This information is current as
of June 17, 2017.
Identification of a New Exo-Endocytic
Mechanism Triggered by
Corticotropin-Releasing Hormone in Mast
Cells
Santiago Balseiro-Gomez, Juan A. Flores, Jorge Acosta, M.
Pilar Ramirez-Ponce and Eva Ales
J Immunol published online 22 July 2015
http://www.jimmunol.org/content/early/2015/07/22/jimmun
ol.1500253
http://www.jimmunol.org/content/suppl/2015/07/22/jimmunol.150025
3.DCSupplemental
Subscription
Information about subscribing to The Journal of Immunology is online at:
http://jimmunol.org/subscription
Permissions
Email Alerts
Submit copyright permission requests at:
http://www.aai.org/About/Publications/JI/copyright.html
Receive free email-alerts when new articles cite this article. Sign up at:
http://jimmunol.org/alerts
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 © 2015 by The American Association of
Immunologists, Inc. All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
Supplementary
Material
Published July 22, 2015, doi:10.4049/jimmunol.1500253
The Journal of Immunology
Identification of a New Exo-Endocytic Mechanism Triggered
by Corticotropin-Releasing Hormone in Mast Cells
Santiago Balseiro-Gomez,1 Juan A. Flores,1 Jorge Acosta, M. Pilar Ramirez-Ponce, and
Eva Ales
M
ast cells (MC) are connective tissue and mucosa cells,
mostly positioned at the interface of the external environment and nerves or blood vessels. Therefore, it is
not surprising that MC have been widely recognized as important
effectors of the immune system, playing a pivotal role in both
innate and acquired immune system processes, inflammation, and
host–pathogen interactions (1–3). Moreover, MC are involved in
numerous diseases that are affected by stress, such as asthma,
inflammatory arthritis, inflammatory bowel disease, and chronic
inflammation, and can significantly promote or suppress various
aspects of the stress response (4–6). When MC are activated to
degranulate, secretory granules (SG) release their contents, which
include a diverse array of biologically active products, many of
which mediate potential immunoregulatory effects (7, 8).
After MC activation, a degranulation process occurs when SGcontaining mediators fuse with the plasma membrane by exocytosis
Departamento de Fisiologı́a Médica y Biofı́sica, Facultad de Medicina, Universidad
de Sevilla, 41009 Seville, Spain
1
S.B.-G. and J.A.F. contributed equally to this work.
Received for publication February 3, 2015. Accepted for publication June 18, 2015.
This work was supported by the Ministerio de Ciencia e Innovación (Instituto de
Salud Carlos III) and by Fondo Europeo de Desarrollo Regional Grant PI11/00257
(to E.A.).
The funders had no role in study design, data collection, analysis, decision to publish,
or preparation of the manuscript.
Address correspondence and reprint requests to Dr. Eva Ales, Departamento de
Fisiologia Medica y Biofisica, Facultad de Medicina, Universidad de Sevilla, Avenida
Sánchez Pizjuán 4, 41009 Seville, Spain. E-mail address: [email protected]
The online version of this article contains supplemental material.
Abbreviations used in this article: a.u., arbitrary unit; Ca2+i, intracellular calcium;
C48/80, compound 48/80; CRH, corticotropin-releasing hormone; ΔF, fluorescence
increase; FM1-43, N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl)pyridinium
dibromide; 5-HT, 5-hydroxytryptamine (serotonin); Imax, maximum oxidation current;
IMC, intestinal mast cell; MC, mast cell; PMC, peritoneal mast cell; PMD, piecemeal
degranulation; Q, charge; SG, secretory granule.
Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1500253
in an exquisitely orchestrated cellular process (9). Studies using
electrophysiological and electrochemical techniques have described this process in detail and established three exocytotic
modes in secretory cells: full-collapse fusion, reversible fusion
(kiss-and-run), and compound exocytosis. Each of these modes
controls the rate and amount of granular content release and thus
controls exocytosis strength. The three exocytotic modes are followed by different endocytotic routes (classical endocytosis, kissand-run, and bulk endocytosis), which ensure sufficient membrane
recycling (10). Compound exocytosis consists of a fusion event of
a giant SG preformed by granule–granule fusion or by fusion of an
SG on an already fused but not collapsed SG. This mode of
exocytosis has been well established in MC (11), and our group
has previously reported that it can be followed by a mode of endocytosis called compound endocytosis (12). In addition to compound exocytosis, it has been shown that MC can release granular
mediators by piecemeal degranulation (PMD), a process in which
small vesicles are budded off from SG and fuse with the plasma
membrane, leading to selective content discharge. Following exocytosis, endocytosis of these small vesicles is initiated to retrieve
them from the plasma membrane. Finally, the vesicles traverse the
cytoplasm to fuse with SG (13–16). This type of exocytosis can be
mediated by different stimuli such as IL-1 or IL-4 (17, 18).
The crosslinking of IgE bound to receptor (FcεRI) by multivalent Ags is traditionally known for being a potent MC activation
stimulus that leads to anaphylactic degranulation (19, 20). However, MC can also be activated by non-Ag triggers such as neuropeptides, complement activation, and certain toxins (1, 8).
Corticotropin-releasing hormone (CRH), a peptide that activates
the hypothalamic/pituitary/adrenal axis under stress conditions, is
also peripherally secreted and has proinflammatory effects (21)
mediated through immune cells, including MC (22). Indeed, it has
been shown that MC synthesize and secrete CRH, and there is
evidence indicating that the main CRH receptors, CRH-R1 and
CRH-R2, are functionally expressed by MC as well (23, 24).
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
The key role of mast cells (MC), either in development of inflammatory pathologies or in response to environmental stress, has been
widely reported in recent years. Previous studies have described the effects of corticotropin-releasing hormone (CRH), which is
released from inflamed tissues by cellular stress signals, on MC degranulation, a process possibly driven by selective secretion of
mediators (piecemeal degranulation). In this study, we introduce a novel granular exo-endocytic pathway induced by CRH on
peritoneal MC. We found that CRH triggers substantial exocytosis, which is even stronger than that induced by Ag stimulation
and is characterized by large quantal size release events. Membrane fluorescence increases during stimulation in the presence of
FM1-43 dye, corroborating the strength of this exocytosis, given that discrete upward fluorescence steps are often observed and
suggesting that secretory granules are preferentially released by compound exocytosis. Additionally, the presence of a depot of large
tubular organelles in the cytoplasm suggests that the exocytotic process is tightly coupled to a fast compound endocytosis. This CRHstimulated mechanism is mediated through activation of adenylate cyclase and an increase of cAMP and intracellular Ca2+, as
evidenced by the fact that the effect of CRH is mimicked by forskolin and 8-bromo-cAMP. Thus, these outcomes constitute new
evidence for the critical role of MC in pathophysiological conditions within a cellular stress environment and an alternative
membrane trafficking route mediated by CRH. The Journal of Immunology, 2015, 195: 000–000.
2
Materials and Methods
Cell culture
Primary MC cultures were prepared from wild-type C57BL/6 mice or
Wistar rats. All rodent studies were conducted under a protocol approved by
the Ethical Committee of the University of Seville and Consejeria de
Agricultura y Pesca (Junta de Andalucia, Spain). MC were isolated from the
peritoneal cavity of 3- to 4-mo-old mice following a procedure described in
detail elsewhere (11, 12). The peritoneal wash fluid contains a large
number of cells, a small fraction of which are MC. Using a Percoll (SigmaAldrich) gradient, fully differentiated connective tissue PMC were separated from other cells (28). Isolated PMC were plated on poly-D-lysine–
treated coverslips and maintained in advanced RPMI 1640 medium (Life
Technologies) supplemented with FBS (10% v/v), penicillin (50 U/ml),
streptomycin (50 mg/ml), L-glutamine (2 mM), recombinant mouse stem
cell factor (30 ng/ml; PeproTech), and recombinant mouse IL-3 (10 ng/ml;
PeproTech). Cultured PMC were kept at 37˚C, 5% CO2 and 95% air for at
least 48 h prior to experimentation. To induce anaphylactic degranulation,
PMC were sensitized with mouse monoclonal anti-DNP IgE (3 mg/ml;
Sigma-Aldrich) in medium (29). The doses of anti-DNP IgE and DNP–
human serum albumin used in this study produced the maximum exocytotic response. To avoid variability between cells influencing the outcomes
of our experiments, the effects of CRH in PMC secretion were alternated
with control experiments (anaphylactic degranulation by Ag stimulation)
under the same conditions. All experiments were performed at room
temperature (22–24˚C) and only 2- to 4-d-old cells were used.
