La Jolla Institute for Molecular Medicine, San Diego, California 92121
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ABSTRACT |
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Stimulation of microvascular endothelial cells with interleukin (IL)-8 leads to cytoskeletal reorganization, which is mediated by combined activation of the CXCR1 and the CXCR2. In the early phase actin stress fibers appear, followed by cortical actin accumulation and cell retraction leading to gap formation between cells. The early response (between 1 and 5 min) is inhibited by an antibody that blocks the CXCR1. The later phase (from about 5 to 60 min), which is associated with cell retraction, is prevented by anti-CXCR2 antibody. Furthermore, anti-CXCR2, but not anti-CXCR1, antibody blocked IL-8-mediated haptotaxis of endothelial cells on collagen. The later phase of the IL-8-mediated actin response is inhibited by pertussis toxin, indicating that the CXCR2 couples to Gi. In contrast, the early phase is blocked by C3 botulinum toxin, which inactivates Rho, and by Y-27632, which inhibits Rho kinase, but not by pertussis toxin. Furthermore, the early CXCR1-mediated formation of stress fibers was prevented by dominant negative Rho. Dominant negative Rac on the other hand initially translocated to actin-rich filopodia after stimulation with IL-8 and later prevented cell retraction by blocking the CXCR2-mediated cytoskeletal response. These results indicate that IL-8 activates both the CXCR1 and the CXCR2 on microvascular endothelial cells, using different signal transduction cascades. The retraction of endothelial cells due to activation of the CXCR2 may contribute to the increased vascular permeability observed in acute inflammation and during the angiogenic response.
C-X-C chemokines; endothelial cells; interleukin-8 receptors; inflammation
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INTRODUCTION |
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INTERLEUKIN
(IL)-8 is a member of the C-X-C family of chemokines that shows
high-affinity binding to the CXCR1 (IL-8 receptor type 1) and the CXCR2
(IL-8 receptor type 2). Although the CXCR1 is selectively activated by
IL-8 only, the CXCR2 responds to several additional chemokines
including growth-related protein- (GRO
), neutrophil-activating
peptide-2, and epithelial-derived neutrophil attractant-78. The common
denominator shared by all chemokines that activate the CXCR2 is a
Glu-Leu-Arg (ELR) sequence in the amino terminus, which appears to
serve as a recognition sequence for receptor binding and activation
(17).
Early investigations concentrated on the effect of IL-8 on neutrophils,
which respond to IL-8 with calcium mobilization (3), actin
polymerization (35), enzyme release, chemotaxis, and a weak respiratory burst. Despite similar affinities for IL-8 and similar
receptor numbers of the CXCR1 and CXCR2, neutrophil chemotaxis is
primarily mediated by the CXCR1 (6, 39). Pertussis toxin blocks all aspects of IL-8-mediated leukocyte activation, indicating that both the CXCR1 and CXCR2 are coupled to Gi in
neutrophils (3) where Gi-2 is very
abundant. It has, however, been shown that IL-8-receptor coupling is
not restricted to Gi. At least under conditions where
G
14 and G
16, were overexpressed, these G
proteins were able to serve as alternate signal-transducing elements of
IL-8-mediated cellular responses (54).
Apart from neutrophils and monocytes (13), numerous sessile cell types have been shown to express IL-8 receptors. These cell types include neurons (19), various cancer cells (30, 32, 36, 51), and endothelial cells (33). Although activation of the CXCR2 can enhance cell proliferation in cancer cells, the physiological role of IL-8 receptors on nonhematopoietic cells is far from clear but is expected to include cell locomotion.
The angiogenic property of IL-8 and related chemokines in vivo has been known for several years (25). Chemokines that do not possess the NH2-terminal ELR sequence or in which this sequence was mutated are devoid of angiogenic activity (47). This ligand usage implies that the CXCR2 is the endothelial cell receptor that mediates the angiogenic response, which has, however, not been shown experimentally. Although an early report demonstrated that IL-8 is a chemotactic factor for endothelial cells (25), there have been difficulties in detecting IL-8 receptors on human umbilical vein endothelial cells (HUVECs) consistently. This led to the conclusion that IL-8 receptors are lost in culture (38), a concept that only recently has been rechallenged by Murdoch et al. (33), who showed expression of both the CXCR1 and CXCR2 on HUVECs. These cells responded with similar chemotactic responses to IL-8 and stroma-derived factor (SDF)-1, a C-X-C chemokine that lacks the NH2-terminal ELR sequence, but only SDF-1 was capable of inducing intracellular calcium mobilization (33). This was a first indication that IL-8 receptors exist on endothelial cells in vitro and that they evoke behaviors that differ from those observed in leukocytes.
