By
From the Laboratory of Tumor Biology and Genetics, Neurosurgery Department, University Hospital (CHUV), 1011 Lausanne, Switzerland
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.
Astrocytomas are the most common and lethal human
primary brain tumors and can be subdivided into low
grade astrocytoma (WHO grade II), anaplastic astrocytoma
(grade III), and glioblastoma (grade IV) according to cellularity, cellular pleomorphism, degree of neovascularization,
and the presence of necrosis (1). Glioblastoma can occur de
novo or as the recurrence of a grade II or III astrocytoma. Little is known about the molecular mediators inducing the
biological changes occurring during this progression. Here
we address two interesting biological features of these tumors: development of tumor-induced neovascularization
and the use of this vascular network by lymphoid/myeloid
cells for tumor infiltration.
As for other tumor types, the progression of astrocytoma
is dependent on the development of new blood supply (2,
3). New blood vessels appear in low grade astrocytoma;
these vessels are anatomically indistinguishable from those
found in the surrounding normal brain. In the malignant
phase of the disease, vessel density increases and the
neovessels acquire an abnormal architecture, becoming extensively convoluted with the formation of vascular glomeruli, showing lumen occlusion, and displaying hyperplasia of the smooth muscle/pericyte and endothelial cell layers
(1, 3). Maximal vessel density is reached in glioblastoma
which is among the most vascularized tumors (4). Paradoxically, this increase in vessels is accompanied by the development of necrosis, the pathognomonic criterion that distinguishes glioblastoma from anaplastic astrocytoma (1, 3).
The precise mechanism(s) at the origin of this tissue death
are unresolved, but at least two factors are believed to contribute to its genesis. One is the outgrowth of blood supply
by a rapidly growing tumor leading to tissue hypoxia/anoxia. The second is thrombotic occlusion of vessels, conducive to tissue ischemia (1).
Parallel to vessel development, astrocytomas are often infiltrated with numerous lymphoid/myeloid cells extravasating from newly formed tumor vessels. These are predominantly macrophages and CD8 T lymphocytes, but, B cells,
NK cells, and CD4 T cells are also present (5, 6). It is unclear whether these infiltrates participate in an antitumor
response or contribute indirectly to tumor expansion by secretion of growth factors or cytokines. Clearly, they are inefficient at eradicating tumor growth and do not appear to
relate to a favorable prognosis (7, 8). The precise mechanism leading to infiltration in astrocytoma is unknown, but
it is likely to involve both adhesion molecules (9, 10) and
chemoattractants (11, 12).
IL-8 is a candidate molecule that may play a role in both
of these processes. Belonging to the subfamily of chemokines blueprinted by a C-X-C amino acid cystein motif (see
review in reference 13), IL-8 is secreted by many different
cell types and is a chemoattractant for neutrophils, T lymphocytes, and basophils (14). Furthermore, recent work
has demonstrated that IL-8 is a mediator of angiogenesis. IL-8 induces endothelial cell chemotactic and proliferative
activity (20) and mediates neovascularization in rat and
rabbit corneas in the absence of inflammation (23), as
well as in the rat mesenteric window assay (25). IL-8 is secreted by a variety of tumor cells (see review in reference
13), promotes growth of bronchogenic carcinoma (26) and
nonsmall cell lung cancer (22), and correlates with metastatic potential of human melanoma cells in nude mice (27).
Three IL-8-binding receptors participate in the biological responses mediated by this cytokine: C-X-C chemokine receptor 1 (CXCR1/IL-8RA), C-X-C chemokine
receptor 2 (CXCR2/IL-8RB), and the Duffy antigen receptor for chemokines (DARC).1 Although IL-8 is the
only chemokine known to bind to CXCR1, CXCR2 is
shared with all C-X-C chemokines carrying the amino acid
motif E-L-R-C-X-C. DARC is a promiscuous receptor for
many C-X-C and C-C chemokines and serves as a site of
anchorage for infection by malaria parasite Plasmodium vivax
(28, 29).
