VAMP8, a member of the soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) family of fusion proteins, initially characterized in endosomal and endosomal-lysosomal fusion, may also function in regulated exocytosis. VAMP8 physiological function in inflammation has not been elucidated. In this paper, we show that deficiency of VAMP8 protects mice from anaphylatoxin (C5a)-induced neutropenia, peritonitis, and systemic inflammation. We show that, in vivo, VAMP8 deletion inhibits neutropenia and phagocyte recruitment. We also show that in macrophages, VAMP8 localizes on secretory granules and degranulation is inhibited in VAMP8-deficient macrophages. Moreover, VAMP8−/− mice show reduced systemic inflammation with inhibition of serum TNF-α levels, whereas IL-1β, IL-6, and MIP1α release are not affected. In wild-type macrophages, TNF-α colocalizes with VAMP8-positive vesicles, and in VAMP8-deficient macrophages, the TNF-α release is inhibited. Furthermore, VAMP8 regulates the release of TNF-α and β-hexosaminidase triggered by fMLP, and VAMP8−/− mice are protected from fMLP-induced peritonitis. These data demonstrate that the VAMP8 vesicle-associated-SNARE is required for the proper trafficking of secretory lysosomal granules for exocytosis in macrophages and for the release of the potent proinflammatory cytokine, TNF-α.

The complement system was discovered more than 100 years ago during the seminal experiments of Jules Bordet, described in 1895. The general understanding of complement activation is that it plays a central role in the innate immune system in host defenses against invading pathogens and in clearance of potentially damaging cell debris (1). However, this view has expanded in the last 10 years because of the discovery of complement receptors and new functional aspects of complement activation products. Thus, complement activation has recently been implicated in the pathogenesis of many inflammatory diseases, such as sepsis (2, 3), acute respiratory distress syndrome (4), rheumatoid arthritis (5), glomerulonephritis (6), multiple sclerosis (7), ischemia-reperfusion injury (8), and asthma (9). Many of the harmful outcomes of complement activation are mediated by the generation of complement protein split products, especially the anaphylatoxins C3a and C5a. It is believed that anaphylatoxins may not be critical factors in the initiation of inflammatory disorders; however, they do play key roles for promoting and perpetuating inflammatory reactions (10). Within the complement activation products, C5a is one of the most potent inflammatory peptides, with a broad spectrum of functions. C5a is a strong chemoattractant for neutrophils and also has chemotactic activity for monocytes and macrophages (11, 12, 13). C5a triggers the phagocytic NADPH-oxidative burst and enhances phagocytosis and the release of granule enzymes from neutrophils and macrophages (12, 13). C5a also triggers the release of proinflammatory cytokines and chemokines from various cell types (12, 13, 14, 15), and has been shown to enhance the expression of cell-adhesion molecule expression on neutrophils (16) and to activate the coagulation pathway (17). C5a mediates its effects through two specific and high-affinity receptors (C5aR and C5L2). Interestingly, simultaneous double-blockade of both C5aR and C5L2 dramatically improved survival in experimental sepsis, suggesting a key position for C5a-mediated responses in sepsis (18). However, not much is known about the intracellular molecular mechanisms that regulate C5a-triggered proinflammatory responses.

Soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors (SNAREs)2 are membrane proteins that are localized on various intracellular organelles. SNARE assembly may promote intracellular membrane fusion, an essential process for vesicular transport within a cell, as well as for the secretion of proteins and other biologically active products from cells (19). Among the SNAREs, endobrevin/VAMP8 was originally identified as an endosomal vesicle-associated-SNARE, involved in fusion between early and late endosomes (20). However, recent studies using VAMP8−/− mice have shown major physiological roles for VAMP8: the secretion of zymogen granules from pancreatic acinar cells (21), platelet activation and granular release reaction (22), the secretion of lysosomal granules from mast cells (23), and a recent study by Cosen-Binker et al. provided evidence that VAMP8 is critical for alcohol-induced pancreatitis (24). In the current study, we sought to evaluate the consequences of VAMP8 blockade on the cytokines generated in response to C5a on macrophages, and in vivo in anaphylatoxin-induced shock. We hypothesized that VAMP8 may be required for the secretion of certain cytokines and preformed proinflammatory mediators triggered by C5a. Thus, targeting VAMP8 might greatly impair the C5a-triggered production and/or secretion of proinflammatory mediators that might result in anaphylatoxin induced shock.

