Vesicular nucleotide transporter mediates ATP release and migration in neutrophils

Abstract

Neutrophils migrate to sites infected by pathogenic microorganisms. This migration is regulated by neutrophil-secreted ATP, which stimulates neutrophils in an autocrine manner through purinergic receptors on the plasma membrane. Although previous studies have shown that ATP is released through channels at the plasma membrane of the neutrophil, it remains unknown whether it is also released through alternate secretory systems involving vesicular mechanisms. In this study, we investigated the possible involvement of vesicular nucleotide transporter (VNUT), a key molecule for vesicular storage and nucleotide release, in ATP secretion from neutrophils. RT-PCR and Western blotting analysis indicated that VNUT is expressed in mouse neutrophils. Immunohistochemical analysis indicated that VNUT mainly colocalized with matrix metalloproteinase-9 (MMP-9), a marker of tertiary granules, which are secretory organelles. In mouse neutrophils, ATP release was inhibited by clodronate, which is a potent VNUT inhibitor. Furthermore, neutrophils from VNUT^−/− mice did not release ATP and exhibited significantly reduced migration in vitro and in vivo. These findings suggest that tertiary granule-localized VNUT is responsible for vesicular ATP release and subsequent neutrophil migration. Thus, these findings suggest an additional mechanism through which ATP is released by neutrophils.

Introduction

Purinergic chemical transmission is involved in regulating the function of many types of blood cells (1). Polymorphonuclear neutrophils (PMNs)³ are primary phagocytic cells that play a crucial role in defense against invading microorganisms such as bacteria, fungi, and protozoa. When these organisms invade, PMNs sense the infection and secrete nucleotides such as ATP, which act as autocrine or paracrine signals to initiate a series of responses including granular release, chemotaxis, the production of superoxide, and the regulation of apoptosis upon binding to purinoceptors in neutrophils (2–7). For example, during chemotaxis, ATP released from chemoattractant-stimulated neutrophils is involved in signal amplification, controlling gradient sensing, and controlling migration speed via the purinergic P2Y2 receptor and A₃ receptor in an autocrine manner (3). Thus, purinergic chemical transmission plays a crucial role in neutrophil function.

Even though ATP signal reception by neutrophils is well understood, the mechanism through which neutrophils secrete ATP is relatively unknown. At least two major mechanisms have been postulated for the release of ATP: one is that ATP release is mediated by ATP channels at the plasma membrane, and another is exocytosis of ATP (8, 9). Connexin-43 and pannexin-1 channels are involved in ATP release from neutrophils, leading to migration (10–12). However, the mechanism of ATP release is controversial; specifically, there are multiple ATP release pathways in neutrophils, including the secretion of granules that store ATP, because the amount of ATP released is dependent on the concentration of the chemoattractant (13). These studies suggest the existence of granule-mediated ATP exocytosis.

Vesicular nucleotide transporter (VNUT) is responsible for the vesicular storage of ATP and plays an essential role in the exocytosis of ATP upon stimulation (14). This protein is a member of the SLC17 anion transporter family and transports nucleotides such as ATP and ADP into secretory vesicles using the membrane potential (Δψ; inside positive) across membranes, which is established by vacuolar H⁺-ATPase (14, 15). Recent studies indicated that VNUT is expressed and functions in neuroendocrine and immune cells, which have been reported to be associated with purinergic tissues or cells (14–22). VNUT^−/− mice were shown to lose vesicular ATP contents and vesicular ATP release, resulting in loss of purinergic chemical transmission in vivo (19). VNUT is primarily responsible for vesicular ATP storage and release and plays an essential role in purinergic chemical transmission.

In this study, we determined whether VNUT is expressed and functions in neutrophils. We showed that VNUT is expressed and localized in the tertiary granules of neutrophils and that neutrophils release ATP in a VNUT-dependent manner. In addition, VNUT-mediated ATP release was shown to be involved in neutrophil migration, which was blocked by the clinically available VNUT inhibitor clodronate.

