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RAPID COMMUNICATION
Vesicular Transport of Histamine in Stimulated Human Basophils
By Ann M. Dvorak, Donald W. MacGlashan, Jr, Ellen S. Morgan, and Lawrence M. Lichtenstein
Human basophils participating in experimentally produced
contact allergy display progressive secretion of electrondense secretory granule contents and retention of cytoplasmic granule containers in the absence of entire granule
extrusion, a process termed piecemeal degranulation (PMD)
and postulated t o be effected by vesicular transport (Dvorak
HF, Dvorak AM: Clin Hematol4651, 1975). Proof of this hypothesis was sought using models of human basophil-stimulated secretion, partially purified human peripheral blood
basophils, and a morphometric analysis of the fraction of
total cellular cytoplasmic vesicles loaded with histamine, a
major proinflammatory mediator present in basophil secretory granules. The subcellular localization of histamine was
accomplished using a new ultrastructural enzyme-affinitygold method based on the affinity of diamine oxidase for its
substrate, histamine (Dvorak et al: J Histochem Cytochem
41:787, 1993).Two models were selected for a kinetic analysis of stimulated vesicle transport of histamine based on
known biochemical and ultrastructural characteristics (MacGlashan et al: J lmmunol 136:2231, 1986; Warner et al: ./
LeukocBio/45:558,1989;Dvorak et al: Am Jfathol141:1309,
1992; Dvorak et al: Lab lnvest W234, 1991). These models
were selected t o include the rapid release reaction stimulated by the bacterial peptide, FMLP, and the slow release
reaction stimulated by the phorbol diester tumor promoter,
TPA. The results of this study showed that the fraction of
histamine-loaded cytoplasmic vesicles (%VG/TV/pm*) in
TPA-stimulated basophils significantly exceeded the fraction
in unstimulated cells, a process that persisted for 45 minutes
after TPA stimulation and was associated with extensive
PMD and no morphologic evidence of recovery. Similarly,
the fraction of histamine-loaded cytoplasmic vesicles after
FMLP stimulation significantly exceeded the fraction in unstimulated cells, a process that persisted for 10 minutes after
FMLP stimulation and was associated with the morphologic
continuum of PMD + anaphylactic degranulation (characterized by extrusion of granules) recovery, a process largely
complete in the IO-minute samples. These studies establish
for the first time that an important proinflammatory mediator, histamine, traffics from secretory granules t o the extracellular milieu in small cytoplasmic vesicles in stimulated
human basophils. The association of this process with the
ultrastructural release reaction defined as PMD produced
primarily by TPA and in part by FMLP establishes vesicular
transport as the mechanism for effecting this type of regulated secretion. Vesicular transport of histamine was also
significant in the more complex stimulated secretory and
recovery model produced by exposure of human basophils
t o the bacterial peptide FMLP.
0 1996 by The American Society of Hematology.
H
entire granules by extrusion, a process well known in various
secretory cells and one that places extruded granule membranes in continuity with plasma membranes.’ (In basophils,
we generically refer to this type of secretion as anaphylactic
degranulation [AND].6) Since our description of PMD of
human basophils in human skin contact allergy,’.5 we have
noted that this form of secretion is the one prevalent in tissue
basophils in humans and animals in various diseases and
experimental models. I.’.‘
Secretion biologists generally divide secretion into two
types: (1) regulated and (2) constit~tive.’.’.~
Regulated secretion occurs in granulated secretory cells when appropriate
secretogogues stimulate granules to fuse with plasma membranes and to extrude their contents. A variation of this
general scheme includes the formation of intracellular degranulation chambers by granule membrane fusions and solubilization of granules before their secretion. Constitutive
secretion occurs continually in many cells, does not involve
large secretory granules, and presumably is a Golgi to plasma
membrane transport event. We have generally regarded
PMD as a form of regulated secretion effected by vesicular
transport directly from secretory storage granules to the
plasma membrane. Recently, we and others have documented such vesicular transport from immature and mature
eosinophilg~“’
and acinar cell granulesl’-13to the plasma membrane.
A better understanding of vesicular transport processes in
basophils requires knowledge of appropriate secretogogues
which stimulate them as well as of vesicular contents within
them. Our early studies emphasized the endocytosis and vesicular transport of serum proteins (examples include exogenous horseradish peroxidase, eosinophil peroxidase) into basophil granules, documenting for the first time that typical
UMAN BASOPHILS primarily remain in the circulatory system but have the capacity to home to tissues
by migrating from the blood through vessels in a variety
of diseases.’,’ Our initial studies of human basophils were
designed to document this capacity in experimentally produced skin contact allergy lesions.’-’ These studies showed
that tissue basophils progressively secreted granule contents
over several days, a process identified in light and electron
microscopic samples as viable cells which retained granule
containers devoid of metachromatically stained or electrondense contents. Large numbers of small, smooth membranebound, cytoplasmic vesicles accompanied this process.
These vesicles often were concentrated in perigranular and
peripheral cytoplasmic areas; some of these were fused to
granule and plasma membranes. We termed this form of
secretion by human basophils ‘‘piecemeal degranulation”
(PMD) to distinguish it from typical regulated secretion of
From the Departments of Pathology, Beth Israel Hospital and
Harvard Medical School, Boston, MA: and the Johns Hopkins
Asthma and Allergy Center, Allergy and Clinical Immunology Division, Baltimore, MD.
Submitted June 18, 1996: accepted September 4, 1996.
Supported by US Public Health Service Grant No. AI-33372.
Address reprint requests to Ann M. Dvorak, MD, Department o j
Pathology, Beth Israel Hospital, 330 Brookline Ave, Boston, MA
02215.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1996 by The American Society of Hematology.
0006-4971/96/8811-0036$3.00/0
4090
+
Blood, Vol 88, No 11 (December I ) , 1996: pp 4090-4101
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VESICULAR TRANSPORT OF HISTAMINE
secretory granules could suhserve a storage and rerelease
function for circulating proteins in humans and
Reverse vesicular traffic of horseradish peroxidase was accomplished in washout experiments in the absence of granule
release and resulting in peroxidase-negative intracytoplasmic
granules, whereas stimulation of AND after granule loading
with horseradish peroxidase resulted in extrusion of peroxidase-containing secretory granules.’
