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Differential expression of pentraxin 3 in neutrophils
Experimental and Molecular Pathology 98 (2015) 33–40
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Differential expression of pentraxin 3 in neutrophils Olga Razvina a, Shuying Jiang a, Koichi Matsubara a, Riuko Ohashi a, Go Hasegawa a, Takashi Aoyama a, Kenji Daigo b, Tatsuhiko Kodama b, Takao Hamakubo c, Makoto Naito a,⁎ a b c
Department of Cellular Function, Division of Cellular and Molecular Pathology, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan Laboratory for Systems Biology and Medicine, Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo, Japan Department of Quantitative Biology and Medicine, The University of Tokyo, Tokyo, Japan
a r t i c l e
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Article history: Received 8 November 2014 Accepted 18 November 2014 Available online 20 November 2014 Keywords: Immunohistochemistry Neutrophils Neutrophil extracellular traps (NETs) Neutrophilic granules Pentraxin 3
a b s t r a c t Pentraxins belong to the superfamily of conserved proteins that are characterized by a cyclic multimeric structure. Pentraxin 3 (PTX3) is a long pentraxin which can be produced by different cell types upon exposure to various inflammatory signals. Inside the neutrophil PTX3 is stored in form of granules localized in the cytoplasm. Neutrophilic granules are divided into three types: azurophilic (primary) granules, specific (secondary) granules and gelatinase (tertiary) granules. PTX3 has been considered to be localized in specific (secondary) granules. Immunofluorescent analyses using confocal laser microscopic examination were performed to clarify the localization of all three groups of granules within the cytoplasm of the mature neutrophils and neutrophils stimulated with IL-8. Furthermore, PTX3 was localized in primary granules of promyelocyte cell line HL-60. As a result, we suggest that PTX3 is localized not only in specific granules, but is also partly expressed in primary and tertiary granules. After the stimulation with IL-8, irregular reticular structures called neutrophil extracellular traps (NETs) were formed, three types of granules were trapped by NETs and PTX3 showed partial colocalization with these granular components. PTX3 localized in all three types of granules in neutrophils may play important roles in host defense. © 2014 Published by Elsevier Inc.
1. Introduction Polymorphonuclear neutrophils (or PMNs) are the most numerous leukocytes in mammals, much more numerous than the longer-lived monocytes/macrophages (Witko-Sarsat et al., 2000). PMNs are fast acting and effective phagocytes, which play a very important role in innate and adaptive immunity (Jena et al., 2012). They also stand as a first-line defense against invading pathogens (Borregaard, 2010). Neutrophils have various types of granules, containing lots of antibacterial proteins which can kill microbes and digest tissues (Borregaard, 2010; Kaplan and Radic, 2012). Neutrophil granules have been divided into three major subsets according to differences in protein content and propensity for mobilization (Hager et al., 2010). These subsets are azurophilic (primary) granules, specific (secondary) granules and gelatinase (tertiary) granules. The primary granules contain myeloperoxidase (MPO), neutrophil elastase, azurocidin and others, secondary granules contain lactoferrin and cathelicidin, and tertiary granules contain gelatinase, cathepsin and others (Jaillon et al., 2007). These granules appear in
⁎ Corresponding author at: Department of Cellular Function, Division of Cellular and Molecular Pathology, Niigata University Graduate School of Medical and Dental Sciences, 1-757 Asahimachi-dori, Chuo-ku, Niigata 951-8510, Japan. E-mail address: [email protected] (M. Naito).
http://dx.doi.org/10.1016/j.yexmp.2014.11.009 0014-4800/© 2014 Published by Elsevier Inc.
the PMNs sequentially as a result of granulocytic differentiation in the bone marrow, from myeloblast to the segmented stage when maturation is reached (Borregaard, 2010; Papayannopoulos and Zychlinsky, 2009). Pentraxins are small pentameric innate immunity effector proteins, which are very important in infectious and inflammatory responses (Bottazzi et al., 2006). Pentraxin 3 (PTX3) is the member of the pentraxin superfamily, which expression is induced in response to inflammatory signals. It is characterized by the presence of the carboxil terminal region, called pentraxin domain. PTX3 is the first identified long pentraxin (Mantovani et al., 2008). A variety of cell types, particularly macrophages, polymorphonuclear neutrophils, dendritic cells, endothelial cells and smooth muscle cells can produce PTX3 upon exposure to primary inflammatory signals, such as lipopolysaccharide and other agonists for Toll-like receptor family, IL-1β, and TNF-α, as well as oxidized low-density-lipoproteins (Bottazzi et al., 2006). Although previous studies demonstrated that PTX3 inside the neutrophil is stored in form of specific (secondary) granules in the cytoplasm (Jaillon et al., 2007), the strict co-localization of PTX3 with other granular proteins in the PMNs is not fully confirmed. In this study, using antibodies for granular components in the three groups of granules, we have investigated the localization of PTX3 and its relation to granules inside the neutrophil and those adhered to degenerated neutrophils called NETs.
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2. Materials and methods 2.1. Isolation of neutrophils Human polymorphonuclear leukocytes (PMLs) were isolated from the peripheral blood of healthy individuals using mono-poly resolving medium (DS Pharma Biomedical Co., Ltd., Osaka, Japan). After centrifugation the lower fraction consisted of PMNs, the percentage of neutrophils was evaluated using May–Giemsa staining. There were no immature myeloid elements. Isolated PMNs were suspended at a concentration of 1 × 106 cells/ml in RPMI 1640 medium with 2% fetal bovine serum and Penicillin Streptomycin (Pen Strep) mixture (Gibco). Freshly isolated cells were collected for immunofluorescent staining and immunoblotting. For immunoblotting cells were placed in a 3 cm dish and incubated, then one group of cells was stimulated using 100 ng/ml of IL-8. After 40 min, the supernatant and cell lysate were collected. Supernatant was also collected for enzyme-linked immunosorbent assay (ELISA), in order to check the level of PTX3 released by activated neutrophils. For immunofluorescent staining of neutrophils without stimulation freshly isolated non-stimulated PMNs were cytospined for 1 min at 1000 rpm and then stained. For immunofluorescent staining with stimulation, PMNs were seeded on glass coverslips treated with 0.001% polylysine and stimulated with 100 ng/ml of IL-8 (R&D Systems Inc., Minneapolis, MI, USA) for 40 min. 2.2. Isolation of HL-60 cell culture HL-60 cells were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum and Penicillin Streptomycin (Pen Strep) mixture (Gibco). Cells were maintained at 37 °C in a 5% CO2/95% air atmosphere and were used for experiments during the exponential phase of growth. The percentage of cells was evaluated using the Giemsa staining. Freshly isolated cells were collected for immunofluorescent staining and immunoblotting. For immunofluorescent staining cells were cytospined for 1 min at 1000 rpm and then stained. 2.3. Differentiation of HL-60 cell culture to neutrophil-like cells HL-60 cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and Penicillin Streptomycin (Pen Strep) mixture (Gibco) and at a density of 1 × 106 cells/ml was stimulated with 1.25% dimethyl sulfoxide DMSO (Sigma-Aldrich, Missouri, USA) for 7 days to
