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Blackwell Munksgaard
# 2008 The Authors
Journal compilation # 2008 Blackwell Munksgaard
doi: 10.1111/j.1600-0854.2008.00838.x
The Rab27a Effectors JFC1/Slp1 and Munc13-4
Regulate Exocytosis of Neutrophil Granules
Agnieszka A. Brzezinska1, Jennifer L. Johnson1,
Daniela B. Munafo1, Karine Crozat2,
Bruce Beutler2, William B. Kiosses3,
Beverly A. Ellis1 and Sergio D. Catz1,*
1
Department of Molecular and Experimental Medicine,
The Scripps Research Institute, 10550 North Torrey
Pines Road, La Jolla, CA 92037, USA
2
Department of Genetics, The Scripps Research
Institute, 10550 North Torrey Pines Road, La Jolla,
CA 92037, USA
3
Core Microscopy Facility, The Scripps Research
Institute, 10550 North Torrey Pines Road, La Jolla,
CA 92037, USA
*Corresponding author: Sergio D. Catz, [email protected]
Neutrophil granules contain secretory molecules that
contribute to the implementation of all neutrophil functions. The molecular components that regulate the
exocytosis of neutrophil granules have not been characterized. In this study, using small interfering RNA genetargeting approaches and granulocytes from genetically
modified mice, we characterized the Rab27a effectors
JFC1/Slp1 and Munc13-4 as components of the exocytic
machinery of granulocytes. Using total internal reflection
fluorescence microscopy analysis, we show that Rab27a
and JFC1 colocalize in predocked and docked vesicles in
granulocytes. Next, we demonstrate that JFC1-downregulated granulocytes have impaired myeloperoxidase
secretion. Using immunological interference, we confirm
that JFC1 plays an important role in azurophilic granule
exocytosis in human neutrophils. Interference with Rab27a but not with JFC1 impaired gelatinase B secretion in
neutrophils, suggesting that a different Rab27a effector
modulates this process. In similar studies, we confirmed
that Munc13-4 regulates gelatinase secretion. Immunofluorescence analysis indicates that Munc13-4 localizes at
secretory organelles in neutrophils. Using neutrophils
from a Munc13-4-deficient mouse model (Jinx), we demonstrate that Munc13-4 plays a central role in the regulation of exocytosis of various sets of secretory organelles.
However, mobilization of CD11b was not affected in
Munc13-4-deficient neutrophils, indicating that secretory
defects in these cells are limited to a selective group of
exocytosable organelles.
Key words: GTPase, inflammation, innate immunity,
integrin, MMP-9, myeloperoxidase, secretion
Received 4 April 2008, revised and accepted for publication 30 September 2008, uncorrected manuscript published online 7 October 2008, published online 30 October
2008
Neutrophils contain several types of secretory organelles
that hold a variety of specialized proteins, which play a central
role in inflammation and host defense. Based on morphology, protein content and density, four types of secretory
organelles have been identified in human neutrophils.
Peroxidase-positive (azurophilic or primary) granules contain
myeloperoxidase (MPO), elastase, a-defensins and other
inflammatory peptides and proteins (1). Specific (secondary)
granules are enriched in the immunomodulators lactoferrin
and matrix metalloproteinase 9 (MMP-9). Gelatinase (tertiary) granules also store MMP-9 but lack lactoferrin. The
fourth granule type the secretory vesicles contain alkaline
phosphatase, albumin and the b2 integrin CD11b/CD18, also
present in specific granules (2). It is generally accepted that
neutrophil secretory organelles have different tendencies to
undergo exocytosis in response to stimuli. The maintenance
of this order in the exocytic process is directly linked to the
different roles played by the luminal and membrane proteins
of neutrophil organelles during the innate immune response.
Unrestricted release of toxic neutrophil granular proteins to
the extracellular milieu is potentially deleterious to the host;
therefore, neutrophil exocytosis should be strictly controlled.
Despite its biological importance, the molecular mechanism
underlying exocytosis of neutrophil secretory organelles
remains relatively unknown. In a previous study, we presented evidence that Rab27a is a key component of the
secretory apparatus of azurophilic granules (3). However,
the molecular details of this mechanism are still elusive, and
the question whether Rab27a regulates the secretion of
other secretory organelles in neutrophils remains unanswered. In this work, we approach these questions by
focusing on the Rab27a effectors.
The function of monomeric guanosine triphosphatases
(GTPases) is regulated by specific Rab effector proteins (4).
In the particular case of Rab27a, 11 potential Rab27a
effectors have been described (5). JFC1 [also named synaptotagmin-like protein 1 (Slp1)], Slp2, Slp3, Granuphilin/Slp4
and Slp5 constitute the family of Rab27a-binding proteins
containing tandem C2 domains in their carboxy terminus.
Another Rab27a effector, Munc13-4, is composed of two C2
domains surrounding two Munc homology domains (MHD).
Melanophilin (Slac2-a) (6), Slac2-b and Slac2-c (7) also bind
Rab27a, but they lack C2 domains, having instead a myosinbinding domain (8,9) and, therefore, they regulate trafficking
in a different fashion. JFC1 was the only Rab27a effector
identified when a leukocyte human library was analyzed
using a two-hybrid system with Rab27a as bait (8). Furthermore, proteomic analysis of neutrophil granules identified
JFC1 and Munc13-4 but not other Rab27a effectors (10).
These data suggest that Munc13-4 and JFC1 are the only
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Rab27a effectors expressed in neutrophils (3,10). Although
both JFC1 and Munc13-4 have in common their ability to bind
Rab27a, their molecular structures are substantially different.
Munc13-4 lacks SHD (Slp homology domains), which is the
Rab-binding domain in JFC1 (5). Instead, Munc13-4 is
suggested to bind to Rab27a by the region composed of
residues 240–543, an undefined domain located between
the C2A domain and the MHD1 domain (11). It is clear then
that the molecular interactions between Rab27a and
Munc13-4 and Rab27a and JFC1 are different.
Munc13-4 was recently identified as a key component of the
secretory machinery of lytic granules in cytotoxic T lymphocytes (CTLs) (12) and is also known to regulate exocytosis
in platelets (13). Deficiency of Munc13-4 in humans causes
the immunodeficiency type 3 familial hemophagocytic lymphohistiocytosis, an autosomal recessive disorder characterized by the malfunction of CTLs and natural killer cells
(12). The observation that in Munc13-4-deficient CTLs, lytic
granules are able to dock at the plasma membrane but fail
to undergo exocytosis in response to stimuli supports a
role for this effector in vesicle priming or fusion rather than
in docking (12). Contrarily, in Rab27a-deficient CTLs, lytic
granules are unable to come in close proximity to the
immunological synapse (14). These observations suggest
that a Rab27a effector different from Munc13-4 may bring
lytic granules to the docking point at the immunological
synapse and support the idea that multiple Rab27a effectors may co-ordinate the secretory pathway of lytic granules in CTLs. Supporting this idea, in a recent report,
Griffiths and collaborators presented evidence for a role
of Slp family members in lytic granule secretion (15).