Considerable progress has been accomplished regarding
the identification of potential secretogogues for PMD by
combining ultrastructural and biochemical kinetic studies of
human basophil release reaction^."^^^ These types of analyses have shown that some stimuli are associated primarily
with PMD19 but that others stimulate a continuum of PMD
-+ AND with kinetics unique for individual stimuli.’* We
have used two of these
with different anatomic
and biochemical kinetics to do extensive studies of the transport of two materials in secretory human basophils. Two
newly developed techniques to label these substances in
small cytoplasmic vesicular structures were applied to the
secretory models. These included the immunogold detection
of Charcot-Leyden crystal (CLC) protein-a specific eosinophil and basophil
(Dvorak et al, unpublished
data, June 1996) and the enzyme-affinity gold detection of
hi~tamine,”.’~
long regarded as a constituent of basophil (and
mast cell) granules implicated in allergic inflammation.’.2629
In the work reported here, we quantitated the proportion of
total cytoplasmic vesicles that were labeled with diamine
oxidase (DA0)-gold, indicating hi~tamine,’~
in human basophils stimulated with either tetradecanoyl phorbol acetate
(TPA) or formyl methionyl leucyl phenylalanine (FMLP),
and we made comparisons of these values to those of unstimulated cells, to different time points for each trigger, and
between similar and disparate times for each secretogogue.
We found that secretory human basophils move histamine
in cytoplasmic vesicles and that the vesicular transport is
similar to that suggested by morphologies and models previously r e ~ o r d e d . ~ ~ l * ~ ’ ~
MATERIALS AND METHODS
Basophil pur$cation. Buffy coat cells, obtained from normal
donors undergoing hemapheresis, were partially purified by countercurrent centrifugal elutriation and then placed on Percoll density
gradients (1.075 and 1.066 gfmL). Basophils were recovered from
the interface between the two Percoll layers.3oTwo preparations,
each containing 32% basophils, were used for these studies.
Histamine release. Cells were suspended in PAG CM (PIPES
[piperazine N,N’-bis-2-ethane sulfonic acid]-albumin-glucose supplemented with 1 mmom CaClz and 1 m m o a MgCI2) and stimulated with either 100 nglmL of the phorbol diester tumor promoter,
12-0-tetradecanoyl-phorbol13-acetate (TPA)3’.32or with 1 pmol/L
of the bacterial peptide, FMLP,” at 37°C for 30 or 60 minutes. The
supernatant was removed and assayed for histamine release with an
automated fluorometric technique.33Buffer-incubated controls were
also assayed, and spontaneous histamine release was subtracted to
give final stimulated histamine release values. Histamine release was
79.1% and 66.2% at 60 minutes in each of two experiments after
TPA stimulation and 33% at 30 minutes and 56% at 60 minutes
after FMLP stimulation. The two secretogogues used were chosen
based on detailed published documentation of the biochemistry and
ultrastructural anatomy of their basophil release reactions that indicated a possible role for vesicular t r a f f i ~ , ” - ~ whereas
~ . ~ ’ . ~ ~similar
4091
initial studies of secretion induced by an IgE-mediated stimulus did
not.”
Electron microscopy. Aliquoted samples of cells stimulated with
100 ng/mL TPA (Sigma Chemical Co, St Louis, MO) or with 1
p m o m FMLP were recovered for electron microscopy at the following times poststimulus: for TPA-0 time, 1, 2, 5, 10, 15, 30, 45
minutes; for FMLP-0 time, 10, 20, 30 seconds, 1, 2, 5 and 10
minutes. Unstimulated buffer-incubated basophils (20 seconds, 1 and
10 minutes) were also prepared for electron microscopy as before.34
Briefly, cell suspensions were fixed by diluting them in a 10-fold
excess of 2% paraformaldehyde, 2.5% glutaraldehyde, and 0.025%
CaCI2 in 0.1 m o m sodium cacodylate buffer, pH 7.4. Cells were
fixed for 1 hour at room temperature, washed, and resuspended
in 0.1 m o m sodium cacodylate buffer, pH 7.4, 4°C. Some cell
preparations were treated with cationized ferritin after fixation to
stain cell surfaces, as previously done.34 Cell samples were suspended in warm 2% agar, rapidly centrifuged to form agar pellets
containing cells, and postfixed for 2 hours at 4°C in 2% aqueous
osmium tetroxide and 1.5% potassium ferrocyanide in 0.1 m o m
sodium phosphate buffer, pH 6.0. Cell pellets were then dehydrated
in a graded series of alcohols and embedded in a propylene oxideEpon (Ted Pella, Inc, Redding, CA) sequence. Enzyme-affinity goldstained thin sections (see next section) were stained with lead citrate
and examined in a Philips 400 electron microscope (Philips Export
B.V., Eindhoven, The Netherlands).
Enzyme-afJinniiy procedure to demonstrare histamine. Thin sections of Epon-embedded material were prepared to demonstrate histamine with the ultrastructural enzyme-affinity method, based on
diamine oxidase-gold (DAO-G) binding.24Briefly, preparation of the
DAO-G reagent was as follows: A colloidal suspension of gold was
prepared according to the method of F r e n ~ Four
. ~ ~ milliliters of an
aqueous 1% solution of sodium citrate was added to a boiling aqueous solution of 100 mL 0.01% tetrachloroauric acid and allowed to
boil for 5 minutes before cooling on ice. The pH of the colloidal gold
suspension was adjusted to 7 with 0.2 m o m potassium carbonate.
Preparation of the DAO-G complex was according to the method
of B e n d a ~ a nThree
. ~ ~ milligrams of DAO was dissolved in 0.3 mL
distilled water and placed in a polycarbonate ultrafuge tube with 10
mL of the gold suspension. The mixture was centrifuged at 25,000
rpm for 30 minutes, 4”C, in a Beckman ultracentrifuge (Beckman
Instruments, Inc, Palo Alto, CA). The DAO-G complex formed a
red sediment that was carefully recovered and resuspended in 3
mL 0.1 molL phosphate-buffered saline (PBS) containing 0.02%
polyethylene glycol, pH 7.6 (final concentration, 1 mg DAO/mL).