induce differentiation. Differentiated cell morphology was monitored by the Giemsa staining. A stationary state was reached at day 7 and isolated cells were collected for immunofluorescent staining. For immunofluorescent staining cells were cytospined for 1 min at 1000 rpm and then stained. 2.4. Immunofluorescent analysis Freshly isolated PMNs and HL-60 cells were cytospined for 1 min at 1000 rpm, blocked with 4% paraformaldehyde for 5 min and 0.5% Triton X-100/PBS for 5 min. Then specimens were blocked with 10% normal goat serum and stained with primary antibodies: anti-PTX3 monoclonal antibodies PPZ-1228 (Savchenko et al., 2008) at a dilution of 1:100 (Perseus Proteomics Inc., Tokyo, Japan), anti-lactoferrin monoclonal antibodies (EPR4338, GenTex Inc., Irvine, CA, USA) at a dilution of 1:100, polyclonal anti-myeloperoxidase antibodies (Abcam plc., Cambridge, UK) at 1:1000, polyclonal anti-gelatinase antibodies (EMD Millipore Corporation, Billerica, MA, USA) at 1:10,000 and monoclonal anti-azurocidin 1 Z6718 at 1:1000 dilution (Daigo and Hamakubo, 2012). Alexa Fluor 568 goat anti-mouse IgG Fab′ fragment (1:250 dilution, Invitrogen, Carlsbad, CA, USA) was used as secondary antibodies for PTX3 detection. A secondary antibody used for the detection of lactoferrin, gelatinase and MPO was Zenon Alexa Fluor 488 labeling kit goat anti-rabbit IgG Fab′ fragment (Invitrogen) at a dilution of 1:500. Monoclonal anti-azurocidin 1 Z6718 antibody was labeled with Z25102: Zenon Alexa Fluor 488 Mouse IgG2a Labeling kit (Invitrogen). DNA detection was carried out using blue fluorescent Hoechst 33342 (MW 615.99, H3570), purchased from Molecular Probes Inc. (Carlsbad, CA, USA). Control experiments were carried out by omitting primary antibodies. Immunofluorescent analysis was performed using the photomultiplier of a multi-photon laser scanning microscope (LSM510 META; Carl Zeiss, Jena, Germany). 2.5. Immunoblotting Grown PMNs were collected and lysed in a solution containing 150 mM NaCl, 50 nM Tris–HCl (pH 8.0), 5 mM ethylenediaminetetraacetic acid, 1% Triton X-100 and 1 mM phenylmethylsulfonyl fluoride, 1 mM leupeptin, 1 mM aprotinin for 10 min on ice and centrifuged at 15,000 rpm at 4 °C for 15 min. The supernatants were used with no dilution. Twenty five micrograms of the lysates and supernatants was
Fig. 1. a Expression and secretion of PTX3 protein in neutrophils. Lysates and supernatants revealed bands corresponding to molecular weight of 90 and 40 kDa. Freshly isolated lysates and supernatants showed PTX3 protein presence, with more intense expression in supernatants. After 40 min of IL-8 stimulation expression of PTX3 in cell lysate appeared to decrease in comparison to the cell lysate without stimulation. However, no clear difference was seen in PTX3 expression between supernatants of neutrophil culture with and without stimulation. b PTX3 levels in supernatant of non-stimulated neutrophils and neutrophils stimulated with IL-8. Supernatant of stimulated neutrophils showed 4 times higher PTX3 concentration compared to non-stimulated ones. c Lactoferrin expression in neutrophils. Lactoferrin expression without stimulation was seen in cell lysate only. Forty minutes after IL-8 stimulation lactoferrin expression was seen in supernatant as well as in cell lysate. Lactoferrin expression in cell lysate of non-stimulated neutrophils is more remarkable than in cell lysate of IL-8 stimulated neutrophils. HEK cells — Human Embryonic Kidney 293 cells.
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applied. The samples were resolved by sodium-dodecyl-sulfatepolyacrylamide-gel electrophoresis. After electrophoresis, the protein was transferred to a PVDF membrane (GE Healthcare, Amersham, UK). The blots were pre-treated with 5% skim milk overnight and then incubated with anti-lactoferrin (1:500 dilution), anti-gelatinase (1:1000), anti-myeloperoxidase (1:500) and anti-azurocidin (1:1000) antibodies. Then they were incubated with secondary anti-mouse IgG horseradish peroxidase-conjugated antibody (GE Healthcare) at a dilution of 1:2000 for 30 min and washed three times with PBS, between each step of the procedure. They were visualized with the ECL detection system (GE Healthcare) according to manufacturer's instructions (Imamura et al., 2007). 2.6. Enzyme-linked immunosorbent assay ELISA was performed using the described protocol (Imamura et al., 2007; Inoue et al., 2007). Pepsin-digested PPMX0104 (100 μl of a 5 μg/ml solution in saline) was coated on a microtiter plate, MaxiS-ope (Nalge Nunc, Penfield, New York, USA) at 4 °C overnight. The wells were blocked with 1% of casein in PBS, pH 7.4 at 4 °C overnight. Supernatant samples were added to the wells and incubated for 1 h. Bound PTX3 was detected using horseradish peroxidaseconjugated PPMX0105 (Perseus Proteomics Inc., Tokyo, Japan).
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Substrate solution was added to each well and incubated for 30 min. Stop solution was added and absorbance at 450 nm was measured with a microplate reader system (Towa Labo, Tokyo, Japan). All assays were carried out in duplicate. 2.7. Electron microscopy analysis Scanning electron microscopy (SEM) was used to observe IL-8 stimulated and non-stimulated PMN samples after fixation with 2% glutaraldehyde. SEM was performed as described (Oyama et al., 2006). Cells were stained using a tannin-osmium method, dehydrated in graded ethanol series and critical point dried. Then, specimens were coated with platinum–palladium using an ion-coater, and microscopically examined with a scanning electron microscope (H700: Hitachi, Tokyo, Japan). 2.8. Cell counting and colocalization analysis Double immunofluorescent staining of cytospined, non-stimulated neutrophils for PTX3 and MPO, azurocidin, lactoferrin, and gelatinase was performed. Then, photographs were taken at a magnification × 2000, using the photomultiplier of a multi-photon laser scanning microscope (LSM510 META; Carl Zeiss). The photographs were printed
Fig. 2. Non-stimulated cytospined neutrophils. a Double immunofluorescent staining for pentraxin 3 (PTX3) (red) and myeloperoxidase (MPO) (green). b Double immunofluorescent staining for pentraxin 3 (PTX3) (red) and azurocidin (green). c Double immunofluorescent staining for pentraxin 3 (PTX3) (red) and lactoferrin (green). d Double immunofluorescent staining for pentraxin 3 (PTX3) (red) and gelatinase (green). Bar (a, b, c, d) 5 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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out, magnified up to ×10,000. All the granules were counted separately: MPO, azurocidin, lactoferrin, and gelatinase — green granules, PTX3 — red granules and co-localized granules — yellow granules (merged image). Thirty neutrophils were counted for each of four groups (MPOPTX3, azurocidin-PTX3, lactoferrin-PTX3, gelatinase-PTX3), totally one hundred twenty neutrophils. 3. Results 3.1. PTX3 expression in PMNs and its levels after stimulation The secreted recombinant PTX3 protein consists of a major 45 kDa form of the monomer and 90 kDa form of dimer. PTX3 expression in neutrophils was also confirmed (Fig. 1a). Immunoblotting of freshly isolated lysates and supernatants obtained from PMN culture showed PTX3 proteins corresponding to molecular weight of 90 and 45 kDa. Expression of PTX3 was stronger in supernatants in comparison with cell lysates, suggesting that PTX3 protein was produced in neutrophils and secreted extracellularly. Under IL-8 stimulation, expression of PTX3 in cell lysate decreased in comparison with non-stimulated cell lysate. There appeared to be no clear difference in PTX3 expression between IL-8 stimulated and non-stimulated supernatants (Fig. 1a), however, ELISA showed 4 times higher PTX3 concentration in the culture medium of neutrophils stimulated with IL-8, compared to non-stimulated ones, indicating that PTX3 released from PMNs is enhanced by IL-8 (Fig. 1b). 3.2. Lactoferrin expression in PMNs after stimulation Lactoferrin expression was confirmed in neutrophils as well. Immunoblotting of isolated lysates and supernatants revealed expression of lactoferrin protein corresponding to molecular weight of 85 kDa (predicted band size stated in the lactoferrin antibody's datasheet is confirmed). Lactoferrin expression was more intense in cell lysates, suggesting that lactoferrin is mostly stored intracellularly. No band was present in supernatant without stimulation with IL-8. Stimulated
supernatant medium showed distinct lactoferrin expression suggesting that lactoferrin granules are released outside the cell under the stimulation (Fig. 1c). Similar expression pattern was observed by immunoblotting using antibodies against azurocidin, MPO and gelatinase (data not shown).