Because chemotaxis depends on a cytoskeletal response, we analyzed filamentous actin (F-actin) filament formation in cultured endothelial cells exposed to IL-8. As shown below, IL-8 caused cytoskeletal rearrangement due to activation of both the CXCR1 and the CXCR2, but both temporal and qualitative differences existed between the behavior of the two receptors. The results suggested that sequential receptor activation through two signaling pathways may regulate endothelial cell cytoskeletal responses to IL-8.
The small G proteins Rac, Rho and Cdc42 regulate the formation of polymerized actin. In fibroblasts, activation of Rac causes the formation of lamellipodia, sheet-like structures consisting of a cross-linked meshwork of actin filaments at the leading edge of migrating cells (41). Activation of Cdc42 induces fingerlike structures called filopodia, and Rho activation leads to the formation of stress fibers, which insert into focal adhesion complexes. In fibroblasts, the activities of these three proteins are arranged in a hierarchical fashion: Cdc42 activates Rac, which in turn activates Rho (34). Depending on cell type and possibly the activation pathway, the relationship between the different small G proteins varies, however, and activated Rac may even block Rho-induced stress fiber formation (50). In their GTP-bound active state, Rho family proteins couple to effector proteins, e.g., Ser/Thr kinases such as Rho kinase or p21-activated kinase (PAK) for downstream signaling, resulting in cytoskeletal responses. It has been shown that in endothelial cells, activation of Rac by thrombin leads to cell retraction (52). Activated PAK, a downstream target of activated Rac and Cdc42, also has been shown to cause endothelial cell contraction (23). Lysophosphatidic acid on the other hand, which activates Rho and stress fiber formation (15), caused actin polymerization in endothelial cells but not cell retraction (52). Here we report that IL-8 initially activates Rho and actin stress fiber formation in endothelial cells due to activation of the CXCR1. At later time points, Rac is activated in a CXCR2-dependent fashion, leading to cell retraction and gap formation between neighboring cells.
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METHODS AND MATERIALS |
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Reagents. Blocking antibodies against the CXCR1 and CXCR2 were purchased from R&D Systems (Minneapolis, MN). Anti-Myc tag 9E10 antibody and anti-Rac and anti-Rho monoclonal antibodies were from Upstate Biotechnology (Lake Placid, NY), FITC-IgG was from BioSource (Camarillo, CA), and pertussis toxin and C3 Clostridium botulinum toxin were purchased from List Biological Laboratories (Campbell, CA). Y-27632 was a gift from Yoshitomi Pharmaceuticals (Osaka, Japan).
IL-8 and GROCell culture. Human lung microvascular endothelial cells (HMVECs) were obtained from Clonetics (San Diego, CA) and used between passages 6 and 8. Immortalized dermal human microvascular endothelial cells (HMECs) (1) were obtained from the Centers for Disease Control (Atlanta, GA). Both cells were grown in EGM-2-MV (Clonetics).
HMECs or HMVECs were seeded at low density on fibronectin-coated coverslips and grown in EGM-2-MV (Clonetics) containing 10% fetal calf serum (FCS). On day 7, when the cells had reached confluence, they were serum starved for 2 h (HMVECs) or 3-4 h (HMECs) and stimulated with IL-8 or GROHaptotaxis of HMECs on collagen. Transwell filters (8-µm2 pore size, Costar) were coated overnight with 230 µl of bovine collagen (10 µg/ml, Cohesion) in PBS and blocked for 30 min with 1% bovine serum albumin (BSA). EGM-2-MV (500 µl/well, Clonetics) containing 0.1% BSA, 0.5 µg/ml hydrocortisone, and 50 µg/ml gentamicin were pipetted into the bottom well, and 5 × 104 HMECs in the same medium were added to the inserts. For antibody inhibition studies, the cells were incubated with 10 µg/ml of anti-CXCR1 or anti-CXCR2 antibody for 30 min. After the addition of IL-8 to the bottom wells, the cells were incubated for 4 h at 37°C in a tissue culture incubator and stained for 10 min with 1 µM calcein acetoxymethyl ester (Molecular Probes). Cells in the upper well were carefully removed with a cotton swab, and transmigrated cells were counted at ×10 magnification on a Leitz Fluovert FS microscope using FITC excitation and emission. Results are expressed as percent of cells transmigrated in the absence of IL-8 (means ± SE of 4 experiments in triplicate).