IMC were isolated from whole colon by enzymatic and mechanical tissue
dispersion. Rat colon was cut in five or six pieces, with adherent mesentery
and fat removed, and then washed with NaCl (0.9%). Tissues were passed to
a Locke’s buffer containing ampicillin (0.5 mg/ml), gentamicin (0.2 mg/ml),
and metronidazole (0.2 mg/ml). Subsequently, tissues were immersed in
a solution with N-acetyl-L-cysteine (1 mg/ml) and EDTA (2 mM) to remove
mucus. Epithelial cells were removed in Locke’s buffer with EDTA (5 mM).
Next, the mucosa of tissues were scraped with a scalpel and placed
in Locke’s buffer containing gelatin (1 mg/ml), MgCl2 (1.23 mM),
DNAse (15 mg/ml), and collagenase D (0.5 mg/ml) and incubated in
a shaking water bath at 37˚C for 20 min. After enzymatic digestion, the
tissue was mechanically dispersed and filtered through a nylon filter
with a 100-mm pore size. Filtrate was centrifuged and the pellet subjected to
a Percoll gradient (55%) to obtain mucosal MC, which were seeded onto
poly-D-lysine–treated coverslips. Experiments were conducted within 24 h
after culture.
Electrochemistry experiments
Amperometric detection of 5-HT release from SG was monitored with
a carbon fiber microelectrode with a tip diameter of 12 mm, fabricated as
previously described (30). Before the experiments were performed, cells
were incubated in an external solution containing 5-HT (1 mM) for 30 min
at 37˚C and then mounted on an all-glass experimental chamber with
5-HT–free external solution (31). Exocytosis was induced by a 5 min application of DNP (Ag) (1 mg/ml) or CRH (10 nM). Carbon fiber was cut
fresh and the electrode backfilled with KCl (1 M) prior to experimentation.
The electrode, placed in close proximity to a cell, was held at 700 mV
versus an Ag/AgCl reference electrode (World Precision Instruments)
using a commercial patch-clamp amplifier (EPC-10 USB; HEKA Electronics). At this voltage, 5-HT is the main secretory product detected in
PMC by amperometry (31, 32). The signal was digitized at 4 kHz and
filtered with an internal low-pass Bessel filter at 2 kHz with the acquisition
software PatchMaster (HEKA Electronics). The signal was displayed in
real time and stored digitally.
Analysis of amperometric data
The amperometric data were analyzed in Igor Pro 6 (version 6.21;
WaveMetrics) using an Igor procedure file designed for analysis of quantal
release and developed by Sulzer and colleagues (33). The amperometric
integral (cumulative Q) was calculated by subtracting the constant direct
current level in the carbon fiber and integrating the whole amperometric
recording. The latency of release that follows stimulation was measured as
the length of time required for completion of 20% of the total secretory
response (final cumulative Q value). The analysis of an individual exocytotic event was performed through the measurement of several parameters (see the graphic in Table I). Only well-resolved spikes with
amplitudes .5 pA were used for our analysis. Each amperometric parameter was statistically analyzed by taking the mean value of the events
from individual cells with .15 spikes during the recording. Amperometric
spike characteristics reveal rich details about the intracellular movement of
the secretory granule toward the cell membrane and fusion with it (34, 35).
Imax represents maximum oxidation current or spike peak. The area under
a spike (spike Q) can be directly related to the number of molecules secreted using Faraday’s law: Q = znF, where Q is the charge, z is the number
of electrons lost in oxidation, F is Faraday’s constant, and n is the number
of electroactive molecules secreted. The cumulative Q for the entire cell is
used to estimate the total amount of 5-HT released per cell. Risem is the
ascending slope of spike calculated from 25 to 75% from Imax. The spike
half-width (t1/2) value is a measurement of the rate of expulsion of
chemical messengers following cell–granule fusion and is measured at the
half-maximum of spike current. Fall time provides total decay time required to return to the baseline amperometric current. Spike number
reveals the number of SG that successfully fuse with the plasma membrane
to initiate exocytosis. The trickling of transmitter through the fusion pore
produces the foot observed before the amperometric spike (34). The criteria to analyze prespike foot signals was restricted to foot amplitudes .2 pA.
The analysis of a foot signal was performed through the measurement
of several parameters (see the graphic in Table II). The foot frequencies
were measured by counting the total number of feet with respect to the
total number of spikes. The foot amplitude is proportional to the fusion
pore conductance (36) and the foot duration is thought to reflect the stability of the fusion pore (37).
Epifluorescence imaging
Exocytosis was imaged on an epifluorescence microscope system including
an inverted microscope (Axiovert 200) equipped with a high-resolution
CCD camera (ORCA-R2 C10600-10B; Hamamatsu Photonics). FM1-43
fluorescence was detected using a standard filter set (XF115-2; Omega
Optical). Images were acquired through a 363 oil immersion objective
(102 nm/image pixel). Images were acquired at 1 Hz with a 0.2-s exposure.
During experiments, labeling solution (4 mM FM1-43), drug solution
(4 mM FM1-43 plus 10 nM CRH, 1 mg/ml DNP, or 100 mg/ml compound
48/80 [C48/80]) and standard external solution (dye- and stimuli-free external solution) were exchanged by a superfusion multibarreled pipette
with a common outlet positioned 50–100 mm from the cell. The solutions
were exchanged using a pinch valve controller (Warner Instruments) that
provides a flow rate that completely replaces the cell surroundings in ,2 s.
Unprocessed images were analyzed with HCImage software (Hamamatsu
Photonics). For measuring the fluorescent exocytotic spots area, we performed a frame-by-frame analysis recording from each cell. Spots were
manually selected and duplicated or non–well-limited ones were discarded. To obtain the increase of fluorescence (ΔF) values, each cell was
manually selected and the membrane fluorescence measured before stimulation (basal labeling) (no. 1 in Figs. 2 and 7) and the final membrane
fluorescence after stimulation (total exocytosis) (no. 4 in Fig. 2A and 2B
and no. 3 in Fig. 7A). All values were corrected by subtracting the
background fluorescence. Finally, we calculated ΔF by subtracting the
basal fluorescence from the final membrane fluorescence.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
Although the mechanism underlying Ag stimulation leading to
anaphylactic degranulation is well understood, the effects of CRH
on MC activation remain unclear. It has been shown that CRH
induces MC degranulation and selective secretion of mediators
stored in cytoplasmic SG, most likely through PMD (22, 24, 25).
Even so, whether there are other exocytotic mechanisms involved
in this process is still unknown. In this study, we stimulated
peritoneal MC (PMC) and intestinal MC (IMC) with CRH. We applied imaging and electrochemical techniques to better understand and
supplement our knowledge about CRH-triggered exocytosis and endocytosis. We observed that CRH increases the 5-hydroxytryptamine
(5-HT, or serotonin) content of SG and the number of release events in
PMC. Imaging of the exo- and endocytosis events during hormone
stimulation in the presence of N-(3-triethylammoniumpropyl)-4(4-(dibutylamino)styryl)pyridinium dibromide (FM1-43) allowed
us to identify multiple steps that reflect compound exocytosis.
This process was tightly coupled to rapid compound endocytosis.
