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Characterization of a Novel
Chemokine-Containing Storage Granule in
Endothelial Cells: Evidence for Preferential
Exocytosis Mediated by Protein Kinase A and
Diacylglycerol
Inger Øynebråten, Nicolas Barois, Kathrine Hagelsteen,
Finn-Eirik Johansen, Oddmund Bakke and Guttorm
Haraldsen
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The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2005 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2005; 175:5358-5369; ;
doi: 10.4049/jimmunol.175.8.5358
http://www.jimmunol.org/content/175/8/5358
The Journal of Immunology
Characterization of a Novel Chemokine-Containing Storage
Granule in Endothelial Cells: Evidence for Preferential
Exocytosis Mediated by Protein Kinase A and Diacylglycerol1
Inger Øynebråten,2* Nicolas Barois,‡ Kathrine Hagelsteen,* Finn-Eirik Johansen,*†
Oddmund Bakke,‡§ and Guttorm Haraldsen*†
C
hemokines are small (8 –10 kDa) soluble proteins that
control leukocyte chemotaxis and extravasation. Extravasation is a multistep process in which the function of the
chemokines is to trigger firm arrest of the leukocyte and enable
transendothelial migration (1, 2). At sites of acute injury or inflammation, leukocyte recruitment may occur rapidly. For instance, granulocytes can extravasate within minutes in a process
that depends on immediate endothelial cell (EC)3 activation (referred to as type I activation) and not on new protein synthesis (3).
Instead, relevant mediators are synthesized and stored in intracellular compartments (i.e., secretory granules), from which they can
be rapidly released to the EC surface upon stimulation in the pro*Laboratory for Immunohistochemistry and Immunopathology and †Department of
Pathology, University of Oslo and Rikshospitalet University Hospital, Oslo, Norway;
‡
Department of Molecular Biosciences, University of Oslo, Oslo, Norway; and §Department of Biomedicine, University of Bergen, Bergen, Norway
Received for publication December 14, 2004. Accepted for publication July 28, 2005.
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 accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This study was supported by the Norwegian Cancer Society Grant B02085, Research Council of Norway Grant 133924/300 and Anders Jahrés Fund. I.Ø. is a Research Fellow of the Norwegian Cancer Society. F.-E.J. and G.H. are Career Investigators of the Research Council of Norway.
2
Address correspondence and reprint requests to Inger Øynebråten, Laboratory for Immunohistochemistry and Immunopathology, Institute of Pathology, Rikshospitalet,
N-0027 Oslo, Norway. E-mail address: [email protected]
3
Abbreviations used in this paper: EC; endothelial cell; AKAP, A kinase-anchoring
protein; 6-Bnz-cAMP, N6-benzoyladenosine-3⬘,5⬘-cyclic monophosphate; CI, confidence interval; IBMX, 3-isobutyl-1-methylxanthine; PAG, protein A-coated colloidal
gold particle; 8-pCPT-2⬘-O-Me-cAMP, 8-(4-chlorophenylthio)-2⬘-O-methyladenosine-3⬘,5⬘-cyclic monophosphate; PKA, protein kinase A; PKC, protein kinase C;
tPA, tissue plasminogen activator; WPB, Weibel-Palade body; VAMP, vesicle-associated membrane protein; vWf, von Willebrand factor.
Copyright © 2005 by The American Association of Immunologists, Inc.
cess of regulated secretion/exocytosis (reviewed in Refs. 4 – 6).
The best characterized compartment for such regulated exocytosis
in ECs of blood vessels is the Weibel-Palade body (WPB), identified by its rod shape (usually 0.2 ⫻ 2–3 ␮m) and its content of
von Willebrand factor (vWf) (4, 7–9). Regulated EC exocytosis is
also reported to originate from smaller, round granules harboring
the anti-coagulant proteins tissue plasminogen activator (tPA)
(10 –13) and protein S (14). In addition, multimerin, a possible
carrier protein for platelet factor V, is stored in small, round granules as well as in elongated structures different from the WPB (15).
It is currently unknown whether tPA, protein S, and multimerin are
sorted to the same granule. Moreover, the localization of tPA is
still a matter of controversy, because t-PA has been described by
some groups to be sorted to the WPB (16 –18) instead of small
granules (10 –13). Finally, the anticoagulant protein tissue factor
pathway inhibitor is released by secretagogue stimulation, but its
main intracellular localization is in caveolae and not in secretory
granules (19 –21).
Regulated exocytosis in ECs is induced by a number of agonists
that can be separated into two distinct groups: those acting by
elevating the level of cytosolic Ca2⫹ and those acting in a Ca2⫹independent manner (reviewed in Refs. 4 and 5). For example,
thrombin (22–24), histamine (25), and purine nucleotides (26, 27)
have been shown to induce granule release in a Ca2⫹-dependent
manner. By contrast, epinephrine and forskolin elevate the level of
cAMP, thereby activating PKA, and resulting in Ca2⫹-independent
regulated exocytosis (28, 29). Regulated exocytosis might also be
induced in a Ca2⫹-independent manner by phorbol esters, either by
activation of PKC, RasGRP, or Munc13 (30 –32), the latter being a
SNARE-associated protein important for the secretory granule to
become fusion competent. The molecular mechanisms of regulated
0022-1767/05/$02.00
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We have recently shown that several proinflammatory chemokines can be stored in secretory granules of endothelial cells (ECs).
Subsequent regulated exocytosis of such chemokines may then enable rapid recruitment of leukocytes to inflammatory sites.