We previously demonstrated that IL-8 is synthesized in
vivo during all stages of astrocytoma progression (12). Here
we examine which physiological mechanisms regulate IL-8
expression during the progression of human astrocytoma,
and elucidate whether this secretion mediates a biological
response in these tumors. We demonstrate that two mechanisms are likely to be involved in IL-8 secretion: early induction by the presence of inflammatory signals such as IL-1
and TNF, and late induction by a change in the physiology of the tumor, namely, a decrease in oxygen levels due to ischemia/hypoxia. Expression of IL-8 receptors CXCR1
and CXCR2 on a subset of infiltrating leukocytes and of
DARC on tumor microvasculature supports a role for IL-8
in leukocyte attraction, activation, and angiogenesis.
Cell Culture, Spheroid Formation, and Anoxic Treatments.
Human
glioblastoma cell lines U87MG (endogenous WTp53, tumorigenic in immunocompromised mice), LN-229 (mutant p53, tumorigenic), LN-Z308 (p53-null, tumorigenic), and T98G (mutant
p53, nontumorigenic) were grown as previously described (30). To
induce anoxia, cells were incubated in an Oxoid gas generating
anaerobic system chamber (Unipath Ltd., Hamshire, UK) using
hydrogen, CO2, and a palladium catalyst to remove all traces of
oxygen (final anoxic conditions: 93% hydrogen, 7% CO2). Cells
(1.5 × 106) were plated in tissue culture dishes 48 h before anoxic
induction. When the cultures reached 80% confluence, fresh culture medium (DMEM-5% FCS) was added and dishes were incubated under normoxic or anoxic conditions for different times.
To allow spheroid formation, LN-229 cells were trypsinized and
2 × 104 cells were seeded in 0.5% agar-coated wells (GIBCO
BRL, Gaithersburg, MD). Culture medium was changed every 2 d
and spheroids were incubated for 4-11 d until necrosis formation.
Spheroids were then embedded in Tissue-Tek OCT4583 (Miles
Inc., Elkhart, IN) and snap frozen in liquid nitrogen. Serial sections of 5-µm thickness were then processed for in situ hybridization or immunohistochemistry.
Preparation of Conditioned Media for Measurement of IL-8 Production by ELISA.
Culture supernatants of LN-229 were collected
after 4, 12, and 24 h under normoxic or hypoxic conditions, centrifuged at 1,000 g, aliquoted, and stored at Tissue Specimens and Immunohistochemistry.
Astrocytoma specimens and nontumoral brain tissue from pharmacoresistant epilepsy patients were snap frozen in refrigerated isopentane and
stored at RNA Extraction and Reverse Transcriptase/PCR.
Total RNA
from resected human astrocytomas (5 grade II, 7 grade III, and 12 grade IV) and from four cell lines (LN-229, T98G, LN-Z308,
and U87MG) were extracted with TRIzol Reagent as described by the manufacturer (GIBCO BRL). The reverse transcription
(RT)/PCR was done with 1 µg of total RNA as previously described (11). The following primer pairs were used; annealing
temperatures, MgCl2 concentrations, and amplicon sizes are indicated in brackets. For IL-8: 5 Northern Blot Analysis.
10 µg of RNA/sample were electrophoresed through a 1% agarose formaldehyde gel and blotted
onto a nitrocellulose membrane (Hybond N; Amersham, Aylesbury, UK) as previously described (11). The blots were hybridized with the following probes labeled by random primed DNA
labeling kit (Boehringer Mannheim, Indianapolis, IN): a 298-bp
PCR fragment of IL-8 cDNA (see above), a 500-bp EcoRI-
BamHI fragment of human vascular endothelial growth factor (VEGF) cDNA (plasmid pBluescript KS-VEGF), a 1,100-bp PstI
fragment of In Situ Hybridization.
In situ hybridization was performed as
previously described (11) using the following cRNA sense and
antisense probes labeled with 35S-UTP during in vitro transcription reactions with T3 or T7 RNA polymerase: a 240-bp PstI-
EcoRI fragment of the IL-8 cDNA, a 477-bp EcoRI-XhoI
CXCR1 cDNA fragment (33), a 1,510-bp EcoRI-XhoI CXCR2
cDNA fragment (34), and, as a positive control, a BamHI-EcoRI VEGF165 cDNA fragment.