Results

VNUT is localized in tertiary granules of neutrophils

As the first step of the study, we isolated neutrophils from the bone marrow of wildtype and VNUT^−/− mice and examined the status of VNUT. Regarding the form of neutrophils, there was no morphological difference between those from wildtype and VNUT^−/− mice, based on Giemsa staining (Fig. 1A). RT-PCR analysis indicated that a 523-bp VNUT-specific transcript was amplified from mouse neutrophils, whereas no such transcript was detected in wildtype neutrophils without the RT reaction or in neutrophils from VNUT^−/− mice (Fig. 1B). Immunoblotting with specific antibodies against VNUT indicated that the membrane fraction of wildtype mouse neutrophils contained an immunoreactive polypeptide with an apparent molecular mass of 70 kDa (Fig. 1C). This was absent in neutrophils from VNUT^−/− mice. The protein levels of other neutrophil granules or vesicle proteins, including V-ATPase, formyl peptide receptor-like 1 (FPRL1), and VAMP2 (vesicle-associated membrane protein 2), were unaffected by the absence of VNUT (Fig. 1C). Furthermore, indirect immunofluorescence microscopy indicated that VNUT was present in mouse neutrophils and exhibited a punctate distribution, whereas control reactions with preabsorbed antibodies resulted in only background levels of staining (Fig. 1D). In VNUT^−/− mice, no VNUT immunoreactivity was observed, similar to that observed in the negative control (Fig. 1D).

Figure 1.

Expression of VNUT in mouse neutrophils. A, neutrophils from the bone marrow of WT and VNUT^−/− (KO) mice were stained with a Giemsa stain. Segmented nuclei were stained purple. Bar, 10 μm. B, RT-PCR analysis was performed using total RNA isolated from bone marrow-derived neutrophils of WT and KO mice (523 bp) after RT reaction (+RT) and without RT reaction (−RT). The PCR product from VNUT cDNA was shown as a positive control. The expression of mouse glyceraldehyde-3-phosphate dehydrogenase (mG3PDH) was also shown for internal RNA quality control (150 bp). C, Western blot of bone marrow-derived neutrophil membrane vesicles (100 μg) prepared from wildtype and VNUT^−/− mice and probed using anti-VNUT antibody. The position of VNUT (70 kDa) is marked with an arrowhead. The expression of V-ATPase subunit A, FPRL1, and VAMP2 is also shown. D, indirect immunofluorescence microscopy revealed that VNUT is expressed in wildtype mouse bone marrow-derived neutrophils. No VNUT immunoreactivity was observed in neutrophils from VNUT^−/− mice. Inset, background signal with preabsorbed anti-mouse VNUT antibody. Bars, 10 μm.

Next, we examined whether VNUT immunoreactivity was associated with granular components in mouse neutrophils. Neutrophils harbor some secretory organelles such as azurophil granules, specific granules, tertiary granules, and secretory vesicles, which contain an abundance of enzymes or receptors (23). Double-labeling immunofluorescence microscopy revealed that VNUT immunoreactivity was colocalized with matrix metalloproteinase-9 (MMP-9), a tertiary granule marker (colocalization coefficients for VNUT: M1: 0.703, M2: 0.764), but not with myeloperoxidase (MPO), an azurophil granule marker (M1: 0.170, M2: 0.378); lactoferrin, a specific granule marker (M1:0.121, M2:0.449); or CD35, a secretory vesicle marker (M1:0.163, M2:0.400) in mouse neutrophils (Fig. 2A). Immunofluorescence microscopy also showed no apparent differences in the fluorescent staining images between wildtype and VNUT^−/− mice (Fig. 2A). VNUT was not colocalized with GM130 (M1: 0.079, M2: 0.581), early endosome antigen 1 (EEA1; M1: 0.229, M2: 0.341), LAMP1 (lysosomal-associated membrane protein 1; M1: 0.240, M2: 0.396), or protein disulfide isomerase (PDI; M1: 0.257, M2: 0.565), markers of the Golgi apparatus, early endosome, lysosome, and endoplasmic reticulum, respectively (Fig. 2B). Furthermore, VNUT was partially colocalized with VAMP2 (M1: 0.659, M2: 0.772), a marker of secretory granules (Fig. 2B). These results suggested that VNUT is localized to tertiary granules in mouse neutrophils.

Figure 2.

Localization of VNUT in mouse neutrophils. A and B, mouse neutrophils derived from bone marrow were double-immunostained with antibodies against VNUT (green, left panels) and granule markers (MPO, lactoferrin, MMP-9, and CD35) (A) and GM130, EEA1, LAMP1, PDI, and VAMP2 (B) (red, middle panels). Merged images (right panels) are also shown. Images of neutrophils from VNUT^−/− mice which were immunostained with antibodies of granule markers are also shown. Areas surrounded by dotted lines are enlarged in insets. Arrowheads, merged regions. Bars, 10 μm. C, summary of digital image analysis (colocalization coefficients) in A and B. M1 (upper panel) and M2 (lower panel) are shown. The data are shown as the mean ± S.E. (n = 3).