We also found that CRH, acting on its membrane receptors,
triggers a strong increase in intracellular calcium (Ca2+i), which
was mimicked by both 8-bromo-cAMP and forskolin, indicating
that cAMP might favor this response. The fact that CRH is able to
trigger mainly large SG fusion indicates that MC release a large
amount of cargo and differs from the selective secretion through
the “shuttling vesicle” hypothesis or PMD (26, 27).
NEW EXO-ENDOCYTIC MECHANISM TRIGGERED BY CRH
The Journal of Immunology
3
Results
FM1-43 fluorescence pattern suggests that preferentially CRH
induces compound exocytosis
To quantify endocytosis, cells were incubated in an external solution
containing FM1-43 (4 mM) and CRH (10 nM) or DNP (1 mg/ml) for 5 min
and washed with standard external solution. Images were captured using
an upright Olympus FV1000 confocal laser scanning microscope equipped
with three visible wavelength lasers (argon-krypton laser with 488, 561,
and 633 nm excitation lines) and a 360 water-immersion objective
(82 nm/image pixel, numerical aperture of 1.2). To illuminate FM1-43–labeled
endocytotic events, cells were exposed to the 561-nm excitation laser. The
bandwidth of emission spectrum was adjusted to 580–620 nm for acquiring
images. Reconstruction of three-dimensional images from 1-mm Z-stacks
were prepared using ImageJ (National Institutes of Health). To detect and
quantify the fluorescent area on raw images from single slices from the
Z-projection of each cell, HCImage software was used. We assumed that
the fluorescent area could be simple (a single spot) or compound (two, three,
or more fused spots, also referred as endocytotic organelles) in its equatorial
plane and were selected based on threshold brightness and a minimum fluorescent area, discarding duplicated events found in more than one slice.
Recordings of Ca2+i signal
Changes in Ca2+i concentration in response to different drugs were monitored by dual excitation microfluorimetry in PMC incubated in an external
solution containing fura 2-AM (5 mM) for 40 min at 37˚C. Then, cells
were washed twice in external solution without the probe at room temperature and used immediately for imaging. Fura 2-AM loading was
usually uniform over the cytoplasm, and compartmentalization of the dye
was never observed. Experiments were conducted using an AxioVert 200
inverted microscope equipped with a standard filter set (XF04-2; Omega
Optical). Dye-loaded cells were given a long application of the stimuli and
then washed. During recordings, cells were excited at 360/380-nm wavelengths (exposure time, 0.5 s; data acquisition at 0.5 Hz) and fluorescence
elicited was collected at 510 nm (ΔF ratio). Data were acquired and stored
using HCImage software and exported to Igor Pro 6.21 to perform analysis. Time to peak represents the time to reach the peak maximum. The
plateau phase (ΔF ratio plateau) was calculated as the difference between
the final fluorescence after washing and the baseline fluorescence. All
values were normalized to the basal fluorescence (baseline).
Solutions and reagents
All experiments were performed in an external solution that contained
140 mM NaCl, 10 mM HEPES, 3 mM KOH, 2 mM MgCl2, and 1 mM CaCl2.
Locke’s buffer contained 140 mM NaCl, 5 mM KCl, 25 mM NaHCO3,
5 mM HEPES, and 11.1 mM glucose. FM1-43 (1 mM stock solution) and
fura 2-AM (1 mM stock solution) were obtained from Life Technologies.
CRH (5 mM stock solution), Ag DNP–human serum albumin (1 mg/ml
stock solution), forskolin (10 mM stock solution), and C48/80 (50 mg/ml
stock solution) were purchased from Sigma-Aldrich. 8-Bromo-cAMP (50 mM
stock solution) and astressin (250 mM stock solution) were obtained from
Tocris Bioscience. All stock solutions were aliquoted and stored at 220˚C to
protect them from light.
Statistical analysis
CRH induces a high number of fusion events with a large
quantal size
Amperometry is a sensitive and very powerful method of measuring secretion by monitoring the release of electroactive secretory products, such as 5-HT, with a carbon-fiber microelectrode
placed very close to the surface of an isolated cell. Following cell
stimulation, a brief current in the form of spikes can be observed as
a consequence of the oxidation of the transmitters released by SG
(31, 38). This technique allows for the detection of differences in
the amount of product secreted per SG and cell (39). Therefore,
we studied the effects of CRH and Ag (DNP) on the characteristics of exocytotic events.
We next sought to verify the strength of the exocytosis evoked by CRH
using a fluorescent dye. We visualized the exocytosis triggered by
CRH (10 nM) and Ag (1 mg/ml) using the fluorescent probe FM1-43
(4 mM) (Fig. 2, Supplemental Videos 1, 2). FM1-43 and similar
amphipathic styryl dyes have been used for imaging exo-endocytosis
pathways in different types of cells. These dyes can reversibly stain
membranes and are more fluorescent when bound to membranes than
when in solution (40). In PMC, both the membrane and the dense
core of SG are stained with FM1-43, making the release of SG visible
as a bright diffraction of limited spots (12).
Ag stimulation generally showed a gradual rising in the fluorescent trace, without discrete upward steps (nos. 2 and 3 in
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
Throughout this study, each variable of the treatment groups was compared
using a Mann–Whitney U test unless otherwise specified. A Student t test
was used to compare data groups when both passed the normality test
(Kolmogorov–Smirnov). Data were considered significant at p # 0.050
and are plotted as the means with SEM. All statistical analyses were
carried out using SPSS Statistics 22 software (IBM).
Fig. 1 shows the typical amperometric recording after a 5-min
application of 1 mg/ml Ag (Fig. 1A) and 10 nM CRH (Fig. 1B).
The temporal course shows that after stimulation, there is an initial
latency phase before amperometric spikes appear. This period was
longer under CRH than Ag stimulation (CRH, 4.95 6 0.58 min,
n = 16; DNP, 2.01 6 0.30 min, n = 18; p = 0.0001 by Student t
test). Even so, the response phase is longer in CRH-treated cells
(8.15 6 0.72 min) than in Ag-treated cells (4.13 6 0.57 min; p =
0.0001 by Student t test). Additionally, the number of successfully
fused SG (the number of spikes; Fig. 1C) is higher in CRH- than
in Ag-treated cells (CRH, 90 6 19; DNP, 54 6 18; p = 0.050,
Fig. 1C). This difference in spike number was more marked when
we measured the cumulative charge (Q) per cell (p = 0.0001, see
Fig. 1D). The cumulative Q is larger in CRH-stimulated cells than
in Ag-stimulated cells (CRH, 1.21 6 0.17 nC; DNP, 0.42 6 0.09
nC). These data indicate that the total amount of 5-HT released per
cell is greater during exocytosis triggered by CRH. We observed
that the average amperometric spike recorded after CRH stimulation (black trace) is larger than that recorded after Ag stimulation (gray trace) (Fig. 1E). CRH evoked spikes characterized by
a large amplitude and large area under the spike. Taking into
account that the spike area defines spike Q, we used logtransformed values for curve fitting of the Q individual spikes
(CRH, n = 1437 spikes; DNP, n = 964 spikes) (Fig. 1F). We observed a shift in the centers of the two distributions (CRH, 0.50
pC; DNP, 0.23 pC), showing that spikes recorded during CRH
stimulation contain more 5-HT than Ag-induced spikes.
To gain insight into a single exocytotic event, we quantified several
parameters of individual spikes (Table I). Spikes triggered by both
stimuli were no different in kinetic parameters (t1/2 and fall time)
with the exception of Risem, which was longer in CRH-evoked
exocytosis. However, in line with our previous observation, there
were differences in quantitative parameters between the two groups.
Spikes recorded after CRH stimulation showed larger Imax and spike
Q than did those recorded after Ag stimulation, suggesting that CRH
triggers exocytosis of large quantal size SG. We next examined foot
signals preceding amperometric spikes because parameters of these
amperometric feet (e.g., their duration, amplitude, and charge)
provide information about properties of the exocytotic event (34).