Previous work established that IL-8 is produced in patients
with all grades of astrocytoma progression. Interestingly,
immunohistochemistry showed predominant IL-8 localization on tumor cells or macrophages in perivascular areas
with leukocytic infiltrates and on pseudopalisading cells
surrounding necrosis (12), a spatial pattern that is compatible with a role in chemotaxis and/or angiogenesis. However, localization of cytokine secretion sites on this basis
may be misleading and be the result of cytokine sequestration rather than production. To unequivocally identify the
IL-8 producer cells in these areas, we performed in situ hybridization with sense and antisense IL-8 cRNA probes on
ex vivo glioblastoma samples. IL-8 messenger RNA (mRNA)
was found in areas surrounding blood vessels, both in those showing a lumen (Fig. 1, A and B) and in those with glomeruloid microvascular proliferation (Fig. 1, C and D) characteristic of glioblastoma (1). This confirms the spatial pattern
of expression observed previously with in situ immunodetection (12). Moreover, elevated IL-8 mRNA levels were
observed in cells surrounding necrosis (Fig. 1, E and F).
These cells form a particular structure called pseudopalisade
and are believed to undergo severe stress due to oxygen
deprivation (1). Controls with sense probes on adjacent sections were negative (Fig. 1, G and H). Staining of these
sections (Fig. 1 A) or adjacent sections (not shown) with an
antibody against GFAP, a glial lineage marker, suggested
that the cells expressing IL-8 mRNA were either of glial
origin (astrocytoma cells or reactive astrocytes) or cells infiltrating the tumor in areas with intricate patterns of GFAP
expression.
The correlation of IL-8 expression and tumor cell proximity to necrosis prompted us to investigate whether there
was a causal relationship. Therefore, we used an experimental model system in which necrosis forms at the center
of a multicellular tumor spheroid (35, 36). These spheroids
can be easily manipulated in vitro, the appearance of necrosis is dependent on spheroid size (150-300 µM diameter),
and measurement of oxygen content in the necrotic region
showed that it was anoxic (35). Another advantage of this
model is that it allows examination of inducing events linked
to necrosis formation without the in vivo complications linked to the presence of nontumoral cells (microvessel
cells, microglia, and infiltrating leukocytes). Spheroids were
produced from glioblastoma cells and analyzed for IL-8
mRNA expression by in situ hybridization and Northern
blotting before and after appearance of central necrosis.
Slight constitutive IL-8 mRNA expression was found in
small spheroids without necrosis (diameter of <150 µm)
under the culture conditions used (Fig. 2, A and B); this
likely reflects serum stimulation of IL-8 expression as previously shown in monolayer cultures (12). In larger spheroids
(>10 d old) with central necrosis, a ring-like augmentation
of IL-8 mRNA levels was observed (Fig. 2, D and E). This
increase in IL-8 mRNA could be the result of an inductive
event in the stressed cells lining necrosis or an inhibition of
IL-8 mRNA expression in the peripheral cell layer at the
contact of culture medium. To discriminate between these
two possibilities, we incubated the spheroids with TNF-
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.
Induction of IL-8 expression could be the direct result of
physiological conditions of minimal oxygen supply or related to the presence of factors produced either by dying
cells or liberated by cell lysis during necrosis. To discriminate between these possibilities, we placed monolayer glioblastoma cell cultures under experimental conditions of oxygen deprivation using anaerobic chambers (see Materials
and Methods). These chambers generate complete anoxia within a time frame of 6-8 h due to oxygen consumption
by a palladium catalyst. Using this system we were able to
demonstrate an increase in IL-8 mRNA levels, as do IL-1
or TNF treatments, in glioblastoma cell lines LN-229 (Fig.