Although IL-8/CXCL8 and eotaxin-3/CCL26 are sorted to the rod-shaped Weibel-Palade body (WPB), we found that GRO␣/
CXCL1 and MCP-1/CCL2 reside in small granules that, similarly to the WPB, respond to secretagogue stimuli. In the present
study, we report that GRO␣ and MCP-1 colocalized in 50- to 100-nm granules, which occur throughout the cytoplasm and at the
cell cortex. Immunofluorescence confocal microscopy revealed no colocalization with multimerin or tissue plasminogen activator,
i.e., proteins that are released from small granules of ECs by regulated exocytosis. Moreover, the GRO␣/MCP-1-containing
granules were Rab27-negative, contrasting the Rab27-positive, WPB. The secretagogues PMA, histamine, and forskolin triggered
distinct dose and time-dependent responses of GRO␣ release. Furthermore, GRO␣ release was more sensitive than IL-8 release
to inhibitors and activators of PKA and PKC but not to an activator of Epac, a cAMP-regulated GTPase exchange factor,
indicating that GRO␣ release is regulated by molecular adaptors different from those regulating exocytosis of the WPB. On the
basis of these findings, we designated the GRO␣/MCP-1-containing compartment the type 2 granule of regulated secretion in ECs,
considering the WPB the type 1 compartment. In conclusion, we propose that the GRO␣/MCP-1-containing type 2 granule shows
preferential responsiveness to important mediators of EC activation, pointing to the existence of selective agonists that would allow
differential release of selected chemokines. The Journal of Immunology, 2005, 175: 5358 –5369.
The Journal of Immunology
5359
Materials and Methods
Reagents
Recombinant human IL-1␤, recombinant human epidermal growth factor,
recombinant human basic fibroblast growth factor, recombinant human
platelet factor 4, and matched Ab pairs for ELISAs of human IL-8 and
GRO␣ were obtained from R&D Systems or PeproTech. FBS, gentamicin,
fungizone, L-glutamine, MCDB 131, lipofectin reagent, and Opti-MEM I
were purchased from Invitrogen Life Technologies, trypsin-EDTA was
purchased from BioWhittaker, and alkaline phosphatase- or HRP-conjugated streptavidin was purchased from Southern Biotechnology Associates
and R&D Systems, respectively. The tetramethylbenzidine microwell peroxidase substrate system was purchased from Kirkegaard & Perry Laboratories and protein A-coated colloidal gold particles were obtained from G.
Posthuma (University Medical Center, Utrecht, The Netherlands). LPS, sodium butyrate, 3-isobutyl-1-methylxanthine (IBMX), and (⫾)-epinephrine
were from the Sigma-Aldrich; 8-(4-Chlorophenylthio)-2⬘-O-methyladenosine3⬘,5⬘-cyclic mono-phosphate (8-pCPT-2⬘-O-Me-cAMP) and N6-benzoyladenosine-3⬘, 5⬘-cyclic monophosphate (6-Bnz-cAMP) were purchased from
BioLog; and H-89, chelerythrine chloride, and Gf109203x/bisindolylmaleimide I were purchased from Calbiochem. Tacrolimus/FK506 was purchased
from Fujisawa. The primary Abs used for immunostaining are listed in Table
I. Unconjugated rabbit anti-mouse IgG⫹M and tetramethylrhodamine isothiocyanate-labeled swine anti-rabbit IgG were purchased from DakoCytomation,
biotinylated horse anti-mouse IgG from Vector Laboratories, streptavidin-Cy2
conjugate was purchased from Amersham Biosciences, Alexa-488 goat antirabbit IgG was purchased from Molecular Probes, and Cy3-conjugated donkey
anti-mouse IgG, Cy3-conjugated donkey anti-rabbit IgG, and Cy3-conjugated
streptavidin were purchased from The Jackson Laboratory.
Cell culture
Umbilical cords were obtained from the Department of Gynecology and
Obstetrics, Rikshospitalet, and HUVECs were isolated as described by
Jaffe et al. (40) and cultured in MCDB 131 containing 7.5% FBS, 10 ng/ml
recombinant human epidermal growth factor, 1 ng/ml recombinant human
basic fibroblast growth factor, 1 ␮g/ml hydrocortisone, 50 ␮g/ml gentamicin, and 250 ng/ml fungizone. The cells were maintained at 37°C in
Table I. Primary Abs used for immunostaining
Human Specificity
EEA1
GRO␣
GRO␣
GRO␣
LAMP-2
MCP-1
MCP-1
MCP-1
Multimerin
Multimerin
Rab 8A
Rab 27
Rab 32
SDF-1
Syntaxin 4
tPA
tPA
tPA
VAMP 1/2
VAMP-8
VAMP-8
vWf
vWf
Irrelevant control
Irrelevant control
Irrelevant control
Designation
Working
Concentration
14
20326
BAF275
500-P92
H4B4
1.2 ␮g/ml
1 ␮g/ml
10 ␮g/ml
5 ␮g/ml
1/200
Mouse IgG1
Mouse IgG
Goat IgG
Rabbit
Mouse IgG1
23007.111
BAF279
500-P34
JS-1
1 ␮g/ml
10 ␮g/ml
5 ␮g/ml
5 ␮g/ml
Mouse IgG2B
Goat IgG
Rabbit
Mouse
3
1/200
1 ␮g/ml
1/100
Rabbit
Rabbit
Mouse IgG1
Rabbit anti-mouse
K15C
2 ␮g/ml
Mouse IgG2a
49
PAM-3
9020 – 0809
ESP-4
FL-118
104 302
1 ␮g/ml
10 ␮g/ml
1/200
10 ␮g/ml
10 ␮g/ml
1/300
1/300
1/200
1/1400
Mouse IgG1
Mouse IgG1
Sheep Ig
Mouse IgG
Rabbit IgG
Rabbit
Rabbit
Mouse IgG1
Rabbit
Mouse IgG1
Mouse IgG2a
Rabbit IgG
F8/86
R0156
MOPC-21
UPC-10
“OD17”
Specification
Source
Transduction Laboratories
R&D Systems
R&D Systems
PeproTech
Drs. J. T. August and J. E. K. Hildreth, (Johns Hopkins
University, Baltimore, MD)
R&D Systems
R&D Systems
PeproTech
Dr. C. P. M. Hayward (McMaster University and the
Hamilton Regional Laboratory Medicine Program,
Ontario, Canada)
Dr. C. P. M. Hayward
Dr. J. Peranen (University of Helsinki, Helsinki, Finland)
Transduction Laboratories
Dr. S. Chen (The State University of New Jersey,
Piscataway, NJ)
Dr. S. Arenzana-Seisdedos (Institute Pasteur, Paris,
France)
Transduction Laboratories
Biopool
Biogenesis
American Diagnostica
Santa Cruz Biotechnology
Synaptic Systems
Dr. R. C. Piper (University of Iowa, Iowa City, IA)
DakoCytomation
DakoCytomation
Sigma-Aldrich
Sigma-Aldrich
Institute of Pathology, University of Oslo, Oslo, Norway
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exocytosis in ECs have been focused mainly on WPB release. For
example, the monomeric GTPase Rab3D is suggested to play a role in
WPB/granule formation (12), whereas Rab27A is associated with the
mature pool of the WPBs (33). Furthermore, vesicle-associated membrane protein (VAMP)-3 and syntaxin4, two members of the SNARE
core complex critical for intracellular fusion events, are involved in
WPB exocytosis (34). Finally, the involvement of the small GTPase
RalA in both thrombin- and epinephrine-induced WPB release suggests that there is convergence downstream of agonists activating distinct transduction pathways (29, 35, 36).