3.3. Double immunofluorescent staining of non-stimulated neutrophils Double immunofluorescent analysis of non-stimulated neutrophils revealed positive expression of PTX3 in the cytoplasm of a neutrophil in a form of diffusely located granules. Double immunofluorescence staining showed that most of the lactoferrin-positive granules were colocalized with PTX3 granules (Figs. 2c, 3c). The mean quantity of colocalized PTX 3 and lactoferrin granules was 72.3 ± 8.2. Azurocidin and myeloperoxidase which belong to the group of primary granules showed partial colocalization with PTX3 granules. The mean number of colocalized MPO and PTX3 granules was 26.2 ± 5.5. The mean number of colocalized azurocidin and PTX3 granules was 30.0 ± 4.9 (Figs. 2a, b, 3a, b). Gelatinase granules were also partly co-localized with PTX3. The mean quantity of colocalized PTX 3 and gelatinase granules was 34.0 ± 4.7 (Figs. 2d, 3d). The highest percentage of co-localization was seen between PTX3 and lactoferrin — 71% (Fig. 3e). Percent of co-localization for other granular components was as follows: MPO — 25%, azurocidin — 31, 5%, and gelatinase — 35%, showing that they are partly colocalized with PTX3 (Fig. 3e).
3.4. Expression of PTX3 and other granular components in stimulated PMNs Neutrophils were stimulated with IL-8, cultured for 40 min and then observed with a scanning electron microscope. Degeneration and cell death of neutrophils can be seen on a scanning electron microscopic image (Fig. 4). It revealed the irregular reticular structure of NETs,
Fig. 3. The mean number of neutrophil granular components, their colocalization with PTX3. Red: PTX3; yellow: granules, showed colocalization with PTX3; green: a MPO, b azurocidin, c lactoferrin, d gelatinase. e Percentage of colocalization with PTX3. No. of double positive granules/No. of PTX3+ granules × 100 = %. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Fig. 4. a Scanning electron micrograph (SEM) of non stimulated PMNs. B Neutrophils activated with IL-8 for 40 min. c Higher magnification of IL-8 stimulated neutrophils showing irregular reticular structure and globular domains aggregated on threads (NETs).
which was supposedly formed by activated PMNs and globular domains aggregated to threads. Immunofluorescent analysis of PMNs stimulated with IL-8 showed the Hoechst 33342-positive irregular reticular structure, considered to be NETs. PTX3 positive granules adhered to this structure were observed. It was demonstrated that PTX3 was partially co-localized with MPO, azurocidin and gelatinase (Fig. 5a, b, d) as well as
lactoferrin (Fig. 5c) not only in neutrophils but also on the surface of NETs. 3.5. Expression of granular components in promyelocytes HL-60 (human promyelocytic leukemia cells) cell line was used, as HL-60 cells contain only primary granules. Double immunofluorescent
Fig. 5. Neutrophils activated with IL-8. a Double immunofluorescent staining for pentraxin 3 (PTX3) (red) and myeloperoxidase (MPO) (green). b Double immunofluorescent staining for PTX3 (red) and azurocidin (green). c Double immunofluorescent staining for PTX3 (red) and lactoferrin (green). d Double immunofluorescent staining for PTX3 (red) and gelatinase (green). Bar (a, b, c, d) 5 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Fig. 6. The HL-60 (human promyelocytic leukemia cells) cell line. a Double immunofluorescent staining for pentraxin 3 (PTX3) (red) and myeloperoxidase (MPO) (green). b Double immunofluorescent staining for PTX3 (red) and lactoferrin (green). c Double immunofluorescent staining for PTX3 (red) and gelatinase (green). Bar (a, b, c) 2.5 μm. d Expression of PTX3, MPO, lactoferrin and gelatinase in HL-60 cell line. Expression of PTX3 (90 and 40 kDa bands) and MPO (84 kDa band) was found to be positive, while expression of lactoferrin and gelatinase was found to be negative.
analysis showed that primary granules (MPO) were positively stained as well as PTX3 (Fig. 6a). Other components of secondary (lactoferrin) and tertiary (gelatinase) granules were stained negatively (Fig. 6b, c). These results were also confirmed by immunoblotting of freshly isolated HL-60 cells. MPO protein was detected as bands corresponding 84 kDa. PTX3 expression was confirmed as well. Bands corresponding to molecular weight of 45 kDa (monomer) and 90 kDa (dimer) were observed, showing that PTX3 may localize not only in specific granules, but in primary granules as well (Fig. 6d).
3.6. Expression of granular components in HL-60-derived neutrophil-like cells Double immunofluorescent analysis showed that PTX3 and all other three groups of granules: primary (MPO), secondary (lactoferrin), and tertiary (gelatinase) granules were present in HL-60 neutrophil-like cells differentiated with DMSO, showing partial colocalization with PTX3 (Fig. 7a, b, c).
4. Discussion In this study, we have confirmed the positive PTX3 expression in mature PMNs, in the form of diffusely located granules in the cytoplasm, using the immunofluorescent staining. PTX3 was mainly colocalized with lactoferrin, which is considered to be the component of secondary (specific) granules as was reported previously (Jaillon et al., 2007; Remijsen et al., 2011). Furthermore we observed partial expression of PTX3 in primary and tertiary granules, though the percentage of PTX3 colocalization in these two granules was lower than that in specific granules. Neutrophils act as the first line defense against microbes and perform several methods of direct killing pathogens: phagocytosis and release of antibacterial substances, including granule proteins. Neutrophil granules are classified into primary (azurophilic) granules (MPO), secondary (specific) granules (lactoferrin), and tertiary (gelatinase) granules. These granules are formed sequentially during granulocytic differentiation in the bone marrow. PTX3 is also a granular component which localizes in specific granules as demonstrated
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Fig. 7. The HL-60-derived neutrophil-like cells. a Double immunofluorescent staining for pentraxin 3 (PTX3) (red) and myeloperoxidase (MPO) (green). B Double immunofluorescent staining for PTX3 (red) and lactoferrin (green). C Double immunofluorescent staining for PTX3 (red) and gelatinase (green). Bar (a, b, c) 5 μm. d HL-60 cell line without any stimulation, which consisted predominantly of promyelocytes with azurophilic granules, large round nuclei and prominent nucleoli. Giemsa staining, 100× magnification. (e) HL-60 cell line stimulated with 1, 25% DMSO for 7 days. HL-60-derived neutrophil-like cells are seen, with decreased nuclear/cytoplasmic ratio and nuclei subdivided into lobes. Giemsa staining, 100× magnification. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