3 B), U87MG, LN-Z308, and T98G (not shown), a panel
representative of the genetic and biological heterogeneity of glioblastoma (37). IL-8 mRNA peaked between 12 and
24 h after induction (not shown) and resulted in augmentation of secreted IL-8 in culture medium as measured by
ELISA (Fig. 3 C). Similar results were obtained in cells incubated under a gas mixture composed of 95% N2 and 5%
CO2 (not shown). Cell counting before and after the 24-h
anoxic treatment showed moderate reduction in cell numbers (by 20-30%) as compared to untreated cells (Fig. 3 D),
due to a reduced proliferation rate under anoxia. Examination of morphology and membrane integrity by exclusion
of trypan blue did not show loss of cell viability and there
was no cell detachment from the monolayer. This demonstrates that IL-8 induction is not due to cell products released from lysing cells, but rather a biological response to
anoxic stress. Furthermore, the induction was obtained irrespective of the endogenous wild-type or mutant p53
gene status of the cell lines analyzed (37), suggesting that it
was not linked to hypoxia-induced p53 (38). This is particularly relevant since consensus binding sites for the p53
transcription factor (39) are present in the IL-8 gene. To
directly evaluate whether WTp53 could increase IL-8
mRNA levels, we used a clone of glioblastoma cell line
T98G in which WTp53 expression can be conditionally induced by dexamethasone (40). No difference in IL-8
mRNA content was found in p53-induced and noninduced cells by Northern blotting (not shown).
The biological relevance of IL-8 expression in glioblastoma depends on the presence of cells with receptors capable of binding IL-8. Therefore, we evaluated whether IL-8
might have autocrine and/or paracrine functions in vivo.
First, we examined CXCR1, CXCR2, and DARC mRNA
expression by RT/PCR. CXCR1 mRNA was found in 5 of 5 grade II, 1 of 7 grade III, and 8 of 12 grade IV astrocytoma. CXCR2 mRNA was found in 5 of 5 grade II, 6 of 7 grade III, and 11 of 12 grade IV. DARC mRNA was
found in 3 of 5 grade II, 5 of 7 grade III, and 10 of 12 grade
IV (results not shown). This suggested that IL-8 may serve
a biological function during the progression of human astrocytoma in vivo, either on tumor cells or on accessory cells.
Next, we examined which cell types expressed IL-8-binding receptors in vivo by immunohistochemistry (representative stainings are shown in Fig. 4). Very interestingly,
DARC was specifically expressed by microvascular cells in
5 of 6 grade II, 5 of 6 grade III, and 14 of 19 grade IV astrocytomas (Fig. 4 A) with a staining pattern similar to the
one obtained for factor VIII, a microvascular marker (Fig. 4
B). DARC expression was also present on 5 of 5 nontumoral brains (Fig. 4 C). Control stainings with preimmune serum was negative on all samples (Fig. 4 D). In contrast,
for both CXCR1 and CXCR2, isolated positive cells were
found surrounding blood vessels. For CXCR1, 2 of 9 grade II, 1 of 3 grade III, and 5 of 18 grade IV were positive (Fig. 4 E), and for CXCR2, 1 of 9 grade II, 0 of 3 grade III, and 5 of 18 grade IV (Fig. 4 G). Staining of adjacent sections for CD3, a specific marker of T lymphocytes
(Fig. 4 F), and CD15, a macrophage marker (not shown), showed infiltrates in these areas, suggesting expression by a subset of T lymphocytes and/or macrophages. Unfrequently,
numerous CXCR2 positive cells were found close to necrosis (Fig. 4 H).
To confirm the CXCR1- and CXCR2-expressing cells
detected by immunohistochemistry, we performed in situ
hybridization on three glioblastomas. Specific signals for
both CXCR1 (Fig. 5, A-D) and CXCR2 (Fig. 5, G-J)
mRNAs were found on small isolated cells surrounding vessels, with a morphology compatible with lymphoid or
myeloid infiltrates. Double staining with an anti-GFAP antibody failed to associate these cells with GFAP filaments
(Fig. 5 A). Control with sense probes for CXCR1 (Fig. 5,
E and F) and CXCR2 (Fig. 5 K) were negative. These data
are consistent with expression by infiltrating leukocytes and
confirm immunostaining results.
