Accepted Manuscript Title: Carp thrombocyte phagocytosis requires activation factors secreted from other leukocytes Author: Takahiro Nagasawa, Tomonori Somamoto, Miki Nakao PII: DOI: Reference:
S0145-305X(15)00105-6 http://dx.doi.org/doi:10.1016/j.dci.2015.05.002 DCI 2391
To appear in:
Developmental and Comparative Immunology
Received date: Revised date: Accepted date:
7-4-2015 7-5-2015 8-5-2015
Please cite this article as: Takahiro Nagasawa, Tomonori Somamoto, Miki Nakao, Carp thrombocyte phagocytosis requires activation factors secreted from other leukocytes, Developmental and Comparative Immunology (2015), http://dx.doi.org/doi:10.1016/j.dci.2015.05.002. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
Short communication
2 3
Carp thrombocyte phagocytosis requires activation factors secreted from other
4
leukocytes
5 6
Takahiro Nagasawa, Tomonori Somamoto *, and Miki Nakao
7
Laboratory of Marine Biochemistry, Department of Bioscience and Biotechnology,
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Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University,
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Fukuoka 812-8581, Japan
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*Corresponding author: Tomonori Somamoto,
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Phone: (81)-92-642-2895; Fax: (81)-92-642-2897
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E-mail: [email protected]
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Highlights
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・Isolated carp thrombocytes lose their phagocytic abilities
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・Thrombocyte phagocytosis is activated by other leukocytes
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・Soluble factors secreted by leukocytes activate thrombocyte phagocytosis
29 30
Abstract
31
Thrombocytes are nucleated blood cells in non-mammalian vertebrates, which were
32
recently focused on not only as hemostatic cells but also as immune cells with potent
33
phagocytic activities. We have analyzed the phagocytic activation mechanisms in
34
common carp (Cyprinus carpio) thrombocytes. MACS-sorted mAb+ thrombocytes
35
showed no phagocytic activity even in the presence of several stimulants. However,
36
remixing these thrombocytes with other anti-thrombocyte mAb− leukocyte populations
37
restored their phagocytic activities, indicating that carp thrombocyte phagocytosis
38
requires
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anti-thrombocyte mAb− leukocytes harvested after PMA or LPS stimulation, but not
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culture supernatant from unstimulated leukocytes, could activate thrombocyte
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phagocytosis. This proposed mechanism of thrombocyte phagocytosis activation
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involving soluble factors produced by activated leukocytes suggests that thrombocyte
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activation is restricted to areas proximal to injured tissues, ensuring suppression of
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excessive thrombocyte activation and a balance between inflammation and tissue repair.
an
appropriate
exogenous
stimulation.
Culture
supernatant
from
45 46
Keywords
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Thrombocyte, phagocytosis, fish immunity, cytokine, platelet.
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2 Page 2 of 18
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List of abbreviations
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ADP: adenosine diphosphate, IFNγ: interferon gamma, LPS: lipopolysaccharide, mAb:
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monoclonal antibody, MACS: magnetic-activated cell sorting, PBL: peripheral blood
52
leukocyte, PMA: phorbol 12-myristate 13-acetate.
53 54
1. Introduction
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Thrombocytes are nucleated blood cells in non-mammalian vertebrates that are
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considered functional homologues of mammalian platelets. Platelets and thrombocytes
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share characteristics involved in hemostasis; in brief, they aggregate in response to
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several damage signals, including collagen, to initiate blood clotting (Belamarich et al.,
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1966; Nakayasu et al., 1997). In addition to their primary functions, platelets and
60
thrombocytes are also important mediators of both the innate and adaptive immune
61
systems (Elzey et al., 2003; Ferdous and Scott, 2015). Particularly, nucleated
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thrombocytes have a potential to become powerful immune cells owing to their
63
phagocytic activities that kill internalized bacteria (Hill and Rowley, 1998; Stosik et al.,
64
2002; Nagasawa et al., 2014). Many studies reported that mammalian platelets may
65
have phagocytic activity; however, their ability to engulf microbes remains
66
controversial (White, 2006; Antczak et al., 2011). On the other hand, nucleated
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thrombocytes express MHC class II molecules (Köllner et al., 2004; St. Paul et al.,
68
2012; Nagasawa et al., 2014, Fink et al., 2015), suggesting that they have the potential
69
to become antigen-presenting cells (e.g., macrophages and dendritic cells), which
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present extracellular antigens. Elucidation of these phagocytic thrombocytes and
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platelets may be important for understanding immune systems, particularly in lower
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vertebrates. 3 Page 3 of 18
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Phagocytosis is the process of engulfing large particles into intracellular vacuoles,
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contributing to the elimination of pathogenic microbes. In professional phagocytes such
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as neutrophils and macrophages, phagocytosis is triggered by the recognition of
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pathogen-associated
77
immunoglobulins and complement components bound to the microbes, which are
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recognized by phagocytosis-promoting receptors (Jutras and Desjardins, 2005). These
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phagocytic abilities are also enhanced by several inflammatory cytokines such as
80
interferon gamma (IFNγ) and tumor necrosis factor (TNF) produced by several immune
81
cells (Shalaby et al., 1985). Similar regulatory mechanisms are also conserved in fish
82
(Grayfer et al., 2009).