We have recently shown that the chemokines eotaxin-3
(CCL26) and IL-8 (CXCL8) are released from the WPB, whereas
GRO␣ (CXCL1) and MCP-1 (CCL2) are both stored in a compartment different from the WPB that is also sensitive to histamine
and PMA (37). Use of distinct granules for regulated exocytosis
might enable the cell to control the acute release of mediators, and
evidence for differential release mechanisms in ECs have indeed
been described previously (11–13, 38, 39). In this respect, we explored whether differential release might be a feature of regulated
chemokine exocytosis. We first showed that GRO␣ and MCP-1
colocalize in small granules distinct from the WPB, and found that
the GRO␣/MCP-1-containing granules were Rab27-negative in
contrast to the Rab27-positive WPBs. Moreover, the responsiveness of the GRO␣/MCP-1-containing granule to relevant secretagogues revealed distinct dose-dependent responses and release kinetics that were nevertheless similar to those obtained for the
WPB. However, specific activation or inhibition of PKA or PKC
revealed interesting differences between the GRO␣/MCP-1-containing compartment and the WPB, suggesting that different molecular mechanisms are involved in their exocytic machineries and
pointing to the existence of agonists that allow selective release of
either compartment.
5360
NOVEL CHEMOKINE STORAGE GRANULE IN ENDOTHELIAL CELLS
humid 95% air/5% CO2 atmosphere and split at ratio 1:3. The cultures were
used at passage level one to six.
Transfection
Transient transfections into HUVECs were performed by lipofection according to the manufacturer’s instruction (Invitrogen Life Technologies),
using 0.6 ␮g of DNA mixed with 1.2 ␮l of lipofectin/well in 8-well LabTek chamber slides (Nalge Nunc International). Alternatively, the
HUVECs were transfected by electroporation according to the protocol
0394 from BTX.
Immunoelectron microscopy
Immunostaining protocols
Monolayers of HUVECs grown on Lab-Tek chamber slides (Nalge Nunc
International) coated with 1% (w/v) gelatin type A from porcine skin, were
briefly submerged in prewarmed PBS (37°C) and fixed in prewarmed 4%
paraformaldehyde (37°C, pH 7.4) for 10 min before washing 2 ⫻ 5 min in
PBS. For immunostaining, the fixed monolayers were incubated with the
primary mouse Ab (see Table I) overnight at 4°C; then with biotinylated
horse anti-mouse IgG (1/200) combined with primary rabbit IgG (Table I)
for 1.5 h at room temperature; and finally with streptavidin-Cy2 conjugate
(1/1000) combined with tetramethylrhodamine isothiocyanate-labeled
swine anti-rabbit IgG (1/80) or Cy3-conjugated donkey anti-rabbit IgG for
1 h at room temperature. In an alternative protocol, the fixed monolayers were
incubated with the primary mouse Ab overnight at 4°C before incubation with
biotinylated horse anti-mouse IgG (1/200) combined with primary rabbit IgG
for 1.5 h at room temperature visualized by streptavidin-Cy3 conjugate (1/
1000) combined with Alexa 488-labeled swine anti-rabbit (1/800). Saponin
(0.1%) was used for permeabilization in all steps during immunostaining, except in the last washing. Irrelevant, concentration-matched primary Abs were
used as negative controls. The immunostained cells were examined by a confocal laser scanning microscope (Leica TCS) equipped with an Ar (488
nm) and a He/Ne (543 and 633 nm) laser. A Plan apochromat ⫻100/1.4
oil objective was used, and the fluorochromes were excited and detected
sequentially.
Secretion experiments
HUVECs were seeded out at confluence (1.6 ⫻ 104 cells/well in 96-well
trays; BD Biosciences) and cultivated for at least two days before being
stimulated by 1 ng/ml IL-1␤ for analysis of IL-8 and GRO␣. To maintain
good culture conditions, we added fresh medium daily. Approximately
35 h after adding the cytokine, the monolayers were washed twice in prewarmed PBS and incubated in fresh medium without cytokine, containing
100 ␮g/ml cycloheximide for the next 3– 4 h. Subsequently, the cells were
washed twice in prewarmed PBS and incubated in growth medium with
100 ␮g/ml cycloheximide and the different agonists. IBMX was added
together with forskolin to inhibit phosphodiesterase activity. All secretion
experiments were performed in duplicate (dose responsiveness of PMA) or
triplicate wells of microtiter plates from which supernatants were harvested
for measurement of GRO␣ and IL-8.
ELISA
The chemokines were analyzed by DuoSet ELISA kits or matched Ab pairs
(R&D Systems or PeproTech) according to the recommendations of the
Results
A novel compartment for regulated exocytosis of chemokines
from endothelial cells
We have recently shown that the chemokines GRO␣ and MCP-1
are released from HUVECs in response to the secretagogues histamine and PMA, indicating that they are stored in a compartment
for regulated secretion (37). Interestingly, immunocytochemistry
and confocal analysis revealed that both chemokines localized in a
compartment different from the WBP. Moreover, this compartment
did not belong to the endocytic pathway and we concluded that
GRO␣ and MCP-1 were sorted to a histamine- and PMA-sensitive
intracellular storage granule distinct from the WPB (37).