Morphological examination of the malignant progression
of astrocytoma shows that the transition to the most malignant form (glioblastoma) is defined by the appearance of
necrosis (1). It is intriguing that a decrease in physiological
oxygen pressure, ultimately lethal to tumor cells, coincides
with maximal tumor aggressiveness. This raises the question as to whether the hypoxic/ischemic conditions increasing at this stage are a consequence or a cause of increased malignancy. The appearance of necrosis is also
closely associated with the ultimate changes in the angiogenic phenotype of glioblastoma. The mRNA for VEGF,
an endothelial-specific angiogenic mitogen, is upregulated
by hypoxia in glioma cells in vitro and is overexpressed in
cells lining necrotic areas in vivo (41, 42, 36). VEGF receptor type 2 mRNA is upregulated on endothelial cells in
glioblastoma, and studies with animal models of glioma have shown that vessel development and tumor growth
were partially inhibited by anti-VEGF antibodies (43),
overexpression of a dominant negative VEGF receptor
mutant (44), or antisense VEGF gene constructs (45, 46).
These results demonstrate that hypoxic regulation of VEGF
is of biological consequence for the angiogenic phenotype
associated with the transition to the most malignant form of
astrocytoma. The factors inducing neovascularization in the
early phases of astrocytoma development are less well characterized (3). The presence of residual tumor upon anti-VEGF treatment further suggests that more than one factor
ensures angiogenic supply to support astrocytoma growth.
Finally, because astrocytoma cells also secrete angiogenic
inhibitors (47) and cytokines such as IL-6 (30) or leukemia inhibitory factor (LIF) (our unpublished results) with antiangiogenic properties (48), the final angiogenic response
will be determined by the balance between positive and
negative regulators (2).
Here we demonstrate that IL-8, another soluble biological mediator with angiogenic and chemotactic properties, is
upregulated in cells surrounding necrosis in glioblastoma
and that IL-8-binding receptors are concomitantly expressed in vivo. IL-8 is the best studied member of the
chemokines, a class of cytokines extensively analyzed for
their ability to attract and activate leukocytes during inflammation (see review in reference 13). It has previously been demonstrated that upon induction by inflammatory
cytokines IL-1 and TNF astrocytoma cells release a variety
of cytokines in vitro, including biologically active IL-8 (see
review in reference 49). In vivo, it was unclear whether cytokine expression resulted from indirect induction by tumor-related inflammatory responses or was directly linked
to tumor growth and progression.
We now confirm the presence of IL-8 expression in astrocytoma and show that it is likely to occur by two distinct mechanisms during the progression of astrocytoma.
Initial upregulation may be mediated by stimulatory signals
such as proinflammatory cytokines (e.g., IL-1 or TNF),
which induce IL-8 in astrocytoma cells in vitro and can be
present in glioma, in glioma-associated cyst fluid, and in
cerebrospinal fluid derived from glioma patients (see review in reference 49). Later in disease progression, strong
IL-8 upregulation is observed in cells surrounding necrotic areas in glioblastoma, suggesting further induction by hypoxia/ischemia. This hypothesis was sustained by increased
IL-8 mRNA and protein expression upon in vitro exposure of glioblastoma cells to anoxia as monolayer cultures
or in the central necrosis of three-dimensional spheroids.
These experiments further showed that this increase was
not dependent on nontumoral accessory cells or on stimulation by factors released by dying tumor cells. Thus, IL-8
increase in late stage astrocytoma is most likely to be due to
physiological changes in oxygen pressure occurring during
tumor growth, although we cannot exclude participation
of accessory cells such as macrophages, microglia, or reactive astrocytes in vivo.
The influence of altered oxygen concentrations on IL-8
expression has previously been demonstrated in the context
of ischemia reperfusion. Human umbilical vein endothelial
cells were shown to upregulate IL-8 during hypoxia (50) as
were monocytes during reoxygenation (51). Here, we
show that similar IL-8-inducing events may occur in the
pathologic process of cancer. Tumor cells and/or tumor-infiltrating macrophages or microglia upregulate IL-8 expression which may play important functions in tumor angiogenesis and tumor immune interactions through paracrine
stimulation of cognate receptors. Upregulation of IL-8 in tumor endothelial cells was not observed in astrocytoma, and
may reflect differences in endothelial subtypes, the abnormal structure of the tumor endothelium, as well as tissue
environment (12) or rapid internalization after binding to
DARC (52).