VNUT-dependent vesicular ATP release from mouse neutrophils

Further, we examined whether the vesicular release of ATP from neutrophils occurs in a VNUT-dependent manner. ATP release was observed in isolated mouse neutrophils upon stimulation with 5 μm A23187, a Ca²⁺ ionophore, or 100 nm W-peptide, a formyl peptide receptor ligand; however, this was almost completely abolished in cells from VNUT^−/− mice (Fig. 3, A and B, left panels). On the other hand, no differences were observed in the release of MPO after stimulation with 5 μm A23187 and 100 nm W-peptide or in the release of MMP-9 upon stimulation with 5 μm A23187 in cells from wildtype and VNUT^−/− mice; however, W-peptide–induced MMP-9 release was reduced in VNUT^−/− cells (Fig. 3, A and B, middle and right panels). ATP release was also blocked by 0.1 or 1 μm clodronate, a potent and selective VNUT inhibitor (24) (Fig. 3C). After adding 0.1 or 1 μm clodronate, neutrophil viability was 97.6 and 96.9%, respectively. Previous studies reported that vesicular ATP release is strongly inhibited by low temperatures or Ca²⁺ chelators (16, 24, 25). In this study, we showed that A23187-stimulated ATP release is significantly reduced when neutrophils are incubated at 20 °C or 4 °C (Fig. 3D). In the presence of an extracellular and intracellular Ca²⁺ chelator EGTA and EGTA-AM, the release of ATP stimulated by 5 μm A23187 was also reduced (Fig. 3E). These results strongly suggested that ATP release from mouse neutrophils can occur via a VNUT-mediated exocytotic mechanism.

Figure 3.

VNUT is involved in ATP release from mouse neutrophils. The data are shown as the mean ± S.E. A and B, the release of ATP (left panels), MPO (middle panels), and MMP-9 (right panels) from bone marrow-derived neutrophils from wildtype (open bars) and VNUT^−/− mice (filled bars) was assessed 3 min (ATP) or 20 min (MPO and MMP-9) after the addition of 5 μm A23187 (A) and 100 nm W-peptide (B, n = 4–10). C, effects of 0.1 or 1 μm clodronate on A23187-induced ATP release from mouse neutrophils. D, temperature dependence for A23187-induced ATP release (n = 8–9). E, calcium dependence for A23187-induced ATP release. Mouse neutrophils were incubated in the presence or absence of 1 mm EGTA (left panel) and 50 μm EGTA-AM (right panel) (n = 7–10). N.D., not detected; N.S., not significant; **, p < 0.01.

Expression of VNUT in human neutrophils

To examine the function of VNUT in greater detail, we prepared human neutrophils from blood samples obtained from volunteers and determined whether VNUT was expressed in human neutrophils (Fig. 4A). Through RT-PCR analysis, a 115-bp VNUT-specific transcript was amplified in human neutrophils (Fig. 4B). Indirect immunofluorescence microscopy further indicated that VNUT immunoreactivity occurred in human neutrophils and exhibited a punctate distribution, whereas control staining with preabsorbed antibodies exhibited only background levels of staining (Fig. 4C). Furthermore, in agreement with our results in mouse neutrophils, double-labeling immunofluorescence microscopy and digital image analysis (colocalization coefficients) indicated that VNUT immunoreactivity was colocalized with MMP-9 (M1: 0.796, M2: 0.639) (Fig. 4D). In contrast, this immunoreactivity was not colocalized with MPO (M1: 0.191, M2: 0.420), lactoferrin (M1: 0.341, M2: 0.354), or CD35 (M1: 0.167, M2: 0.407). Taken together, it can be suggested that VNUT is also localized in tertiary granules in human neutrophils.

Figure 4.