To characterize the GRO␣- and MCP-1-containing granules in
more detail, we performed paired immunostaining and confocal
analysis, finding that the two chemokines colocalized in the Golgi
and in the majority of granules (Fig. 1A). Next, we analyzed the
ultrastructure of these granules by means of immunoelectron microscopy on ultrathin cryosections of IL-1␤-treated HUVECs.
MCP-1 was found in granules close to the cell membrane as well
as in the trans-Golgi network and tubulovesicular structures (Fig.
1B). In contrast to the large and elongated WPBs (see inset Fig.
1C, upper right corner), the MCP-1-containing granules were
round, 50 –100 nm in diameter, and moderately electron dense. In
general, no coat was observed on these granules. The same intracellular distribution was found for GRO␣ (Fig. 1C) and double
labeling for GRO␣ and MCP-1 confirmed colocalization in such
granules as well as in the Golgi stack (Fig. 1C). By contrast, the
mononuclear cell-recruiting chemokine SDF-1/CXCL12, that was
also found in a punctuate, perinuclear pattern, failed to colocalize
with GRO␣ (Fig. 1D). Based on the colocalization of GRO␣ and
MCP-1, we focused on GRO␣ detection in the further studies of
this novel chemokine-storage granule.
We next asked whether sorting to these granules might depend
on the synthesis-activating stimulus. In agreement with Wen et al.
(42), we found that LPS is a strong inducer of GRO␣ synthesis,
and that LPS-activated cells contained GRO␣ and MCP-1-positive
granules of similar size and distribution as those induced by IL-1␤
(data not shown). Moreover, LPS-induced granules were released
in response to PMA in a similar manner (data not shown).
Furthermore, we costained for GRO␣ and multimerin or tPA,
two proteins reported to be stored by ECs in secretory granules
distinct from the WPB (10 –13, 15). Multimerin was found in rodshaped structures and small, round granules consistent with a previous report (15) but did not colocalize with GRO␣ (Fig. 1E). To
detect tPA, HUVECs were either treated with 3 mM sodium nbutyrate for 20 h to increase the endogenous expression (data not
shown) or transfected with pcDNA3-tPA (Fig. 1F). These treatments enabled immunodetection of tPA in small granules that were
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IL-1␤-treated cells were fixed for 3 h at room temperature in 0.1 M phosphate buffer containing 4% paraformaldehyde or 0.1% glutaraldehyde. After washing in 1⫻ PBS, the cells were scraped off and spun down. Cell
pellets were embedded in 1⫻ PBS with 12% gelatin, infiltrated with 2.3 M
sucrose overnight at 4°C (41), and then cut in small blocks which were
mounted on pins and frozen in liquid nitrogen. Ultrathin cryosections of
⬃60- to 70-nm thickness were obtained by cutting at ⫺120°C with a
Reichert Ultracut S ultracryomicrotome from Leica and were picked up in
a 1:1 mixture of 2% methylcellulose and 2.3 M sucrose. Cryosections were
then single or double immunostained and the labeling detected using protein A-coated colloidal gold particles (PAGs) of different sizes. For the
single labeling, cryosections were sequentially incubated for 30 min at
room temperature with the rabbit anti-MCP-1, then PAG in 1⫻ PBS with
1% BSA. For the double labeling, cryosections were sequentially incubated
with the rabbit anti-GRO␣ and PAG, then postfixed in 1⫻ PBS containing
1% glutaraldehyde before performing the second labeling with the mouse
anti-MCP-1, followed by the rabbit anti-mouse IgG/IgM, and PAG. Finally, cryosections were contrasted with a 1:9 mixture of 3% uranyl-acetate
and 2% methylcellulose and examined in a Philips CM100 transmission
electron microscope from FEI. Pictures were obtained with a MegaView III
1000*1000 pixel digital camera from Soft Imaging System (SIS).
manufacturer’s with the following modifications: microtiter plates were
incubated overnight with the coat Abs diluted in PBS (all steps were performed at room temperature), washed in H2O or PBS with 0.05% Tween
20, and blocked by 1% (w/v) BSA in PBS for 2 h. Before each of the
following incubation steps, the plates were washed four times in PBS with
0.05% Tween 20. Samples (50 ␮l/well) were incubated overnight followed
by detection Abs (1.5 h) and alkaline phosphatase-conjugated streptavidin
(1/3000, 1.5 h) or HRP-conjugated streptavidin (1/200, 1.5 h). p-Nitrophenyl phosphate in diethanolamine buffer or peroxidase substrate was developed for 5– 40 min, and the absorbance was measured at 405 or 450 nm,
respectively, with a Tecan Sunrise Microplate Reader (Tecan Austria Gesellschaft). Standard curves were generated from 3-fold dilutions of recombinant chemokines (R&D Systems). Sigmoidal dose-response curves
were fitted to the Hill equation by means of Graph Pad Prism to estimate
EC50.
The Journal of Immunology
5361
mainly perinuclear after n-butyrate stimulation but were distributed throughout the whole cell after transfection, probable reflecting differences in the expression levels. In addition, tPA was found
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FIGURE 1. Ultrastructural characteristics and immunofluorescent analysis of the GRO␣/MCP-1-containing storage granule. A, Paired immunostaining for
GRO␣ and MCP-1 in IL-1␤-activated HUVECs
showed colocalization in the Golgi complex and in
evenly distributed granules. B, Single immunogold labeling for MCP-1 (10-nm gold particles) in IL-1␤treated HUVECs showed MCP-1 in granules close to
the plasma membrane (inset, right corner), in the Golgi
complex, and in tubulovesicular structures (middle inset). C, Double immunogold labeling for GRO␣ (10 nm
of gold particles) and MCP-1 (15-nm gold particles)
showed single- and double-positive granules close to
the plasma membrane and colocalization in the Golgi
complex. Inset, upper right corner, A WPB after immunogold labeling of vWf (5-nm gold particles).