Foot frequency indicates that ∼50% of CRH-induced spikes had
a prespike foot. We observed that foot duration was no different
between CRH- and Ag-treated cells. In contrast, foot amplitude and
foot Q are significantly different (Table II) between the two stimuli,
further showing that CRH does affect the amount of 5-HT release.
Altogether, our electrochemical measurements indicate that the
intense exocytosis observed after CRH stimulation in PMC is
stronger than the Ag-induced response and is a consequence of
a higher number of SG fused with the plasma membrane and a large
amount of transmitter released by an exocytotic event.
Live-cell confocal microscopy
4
NEW EXO-ENDOCYTIC MECHANISM TRIGGERED BY CRH
Fig. 2A). However, CRH stimulation resulted in upward steps that
corresponded with the presence of large fluorescent spots (nos. 2
and 3 in Fig. 2B). The examination of the fluorescent spot area
corroborated that CRH induces exocytosis of giant SG (CRH,
3.29 6 0.40 mm2, n = 26 cells; DNP, 1.62 6 0.08 mm2, n = 48 cells)
(Fig. 2C). Additionally, we measured the fluorescence intensity
increase (ΔF) by subtracting the basal membrane fluorescence
(no. 1 in Fig. 2A, 2B) to the final fluorescence (no. 4 in Fig. 2A,
2B). The analysis showed that exocytosis induced by CRH
(1073 6 131 arbitrary units [a.u.], n = 26) is stronger than that
evoked by Ag (775 6 95 a.u., n = 48), (Fig. 2D). Altogether,
these experimental data suggest that the strong exocytosis triggered
by CRH is likely due to a compound exocytosis process (41).
CRH stimulation leads to the formation of large endosome-like
structures
We next examined whether CRH and/or Ag response could also
induce a compensatory endocytosis coupled to the observed
exocytosis. To test this, we performed live cell confocal microscopy
for a quantitative assessment of the total endocytosed probe (see
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
FIGURE 1. Effects of CRH and Ag (DNP) on
secretory response of individual MC. (A) Representative amperometric trace and cumulative Q
(black trace) obtained during a 5-min application
(arrow) of 1 mg/ml DNP. The split line represents
the final period in which amperometric spikes
were not detected. (B) Representative amperometric trace and cumulative Q (black trace)
recorded during a 5-min application of 10 nM
CRH (arrow). Calibration for time, amperometric
current, and cumulative Q are indicated with solid
bars. (C) The bar histogram shows the number of
spikes pooled from cells stimulated with CRH
(filled) and DNP (open). *p , 0.05, Mann–
Whitney U test. (D) The bar histogram shows the
average of total amount of 5-HT released per cell
(cumulative Q) stimulated with CRH (filled) or
DNP (open). ***p , 0.001, Mann–Whitney U
test. (E) Average spikes generated from the data
from Tables I and II, obtained from stimulation
with CRH (black spike) and DNP (gray spike).
(F) Frequency histograms of the log-transformed
values of individual spikes Q from CRH-treated
cells. The solid line represents the Gaussian
function fitted to the data from CRH-treated cells.
The dashed line represents the Gaussian function
fitted to the data from DNP-treated cells. Data in
(C) and (D) are represented as the means 6 SEM.
All data shown are representative of four independent cultures (DNP, n = 18 cells; CRH, n = 15
cells).
Materials and Methods). As indicated above, FM1-43 can be used
as a reliable reporter of exocytosis in PMC. After washing, the
internalized fluorescence observed in cells is a consequence of an
endocytotic process. A 5-min exposure to both stimuli (10 nM
CRH or 1 mg/ml Ag) resulted in a rapid compensatory endocytotic
response (Fig. 3). However, CRH-stimulated cells took up a
greater amount of the dye than did Ag-stimulated cells, indicating
that CRH triggers a more intense endocytosis (Fig. 3A). Indeed, in
CRH-stimulated cells, the total area occupied by the endocytotic
events per cell was larger (16.24 6 1.95 mm2, n = 38) than in Agstimulated cells (11.57 6 2.63 mm2, n = 42) (Fig. 3B). To test
whether these spots represent a single endocytotic event or a
compound event (multiple fused SG), single sections gradually
obtained in the Z plane from a CRH-stimulated cell were analyzed
(Fig. 3C). A detailed view of the data allowed us to describe the
presence of large tubular endocytotic organelles that have a several-fold higher area than a single spot (Fig. 3C, inset). Remarkably, this type of tubular endocytotic structure was not often observed
after Ag stimulation. On the whole, the size of the endocytotic events
are larger in CRH-stimulated cells (4.38 6 1.57 mm2, n = 38) than
The Journal of Immunology
5
Table I. Effects of 10 nM CRH and 1 mg/ml DNP on secretory spike parameters
in Ag-stimulated cells (1.55 6 0.24 mm2, n = 42) (Fig. 3D).
Overall, the presence of these large endocytotic organelles suggests that CRH evokes a compound exocytosis that is tightly
coupled to the compound endocytosis of several SG by a single
fission event and results in a rapid and efficient compensation of
the membrane excess caused by intense exocytosis.
CRH and Ag stimulation trigger different Ca2+i signals
It has long been apparent that the elevation of Ca2+i is important in
the regulation of MC degranulation (42). Indeed, it has been
shown that activation by Ag stimulation induces an increase of
Ca2+i (19, 20). Activation of CRH-R can elevate Ca2+i in different
cells (43), including MC, and there is some evidence that Ca2+i
increases through CRH-R1 and hence leads to a degranulation
process (44). We measured and compared Ca2+i signals evoked by
a long application of both stimuli (10 nM CRH and 1 mg/ml Ag)
using the fura 2-AM probe (45) (Fig. 4). CRH addition caused
a rapid peak increase in Ca2+i followed by a decrease to a stable
plateau phase (Fig. 4A, black trace). Ag stimulation also evoked
an increase but at a slower rate and with a higher plateau phase
(Fig. 4A, gray trace). The amplitude of the Ca2+i signal (ΔF ratio)
was no different between the two stimuli (CRH, 0.128 6 0.009 a.u.,
n = 49; DNP, 0.127 6 0.005 a.u., n = 103) (Fig. 4B). In contrast, the
plateau phase was lower in CRH-stimulated cells (0.025 6 0.001 a.u.)
Table II. Parameters of the foot signal from spikes induced by 10 nM CRH and 1 mg/ml DNP
The analyzable prespikes foot parameters were as follows: foot amplitude, maximum oxidation current; foot frequency, percentage of spikes
that presented a foot preceding the spike; foot Q, foot area or foot net charge. Data are presented as means 6 SEM. All values obtained were
compared using a Mann–Whitney U test (p value).
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
Parameters of secretory spikes elicited by a 5-min application of 10 nM CRH or 1 mg/ml DNP (Ag) in MC. The following
parameters were analyzed from the detected spikes: fall time, total decay time; Risem, ascending slope of spike calculated from
25 to 75% from Imax; spike Q, area of spike or spike net charge; t1/2, spike width at half height. Data are presented as means 6
SEM. The pairs of data sets were compared using a Mann–Whitney U test (p value).
6
NEW EXO-ENDOCYTIC MECHANISM TRIGGERED BY CRH
induced by Ag stimulation but faster and with a lower plateau
phase.
Effects of 8-bromo-cAMP and forskolin on Ca2+i levels in MC
cAMP alone is sufficient to elicit exocytosis in PMC
FIGURE 2. FM1-43 reveals differences in the exocytotic mechanisms
triggered by CRH and Ag (DNP). (A) Raw fluorescent images of a MC
bathed in 4 mM FM1-43 (upper panel, no. 1) and during stimulation with
1 mg/ml DNP (nos. 2–4). Trace FM1-43 fluorescence of the cell shown in the
upper panel is detailed in the bottom panel. The black arrow in the trace
indicates the beginning of the Ag application. (B) Raw fluorescent images of
a MC bathed in 4 mM FM1-43 (upper panel, no. 1) and during stimulation
with 10 nM CRH (nos. 2–4). Trace FM1-43 fluorescence of the cell shown in
the upper panel is detailed in the bottom panel. The black arrow in the trace
indicates the beginning of the drug application. Note the different size of the
released granules for both stimuli [white arrows in upper panels in (A) and (B)].