Similar to lytic granules and melanosomes, azurophilic
granules belong to a group of secretory vesicular compartments called lysosome-related organelles or secretory
lysosomes (16). These organelles share a similar biosynthetic pathway (17). However, as learned from melanosomes and lytic granules, these organelles differ
substantially in the way their secretion is regulated, despite the observation that they have Rab27 GTPases as part
of their secretory machinery (14,18). The mechanism of
azurophilic granule exocytosis remains elusive, and the
role of JFC1 and Munc13-4 in the regulation of the exocytosis of other neutrophil granules is currently unknown.
In this work, using a combination of immunological exocytosis interference, small interfering RNA gene-targeting
approaches and granulocytes from genetically modified
mice, we characterized JFC1 and Munc13-4 as components of the exocytic machinery of granulocytes.
Results
JFC1/Slp1 regulates azurophilic granule dynamics
and secretion in HL-60 granulocytes
To analyze the hypothesis that JFC1 is an important component of the molecular secretory machinery of azurophilic
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granules, we utilized both human promyelocytic leukemia
cells (HL-60) granulocytes and human neutrophils. HL-60
cells undergo azurophilic granule exocytosis in response to
a variety of physiological stimuli (19,20) and express both
JFC1 and Rab27a (3). The observation that these secretory
components are distributed in secretory organelles in
HL-60 granulocytes and the finding that downregulation
of Rab27a impairs secretion of MPO in HL-60 cells (3)
support the idea that the Rab27a secretory machinery is
functional in these cells. In this study, to further validate
the use of HL-60 cells for studying the mechanism of
exocytosis of azurophilic granules, we have utilized total
internal reflection fluorescence microscopy (TIRFM) and
analyzed the dynamics of vesicles containing Rab27a and
JFC1 in granulocytes. Using dual-color live cell TIRFM, we
simultaneously analyzed the dynamics of both JFC1- and
Rab27a-positive vesicles. Our analysis shows that JFC1colocalizes with Rab27a on virtually all vesicles in the TIRFM
plane in HL-60 granulocytes (Figure 1A,B and Movies
S1–S3). These data indicate, first, that JFC1 and Rab27a
undergo true colocalization as validated by the similar
temporal distribution of JFC1 and Rab27a molecules;
second, that most vesicles in the docking zone (proximity
to the plasma membrane 100 nm) contain both JFC1 and
Rab27a prior to stimulation; third, that various populations
of secretory vesicles could be identified according to their
movement: fast-moving or docked vesicles (Figure 1B,
Movie S4 and Table S1), motile granules defined as those
with displacement larger than 0.5 mm and a velocity of
0.1 mm/second or greater (21). The results presented in
this study, together with our previous study showing
decreased exocytosis of azurophilic granules in Rab27adownregulated HL-60 granulocytes (3), indicate that these
cells constitute a good cellular system for the analysis of
the Rab27a-dependent secretory machinery. They also
support the hypothesis that JFC1 is a Rab27a effector in
granulocytes. Next, we demonstrate that enhanced green
fluorescent protein (EGFP)-Rab27a-containing vesicles are
mobilized to the plasma membrane upon stimulation in
HL-60 granulocytes, further supporting the idea that the
Rab27a secretory machinery is functionally linked to exocytosable organelles in these cells (Figure 1C). In these
experiments, we observed fusion of both predocked
vesicles and newly arriving EGFP-Rab27a-containing organelles, supporting the idea that vesicle trafficking from
the site of storage to the plasma membrane as well as
exocytosis are functional in these cells.
Downregulation of Rab27a decreases MPO secretion in
HL-60 cells (3). Immunoprecipitation analysis showed that
endogenous JFC1 binds to Rab27a in HL-60 cells, suggesting that they are components of the same exocytic
machinery (3). To demonstrate that JFC1 plays a significant
role in MPO exocytosis, we took a short-hairpin RNA
(shRNA) gene-targeting approach. We generated three
clonal HL-60 cell lines expressing different shRNAs specific for JFC1 (shRNA sequences are shown in Figure 2A).
Two of these clones showed specific downregulation
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Figure 1: Dual-color live cell imaging to simultaneously visualize the colocalization of JFC1/Slp1 and Rab27a in a spatiotemporal manner in granulocytes. HL-60 granulocytes were cotransfected with vectors for the expression of EGFP-Rab27 and DsREDJFC1 and analyzed by TIRFM. Laser illumination was adjusted to impinge on the coverslip at an angle to yield a calculated evanescent field
depth of 108 nm. Images were analyzed using IMARIS software. A) Rab27a (green), JFC1 (red) and overlaid (merged) images showing true
colocalization of Rab27a and JFC1 on most vesicles. The dynamics of the labeled vesicles and the spatio-temporal displacement of JFC1
and Rab27a were followed for 1 min and can be viewed in associated Movies S1–S3. B) Analysis of vesicle dynamics using IMARIS software.
Left panel, each sphere represents a vesicle. Right panel, the tracks indicate the trajectory of the associated vesicles occurring during the
analysis. Increasing colour in a given track is indicative of time of appearance and residence in the TIRF zone. Tracks without
a corresponding vesicle indicate that the vesicle appeared in the TIRF zone at a different frame from that shown in (A). Vesicles without
associated tracks represent docked vesicles. The dynamics of these vesicles during the length of the analysis can be observed in
associated Movie S4 and can be found in the data analysis chart (Table S1). All vesicles (n ¼ 254) that appeared in the TIRFM zone during
the length of the study (1 min) were included in the analysis. C) HL-60 granulocytes were transfected with a vector for the expression of
EGFP-Rab27a and analyzed by TIRFM. The cells were stimulated with PMA (0.1 mg/mL), and vesicle dynamics were continuously
monitored for 7 min. Images were collected at one frame per second. The white arrows indicate a docked vesicle finishing the fusion
process. The black arrows indicate a vesicle appearing in the TIRFM zone 26 seconds after the addition of the stimulus and subsequently
undergoing fusion. Synchronous increase in total intensity and a concomitant increase in the width of fluorescence are consistent with
fusion of the vesicle to the plasma membrane (44). Scale bar ¼ 1 mm.
of JFC1 as evaluated by western blot (Figure 2B).
The expression of other secretory proteins including Rab27a
was not decreased in JFC1-deficient cells (Figure 2C). To
analyze whether JFC1 plays a significant role in azurophilic
granule exocytosis, we stimulated JFC1-downregulated cells
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with the formylated peptide fMLP (formyl-Met-Leu-Phe),
a stimulus that activates granulocyte exocytosis through
a G-protein-coupled membrane receptor. Functional analysis
shows that JFC1 downregulation induces a severe impairment in MPO exocytosis in response to this physiological
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phorbol 12-myristate-13-acetate (PMA) in wild-type HL-60
granulocytes. Secretion was also increased in JFC1-downregulated cells (Figure 3). However, MPO secretion in
granulocytes downregulated for JFC1 did not reach the
high level of degranulation observed in control cells even
after cytochalasin D treatment, suggesting that actin depolymerization does not fully compensate for the secretory
defect caused by JFC1 deficiency.