For cytochemical labeling, section-containing grids were inverted
and floated on PBS drops for 5 minutes, followed by incubation on
a drop of DAO-G at 37°C for 60 minutes. The grids were vigorously
washed in distilled water and stained with dilute lead citrate for 10
minutes before viewing in a Philips 300 or 400 electron microscope.
Specificity controls for the enzyme-affinity gold method included
prior digestion of samples with diamine oxidase or absorption of
the diamine oxidase-gold reagent by solid-phase histamine.
Ultrastructural data collection and analysis. One-micron sections of Epon blocks containing cell suspensions from each time
point for each secretogogue, as well as for buffer-incubated controls,
were prepared and viewed by light microscopy. Random thin sections of each block were made for the enzyme-affinity gold preparations, which were then viewed and sampled randomly by electron
microscopy. Counts were done on 10 micrographs of basophils (and
adjacent Epon background) imaged in 8 x 10 prints at each interval
after TPA or FMLP stimulation; this was also done with unstimulated basophils.
A total of 170 basophils comprised the morphometric sample. The
total number of cytoplasmic, smooth membrane-bound vesicles and
gold-labeled vesicles for each basophil was determined and expressed as the average number/wmZand as percent of total vesicles
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DVORAK ET AL
4092
secretory granules were shown in unstimulated cells with an
enzyme-affinity ultrastructural cytochemical t e c h n i q ~ e * ~ ~ ~ ’ ~ ~ ’
(Fig 1A). Granules that contained homogeneously dense
CLCs’* within their particle matrix showed gold-labeled histamine only in the particulate matrix, not in the CLC (Fig
1B). Specificity controls for the diamine-oxidase affinitygold technique abrogated label for histamine when the DAOG reagent was absorbed with histamine-bound agarose beads
before staining sections (Fig IC) or when sections were
digested with diamine oxidase to remove granule histamine
before staining with DAO-G (Fig 1D).
Vesicular transport of histamine in TPA-stimulated human
basophils. The ultrastructural changes in histamine-releasing human basophils that were induced by exposure to TPA
and sampled at 0, 1,2,5, 10, 15,30, and 45 minutes thereafter were similar to those previously reported.” Briefly, the
predominant secretory response of human basophils to TPA
was PMD, which extended over the 45-minute kinetic interval tested. No evidence of recovery from secretion’‘..’* was
evident during this time interval. Between 1 and 15 minutes,
ultrastructural changes (termed a forme fruste of AND”)
appeared in a small number of cells. Characteristically, granule-plasma membrane fusions and release of granule contents without exteriorization of the granule containers occurred. By 30 minutes after exposure to TPA, a small number
of cells showed classic AND with extrusion of granules and
exteriorization of granule containers.
The mechanism for effecting PMD in human basophils
has been postulated to be by vesicular transport of stored
granule contents.’ We used a morphometric analysis of
DAO-G-labeled, TPA-stimulated cells to quantitate this process here. Table 1 shows the average cell area, total number
and average number of cytoplasmic vesicles, total number
and average number of gold-labeled (histamine-carrying)
vesicles, and the number of total vesicles/pn2 and of goldlabeled vesicles/pm* for unstimulated, buffer-incubated cells
(20 seconds, 1 and IO minutes) as well as for TPA-stimulated
cells collected at 0 time and I , 2, 5, IO, 15, 30, and 45
minutes thereafter. During this kinetic sequence of changes
stimulated in human basophils by TPA, the mean cell area
did not differ significantly at individual times after stimulation compared with unstimulated cells (P = not significant
[NS]), although a statistically insignificant trend toward
Fig 1. Unstimulated human basophil granules labeled for histamine with DAO-gold. In (A) and (B), the gold label overlies the electron-denseparticulate granule matrix and spares the homogeneously
dense, intragranularCharcot-Leyden crystal (arrowhead in B). Larger
electron-dense glycogen particles (arrows in B) reside in the perigranular cytoplasm. Unstimulated basophil granules are not labeled in
specificity controls IC and D). In (C), DAO-G was absorbed with histamine-agarose before staining; in (DI, the section was digested with
DAO before staining with DAO-G. Electron-dense glycogen particles
remain in the cytoplasm (arrows). Original magnifications(OMS):(A),
x 57,000; (B), x 55,000; IC and D), x 50,500.
that were gold-labeled(% V G N ) . Statistical comparisons were made
using the Kruskall-Wallis nonparametric A N O V A and
tests.
x2
RESULTS
Subcellular localization of histamine in human basophils.
Histamine-labeled, dense particle-packed human basophil
Table 1. Total Cytoplasmic Vesicles and Histamine-LoadedVesicles in TPA-Activated Human Basophils
Total Vesicles
Gold-Labeled Vesicles
Average
Cell
Area
Total
Average
Average
cSE
No.
No.
?SE
Total
Vesicleslpm’
Total
TPA
zSE
No.
No.
=SE
Total GoldLabeled
Vesicleslpm’
5SE
Control
0 time
1 min
2 min
5 min
10 min
15 min
30 min
45 min
22.48
24.15
28.36
20.38
22.67
21.91
22.81
26.24
16.19
1.77
1.29
3.44
1.11
1.25
1.33
0.96
1.37
1.51
393
309
353
280
193
334
199
137
155
39.3
30.9
35.3
27.0
19.3
34.4
19.9
13.8
15.5
3.29
4.37
4.46
3.43
3.98
3.75
4.15
1.36
2.27
1.82
1.28
1.39
1.33
0.93
1.60
0.91
0.56
0.97
0.18
0.16
0.23
0.18
0.27
0.22
0.22
0.06
0.10
86
74
105
80
40
151
112
54
85
8.6
7.4
10.5
8.0
4.0
15.0
11.2
5.4
8.5
1.19
1.54
2.35
0.73
0.74
1.95
2.48
0.87
1.51
0.38
0.31
0.37
0.39
0.18
0.69
0.49
0.21
0.53
0.05
0.06
0.07
0.04
0.03
0.12
0.13
0.03
0.06
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VESICULAR TRANSPORT OF HISTAMINE
4093
Table 2. Histamine-LoadedCytoplasmic Vesicles in TPA-Activated Human Basophils
TPA
Stimulation
Time
%VglTV/pm’
UN
Unstimulated
0 time
1 min
2 min
5 min
10 min
15 min
30 min
45 min
22
24
30
29
21
45
56
39
55
P=NS
P < .025
Pc.05
P=NS
P < .001
P < ,001
P < .001
P < .001
Significance To
0 time
P = NS
P=NS
P=NS
P c .001
P < ,001
P < .001
P < .001
P=NS
P < .025
P < .001
P < .001
P < .05
P < .001
smaller cells in the 45-minute sample ( 16.19 pm) compared
with unstimulated cells (22.48 pm) was present (P = NS)
(Table I). From these data, the fraction of available total
cellular vesicles carrying histamine (gold-labeled) was calculated (Table 2, Fig 2).