Calibration for fluorescence is indicated with a solid bar, expressed in arbitrary
fluorescence units (a.u.). All traces are on the same fluorescence intensity
scale. Scale bar, 5 mm. (C) Analysis of the fluorescent spot area observed
after CRH (filled bar) and DNP application (open bar). *p , 0.05,
Mann–Whitney U test. (D) Significant differences were found for ΔF
induced by CRH (filled bar) and DNP (open bar). Data in (C) and (D) are
represented as the means 6 SEM. All data shown are representative of
six independent cultures (DNP, n = 48 cells; CRH, n = 26 cells). See also
Supplemental Videos 1, 2. *p , 0.05, Mann–Whitney U test.
than in Ag-stimulated cells (0.091 6 0.004 a.u.) (Fig. 4C). The time
to reach the maximum peak after stimulation (time to peak) was
shorter in CRH-stimulated cells (14 6 1 s) than in Ag-stimulated
cells (25 6 1 s) (Fig. 4D). Taken together, these results suggest
that the increase of Ca2+i induced by CRH is as strong as that
We next determinate whether cAMP elevation is sufficient to elicit
exocytosis in MC. We labeled PMC with FM1-43 and stimulated
them with 8-bromo-cAMP (Fig. 6). We compared the cAMPinduced exocytosis with the cellular response to CRH. We found
that ΔF under 8-bromo-cAMP stimulation is similar to that
obtained with CRH (Fig. 6) (CRH, 1098 6 144 a.u., n = 8; 8-bromocAMP, 1075 6 108 a.u., n = 21). To further confirm that compound fusion is the preferential mode of cAMP-induced exocytosis, we also examined the fluorescent area of single spots. Our
analysis revealed that the average spot area is not significantly
different between CRH-treated cells (CRH, 3.29 6 0.40 mm2) and
8-bromo-cAMP-treated cells (8-bromo-cAMP, 3.07 6 0.71 mm2;
p = 0.374). We next compared the effects of astressin on exocytosis by direct observation of FM1-43 fluorescence in CRHstimulated cells. We found that exocytosis is completely abolished when CRH-R are blocked, suggesting that this hormone
triggers exocytosis through CRH-R (Fig. 6) (CRH plus astressin,
53 6 22 a.u., n = 6).
FM1-43 imaging suggests that CRH evokes PMD in IMC
Because it has been shown that CRH leads to selective secretion of
vascular endothelial growth factor by PMD (24), we wanted to
determine whether the type of degranulation induced by CRH
(piecemeal or complete exocytosis) might simply depend on
phenotypical differences between connective tissue MC from the
peritoneal cavity and mucosal MC from the intestinal lamina
propria. We therefore cultured IMC and imaged exocytosis in the
presence of 4 mM FM1-43 (Fig. 7). The scheme in Fig. 7A represents different images acquired during a representative experiment with CRH-stimulated IMC (no. 1 basal state, nos. 2 and 3
exocytotic, and no. 4 endocytotic fluorescence). The time course
of FM1-43 fluorescence is represented in Fig. 7B. The increase in
fluorescence was gradual, and after 10 min of recording, ΔF
reached an average value of 674 6 116 a.u. This ΔF is lower than
that obtained in PMC (1073 6 131 a.u., p = 0.030). After washing
off the extracellular dye, the internalized fluorescence can be
observed as tiny spots whose area is 0.27 6 0.09 mm2 (Fig. 7C).
This spot area is smaller than that obtained in Ag-stimulated PMC
(Fig. 3D, 1.55 6 0.24 mm2, p = 0.0001 by Student t test) and IMC
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
CRH binding to CRH-R typically activates adenylate cyclase,
leading to increased intracellular concentrations of cAMP (46).
Hence, we investigated whether the Ca2+i increase is mediated by
CRH-induced cAMP. We tested whether the cell-permeable cAMP
analog 8-bromo-cAMP and the adenylate cyclase activator forskolin could increase Ca2+i in PMC (Fig. 5). PMC were stimulated
with 10 nM CRH (Fig. 5A), 1 mM 8-bromo-cAMP (Fig. 5B), and
10 mM forskolin (Fig. 5C). We found that both 8-bromo-cAMP
and forskolin mimicked Ca2+i signals induced by CRH. The addition of the drugs produced a remarkably similar peak increase in
Ca2+i as was obtained with CRH stimulation (8-bromo-cAMP,
98 6 5%, n = 33; forskolin, 89 6 14%, n = 12) (Fig. 5E). We
then stimulated PMC with CRH in the presence of astressin,
a nonspecific CRH-R antagonist. The effect of CRH on Ca2+i
increase was completely abolished with astressin at the concentration of 1 mM (Fig. 5D, 5E). These observations suggest that
CRH induces a rise in Ca2+i through CRH-R–dependent activation
of adenylate cyclase and increased cAMP. These results were also
confirmed in rat PMC.
The Journal of Immunology
7
stimulated with 100 mg/ml C48/80 (Fig. 7C, 1.19 6 0.23 mm2). A
plausible explanation for these observations, based on background
literature, could be that IMC undergo PMD, whereas PMC complete exocytosis in response to CRH.
Discussion
It has been suggested that CRH-induced activation of MC causes
differential release by PMD (24, 47). This mechanism has been
proposed as a general secretory mechanism for the slow release of
FIGURE 4. Ca2+i signals triggered by CRH
and Ag (DNP). (A) Average traces, normalized
at baseline, showing the effect of 10 nM CRH
(black circles trace) and 1 mg/ml DNP (gray
triangles trace). Note the shift in time to peak
and the presence of different plateau phase
levels. The arrow indicates the initiation of
stimulation. Significant differences were not
found in the ΔF ratio during the peak phase (B)
between CRH (filled bar) and DNP (open bar)
stimulation. However, both plateau phase ΔF (C)
and time to peak (D) showed significant differences in CRH-treated (filled bar) and DNPtreated (open bar) cells. Data in (B)–(D) are
represented as the means 6 SEM. All data
shown are representative of five independent
cultures (DNP, n = 103 cells; CRH, n = 49 cells).
***p , 0.001, Mann–Whitney U test.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
FIGURE 3. Confocal microscopy allows for
the identification of large FM1-43-labeled endocytotic organelles after CRH stimulation. (A)
Left images show brightfield single confocal
sections, whereas right images show Z-stack
projections of endocytotic granules labeled with
4 mM FM1-43. (B) Analysis of the total endocytotic area per cell showed significant differences between treatment with 10 nM CRH
(filled bar) and 1 mg/ml DNP (Ag) (open bar).
**p , 0.01, Mann–Whitney U test. (C) Sequence gradually obtained at 2-mm separation
from the CRH-stimulated cell shown in (A). The
lower images show an enlarged view of groups
of large endocytotic organelles marked with
dashed lines and enclosed by a box in images 1–
4. Scale bar, 5 and 1 mm. (D) Total FM1-43 area
per spot is larger in CRH-stimulated cells (filled
bar) compared with DNP-treated cells (open
bar). Data in (B) and (D) are represented as the
means 6 SEM. All data shown are representative of four independent cultures (DNP, n = 42
cells; CRH, n = 38 cells). *p , 0.05, Mann–
Whitney U test.