by immunofluorescence observation (Jaillon et al., 2007) and proteomics analysis (Remijsen et al., 2011). Using western blotting we have also confirmed that both PTX3 and lactoferrin proteins were found in PMNs and released extracellularly, suggesting both proteins are present in the same granules. As to granule biogenesis, primary granules are formed at the stage of promyelocytes and secondary and tertiary granules are produced at the stage of myelocytes and metamyelocytes. Jallion et al. reported that PTX3 mRNA expression is positive in progenitor neutrophils (promyelocytes, myelocytes, metamyelocytes), but it is absent in mature neutrophils (Garlanda et al., 2002), suggesting that PTX3 is produced during the maturation course of neutrophils and stored in mature neutrophils in the form of ready-to-use specific granules (Jaillon et al., 2007; Moalli et al., 2011; Papayannopoulos and Zychlinsky, 2009). We have demonstrated that the human promyelocytic cell line HL-60 expresses MPO and azurocidin, markers of primary granules, does not express lactoferrin, a marker of specific granules, and gelatinase, a marker of tertiary granules. However, immunostaining and immunoblotting demonstrated that PTX3 protein was expressed in HL-60 cell line. These findings indicate that PTX3 may localize in primary granules as well as in specific granules. We have also stimulated HL-60 cells with DMSO in order to differentiate them to neutrophil-like cells. These differentiated cells showed positive expression and colocalization of PTX3 and other granular
components: primary (MPO), secondary (lactoferrin), and tertiary (gelatinase) granules. The quantity of MPO and PTX3 granules considerably increased in comparison with non-stimulated HL-60 cells, while lactoferrin (secondary) and gelatinase (tertiary granules) turned to be positive, which confirms neutrophilic granule biogenesis theory. We have found that in mature neutrophils PTX3 is partly colocalized with MPO and azurocidin, which belong to primary granules, and gelatinase, which belongs to tertiary granules. Combining the present and previously reported results, we suggest that PTX3 not only localizes in specific granules, but is also expressed in primary and tertiary granules. Neutrophil extracellular traps (NETs) are chromatin structures loaded with antimicrobial molecules first reported in 2004 (Brinkmann et al., 2004). This reticular structure is composed of nuclear chromatin fibers, nuclear histones and granules with antimicrobial proteins and is released as a response to different inflammatory stimuli (Fuchs et al., 2007; Imamura et al., 2007). Netting neutrophils do not display “eat me” signals as apoptotic cells do, but it may help them to escape clearance by phagocytes (Borregaard et al., 2012; Bottazzi et al., 2006). NETs can possibly trap and kill pathogens (bacteria, fungi) by means of bactericidal proteins, stored in neutrophil granules, which are connected to DNA fibers, forming a special microenvironment (Lominadze et al., 2005; Mantovani et al., 2008). The present study revealed that after the stimulation three types of granules were localized extracellularly, trapped by NETs and PTX3 was
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partially colocalized with these granular components. PTX3 adhered to NET structure may play a great role in suppression of pathogens (Bottazzi et al., 2006; Deban et al., 2011; Papayannopoulos and Zychlinsky, 2009). It has been reported in the study using PTX3-null mice that PTX3 can directly bind to microbial pathogens, such as Aspergillus fumigatus, Pseudomonas aeruginosa, Salmonella typhimurium and others, thereby representing a mechanism of innate resistance amplification against pathogens. PTX3 and granular components adhered to NETs may act locally at the site of infection and inflammation (Garlanda et al., 2002). Recently, it has been identified that PTX3 may enhance the antibacterial efficiency of azurocidin and MPO. A biochemical analysis demonstrated the direct binding of azurocidin and MPO to PTX3 (Daigo and Hamakubo, 2012). It is not clear whether these PTX3-MPO and PTX3azurocidin complexes would occur after the release of granules or they are already formed in neutrophil granules. For understanding the precise mechanism of PTX3 and azurocidin or MPO colocalization in neutrophils and NETs further studies are required. Conflict of interest statement The authors declare that there are no conflicts of interest. Acknowledgments The authors would like to thank Mr. Kenji Oyauchi, Mrs. Yasue Honma and all the stuff of the Department of Cellular Function, Division of Cellular and Molecular Pathology, Niigata University Graduate School of Medical and Dental Sciences, for their excellent technical assistance. This study was supported in part by Ministry of Scientific Culture, Sports, Science and Technology Grant-in-aid for Scientific Research 25220205. References Borregaard, N., 2010. Neutrophils, from marrow to microbes. Immunity 33 (5), 657–670. http://dx.doi.org/10.1016/j.immuni.2010.11.011 (Nov 24). Borregaard, N., Stenger, S., Sonawane, A., Sorensen, O.E., 2012. Azurophil granule proteins constitute the major mycobactericidal proteins in human neutrophils and enhance the killing of mycobacteria in macrophages. PLoS One 7 (12), e50345. http://dx.doi. org/10.1371/journal.pone.0050345. Bottazzi, B., Garlanda, C., Salvatori, G., Jeannin, P., Manfredi, A., Mantovani, A., 2006. Pentraxins as a key component of innate immunity. Curr. Opin. Immunol. 18 (1), 10–15. Brinkmann, V., Reichard, U., Goosmann, C., Fauler, B., Uhlemann, Y., Weiss, D.S., Weinrauch, Y., Zychlinsky, A., 2004. Neutrophil extracellular traps kill bacteria. Science 303 (5663), 1532–1535. Daigo, K., Hamakubo, T., 2012. Host-protective effect of circulating pentraxin 3 (PTX3) and complex formation with neutrophil extracellular traps. Front. Immunol. 3, 378. http://dx.doi.org/10.3389/fimmu.2012.00378.
Deban, L., Jaillon, S., Garlanda, C., Bottazzi, B., Mantovani, A., 2011. Pentraxins in innate immunity: lessons from PTX3. Cell Tissue Res. 343 (1), 237–249. http://dx.doi.org/10. 1007/s00441-010-1018-0. Fuchs, T.A., Abed, U., Goosmann, C., Hurwitz, R., Schulze, I., Wahn, V., Weinrauch, Y., Brinkmann, V., Zychlinsky, A., 2007. Novel cell death program leads to neutrophil extracellular traps. J. Cell Biol. 176 (2), 231–241. Garlanda, C., Hirsch, E., Bozza, S., Salustri, A., De Acetis, M., Nota, R., Maccagno, A., Riva, F., Bottazzi, B., Peri, G., Doni, A., Vago, L., Botto, M., De Santis, R., Carminati, P., Siracusa, G., Altruda, F., Vecchi, A., Romani, L., Mantovani, A., 2002. Non-redundant role of the long pentraxin PTX3 in anti-fungal innate immune response. Nature 420 (6912), 182–186. Hager, M., Cowland, J.B., Borregaard, N., 2010. Neutrophil granules in health and disease. Intern. Med. 268 (1), 25–34. http://dx.doi.org/10.1111/j.1365-2796.2010.02237.x. Imamura, M., Kawasaki, T., Savchenko, A.S., Ohashi, R., Jiang, S., Miyamoto, K., Ito, Y., Iwanari, H., Sagara, M., Tanaka, T., Hamakubo, T., Kodama, T., Uchiyama, M., Naito, M., 2007. Lipopolysaccharide induced expression of pentraxin 3 in human neutrophils and monocyte-derived macrophages. Cell. Immunol. 248 (2), 86–94. Inoue, K., Sugiyama, A., Reid, P.C., Ito, Y., Miyauchi, K., Mukai, S., Sagara, M., Miyamoto, K., Satoh, H., Kohno, I., Kurata, T., Ota, H., Mantovani, A., Hamakubo, T., Daida, H., Kodama, T., 2007. Establishment of a high sensitivity plasma assay for human pentraxin3 as a marker for unstable angina pectoris. Arterioscler. Thromb. Vasc. Biol. 27 (1), 161–167. Jaillon, S., Peri, G., Delneste, Y., Frémaux, I., Doni, A., Moalli, F., Garlanda, C., Romani, L., Gascan, H., Bellocchio, S., Bozza, S., Cassatella, M.A., Jeannin, P., Mantovani, A., 2007. The humoral pattern recognition receptor PTX3 is stored in neutrophil granules and localizes in extracellular traps. J. Exp. Med. 204 (4), 793–804. Jena, P., Mohanty, S., Mohanty, T., Kallert, S., Morgelin, M., Lindstrom, T., Borregaard, N., Stenger, S., Sonawane, A., Sorensen, O.E., 2012. Azurophil granule proteins constitute the major mycobactericidal proteins in human neutrophils and enhance the killing of mycobacteria in macrophages. PLoS One 7 (12), e50345. http://dx.doi.org/10.1371/ journal.pone.0050345. Kaplan, M.J., Radic, M., 2012. Neutrophil extracellular traps: double-edged swords of innate immunity. J. Immunol. 