molecular
patterns
(PAMPs)
and
opsonins
such
as
83
Platelet activation for hemostasis has been well studied and is induced by various
84
factors, including collagen, adenosine diphosphate (ADP), and thrombin. Upon
85
activation, platelets morph into a stellate form and secrete various factors to trigger
86
coagulation and inflammation (Weyrich and Zimmerman, 2004). Furthermore, although
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nucleated thrombocytes are activated by collagen and other molecules to facilitate
88
coagulation, fish thrombocytes responded differently, including being unresponsive to
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ADP (Belamarich et al., 1966; Matsushita et al., 2004). However, whether phagocytosis
90
activation pathway is similar to typical hemostasis pathway remains unknown. For
91
understanding the immune functions of thrombocytes, the activation mechanism for
92
their phagocytic activity is required. In the present study, we evaluated activation
93
mechanism of fish thrombocyte phagocytosis, including cell exposure to several
94
stimulants and the influence of other leukocytes. Thrombocyte phagocytic activity was
95
not induced by typical platelet stimulants, but was dramatically triggered by the
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presence of other leukocytes and by activated leukocyte cell culture supernatants. These 4 Page 4 of 18
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results reveal mechanisms regulating thrombocyte phagocytosis in immune system and
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tissue maintenance.
99 100
2. Materials and methods
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2.1. Fish
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Common carp (Cyprinus carpio, approximately 100 g) were maintained in our
103
laboratory at 25°C and fed with commercial pellets. All animal experiments were
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performed in accordance with the guidelines of the Animal Experiments Committee at
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Kyushu University.
106 107
2.2. Thrombocyte isolation and purification
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Carp peripheral blood samples were collected from caudal veins using heparinized
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syringes, diluted with medium (RPMI-1640; Nissui Pharmaceutical), and overlaid onto
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Percoll adjusted to a concentration of 1.08 g/mL (BD Biosciences). Samples were then
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centrifuged at 500 × g for 30 min at 4°C to isolate peripheral blood leukocytes (PBLs).
112
PBLs were harvested from the top of the Percoll layer, washed twice with the medium
113
by centrifugation at 500 × g for 10 min at 4°C, and adjusted to a concentration of 1 ×
114
107 cells/mL with medium. The PBLs were then incubated with an HB8 monoclonal
115
antibody (mAb) specific to carp thrombocytes (Nakayasu et al., 1997) for 30 min on ice.
116
After washing with medium twice, the PBLs were incubated with MACS microbeads
117
coupled to a goat anti-mouse IgG antibody (Miltenyi Biotec). After washing with
118
medium twice, the PBLs were resuspended in RPMI-1640 containing 2 mM EDTA and
119
5% fetal bovine serum (FBS), and then loaded on a mini MACS column (Miltenyi) to
120
purify the thrombocytes. Samples were passed through the column twice to obtain 5 Page 5 of 18
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thrombocytes at high purity. Purified thrombocytes were stained with phycoerythrin
122
(PE)-conjugated goat anti-mouse IgG antibody (Sigma–Aldrich) for 30 min at 4°C.
123 124
2.3. Thrombocyte stimulation
125
The purified thrombocytes (1 × 107 cells/mL) were incubated for 30 min at 25°C
126
with medium containing lipopolysaccharides (LPS from Escherichia coli O55, 10
127
μg/mL; Wako Pure Chemical), phorbol 12-myristate 13-acetate (PMA, 1 μg/mL; Sigma),
128
collagen type I (1 μg/mL; Wako), ADP (1 μg/mL; Tokyo Chemical Industry), or
129
thrombin (1 unit/mL; from bovine, Sigma). Next, after washing twice, the cells were
130
resuspended in medium at the same concentration.
131 132
2.4. Preparation of leukocyte culture supernatants
133
HB8 mAb-negative (HB8−) leukocytes purified with MACS microbeads were
134
stimulated with either LPS or PMA in the same manner as described above, washed 3
135
times, adjusted to 5 × 107 cells/mL with medium, and then incubated for 60 min at 25°C.
136
The culture supernatants were collected by centrifugation at 1,000 × g for 10 min at 4°C
137
3 separate times to remove all leukocytes.
138 139
2.5. Phagocytosis assay
140
Stimulated thrombocytes (1 × 107 cells/mL) were incubated with 1 μm fluorescent
141
latex beads (Fluoresbrite Yellow Green Microspheres; Polysciences, Warrington, PA,
142
USA) at a cell-to-bead ratio of 1:5 for 3 h at 25°C in 100 μL of medium. To evaluate the
143
effect of other leukocytes on activation, thrombocytes and HB8− leukocytes were mixed
144
at 5 × 106 cells/mL each and incubated as previously described. To evaluate the effect of 6 Page 6 of 18
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soluble factors produced by leukocytes, thrombocytes were resuspended in 50% HB8−
146
leukocyte culture supernatant diluted in medium and incubated with fluorescent beads
147
as previously described. Flow cytometry (Epics XL, Beckman Coulter) was used to
148
measure the percentage of phagocytic thrombocytes in each experimental condition.
149 150
2.6. Bacterial phagocytosis
151
FITC-conjugated Escherichia coli (DH5α strain) were incubated with 10% normal
152
carp serum in PBS in the presence of 2 mM Mg2+ and 2 mM Ca2+ for 30 min at 25°C.
153
As a control, bacteria were incubated with heat-inactivated (20 min, 50ºC) carp serum
154
containing 10 mM EDTA under the same conditions. After washing with PBS 3 times,
155
the treated bacteria were adjusted to OD600nm = 0.5 and incubated with the carp
156
thrombocytes using the same conditions as described for the incubation with the beads.
157
The percentage of phagocytic thrombocytes was measured by flow cytometry.