All materials, unless stated otherwise, were bought from Sigma- Aldrich. The Abs for immunostaining were purchased from Santa Cruz Biotechnology.

The wild-type (WT), VAMP8+/−, and VAMP8−/− mice were bred in Institute of Molecular and Cell Biology (IMCB) Singapore and maintained on a mixed genetic background of 129 SvJ and C57/BL6. Mice were transferred from the IMCB to the National University of Singapore (NUS) for this study. The study was conducted according to the NUS guidelines for animal experimentation.

C5a-induced neutropenia was performed as previously described (24). Briefly, anesthetized mice were given a bolus i.v. dose of recombinant human C5a (hC5a; 4 μg/mouse in a final volume of 100 μl), and blood samples were collected at regular intervals over a 2-h observation period. Polymorphonuclear neutrophils (PMNs) were isolated and counted as previously reported (25) and expressed as a percentage of the PMN concentration before C5a challenge.

Acute peritonitis was induced by an i.p. injection of recombinant hC5a (4 μg/mouse in a final volume of 100 μl), or fMLP (50 ng/mouse). Control mice were injected with 100 μl of PBS i.p. At the indicated times, mice were killed and their peritoneal cavity was washed with 2 ml of ice-cold PBS, 0.2% BSA. The recovered peritoneal lavage fluid was analyzed for different cell infiltrates, measured as previously reported (25).

For permeability analysis, 100 μl Evans blue dye (10 mg/ml) was i.v. administered 10 min before the C5a or PBS i.p. administration. At the indicated times, mice were killed and their peritoneal cavity was washed with 2 ml of ice-cold PBS with 0.2% BSA. The cells were spun down and the OD of the supernatant, indicating Evans blue leakage into the peritoneal cavity, was measured as previously described (25).

IL-1β, IL-6, TNF-α, and MIP-1α levels were analyzed from serum and peritoneal lavage using ELISA kits (R&D Systems), following the manufacturer’s instructions.

For morphological investigation, the lungs and peritoneal membranes of WT and VAMP8−/− mice were carefully dissected out and immersed in 10% formalin fixative for 1 day. The specimens were then dehydrated through an ascending series of ethanol and cleared in toluene before being embedded in paraffin. The tissue blocks were cut at 4-μm thickness by means of a Leica Rotary Microtome (Model 2165; Leica Microsystems) and processed for H&E staining, as described before (26).

Lungs and peritoneal membrane sections were deparaffinized in two changes of xylene, 5 min each. Then they were hydrated in two changes of 100% ethanol for 3 min each, 95% and 80% ethanol for 1 min each, and rinsed in distilled water. Next, the sections were incubated with 2N HCl solution for 20 min for Ag retrieval. The tissues were marked with a circle using a DAKO pen (DAKO Biotech). Then they were permeabilized using 0.2% Saponin for 10 min and washed in 1× TBS, three times for 5 min. Goat serum (1/30) was added for 30 min at room temperature and drained with blotting paper. The tissues were incubated overnight in a humidity chamber at 4°C with the primary Ab (Santa Cruz Biotechnology), for the respective cell adhesion molecule to be probed. Then they were washed with 1× TBS, three times for 5 min. The corresponding secondary Ab (DAKO Biotech), labeled either with FITC or tetramethylrhodamine isothiocyanate, was added at room temperature for 1 h. The tissues were then washed with 1× TBS, three times 5 min. Finally, the slides were mounted with a cover slip, using FluorSave (Calbiochem).