Expression and localization of VNUT in human neutrophils. A, neutrophils from human peripheral blood samples were stained with Giemsa stain. Segmented nuclei were stained purple. Bar, 10 μm. B, RT-PCR analysis was performed using total RNA isolated from neutrophils of human peripheral blood (115 bp) after RT reaction (+RT) and without RT reaction (−RT). The PCR product from VNUT cDNA is shown as the positive control. Expression of the human glyceraldehyde-3-phosphate dehydrogenase (hG3PDH) gene was also shown as an internal RNA quality control (115 bp). C, indirect immunofluorescence microscopy revealed that VNUT was expressed in human neutrophils. Inset, background signal with preabsorbed anti-human VNUT antibody. Bars, 10 μm. D, human neutrophils were double-immunostained with antibodies against human VNUT (green, left panels) and granule markers (MPO, lactoferrin, MMP-9, and CD35). Merged images (right panels) are also shown. Areas surrounded by dotted lines are enlarged in insets. Arrowheads, merged regions. Bars, 10 μm. E, summary of digital image analysis (colocalization coefficients) in D. M1 (upper) and M2 (lower) are shown. The data are shown as the means ± S.E. (n = 3).

VNUT^−/− mice and VNUT inhibitors result in impaired neutrophil migration

Finally, we studied the role of VNUT-independent ATP release on neutrophil function. To assess neutrophil migration in vitro using cells isolated from the bone marrow of wildtype and VNUT^−/− mice, we performed Transwell assays consisting of upper wells with neutrophils and lower wells with 100 nm W-peptide separated by a filter with 3-μm pores. Consistent with the results of ATP release, we found that the W-peptide–induced migration of neutrophils from VNUT^−/− mice was decreased by 54% compared with that of cells from wildtype mice (Fig. 5A). No effect on exogenous ATP was observed in the W-peptide–induced migration of neutrophils from VNUT^−/− mice, whereas the addition of adenosine or IB-MECA, an A₃ receptor agonist, increased the migration of neutrophils from VNUT^−/− mice (Fig. 5, A and B). The addition of 1 μm clodronate also inhibited W-peptide–induced neutrophil migration by 41% (Fig. 5C). To confirm the importance of VNUT for neutrophil migration in vivo, we assessed cell recruitment to the hind paw of wildtype control and VNUT^−/− mice after a subcutaneous injection of 20 μl of 1 mg/ml complete Freund's adjuvant (CFA). As shown in Fig. 5 (D and E), CFA-induced inflammatory and neutrophil recruitment was observed in the hind paw of wildtype mice, but this was reduced in VNUT^−/− mice. Compared with neutrophil numbers in wildtype mice, those in VNUT^−/− mice were decreased by 73, 46, 38, and 45% at 6, 12, 24, and 48 h, respectively, after injecting CFA (Fig. 5E). These results indicated that VNUT is important for neutrophil migration both in vitro and in vivo.

Figure 5.

VNUT is involved in mouse neutrophil migration. All data are shown as the means ± S.E. A and B, Transwell migration assays, toward 100 nm W-peptide, were performed with neutrophils from wildtype (open bars) and VNUT^−/− mice (filled bars). Migration was expressed as a percentage of the response to W-peptide in wildtype mice. Effects of 100 μm ATP (A) and 1 μm adenosine, and 1 μm IB-MECA (B) on W-peptide–induced migration of wildtype and VNUT^−/− mice neutrophils are shown (n = 4). C, Transwell migration assays were performed in the presence of 1 μm clodronate (n = 8–10). D, CFA-induced neutrophil recruitment into hind paws of wildtype and VNUT^−/− mice. The typical dorsa of hind paw sections from wildtype and VNUT^−/− mice at 24 and 48 h after subcutaneous injection of CFA are shown. Photographs were analyzed by BZ-X Analyzer software for counting. The original image of Gr-1 immunohistochemistry staining (upper row) and markup image after analysis with red (lower row) are shown. Bars, 10 μm. E, neutrophil migration into the dorsa of wildtype (open squares) and VNUT^−/− mouse (filled squares) hind paws 3, 6, 12, 24, and 48 h after subcutaneous injection of CFA was assessed (n = 3–6 animals; two sections per animal) for each group. *, p < 0.05; **, p < 0.01; N.S., not significant.

Discussion

How and where ATP is stored in neutrophil granules and how vesicular ATP release is regulated during neutrophil migration are unresolved questions. In the present study, we found that VNUT is involved in the vesicular release of ATP from neutrophils. VNUT is expressed and associated with granules in neutrophils. In vitro, neutrophils were found to release ATP and migrate upon W-peptide stimulation, which is abolished by a VNUT inhibitor and in VNUT^−/− mice. In vivo, CFA induced neutrophil migration into the footpad of wildtype mice, whereas this migration was reduced in VNUT^−/− mice. These observations indicated that VNUT is involved in ATP release and migration in neutrophils.