8
NEW EXO-ENDOCYTIC MECHANISM TRIGGERED BY CRH
bioactive materials of granulated secretory cells (14) and produces
ultrastructural changes characterized by empty or partially empty
SG chambers that do not fuse with the plasma membrane (14, 48,
49). The aim of the present research was to understand how CRH
stimulates MC and evokes transmitter release using electrochemical and fluorescent techniques. Surprisingly, CRH treatment
leads to a large and explosive 5-HT release and intense degranulation in PMC that does not agree with PMD. Our results indicate
that CRH induces secretion of the SG contents by extrusion via
exocytosis. The number of exocytotic events and the total amount
of 5-HT are larger than the Ag-stimulation response. The increase
in 5-HT is caused by a gain in the granular content. Table I shows
that Q increases by 50%. The effect of CRH on SG charge occurs
within 2 to 5 min. A comparison of the data from Fig. 1 indicates
that the increase in the total amount of 5-HT secretion comes
mainly from a net enlargement in Q, not from the frequency of
firing. The rapid changes observed in granule content are unlikely
to be caused by an increase in 5-HT synthesis. However, the
augmentation of the granular content could be a result of compound fusion (i.e., two or more SG that fuse before exocytosis)
(50). Previous analysis of amperometric spikes has indicated that
different modes of exocytosis could be reflected in several characteristics, such as the spike half-width or spike rise (39, 51, 52).
Moreover, a previous study suggested that amperometric feet
correspond to SG with higher content (35), similar to what we
observed in our study (Table II). In either case, this method cannot
conclusively address the changes observed in spike shape to
compound fusion. However, live-cell imaging offers the possibility of identifying different modes of exocytosis and endocytosis
(40, 53). Thus, during CRH stimulation in the presence of the
fluorescent dye FM1-43 we observed large steps reflecting multiple SG that exocytose through a single fusion event (41) (Fig. 2).
SG that undergo compound exocytosis could appear as giant
spherical shape events, explaining the large increase in fluorescence observed.
When FM1-43 is washed from the bath, some fluorescent signals
remain associated to the same location as the previous spots, indicating the retrieval of FM1-43–stained SG dense cores (Fig. 3).
The estimation of the size of the internalized membrane (area
of endocytotic organelles) is larger in CRH-treated cells than in
Ag-treated cells. Endocytosis occurs rapidly after CRH-induced
stimulation, as evidenced by the ability to retain FM dye into
SG cores just 5 min after exocytosis. An endocytotic mechanism
that can explain the rapid and extensive membrane retrieval in MC
is compound endocytosis (12). Such a mechanism is a rapid
process because fused SG are not obliged to flatten but are retrieved nearly intact. This mechanism also results in a massive
internalization of the membrane because it involves the recapture
of the compound cavity formed by the fused SG. Therefore,
compound exocytosis should be followed by compound endocytosis in PMC under CRH stimulation. As a result, large endocytotic cavities with reduced contents should appear when MC are
stimulated by CRH. It is curious that PMD can be imaged by
electron microscopy as a uniform decrement of granule electron
density. Additionally, during the process of PMD, SG sometimes
maintain their original size but more often become enlarged (48,
54). Because PMD identification is dependent on ultrastructural
criteria, the possibility cannot be ruled out that the rapid exo-
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
FIGURE 5. Calcium signals evoked by
8-bromo-cAMP and forskolin. (A–D) Representative traces, normalized at baseline, of the Ca2+i
signals evoked by 10 nM CRH (A), 1 mM
8-bromo-cAMP (8-Br-cAMP) (B), 10 mM forskolin
(Forsk) (C), and 1 mM astressin (Ast)-treated
cells stimulated with 10 nM CRH (D). (E) ΔF
ratio measured in cells treated with 8-Br-cAMP,
Forsk, and CRH plus Ast in relationship to CRH
response. Significant differences were only found
between CRH and CRH plus Ast. Data in (E) are
represented as the means 6 SEM. All data shown
are representative of two independent cultures
(CRH, n = 28 cells; 8-Br-cAMP, n = 33 cells;
Forsk, n = 12; CRH plus Ast, n = 39). ***p ,
0.001, Mann–Whitney U test.
The Journal of Immunology
9
FIGURE 6. cAMP increases FM1-43 fluorescence in MC. (A) Raw
fluorescent images of a MC bathed in 4 mM FM1-43 and stimulated with
10 nM CRH (upper panel), 1 mM 8-bromo-cAMP (8-Br-cAMP, middle
panel), or 10 nM CRH plus astressin (1 mM) (CRH + Ast) (lower panel).
Left panels (no. 1) show an initial stage of recording, and right panels (no.
2) show a late stage of recording. Scale bar, 5 mm. (B) Average membrane
fluorescence shows that there is no significant difference between the CRH
and 8-bromo-cAMP response. Astressin completely inhibited the degranulation induced by CRH. PMC were incubated for 10 min with 1 mM
astressin and then stimulated with 10 nM CRH. Data in (B) are represented
as the means 6 SEM. All data shown are representative of two independent cultures (CRH, n = 8 cells; 8-Br-cAMP, n = 21 cells; CRH + Ast,
n = 6 cells). ***p , 0.001, Mann–Whitney U test.
endocytosis mode we have identified after CRH exposure is responsible for the previously reported PMD phenotype. In contrast,
we observed that CRH induces a gradual increase in fluorescence
in IMC (Fig. 7) by the appearance of many tiny spots, suggesting
the fusion of small vesicles. Two scenarios could, in principle, be
envisioned for these data. First, CRH leads to differential release
by PMD, as previously described. Second, CRH induces complete
exocytosis, as we observed in PMC. Given that the size of the
fluorescent events are more discrete than those obtained during
complete fusion, a likely explanation is that IMC perform PMD in
response to CRH, which would support the scenario of PMD.
Hence, the measurement of membrane fluorescence using FM
dyes can be used as an in vivo technique that allows for the study
of PMD. Indeed, our experimental data show that after this exocytotic mechanism is induced, a fast and efficient membrane retrieval event occurs.
We examined the Ca2+i signals triggered by CRH in PMC
(Fig. 4). Within seconds, CRH evokes a rapid increase in Ca2+i,
followed by a decrease to a long-lasting plateau. Compared to
CRH, the addition of DNP induced a rapid peak increase in Ca2+i
at ∼25 s with a similar maximum value of Ca2+i but an elevated
plateau phase. Urocortin, a peptide that is structurally related to
CRH, has previously been reported to induce a similar Ca2+i
signaling pattern in rat lung MC (44). The rapid peak increase in
Ca2+i concentration observed after CRH administration does not
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
FIGURE 7. The exocytotic response induced by CRH in IMC measured
by FM1-43 fluorescence. (A) Raw fluorescent images of an IMC bathed in
4 mM FM1-43 (upper panels, no. 1), during stimulation with 10 nM CRH
(nos. 2 and 3), and after washing (no. 4). Scale bar, 5 mm. (B) Trace of
FM1-43 fluorescence of the cell shown in the upper panel images. Black
arrow in trace indicates the beginning of drug application. (C) Analysis of
the fluorescent spot area observed after CRH (filled bar) and 100 mg/ml
C48/80 applications (open bar). Significant differences were found for ΔF
(C) evoked by CRH (filled bar) and C48/80 (open bar). Data in (C) are
represented as the means 6 SEM. All data shown are representative of
three independent cultures (CRH, n = 15 cells; C48/80, n = 14 cells).
**p , 0.005, Student t tests.
10
Acknowledgments
We thank Dolores Gutierrez for technical assistance.
Disclosures
The authors have no financial conflicts of interest.
References
1. Metcalfe, D. D., D. Baram, and Y. A. Mekori. 1997. Mast cells. Physiol. Rev. 77:
1033–1079.
2. Galli, S. J., S. Nakae, and M. Tsai. 2005. Mast cells in the development of
adaptive immune responses. Nat. Immunol. 6: 135–142.
3. Beghdadi, W., L. C. Madjene, M. Benhamou, N. Charles, G. Gautier, P. Launay,
and U. Blank. 2011. Mast cells as cellular sensors in inflammation and immunity.
Front. Immunol. 2: 37.
4. Theoharides, T. C., and D. Kalogeromitros. 2006. The critical role of mast cells
in allergy and inflammation. Ann. N. Y. Acad. Sci. 1088: 78–99.