158 159
3. Results and Discussion
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3.1. Purified thrombocytes are not phagocytic
161
As previously described (Nagasawa et al. 2014), carp thrombocytes in peripheral
162
leukocyte pool actively ingest latex beads. In the present study, purified thrombocytes
163
were used to assess the direct influence of several different stimulants individually on
164
thrombocyte phagocytic activity. Purified carp thrombocytes incubated with 1-μm beads
165
in the same manner as PBL incubation rarely ingested the beads (2.1 ± 0.7%; Fig. 1A,
166
right). The phagocytic capacity of thrombocytes in the blood leukocyte samples was
167
verified just before cell sorting, indicating that the anti-thrombocyte mAb staining did
168
not inhibit thrombocyte phagocytosis (23.9 ± 5.1%; Fig. 1A, left). Based on these 7 Page 7 of 18
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findings, we hypothesized that thrombocyte phagocytosis may be triggered by activators
170
derived from other leukocytes.
171
Next, we individually assessed the ability of several potential stimulants to enhance
172
purified thrombocyte phagocytic activity. For this assessment, thrombocytes were
173
preincubated with different chemicals before mixing with the beads. Although the
174
thrombocytes were exposed to typical platelet stimulators such as collagen, ADP, bovine
175
thrombin, LPS, and PMA, none of them enhanced the thrombocyte bead ingestion (Fig.
176
1B). Moreover, the effects of these stimulants on thrombocyte morphology were
177
examined by microscopy. In the absence of these reagents, thrombocytes formed a
178
typical spindle shape (see Fig. S1A). Upon incubation with collagen, thrombocytes
179
immediately adhered to the culture plate (Fig. S1B). In the presence of PMA, known to
180
induce platelet activation associated with CD62P expression (Baudouin-Brignole et al.,
181
1997), thrombocytes assumed round or oval forms with many cells adhering to the
182
culture plate (Fig. S1C). ADP only weakly induced similar morphological change of the
183
thrombocytes (Fig. S1D). These results suggest that thrombocyte phagocytic activity is
184
activated in a manner that is distinct from hemostatic aggregation. LPS and thrombin
185
did not induce morphological changes in the thrombocyte (data not shown). Mammalian
186
platelets express TLR4 to sense LPS, which does not induce a typical platelet activation
187
and aggregation (Cognasse et al., 2005; Ward et al., 2005); however, LPS enhances the
188
secretion of several cytokines in platelets and expression of them in chicken
189
thrombocytes (Cognasse et al., 2008; Scott and Owens, 2008). These data suggest that
190
thrombocyte phagocytosis is activated through a different mechanism than that by
191
which cytokine production is activated. The finding that bovine thrombin did not affect
192
carp thrombocytes may be the result of interspecies incompatibility. 8 Page 8 of 18
193 194
3.2. Thrombocyte phagocytosis is activated by co-incubation with leukocytes
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Next, we evaluated the effects of other leukocytes on thrombocyte phagocytosis.
196
Purified thrombocytes were remixed with leukocytes (HB8 mAb− fraction) and
197
incubated with fluorescent beads. Notably, the remixed thrombocytes recovered their
198
phagocytic abilities (24.9 ± 5.2%; Fig. 2A), suggesting that co-incubation with other
199
leukocytes triggered thrombocyte phagocytic activity. This result also showed that cell
200
purification did not irreversibly harm the ability of thrombocytes to ingest particles.
201
Thrombocytes were also incubated with leukocytes that had been preincubated with
202
LPS or PMA. These stimulated HB8− leukocytes ingested the beads more actively than
203
control leukocytes incubated without stimulation (Fig. 2A). Remarkably, co-incubation
204
with LPS-stimulated leukocytes enhanced thrombocyte phagocytic activity more
205
strongly (35.5 ± 5.5%) than co-incubation with unstimulated leukocytes, suggesting that
206
LPS-stimulation improved the leukocyte abilities to activate the thrombocyte
207
phagocytosis.
208
We also assessed the effect of opsonization on purified thrombocyte phagocytosis.
209
In a previous study, carp thrombocyte phagocytosis was enhanced by serum
210
opsonization of bacterial particles in the presence of other leukocytes (Nagasawa et al.,
211
2014). In the flow cytometry analysis, very few thrombocytes interacted with bacteria
212
even preincubated with serum (control, 1.3 ± 0.3%; opsonized, 1.4 ± 0.5%; Fig. 2B),
213
whereas the ingestion of thrombocytes that had been remixed with HB8− PBLs was
214
enhanced by opsonization (control, 14.4 ± 1.3%; opsonized, 17.9 ± 1.7%). These results
215
suggested that phagocytosis-promoting receptors such as complement receptors may not
216
be expressed on the surface of thrombocytes in the resting phase; however, after 9 Page 9 of 18
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activation, the thrombocytes express these receptors and are able to recognize the
218
opsonized antigens.
219 220
3.3. Phagocytic thrombocytes activated by leukocyte secretary components
221
To assess whether thrombocyte activation by other leukocytes was mediated by
222
soluble factors released from the leukocytes or rather was dependent on cell-to-cell
223
contacts, purified thrombocytes were incubated with labeled beads in the presence of
224
HB8− leukocyte cell culture supernatant. When the thrombocytes were incubated with
225
unstimulated leukocyte supernatant, thrombocyte phagocytosis did not increase (2.1 ±
226
1.4%; Fig. 2C).
227
Remarkably, when the thrombocytes were incubated with labeled beads in the
228
presence of LPS- or PMA-stimulated leukocyte cell culture supernatant, the number of
229
phagocytic thrombocytes dramatically increased (LPS, 13.9 ± 1.8%; PMA, 9.9 ± 2.0%).