Neutrophils possess several distinct granule subsets, namely azurophil granules, specific granules, and tertiary granules, and secretory vesicles (23). These granules and vesicles are secretory organelles that are classified based on their size, contents, and other parameters. It remains unknown whether ATP is stored in neutrophil granules and subsequently released through granule exocytosis. Our immunohistochemical analyses indicated that VNUT is colocalized with MMP-9, indicating that VNUT-containing granules are tertiary granules that contain V-ATPase as the driving force of ATP transport (Δψ) (26). These granules also contain matrix-degrading enzymes (such as MMPs) and membrane receptors (23). Exocytosis of these enzymes from tertiary granules is essential for interstitial structures during neutrophil migration. Upon stimulation, the formyl-Met-Leu-Phe receptor, which is localized to tertiary granules, translocates to the plasma membrane through exocytosis (27).receptors are involved in signal amplification These results suggested that the translocation of the formyl-Met-Leu-Phe receptor to the leading edge of the plasma membrane coincides with vesicular ATP release and promotes more efficient migration in neutrophils. Identification of VNUT-containing granules will be helpful for understanding the mechanisms that regulate ATP release and migration in neutrophils.

We found that W-peptide–stimulated ATP release is abolished in neutrophils derived from VNUT^−/− mice, whereas nonstimulated cells constantly release ATP (Fig. 3B). Previous inhibitor-based studies have shown that ATP release is also dependent on pannexin 1 hemichannels and connexin 43 from leukotriene B4 (LTB4)-stimulated neutrophils, as well as vesicular release (8–12). However, we previously found that the disruption of ATP release using inhibitors of hemichannels also inhibits VNUT (28). In addition, the amount of ATP released varies according to the stimulation intensity (13), suggesting that neutrophils have multiple ATP release pathways. Neutrophils might use multiple ATP release pathways depending on the type and concentration of chemoattractant. Therefore, the characterization of ATP release pathways might help to elucidate how VNUT-mediated and channel-mediated ATP release is involved in the pathogenic mechanisms of inflammatory diseases.

In the present study, it was determined that A23187-stimulated MMP-9 release from VNUT^−/− mice was the same as that from wildtype mice (Fig. 3A). This suggests that VNUT is involved in the vesicular storage and release of ATP but not granular accumulation or the release of other contents. On the other hand, W-peptide–stimulated MMP-9 release was reduced in neutrophils derived from VNUT^−/− mice (Fig. 3B). Extracellular ATP caused an increase in intracellular Ca²⁺ through the P2Y2 receptor, leading to degranulation (11). These results suggested that MMP-9 release is dependent on physiological stimuli and is positively controlled by VNUT-mediated ATP release. A similar mechanism of autocrine regulation of ATP has been reported in connection with the release of adrenaline from chromaffin cells (19). VNUT might be a key molecule for autocrine regulation of ATP in neutrophils. Another possibility of this observation is that VNUT regulates tertiary granules secretion independent of ATP secretion under physiological stimulation. We also found that neutrophil migration was not rescued by the addition of ATP, but migration was rescued by adding adenosine and IB-MECA, an A₃ agonist, in VNUT^−/− cells in vitro. (Fig. 5, A and B). Previous reports showed that A₃ receptors promote neutrophil migration and P2Y2 receptors are involved in signal amplification, gradient sensing and cell motility (29). Our results suggest that exogenous ATP access P2Y2 receptors omnidirectionaly, resulting in a loss of gradient sensing and polarity in neutrophils.

Recently, we identified a potent allosteric inhibitor of VNUT, clodronate (24). VNUT mediated-ATP transport activity is allosterically regulated by Cl⁻ and inhibited competitively and reversibly by keto acids such as acetoacetate and glyoxylate (22, 30). A previous study indicated that clodronate inhibits vesicular ATP release and decreases the release of inflammatory mediators, thereby attenuating chronic inflammation (24). In the present study, we found that clodronate also inhibits vesicular ATP release from neutrophils and reduces neutrophil migration, indicating a novel mechanism through which VNUT mediates its anti-inflammatory effects (Fig. 5C).