IL-8 induction is relevant to the biology of the tumor
since we detected the expression of IL-8 receptor mRNA
and protein. Tumor cells did not express IL-8 receptors in
vivo demonstrating that IL-8 does not participate in an autocrine growth regulatory loop mediated by any of these
three receptors. However, IL-8 receptor expression pattern
suggests two functions for IL-8 in astrocytoma.
First, the constitutive expression of the DARC receptor
on normal brain and tumor microvasculature supports an
angiogenic function for IL-8 in astrocytoma. This is sustained by a recent report in which the angiogenic factors
present in the supernatant of glioma cell lines were evaluated in an in vitro angiogenic assay using human microvascular endothelial cells. The authors showed with antibody
neutralization assays that the angiogenic response was primarily due to IL-8 or the combination of VEGF and
bFGF, depending on the cell line analyzed (53). Clearly, further in vivo studies will have to establish whether IL-8
and other angiogenic chemokines are essential contributors
to the angiogenic response seen in astrocytoma, whether
this response is mediated by DARC, and whether they act
in synergy with VEGF or represent alternative angiogenic
routes. This is, to our knowledge, the first report of DARC
expression on tumor vasculature. The presence of DARC
on normal endothelial cells lining postcapillary venules in
kidney, spleen, lung, and brain (32, 29, 54), cells potentially constitutively responsive to IL-8, may implicate DARC in early tumor angiogenesis. It is noteworthy that
this vascular staining is not the result of vessel cell or extracellular matrix binding of DARC released from erythrocytes, since this staining was maintained in individuals lacking erythroid DARC gene expression, due to a mutation
disrupting a binding site for the GATA1 erythroid transcription factor in the DARC gene promoter (52, 55). Furthermore, these results designate DARC as a prime candidate endothelial receptor to explain endothelial cell binding
and direct angiogenic responses mediated by IL-8 (20,
56, 57). This should stimulate the deciphering of the
downstream effector mechanisms, likely the demonstration
that DARC elicits an intracellular signal upon ligand binding in microvascular cells. Lastly, due to the promiscuous nature of the DARC receptor, a variety of C-C and C-X-C
chemokines expressed by astrocytoma (see review in reference 49) may bind tumor microvascular cells. The convergence of a large panel of potentially angiogenic cytokines
(58) in the use of a single microvascular receptor, might
designate DARC as a target of choice for therapeutic interference at what might become the "Achilles' heel" of
chemokine redundancy.
Second, the expression of CXCR1 and CXCR2 on a
subset of infiltrating lymphoid and/or myeloid cells is compatible with a role for IL-8 as chemotactic and activating
agent for leukocytes in astrocytoma, as suggested in other
systems (14, 15, 17). It will be of interest to further
characterize this leukocyte subset and to study its function
in the tumor immune system relationship and as an indirect
elicitor of angiogenesis through cytokine release (59, 60). It
should also be mentioned that if IL-8 chemoattracts T lymphocytes, other astrocytoma-expressed leukocyte chemoattractants, such as MCP-1 (11, 61) are certainly involved,
since CXCR1 and CXCR2 expression was found on only
a fraction of leukocytic infiltrates. The reasons for the rare
occurence of neutrophils in astrocytoma/glioblastoma despite functional IL-8 inducing signals are unknown and
were previously discussed (12). Additional hypotheses include inappropriate expression of homing signals, such as
specific selectins (62), potential killing of Fas-expressing
neutrophils (63) by Fas ligand expressed by tumor cells (64,
65), disappearance/physiological downregulation by receptor desensitization (66), excessive release of IL-8 in circulation
leading to saturation (67), or inactivation of chemoattractant ability due to cleavage by aminopeptidase N (CD13) (68).