HUVECs were IL-1␤-activated and immunostained for
GRO␣ and SDF-1 (D) or GRO␣ and multimerin (E). F,
GRO␣ and tPA were detected in HUVECs transfected
by pcDNA3-tPA. Corner insets, High magnification of
framed areas. The immunofluorescent pictures were acquired with original magnification, ⫻100. Bars on the
electron microscopy pictures indicate 500 nm; e, endosome; G, Golgi complex; m, mitochondria; pm, plasma
membrane.
diffusely throughout the cytoplasm. With the exception of a very
few double positive vesicles, we did not observe overt colocalization of GRO␣ and tPA (Fig. 1F) nor did we observe colocalization
5362
NOVEL CHEMOKINE STORAGE GRANULE IN ENDOTHELIAL CELLS
with vWf (data not shown). On the basis of these data, we concluded that GRO␣ and MCP-1 were stored in a hitherto undescribed granule in HUVECs designated for regulated secretion and
propose to refer to it as the type 2 compartment for regulated
secretion in ECs, considering the WPB the type 1 compartment.
The GRO␣/MCP-1-containing type 2 granule is Rab27 negative
in contrast to the WPB
Secretagogue-specific release kinetics of the type 2 granule
Use of distinct granules for storage would enable the EC to control
the release of molecules in a differential manner. Such differential
release is known for secretory granules in other cell types and has
been described for vWf and t-PA in ECs (11–13, 39), as well as for
the WPB and small granules of unknown identity (38). To examine
whether the GRO␣- and MCP-1-containing granule and the IL-8containing WPB are differentially released, we compared the release kinetics of GRO␣ and IL-8 in response to PMA, histamine,
and forskolin, each known to induce release of the WPB by different signal transduction pathways (4, 5, 29). First, we examined
the response to 100 ng/ml PMA (Fig. 3), observing release of both
GRO␣ and IL-8 as early as 2 min poststimulation and finding that
about half of the releasable chemokines were secreted within the
first 10 min (45% of GRO␣, 38 –53% of IL-8, n ⫽ 3). When
examining the release kinetics in the presence of 100 ␮M histamine, we observed an immediate, faster release of both chemokines compared with that in response to PMA (Fig. 3). Within 2
and 10 min more than 40 and 60% of both chemokines were released relative to the amount released after 30 min, respectively.
Finally, we examined the response to the cAMP-elevating mediators epinephrine and forskolin. In initial experiments we observed
that epinephrine combined with IBMX (a nonspecific inhibitor of
phosphodiesterases) gave variable but weak responses (data not
shown), consistent with a previous study examining the release of
vWf (28). Thus, we proceeded with forskolin combined with
IBMX, and observed a substantially slower time course of release
for both chemokines that only became apparent after 10 min.
Therefore, we concluded that the GRO␣-containing compartment
and the WPB showed similar release kinetics in response to all
three secretagogues, but each secretagogue nevertheless induced
distinct release kinetics because histamine clearly induced the fastest response while forskolin induced a slower and smaller response
than the other agonists.
Dose responsiveness of the type 2 endothelial secretory granule
We next assessed the dose response to PMA, histamine, and forskolin. When examining chemokine release in response to increasing concentrations of PMA (Fig. 4), we found that the half-maximum release (EC50) was reached at 1.5 ng/ml (95% confidence
interval (CI): 0.4 – 6.0) for GRO␣. By analyzing another fraction
of the same supernatants, we found EC50 for IL-8 to be reached at
a somewhat higher concentration of PMA (2.6 ng/ml, 95% CI:
0.4 –17.4). However, the threshold for release of GRO␣ and IL-8,
defined as the intercept with the abscissa of the extrapolated slope
of the dose-response curve (indicated by the stippled line), was 0.5
ng/ml for GRO␣ and 0.1 ng/ml for IL-8. Moreover, while plateau
and maximum release was obtained at 10 –100 ng/ml for GRO␣,
release of IL-8 did not reach a plateau at the tested concentrations
of PMA. In addition, the released amount of GRO␣ differed from
that of IL-8. Average release above the level of constitutive secretion was 298 pg/ml (range, 188 – 454 pg/ml, n ⫽ 4) for GRO␣
and 502 pg/ml (range, 407– 691 pg/ml, n ⫽ 4) for IL-8 at 100
ng/ml PMA, showing that the releasable pool of GRO␣ was
smaller than that of IL-8 in response to PMA.
In the next series of experiments, we examined the dose response to histamine (Fig. 4). We calculated the EC50 to 1.9 ␮M
(95% CI: 0.5–7.1) for GRO␣ and to 5.0 ␮M (95% CI: 2.6 –9.6) for
IL-8, thus indicating a trend to a higher agonist sensitivity of the
GRO␣-granules. Moreover, the threshold of release in response to
histamine was 0.08 ␮M for GRO␣ and 0.7 ␮M for IL-8. Thus,
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To further explore whether there were differential mechanisms involved in GRO␣ and IL-8 release, we examined the intracellular
distribution of SNARE core complex-associated proteins relative
to that of GRO␣ and vWf. We chose to study adaptors known to
be involved in regulated secretory granule release but excluded
candidates that were not expressed at substantial mRNA levels in
cultured HUVECs (M. Veuger and G. Haraldsen, unpublished
observations).
Due to lack of a well-working Ab to Rab4, which is involved in
release of ␣-granules but not dense granules from platelets (43,
44), we transfected Rab4 fused to YFP into HUVECs. We observed yellow fluorescence in the Golgi region, in structures outside the Golgi and in a punctate pattern in the periphery of the cell.
However, immunostaining showed no colocalization with GRO␣
(Fig. 2A). Instead Rab4 colocalized with EEA1 and partially with
LAMP-2 (data not shown). Furthermore, we evaluated Rab8,
Rab27, and Rab32 by paired immunostaining and confocal analysis or conventional fluorescence microscopy. Rab8 is associated
with the ␣-granule of platelets (45) and we observed a fluorescent
signal for this protein in the Golgi region, in scattered granules
throughout the cytoplasm, as well as in elongated structures at the
cell periphery. However, neither GRO␣ nor vWf colocalized with
Rab8 outside the Golgi region (Fig. 2B and data not shown). We
next costained for GRO␣ and Rab27, which is associated with
regulated exocytosis in a number of cells (reviewed in ref (46).