Mice were killed and immediately their peritoneal cavity was washed with 2 ml ice-cold PBS, 0.2% BSA. The recovered peritoneal lavage fluid was spun down and the cells were resuspended in culture medium containing RPMI 1640 (Invitrogen Life Technologies) supplemented with 10% FCS, 2 mM glutamine, 10 U/ml penicillin, and 10 mg/ml streptomycin, and kept at 37°C, 5% CO2 in a water-saturated atmosphere for 2 h, after which nonadherent cells were removed by washing the wells twice with RPMI 1640. Viability, determined by trypan blue exclusion, was ∼95% in all experiments.

Peritoneal macrophages (1 × 105 cells/well), were seeded in a 12-well plate containing a cover slip/well and cultured overnight in RPMI 1640 medium supplemented with 10% FCS and PenStrep at 37°C in a 5% CO2-containing atmosphere. Next, the cover slips with cells attached to them were recovered and treated with a mixture of methanol and acetone, 1:1, to fix and permeabilize the cells, then incubated with primary Abs for 2 h at room temperature and washed and stained with secondary Abs and 4′,6′-diamidino-2-phenylindole for 1 h at room temperature in the dark. Cells were then washed and mounted on slides. The slides were viewed using either a Zeiss LSM510 confocal microscope or a Leica SP5 confocal microscope. Abs used: primary goat anti-VAMP8 (SC-19367; Santa Cruz Biotechnologies), secondary anti-goat-FITC (F9012; Sigma-Aldrich), primary rat anti-TNF-α (AMC3814; Invitrogen), secondary goat anti-rat IgG-Alexa Fluor 594 (A-11007; Invitrogen), primary rabbit anti-β-hexosaminidase (11317–1-AP; Proteintech), and secondary goat anti-rabbit IgG-Alexa Fluor 594 (A-11012; Invitrogen).

Macrophage degranulation was measured using a previously described method for the release of β-hexosaminidase (12).

A total of 2 × 106 cells per sample were stimulated by the addition of 5 nM hC5a or 10 nM fMLP for 24 h. Following stimulation, the supernatants were collected and stored at −20°C until used. TNF-α, IL-1β, IL-6, and MIP-1α levels in the supernatants were evaluated using ELISA (R&D Systems).

Data from experimental groups was analyzed by unpaired Student’s t test.

In our present study, we have investigated the role of endobrevin/VAMP8 in anaphylatoxin C5a-induced inflammatory responses. We show that i.v. administration of recombinant hC5a in mice resulted in neutropenia, as seen by the rapid decrease of circulating PMNs, dropping to 30 ± 7% of the levels observed in both WT and VAMP8+/− mice at 5 min after the C5a i.v., then returning to normal/control values by 1 h. However, in VAMP8−/− mice, the PMN levels were similar to that of the unstimulated WT mice (Fig. 1,a). Analysis of lung sections indicates that the PMNs emigrate from the blood vessels and infiltrate the lung tissues after the C5a i.v. administration (Fig. 1,b). However, the infiltration of PMNs was blocked in VAMP8−/− mice (Fig. 1,b). Moreover, injection of recombinant hC5a into the peritoneal cavity caused a rapid influx of neutrophils into the peritoneal cavity, reaching a peak at 2 h, but then dropping rapidly by 6 h (Fig. 2,a). This was later followed by monocyte infiltration into the cavity, observed only after 4 h, and continuing to increase by 6 h (Fig. 2,b). However, in VAMP8−/− mice, there was a significant reduction of neutrophil and monocyte infiltration at all time points (Fig. 2, a and b). Furthermore, analysis of vascular permeability reveals that in VAMP8−/− mice, the C5a-mediated vascular leakage was very much inhibited (Fig. 2,c). Interestingly, analysis of heterozygote mice revealed a dosage phenomenon in the role of VAMP8 on the inflammatory parameters (Fig. 2).

FIGURE 1.