230
This finding indicates that the stimulated leukocytes secreted factors that triggered
231
thrombocytes to ingest the labeled beads. This result also suggests that only activated
232
leukocytes release the thrombocyte activation factors that trigger phagocytic activity.
233
Macrophages are traditional phagocytic cells that are differentially activated by
234
microbial stimulants and the presence or absence of cytokines excreted by other
235
leukocyte subtypes such as natural killer cells and Th1/Th2 cells for regulation which
236
contributes to prevent them from damaging host tissues (Mantovani et al., 2004; Gordon
237
and Taylor, 2005; Mosser and Edwards, 2008; Forlenza et al., 2011). Likewise,
238
thrombocyte phagocytosis also may be regulated by inflammatory signals released by
239
other leukocytes. Thus, thrombocytes may only be recruited as phagocytes in the acute
240
phase of inflammation or pathogen invasion. 10 Page 10 of 18
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Our results suggest that even though the carp thrombocytes recognized antigens,
242
they did not become phagocytic in the absence of inflammatory signals originating from
243
other immune components. In mammals, generalized platelet activation often causes
244
serious thrombocytopenia, similar to that in sepsis (Lupu et al., 2014). Even in carp, it
245
was reported that infection with the parasite Trypanoplasma borreli dramatically
246
reduced the number of thrombocytes in the host blood (Fink et al., 2015). These
247
thrombocytopenic conditions can cause a serious decline in host health. Thus, a
248
thrombocyte regulatory system may help prevent excessive activation.
249 250
4. Conclusions
251
Our results indicate that the initiation of thrombocyte phagocytosis requires soluble
252
factors secreted from activated leukocytes. Isolated thrombocytes did not actively ingest
253
particles; however, their phagocytic activity was restored by incubation with either
254
HB8− leukocytes or stimulated leukocyte cell culture supernatant. Cell culture
255
supernatant from unstimulated PBLs did not induce phagocytosis, indicating that the
256
activating factors are produced upon immune system activation. These results indicate
257
that thrombocyte phagocytic activity requires a different activation pathway than the
258
typical route for hemostatic activation. These regulatory mechanisms may contribute to
259
the control of tissue repair and inflammation. Future studies will help establish the
260
details of thrombocyte phagocytic activation, such as the cell types and molecules
261
capable of inducing phagocytosis.
262 263
Acknowledgements
264
This research was supported in part by a Grant-in-Aid for Scientific Research (B) 11 Page 11 of 18
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(Grant Number 25292127) from the Japan Society for the Promotion of Science (JSPS).
266 267
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Figure legends
371
Fig. 1. (A) Flow cytometry scatter plot of unsorted carp PBLs and purified
372
thrombocytes incubated with 1 μm beads. Data are representative of 3 independent
373
experiments. (B) Percentages of phagocytic thrombocytes after preincubation with
374
collagen (1 μg/ml), adenosine diphosphate (ADP; 10 μg/ml), thrombin (10 units/ml),
375
lipopolysaccharide (LPS from Escherichia coli O55; 10 μg/ml) or phorbol 12-myristate
376
13-acetate (PMA; 1 μg/ml). For co-incubation (thrombin/PBLs), thrombocytes, and
377
sorted HB8− PBLs (5 × 106 cells/ml each) were mixed and then incubated with the
378
labeled beads. PBLs: peripheral blood leukocytes. The asterisks (*) indicate a
379
significant difference from the control as analyzed by Tukey’s test (**P < 0.01). Data
380
are the average rates from 3 independent experiments, analyzed at the same time as the
381
assays in Figs. 1B, 2A, and 2C.
382 383
Fig. 2. (A) Percentages of phagocytic cells after cells were remixed and then incubated
384
with fluorescent beads. Thrombocytes (5 × 106 cells/ml) were incubated with the beads
385
in the presence of HB8− PBLs (5 × 106 cells/ml) preincubated with cells alone or with
386
LPS (10 μg/ml) or PMA (1 μg/ml). Peripheral blood leukocytes (PBLs) in the HB8−
387
fraction. Different letters indicate significant differences as analyzed by Tukey's test (P
388
< 0.05). (B) Percentages of phagocytic cells incubated with fluorescein isothiocyanate
389
(FITC) -conjugated bacteria. Thrombocytes (5 × 106 cells/ml alone or 5 × 106 cells/ml
390
with 5 × 106 cells/ml of HB8− leukocytes) were incubated with FITC-E. coli (OD600nm =
391
0.5) pretreated with normal carp serum (op) or heat-inactivated carp serum (con).
392
Peripheral blood leukocytes (PBLs) in the HB8− fraction. The asterisks (*) indicate a
393
significant difference from the control group (thrombin/PBL con) as analyzed by 17 Page 17 of 18
394
Tukey’s test (*P < 0.05). (C) Percentages of phagocytic thrombocytes after stimulation
395
with cell culture supernatants from other leukocytes. Thrombocytes (1 × 107 cells/ml)
396
were incubated with 1 μm beads in the presence of culture supernatants from HB8−
397
leukocytes incubated alone (con) or with LPS (10 μg/ml) or PMA (1 μg/ml). Peripheral
398
blood leukocytes (PBLs) in the HB8− fraction. The asterisks (*) indicate significant
399
differences from the control group (throm. only) as analyzed by Tukey’s test (*P < 0.05;
400
**P < 0.01). Data are the average rates from 3 independent experiments, analyzed at the
401
same time as the assays in Figs. 1B, 2A, and 2C.