Because HUVECs express mRNA encoding Rab27a but not
Rab27b (33), we considered our anti-Rab27 reagent to detect
Rab27a. For the latter protein, we observed staining in the nucleus,
in cigar-shaped structures, as well as in a punctate pattern throughout the cytoplasm, but there was no colocalization with GRO␣
(Fig. 2C). Consistent with a previous report (33), the Rab27a-positive cigar-shaped structures were vWf-positive (Fig. 2C and data
not shown). Finally, we stained for Rab32, which is associated
with platelet granules and melanosomes (47, 48), observing Rab32
in cytoplasmic peripheral granules negative for GRO␣ (Fig. 2D).
Next, we examined the localization of VAMP-3 and VAMP-8,
which have been associated with WPB release and granule platelet
exocytosis, respectively (34, 49). We first used the Ab used by
Matsushita et al. (34), which recognizes VAMP-1, -2, and -3.
However, due to the low expression of VAMP-2 and the absence
of VAMP-1 in HUVECs, the staining pattern obtained by this Ab
would be expected to mainly represent VAMP-3. Accordingly, we
observed VAMP-3 staining in the nucleus, in elongated, spikeshaped structures close to the cell periphery. None of these structures costained for GRO␣ (Fig. 2E) or vWf (data not shown).
Furthermore, two primary Abs were used for detection of
VAMP-8. The first Ab, from Synaptic Systems, stained perinuclear
round vesicles, while the second (kind gift of Dr. R. C. Piper,
University of Iowa, Iowa City, IA) stained the nucleus, donutshaped structures mainly in the perinuclear area, and elongated
structures in the periphery of the cells. However, none of the
VAMP-8 patterns colocalized with GRO␣ (Fig. 2F and data not
shown). Finally, we examined the subcellular distribution of syntaxin4 with an Ab that has been shown to affect WPB release in a
streptolysin-O assay (34). Staining against syntaxin4 resulted in a
diffuse signal throughout the cytoplasm as well as a weak granular
signal, but revealed no colocalization with GRO␣ (Fig. 2G).
The Journal of Immunology
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FIGURE 2. Paired immunostainings for GRO␣ and
markers of the molecular sorting machinery. A, The localization of GRO␣ vs Rab4 was examined by transfection of Rab4YFP and IL-1␤ treatment before fixation and immunostaining for GRO␣ (clone ID2/A12).
B–G, IL-1␤-activated HUVECs were immunostained
for GRO␣ (clone ID2/A12) and Rab8, Rab27, Rab32,
VAMP-3, VAMP-8 (Synaptic Systems), or syntaxin4.
Original magnification in all panels, ⫻100. Corner insets, High magnification of framed areas.
5363
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NOVEL CHEMOKINE STORAGE GRANULE IN ENDOTHELIAL CELLS
GRO␣ release appeared to be triggered at lower concentrations of
histamine than IL-8 release. Furthermore, both chemokines were
released to a lesser extent by histamine than by PMA and similar
to PMA, histamine released less GRO␣ than IL-8 (Fig. 4). Therefore, we concluded that a smaller pool of both chemokines was
responsive to histamine compared with PMA.
Next, we assessed the dose response of forskolin. Although forskolin yielded more reproducible results than epinephrine, we nevertheless observed only weak responsiveness in the range of
0.001–100 ␮M (Fig. 4). Moreover, the dose response did not reach
sigmoid saturation but appeared to transiently peak at 5 ␮M for
both chemokines. The reduced release observed at higher concentrations of forskolin was not caused by cell damage as the monolayer remained trypan blue-impermeable (data not shown). Instead
the bell shaped dose response was similar to that reported for tPA
and vWf release in response to the cAMP-elevating, ␤-adrenergic
isoproterenol (50). Although forskolin appeared to release even
less chemokine than histamine, we again found that more IL-8 than
GRO␣ was released (Fig. 4).
Finally, we analyzed the possible effect of TNF-␣ on regulated
EC secretion as this cytokine is a rapidly releasable agonist stored
in mast cells and therefore of putative importance in inducing rapid
cellular responses in analogy to for example mast cell-derived histamine. However, TNF-␣ did not promote substantial secretion of
either chemokine in the range of 1–100 ng/ml over 1 h, despite
readily detectable effects in the detection of E-selectin and
VCAM-1 (data not shown).
Additive agonist effects on GRO␣/MCP-1 release
Synergism between cAMP activating and calcium-raising agents
has been described in for example pancreatic ␤ cells (51–53). To
examine whether such mechanisms are involved in release of the
GRO␣/MCP-1-compartment, we combined forskolin (10 ␮M) and
IBMX (100 ␮M) with increasing doses of histamine (1–100 ␮M)
for 30 min (Fig. 5, top panels). The release of both GRO␣ and IL-8
in the presence of both agonists were similar to the sum of released
chemokine in response to either agonist alone, suggesting that his-
tamine and forskolin mediate exocytosis of GRO␣ and IL-8
through additive independent, noninteracting signaling pathways.
In a separate experiment, we added forskolin 25 h before stimulation with histamine (data not shown), but a priming effect of
forskolin was not observed.
Furthermore, we assessed the possible involvement of the calcium/calmodulin dependent protein phosphatase 2B/calcineurin in
the release of the chemokines as protein phosphatase 2B is known
to affect regulated exocytosis in a cell-type dependent manner (54,
55). For this purpose, we used the immunosuppressant FK-506/
tacrolimus that in complex with immunophilins binds to calcineurin and inhibits its dephosphorylation activity (56, 57). The
release in response to FK506 did vary, possibly due to differences
between the cultures and their activation status of the cells as well
as the fact that protein phosphatase 2B/calcineurin is part of a
complex network of regulatory molecules. Nevertheless, we observed a weak but reproducible, dose-dependent secretion of both
GRO␣ and IL-8 induced by FK-506 alone that was further enhanced in an additive manner by coincubation with forskolin (5 or
10 ␮M, Fig. 5, bottom panels).