In conclusion, our results support a model where early in
astrocytoma development tumor-related inflammatory responses trigger IL-8 and VEGF release. This would elicit
direct and/or indirect angiogenic responses through binding of IL-8 to the DARC receptor expressed constitutively
on brain microvascular cells and CXCR1 and CXCR2 receptors on leukocytes. Chemoattracted leukocytes infiltrate the tumor and their activation induces release of cell products/cytokines with angiogenic potential. VEGF release induced by inflammatory signals might exert its first action
when VEGFR1 expression appears in low grade astrocytoma (3). Later in astrocytoma progression and in de novo
glioblastoma, induction of IL-8 and VEGF production by
reduced oxygen pressure and appearance of expression of
VEGFR2 (3) would further contribute, besides other factors, to the florid microvascular proliferation characteristic
of glioblastoma. The understanding of the molecular
mechanisms at the origin of tumor neovascularization, especially at early disease stages, should permit the development of new therapeutic modalities targeted at specific angiogenic effectors.
80°C until tested.
ELISA was done in duplicate using a commercially available assay
system according to the manufacturer's instructions (R&D Sys.
Inc., Minneapolis, MN). The minimal detection limit was 3 pg/
ml of recombinant human IL-8 in the culture medium.
80°C. Cyostat tissue or spheroid sections were incubated with optimal concentrations of the following primary antibodies: anti-IL-8 hybridoma 46E5 (31), anti-CXCR1/IL-8RA hybridoma 5A12-5, anti-CXCR2/IL-8RB hybridoma 6C6-1C
(18), anti-DARC polyclonal antibody 6615 and corresponding
rabbit preimmune serum (32), and purified monoclonal antibody
preparations against glial fibrillary acidic protein (GFAP) clone
6F2 (Dako, Copenhagen, Denmark), T cell marker CD3 clone
SK7 (Becton Dickinson, Basel, Switzerland), and isotype-matched
mouse immunoglobulins as negative controls (Dako). The sections were then incubated for 25 min with a biotinylated anti-
mouse IgG horse antibody (Vector Labs., Burlingame, CA), followed by a 25-min incubation with peroxidase-labeled avidin. The chromogens used were 3-amino-9-ethylcarbazole (Sigma
Chemical Co., St. Louis, MO) or 3,3
-diaminobenzidine (Fluka
Chemie AG, Buchs, Switzerland). Counterstaining was done with
hematoxylin for 30 s.
primer (5
ATG ACT TCC AAG
CTG GCC GTG 3
), and 3
primer (5
CTC TTC AAA AAC
TTC TCC CGA CTC TTA AGT ATT 3
) [65°C, 1.5 mM, 298 bp]; for CXCR1: primer A (5
GAG GTT GTG TGT GGA
AGG TG 3
), and primer B (5
AGG TTG ATG TTT TGG
CAG TG 3
) [64°C, 1 mM, 476 bp]; for CXCR2: primer A (5
GCT CTA GAG CTG GGC AAC AAT ACA GCA AACT 3
)
and primer B (5
CCA TCG ATG GGC ACT TAG GCA GGA
GGT CTTA 3
) [60°C, 1.5 mM, 493 bp]. RT controls included reactions without mouse Moloney leukemia virus (MMLV) reverse transcriptase and without RT product.
actin cDNA (plasmid pAL41), and a 280-bp EcoRI
fragment of the bovine 28S ribosomal DNA gene.
Fig. 1.
Detection of IL-8 mRNA in
glioblastoma sections by in situ hybridization. (A-F) Antisense IL-8 cRNA probe,
(G and H) sense IL-8 cRNA probe (negative control). Sections were derived from glioblastoma No. 921 for A and C (bright
field) and B and D (dark field), and glioblastoma No. 882 for E and G (bright field) and F and H (dark field). (A and B were also
used for immunohistochemistry with an
anti-GFAP antibody (A, red filaments; B,
green filaments). (E-H) Pseudopalisading cells
separate a necrotic tumor area with numerous dying cells with pyknotic nuclei (top)
from a viable richly vascularized tumor area
(bottom). Bright areas in H are tumor vessels.
[View Larger Version of this Image (116K GIF file)]
,
an inflammatory cytokine known to induce IL-8 mRNA production. Increase of IL-8 mRNA was now observed both
in the cells lining necrosis and in those at the periphery of
the spheroid (Fig. 2, F and G), showing that the peripheral
cells have not become refractory to an IL-8-inducing stimulus. In situ hybridization of these spheroids with a VEGF
antisense probe (Fig. 2 I) showed that the mRNA increases observed for both IL-8 and VEGF were similar in both intensity and perinecrotic localization. Control sense cRNA
probes for IL-8 (Fig. 2, C and H) and VEGF (Fig. 2 J) were
negative.