189 (6), 2689–2695. http://dx.doi.org/10.4049/ jimmunol.1201719. Lominadze, G., Powell, D.W., Luerman, G.C., Link, A.J., Ward, R.A., McLeish, K.R., 2005. Proteomic analysis of human neutrophil granules. Mol. Cell. Proteomics 4 (10), 1503–1521 (Oct). Mantovani, A., Garlanda, C., Doni, A., Bottazzi, B., 2008. Pentraxins in innate immunity: from C-reactive protein to the long pentraxin PTX3. J. Clin. Immunol. 28 (1), 1–13. Moalli, F., Jaillon, S., Inforzato, A., Sironi, M., Bottazzi, B., Mantovani, A., Garlanda, C., 2011. Pathogen recognition by the long pentraxin PTX3. J. Biomed. Biotechnol. 2011, 830421. http://dx.doi.org/10.1155/2011/830421. Oyama, T., Abe, H., Ushiki, T., 2006. The connective tissue and glial framework in the optic nerve head of the normal human eye: light and scanning electron microscopic studies. Arch. Histol. Cytol. 69 (5), 341–356. Papayannopoulos, V., Zychlinsky, A., 2009. NETs: a new strategy for using old weapons. Trends Immunol. 30 (11), 513–521. http://dx.doi.org/10.1016/j.it.2009.07.011. Remijsen, Q., Kuijpers, T.W., Wirawan, E., Lippens, S., Vandenabeele, P., Vanden Berghe, T., 2011. Dying for a cause: NETosis, mechanisms behind an antimicrobial cell death modality. Cell Death Differ. 18 (4), 581–588. http://dx.doi.org/10.1038/cdd.2011.1. Savchenko, A., Imamura, M., Ohashi, R., Jiang, S., Kawasaki, T., Hasegawa, G., Emura, I., Iwanari, H., Sagara, M., Tanaka, T., Hamakubo, T., Kodama, T., Naito, M., 2008. Expression of PTX3 in human atherosclerotic lesions. J. Pathol. 215 (1), 48–55. http://dx.doi. org/10.1002/path.2314. Witko-Sarsat, V., Rieu, P., Descamps-Latscha, B., Lesavre, P., Halbwachs-Mecarelli, L., 2000. Neutrophils: molecules, functions and pathophysiological aspects. Lab. Investig. 80 (5), 617–653.
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Experimental and Molecular Pathology journal homepage: www.elsevier.com/locate/yexmp
Differential expression of pentraxin 3 in neutrophils Olga Razvina a, Shuying Jiang a, Koichi Matsubara a, Riuko Ohashi a, Go Hasegawa a, Takashi Aoyama a, Kenji Daigo b, Tatsuhiko Kodama b, Takao Hamakubo c, Makoto Naito a,⁎ a b c
Department of Cellular Function, Division of Cellular and Molecular Pathology, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan Laboratory for Systems Biology and Medicine, Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo, Japan Department of Quantitative Biology and Medicine, The University of Tokyo, Tokyo, Japan
a r t i c l e
i n f o
Article history: Received 8 November 2014 Accepted 18 November 2014 Available online 20 November 2014 Keywords: Immunohistochemistry Neutrophils Neutrophil extracellular traps (NETs) Neutrophilic granules Pentraxin 3
a b s t r a c t Pentraxins belong to the superfamily of conserved proteins that are characterized by a cyclic multimeric structure. Pentraxin 3 (PTX3) is a long pentraxin which can be produced by different cell types upon exposure to various inflammatory signals. Inside the neutrophil PTX3 is stored in form of granules localized in the cytoplasm. Neutrophilic granules are divided into three types: azurophilic (primary) granules, specific (secondary) granules and gelatinase (tertiary) granules. PTX3 has been considered to be localized in specific (secondary) granules. Immunofluorescent analyses using confocal laser microscopic examination were performed to clarify the localization of all three groups of granules within the cytoplasm of the mature neutrophils and neutrophils stimulated with IL-8. Furthermore, PTX3 was localized in primary granules of promyelocyte cell line HL-60. As a result, we suggest that PTX3 is localized not only in specific granules, but is also partly expressed in primary and tertiary granules. After the stimulation with IL-8, irregular reticular structures called neutrophil extracellular traps (NETs) were formed, three types of granules were trapped by NETs and PTX3 showed partial colocalization with these granular components. PTX3 localized in all three types of granules in neutrophils may play important roles in host defense. © 2014 Published by Elsevier Inc.
1. Introduction Polymorphonuclear neutrophils (or PMNs) are the most numerous leukocytes in mammals, much more numerous than the longer-lived monocytes/macrophages (Witko-Sarsat et al., 2000). PMNs are fast acting and effective phagocytes, which play a very important role in innate and adaptive immunity (Jena et al., 2012). They also stand as a first-line defense against invading pathogens (Borregaard, 2010). Neutrophils have various types of granules, containing lots of antibacterial proteins which can kill microbes and digest tissues (Borregaard, 2010; Kaplan and Radic, 2012). Neutrophil granules have been divided into three major subsets according to differences in protein content and propensity for mobilization (Hager et al., 2010). These subsets are azurophilic (primary) granules, specific (secondary) granules and gelatinase (tertiary) granules. The primary granules contain myeloperoxidase (MPO), neutrophil elastase, azurocidin and others, secondary granules contain lactoferrin and cathelicidin, and tertiary granules contain gelatinase, cathepsin and others (Jaillon et al., 2007). These granules appear in
⁎ Corresponding author at: Department of Cellular Function, Division of Cellular and Molecular Pathology, Niigata University Graduate School of Medical and Dental Sciences, 1-757 Asahimachi-dori, Chuo-ku, Niigata 951-8510, Japan. E-mail address: [email protected] (M. Naito).
http://dx.doi.org/10.1016/j.yexmp.2014.11.009 0014-4800/© 2014 Published by Elsevier Inc.
the PMNs sequentially as a result of granulocytic differentiation in the bone marrow, from myeloblast to the segmented stage when maturation is reached (Borregaard, 2010; Papayannopoulos and Zychlinsky, 2009). Pentraxins are small pentameric innate immunity effector proteins, which are very important in infectious and inflammatory responses (Bottazzi et al., 2006). Pentraxin 3 (PTX3) is the member of the pentraxin superfamily, which expression is induced in response to inflammatory signals. It is characterized by the presence of the carboxil terminal region, called pentraxin domain. PTX3 is the first identified long pentraxin (Mantovani et al., 2008). A variety of cell types, particularly macrophages, polymorphonuclear neutrophils, dendritic cells, endothelial cells and smooth muscle cells can produce PTX3 upon exposure to primary inflammatory signals, such as lipopolysaccharide and other agonists for Toll-like receptor family, IL-1β, and TNF-α, as well as oxidized low-density-lipoproteins (Bottazzi et al., 2006). Although previous studies demonstrated that PTX3 inside the neutrophil is stored in form of specific (secondary) granules in the cytoplasm (Jaillon et al., 2007), the strict co-localization of PTX3 with other granular proteins in the PMNs is not fully confirmed. In this study, using antibodies for granular components in the three groups of granules, we have investigated the localization of PTX3 and its relation to granules inside the neutrophil and those adhered to degenerated neutrophils called NETs.