PKA and PKC are potential regulators of differential chemokine
release
Finally, we compared the extent to which Epac, PKA, or PKC
were involved in the regulated exocytosis of the type 1 and 2
compartment. Epac, a cAMP-regulated exchange factor for the
small GTPases Rap1 and Rap2 is a PKA-independent activator of
regulated exocytosis in both pancreatic ␤ cells (reviewed in Ref.
58) and ECs (29). In the presence of the Epac-specific activator
8-pCPT-2⬘-O-Me-cAMP (59), GRO␣ and IL-8 were secreted at
levels similar to those observed in response to forskolin (Fig. 6A).
Also, the PKA-specific activator 6-Bnz-cAMP induced release of
GRO␣ and IL-8 (Fig. 6B), suggesting that both Epac and PKA
mediate regulated exocytosis of GRO␣ and IL-8. However, 6-BnzcAMP released substantially less IL-8 than GRO␣ (relative to the
amount released by forskolin), suggesting a stronger involvement
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FIGURE 3. Release kinetics of GRO␣ and IL-8 in response to PMA, histamine, and forskolin. The time course of release from IL-1␤-treated, confluent
HUVECs was measured in absence (open symbols) or presence (filled symbols) of 100 ng/ml PMA, 100 ␮M histamine, or 10 ␮M forskolin by ELISA.
Each pair of circles (GRO␣) or squares (IL-8) represent means ⫾ SD of triplicate wells from an individual HUVEC culture. Data represent one of three
similar experiments.
The Journal of Immunology
5365
FIGURE 4. Dose responsiveness of GRO␣ and IL-8 release in response to PMA, histamine, and forskolin. Secretion from IL-1␤-treated, confluent
HUVECs of GRO␣ and IL-8 in response to increasing concentrations of PMA (incubation time 15 min), histamine (15 min), or forskolin (30 min) was
measured by ELISA. Four experiments were performed for each secretagogue. All secretagogue concentrations were not included in all experiments;
therefore, each circle or square represents mean values of two to four experiments ⫾ SEM. The secretion of chemokine in absence of secretagogue was
set to 100%.
Discussion
We recently discovered that the chemokines GRO␣ and MCP-1
are stored in ECs and released from intracellular compartments
distinct from the IL-8/eotaxin-3-containing WPB (37). Because the
GRO␣/MCP-1-containing granules failed to exhibit markers of the
endocytic pathway and disappeared after PMA stimulation, we
concluded that they belong to the secretory pathway (37). Here, we
found that both chemokines are sorted to the same compartment of
small, Rab27-negative, coatless granules, 50 –100 nm in diameter
that occur throughout the cytosol and close to the plasma membrane. These structures resemble secretory granules previously reported to harbor other EC mediators, i.e., protein S (14), tPA (10 –
13), and multimerin (15). We found that HUVECs expressed only
low levels of mRNA transcripts for protein-S (M. Veuger and G.
Haraldsen, unpublished observation), and efforts to immunostain
these monolayers with three different Abs were unsuccessful (I.
Øynebråten, M. Veuger, and G. Haraldsen, unpublished data).
However, the transient release reported for protein S (14) is different from the release kinetics observed for GRO␣ (see below),
and it is therefore unlikely that protein S and GRO␣ originate from
the same storage compartment. Moreover, tPA has been reported
by some groups to localize in the WPB of cultured HUVECs (16 –
18) and by others in small granules (10 –13). We observed tPA in
small granules of both primary and advanced-passage level cultures of HUVECs, thus supporting the studies by Emeis and Knop
(10 –13) with no evidence for vWf or GRO␣ colocalization. Given
that multimerin also failed to colocalize with GRO␣, our data suggested that GRO␣ and MCP-1 are released from a novel regulated
secretory granule in HUVECs. Finally, because tissue factor pathway inhibitor is found in caveoli rather than coatless granules (19 –
21), it is interesting to note that chemokines stored in ECs do not
appear to reside in compartments containing anticoagulant
proteins.
Differential release of various granule populations is a wellknown feature of certain regulated secretory cells (reviewed in
Refs. 6 and 60), and it has also been described for ECs with respect
to the WPB and a small tPA-containing granule (11–13, 39) as
well as for large vs small vesicles measured by whole-cell patch
clamp technique (38). In the present study, interesting trends were
observed for GRO␣ and IL-8 in terms of EC50 and threshold concentrations of release in response to both PMA and histamine.
However, the most apparent differences were observed at the level
of intracellular mediators. Thus, release of GRO␣ varied substantially from that of IL-8 in response to the PKA-specific activator
6-Bnz-cAMP and the PKA inhibitor H-89. Consistent with previous work (61), PKA was involved in forskolin-mediated WPB
exocytosis as tested by the release of IL-8, but our data suggested
that PKA was even more crucially involved in release of the
GRO␣-containing compartment. In a more general context, this
finding points to a novel pathway in differential release of secretory granules, which until now has been associated with regulation
at the level of calcium signaling (reviewed in Ref. 6).
We also observed a stronger effect on PMA-induced GRO␣ release compared with that of IL-8 release in the presence of the
broadly acting PKC inhibitor Gf109203x or chelerythrine chloride,
suggesting another level of regulating differential release of secretory granules from ECs. However, although PKC has been shown
to be important in WPB release (24), our data clearly imply that the
diacylglycerol-mimetic phorbol ester PMA also exhibits PKC-independent effects in regulated secretion in ECs. Such possible
mechanisms may include, but not be limited to, the diacylglycerol/
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of PKA in GRO␣ release than in IL-8 release (Fig. 6B). This possibility was further supported by the observation that the PKA
inhibitor H-89 affected the release of GRO␣ more efficiently than
that of IL-8 (Fig. 6C). Incubation with the inhibitor alone did not
decrease the levels of constitutively released GRO␣ or IL-8 (data
not shown).