Fig. 2.
Detection of IL-8 and VEGF
mRNAs on spheroid sections of glioblastoma
cell line LN-229 by in situ hybridization. (A, B,
and D-G) Antisense IL-8 cRNA probe, (C
and H) sense IL-8 cRNA probe (negative
control). Green area in H corresponds to necrosis. (I) Antisense VEGF cRNA probe. (J)
VEGF cRNA probe (negative control). (A-
C) 4-d-old spheroid, magnification of 20; (D-
J) 10-d-old spheroids with central necrosis,
magnification of 10. Spheroid in F and G was
incubated for 24 h with TNF (100 U/ml) before sectioning.
[View Larger Version of this Image (122K GIF file)]
Fig. 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-1 treatment at 10 U/ml for 24 h; TNF, TNF-
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.
[View Larger Versions of these Images (24 + 47 + 24K GIF file)]
Fig. 4.
Detection of IL-8-binding
receptors in glioblastoma by immunohistochemistry. DARC (A) and factor VIII
(B) expression on microvascular cells of
glioblastoma No. 1069. DARC (C) and
preimmune serum (D) staining on nontumoral brain No. T265CN. CXCR1 (E)
and CD3 (F) staining on glioblastoma No.
906. CXCR2 staining on sections with vessels (G) or in a perinecrotic region (H)
of glioblastoma No. 842. Examples of positive cells are shown by arrows.
[View Larger Version of this Image (119K GIF file)]
Fig. 5.
Detection of IL-8-binding receptors CXCR1 and CXCR2 mRNAs in
glioblastoma sections by in situ hybridization.
(A-D) Antisense CXCR1 cRNA probe, (E
and F) negative control sense CXCR1 cRNA probe, (G-J) antisense CXCR2 cRNA
probe, (K) negative control sense CXCR2
cRNA probe. Sections were derived from
glioblastoma No. 921 for A and G (bright
field) and B and H (dark field), glioblastoma No. 892 for C and E (bright field) and D and
F (dark field), and glioblastoma No. 882 for I
(bright field), J and K (dark field). Immunohistochemistry with an anti-GFAP antibody
was used for sections A and B (A, red filaments,
and B, green-brown filaments) and with an antismooth muscle actin antibody for E and F.
[View Larger Version of this Image (94K GIF file)]
Address correspondence to Erwin G. Van Meir, Laboratory of Tumor Biology and Genetics, Neurosurgery Department, University Hospital (CHUV), 1011 Lausanne, Switzerland. Phone: +41-21-314-2582; FAX: +41-21-314-2587; E-mail: evanmeir{at}hola.hospvd.ch
Received for publication 6 May 1997 and in revised form 23 July 1997.
1 Abbreviations used in this paper: DARC, Duffy antigen receptor for chemokines; GFAP, glial fibrillary acidic protein; mRNA, messenger RNA; RT, reverse transcription; VEGF, vascular endothelial growth factor.We would like to thank Drs. M. Buckingham, J.F. Brunet, W.E. Holmes, H. Marti, P.M. Murphy, A.O. Pogo, S.X. Qin, M. Sticherling, and H. Weich for plasmids, antibodies, and nontumoral brain samples. We would also like to show our appreciation to Drs. P.-Y. Dietrich, M. Gassmann, R. Kessler, M. Pepper, A.O. Pogo, E. Reichmann, and P.R. Walker for helpful advice and reading the manuscript.
This work was supported by Swiss National Science Foundation grants no. 31-39634.93 (to N. de Tribolet), 31-39356, and 4037-044729 (to E.G. Van Meir), by the Swiss Cancer Research Foundation grant KFS172-9-1995 (to E.G. Van Meir), by the Swiss and Vaud Anti-Cancer Leagues grants FOR254 and SKL116-7-1995 (to N. de Tribolet) and the San Salvatore Foundation (to E.G. Van Meir).
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