Neutrophils migrate to the sites of pathogenic invasion or inflammation and release cytokines in response to a concentration gradient of chemoattractants from microbial pathogens or injured cells. Interestingly, purinergic chemical transmission is involved not only in migration but also in phagocytosis and the production and release of inflammatory mediators (31, 32). It has been reported that adenosine, which is a product of ATP degradation, affects phagocytosis via the A₁ or A_2A receptor and modulates inflammatory mediators such as tumor necrosis factor via the A_2A receptor (31, 32). Neutrophils generate reactive oxygen species to kill pathogens targeted for phagocytosis; however, the release of such compounds from overactivated neutrophils can also damage host tissues in inflammatory diseases. Purinergic chemical transmission also affects the generation of reactive oxygen species in neutrophils (31, 33). VNUT can be a major source of extracellular ATP and is involved in neutrophil activation; it might also be the cause of some inflammatory diseases.

It is noteworthy that VNUT could be a good target for the treatment of chronic inflammatory diseases such as chronic obstructive pulmonary disease and cystic fibrosis. In these diseases, controlling of neutrophil function including recruitment would be an efficient therapy to treat inflammation (34). Clodronate is a specific inhibitor of VNUT and is currently used as an anti-osteoporotic drug (24, 35). Our previous study on the biochemistry and pharmacology of clodronate showed that the compound has an anti-inflammatory effect. Therefore, our findings support the clinical significance of clodronate for the treatment of acute sepsis and chronic inflammatory diseases. Further pathological studies regarding the inhibitory effects of clodronate on neutrophil migration are currently in progress in our laboratories.

In conclusion, we demonstrated that VNUT is localized to tertiary granules and is responsible for vesicular ATP release from neutrophils, which is blocked by a clinically available VNUT inhibitor, clodronate. These results strongly suggest that VNUT represents the missing link in the ATP release pathway of neutrophils.

Experimental procedures

Materials

Nucleotides, dichloromethylenediphosphonic acid disodium salt (clodronate), CFA, 3,3′-diaminobenzidine (DAB), A23187, and DMSO were obtained from Sigma–Aldrich. W-peptide (Trp-Lys-Tyr-Met-Val-Met-NH₂) was obtained from Phoenix Pharmaceuticals (Belmont, CA). Giemsa's azur eosin methylene blue solution was obtained from Merck. EGTA was obtained from Wako (Tokyo, Japan). IB-MECA was obtained from Abcam (Cambridge, UK). EGTA-tetraacetoxymethyl ester (AM) was obtained from AnaSpec Inc. (Fremont, CA).

Animals

C57BL/6 mice were obtained from Japan SLC (Shizuoka, Japan). VNUT^−/− mice were generated as previously described (19). All animal procedures and care were approved by the Institutional Animal Care and Use Committee and were performed according to the guidelines of the Okayama University. For the in vivo experiment, 20 μl of 1 mg/ml CFA was injected subcutaneously into the plantar region of the left hind paw of mice (male, 9–13 weeks old) using a 100-μl Hamilton microsyringe with a 27-gauge needle.

Isolation of mouse PMNs from bone marrow and of human PMNs from peripheral blood

Neutrophils were isolated from the bone marrow of mice as previously described (36). Briefly, tibias and femurs were removed and stripped of their muscles. The bone marrow was flushed using neutrophil isolation buffer containing 0.4% sodium citrate in Hanks' balanced salt solution (HBSS). Neutrophils were separated by density centrifugation using a Percoll (62% (v/v) in neutrophil isolation buffer; GE Healthcare) gradient at 1,000 × g for 30 min at 4 °C. PMNs were recovered as a pellet at the bottom of the 62% Percoll gradient and washed with PBS. Erythrocytes were removed by hypotonic lysis.

Human PMNs were isolated from peripheral blood samples using Histopaque-1119 and Histopaque-1077 (density: 1.119 g/ml and 1.077 g/ml, respectively; Sigma–Aldrich) double-density gradient centrifugation. Peripheral blood was obtained from healthy volunteer donors in compliance with the guidelines of the ethics committee of Okayama University (permit number 1388). Blood samples (36 ml) were mixed with 4 ml of 3.8% sodium citrate immediately after drawing, layered on a Histopaque gradient, and centrifuged at 700 × g for 30 min at room temperature. The PMN fraction was collected and washed with PBS. The purity of mouse and human neutrophil populations was >90 and >93%, respectively, and cell viability was >97 and >93%, respectively, as determined by Giemsa and Trypan blue staining. The specimens were observed using an Olympus IX83 microscope and Olympus DP80 camera.