The putative involvement of PKC was studied by means of
Gf109203x, an inhibitor of the PKC isoforms ␣, ␤I, ␤II, ␥, ␦, and
⑀, or by means of chelerythrine chloride, which inhibits all PKC
isoforms. Gf109203x inhibited PMA-induced GRO␣ release in a
dose-dependent manner and at 10 ␮M the release was reduced by
32% (Fig. 6D). By contrast, we observed a 20% reduction of IL-8
release at all concentrations of Gf109203x tested in these experiments (0.1, 1, and 10 ␮M) (Fig. 6D). Cell viability was at all doses
excellent as judged by trypan blue. In the presence of 10 ␮M
chelerythrine chloride, PMA-induced release of GRO␣ was more
strongly reduced (50%) compared with that of IL-8 (25%) (Fig.
6E). Moreover, chelerythrine chloride reduced the constitutive release of GRO␣ by 15% but did not affect the constitutive IL-8
release (data not shown) while Gf109203x alone affected release of
neither (data not shown).
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NOVEL CHEMOKINE STORAGE GRANULE IN ENDOTHELIAL CELLS
PMA-responsive Munc13, a crucial protein for phorbol ester-induced PKC-independent presynaptic neurotransmitter release (30).
Moreover, the known link between Munc13-4 and Rab27 in platelets (62) and basophils (63), makes it tempting to assume that
Rab27 on WPB provide a less PKC-dependent molecular machinery of secretion compared with the type 2 Rab27-negative granule.
Thus, a more careful dissection of the PKC involvement in GRO␣
release as well as other diacylglycerol targets deserves further attention. Moreover, the differential effects of both PKA and PKC on
GRO␣ and IL-8 release, makes it tempting to suggest possible
molecular mechanisms of convergence, such as the binding of both
kinases to A kinase-anchoring proteins (AKAPs) (64, 65), one of
which (AKAP79) has been demonstrated to regulate release of
insulin from pancreatic ␤ cells (54, 66). Furthermore, because molecules such as AKAPs contribute to target effects of second messengers to specific compartments, it may also be assumed that such
anchoring molecules are differentially distributed among subsets of
granules.
Both GRO␣ and IL-8 release responded more rapidly to histamine than to PMA and forskolin. For IL-8, this difference accorded
with recently published real-time imaging data of the WPB (67).
The late response to PMA compared with that of histamine could
reflect a delay in the intracellular mechanisms involved in exocytosis, or alternatively, suggest that PMA must penetrate the cell
membrane to induce effector mechanisms. The substantially
slower response to forskolin has been suggested to reflect a dif-
ference in the physiological function between Ca2⫹-raising and
cAMP-elevating agonists. Although, for instance, the Ca2⫹-raising
agonist thrombin might act as a local emergency signal, the cAMPraising agonist epinephrine could act more systemically in respect
to vWf release, perhaps regulating the vWf plasma levels (28, 68).
Moreover, histamine released more GRO␣ and IL-8 than forskolin. This was consistent with a previous report finding that while
histamine releases both peripheral and centrally located granules,
forskolin primarily involves release of vesicles located in the periphery (68). In release of, for instance, amylase and insulin from
exocrine and endocrine pancreatic cells, respectively, calciumraising agents and hormones raising the level of cAMP have been
described to have synergistic effects (51–53). However, when forskolin was added together with histamine we only observed additive effects related to GRO␣ and IL-8 release and our data do not
support the existence of synergistic, interacting cAMP- and histamine/calcium-mediated pathways in ECs. This is consistent with
results reported for vWf release from HUVECs in the presence of
thrombin combined with forskolin or epinephrine (28). Furthermore, cAMP-mediated release might be regulated by the protein
phosphatase 2B/calcineurin. To inhibit the potential activity of this
phosphatase, we used FK-506/tacrolimus that alone induced release of both GRO␣ and IL-8. This is well in line with the insulin
release stimulated by short-term exposure to cyclosporine A (another inhibitor of calcineurin) in pancreatic ␤ cells (69). The
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FIGURE 5. Additive agonist effects on GRO␣ and IL-8 release. Different concentrations of histamine (upper panel, open symbols) or FK-506 (bottom
panel, open symbols) was added alone to IL-1␤-treated, confluent HUVECs, or together with forskolin (10 ␮M) (filled symbols) for 30 min before
harvesting and ELISA of GRO␣ and IL-8 in the supernatants. Each pair of circles (GRO␣) or squares (IL-8) represent means ⫾ SD of triplicate wells from
an individual HUVEC culture. Data represent one of two similar experiments.
The Journal of Immunology
5367
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FIGURE 6. Effect of Epac and PKA and PKC modulators on GRO␣ and IL-8 release. IL-1␤-stimulated, confluent HUVECs were incubated with
different concentrations of 8-pCPT-2⬘-O-Me-cAMP for 1 h (A), 6-Bnz-cAMP for 1 h (B), H-89 in combination with 10 ␮M forskolin for 40 min (C),
Gf109203x together with 100 ng/ml PMA for 30 min (D), and chelerythrine chloride together with 100 ng/ml PMA for 30 min (E) before harvesting and
ELISA of the supernatants. The secretion of chemokine in presence of secretagogue was set to 100%. Each bar represent mean values ⫾ SEM of two or
three similar experiments, each set up in triplicate wells.
enhancing effect of FK-506 on forskolin-induced chemokine secretion is also similar to the response reported in ␤ cells (54).
Furthermore, while FK-506 and forskolin appear to act in a syn-
ergistic fashion in insulin release, we found only additive-effects
on chemokine release, not allowing us to speculate on common
targets as has been demonstrated in ␤ cells.
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NOVEL CHEMOKINE STORAGE GRANULE IN ENDOTHELIAL CELLS
In conclusion, we have characterized the ultrastructure and release properties of a novel chemokine-containing granule in ECs
that appears to be distinct from compartments containing anticoagulant proteins. Furthermore, this GRO␣/MCP-1-containing
granule showed distinct release properties related to both PKA and
PKC compared with that of the IL-8/eotaxin-3-containing WPB,
suggesting that these two granules may be released differentially in
response to currently unknown extracellular signals.
Acknowledgments
Disclosures
The authors have no financial conflict of interest.
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