The percent of VGiTV was similar in unstimulated cells
and in TPA-stimulated cells obtained at time 0 (P = NS).
At I minute stimulation with TPA, 30% of the available
vesicles were gold-labeled (P < .025 compared with unstimulated cells, 22%); at 2 minutes, 29% were gold-labeled (P
< .OS, compared with unstimulated cells); at I O minutes,
45%; 15 minutes, 56%; 30 minutes, 39%; and at 45 minutes,
55%; values all significantly greater than unstimulated cells
(P < .001). Thus, the vesicles carrying histamine in TPAstimulated cells increased and remained elevated in the same
time frame during which histamine release was measured
(79.1%, 66.2% at 60 minutes in each of two experiments
after TPA stimulation). Additional significance values for
inter-sample comparisons are listed in Table 2. For example,
all stimulated samples from I O minutes on had significantly
TPA
un
1
0
2
5
10 15 30 45
Minutes
* p < .025
**
p < .05
m
2 min
1 min
p < .001
Fig 2. Percentage of total cytoplasmic vesicles that are labeled
with gold, indicating the presence of histamine in unstimulated human basophils and in human basophils stimulated with TPA (0 to 45
minutes). * P c .025; x x P < .05; rrrP c .001 compared with unstimulated cells.
P
P
P
P
P
5 min
10 min
15 min
P < ,025
P = NS
P < .05
P < .025
30 min
= NS
< .001
< .001
< .05
P < .001
P < .001
P < .001
< .001
P < .001
P = NS
P < .01
more VGiTV than at 0 time (P < .OOl); the 5- ( P < ,025).
IO- and 15- (P < .OOl), 30- (P < .OS). and 4.5-minute ( P
< .001) samples all exceeded the one-minute sample; the
IO-, 15- (P < .OOl), 30- ( P < .OS), and 45-minute ( P <
,001) samples exceeded the 2-minute sample; all samples
from I O minutes on ( P < .001) exceeded the 5-minute sample; the IS-minute ( P < .025) and 45-minute ( P < .OS)
samples exceeded the IO-minute sample; the 30-minute sample ( P < .025) was less than the IS-minute sample; the 45minute (P < .Ol) sample exceeded the 30-minute sample.
Thus, progressive significant increases in histamine-carrying
vesicles occurred in TPA-stimulated cells concomitant with
the development and persistence of PMD without evidence
of recovery of basophils either by synthetic or endocytotic
mechanisms or by a mixture of these
Ultrastructural identification of histamine-labeled cytoplasmic vesicles was possible using the DAO-G enzymeaffinity technique (Fig 3). At all times after exposure to TPA.
it was possible to identify gold-loaded cytoplasmic vesicles
primarily in the perigranular and peripheral cytoplasmic
areas. These vesicles were bounded by smooth membranes
and displayed either electron-lucent interiors or contained
electron-dense particles analogous to the particles filling the
much larger secretory granules. Some gold-labeled electronlucent vesicles were surrounded by masses of glycogen (Fig
3B). Specificity controls showed abrogation of label in vesicles and granules (data not shown). Rarely, fusion of goldlabeled vesicles with the plasma membrane was visible (Fig
3G). Gold-loaded, perigranular cytoplasmic vesicles adjacent to particle-packed granules (Fig 3C, E, and F) as well
as adjacent to empty granules devoid of their electron-dense
contents (Fig 3H and L) were noted, persisting to the 45minute time interval after TPA activation (Fig 3L).
Vesicular transport of histamine in f-Met peptide-stimuluted human basophils. The ultrastructural changes in histamine-releasing human basophils that were induced by exposure to FMLP and sampled at 0, 10, 20, and 30 seconds
and I , 2, 5, and I O minutes thereafter were similar to those
previously reported.“ Briefly, FMLP stimulated a rapid continuum of secretory and recovery responses in the IO-minute
study interval corresponding to the biochemical kinetics of
mediator (histamine) relea~e.”.’~
This continuum included
an early PMD phase, followed by an AND phase with cytoplasmic granule recovery by I O minutes in these sample$.
The majority of cells participated in this anatomic continuum.
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4094
DVORAK ET AL
Fig 3. TPA-stimulated human basophils prepared with DAO-gold t o detect histamine studied at 0 time (A), 2 minutes (B through D), 5
minutes (E through G), 10 minutes (H and I), 15 minutes (J), 30 minutes (K), and 45 minutes (L) after activation. In (A), at 0 time, the basophil
granule (01 is labeled with DAO-G. In (B),at 2 minutes, three cytoplasmic vesicles near the cell surface (open arrowhead) are labeled for
histamine. One gold-labeled vesicle is electron-lucent and is encased in electron-dense glycogen particles (closed arrowhead). Another goldlabeled vesicle also contains granule particles (long arrow)and a thirdgold-labeled vesicle is electron-lucent (short arrow). In (C), at 2minutes,
note the gold-labeled vesicle filled with granule particles adjacent to the labeled granule ( G ) . In (D), also at 2 minutes, the electron-lucent
vesicle contains DAO-gold. In (E through G), at 5 minutes, the cell surface is indicated by open arrowheads. Note in panels (E) and (F) the
granule particle-filled, DAO-gold-labeled perigranular vesicles (open arrows) adjacent to thecell surfaces. The adjacent granules ( G ) also label
for histamine; the granule in (F) has a focal, electron-lucent region (arrow) typical forPMD. Cytoplasmic glycogen particles (arrows in E) are
electron-dense and generally larger than the -20-nm gold label. In (G), at 5 minutes, a DAO-gold-labeled vesicle is fused to the cell surface
(open arrowhead). In (H) and (I), at l 0 minutes, electron-lucent (H) and particle-filled (I)cytoplasmic vesicles (arrows) contain histamine. In
(HI, a large empty granule (EG)devoid of gold label and granule particles is typical forPMD. In (I), the cell surface is coated with cationized
ferritin (used in cell processing for electron microscopy) and is indicated by theopen arrow. The granule particle-filled cytoplasmic vesicles
are labeled with DAO-gold (arrows) at15 IJ), 30 (K), and 45 (L) minutes after TPA activation. The cell surface is indicated byopen arrowheads
in (K) and (L); an empty granule (EG)typical for PMD is present at 45 minutes poststimulation in (L). OMS: (A and B), x 55,000; (C), x 117,500;
(D), x 69,000; (Eand K), x 71,500; (F), x 61,000; (G), x 80,000; (H), x 67,500; (I and J), x 113,000; (L), x 54,000.