18 Page 18 of 18
S0145-305X(15)00105-6 http://dx.doi.org/doi:10.1016/j.dci.2015.05.002 DCI 2391
To appear in:
Developmental and Comparative Immunology
Received date: Revised date: Accepted date:
7-4-2015 7-5-2015 8-5-2015
Please cite this article as: Takahiro Nagasawa, Tomonori Somamoto, Miki Nakao, Carp thrombocyte phagocytosis requires activation factors secreted from other leukocytes, Developmental and Comparative Immunology (2015), http://dx.doi.org/doi:10.1016/j.dci.2015.05.002. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
Short communication
2 3
Carp thrombocyte phagocytosis requires activation factors secreted from other
4
leukocytes
5 6
Takahiro Nagasawa, Tomonori Somamoto *, and Miki Nakao
7
Laboratory of Marine Biochemistry, Department of Bioscience and Biotechnology,
8
Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University,
9
Fukuoka 812-8581, Japan
10 11 12 13 14 15 16 17 18 19 20
*Corresponding author: Tomonori Somamoto,
21
Phone: (81)-92-642-2895; Fax: (81)-92-642-2897
22
E-mail: [email protected]
23 24 1 Page 1 of 18
25
Highlights
26
・Isolated carp thrombocytes lose their phagocytic abilities
27
・Thrombocyte phagocytosis is activated by other leukocytes
28
・Soluble factors secreted by leukocytes activate thrombocyte phagocytosis
29 30
Abstract
31
Thrombocytes are nucleated blood cells in non-mammalian vertebrates, which were
32
recently focused on not only as hemostatic cells but also as immune cells with potent
33
phagocytic activities. We have analyzed the phagocytic activation mechanisms in
34
common carp (Cyprinus carpio) thrombocytes. MACS-sorted mAb+ thrombocytes
35
showed no phagocytic activity even in the presence of several stimulants. However,
36
remixing these thrombocytes with other anti-thrombocyte mAb− leukocyte populations
37
restored their phagocytic activities, indicating that carp thrombocyte phagocytosis
38
requires
39
anti-thrombocyte mAb− leukocytes harvested after PMA or LPS stimulation, but not
40
culture supernatant from unstimulated leukocytes, could activate thrombocyte
41
phagocytosis. This proposed mechanism of thrombocyte phagocytosis activation
42
involving soluble factors produced by activated leukocytes suggests that thrombocyte
43
activation is restricted to areas proximal to injured tissues, ensuring suppression of
44
excessive thrombocyte activation and a balance between inflammation and tissue repair.
an
appropriate
exogenous
stimulation.
Culture
supernatant
from
45 46
Keywords
47
Thrombocyte, phagocytosis, fish immunity, cytokine, platelet.
48
2 Page 2 of 18
49
List of abbreviations
50
ADP: adenosine diphosphate, IFNγ: interferon gamma, LPS: lipopolysaccharide, mAb:
51
monoclonal antibody, MACS: magnetic-activated cell sorting, PBL: peripheral blood
52
leukocyte, PMA: phorbol 12-myristate 13-acetate.
53 54
1. Introduction
55
Thrombocytes are nucleated blood cells in non-mammalian vertebrates that are
56
considered functional homologues of mammalian platelets. Platelets and thrombocytes
57
share characteristics involved in hemostasis; in brief, they aggregate in response to
58
several damage signals, including collagen, to initiate blood clotting (Belamarich et al.,
59
1966; Nakayasu et al., 1997). In addition to their primary functions, platelets and
60
thrombocytes are also important mediators of both the innate and adaptive immune
61
systems (Elzey et al., 2003; Ferdous and Scott, 2015). Particularly, nucleated
62
thrombocytes have a potential to become powerful immune cells owing to their
63
phagocytic activities that kill internalized bacteria (Hill and Rowley, 1998; Stosik et al.,
64
2002; Nagasawa et al., 2014). Many studies reported that mammalian platelets may
65
have phagocytic activity; however, their ability to engulf microbes remains
66
controversial (White, 2006; Antczak et al., 2011). On the other hand, nucleated
67
thrombocytes express MHC class II molecules (Köllner et al., 2004; St. Paul et al.,
68
2012; Nagasawa et al., 2014, Fink et al., 2015), suggesting that they have the potential
69
to become antigen-presenting cells (e.g., macrophages and dendritic cells), which
70
present extracellular antigens. Elucidation of these phagocytic thrombocytes and
71
platelets may be important for understanding immune systems, particularly in lower
72
vertebrates. 3 Page 3 of 18
73
Phagocytosis is the process of engulfing large particles into intracellular vacuoles,
74
contributing to the elimination of pathogenic microbes. In professional phagocytes such
75
as neutrophils and macrophages, phagocytosis is triggered by the recognition of
76
pathogen-associated
77
immunoglobulins and complement components bound to the microbes, which are
78
recognized by phagocytosis-promoting receptors (Jutras and Desjardins, 2005). These
79
phagocytic abilities are also enhanced by several inflammatory cytokines such as
80
interferon gamma (IFNγ) and tumor necrosis factor (TNF) produced by several immune
81
cells (Shalaby et al., 1985). Similar regulatory mechanisms are also conserved in fish
82
(Grayfer et al., 2009).