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2. Materials and methods 2.1. Isolation of neutrophils Human polymorphonuclear leukocytes (PMLs) were isolated from the peripheral blood of healthy individuals using mono-poly resolving medium (DS Pharma Biomedical Co., Ltd., Osaka, Japan). After centrifugation the lower fraction consisted of PMNs, the percentage of neutrophils was evaluated using May–Giemsa staining. There were no immature myeloid elements. Isolated PMNs were suspended at a concentration of 1 × 106 cells/ml in RPMI 1640 medium with 2% fetal bovine serum and Penicillin Streptomycin (Pen Strep) mixture (Gibco). Freshly isolated cells were collected for immunofluorescent staining and immunoblotting. For immunoblotting cells were placed in a 3 cm dish and incubated, then one group of cells was stimulated using 100 ng/ml of IL-8. After 40 min, the supernatant and cell lysate were collected. Supernatant was also collected for enzyme-linked immunosorbent assay (ELISA), in order to check the level of PTX3 released by activated neutrophils. For immunofluorescent staining of neutrophils without stimulation freshly isolated non-stimulated PMNs were cytospined for 1 min at 1000 rpm and then stained. For immunofluorescent staining with stimulation, PMNs were seeded on glass coverslips treated with 0.001% polylysine and stimulated with 100 ng/ml of IL-8 (R&D Systems Inc., Minneapolis, MI, USA) for 40 min. 2.2. Isolation of HL-60 cell culture HL-60 cells were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum and Penicillin Streptomycin (Pen Strep) mixture (Gibco). Cells were maintained at 37 °C in a 5% CO2/95% air atmosphere and were used for experiments during the exponential phase of growth. The percentage of cells was evaluated using the Giemsa staining. Freshly isolated cells were collected for immunofluorescent staining and immunoblotting. For immunofluorescent staining cells were cytospined for 1 min at 1000 rpm and then stained. 2.3. Differentiation of HL-60 cell culture to neutrophil-like cells HL-60 cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and Penicillin Streptomycin (Pen Strep) mixture (Gibco) and at a density of 1 × 106 cells/ml was stimulated with 1.25% dimethyl sulfoxide DMSO (Sigma-Aldrich, Missouri, USA) for 7 days to
induce differentiation. Differentiated cell morphology was monitored by the Giemsa staining. A stationary state was reached at day 7 and isolated cells were collected for immunofluorescent staining. For immunofluorescent staining cells were cytospined for 1 min at 1000 rpm and then stained. 2.4. Immunofluorescent analysis Freshly isolated PMNs and HL-60 cells were cytospined for 1 min at 1000 rpm, blocked with 4% paraformaldehyde for 5 min and 0.5% Triton X-100/PBS for 5 min. Then specimens were blocked with 10% normal goat serum and stained with primary antibodies: anti-PTX3 monoclonal antibodies PPZ-1228 (Savchenko et al., 2008) at a dilution of 1:100 (Perseus Proteomics Inc., Tokyo, Japan), anti-lactoferrin monoclonal antibodies (EPR4338, GenTex Inc., Irvine, CA, USA) at a dilution of 1:100, polyclonal anti-myeloperoxidase antibodies (Abcam plc., Cambridge, UK) at 1:1000, polyclonal anti-gelatinase antibodies (EMD Millipore Corporation, Billerica, MA, USA) at 1:10,000 and monoclonal anti-azurocidin 1 Z6718 at 1:1000 dilution (Daigo and Hamakubo, 2012). Alexa Fluor 568 goat anti-mouse IgG Fab′ fragment (1:250 dilution, Invitrogen, Carlsbad, CA, USA) was used as secondary antibodies for PTX3 detection. A secondary antibody used for the detection of lactoferrin, gelatinase and MPO was Zenon Alexa Fluor 488 labeling kit goat anti-rabbit IgG Fab′ fragment (Invitrogen) at a dilution of 1:500. Monoclonal anti-azurocidin 1 Z6718 antibody was labeled with Z25102: Zenon Alexa Fluor 488 Mouse IgG2a Labeling kit (Invitrogen). DNA detection was carried out using blue fluorescent Hoechst 33342 (MW 615.99, H3570), purchased from Molecular Probes Inc. (Carlsbad, CA, USA). Control experiments were carried out by omitting primary antibodies. Immunofluorescent analysis was performed using the photomultiplier of a multi-photon laser scanning microscope (LSM510 META; Carl Zeiss, Jena, Germany). 2.5. Immunoblotting Grown PMNs were collected and lysed in a solution containing 150 mM NaCl, 50 nM Tris–HCl (pH 8.0), 5 mM ethylenediaminetetraacetic acid, 1% Triton X-100 and 1 mM phenylmethylsulfonyl fluoride, 1 mM leupeptin, 1 mM aprotinin for 10 min on ice and centrifuged at 15,000 rpm at 4 °C for 15 min. The supernatants were used with no dilution. Twenty five micrograms of the lysates and supernatants was
Fig. 1. a Expression and secretion of PTX3 protein in neutrophils. Lysates and supernatants revealed bands corresponding to molecular weight of 90 and 40 kDa. Freshly isolated lysates and supernatants showed PTX3 protein presence, with more intense expression in supernatants. After 40 min of IL-8 stimulation expression of PTX3 in cell lysate appeared to decrease in comparison to the cell lysate without stimulation. However, no clear difference was seen in PTX3 expression between supernatants of neutrophil culture with and without stimulation. b PTX3 levels in supernatant of non-stimulated neutrophils and neutrophils stimulated with IL-8. Supernatant of stimulated neutrophils showed 4 times higher PTX3 concentration compared to non-stimulated ones. c Lactoferrin expression in neutrophils. Lactoferrin expression without stimulation was seen in cell lysate only. Forty minutes after IL-8 stimulation lactoferrin expression was seen in supernatant as well as in cell lysate. Lactoferrin expression in cell lysate of non-stimulated neutrophils is more remarkable than in cell lysate of IL-8 stimulated neutrophils. HEK cells — Human Embryonic Kidney 293 cells.
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applied. The samples were resolved by sodium-dodecyl-sulfatepolyacrylamide-gel electrophoresis. After electrophoresis, the protein was transferred to a PVDF membrane (GE Healthcare, Amersham, UK). The blots were pre-treated with 5% skim milk overnight and then incubated with anti-lactoferrin (1:500 dilution), anti-gelatinase (1:1000), anti-myeloperoxidase (1:500) and anti-azurocidin (1:1000) antibodies. Then they were incubated with secondary anti-mouse IgG horseradish peroxidase-conjugated antibody (GE Healthcare) at a dilution of 1:2000 for 30 min and washed three times with PBS, between each step of the procedure. They were visualized with the ECL detection system (GE Healthcare) according to manufacturer's instructions (Imamura et al., 2007). 2.6. Enzyme-linked immunosorbent assay ELISA was performed using the described protocol (Imamura et al., 2007; Inoue et al., 2007). Pepsin-digested PPMX0104 (100 μl of a 5 μg/ml solution in saline) was coated on a microtiter plate, MaxiS-ope (Nalge Nunc, Penfield, New York, USA) at 4 °C overnight. The wells were blocked with 1% of casein in PBS, pH 7.4 at 4 °C overnight. Supernatant samples were added to the wells and incubated for 1 h. Bound PTX3 was detected using horseradish peroxidaseconjugated PPMX0105 (Perseus Proteomics Inc., Tokyo, Japan).