The relationship of cytoplasmic vesicles and their contents
to the complex secretory response stimulated in human basophils by FMLP was examined using a morphometric analysis
of DAO-G-labeled cells. Table 3 shows the average cell
area, total and average numbers of cytoplasmic vesicles, total
and average numbers of gold-labeled (histamine-carrying)
vesicles, and the numbers of total and gold-labeled vesicles/
pm2 for unstimulated, buffer-incubated cells (20 seconds, 1
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VESICULAR TRANSPORT OF HISTAMINE
4095
Table 3. Total Cytoplasmic Vesicles and Histamine-Loaded Vesicles in FMLP-ActivatedHuman Basophils
Gold-Labeled Vesicles
Total Vesicles
FMLP
Control
0 time
10 s
20 s
30 s
1 min
2 min
5 min
10 m i n
Average
Cell
Area
22.48
27.69
28.24
21.10
24.37
21.56
18.21
26.73
22.64
+SE
1.77
1.24
1.37
0.69
1.64
1.76
2.00
1.61
1.01
Total
No.
Average
No.
393
415
405
471
447
307
158
276
287
39.3
41.5
40.5
47.1
44.7
30.7
15.8
27.6
28.7
?SE
3.29
5.10
5.13
5.01
6.84
2.63
3.81
4.41
4.53
Total
Vesicles/pm2
1.82
1.55
1.48
2.28
1.94
1.52
0.81
1.03
1.28
and 10 minutes) as well as for FMLP-stimulated cells collected at 0 time and at 10, 20, and 30 seconds and 1, 2, 5,
and 10 minutes thereafter. During this kinetic sequence of
changes stimulated in human basophils by FMLP, the mean
cell area did not differ significantly at individual times after
stimulation compared with unstimulated cells ( P = NS).
From these data, the fraction of available total cellular vesicles carrying histamine (gold-labeled) was calculated (Table
4, Fig 4).
The percent of VG/TVl,um2 was significantly elevated in
all FMLP-stimulated samples compared with unstimulated
cells ( P < ,001) (Fig 4). Thus, the vesicles carrying histamine in FMLP-stimulated cells increased and remained elevated during the same time frame in which histamine release
can be mea~ured'~
and in which anatomic recovery of histamine-secreting cells takes place.'8 Significance values for
inter-sample comparisons are also listed in Table 4. These
comparisons showed that the lo-, 20-second ( P < .001), 30second ( P < .005), 1-minute ( P < .025), and 5-minute ( P
< .001) samples all exceeded the 0 time point of stimulated
cells; the 20-second ( P < .001), 30-second ( P < .005), and
1-, 2-, 5-, and 10-minute ( P < .001) samples all exceeded
the 10-second sample; the 30-second ( P < .001), 2-minute
( P < .025), and 10-minute ( P < ,001) samples exceeded
the 20-second sample; the 1-minute ( P < .001), 2-minute
( P < .005) and 5-minute ( P < .001) samples exceeded the
30-second sample; the 10-minute ( P < .005) sample exceeded the 1-minute sample; the 5-minute ( P < .025) sample
exceeded the 2-minute sample, and the 10-minute ( P < .001)
?SE
Total
No.
Average
No.
0.18
0.21
0.21
0.29
0.37
0.20
0.18
0.14
0.21
86
243
156
339
218
206
98
199
160
8.6
24.3
15.6
33.9
21.8
20.6
9.8
19.9
16.0
+SE
Total GoldLabeled
Vesicles/pm'
+SE
1.19
2.64
2.52
4.04
4.37
2.28
2.74
3.17
1.93
0.38
0.90
0.58
1.65
0.96
0.98
0.49
0.75
0.74
0.05
0.1 1
0.1 1
0.24
0.23
0.10
0.1 1
0.1 1
0.1 1
sample exceeded the 5-minute sample. Thus, progressive
significant increases in histamine-carrying vesicles occurred
in FMLP-stimulated cells concomitant with the development
of the anatomic secretory continuum PMD + AND recovery. Recovery mechanisms for human basophils after FMLP
stimulation include vesicular trafficking of synthetic and endocytotic
Ultrastructural identification of histamine-labeled cytoplasmic vesicles was done using the DAO-G technique (Fig
5). Gold-labeled vesicles were present in all samples of
FMLP-stimulated cells. The structures were smooth membrane-bound and either displayed an electron-lucent interior
or contained dense particles identical to those in the much
larger secretory granules. In addition to the presence of these
gold-labeled vesicles in the perigranular and peripheral cytoplasmic areas, and in contrast to TPA-activated cells, some
were also present in the cytoplasm containing Golgi structures. Some gold-labeled vesicles rested beneath the plasma
membrane, through which granules had been extruded during
the AND phase of the degranulation continuum (Fig 5K). The
FMLP-stimulated samples contained particle-filled granules
that labeled for histamine (Fig 5E and F); these samples also
contained granules (partially and completely devoid of electron-dense materials) with reduced or absent histamine stores
(Fig 5C and F). Specificity controls for the DAO-G technique
in these samples showed abrogation of granule and vesicle
label when the DAO-G reagent was absorbed by histamine
beads before staining or when DAO digestion of sections was
done before DAO-G staining (Fig 5D and G).