Fluorescence microscopy. For F-actin localization, cells were fixed for 20 min in 3% paraformaldehyde in PBS, put on ice, and permeabilized for 5 min in 0.2% Triton X-100, incubated with 5 mU/ml of rhodamine-phalloidin or FITC-phalloidin (Molecular Probes) for 30 min, washed three times with PBS, and mounted with Antifade (Molecular Probes). For immune detection of Myc-tagged T17NRac and T19NRho, the same fixed and permeabilized cells were incubated with a 1:400 dilution of anti-Myc (9E10 monoclonal antibody from Upstate Biotechnology) for 30 min, washed three times with PBS containing 0.5% FCS, followed by incubation with a 1:200 dilution of FITC-labeled goat anti-mouse IgG (Biosource) for 30 min and three more washes in PBS. The same procedure was followed for staining with anti-Rac or anti-Rho antibody, except that a 1:200 dilution was used for the first antibody.
Fluorescence microscopy was performed on a Zeiss Axiovert 100 microscope with the Spot 32 program to obtain digital images. Images were processed with Adobe Photoshop and Scion Image software was used for the quantification of F-actin in the fluorescent images. ![]() |
RESULTS |
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Cytoskeletal effects of IL-8 on microvascular endothelial cells.
The addition of IL-8 to HMVECs or HMECs caused increased
F-actin formation in both cell types (Fig.
1). This cytoskeletal response was a
consistent finding in over 20 experiments with microvascular
endothelial cells. HUVECs showed a less reproducible response (results
not shown). In unstimulated serum-starved HMVECs, F-actin staining of
low intensity was concentrated in the cell periphery where adjacent
cells touched each other. After the addition of IL-8, prominent stress
fibers appeared within 1 min of activation. In contrast to the
transient actin polymerization observed in leukocytes, which lasts for
about 2 min (35), endothelial cells responded to IL-8 with
prolonged cytoskeletal activation, which was still maximal between 15 and 30 min (Fig. 1) and had not returned to baseline at 1 h.
Starting between 5 and 10 min, the cells retracted, leaving denuded
surface areas between adjacent cells, as indicated by arrows in Fig.
1D.
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Effect of IL-8 and anti-CXCR antibodies on HMEC haptotaxis.
Because formation of F-actin is a prerequisite for cell migration, HMEC
haptotaxis on collagen was determined in response to IL-8 and GRO.
As expected from the preceding F-actin staining studies, both IL-8 and
GRO
were chemotactic for HMECs (Fig. 5). Anti-CXCR2
antibody, but not anti-CXCR1 antibody, prevented this behavior (Fig.
5).
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Effect of CXCR and G protein inhibitors on cytoskeletal response.
Pertussis toxin caused moderate formation of stress fibers in
unstimulated HMECs (Fig. 6). It
prevented, however, the response to GRO overall and the response to
IL-8 at the later time points (Table 1). In contrast, it showed little
effect early on after IL-8 stimulation (Fig. 6 and Table 1) during the
time points when anti-CXCR1 antibody was most effective. Because
pertussis toxin blocks CXCR1- and CXCR2-mediated responses in
neutrophils, this response in endothelial cells was unexpected,
although it had been shown previously that the CXCR1 has the potential
to couple to G proteins other than Gi (54).
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Role of Rac and Rho. To assess the role of the small G proteins in the cytoskeletal response directly, two approaches were taken: 1) HMECs were transiently transfected with dominant negative Rac (T17NRac) or dominant negative Rho (T19NRho) followed by stimulation with IL-8 and detection of the Myc-tagged Rac or Rho with anti-Myc antibody in parallel with F-actin staining and 2) cellular Rac and Rho were detected in untransfected cells by indirect immunofluorescence staining with the respective antibody, again in combination with F-actin detection.
T19NRho prevented the early CXCR1-mediated formation of stress fibers in the presence of IL-8 (Fig. 7, A-C). In contrast, cells transfected with constitutively active Rho (Q63LRho) demonstrated stress fibers in the absence of any stimulus (results not shown). Direct Rho staining was diffuse but appeared to increase in the cell periphery after IL-8 stimulation (results not shown).
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DISCUSSION |
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Although it has been known for several years that IL-8 and other ELR-containing C-X-C chemokines are angiogenic factors in vivo (25), the mechanism involved has remained unresolved because the presence of IL-8 receptors on cultured endothelial cells has been disputed (38). Here we report that microvascular endothelial cells responded to IL-8 on all occasions in vitro and that this response resulted from combined activation of the CXCR1 and the CXCR2. These functional studies are in agreement with the recent findings of Murdoch et al. (33), who detected the CXCR1 and the CXCR2 on endothelial cells by RT-PCR and fluorescence-activated cell-sorter analysis with specific anti-receptor antibodies.