C5a-induced neutropenia in WT, VAMP8+/−, and VAMP8−/− mice. a, Neutrophil blood levels. The blood was collected at the times indicated in the figure following the i.v. injection of vehicle/PBS (WT PBS) or C5a in WT (WT C5a), VAMP8 +/− (VAMP8+/− C5a), and VAMP8−/− (VAMP8−/− C5a) mice. Data shown as means ± SD (n = 5). Student’s t test p values compared with C5a-induced WT mice; **, p < 0.01. b, H&E staining of lungs following the i.v. injection of vehicle/PBS or C5a at the times indicated in the figure in WT, VAMP8+/−, and VAMP8−/− mice. Microscopy magnifications are 40× and 100×, as indicated in the histological pictures. The green arrows indicate no or little infiltration of immune cells from the blood vessel into the surrounding lung tissues. The red arrows indicate extensive infiltration of immune cells from the blood vessel into the surrounding tissues of the lungs. Data shown is representative of at least three separate experiments and five mice. Student’s t test p-value compared with C5a-induced WT mice.

FIGURE 1.

C5a-induced neutropenia in WT, VAMP8+/−, and VAMP8−/− mice. a, Neutrophil blood levels. The blood was collected at the times indicated in the figure following the i.v. injection of vehicle/PBS (WT PBS) or C5a in WT (WT C5a), VAMP8 +/− (VAMP8+/− C5a), and VAMP8−/− (VAMP8−/− C5a) mice. Data shown as means ± SD (n = 5). Student’s t test p values compared with C5a-induced WT mice; **, p < 0.01. b, H&E staining of lungs following the i.v. injection of vehicle/PBS or C5a at the times indicated in the figure in WT, VAMP8+/−, and VAMP8−/− mice. Microscopy magnifications are 40× and 100×, as indicated in the histological pictures. The green arrows indicate no or little infiltration of immune cells from the blood vessel into the surrounding lung tissues. The red arrows indicate extensive infiltration of immune cells from the blood vessel into the surrounding tissues of the lungs. Data shown is representative of at least three separate experiments and five mice. Student’s t test p-value compared with C5a-induced WT mice.

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FIGURE 2.

C5a-induced immune cell recruitment into the peritoneal cavity in WT, VAMP8+/−, and VAMP8−/− mice. a, Neutrophil infiltration. Neutrophil numbers in the peritoneum lavage, following the i.p. injection of vehicle/PBS (WT PBS) or C5a, at the times indicated in the figure in WT (WT C5a), VAMP8+/− (VAMP8+/− C5a), and VAMP8−/− (VAMP8−/− C5a) mice. b, Monocyte infiltration. Monocyte numbers in the peritoneum lavage, following the i.p. injection of vehicle/PBS (WT PBS) or C5a, at the times indicated in the figure in WT, VAMP8+/−, and VAMP8−/− mice. c, Vascular permeability. Evans blue extravasation in the peritoneal lavage, following the i.p. injection of vehicle/PBS/Evans blue (WT PBS) or C5a/Evans blue, at the times indicated in the figure, in WT, VAMP8+/−, and VAMP8−/− mice. The absorbance was measured at the indicated times. Data shown as means ± SD (n = 5). *, p < 0.05 and **, p< 0.01; Student’s t test p values compared with C5a-induced WT mice.

FIGURE 2.

C5a-induced immune cell recruitment into the peritoneal cavity in WT, VAMP8+/−, and VAMP8−/− mice. a, Neutrophil infiltration. Neutrophil numbers in the peritoneum lavage, following the i.p. injection of vehicle/PBS (WT PBS) or C5a, at the times indicated in the figure in WT (WT C5a), VAMP8+/− (VAMP8+/− C5a), and VAMP8−/− (VAMP8−/− C5a) mice. b, Monocyte infiltration. Monocyte numbers in the peritoneum lavage, following the i.p. injection of vehicle/PBS (WT PBS) or C5a, at the times indicated in the figure in WT, VAMP8+/−, and VAMP8−/− mice. c, Vascular permeability. Evans blue extravasation in the peritoneal lavage, following the i.p. injection of vehicle/PBS/Evans blue (WT PBS) or C5a/Evans blue, at the times indicated in the figure, in WT, VAMP8+/−, and VAMP8−/− mice. The absorbance was measured at the indicated times. Data shown as means ± SD (n = 5). *, p < 0.05 and **, p< 0.01; Student’s t test p values compared with C5a-induced WT mice.