+
Table 4. Histamine-LoadedCytoplasmic Vesicles in FMLP-Activated Human Basophils
FMLP
Stimulation
Time
Significance To
%VgTTV/pm2
UN
0 time
10 s
20 s
30 s
1 min
2 min
5 min
Unstimulated
0 time
10 s
20 s
30 s
1 min
2 min
5 min
10 min
22
59
39
72
49
67
62
72
56
P i ,001
P < ,001
P < ,001
P < ,001
P < ,001
P < ,001
P < ,001
P < ,001
P < ,001
P < ,001
P < ,005
P < ,025
P = NS
P < ,001
P = NS
P < .001
P < ,005
P < ,001
P < ,001
P < ,001
P < ,001
P < ,001
P = NS
P < ,025
P = NS
P < ,001
P < ,001
P < .005
P < ,001
P = NS
P = NS
P = NS
P < ,005
P < ,025
P = NS
P < .001
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DVORAK ET AL
4096
80-
FMLP
*
60-
L
3
40-
8
11111111~
81I
0
-
un
0
10 20 30 1
Seconds
*
*
netics). Significantly larger numbers of total vesicles were
gold-loaded in the FMLP-triggered cells than in the TPAtriggered cells ( P < ,001 for 0 time, I-, 2-, and 5-minute
samples, and P < .01 for the 10-minute samples (Fig 6).
Thus, a secretogogue that rapidly induced the anatomic continuum of PMD "* AND -+ recovery also induced more
histamine-loaded vesicles, compared with similar times after
stimulation, than a secretogogue which slowly induced
PMD, persisting to involve -50% of the cells by 45 minutes
and showing only minor amounts of AND and no morphologic evidence of recovery.
Comparison of the fraction of total vesicles carrying histamine at disparate times after stimulation with TPA or FMLP
was also of interest (Table 5). For example, the peak value
for the %VG/TV/pm2for TPA was at 15 minutes. This value
(56%) was significantly less than the peak value (72%) for
FMLP, which occurred at 20 seconds after stimulation ( P <
.001). TPA stimulation induced elevated VG/TV/pm2 at 45
minutes ( 5 5 % ) in samples predominantly still undergoing
PMD (and withsmall amounts of AND and with no anatomic
signs of recovery), whereas predominantly recovering cells
(studied 5 minutes after FMLP stimulation) had significantly
more VG/TV/pm2 (72%; P < .001). End times of kinetic
samples for each secretogogue showed no significant difference in the %VG/TV/pm2 (TPA, 45 minutes 55% v FMLP,
10 minutes 56%, P = NS).
2
5
10
Minutes
p < ,001
Fig 4. Percentage of total cytoplasmic vesicles that are labeled
with gold, indicating the presence of histamine in unstimulated human basophils and in human basophils stimulated with FMLP (0 to
10 minutes). * P < .001 compared with unstimulated cells.
Cytoplasmic vesicles attached by fusion to large secretory
granules were evident in the 0 time FMLP-stimulated sample
(Fig 5C). These fused vesicles were attached to virtually
empty granules with reduced gold label (Fig 5C) as well as
to granules with particles and gold-labeled histamine (Fig
5E). Some cytoplasmic vesicles containing gold label for
histamine were surrounded by glycogen aggregates (Fig 5C,
inset).
Vesicular transport of histamine in the basophils of the
same donors has different kinetics, analogous to biochemically measured release of histamine, when TPA and FMLP
activation arecompared. In Table 5 , comparisons of the
%VGfT'V/pm2 are listed for similar (and several disparate)
times tested when the basophils from the same donors were
stimulated by either TPA (slow kinetics) or FMLP (fast ki-
DISCUSSION
We have used a new enzyme-affinity gold method to localize histamine in subcellular organelles24to quantitate the
number of cytoplasmic vesicles in secreting human basophils
that contain this pro-inflammatory mediator. By examining
multiple time points after stimulation with two different secretogogues known to have different biochemical histamine
release and morphologic kinetics, we have compared the
proportion of total vesicles transporting histamine with those
in unstimulated cells, as well as between different times
after exposure to an individual secretogogue, and between
identical, or disparate, times after exposure to each of two
*
Fig 5. FMLP-stimulated human basophils preparedwith DAO-gold to detect histamine (A through C, E, F, H through L) or with specificity
controls (D and G ) at 0 time (A through D), 10 seconds (E through G), 20 seconds (H), 30 seconds (I),1 minute (J and K), and 10 minutes (L)
after activation. The open arrowhead in all panels indicatesthe call surface; G, granule, EO, empty granule (typical for PMD); N, nucleus. At 0
time and in the same activated cell, both empty, electron-lucent (arrow in A) and full, electron-dense (arrow in B) vesicles are gold-labeled.
Cytoplasmic glycogen particles are electron-dense (A). In (C), a neerly empty granule (typical for PMD) retains some DAO-gold label. One
cytoplasmic vesicle is also labeled. Note the vesicle attachedto the empty granule. Electrondense glycogen particles (arrows) are attached
to the narrowed neck of the fused vesicle. The inset in (C) shows a free, electron-lucent vesicle, labeled with gold for histamine (arrow),
encased in surrounding electron-dense glycogen aggregates, present in the cytoplasm of the tame basophil. The specificity control in (D)
(DAO-gold absorbedwith histamine-agarose before staining) is negative for histamine in the large granule and adjacent vesicles. Electronwith DAO-gold as follows: (11 granule
dense glycogenparticles (arrows) remain visible. In (E), at 10 second., three subcellular aites are labeled
matrix ( G ) but notthe intragranular Charcot-Leyden crystal (CLC); (21 cytoplaarnic, electron-lucent, perigranular veeicle (arrow);(3) polyamines
in the nucleus (NI(see ref 23). A vesicle is attached to the histamine-labeled granule (arrowhead). In IF), also at 10 seconds, DAO-gold
extensively labels the granule matrix ( G ) of the full granule and shows less label in the empty granule (EO)typical for PMD. The specificity
control in ( G ) (digestion of the section with DAO before staining with DAO-gold) is negative for histamine in the large granules( G ) , adjacent
perigranular vasicles, and nucleus(N). Electron-dense glycogen particle6(arrow) remain viaiblein the cytoplasm. Granuleparticlefilled, DAOgold-labeled vesiclesrest just beneath the plasma membrane (open arrowheads)a t 20 seconds (H) and 30 seconds (I)po.btimulus. In (J), at
1 minute, similar particle-filled cytoplasmic vesicles contain hirtamine. Also, at 1 minute after stimulation (K), extrusion of granule particles
to the cell surface is visible (open arrowhead). Theunderlying cytoplaun contains gold-labeled electron-lucent vesides (errows).In (L), at 10
minutes, DAO-gold is attached to the cell surface (open arrowhead),within underlying electron-lucent and granulepartick-filled vesides, and
to an adjacent recovered granule ( G ) . OMS: (A), x 66,000; (B), x 105,OOO; (C), x 59,000; (D), x 55,000; (E), x 50,000; (F), x 54.000; (G), x 54.000;
(H through K), x 113,000; (L), x 78,000.