Figure 2: Downregulation of JFC1/Slp1 impairs MPO secretion in HL-60 cells. A) Sequences of the gene-targeting shRNA
used in this work. The nucleotides of JFC1 targeted by these
interference molecules are indicated between parentheses.
B) HL-60 cells were stably transfected with three vectors expressing shRNAs specific for JFC1 or with a control empty vector (pRS;
OriGene). After differentiation, the expression of JFC1 was
analyzed by western blot. PGK1 was used as control for equal
loading. C) Expression of Rab27a in JFC1-downregulated cells
was verified by western blot. D) Clones 70 and 71 and cells
transfected with the control vector (control) were stimulated with
1 mM fMLP (red columns) or left untreated (black columns).
Secreted MPO was detected by ELISA. Results represent the
mean SEM from three independent experiments. The unstimulated control was designated as 100%. *p < 0.03.
To explore the mechanism underlying JFC1 regulation of
secretory granule dynamics, we investigated granule movement in JFC1-downregulated cells using TIRFM. To this
end, we used granulocytes downregulated for the expression of JFC1 while expressing lysosome-associated membrane protein (LAMP)-3 as a GFP fusion protein and
followed vesicle dynamics using a quantitative microscopy
approach. In Figure 4 and Movies S5 and S6, we show that
JFC1-downregulated cells have a significant increase in
the number of granules undergoing long distance (Figure
4B) and fast movement (Figure 4C), which are types of
movement previously linked to microtubule-associated trafficking (21). Average granule velocity was also increased in
JFC1-downregulated cells (Figure 4B). These data positively
correlate with a previous study showing an increase in the
number of vesicles undergoing fast movement in mast cells
that are deficient in Rab27a and Rab27b (21). However,
contrary to that described for Rab27-knockout mast cells,
we found that JFC1-downregulated cells have an increase
rather than a decrease in the percentage of motile granules
(Figure 4B). Altogether, these data suggest that JFC1 might
play a role in vesicle transition from microtubule-associated
movement (fast movement) (21) to actin-based movement,
tethering or docking. Thus, in the absence of JFC1, the
number of vesicles undergoing fast movement increases.
JFC1/Slp1 regulates the secretion of azurophilic
granules in human neutrophils
We have previously demonstrated that Rab27a localizes on
azurophilic granules in human neutrophils (3). Additionally,
stimulus (Figure 2D). These results demonstrate for the first
time that JFC1 is directly involved in exocytosis in granulocytes. Because endogenous Rab27a interacts with endogenous JFC1 in these cells (3) and both seem to regulate the
same secretory process, these data strongly support the idea
that JFC1 and Rab27a are constituents of the secretory
machinery that controls azurophilic granule exocytosis.
To test whether JFC1 could potentially mediate its exocytic
function through interaction with the actin cytoskeleton,
JFC1-downregulated and control cells were treated with
cytochalasin D before stimulation with fMLP and subsequently evaluated for MPO secretion. Cytochalasin D treatment induces actin depolymerization, thus increasing
vertical vesicle mobility and facilitating vesicle access to
the docking point at the plasma membrane (22). Consistent
with that shown in neutrophils (23), cytochalasin D treatment enhanced the secretory response to both fMLP and
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Figure 3: Cytoskeleton disruption does not fully compensate
for the secretory defects observed in JFC1/Slp1-downregulated cells. JFC1-downregulated and control cells were treated
with cytochalasin D (10 mg/mL) for 15 min before addition of
stimulus. Next, the cells were stimulated with 1 mM fMLP, 0.1 mg/mL
PMA or left untreated (NS, non-stimulated). Secreted MPO was
detected by ELISA. Results represent the mean SEM from two
independent experiments.
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Figure 4: Analysis of vesicle dynamics
in JFC1-downregulated granulocytes.
A) Control and JFC1-downregulated cells
were transfected with a vector for the
expression of EGFP-LAMP-3 and analyzed by TIRFM as described under ‘Materials and Methods’. One representative
cell from each type is shown. Vesicle
dynamics was followed for 30 seconds
and are presented in associated Movies
S5 and S6. Scale bar ¼ 5 mm. B and
C) Analysis of vesicle dynamics in JFC1downregulated and control cells. Motile
granules were defined as those moving
at least 0.5 mm with average velocity
>0.1 mm/second. A total of 831 granules
from six control and 949 granules from six
JFC1-downregulated cells were analyzed.
Data are expressed as mean SEM.
Statistical analysis was performed using
unpaired t-test. B) *p < 0.05, **p < 0.03
(shRNA control versus shRNA JFC1).
C) *p < 0.05 (shRNA control versus
shRNA JFC1).
we have shown that endogenous JFC1 colocalizes with
endogenous Rab27a in neutrophils (3). Using immunofluorescence and confocal microscopy analysis, we further
demonstrate that endogenous JFC1 and MPO colocalize in
punctate structures in neutrophils (Figure 5). This result is
consistent with another study that demonstrated, by
proteomic analysis, that JFC1 is detected in the azurophilic
granule fraction (10). Although these data suggest that
JFC1 may operate as a Rab27a effector to regulate the
secretion of MPO in neutrophils, this has not yet been
demonstrated, and the role of JFC1 in exocytosis has
remained elusive so far. To analyze whether JFC1 plays
a significant role in MPO exocytosis in neutrophils, we
utilized streptolysin O (SLO)-permeabilized human polymorphonuclear leukocytes. Cockcroft demonstrated that
SLO-permeabilized neutrophils undergo exocytosis in the
presence of GTP analogs and Ca2þ and that the efficiency
of the exocytic process in this system is functionally
coupled to the fMLP receptor (24). Yaffe and collaborators
further demonstrated that the ultrastructure of neutrophils
is largely preserved in SLO-permeabilized cells. In this
study, using transmission electron microscopy, we further
demonstrate that the granular distribution is also preserved in SLO-treated neutrophils. In this way, intracellular
granules from permeabilized neutrophils conserve a separation distance of 80–180 nm from the plasma membrane,
which is frequently observed in nonpermeabilized cells
(Figure 6A). This separation is usually attributed to the
actin cortex, which acts as a barrier, preventing vesicle
docking at the plasma membrane (25). To analyze whether
JFC1 regulates azurophilic granule exocytosis in human
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neutrophils, we performed molecular interference by introducing specific antibodies directed to the amino terminal
domain of JFC1 in SLO-permeabilized neutrophils. This
approach has been previously utilized to demonstrate the
participation of the Rab27a/b effector Myrip/Slac-c2 in
amylase release in parotid acinar cells (26). Anti-JFC1
Figure 5: JFC1/Slp1 colocalizes with MPO in neutrophil azurophilic granules. Immunofluorescence analysis of the distribution of JFC1 and MPO in human neutrophils. Endogenous JFC1/
Slp1 was detected using a specific antibody raised in rabbit. For
the detection of MPO, we used an antibody raised in goat. The
nucleus was visualized by staining DNA with 40 , 6-diamidino-2phenylindole, dihydrochloride. The selected areas in the upper
panel are magnified in the lower panel to facilitate the visualization
of the colocalized proteins. Scale bar ¼ 5 mm.