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Substrate solution was added to each well and incubated for 30 min. Stop solution was added and absorbance at 450 nm was measured with a microplate reader system (Towa Labo, Tokyo, Japan). All assays were carried out in duplicate. 2.7. Electron microscopy analysis Scanning electron microscopy (SEM) was used to observe IL-8 stimulated and non-stimulated PMN samples after fixation with 2% glutaraldehyde. SEM was performed as described (Oyama et al., 2006). Cells were stained using a tannin-osmium method, dehydrated in graded ethanol series and critical point dried. Then, specimens were coated with platinum–palladium using an ion-coater, and microscopically examined with a scanning electron microscope (H700: Hitachi, Tokyo, Japan). 2.8. Cell counting and colocalization analysis Double immunofluorescent staining of cytospined, non-stimulated neutrophils for PTX3 and MPO, azurocidin, lactoferrin, and gelatinase was performed. Then, photographs were taken at a magnification × 2000, using the photomultiplier of a multi-photon laser scanning microscope (LSM510 META; Carl Zeiss). The photographs were printed
Fig. 2. Non-stimulated cytospined neutrophils. a Double immunofluorescent staining for pentraxin 3 (PTX3) (red) and myeloperoxidase (MPO) (green). b Double immunofluorescent staining for pentraxin 3 (PTX3) (red) and azurocidin (green). c Double immunofluorescent staining for pentraxin 3 (PTX3) (red) and lactoferrin (green). d Double immunofluorescent staining for pentraxin 3 (PTX3) (red) and gelatinase (green). Bar (a, b, c, d) 5 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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out, magnified up to ×10,000. All the granules were counted separately: MPO, azurocidin, lactoferrin, and gelatinase — green granules, PTX3 — red granules and co-localized granules — yellow granules (merged image). Thirty neutrophils were counted for each of four groups (MPOPTX3, azurocidin-PTX3, lactoferrin-PTX3, gelatinase-PTX3), totally one hundred twenty neutrophils. 3. Results 3.1. PTX3 expression in PMNs and its levels after stimulation The secreted recombinant PTX3 protein consists of a major 45 kDa form of the monomer and 90 kDa form of dimer. PTX3 expression in neutrophils was also confirmed (Fig. 1a). Immunoblotting of freshly isolated lysates and supernatants obtained from PMN culture showed PTX3 proteins corresponding to molecular weight of 90 and 45 kDa. Expression of PTX3 was stronger in supernatants in comparison with cell lysates, suggesting that PTX3 protein was produced in neutrophils and secreted extracellularly. Under IL-8 stimulation, expression of PTX3 in cell lysate decreased in comparison with non-stimulated cell lysate. There appeared to be no clear difference in PTX3 expression between IL-8 stimulated and non-stimulated supernatants (Fig. 1a), however, ELISA showed 4 times higher PTX3 concentration in the culture medium of neutrophils stimulated with IL-8, compared to non-stimulated ones, indicating that PTX3 released from PMNs is enhanced by IL-8 (Fig. 1b). 3.2. Lactoferrin expression in PMNs after stimulation Lactoferrin expression was confirmed in neutrophils as well. Immunoblotting of isolated lysates and supernatants revealed expression of lactoferrin protein corresponding to molecular weight of 85 kDa (predicted band size stated in the lactoferrin antibody's datasheet is confirmed). Lactoferrin expression was more intense in cell lysates, suggesting that lactoferrin is mostly stored intracellularly. No band was present in supernatant without stimulation with IL-8. Stimulated
supernatant medium showed distinct lactoferrin expression suggesting that lactoferrin granules are released outside the cell under the stimulation (Fig. 1c). Similar expression pattern was observed by immunoblotting using antibodies against azurocidin, MPO and gelatinase (data not shown).
3.3. Double immunofluorescent staining of non-stimulated neutrophils Double immunofluorescent analysis of non-stimulated neutrophils revealed positive expression of PTX3 in the cytoplasm of a neutrophil in a form of diffusely located granules. Double immunofluorescence staining showed that most of the lactoferrin-positive granules were colocalized with PTX3 granules (Figs. 2c, 3c). The mean quantity of colocalized PTX 3 and lactoferrin granules was 72.3 ± 8.2. Azurocidin and myeloperoxidase which belong to the group of primary granules showed partial colocalization with PTX3 granules. The mean number of colocalized MPO and PTX3 granules was 26.2 ± 5.5. The mean number of colocalized azurocidin and PTX3 granules was 30.0 ± 4.9 (Figs. 2a, b, 3a, b). Gelatinase granules were also partly co-localized with PTX3. The mean quantity of colocalized PTX 3 and gelatinase granules was 34.0 ± 4.7 (Figs. 2d, 3d). The highest percentage of co-localization was seen between PTX3 and lactoferrin — 71% (Fig. 3e). Percent of co-localization for other granular components was as follows: MPO — 25%, azurocidin — 31, 5%, and gelatinase — 35%, showing that they are partly colocalized with PTX3 (Fig. 3e).
3.4. Expression of PTX3 and other granular components in stimulated PMNs Neutrophils were stimulated with IL-8, cultured for 40 min and then observed with a scanning electron microscope. Degeneration and cell death of neutrophils can be seen on a scanning electron microscopic image (Fig. 4). It revealed the irregular reticular structure of NETs,
Fig. 3. The mean number of neutrophil granular components, their colocalization with PTX3. Red: PTX3; yellow: granules, showed colocalization with PTX3; green: a MPO, b azurocidin, c lactoferrin, d gelatinase. e Percentage of colocalization with PTX3. No. of double positive granules/No. of PTX3+ granules × 100 = %. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Fig. 4. a Scanning electron micrograph (SEM) of non stimulated PMNs. B Neutrophils activated with IL-8 for 40 min. c Higher magnification of IL-8 stimulated neutrophils showing irregular reticular structure and globular domains aggregated on threads (NETs).
which was supposedly formed by activated PMNs and globular domains aggregated to threads. Immunofluorescent analysis of PMNs stimulated with IL-8 showed the Hoechst 33342-positive irregular reticular structure, considered to be NETs. PTX3 positive granules adhered to this structure were observed. It was demonstrated that PTX3 was partially co-localized with MPO, azurocidin and gelatinase (Fig. 5a, b, d) as well as
lactoferrin (Fig. 5c) not only in neutrophils but also on the surface of NETs. 3.5. Expression of granular components in promyelocytes HL-60 (human promyelocytic leukemia cells) cell line was used, as HL-60 cells contain only primary granules. Double immunofluorescent
Fig. 5. Neutrophils activated with IL-8. a Double immunofluorescent staining for pentraxin 3 (PTX3) (red) and myeloperoxidase (MPO) (green). b Double immunofluorescent staining for PTX3 (red) and azurocidin (green). c Double immunofluorescent staining for PTX3 (red) and lactoferrin (green). d Double immunofluorescent staining for PTX3 (red) and gelatinase (green). Bar (a, b, c, d) 5 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Fig. 6. The HL-60 (human promyelocytic leukemia cells) cell line. a Double immunofluorescent staining for pentraxin 3 (PTX3) (red) and myeloperoxidase (MPO) (green). b Double immunofluorescent staining for PTX3 (red) and lactoferrin (green). c Double immunofluorescent staining for PTX3 (red) and gelatinase (green). Bar (a, b, c) 2.5 μm. d Expression of PTX3, MPO, lactoferrin and gelatinase in HL-60 cell line. Expression of PTX3 (90 and 40 kDa bands) and MPO (84 kDa band) was found to be positive, while expression of lactoferrin and gelatinase was found to be negative.
analysis showed that primary granules (MPO) were positively stained as well as PTX3 (Fig. 6a). Other components of secondary (lactoferrin) and tertiary (gelatinase) granules were stained negatively (Fig. 6b, c). These results were also confirmed by immunoblotting of freshly isolated HL-60 cells. MPO protein was detected as bands corresponding 84 kDa. PTX3 expression was confirmed as well. Bands corresponding to molecular weight of 45 kDa (monomer) and 90 kDa (dimer) were observed, showing that PTX3 may localize not only in specific granules, but in primary granules as well (Fig. 6d).