5. Metz, M., M. A. Grimbaldeston, S. Nakae, A. M. Piliponsky, M. Tsai, and
S. J. Galli. 2007. Mast cells in the promotion and limitation of chronic inflammation. Immunol. Rev. 217: 304–328.
6. Larauche, M., C. Kiank, and Y. Tache. 2009. Corticotropin releasing factor
signaling in colon and ileum: regulation by stress and pathophysiological
implications. J. Physiol. Pharmacol. 60(Suppl 7): 33–46.
7. Stevens, R. L., and R. Adachi. 2007. Protease-proteoglycan complexes of mouse
and human mast cells and importance of their b-tryptase-heparin complexes in
inflammation and innate immunity. Immunol. Rev. 217: 155–167.
8. Wernersson, S., and G. Pejler. 2014. Mast cell secretory granules: armed for
battle. Nat. Rev. Immunol. 14: 478–494.
9. Blank, U. 2011. The mechanisms of exocytosis in mast cells. Adv. Exp. Med.
Biol. 716: 107–122.
10. Wu, L.-G., E. Hamid, W. Shin, and H.-C. Chiang. 2014. Exocytosis and endocytosis: modes, functions, and coupling mechanisms. Annu. Rev. Physiol. 76:
301–331.
11. Alvarez de Toledo, G., and J. M. Fernandez. 1990. Compound versus multigranular exocytosis in peritoneal mast cells. J. Gen. Physiol. 95: 397–409.
12. Cabeza, J. M., J. Acosta, and E. Alés. 2013. Mechanisms of granule membrane
recapture following exocytosis in intact mast cells. J. Biol. Chem. 288: 20293–
20305.
13. Dvorak, A. M., R. S. McLeod, A. Onderdonk, R. A. Monahan-Earley,
J. B. Cullen, D. A. Antonioli, E. Morgan, J. E. Blair, P. Estrella, R. L. Cisneros,
et al. 1992. Ultrastructural evidence for piecemeal and anaphylactic degranulation of human gut mucosal mast cells in vivo. Int. Arch. Allergy Immunol. 99:
74–83.
14. Crivellato, E., B. Nico, F. Mallardi, C. A. Beltrami, and D. Ribatti. 2003.
Piecemeal degranulation as a general secretory mechanism? Anat. Rec. A Discov.
Mol. Cell. Evol. Biol. 274: 778–784.
15. Dvorak, A. M. 2005. Ultrastructural studies of human basophils and mast cells.
J. Histochem. Cytochem. 53: 1043–1070.
16. Blank, U., I. K. Madera-Salcedo, L. Danelli, J. Claver, N. Tiwari, E. SánchezMiranda, G. Vázquez-Victorio, K. A. Ramı́rez-Valadez, M. Macias-Silva, and
C. González-Espinosa. 2014. Vesicular trafficking and signaling for cytokine and
chemokine secretion in mast cells. Front. Immunol. 5: 453.
17. Dvorak, A. M., R. I. Tepper, P. F. Weller, E. S. Morgan, P. Estrella,
R. A. Monahan-Earley, and S. J. Galli. 1994. Piecemeal degranulation of mast
cells in the inflammatory eyelid lesions of interleukin-4 transgenic mice. Evidence of mast cell histamine release in vivo by diamine oxidase-gold enzymeaffinity ultrastructural cytochemistry. Blood 83: 3600–3612.
18. Kandere-Grzybowska, K., R. Letourneau, D. Kempuraj, J. Donelan,
S. Poplawski, W. Boucher, A. Athanassiou, and T. C. Theoharides. 2003. IL-1
induces vesicular secretion of IL-6 without degranulation from human mast
cells. J. Immunol. 171: 4830–4836.
19. Blank, U., and J. Rivera. 2004. The ins and outs of IgE-dependent mast-cell
exocytosis. Trends Immunol. 25: 266–273.
20. Kraft, S., and J.-P. Kinet. 2007. New developments in FcεRI regulation, function
and inhibition. Nat. Rev. Immunol. 7: 365–378.
21. Baigent, S. M. 2001. Peripheral corticotropin-releasing hormone and urocortin in
the control of the immune response. Peptides 22: 809–820.
22. Theoharides, T. C., L. K. Singh, W. Boucher, X. Pang, R. Letourneau,
E. Webster, and G. Chrousos. 1998. Corticotropin-releasing hormone induces
skin mast cell degranulation and increased vascular permeability, a possible
explanation for its proinflammatory effects. Endocrinology 139: 403–413.
23. Kempuraj, D., N. G. Papadopoulou, M. Lytinas, M. Huang, K. KandereGrzybowska, B. Madhappan, W. Boucher, S. Christodoulou, A. Athanassiou, and
T. C. Theoharides. 2004. Corticotropin-releasing hormone and its structurally
related urocortin are synthesized and secreted by human mast cells. Endocrinology 145: 43–48.
24. Cao, J., N. Papadopoulou, D. Kempuraj, W. S. Boucher, K. Sugimoto,
C. L. Cetrulo, and T. C. Theoharides. 2005. Human mast cells express
corticotropin-releasing hormone (CRH) receptors and CRH leads to selective
secretion of vascular endothelial growth factor. J. Immunol. 174: 7665–7675.
25. Singh, L. K., W. Boucher, X. Pang, R. Letourneau, D. Seretakis, M. Green, and
T. C. Theoharides. 1999. Potent mast cell degranulation and vascular permeability triggered by urocortin through activation of corticotropin-releasing hormone receptors. J. Pharmacol. Exp. Ther. 288: 1349–1356.
26. Dvorak, H. F., and A. M. Dvorak. 1975. Basophilic leucocytes: structure,
function and role in disease. Clin. Haematol. 4: 651–683.
27. Theoharides, T. C., D. Kempuraj, M. Tagen, P. Conti, and D. Kalogeromitros.
2007. Differential release of mast cell mediators and the pathogenesis of inflammation. Immunol. Rev. 217: 65–78.
28. Kovarova, M. 2013. Isolation and characterization of mast cells in mouse models
of allergic diseases. Methods Mol. Biol. 1032: 109–119.
29. Liu, F. T., J. W. Bohn, E. L. Ferry, H. Yamamoto, C. A. Molinaro,
L. A. Sherman, N. R. Klinman, and D. H. Katz. 1980. Monoclonal dinitrophenylspecific murine IgE antibody: preparation, isolation, and characterization. J.
Immunol. 124: 2728–2737.
30. Kawagoe, K. T., J. B. Zimmerman, and R. M. Wightman. 1993. Principles of
voltammetry and microelectrode surface states. J. Neurosci. Methods 48: 225–240.
31. Alvarez de Toledo, G., R. Fernández-Chacón, and J. M. Fernández. 1993. Release of secretory products during transient vesicle fusion. Nature 363: 554–558.
32. Tatham, P. E. R., M. R. Duchen, and J. Millar. 1991. Monitoring exocytosis from
single mast cells by fast voltammetry. Pflugers Arch. 419: 409–414.
33. Mosharov, E. V., and D. Sulzer. 2005. Analysis of exocytotic events recorded by
amperometry. Nat. Methods 2: 651–658.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
correlate with MC degranulation. However, this early Ca2+i signal
may account for the fusion between unit SG to generate larger SG
with high amounts of material able to fuse with the plasma
membrane and release their contents to the external medium. This
mechanism would provide reliable communication machinery for
adaptation to environmental insults. Increases in Ca2+i and its
sensor for vesicle fusion, synaptotagmin, are required for compound fusion (55) and other proteins such as Rab5 and VAMP8,
are known to play a role in homotypic secretory granule fusion in
MC (56).