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of exocytosis in neutrophils. Interestingly, although interference with JFC1 impaired fMLP-dependent secretion, no
inhibition was observed in cells primed with lipopolysaccharide (LPS) and then stimulated with fMLP even though
interference with Rab27a by means of specific anti-Rab27a
antibodies inhibited exocytosis in both primed and unprimed cells (Figure 6B). Because LPS treatment does not
induce azurophilic granule exocytosis but instead sensitizes neutrophils so that the azurophilic granule secretory
response to a subsequent stimulus is amplified [(27) and
data not shown], it is possible that LPS regulates an early
step in the exocytic process, a step that can bypass JFC1
function and, therefore, compensate for the decreased
availability of JFC1 in our assay. Alternatively, it is possible
that the LPS-induced priming mechanism triggers structural changes in the JFC1 molecule so that it becomes
resistant to antibody-mediated inhibition. For example,
LPS may induce JFC1 phosphorylation and, subsequently,
phospho-JFC1 may recognize counterpart molecules even
in the presence of antibody. More studies are necessary to
clarify which mechanism operates during LPS-dependent
priming of neutrophil exocytosis.
Figure 6: Interference with JFC1/Slp1 inhibits MPO secretion
in permeabilized human neutrophils. A) Electron micrographs of
neutrophils permeabilized with SLO (þSLO) or untreated control
(SLO). The upper panel shows that granule distribution is not
affected by SLO treatment. Granules appear separated from the
plasma membrane by distance of 80–180 nm around the perimeter
of both SLO-treated or untreated cells. The lower panel shows that
the plasma membrane is not grossly damaged in SLO-treated cells.
B) MPO exocytosis was evaluated in SLO-permeabilized neutrophils in the presence of 1 mM Mg2þ/ATP and 100 mM Ca2þ after
incubation with anti-JFC1 or anti-Rab27a antibodies. The cells were
stimulated with 1 mM fMLP. Where indicated, the cells were treated
with 100 ng/mL LPS before stimulation. Results represent the
mean SEM from three independent experiments. *p < 0.05
versus control fMLP and **p < 0.05 versus control LPS þ fMLP.
antibodies impaired the exocytosis of azurophilic granules
in permeabilized neutrophils (Figure 6B). These results
confirmed our observations performed in HL-60 granulocytes and strongly support a role for JFC1 in the regulation
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Rab27a but not JFC1/Slp1 regulates exocytosis
of MMP-9-containing granules in neutrophils
In human neutrophils, the microbicidal molecules elastase,
MPO, cathepsin G and defensins are stored in azurophilic
granules, while MMP-9, a protein involved in migration and
angiogenesis, is compartmentalized in a different set of
secretory organelles. We have previously demonstrated
that Rab27a localizes in a subpopulation of MPO-containing granules in neutrophils (3). Proteomic analysis showed
that Rab27a localizes with a group of secretory organelles
of heterogeneous density in human neutrophils (10). In this
paper, using immunofluorescence analysis, we show that
endogenous Rab27a colocalizes with MMP-9 in punctate
structures in human neutrophils (Figure 7A). We also show
a lack of colocalization between JFC1 and MMP-9 at
neutrophil granules (Figure 7B). To evaluate whether
JFC1 and Rab27a play a significant role in the exocytosis
of MMP-9-containing organelles, we used the SLO permeabilization approach described above. In Figure 7C, we
show that antibodies that interfere with Rab27a but not
with JFC1 inhibit the exocytosis of gelatinase granules
independently of the stimulus used. These results indicated that Rab27a regulates the secretion of gelatinase
and azurophilic granules in a different fashion and suggest
that Rab27a utilizes an effector molecule different from
JFC1 to regulate MMP-9 exocytosis.
The Rab27a effector Munc13-4 is a general
regulator of exocytosis in granulocytes
To further understand the mechanism of granule exocytosis in neutrophils, we focused on Munc13-4, the only
other Rab27a effector identified in neutrophils so far. We
first analyzed its subcellular distribution in human and
murine neutrophils. Using cell fractionation analysis, we
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JFC1 and Munc13-4 Regulate Neutrophil Exocytosis
show that Munc13-4 cofractionates with Rab27a-containing
granules (Figure 8A) in a fraction that colocalizes with a
subpopulation of low-density organelles containing MPO
(Figure 8A) (3). Unlike Rab27a, a significant fraction of
Munc13-4 was detected in the soluble fraction (Figure 8A,
fraction 1), suggesting that two pools of Munc13-4 coexist
in neutrophils, a membrane-associated pool and a soluble,
cytosolic pool. Immunofluorescence analysis demonstrates
that endogenous Munc13-4 distributes to punctate structures that resemble granules in both murine (Figure 8B) and
human neutrophils (Figure 8C,D). Furthermore, we show
that Munc13-4 colocalizes with MPO and MMP-9 at some
neutrophil granular structures; however, we also observed
both granules containing Munc13-4 but lacking MPO or
MMP-9 staining and granules containing MMP-9 or MPO
but lacking Munc13-4 (Figures 8C,D).
Next, to shed light on the mechanisms regulated by
Munc13-4 in neutrophils, we utilized the SLO permeabilization assay together with antibodies directed to the carboxy
terminal domain of Munc13-4. In these experiments, we
found that interference with Munc13-4 significantly inhibits
fMLP-dependent MPO secretion in LPS-primed neutrophils
(Figure 8E). These results, together with those shown in
Figure 6, suggest that azurophilic granule secretion is coordinated by both Rab27a effectors, JFC1 and Munc13-4.
The observation that in neutrophils preincubated with LPS
before fMLP stimulation, azurophilic granule secretion is
affected by interference with Munc13-4 but not with JFC1
suggests that these factors are not redundant Rab27a
effectors. These results, together with the observation that
MPO secretion was abolished in Rab27a-deficient neutrophils even under primed conditions, suggest that different
molecular mechanisms co-ordinate the process of Rab27adependent azurophilic granule exocytosis in primed or
unprimed neutrophils. Whether these mechanisms utilize
other Rab27a effectors together with JFC1 and Munc13-4 is
currently unknown and is subject of investigation in our
laboratory.
Figure 7: Interference with Rab27a but not JFC1/Slp1 impairs MMP-9 secretion in neutrophils. A) Immunofluorescence
analysis of the distribution of Rab27a and MMP-9 in human
neutrophils. Endogenous Rab27a was detected using a specific
antibody raised in rabbit, and MMP-9 was detected using an
antibody raised in goat. The arrows show areas of colocalization in
the upper panel. The lower panel shows a magnification of the
area of colocalization indicated in the upper panel. B) Immunofluorescence analysis of the distribution of JFC1 and MMP-9 in
neutrophils was performed as described in (A). The inset shows
lack of colocalization between JFC1 and MMP-9 in most vesicles.
The exception is indicated with an arrow. Scale bar ¼ 5 mm.
C) MMP-9 exocytosis was evaluated in SLO-permeabilized neutrophils as described under Figure 6. For molecular interference,
we used anti-JFC1 or anti-Rab27a antibodies raised in rabbit.