molecular
patterns
(PAMPs)
and
opsonins
such
as
83
Platelet activation for hemostasis has been well studied and is induced by various
84
factors, including collagen, adenosine diphosphate (ADP), and thrombin. Upon
85
activation, platelets morph into a stellate form and secrete various factors to trigger
86
coagulation and inflammation (Weyrich and Zimmerman, 2004). Furthermore, although
87
nucleated thrombocytes are activated by collagen and other molecules to facilitate
88
coagulation, fish thrombocytes responded differently, including being unresponsive to
89
ADP (Belamarich et al., 1966; Matsushita et al., 2004). However, whether phagocytosis
90
activation pathway is similar to typical hemostasis pathway remains unknown. For
91
understanding the immune functions of thrombocytes, the activation mechanism for
92
their phagocytic activity is required. In the present study, we evaluated activation
93
mechanism of fish thrombocyte phagocytosis, including cell exposure to several
94
stimulants and the influence of other leukocytes. Thrombocyte phagocytic activity was
95
not induced by typical platelet stimulants, but was dramatically triggered by the
96
presence of other leukocytes and by activated leukocyte cell culture supernatants. These 4 Page 4 of 18
97
results reveal mechanisms regulating thrombocyte phagocytosis in immune system and
98
tissue maintenance.
99 100
2. Materials and methods
101
2.1. Fish
102
Common carp (Cyprinus carpio, approximately 100 g) were maintained in our
103
laboratory at 25°C and fed with commercial pellets. All animal experiments were
104
performed in accordance with the guidelines of the Animal Experiments Committee at
105
Kyushu University.
106 107
2.2. Thrombocyte isolation and purification
108
Carp peripheral blood samples were collected from caudal veins using heparinized
109
syringes, diluted with medium (RPMI-1640; Nissui Pharmaceutical), and overlaid onto
110
Percoll adjusted to a concentration of 1.08 g/mL (BD Biosciences). Samples were then
111
centrifuged at 500 × g for 30 min at 4°C to isolate peripheral blood leukocytes (PBLs).
112
PBLs were harvested from the top of the Percoll layer, washed twice with the medium
113
by centrifugation at 500 × g for 10 min at 4°C, and adjusted to a concentration of 1 ×
114
107 cells/mL with medium. The PBLs were then incubated with an HB8 monoclonal
115
antibody (mAb) specific to carp thrombocytes (Nakayasu et al., 1997) for 30 min on ice.
116
After washing with medium twice, the PBLs were incubated with MACS microbeads
117
coupled to a goat anti-mouse IgG antibody (Miltenyi Biotec). After washing with
118
medium twice, the PBLs were resuspended in RPMI-1640 containing 2 mM EDTA and
119
5% fetal bovine serum (FBS), and then loaded on a mini MACS column (Miltenyi) to
120
purify the thrombocytes. Samples were passed through the column twice to obtain 5 Page 5 of 18
121
thrombocytes at high purity. Purified thrombocytes were stained with phycoerythrin
122
(PE)-conjugated goat anti-mouse IgG antibody (Sigma–Aldrich) for 30 min at 4°C.
123 124
2.3. Thrombocyte stimulation
125
The purified thrombocytes (1 × 107 cells/mL) were incubated for 30 min at 25°C
126
with medium containing lipopolysaccharides (LPS from Escherichia coli O55, 10
127
μg/mL; Wako Pure Chemical), phorbol 12-myristate 13-acetate (PMA, 1 μg/mL; Sigma),
128
collagen type I (1 μg/mL; Wako), ADP (1 μg/mL; Tokyo Chemical Industry), or
129
thrombin (1 unit/mL; from bovine, Sigma). Next, after washing twice, the cells were
130
resuspended in medium at the same concentration.
131 132
2.4. Preparation of leukocyte culture supernatants
133
HB8 mAb-negative (HB8−) leukocytes purified with MACS microbeads were
134
stimulated with either LPS or PMA in the same manner as described above, washed 3
135
times, adjusted to 5 × 107 cells/mL with medium, and then incubated for 60 min at 25°C.
136
The culture supernatants were collected by centrifugation at 1,000 × g for 10 min at 4°C
137
3 separate times to remove all leukocytes.
138 139
2.5. Phagocytosis assay
140
Stimulated thrombocytes (1 × 107 cells/mL) were incubated with 1 μm fluorescent
141
latex beads (Fluoresbrite Yellow Green Microspheres; Polysciences, Warrington, PA,
142
USA) at a cell-to-bead ratio of 1:5 for 3 h at 25°C in 100 μL of medium. To evaluate the
143
effect of other leukocytes on activation, thrombocytes and HB8− leukocytes were mixed
144
at 5 × 106 cells/mL each and incubated as previously described. To evaluate the effect of 6 Page 6 of 18
145
soluble factors produced by leukocytes, thrombocytes were resuspended in 50% HB8−
146
leukocyte culture supernatant diluted in medium and incubated with fluorescent beads
147
as previously described. Flow cytometry (Epics XL, Beckman Coulter) was used to
148
measure the percentage of phagocytic thrombocytes in each experimental condition.
149 150
2.6. Bacterial phagocytosis
151
FITC-conjugated Escherichia coli (DH5α strain) were incubated with 10% normal
152
carp serum in PBS in the presence of 2 mM Mg2+ and 2 mM Ca2+ for 30 min at 25°C.
153
As a control, bacteria were incubated with heat-inactivated (20 min, 50ºC) carp serum
154
containing 10 mM EDTA under the same conditions. After washing with PBS 3 times,
155
the treated bacteria were adjusted to OD600nm = 0.5 and incubated with the carp
156
thrombocytes using the same conditions as described for the incubation with the beads.
157
The percentage of phagocytic thrombocytes was measured by flow cytometry.