Activation of the CXCR2 on endothelial cells by IL-8 or GRO led to
cell retraction and gap formation between adjacent cells. This behavior
leads to increased permeability of the endothelial cell monolayer as
has been described for thrombin (52), which stimulates
endothelial cells in a fashion similar to IL-8. In vivo endothelial
cell retraction causes increased vascular permeability, a hallmark of
acute inflammation, which is observed in disease states in which IL-8
is implicated to play a role (31) and where increased
vascular permeability is partially independent of neutrophils (56). Future experiments will have to show the relevance
of our in vitro observations to the in vivo situation.
Angiogenesis, which can be induced by ELR chemokines, is similarly associated with a highly permeable vasculature (10). The role of endothelial cell IL-8 receptors in these in vivo situations deserves future exploration because interruption of the activation of endothelial cell IL-8 receptors appears to be a possible therapeutic intervention in acute inflammatory disease. Furthermore, the same approach also appears promising for the development of antiangiogenic therapies for cancers that produce high concentrations of ELR chemokines, which often have a poor prognosis and are characterized by high vascular density rather than neutrophil accumulation (45).
The CXCR1 and CXCR2 are G protein-coupled seven-transmembrane
receptors coupled to Gi. Although it has been shown that
the CXCR1 can also interact with G14 and
G
16 but not with Gq (54), IL-8-induced activation of leukocytes, which are rich in
Gi, is abolished by pertussis toxin (3). In
contrast, the early CXCR1-mediated response to IL-8 in endothelial
cells was insensitive to pertussis toxin, indicating that the CXCR1
couples to another G protein family in these cells. Besides
Gq, which does not interact with the CXCR1
(54), only G
12 and G
13
promote stress fiber formation in a pertussis toxin-insensitive way
(15). The G
12- or
G
13-mediated cytoskeletal response is blocked by C3
botulinum toxin (4) as was the case for the early
IL-8-mediated response in microvascular endothelial cells described
here. The response to IL-8 in HMECs preincubated with anti-CXCR2
antibody was short-lived as has been described previously for
cytoskeletal changes caused by lysophosphatidic acid and endothelin,
two mediators that couple to G
13 and G
12, respectively, and signal through Rho stimulation (14).
Inhibition of Rho kinase (29), a downstream effector of
Rho, similarly prevented the early response to IL-8. Activation of the
Rho/Rho kinase pathway is associated with stimulation of phospholipase D (42). In accordance, it has been shown previously that
phospholipase D is activated by stimulation of the CXCR1 but not of the
CXCR2 (28). Thus it appears that the CXCR1 in endothelial
cells couples to G
12 or G
13 followed by
activation of the Rho/Rho kinase cascade.
It has been shown that activation of Rho in endothelial cells induces clustering of E-selectin and intercellular and vascular cell adhesion molecules, which leads to increased monocyte adhesion (53). By this mechanism, activation of the CXCR1 on endothelial cells may actively recruit leukocytes to an area of inflammation.
The relationship between Rac and Rho activation and cytoskeletal responses such as cell retraction and cell spreading is complex, not fully understood, and variable depending on cell type and specific experimental conditions (26). Furthermore, when small G proteins are stimulated due to receptor activation, additional signal transduction pathways, such as increases in intracellular free calcium, often contribute to the cytoskeletal response. Mobilization of calcium does not appear to have a role in the activation of endothelial cells by IL-8 (33). Because calcium/calmodulin-dependent myosin light chain kinase phosphorylates the myosin light chain (55), calcium flux as it is observed in endothelial cells stimulated with thrombin may contribute to the Rho-dependent cell retraction seen in these cells (12). In contrast, we could not detect Rho-mediated cell retraction in the presence of IL-8.