3.6. Expression of granular components in HL-60-derived neutrophil-like cells Double immunofluorescent analysis showed that PTX3 and all other three groups of granules: primary (MPO), secondary (lactoferrin), and tertiary (gelatinase) granules were present in HL-60 neutrophil-like cells differentiated with DMSO, showing partial colocalization with PTX3 (Fig. 7a, b, c).
4. Discussion In this study, we have confirmed the positive PTX3 expression in mature PMNs, in the form of diffusely located granules in the cytoplasm, using the immunofluorescent staining. PTX3 was mainly colocalized with lactoferrin, which is considered to be the component of secondary (specific) granules as was reported previously (Jaillon et al., 2007; Remijsen et al., 2011). Furthermore we observed partial expression of PTX3 in primary and tertiary granules, though the percentage of PTX3 colocalization in these two granules was lower than that in specific granules. Neutrophils act as the first line defense against microbes and perform several methods of direct killing pathogens: phagocytosis and release of antibacterial substances, including granule proteins. Neutrophil granules are classified into primary (azurophilic) granules (MPO), secondary (specific) granules (lactoferrin), and tertiary (gelatinase) granules. These granules are formed sequentially during granulocytic differentiation in the bone marrow. PTX3 is also a granular component which localizes in specific granules as demonstrated
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Fig. 7. The HL-60-derived neutrophil-like cells. a Double immunofluorescent staining for pentraxin 3 (PTX3) (red) and myeloperoxidase (MPO) (green). B Double immunofluorescent staining for PTX3 (red) and lactoferrin (green). C Double immunofluorescent staining for PTX3 (red) and gelatinase (green). Bar (a, b, c) 5 μm. d HL-60 cell line without any stimulation, which consisted predominantly of promyelocytes with azurophilic granules, large round nuclei and prominent nucleoli. Giemsa staining, 100× magnification. (e) HL-60 cell line stimulated with 1, 25% DMSO for 7 days. HL-60-derived neutrophil-like cells are seen, with decreased nuclear/cytoplasmic ratio and nuclei subdivided into lobes. Giemsa staining, 100× magnification. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
by immunofluorescence observation (Jaillon et al., 2007) and proteomics analysis (Remijsen et al., 2011). Using western blotting we have also confirmed that both PTX3 and lactoferrin proteins were found in PMNs and released extracellularly, suggesting both proteins are present in the same granules. As to granule biogenesis, primary granules are formed at the stage of promyelocytes and secondary and tertiary granules are produced at the stage of myelocytes and metamyelocytes. Jallion et al. reported that PTX3 mRNA expression is positive in progenitor neutrophils (promyelocytes, myelocytes, metamyelocytes), but it is absent in mature neutrophils (Garlanda et al., 2002), suggesting that PTX3 is produced during the maturation course of neutrophils and stored in mature neutrophils in the form of ready-to-use specific granules (Jaillon et al., 2007; Moalli et al., 2011; Papayannopoulos and Zychlinsky, 2009). We have demonstrated that the human promyelocytic cell line HL-60 expresses MPO and azurocidin, markers of primary granules, does not express lactoferrin, a marker of specific granules, and gelatinase, a marker of tertiary granules. However, immunostaining and immunoblotting demonstrated that PTX3 protein was expressed in HL-60 cell line. These findings indicate that PTX3 may localize in primary granules as well as in specific granules. We have also stimulated HL-60 cells with DMSO in order to differentiate them to neutrophil-like cells. These differentiated cells showed positive expression and colocalization of PTX3 and other granular
components: primary (MPO), secondary (lactoferrin), and tertiary (gelatinase) granules. The quantity of MPO and PTX3 granules considerably increased in comparison with non-stimulated HL-60 cells, while lactoferrin (secondary) and gelatinase (tertiary granules) turned to be positive, which confirms neutrophilic granule biogenesis theory. We have found that in mature neutrophils PTX3 is partly colocalized with MPO and azurocidin, which belong to primary granules, and gelatinase, which belongs to tertiary granules. Combining the present and previously reported results, we suggest that PTX3 not only localizes in specific granules, but is also expressed in primary and tertiary granules. Neutrophil extracellular traps (NETs) are chromatin structures loaded with antimicrobial molecules first reported in 2004 (Brinkmann et al., 2004). This reticular structure is composed of nuclear chromatin fibers, nuclear histones and granules with antimicrobial proteins and is released as a response to different inflammatory stimuli (Fuchs et al., 2007; Imamura et al., 2007). Netting neutrophils do not display “eat me” signals as apoptotic cells do, but it may help them to escape clearance by phagocytes (Borregaard et al., 2012; Bottazzi et al., 2006). NETs can possibly trap and kill pathogens (bacteria, fungi) by means of bactericidal proteins, stored in neutrophil granules, which are connected to DNA fibers, forming a special microenvironment (Lominadze et al., 2005; Mantovani et al., 2008). The present study revealed that after the stimulation three types of granules were localized extracellularly, trapped by NETs and PTX3 was
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partially colocalized with these granular components. PTX3 adhered to NET structure may play a great role in suppression of pathogens (Bottazzi et al., 2006; Deban et al., 2011; Papayannopoulos and Zychlinsky, 2009). It has been reported in the study using PTX3-null mice that PTX3 can directly bind to microbial pathogens, such as Aspergillus fumigatus, Pseudomonas aeruginosa, Salmonella typhimurium and others, thereby representing a mechanism of innate resistance amplification against pathogens. PTX3 and granular components adhered to NETs may act locally at the site of infection and inflammation (Garlanda et al., 2002). Recently, it has been identified that PTX3 may enhance the antibacterial efficiency of azurocidin and MPO. A biochemical analysis demonstrated the direct binding of azurocidin and MPO to PTX3 (Daigo and Hamakubo, 2012). It is not clear whether these PTX3-MPO and PTX3azurocidin complexes would occur after the release of granules or they are already formed in neutrophil granules. For understanding the precise mechanism of PTX3 and azurocidin or MPO colocalization in neutrophils and NETs further studies are required. Conflict of interest statement The authors declare that there are no conflicts of interest. Acknowledgments The authors would like to thank Mr. Kenji Oyauchi, Mrs. Yasue Honma and all the stuff of the Department of Cellular Function, Division of Cellular and Molecular Pathology, Niigata University Graduate School of Medical and Dental Sciences, for their excellent technical assistance. This study was supported in part by Ministry of Scientific Culture, Sports, Science and Technology Grant-in-aid for Scientific Research 25220205. References Borregaard, N., 2010. Neutrophils, from marrow to microbes. Immunity 33 (5), 657–670. http://dx.doi.org/10.1016/j.immuni.2010.11.011 (Nov 24). Borregaard, N., Stenger, S., Sonawane, A., Sorensen, O.E., 2012. Azurophil granule proteins constitute the major mycobactericidal proteins in human neutrophils and enhance the killing of mycobacteria in macrophages. PLoS One 7 (12), e50345. http://dx.doi. org/10.1371/journal.pone.0050345. Bottazzi, B., Garlanda, C., Salvatori, G., Jeannin, P., Manfredi, A., Mantovani, A., 2006. Pentraxins as a key component of innate immunity. Curr. Opin. Immunol. 18 (1), 10–15. Brinkmann, V., Reichard, U., Goosmann, C., Fauler, B., Uhlemann, Y., Weiss, D.S., Weinrauch, Y., Zychlinsky, A., 2004. Neutrophil extracellular traps kill bacteria. Science 303 (5663), 1532–1535. Daigo, K., Hamakubo, T., 2012. Host-protective effect of circulating pentraxin 3 (PTX3) and complex formation with neutrophil extracellular traps. Front. Immunol. 3, 378. http://dx.doi.org/10.3389/fimmu.2012.00378.
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