158 159
3. Results and Discussion
160
3.1. Purified thrombocytes are not phagocytic
161
As previously described (Nagasawa et al. 2014), carp thrombocytes in peripheral
162
leukocyte pool actively ingest latex beads. In the present study, purified thrombocytes
163
were used to assess the direct influence of several different stimulants individually on
164
thrombocyte phagocytic activity. Purified carp thrombocytes incubated with 1-μm beads
165
in the same manner as PBL incubation rarely ingested the beads (2.1 ± 0.7%; Fig. 1A,
166
right). The phagocytic capacity of thrombocytes in the blood leukocyte samples was
167
verified just before cell sorting, indicating that the anti-thrombocyte mAb staining did
168
not inhibit thrombocyte phagocytosis (23.9 ± 5.1%; Fig. 1A, left). Based on these 7 Page 7 of 18
169
findings, we hypothesized that thrombocyte phagocytosis may be triggered by activators
170
derived from other leukocytes.
171
Next, we individually assessed the ability of several potential stimulants to enhance
172
purified thrombocyte phagocytic activity. For this assessment, thrombocytes were
173
preincubated with different chemicals before mixing with the beads. Although the
174
thrombocytes were exposed to typical platelet stimulators such as collagen, ADP, bovine
175
thrombin, LPS, and PMA, none of them enhanced the thrombocyte bead ingestion (Fig.
176
1B). Moreover, the effects of these stimulants on thrombocyte morphology were
177
examined by microscopy. In the absence of these reagents, thrombocytes formed a
178
typical spindle shape (see Fig. S1A). Upon incubation with collagen, thrombocytes
179
immediately adhered to the culture plate (Fig. S1B). In the presence of PMA, known to
180
induce platelet activation associated with CD62P expression (Baudouin-Brignole et al.,
181
1997), thrombocytes assumed round or oval forms with many cells adhering to the
182
culture plate (Fig. S1C). ADP only weakly induced similar morphological change of the
183
thrombocytes (Fig. S1D). These results suggest that thrombocyte phagocytic activity is
184
activated in a manner that is distinct from hemostatic aggregation. LPS and thrombin
185
did not induce morphological changes in the thrombocyte (data not shown). Mammalian
186
platelets express TLR4 to sense LPS, which does not induce a typical platelet activation
187
and aggregation (Cognasse et al., 2005; Ward et al., 2005); however, LPS enhances the
188
secretion of several cytokines in platelets and expression of them in chicken
189
thrombocytes (Cognasse et al., 2008; Scott and Owens, 2008). These data suggest that
190
thrombocyte phagocytosis is activated through a different mechanism than that by
191
which cytokine production is activated. The finding that bovine thrombin did not affect
192
carp thrombocytes may be the result of interspecies incompatibility. 8 Page 8 of 18
193 194
3.2. Thrombocyte phagocytosis is activated by co-incubation with leukocytes
195
Next, we evaluated the effects of other leukocytes on thrombocyte phagocytosis.
196
Purified thrombocytes were remixed with leukocytes (HB8 mAb− fraction) and
197
incubated with fluorescent beads. Notably, the remixed thrombocytes recovered their
198
phagocytic abilities (24.9 ± 5.2%; Fig. 2A), suggesting that co-incubation with other
199
leukocytes triggered thrombocyte phagocytic activity. This result also showed that cell
200
purification did not irreversibly harm the ability of thrombocytes to ingest particles.
201
Thrombocytes were also incubated with leukocytes that had been preincubated with
202
LPS or PMA. These stimulated HB8− leukocytes ingested the beads more actively than
203
control leukocytes incubated without stimulation (Fig. 2A). Remarkably, co-incubation
204
with LPS-stimulated leukocytes enhanced thrombocyte phagocytic activity more
205
strongly (35.5 ± 5.5%) than co-incubation with unstimulated leukocytes, suggesting that
206
LPS-stimulation improved the leukocyte abilities to activate the thrombocyte
207
phagocytosis.
208
We also assessed the effect of opsonization on purified thrombocyte phagocytosis.
209
In a previous study, carp thrombocyte phagocytosis was enhanced by serum
210
opsonization of bacterial particles in the presence of other leukocytes (Nagasawa et al.,
211
2014). In the flow cytometry analysis, very few thrombocytes interacted with bacteria
212
even preincubated with serum (control, 1.3 ± 0.3%; opsonized, 1.4 ± 0.5%; Fig. 2B),
213
whereas the ingestion of thrombocytes that had been remixed with HB8− PBLs was
214
enhanced by opsonization (control, 14.4 ± 1.3%; opsonized, 17.9 ± 1.7%). These results
215
suggested that phagocytosis-promoting receptors such as complement receptors may not
216
be expressed on the surface of thrombocytes in the resting phase; however, after 9 Page 9 of 18
217
activation, the thrombocytes express these receptors and are able to recognize the
218
opsonized antigens.
219 220
3.3. Phagocytic thrombocytes activated by leukocyte secretary components
221
To assess whether thrombocyte activation by other leukocytes was mediated by
222
soluble factors released from the leukocytes or rather was dependent on cell-to-cell
223
contacts, purified thrombocytes were incubated with labeled beads in the presence of
224
HB8− leukocyte cell culture supernatant. When the thrombocytes were incubated with
225
unstimulated leukocyte supernatant, thrombocyte phagocytosis did not increase (2.1 ±
226
1.4%; Fig. 2C).
227
Remarkably, when the thrombocytes were incubated with labeled beads in the
228
presence of LPS- or PMA-stimulated leukocyte cell culture supernatant, the number of
229
phagocytic thrombocytes dramatically increased (LPS, 13.9 ± 1.8%; PMA, 9.9 ± 2.0%).