RT-PCR analysis

An RNeasy mini kit (Qiagen) was used for RNA extraction from neutrophils, according to the manufacturer's instructions. cDNA was generated using PrimeScript RT Master Mix (Takara Bio, Shiga, Japan) with total RNA as the template. RT-PCR was performed as previously described (37). For PCR amplification, cDNA was added to the reaction buffer containing 0.2 mm total dNTP (50.0 μm each dNTP), 10 pmol primers, and 1.0 unit of Ex Taq (Takara Bio). The reaction was conducted over 35 cycles as follows: for human neutrophils, denaturation at 95 °C for 30 s, annealing at 62 °C for 35 s, and extension at 72 °C for 20 s; and for mouse neutrophils, denaturation at 95 °C for 30 s, annealing at 54 °C for 30 s, and extension at 72 °C for 1 min. The amplification products were analyzed using a 10% polyacrylamide gel electrophoresis.

Quantitative PCR was carried out with 400 nm specific forward and reverse primers and SYBR Premix Ex TaqII (Takara Bio). Quantitative PCR conditions included an initial denaturation step of 95 °C for 30 s, followed by 35 cycles of denaturation at 95 °C for 15 s and annealing/extension at 60 °C for 30 s. The primer sets used were as follows: for human VNUT (115 bp), 5′-TGGTCTTTGCATCAGCCTCCATCGG-3′ and 5′-GTGTTGGCCACACCAAACAGAAAGC-3′; for mouse VNUT (523 bp), 5′-GGTCTGCTCCAAGGTGTCTAC-3′ and 5′-GACTGATAAGGCGGTCGGAG-3′; for human glyceraldehyde-3-phosphate dehydrogenase (G3PDH; 115 bp), 5′-GGTGAAGGTCGGAGTCAACGG-3′ and 5′-GTTGAGGTCAATGAAGGGGTC-3′; for mouse G3PDH (150 bp), 5′-TGTGTCCGTCGTGGATCTG-3′ and 5′-TTGCTGTTGAAGTCGCAGG-3′.

Western blotting analysis

For immunoblotting, mouse neutrophils were suspended in 25 ml of 20 mm MOPS-Tris (pH 7.0) containing 0.3 m sucrose, 5 mm EDTA, 10 μg/ml pepstatin A, and 10 μg/ml leupeptin and then pressurized with N₂ for 20 min at 350 psi with constant stirring at 4 °C (38). The cavitate was centrifuged at 500 × g (mouse neutrophils) for 10 min. The resulting supernatant was centrifuged at 250,000 × g for 1 h. The pellet (membrane fraction) was suspended in the same buffer and then denatured with sample buffer containing 1% SDS and 10% β-mercaptoethanol. Polyacrylamide gel electrophoresis and Western blotting were performed as described previously (39). Immunoreactivity was visualized by ECL amplification according to the manufacturer (GE Healthcare). Protein concentrations were determined using a protein assay kit (Bio-Rad) with BSA as a standard.

Immunofluorescence microscopy

Cells on poly-l-lysine–coated coverslips were fixed with 4% paraformaldehyde in PBS for 30 min at room temperature. The cells were washed with PBS and incubated with the same buffer containing 0.1% Triton X-100 for 20 min and then further incubated with 2% goat serum and 0.5% BSA in the same buffer for 30 min at room temperature. Primary antibody treatment was performed using antibodies specific for VNUT (1:100), MPO (1:100, 1:50), lactoferrin (1:100, 1:50), MMP-9 (1:50), and CD35 (1:50) in PBS containing 0.5% BSA for 1 h at room temperature. The secondary antibodies used were Alexa Fluor 488-labeled anti-rabbit IgG (1:500) and Alexa Fluor 568-labeled anti-mouse IgG (1:1000) (Molecular Probes, Eugene, OR). The specimens were observed using an Olympus FV300 confocal laser microscope. Quantitative analysis of colocalization was performed with M1 and M2 coefficients using the JACoP plugin in ImageJ.