Results represent the mean SEM from three independent
experiments. *p < 0.03 versus control fMLP and **p < 0.05
versus control LPS þ fMLP.
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Our results also suggested that Rab27a but not JFC1
regulates exocytosis of MMP-9-containing granules in
neutrophils (Figure 7C). To better understand the mechanism of MMP-9 secretion, we next explored a possible role
of Munc13-4 in this process. To this end, we analyzed the
secretion of MMP-9 in permeabilized neutrophils after
molecular interference with Munc13-4. Different to that
observed for JFC1, interfering with Munc13-4 function
impaired MMP-9 secretion (Figure 8F). Altogether, these
results reveal that the secretory machineries that regulate
azurophilic and gelatinase granules in neutrophils are
different, one being dependent and the other independent
of JFC1 function.
To evaluate the role that Munc13-4 plays in neutrophil
exocytosis in further detail, we took advantage of a unique
biological system available to us, a Munc13-4-deficient
mouse (Munc13-4Jinx/Jinx) (28). The anomalous transcript
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Figure 8: Subcellular localization of Munc13-4 in neutrophils. A) Human neutrophils were disrupted by nitrogen cavitation and
fractionated using a continuous sucrose gradient. Samples were analyzed by western blot for the presence of the indicated secretory
proteins. B) Endogenous Munc13-4 was detected by immunofluorescence and confocal microscopy analysis in murine neutrophils.
Munc13-4 was detected using a specific rabbit polyclonal antibody directed to a peptide located in the carboxy terminal domain of human
Munc13-4. IgG from pre-bleeds was used as control. The nuclei were visualized by staining the DNA with 40 , 6-diamidino-2-phenylindole,
dihydrochloride. The scale bar represents 5 mm. C and D) Immunofluorescence analysis of the colocalization of MPO or MMP-9 and
Munc13-4 in human neutrophils. The insets show areas of colocalization indicated by arrows. Arrowheads indicate structures that contain
MMP-9, MPO or Munc13-4 in the absence of the counterpart protein. E and F) MPO and MMP-9 exocytosis was evaluated using SLOpermeabilized neutrophils as described under Figure 6. For molecular interference, we used anti-Munc13-4 antibodies directed to the
carboxy terminal domain of Munc13-4. Results in (E and F) represent the mean SEM from three independent experiments. *p < 0.05
versus control (IgG) LPS þ fMLP.
in Jinx is expected to generate a truncated protein of 859
amino acids. However, this truncation is undetectable after
western blot analysis, suggesting that Jinx is essentially
a null phenotype (Figure 9A). Initially, we analyzed the
hematologic parameters of Jinx and observed that total
white blood cell count in this mouse model is not significantly different from that of wild-type controls in the
absence of infection (Figure 9B). We confirmed that these
2158
mice are not neutropenic, suggesting that they express
a useful phenotype for the study of neutrophil function
(Figure 9B). Transmission electron microscopy analysis
confirmed that no gross granular abnormalities are present
in Jinx granulocytes (Figure 9C). Furthermore, no quantitative difference in the distribution of granules was
observed in peritoneal Munc13-4-deficient neutrophils
when compared with wild-type granulocytes.
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JFC1 and Munc13-4 Regulate Neutrophil Exocytosis
Figure 9: Characterization of Munc13-4Jinx/Jinx neutrophils.
A) The Munc13-4 predicted truncation is undetectable in Jinx
neutrophils. Peritoneal neutrophils were isolated from wild-type
(WT) or Jinx mice and analyzed for the expression of Munc13-4 by
western blot using an antibody raised against amino acids 1–262
of human Munc13-4 (a gift from Dr Shirakawa, Japan). The
homology between human and mouse Munc13-4 in this region
is 91%. The antibody detects wild-type murine Munc13-4 in WT
neutrophils (white arrow) but fail to detect the Munc13-4 truncation (859 aa) at the expected migration distance (99 kDa, black
arrow). NS, non-specific bands detected in both WT and Jinx. MW
markers, SeeBlue plus 2 (Invitrogen). B) Differential leukocyte
count in Munc13-4-deficient mice. Blood was obtained by cardiac
puncture from wild-type (WT) or Munc13-4Jinx/Jinx mice, and
leukocytes were counted using a HEMAVET-950 Hematology
system (n ¼ 3). No significant difference was observed between
WT and mutant mice. C) Granules of Jinx neutrophils do not
present abnormal morphology. Peritoneal neutrophils from wildtype or Jinx mice were lightly fixed in phosphate-buffered PFA.
Ultrathin sectioning was carried out on a Reichert Ultracut E
freezing ultramicrotome, mounted on parlodion-coated copper
grids and subsequently analyzed using a Philips CM100 electron
microscope. Granules were identified by their morphological
appearance and in relationship to (31). Az, azurophilic granule;
MVB, multivesicular body; Sp, specific granule.
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In subsequent studies, we analyzed the participation of
Munc13-4 in the secretion of MPO. To this end, peritoneal
neutrophils, which respond to physiological stimuli by
secreting MPO from azurophilic granules in the absence
of cytoskeleton-disrupting agents (unpublished data), were
isolated from Jinx and wild-type mice. To stimulate these
cells, we used Gram-positive heat-killed Listeria monocytogenes (HKLM), previously shown by our group to stimulate MPO secretion in murine and human neutrophils.
Results, presented in Figure 10A, show that Jinx neutrophils have a marked defect in the secretion of MPO. Next,
we evaluated exocytosis in Jinx neutrophils by following the
surface appearance of the interleukin-10 receptor (IL10R)
and LAMP-2, proteins previously demonstrated to localize
to specific granules and multivesicular bodies in human
neutrophils (29,30). Our results indicate that the mobilization
of IL10R- and LAMP-2-containing vesicles is also impaired in
Munc13-4 deficiency (Figure 10B,C). Although we attempt
to measure MMP-9 secretion in mouse neutrophils, we
were not able to detect murine MMP-9 using commercially
available antibodies. Finally, we evaluated the mobilization
of secretory organelles that contain the b2 integrin subunit
CD11b, a molecule that plays an important role in neutrophil
adhesion to the activated endothelium during the innate
immune response. We show that, different to that observed
for MPO-, IL10R- and LAMP-2-cointainig secretory organelles, the mobilization of CD11b is not impaired in Munc13-4
deficiency (Figure 10D). This result concurs with studies
from our group showing that Rab27a is not involved in the
control of CD11b-containing granule exocytosis (Figure S1).
These data also indicate that the secretory deficiency of
neutrophil exocytosis observed in Munc13-4-null murine
cells is limited to a selective group of secretory organelles
excluding the readily mobilizable pool of secretory vesicles
containing CD11b.
Discussion
In neutrophils, Rab27a controls the secretion of azurophilic
granules, but the mechanism of this regulation is currently
unknown. In this work, we identified the Rab27a effectors
JFC1 and Munc13-4 as the Rab27a molecular partners that
co-ordinate the secretion of azurophilic granules. Our
results also indicate that Rab27a and Munc13-4 but not
JFC1 regulate the secretion of a different set of secretory
organelles containing MMP-9 in these cells, suggesting
that the differences in the dynamics of exocytosis
between azurophilic and gelatinase granules may be
explained, at least in part, by the Rab27a effectors utilized
by the different secretory organelles.