The later effect of IL-8 or the effect of GRO, which stimulates the
CXCR2 only, was blocked by pertussis toxin, indicating that the CXCR2
in endothelial cells couples to Gi. In contrast to the
transient response in leukocytes, which lasts for less than 5 min,
activation of the CXCR2 on endothelial cells lasted for more than
1 h. This prolonged activation may be a consequence of integrin
activation, which enhances various signaling pathways in adherent cells
(44). In particular, adhesion to the extracellular matrix
results in Rac translocation to the plasma membrane, followed by PAK
activation (9). This translocation of Rac to the plasma membrane was characteristic of IL-8- or GRO
-induced endothelial cell
activation. Because GRO
was capable of causing translocation, this
Rac response is a function of activation of the CXCR2. Rac translocation to the plasma membrane is associated with activation of
PAK, which has been shown to lead to cell retraction in endothelial cells (57). Similarly, activation of the NADPH oxidase
pathway, which causes the respiratory burst, involves translocation of Rac to the plasma membrane (24). Although this pathway is
most prominent in leukocytes, it is operative in endothelial cells and
whether IL-8 can induce superoxide anion generation in endothelial cells deserves future investigation.
Retraction of endothelial cells by IL-8 was mediated by activation of
the CXCR2 in a Rac-dependent fashion. This Rac-mediated retraction of
endothelial cells has been observed previously in thrombin-activated
endothelial cells (52). Particularly when cells are plated
on collagen, Rac has been shown to promote cell migration
(21). In accordance, HMECs migrated toward a source of
IL-8 or GRO in the presence of collagen (Fig. 5). Interestingly, this behavior was a function of the CXCR2 and could be inhibited by
anti-CXCR2 antibody. This contrasts with the situation in neutrophils, where chemotaxis is primarily, but not exclusively, mediated by the
CXCR1 (7, 8, 16, 39) despite similar affinities and
receptor numbers for the CXCR1 and CXCR2. It therefore appears that
chemotaxis is not determined simply by the receptor sequence but
depends on the specific downstream effector interplay that differs in
different cell types.
Activation of the CXCR2 through Rac may represent a mechanism for chemokine-dependent endothelial cell migration in vivo, which is necessary for new vessel growth during angiogenesis. Because all ELR chemokines have an angiogenic potential (47), it appears that activation of the CXCR2 is involved in the angiogenic response. This does not exclude, however, that the CXCR1, which activates the Rho cascade, also has a role in this process because Rho blockade has been shown to inhibit angiogenesis (48).
Various inflammatory stimuli, including tumor necrosis factor- and
IL-1
, induce the production of ELR chemokines in endothelial cells
(22). Furthermore, IL-8 is prestored in
Weibel-Palade bodies and released from endothelial cells on stimulation
(49). Under our conditions in which the endothelial cells
were serum starved for 2-4 h, concentrations of IL-8 and GRO
were in the 10
11 to 10
10 M range, which is
below the concentration range that activated the endothelial cells.
Furthermore the growth medium in which the HMECs were grown contains
hydrocortisone that suppresses production of ELR chemokines by
endothelial cells (37). It is, however, likely that
activated endothelial cells produce ELR chemokines and stimulate their
own IL-8 receptors in an autocrine fashion, which may lead to
continuous activation of the endothelium. Activation of endothelial
cell Rac and Rho as experienced when endothelial cell IL-8 receptors
are stimulated induces nuclear factor-
B-dependent gene expression
including synthesis of IL-8 (18), thus enhancing this
autocrine stimulation pathway. Under conditions of autocrine stimulation, functions of the CXCR2 are activated continuously, but
surface expression of the receptor is minimal (5). In
addition, surrounding cells including fibroblasts, normal epithelial
cells, and cancer cells may provide the source of chemokine for the
endothelium (2, 46). This paracrine supply of endothelial
cells with chemokines may be particularly important for angiogenesis in
the proximity of cancer cells that produce high concentrations of ELR chemokines.
In summary, IL-8 activates both the CXCR1 and the CXCR2 on endothelial cells. The two receptors use different signal transduction cascades that result in the activation of small G proteins and evoke responses that deserve to be further investigated. These chemokine-mediated endothelial cell responses may contribute to increased vascular permeability and leukocyte adhesiveness as observed during acute inflammation on the one hand and endothelial cell migration and proliferation during the angiogenic process on the other hand.
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ACKNOWLEDGEMENTS |
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We thank G. Bokoch for expression vector constructs and Yoshitomi Pharmaceuticals for the gift of Y-27632.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute Grant HL-55657 to I. U. Schraufstatter and Deutsche Forschungsgemeinschaft Fellowship SA-632-2-1 to M. Burger.
Present address of M. Burger: Dept. of Nephrology, Univ. of Freiburg, 79106 Freiburg, Germany.
Address for reprint requests and other correspondence: I. Schraufstatter, La Jolla Institute for Molecular Medicine, 4570 Executive Dr., San Diego, CA 92121 (E-mail: ingrid{at}ljimm.org).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 6 November 2000; accepted in final form 14 December 2000.
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