In MC from peripheral tissues, the physiological actions of CRH
involve coupling of both CRH-R1 and CRH-R2 to Gas proteins
that stimulate cAMP-mediated signaling cascades (46). In chromaffin cells (50) and lactotrophs (57), cAMP modulates exocytotic kinetics and increases the size of exocytotic events and
quantal size. We found that CRH-induced compound exocytosis
is dependent on cAMP because 8-bromo-cAMP and forskolin
mimicked Ca2+i signals triggered by CRH (Fig. 5). Thus, elevated
CRH-induced cAMP stimulates Ca2+i increase and calcium entry,
which has also been described as causing the aggregation of
granules (58). Moreover, imaging of PMC bathed in FM dye and
stimulated with 8-bromo-cAMP reproduced the exocytotic response observed after CRH exposition (Fig. 6). The simplest explanation for these data is that this type of exocytosis involves
a cAMP-dependent Ca2+i increase. Ca2+i signaling is also required
in endocytosis (10). Therefore, it is also possible that the Ca2+i
plateau phase is the driving force for the compensatory endocytosis that we have described. Continuous exocytosis followed by
membrane retrieval would permit MC to efficiently maintain secretory activity for long periods of time and keep the cell membrane area constant.
The results of our study demonstrate that CRH is one of the most
potent triggers of PMC. We found that CRH stimulation induces
giant amperometric spikes and large fluorescence spots, reflecting
exocytosis of large SG. This process is followed by retrieval of
giant membrane organelles, reflecting compound endocytosis that
is an efficient mechanism for membrane retrieval and therefore
results in a relatively short SG recycling time. Overall, these results
suggest a new route of CRH-induced exocytosis and endocytosis,
shedding new light on the biology of MC and their proinflammatory
effects on numerous pathologies under stress conditions in
which this multifaceted immune cell is a major player (3, 21, 59).
MC contribute to immune regulation, innate immunity, parasite
rejection, wound repair, and other biological processes. Their
actions may be critical in any given stressful condition and
therefore the different responses to CRH observed in PMC and
IMC may imply specific immunomodulatory roles for each MC
subtype.
NEW EXO-ENDOCYTIC MECHANISM TRIGGERED BY CRH
The Journal of Immunology
48. Dvorak, A. M., and S. Kissell. 1991. Granule changes of human skin mast cells
characteristic of piecemeal degranulation and associated with recovery during
wound healing in situ. J. Leukoc. Biol. 49: 197–210.
49. Dvorak, A. M. 1998. Cell biology of the basophil. Int. Rev. Cytol. 180: 87–236.
50. Machado, J. D., A. Morales, J. F. Gomez, and R. Borges. 2001. cAmp modulates
exocytotic kinetics and increases quantal size in chromaffin cells. Mol. Pharmacol. 60: 514–520.
51. Graham, M. E., D. W. O’Callaghan, H. T. McMahon, and R. D. Burgoyne. 2002.
Dynamin-dependent and dynamin-independent processes contribute to the regulation of single vesicle release kinetics and quantal size. Proc. Natl. Acad. Sci.
USA 99: 7124–7129.
52. Bretou, M., C. Anne, and F. Darchen. 2008. A fast mode of membrane fusion
dependent on tight SNARE zippering. J. Neurosci. 28: 8470–8476.
53. Angleson, J. K., A. J. Cochilla, G. Kilic, I. Nussinovitch, and W. J. Betz. 1999.
Regulation of dense core release from neuroendocrine cells revealed by imaging
single exocytic events. Nat. Neurosci. 2: 440–446.
54. Dvorak, A. M. 1997. New aspects of mast cell biology. Int. Arch. Allergy
Immunol. 114: 1–9.
55. He, L., L. Xue, J. Xu, B. D. McNeil, L. Bai, E. Melicoff, R. Adachi, and
L.-G. Wu. 2009. Compound vesicle fusion increases quantal size and potentiates synaptic transmission. Nature 459: 93–97.
56. Azouz, N. P., N. Zur, A. Efergan, N. Ohbayashi, M. Fukuda, D. Amihai,
I. Hammel, M. E. Rothenberg, and R. Sagi-Eisenberg. 2014. Rab5 is a novel
regulator of mast cell secretory granules: impact on size, cargo, and exocytosis.
J. Immunol. 192: 4043–4053.
57. Cochilla, A. J., J. K. Angleson, and W. J. Betz. 2000. Differential regulation of
granule-to-granule and granule-to-plasma membrane fusion during secretion
from rat pituitary lactotrophs. J. Cell Biol. 150: 839–848.
58. Caohuy, H., M. Srivastava, and H. B. Pollard. 1996. Membrane fusion protein
synexin (annexin VII) as a Ca2+/GTP sensor in exocytotic secretion. Proc. Natl.
Acad. Sci. USA 93: 10797–10802.
59. Galli, S. J., and M. Tsai. 2010. Mast cells in allergy and infection: versatile
effector and regulatory cells in innate and adaptive immunity. Eur. J. Immunol.
40: 1843–1851.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
34. Chow, R. H., L. von R€
uden, and E. Neher. 1992. Delay in vesicle fusion revealed
by electrochemical monitoring of single secretory events in adrenal chromaffin
cells. Nature 356: 60–63.
35. Amatore, C., S. Arbault, I. Bonifas, Y. Bouret, M. Erard, A. G. Ewing, and
L. A. Sombers. 2005. Correlation between vesicle quantal size and fusion pore
release in chromaffin cell exocytosis. Biophys. J. 88: 4411–4420.
36. Albillos, A., G. Dernick, H. Horstmann, W. Almers, G. Alvarez de Toledo, and
M. Lindau. 1997. The exocytotic event in chromaffin cells revealed by patch
amperometry. Nature 389: 509–512.
37. Lindau, M., and G. Alvarez de Toledo. 2003. The fusion pore. Biochim. Biophys.
Acta 1641: 167–173.
38. Wightman, R. M., J. A. Jankowski, R. T. Kennedy, K. T. Kawagoe, T. J. Schroeder,
D. J. Leszczyszyn, J. A. Near, E. J. Diliberto, Jr., and O. H. Viveros. 1991. Temporally
resolved catecholamine spikes correspond to single vesicle release from individual
chromaffin cells. Proc. Natl. Acad. Sci. USA 88: 10754–10758.
39. van Kempen, G. T. H., H. T. vanderLeest, R. J. van den Berg, P. Eilers, and
R. H. S. Westerink. 2011. Three distinct modes of exocytosis revealed by amperometry
in neuroendocrine cells. Biophys. J. 100: 968–977.
40. Cochilla, A. J., J. K. Angleson, and W. J. Betz. 1999. Monitoring secretory
membrane with FM1-43 fluorescence. Annu. Rev. Neurosci. 22: 1–10.
41. Johnson, J. M., and W. J. Betz. 2008. The color of lactotroph secretory granules
stained with FM1-43 depends on dye concentration. Biophys. J. 94: 3167–3177.
42. Penner, R., and E. Neher. 1988. The role of calcium in stimulus-secretion coupling in excitable and non-excitable cells. J. Exp. Biol. 139: 329–345.
43. Tse, A., A. K. Lee, and F. W. Tse. 2012. Ca2+ signaling and exocytosis in pituitary corticotropes. Cell Calcium 51: 253–259.
44. Wu, Y., J. Hu, R. Zhang, C. Zhou, Y. Xu, X. Guan, and S. Li. 2008. Enhanced
intracellular calcium induced by urocortin is involved in degranulation of rat
lung mast cells. Cell. Physiol. Biochem. 21: 173–182.
45. Grynkiewicz, G., M. Poenie, and R. Y. Tsien. 1985. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J. Biol. Chem. 260: 3440–3450.
46. Grammatopoulos, D. K. 2012. Insights into mechanisms of corticotropinreleasing hormone receptor signal transduction. Br. J. Pharmacol. 166: 85–97.
47. Santos, J., P. C. Yang, J. D. Söderholm, M. Benjamin, and M. H. Perdue. 2001.
Role of mast cells in chronic stress induced colonic epithelial barrier dysfunction
in the rat. Gut 48: 630–636.
11