Previous works had identified some of the components
involved in neutrophil exocytosis. For example, the participation of the Rho GTPase Rac2 in the regulation of
azurophilic granule exocytosis was demonstrated (31),
and a role for t-SNAREs in the regulation of specific
and azurophilic granule fusion was described (32,33).
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Brzezinska et al.
Figure 10: Munc13-4-deficient neutrophils show impaired mobilization
of secretory organelles. A–C) Neutrophils were isolated from the peritoneum
of wild-type (WT) or Munc13-4Jinx/Jinx
(JINX) mice 4 h after intraperitoneal
thioglycolate injections. The cells were
exposed to HKLM or incubated at 378C
in the absence of stimuli (NS, nonstimulated). Next, cell surfaces were
labeled with anti-MPO, anti-LAMP-2 or
anti-IL10R. The cells were fixed and
analyzed by flow cytometry. D) Exocytosis of readily mobilizable organelles
was analyzed by following the surface
expression of CD11b in PMA-stimulated
neutrophils. Each symbol represents the
result obtained using an individual
mouse. *p < 0.05, **p < 0.01.
However, the molecular mechanism of exocytosis of
neutrophil secretory organelles remains elusive. In a previous work, using a Rab27a-deficent mouse model, we
demonstrated that Rab27a plays a central role in the
secretion of azurophilic granules in neutrophils (3). Based
on previous studies showing that endogenous JFC1 binds
to Rab27a and localizes at Rab27a- and MPO-containing
granules, we hypothesized that JFC1 is the Rab27a
effector controlling azurophilic granule secretion. To test
this hypothesis, we knocked down JFC1 using specific
shRNAs and demonstrated that MPO secretion is significantly decreased in JFC1-downregulated granulocytes.
Next, using a model of molecular interference in permeabilized cells, we further demonstrated that JFC1 regulates
azurophilic granule secretion in human neutrophils.
Vesicle trafficking to the plasma membrane requires the
actin cytoskeleton (22), and disruption of the actin cytoskeleton facilitates the exocytosis of azurophilic granules and
other neutrophil secretory organelles (23). Some Rab27a
effectors are directly involved in actin-mediated trafficking.
For example, melanophilin bridges the indirect interaction
between Rab27a and the actin-binding motor protein
myosin-Va, facilitating melanosome transport (34). However, the expression of melanophilin is restricted to melanocytes, and whether members of the Slp family of Rab27a
effectors can directly or indirectly bind to actin is still unclear.
To increase our understanding of the role of JFC1 in
exocytosis, we induced actin depolymerization in JFC1deficient granulocytes using cytochalasin D and we dem2160
onstrate that, although exocytosis is increased under these
experimental conditions, the level of secretion in JFC1deficient cells is significantly lower than that observed in
control cells expressing JFC1. Because actin depolymerization is known to facilitate docking by increasing average
vesicle displacement and organelle motion, these results
suggest that JFC1 regulates exocytosis through mechanisms that are independent of actin-mediated transport.
Based on previous observations that JFC1 binds to plasma
membrane-localized specialized phospholipids, we speculate that JFC1 may play a role in vesicle docking. However,
the observation that some vesicles expressing both
Rab27a and JFC1 fluorescent proteins undergo fast movement and some remained basically motionless during the
TIRFM analysis challenged this hypothesis. A possible
simple explanation is that the vesicles in the TIRFM zone
outnumber the available docking domains at the plasma
membrane in unstimulated cells so that JFC1-positive
vesicles remain undocked. In this paper, to more directly
analyze whether JFC1 might regulate vesicle movement or
docking, we performed TIRFM analysis in JFC1-downregulated cells. We show that cells downregulated for the
expression of JFC1 have a significantly increased number
of fast moving vesicles, supporting the idea that JFC1 may
be involved in tethering or docking. Further studies are
necessary to confirm this hypothesis.
Recent studies have paid a great deal of attention to
Munc13-4, a Rab27a effector proposed to control the
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JFC1 and Munc13-4 Regulate Neutrophil Exocytosis
mechanism of lytic granule fusion in CTLs (11,12). Proteomic analysis demonstrated that Munc13-4 is expressed in
neutrophils (10); however, its function in granulocytes has
not been explored so far. Using Munc13-4-deficient neutrophils, we show that azurophilic granule exocytosis is
dramatically impaired in the absence of Munc13-4. This
result, together with the observation that molecular interference with JFC1 prevents MPO secretion, supports the
idea that both JFC1 and Munc13-4 regulate the exocytosis
of Rab27a-containing azurophilic granules. The experiments using permeabilized neutrophils presented in this
study suggest that JFC1 and Munc13-4 functions are not
redundant. Furthermore, the observation that Munc13-4
co-ordinates post-docking events in other cellular systems
(12) suggests that JFC1 regulates an early step and
Munc13-4 a late step in the mechanism of azurophilic
granule exocytosis. Interestingly, we observed that
Munc13-4-deficient neutrophils have several exocytic defects beyond the impairment in MPO secretion. This
includes decreased mobilization of IL10R- and LAMP-2containing organelles, which were shown to localize at
peroxidase-negative granules in human neutrophils. However, the subcellular localization of these granule markers
in murine neutrophils is currently unknown. Independently
of these results, our experiments showing that molecular
interference with Munc13-4 impairs MMP-9 secretion
further support the idea that Munc13-4 regulates exocytosis of peroxidase-negative granules in human neutrophils. Altogether, our results demonstrate that Munc13-4
function is important for the exocytic mechanism of
a variety of structurally and functionally different secretory
organelles in neutrophils and suggest that, unlike JFC1,
Munc13-4 would play a rather general role in the control of
exocytosis in these cells.
In this work, we show that JFC1 and Munc13-4 play
significant roles in neutrophil exocytosis and suggest that
these Rab27a effectors are important for accurate neutrophil function. The recent association between the neutrophil
secretory protein MMP-9 and the development of angiogenic processes (35) as well as the discovery of the potent
antiviral activity of neutrophil a-defensins, which localizes in
azurophilic granules (36), strongly support the importance of
the study of the mechanism of neutrophil exocytosis
beyond the participation of exocytosis in the bactericidal
mechanisms. The identification of JFC1 and Munc13-4 as
components of the secretory machinery in granulocytes
may have implications in the control of several inflammatory
and infectious diseases in which the secretion of granulocytic cargo proteins has been involved.
cerevisiae) was from Invitrogen. Paraformaldehyde (PFA) was from Electron Microscopy Science.