Antibodies

The specificity of rabbit polyclonal antibodies against human and mouse VNUT and V-ATPase prepared in-house were determined as described previously (14, 39). The following antibodies were obtained commercially: anti-human MPO mouse monoclonal (MS-1439; NeoMarkers, Portsmouth, NH), anti-mouse MPO mouse monoclonal (GTX16886; GeneTex, Irvine, CA), anti-lactoferrin mouse monoclonal (ab166803; Abcam), anti-MMP-9 mouse monoclonal (ab119906; Abcam), anti-CD35 mouse monoclonal (MA5–13122; Thermo Fisher Scientific), anti-FPRL1 rabbit polyclonal (bs-3654R; Bioss Antibodies, Woburn, MA), anti-GM130 mouse monoclonal (610822; BD Biosciences, San Jose, CA), anti-EEA1 mouse monoclonal (610456; BD Biosciences), anti-LAMP1 mouse monoclonal (SMC-140C/D; StressMarq Biosciences, Victoria, Canada), anti-PDI mouse monoclonal (ab2792; Abcam), anti-VAMP2 mouse monoclonal (104211; Synaptic Systems, Göttingen, Germany), and anti-mouse Ly-6G/Ly-6c (Gr-1) rat monoclonal (MAB1037; R&D Systems, Minneapolis, MN).

ATP release assay

Mouse neutrophils (1.0 × 10⁶ cells) were preincubated in Krebs–Ringer solution consisting of 128 mm NaCl, 1.9 mm KCl, 1.2 mm KH₂PO₄, 1.3 mm MgSO₄, 26 mm NaHCO₃, 2.4 mm CaCl₂, 10 mm d-glucose, 10 mm HEPES-Tris (pH 7.4), and 0.2% BSA in the absence or presence of inhibitors for 30 min at 37 °C. The cells were stimulated through the addition of 5 μm A23187 (Ca²⁺ ionophore) or 100 nm W-peptide at 37 °C. After aliquots were taken at the indicated times, the amount of ATP was measured using an ATP bioluminescent assay kit (Sigma–Aldrich) based on a luciferin–luciferase reaction and with Varioskan Flash Microplate Readers (Thermo Scientific).

MPO assay and MMP-9 assay

MPO and MMP-9 supernatants were analyzed by an ELISA using the mouse MPO ELISA kit (Abcam) and the mouse total MMP-9 quantikine ELISA kit (R&D Systems), according to the manufacturer's instructions.

Transwell migration assay

Migration assays using a Transwell system were performed with a 96-well multiscreen MIC plate (Millipore, Billerica, MA) with a pore size of 3.0 μm, as described previously (3). A 100-μl suspension of 1 × 10⁶ cells in HBSS +10% FBS, with or without inhibitors, with nucleotides or agonists at the indicated concentrations, was added to each well of the upper filter plate. Chemoattractants in HBSS +10% FBS (150 μl) were added to each of the lower wells. After incubation at 37 °C for 50 min, the upper plate was removed, and cells in the lower plate were counted.

Assessment of neutrophil migration into footpads

CFA-treated footpads were decalcified with 19% EDTA, fixed in 4% paraformaldehyde in PBS, and cut into 10-μm-thick sections. Immunohistochemical analysis was performed using the horseradish peroxidase–DAB method, as described previously (37). In brief, samples were quenched of endogenous peroxidase activity with 0.3% H₂O₂ and incubated in PBS containing 1.5% goat serum for 30 min. The samples were then incubated with anti-mouse Gr-1 antibodies (1:500) in PBS containing 0.1% BSA and 0.05% Tween 20 for 1 h at room temperature. The samples were washed four times with PBS containing 0.05% Tween 20, and then incubated with biotinylated-labeled anti-rat IgG (1:200; Vector Laboratories, Burlingame, CA) as the secondary antibody for 30 min at room temperature. Samples were washed four times with PBS containing 0.05% Tween 20, incubated with Vectastain ABC reagent (Vector Laboratories) for 30 min, and then incubated with a peroxidase substrate solution consisting of 0.02% DAB and 0.005% H₂O₂. Finally, samples were mounted with Mount-Quick (Daido Sangyo, Tokyo, Japan) and observed using a BZ-X700 microscope (Keyence, Osaka, Japan). Stained and migrated neutrophils in the footpads were quantified using BZ-X Analyzer software (Keyence).

Data analysis

All numerical values are shown as the means ± S.E. unless otherwise specified. Statistical significance was determined by performing a two-tailed paired Student's t test or Dunnett's test for multiple comparisons after analysis of variance. These tests were performed using GraphPad Prism version 6 software (GraphPad Software, La Jolla, CA). Differences were considered significant at p < 0.05.

Author contributions

Y. H. performed the majority of the experiments, designed the experiments, analyzed the data, and wrote the paper. Y. K. performed and designed the experiments. T. M., H. O., Y. M., and M. H. designed the experiments, analyzed the data, and wrote the paper.