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VESICULAR TRANSPORT
OF HISTAMINE
secretogogues. Similar to previous reports, DAO-gold labeled histamine in human basophil secretory granules and
cytoplasmic
cytoplasmic granules in activated
cells partially or completely devoid of electron-dense matrix
had reduced or absent histamine stores.”
The phorbol ester, TPA, and the bacterial peptide, FMLP,
4097
each stimulate the release of pro-inflammatory mediators
from human basophils; the kinetics and products of release
are unique, however, for each ~ecretogogue.”.~’.~~
Generally,
the release of histamine from FMLP-stimulated human basophils is extremely rapid and essentially complete in -2 to 3
minutes. The major product of arachidonic acid metabolism
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DVORAK ET AL
4098
sample. The value at 45 minutes was equivalent to the 10minutesample andwassignificantly
less thanthatat
15
minutes. Thus, as histamine release increased and persisted
Condition
Time
Stlmulus
%VgTTVIpm2
Significance
and PMD developed,so did the transport of histamine-loaded
vesicles. AND began in the 30-minute sample, and basophils
TPA
0
24
P < ,001
FMLP
59
0
revealingthisanatomy
had diminished cytoplasmic vesi1 min
TPA
30
c l e ~ . * ' ~However,
~'
the proportion of these vesicles carrying
1 min
FMLP
67
P < ,001
histamine at 30 minutes still significantly exceeded values
2 min
29
TPA
for controls and early samples (eg, 0 to 5 minutes).
2 rnin
FMLP
P < .001
62
The principal findings and interpretations for the DAO-gold
5 min
TPA
21
labeling of cytoplasmic vesicles in secreting human basophils
5 min
FMLP
72
P < ,001
stimulated by FMLP are as follows: (1) histamine-labeled vesi10 min
TPA
45
cles
in all stimulated samples exceeded those in unstimulated
10 min
FMLP
56
P < .01
samples (thus, histamine is transported in vesicles in cells releas15 min
TPA
56
ing histamine and undergoing PMD + AND recovery); (2)
FMLP
72
20 S
P < ,001
45 rnin
TPA
55
comparisons of timed samples generally revealed increases in
5 min
FMLP
P < .001
72
later samples compared with earlier samples. For example, all
45 min
TPA
55
samples between 20 seconds and 10 minutes exceeded the 1010 min
FMLP
56
P = NS
second value. The increased VGmV indicating histamine transport was accompanied primarily by the morphology of PMD at
early times (1 0 seconds to 1 minute), indicating stimulated secretory transport of vesicle-packaged histamine. At later times (2
produced by human basophils, leukotriene C4 (LTC4), lags
to 10 minutes), the increased VGKV was associated with the
behindhistaminerelease,being
complete for thissecretomorphology of recovery of granule contents, indicating transport
gogue in -7 minutes poststimu1us.'7.4" TPA-stimulated huof vesicle-packaged histamine from two possible sources (endoman basophils do not generate LTC4at all and release histacytosis of released histamine, synthesis in Golgi structures) as
mine slowly (maximum release 1 hour after tim mu la ti on)."."^^"
mechanisms for effecting granule content reconstitution.
Theseunique biochemicalcharacteristics
for release reQuantitativecomparisons of the fraction of total cytosponses stimulated by FMLP and TPA are accompanied by
plasmic vesicles loaded with histamine in these two models
characteristic, unique morphologies for each trigger, as deof human basophil activation were interesting. Because the
termined by routine electron micros copy.'*^'" Multiple samples examined after FMLP stimulation, for example, showed samples for each secretogogue were aliquots of peripheral
blood basophils purified from the same donors and on the
a rapid continuum of changes which proceeded from PMD
same days, interindividual donor anddaily variation in secre+ AND
recovery, a process essentially complete by 10
tory response was eliminated in this analysis. The principal
minutes.'* Incontrast, multiple samples examined after TPA
findings and interpretations of these comparisons are as folstimulation showed extensive PMD extending over
a 45minute interval and involving -50% of the granules in cells
by that time." To a small extent, typical AND was present
and unique interactions of granule and plasma membranes
also occurred with TPA stimulation-aprocesstermed
forme fruste of AND. In the latter case, granule chambers
fused to plasma membranes and emptied their contents, but
the chamber membranes did not evert. No morphologic evidence of granule recovery occurred at 45 minutes after TPA
activation. Thesetwo well-described models of disparate
biochemical and morphological human basophil release reactions were used in the present
study to examine the hypothesis that cytoplasmic vesicle transport of histamine could be
stimulated (eg, regulatedsecretion) and thatthistransport
system proceeded from granule to plasma membranes as a
mechanism for effecting PMD.