Antibodies
The antibodies phycoerythrin (PE)-conjugated anti-mouse CD11b, PEconjugated anti-mouse CD210 (IL-10R) and PerCP- or fluorescein isothiocyanate (FITC)-conjugated anti-mouse Gr-1 (Ly-6G and Ly-6C) were
obtained from BD Pharmingen. We also used FITC-conjugated anti-mouse
MPO (HyCult biotechnology), Alexa Fluor 647-conjugated anti-mouse
LAMP-2 (CD107b) (Biolegend) and anti-human anti-MPO (HyCult biotechnology). The polyclonal antibodies directed against JFC1 and Rab27a used
in this study were described previously (3). The polyclonal antibody directed
to the carboxy terminal peptide of human Munc13-4 was raised in rabbits
using the synthetic peptide C-1031SEEPGEVPQTRLPL1044. The antibody
directed to the amino acids 1–262 of human Munc13-4 was a generous gift
from Dr Shirakawa, Japan, and was described before (13). Other antibodies
used were goat polyclonal anti-human MMP-9 and goat polyclonal antihuman MPO (Santa Cruz Biotechnology).
Neutrophil isolation and fractionation
Neutrophils were isolated from normal donor’s blood by Ficoll density
centrifugation as previously described (37). Cell fractionation assays using
sucrose gradients were performed exactly as depicted before (3).
Cell culture and transfection
The human promyelocytic leukemia cell line HL-60 (American Type Culture
Collection) was cultured in DMEM (Gibco-BRL) supplemented with 20%
FBS (Hyclone), 0.292 mg/mL glutamine, 50 units/mL penicillin and 50 mg/mL
streptomycin at 378C in 5% CO2/air. HL-60 cells were transfected by
nucleoporation using a nucleoporator apparatus (Amaxa Biosystems) as
described before (3), except that the electrical setting X-01 was used. To
generate stably transfected JFC1-downregulated cells, we used the shRNAdelivering vectors pRS shRNA HuSH TI305069 (clone 69), HuSH TI305070
(clone 70) and HuSH TI305071 (clone 71) from Origene Technologies Inc.
Targeting sequences were provided in Figure 2. The transfected cells were
selected using puromycin as described by the manufacturer.
Stimulated secretion in SLO-permeabilized
human neutrophils
Secretory assays using SLO-permeabilized cells were performed as previously described (26). Briefly, neutrophils were washed twice with PBS
and resuspended in permeabilization medium (26). Human neutrophils
(2.5 106) were resuspended in permeabilization buffer (100 mL) and
transferred to a tube containing 2 mL of 2500 units/mL SLO in the presence
of the indicated antibodies (50 mg/mL) and incubated at 378C for 5 min.
Where indicated, the cells were incubated with LPS (100 ng/mL) for 30 min
and stimulated with fMLP (1 mM) for additional 30 min. The reactions were
stopped by transferring the samples to ice and immediately centrifuged at
16000 g at 48C for 5 min. Supernatants were stored at 808C until the
assays were performed. The condition medium was diluted (1:200), and the
concentrations of MPO and MMP-9 in the medium were determined by
enzyme-linked immunosorbent assay (ELISA) (Assay Design and Calbiochem, respectively) following the manufacturer instructions.
Experimental animal model
Materials and Methods
The Munc13-4-deficient mouse model Jinx was generated by random germline mutagenesis using the alkylating agent N-ethyl-N-nitrosourea by Dr Bruce
Beutler (28). Munc13-4Jinx/Jinx was maintained as a homozygous stock for use
in these studies. C57BL/6J control mice were obtained from the animal
resource center at Scripps. All studies were performed using 6- to 8-week old
mice and conducted according to National Institutes of Health and institutional
guidelines and with approval of the institutional animal review board.
Materials
Isolation of murine neutrophils
LPS (Escherichia coli, serotype R515) was obtained from Alexis Biochemicals. HKLM was obtained from InvivoGen, and Zymosan (Saccharomyces
Peripheral murine neutrophils were obtained from blood collected by cardiac puncture in K3EDTA MiniCollect tubes (Greiner Bio-One). Erythrocytes
Traffic 2008; 9: 2151–2164
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Brzezinska et al.
were removed by lysis using a solution consisting of 168 mM NH4Cl, 10 mM
KHCO3 and 0.097 mM ethylenediaminetetraacetic acid (EDTA). Neutrophils
were further isolated using a Percoll gradient fractionation system previously described (38). For the isolation of mouse peritoneal neutrophils,
animals were injected intraperitoneally with 1 mL of a sterile 4% thioglycollate solution. Polymorphonuclear cells were harvested 4 h after injection
by lavage of the peritoneal cavity with RPMI medium. After isolation, cells
were kept on ice until used. Purity was analyzed by morphology, Giemsa
staining and fluorescence-activated cell sorter (FACS) using anti-Ly6G (Gr-1)
monoclonal antibody.
Mobilization of CD11b in murine neutrophils
Peripheral blood was collected by cardiac puncture in K3EDTA MiniCollect
tubes. To avoid activation during isolation, the cells were stimulated in
whole blood (50 mL) using HKLM (108 particles/mL) or PMA (0.1 mg/mL).
After 1 h, reactions were terminated by placing tubes on ice, and cells were
immunostained for 40 min at 48C with antibodies anti-CD11b-PE and anti
Ly6G (Gr-1)-PerCP. Mouse immunoglobulin G (IgG)1 and IgG2b conjugated
with the appropriate fluorophore were used as isotype controls. Murine
erythrocytes were lysed using a hypotonic solution consisting of 168 mM
NH4Cl, 10 mM KHCO3 and 0.097 mM EDTA for 2 min, and leukocytes were
subsequently fixed with 1% PFA. Neutrophils were gated based on staining
with the granulocyte marker Gr-1 and then CD11b-specific staining was
measured in Gr-1-positive cells. Data were collected using FACSCalibur
flow cytometer (BD Biosciences) and analyzed using CELLQUEST software.
Mobilization of azurophilic granules and other
secretory markers in murine neutrophils
To follow azurophilic granule mobilization in murine neutrophils, we took
advantage of the ability of some neutrophil granule proteins to bind to the
outer cellular surface after exocytosis. This property has been shown for
MPO (39) as well as for other granular proteins including elastase (40) and
MMP-9 (41). Peritoneal neutrophils were activated using HKLM as
described above and subsequently fixed with 1% PFA. The surface
increase of MPO in Gr-1-positive cells was detected by flow cytometry
analysis with FITC-conjugated anti-mouse MPO antibodies (HyCult biotechnology). The analysis of MPO release by flow cytometry correlated
with data obtained using an anti-murine MPO ELISA kit (HyCult biotechnology). The quantitative analysis of MPO released to the conditioned
medium by ELISA was performed according to manufacturer instructions.
The surface expression of IL10R and LAMP-2 was analyzed using antiIL10R-PE and anti-LAMP-2 Alexa Fluor 647 or appropriate isotype controls.
IL10R- and LAMP-2-specific staining was measured in Gr-1-positive cells by
flow cytometry.
Gel electrophoresis and western blotting
Proteins were separated by gel electrophoresis using NuPAGE gels and
MOPS buffer (Invitrogen). Proteins were transferred onto nitrocellulose
membranes for 150 min at 60 V and 48C. The membranes were blocked
with PBS containing 5% (w/v) blotting grade nonfat dry milk blocker (BioRad) and 0.05% (v/v) Tween-20. The proteins were detected by probing the
membranes with the indicated primary antibodies at appropriate dilutions.