230
This finding indicates that the stimulated leukocytes secreted factors that triggered
231
thrombocytes to ingest the labeled beads. This result also suggests that only activated
232
leukocytes release the thrombocyte activation factors that trigger phagocytic activity.
233
Macrophages are traditional phagocytic cells that are differentially activated by
234
microbial stimulants and the presence or absence of cytokines excreted by other
235
leukocyte subtypes such as natural killer cells and Th1/Th2 cells for regulation which
236
contributes to prevent them from damaging host tissues (Mantovani et al., 2004; Gordon
237
and Taylor, 2005; Mosser and Edwards, 2008; Forlenza et al., 2011). Likewise,
238
thrombocyte phagocytosis also may be regulated by inflammatory signals released by
239
other leukocytes. Thus, thrombocytes may only be recruited as phagocytes in the acute
240
phase of inflammation or pathogen invasion. 10 Page 10 of 18
241
Our results suggest that even though the carp thrombocytes recognized antigens,
242
they did not become phagocytic in the absence of inflammatory signals originating from
243
other immune components. In mammals, generalized platelet activation often causes
244
serious thrombocytopenia, similar to that in sepsis (Lupu et al., 2014). Even in carp, it
245
was reported that infection with the parasite Trypanoplasma borreli dramatically
246
reduced the number of thrombocytes in the host blood (Fink et al., 2015). These
247
thrombocytopenic conditions can cause a serious decline in host health. Thus, a
248
thrombocyte regulatory system may help prevent excessive activation.
249 250
4. Conclusions
251
Our results indicate that the initiation of thrombocyte phagocytosis requires soluble
252
factors secreted from activated leukocytes. Isolated thrombocytes did not actively ingest
253
particles; however, their phagocytic activity was restored by incubation with either
254
HB8− leukocytes or stimulated leukocyte cell culture supernatant. Cell culture
255
supernatant from unstimulated PBLs did not induce phagocytosis, indicating that the
256
activating factors are produced upon immune system activation. These results indicate
257
that thrombocyte phagocytic activity requires a different activation pathway than the
258
typical route for hemostatic activation. These regulatory mechanisms may contribute to
259
the control of tissue repair and inflammation. Future studies will help establish the
260
details of thrombocyte phagocytic activation, such as the cell types and molecules
261
capable of inducing phagocytosis.
262 263
Acknowledgements
264
This research was supported in part by a Grant-in-Aid for Scientific Research (B) 11 Page 11 of 18
265
(Grant Number 25292127) from the Japan Society for the Promotion of Science (JSPS).
266 267
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Figure legends
371
Fig. 1. (A) Flow cytometry scatter plot of unsorted carp PBLs and purified
372
thrombocytes incubated with 1 μm beads. Data are representative of 3 independent
373
experiments. (B) Percentages of phagocytic thrombocytes after preincubation with
374
collagen (1 μg/ml), adenosine diphosphate (ADP; 10 μg/ml), thrombin (10 units/ml),
375
lipopolysaccharide (LPS from Escherichia coli O55; 10 μg/ml) or phorbol 12-myristate
376
13-acetate (PMA; 1 μg/ml). For co-incubation (thrombin/PBLs), thrombocytes, and
377
sorted HB8− PBLs (5 × 106 cells/ml each) were mixed and then incubated with the
378
labeled beads. PBLs: peripheral blood leukocytes. The asterisks (*) indicate a
379
significant difference from the control as analyzed by Tukey’s test (**P < 0.01). Data
380
are the average rates from 3 independent experiments, analyzed at the same time as the
381
assays in Figs. 1B, 2A, and 2C.
382 383
Fig. 2. (A) Percentages of phagocytic cells after cells were remixed and then incubated
384
with fluorescent beads. Thrombocytes (5 × 106 cells/ml) were incubated with the beads
385
in the presence of HB8− PBLs (5 × 106 cells/ml) preincubated with cells alone or with
386
LPS (10 μg/ml) or PMA (1 μg/ml). Peripheral blood leukocytes (PBLs) in the HB8−
387
fraction. Different letters indicate significant differences as analyzed by Tukey's test (P
388
< 0.05). (B) Percentages of phagocytic cells incubated with fluorescein isothiocyanate
389
(FITC) -conjugated bacteria. Thrombocytes (5 × 106 cells/ml alone or 5 × 106 cells/ml
390
with 5 × 106 cells/ml of HB8− leukocytes) were incubated with FITC-E. coli (OD600nm =
391
0.5) pretreated with normal carp serum (op) or heat-inactivated carp serum (con).
392
Peripheral blood leukocytes (PBLs) in the HB8− fraction. The asterisks (*) indicate a
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significant difference from the control group (thrombin/PBL con) as analyzed by 17 Page 17 of 18
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Tukey’s test (*P < 0.05). (C) Percentages of phagocytic thrombocytes after stimulation
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with cell culture supernatants from other leukocytes. Thrombocytes (1 × 107 cells/ml)
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were incubated with 1 μm beads in the presence of culture supernatants from HB8−
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leukocytes incubated alone (con) or with LPS (10 μg/ml) or PMA (1 μg/ml). Peripheral
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blood leukocytes (PBLs) in the HB8− fraction. The asterisks (*) indicate significant
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differences from the control group (throm. only) as analyzed by Tukey’s test (*P < 0.05;
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**P < 0.01). Data are the average rates from 3 independent experiments, analyzed at the
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same time as the assays in Figs. 1B, 2A, and 2C.
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