The principal findings and interpretations of the specific
enzyme-affinity gold labeling of cytoplasmic vesicles in secreting human basophils stimulated by TPA in the current
study are as follows: ( 1 ) the fraction of gold-labeled vesicles
(indicating histamine) of the total vesicles was elevated in
all stimulated samples compared with unstimulated samples
(thus, histamine is transported in vesicles in cells undergoing
PMD andreleasing histamine); (2) comparison of timed samples show a progressive increasein gold-labeledvesicles
over time persisting to 45 minutes, except in the 30-minute
Table 5. Comparisons of Histamine-Loaded Vesicles in Human
Basophils From the Same Donors Stimulated With TPA or FMLP
+
~
-
+
~~~~~
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4099
VESICULAR TRANSPORT OF HISTAMINE
lows: (1) at identical times poststimulation (eg, 0 time, 1, 2,
5, and 10 minutes), the fraction of VGRV stimulated by
FMLP exceeded that stimulated by TPA (ie, the rapid secretory event characterized by the morphological continuum
PMD AND -+ recovery [FMLP] was associated generally
with more histamine-loaded vesicular traffic than was the
slow secretory event characterized by persistent PMD, a
forme fruste of AND and no recovery [TPA]); ( 2 ) the peak
value of %VG/TV for the slow trigger (TPA) at 15 minutes
(56%) was less than that value for the fast trigger (FMLP)
at 20 seconds (72%) (thus, speed of secretion was associated
with greater histamine-loaded vesicular traffic at peak histamine-loaded vesicle times); (3) at 45 minutes after TPA
stimulation, fewer TV were carrying histamine (55%) than
at 5 minutes after FMLP stimulation (73%) (thus, continuing
PMD and no recovery is associated with less traffic of histamine in vesicles than in samples displaying extensive and
ongoing morphological evidence of recovery, as seen at 5
minutes after FMLP stimulation); (4) the percent of VGRV
was identical in the end time samples for each stimulant of
human basophil secretion-eg, TPA, 45 minutes, 55% and
FMLP, 10 minutes, 56%, P = NS (thus, elevated histamine
vesicular traffic [over unstimulated cells] persists in cells
that are still secreting by PMD but not recovering [eg, TPA
stimulation] as well as in cells that are recovering granule
materials by endocytosis and synthesis, but are no longer
secreting by PMD [eg, FMLP stimulation]).
The ability to correlate directly the fraction of histamineloaded vesicles (%VGN) in human basophils, as determined
with this new ultrastructural imaging technique, to the biochemical measurements of histamine release in the supematants of challenged cells is problematic. To do so with reasonable assurance that these data may be correlated, one
would like to know the relative biochemical concentrations
of histamine in granules and vesicles. The enzyme cytochemical method, by definition a postembedding ultrastructural
label, only accesses histamine at the cut surfaces of vesicles
and granules and not throughout these organelles. Therefore,
the real histamine content of these structures is not discemible with this method and, therefore, their relative contributions to the released histamine measured biochemically are
not known. Also, to compare directly the cytochemical data
with the histamine release data for each of two triggers with
vastly different morphologies and kinetics, reflecting the mechanics of secretion, is difficult. For example, the slow-release model (TPA) essentially shows higher histamine release values (but lower histamine-loaded vesicles) than the
rapid-release model (FMLP). A possible interpretation of
these apparently disparate data is that, in the TPA model,
the efficiency of vesicular transport of histamine that is still
ongoing at 45 minutes (and in the absence of any recovery
from secretion) contributes significantly to the higher biochemical values for histamine release. In the FMLP model,
histamine secretion (as determined biochemically) might be
less than that in the TPA model because the entire secretionrecovery sequence takes place rapidly. That is, the larger
percent VG/V in the PMD phase of this secretory continuum
evolves into a definitive granule extrusion phase with subsequent recovery. However, these interpretations must remain
hypothetical until direct biochemical measurements of the
-+
histamine content in individual cytoplasmic vesicles and
granules of basophils are possible.
Basophils of multiple species are characterized by numerous small, cytoplasmic, smooth membrane-bound vesicles?'
These structures are uniquely present in the perigranular and
subplasma membrane cytosol in activated cells, and ultrastructural studies have detected similar vesicles attached to
granules and fused to plasma membranes. Glycogen particles
and aggregates are frequently closely associated with these
vesicles and have been proposed as a possible energy source
for their t r a n s p ~ r t . ~ ,These
~ * ~ " images prompted a generic
degranulation model for basophils and mast cells to be put
forth in 1975.5Simply stated, this model proposed a continuum of secretion, encompassing PMD AND, mediated by
vesicular transport of granule materials out of cells closely
coupled to endocytotic vesicular traffic into cells. This coupled stimulus-secretion model also proposed that the morphologic expression of PMD or AND depended on the rate of
vesicular traffic in that, at sufficiently slow rates, individual
vesicles would traffic individually while exchanging granular contents, but that, as the traffic rate increased, discrete
vesicles no longer would retain their individuality and would
fuse to form channels connected to adjacent granules and to
the plasma membrane, thereby creating the anatomy of
AND. (An appropriate analogy would be the rate of water
flow from a faucet which can be regulated to produce individual drops at slow speed, whereas fusion of drops would
produce a stream of water at fast speed.)
The studies reported here support this model of basophil
degranulation. For example, the slow rate of secretion of
histamine stimulated by TPA was accompanied primarily by
the morphology of PMD, effected by vesicular traffic of
individual discrete vesicles loaded with histamine; little evidence of the morphology of AND occurred in this slowsecretion model. On the other hand, extremely rapid secretion of histamine, stimulated by FMLP, was accompanied
by the morphology of PMD, effected by vesicular traffic at
early times, and followed by the morphology of AND, with
reduced numbers of vesicles.
Several recent studies of cytochemical and immunogoldlabeled vesicles in stimulated secretory cells also support the
use of these small containers in secretion from granulated
secretory ce11s.9.in.i3.z3.39.43 The secreted materials packaged
in transport vesicles include eosinophil peroxidase in interleukin-5-stimulated human eosinophilic myelocytes' and
mature eosinophils," amylase in norepinephrine-stimulated,
isolated rat parotid gland acinar cells,I3 CLC protein in
FMLP- (Dvorak et al, unpublished data, June 1996) and
TPA-'3 stimulated human basophils, and histamine in FMLPstimulated human basophils." Together with the new studies
we report here, the transport of large granule secretory contents in small vesicular containers during stimulated secretion from inflammatory cells (eosinophils, basophils) and
exocrine secretory cells (parotid gland acinar cells) is established.
+
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4100
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VESICULAR TRANSPORT OF HISTAMINE
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1996 88: 4090-4101
Vesicular transport of histamine in stimulated human basophils
AM Dvorak, DW Jr MacGlashan, ES Morgan and LM Lichtenstein
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