 h. (C) Measurement of IL-8 by ELISA (picogram per milligram  of total cellular protein) in conditioned media of LN-229 cells after 24 h  of normoxic (white column) or anoxic (black column) treatment. Standard  deviations (vertical bars) of triplicates were calculated. (D) Analysis of cell  number and viability of glioblastoma cell lines upon anoxic treatment.  LN-229 (left) and LN-Z308 (right) glioblastoma cells were stained with  trypan blue and counted before (hatched columns) and after a 24-h treatment under normoxic (white columns) or anoxic (black columns) conditions.  Standard deviations of triplicates (vertical bars) were calculated. The  amount of cells permeable to trypan blue was insignificant and is not presented in the graph.
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Figure 3: Measurement of IL-8 mRNA and protein levels in glioblastoma cells exposed to anoxia. (A) Time course of IL-8 mRNA levels in LN-229 glioblastoma cell spheroids by Northern blotting (left) and graphical display of relative mRNA levels (right). 10 μg of total RNA extracted from 4-, 7-, and 11-d-old spheroids is displayed in each lane. (This normalizes for cell content in spheroids.) (B) Northern blot with RNA extracted from LN-229 glioblastoma cells, incubated for 24 h in an anoxia-generating chamber system. N, normoxia; A, anoxia; IL-1, simultaneous IL-1β treatment at 10 U/ml for 24 h; TNF, TNF-α treatment at 100 U/ ml for 24 h. (C) Measurement of IL-8 by ELISA (picogram per milligram of total cellular protein) in conditioned media of LN-229 cells after 24 h of normoxic (white column) or anoxic (black column) treatment. Standard deviations (vertical bars) of triplicates were calculated. (D) Analysis of cell number and viability of glioblastoma cell lines upon anoxic treatment. LN-229 (left) and LN-Z308 (right) glioblastoma cells were stained with trypan blue and counted before (hatched columns) and after a 24-h treatment under normoxic (white columns) or anoxic (black columns) conditions. Standard deviations of triplicates (vertical bars) were calculated. The amount of cells permeable to trypan blue was insignificant and is not presented in the graph.
Mentions: To obtain a quantitative estimate of the increase in IL-8 mRNA production as a result of necrosis development over time, we performed Northern blotting on 10 μg total RNA (thus normalizing for differences in cell numbers between young and old spheroids) extracted from 4-, 7-, and 11-d-old spheroids. Total IL-8 mRNA content of spheroids increased significantly from days 4 to 7 by 2-fold, and from days 4 to 11 by 7.5-fold (Fig. 3 A). These results demonstrate that establishment of a necrotic area in glioblastoma spheroids is sufficient to generate an increase in IL-8 mRNA steady state levels in glioblastoma cells, and that this induction is not dependent on nontumoral accessory cells.

Bottom Line: In glioblastoma, it further localizes to oxygen-deprived cells surrounding necrosis.Hypoxic/anoxic insults on glioblastoma cells in vitro using anaerobic chamber systems or within spheroids developing central necrosis induced an increase in IL-8 messenger RNA (mRNA) and protein expression. mRNA for IL-8-binding chemokine receptors CXCR1, CXCR2, and the Duffy antigen receptor for chemokines (DARC) were found in all astrocytoma grades by reverse transcription/PCR analysis.These results support a model where IL-8 expression is initiated early in astrocytoma development through induction by inflammatory stimuli and later in tumor progression increases due to reduced microenvironmental oxygen pressure.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Tumor Biology and Genetics, Neurosurgery Department, University Hospital (CHUV), Lausanne, Switzerland.

ABSTRACT
Leukocyte infiltration and necrosis are two biological phenomena associated with the development of neovascularization during the malignant progression of human astrocytoma. Here, we demonstrate expression of interleukin (IL)-8, a cytokine with chemotactic and angiogenic properties, and of IL-8-binding receptors in astrocytoma. IL-8 expression is first observed in low grade astrocytoma in perivascular tumor areas expressing inflammatory cytokines. In glioblastoma, it further localizes to oxygen-deprived cells surrounding necrosis. Hypoxic/anoxic insults on glioblastoma cells in vitro using anaerobic chamber systems or within spheroids developing central necrosis induced an increase in IL-8 messenger RNA (mRNA) and protein expression. mRNA for IL-8-binding chemokine receptors CXCR1, CXCR2, and the Duffy antigen receptor for chemokines (DARC) were found in all astrocytoma grades by reverse transcription/PCR analysis. In situ hybridization and immunohistochemistry localized DARC expression on normal brain and tumor microvascular cells and CXCR1 and CXCR2 expression to infiltrating leukocytes. These results support a model where IL-8 expression is initiated early in astrocytoma development through induction by inflammatory stimuli and later in tumor progression increases due to reduced microenvironmental oxygen pressure. Augmented IL-8 would directly and/or indirectly promote angiogenesis by binding to DARC and by inducing leukocyte infiltration and activation by binding to CXCR1 and CXCR2.

Show MeSH
Related in: MedlinePlus