The detection system used horseradish peroxidase-conjugated secondary
antibodies (Bio-Rad) and the LumiGlo chemiluminescence substrate system (Upstate Biotechnology). Transferred proteins were visualized using
Hyperfilm (Amersham Bioscience).
TIRF microscopy analysis
HL-60 cells were transfected by nucleofection as described above using
2 mg of the vectors for the expression of EGFP-Rab27a, DsRED-JFC1 (42),
EGFP-LAMP-3 (a gift from Dr G. Griffiths) or control vectors EGFP or DsRed
(Clontech). DMEM (300 mL) was added to the transfection cuvette immediately after the electrical pulse, and cells were transferred to poly-lysinecoated Lab-Tek chambered cover glass 1.5 (Nunc). The cells were
recovered for 24–48 h. Two hours before experiments, medium was
replaced with phenol red-free DMEM. TIRFM experiments were performed
2162
using a 100 1.45 numerical aperture (NA) TIRF objective (Nikon) on
a Nikon TE2000U microscope custom modified with a TIRF illumination
module. Cells were maintained at 308C during observations (Bionomic
controller, BC-100; 20/20 Technologies Inc.). Laser illumination (488 and
568 nm laser lines) was adjusted to impinge on the coverslip at an angle to
yield a calculated evanescent field depth of 108 nm. Images were acquired
on a 14 bit, cooled charge-coupled device camera (Hamamatsu) controlled
through METAMORPH software (Molecular Devices Inc.). The images were
recorded using 500 milliseconds exposures at 1-second intervals. Images
were analyzed using IMAGEJ and IMARIS software. All data analysis was
performed by tracking granule movement through all frames of the movies.
All vesicles that appeared in the TIRFM zone during the length of the study
were included in the analysis.
Immunofluorescence and confocal
microscopy analysis
Human or murine neutrophils were seeded at 70% confluence in an eightwell chambered cover glass (pretreated with poly-L-lysine at 0.01% in PBS),
fixed with 3.7%PFA, permeabilized with 0.01% saponin and blocked with
a solution of 1% BSA in PBS. To stain the nucleus, some samples were
incubated with 40 , 6-diamidino-2-phenylindole, dihydrochloride for 5 min at
218C. Samples were labeled with the indicated primary antibodies overnight at 48C and then the appropriate combination of the secondary
antibodies (488 and/or 594 nm) Alexa-Fluor-conjugated donkey anti-rabbit,
anti-mouse and/or anti-goat (Molecular Probes). Cells were stored in
Fluoromount-G (Southern Biotechnology) and analyzed using a Bio-Rad
(Zeiss) 2100 Rainbow Radiance laser scanning confocal microscopy
attached to a Nikon TE2000-U microscope at 218C with a 60 oil Plan
Apo 1.4 NA objective. For visualization, fluorescence associated with Alexa
Fluor 594-labelled secondary antibody was excited using the 543 nm laser
line and collected using a standard Texas Red filter. Fluorescence associated with Alexa-Fluor-488-labeled secondary antibodies was visualized
using the 488 nm laser line and collected using a standard FITC filter set.
Images were collected using the Bio-Rad LASERSHARP 2000 (version 3.2)
software and processed using IMAGEJ and ADOBE PHOTOSHOP CS.
Transmission electron microscopy
Human neutrophils (2 106) were incubated for 10 min at 378C in the
presence or absence of SLO as described above. Human or murine
neutrophils were processed for transmission electron microscopy using
a modification of the protocol of Gilula et al. (43). The samples were fixed in
2.5% glutaraldehyde in 0.1 M Na cacodylate buffer (pH 7.3), buffer washed
and fixed in 1% osmium tetroxide in 0.1 M Na cacodylate buffer. They were
subsequently treated with 0.5% tannic acid followed by 1% sodium sulfate.
The pellets were treated with propylene oxide and embedded in Epon/
Araldite. Thin sections (70 nm) of the pelleted samples were cut on a
Reichert Ultracut E (Leica) using a diamond knife (Diatome; Electron
Microscopy Sciences), mounted on parlodion-coated copper slot grids
and stained in uranyl acetate and lead citrate. Sections were examined on
a Philips CM100 TEM (FEI) and documented on Kodak SO-163 film for
later analysis. Negatives were scanned at 600 lpi using a Fuji FineScan
2750xl and then converted to tif format for subsequent handling in ADOBE
PHOTOSHOP.
Statistical analysis
Results are presented as mean SEM. Statistical analysis was calculated
using the Student’s t-test. A value of p < 0.05 was considered as
statistically significant.
Acknowledgments
The study was financially supported by U.S. Public Health Service Grant AI024227 and by the Sam and Rose Stein Endowment Fund. We thank Dr G.
Danuser for assistance with TIRF microscopy and Dr M. Wood for
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JFC1 and Munc13-4 Regulate Neutrophil Exocytosis
assistance with electron microscopy analysis. We also thank Dr Shirakawa
and Dr G. Griffiths for generously contributing the anti-Munc13-4 antibody
and the EGFP-LAMP-3 expression vector, respectively.
Supporting Information
Additional Supporting Information may be found in the online version of
this article:
Movie S1: Dynamics of EGFP-Rab27a-containing vesicle mobilization
visualized by TIRFM.
Movie S2: Dynamics of DsRed-JFC1-containing vesicle mobilization visualized by TIRFM.
Movie S3: Dynamics of vesicles expressing both EGFP-Rab27a and
DsRed-JFC1visualized by TIRFM.
Movie S4: Analysis of vesicle mobilization in HL-60 cells expressing EGFPRab27a and DsRed-JFC1 using IMARIS software.
Movie S5: Dynamics of EGFP-LAMP-3-containing granules in a wild-type
granulocyte.
Movie S6: Dynamics of EGFP-LAMP-3-containing granules in a JFC1downregulated granulocyte.
Figure S1: Mobilization of CD11b from intracellular granules is not
affected in Rab27a2/2 neutrophils. Leukocytes were isolated from
peripheral blood of wild-type or ashen mice. The cells were stimulated
with fMLP or left untreated (NS, non-stimulated). After stimulation, cells
were washed with PBS containing 0.5% BSA and double labeled for the
granulocyte marker Gr-1 (FITC) and for CD11b (PE) or incubated with
isotype controls (BD Biosciences) at 48C for 40 min in a final volume of
50 mL. Next, cells were washed and resuspended in 1% PFA. Data were
collected using a FACSCalibur flow cytometer (BD Biosciences) and
analyzed using CELLQUEST software. No shift was observed in the isotype
controls (data not shown). The data are representative of three different
experiments with similar results.
Table S1: Quantitative analysis of JFC1- and Rab27a-containing vesicle
dynamics. Vesicles expressing both JFC1 and Rab27a (n ¼ 254) were
analyzed over 1 min. All granules that appeared during this time frame were
considered in the analysis. Motile granules were defined as those with
displacement larger than 0.5 mm and a velocity of 0.1 mm/second or greater
(21). According to this classification, 46.3% of total granules were motile.
Please note: Wiley-Blackwell are not responsible for the content or
functionality of any supporting materials supplied by the authors. Any
queries (other than missing material) should be directed to the corresponding author for the article.
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