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Subsequently, we investigated the expression of vascular CAMs in both lungs and peritoneum, in an attempt to further investigate the potential mechanisms by which VAMP8 mediates immune cell extravasation during inflammation. The immunofluorescence staining of lungs of C5a-stimulated WT mice show the up-regulation of P-selectin, VCAM-1, and ICAM-1 in the alveolar epithelium and the endothelium of the lungs (Fig. 3,a). However, in VAMP8−/− mice the expression of P-selectin, VCAM-1, and ICAM-1 was similar to that of unstimulated WT control mice (Fig. 3,a). Moreover, immunofluorescence of the peritoneum of C5a-challenged mice generated similar results, which are a reduction in the CAMs expression levels in VAMP-null mice (Fig. 3 b).

FIGURE 3.

Immunofluorescence detection of the P-selectin, VCAM-1, and ICAM-1 expression in the lungs and the peritoneum of WT, VAMP8 +/−, and VAMP8 −/− mice. a, Lung sections stained for the CAMs following the i.v. injection of vehicle/PBS (WT PBS) or C5a, in WT (WT C5a), VAMP8+/− (VAMP8+/− C5a), and VAMP8−/− (VAMP8−/− C5a) mice. Arrows indicate CAMs. b, Peritoneum sections stained for the CAMs following the i.v. injection of vehicle/PBS or C5a, in WT, VAMP8+/−, and VAMP8−/− mice. The green arrows indicate basal or low levels of CAMs. The white arrows indicate up-regulation of CAMs cell surface expression. Data shown is representative of at least three separate experiments and five mice.

FIGURE 3.

Immunofluorescence detection of the P-selectin, VCAM-1, and ICAM-1 expression in the lungs and the peritoneum of WT, VAMP8 +/−, and VAMP8 −/− mice. a, Lung sections stained for the CAMs following the i.v. injection of vehicle/PBS (WT PBS) or C5a, in WT (WT C5a), VAMP8+/− (VAMP8+/− C5a), and VAMP8−/− (VAMP8−/− C5a) mice. Arrows indicate CAMs. b, Peritoneum sections stained for the CAMs following the i.v. injection of vehicle/PBS or C5a, in WT, VAMP8+/−, and VAMP8−/− mice. The green arrows indicate basal or low levels of CAMs. The white arrows indicate up-regulation of CAMs cell surface expression. Data shown is representative of at least three separate experiments and five mice.

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We next investigated the role of VAMP8 in cytokine production and release following C5a challenge. We demonstrate that i.p. administration of C5a or a bolus i.v. dose of C5a in WT mice resulted in the elevation of various proinflammatory cytokines in the peritoneal cavity and serum, respectively, with TNF-α in the peritoneum reaching a peak of 600 ng/ml at 2 h after the C5a injection (Fig. 4,a). However, in VAMP8−/− mice no significant elevation of the TNF-α level was observed after C5a administration (Fig. 4,a). Similarly, the levels of TNF-α in the serum were substantially elevated following the i.v. administration of C5a in WT mice, but these TNF-α levels were reduced in VAMP8+/− and even further reduced in the VAMP8−/− mice (Fig. 4,b). IL-1β, IL-6, and MIP1α are also released early in an inflammatory response. In this paper, we show that the i.p or i.v. administration of recombinant C5a resulted in the elevation of IL-1β, IL-6, and MIP1α in the peritoneum, as well as in the serum, of WT mice (Fig. 4). Interestingly, this elevation of peritoneum and serum levels of IL-1β, IL-6, and MIP1α were unaffected in VAMP8-deficient mice (Fig. 4). Thus, to further investigate this issue, we performed in vitro studies in primary mouse macrophages. Our results show that C5a stimulated the secretion of IL-1β, IL-6, MIP1α, and TNF-α from WT macrophages (Fig. 5,a). However, the C5a-triggered TNF-α secretion, but not IL-1β, IL-6, or MIP1α, was inhibited in VAMP8-deficient macrophages when compared with WT ones (Fig. 5 a), proving that in the C5a-mediated responses VAMP8 is indeed required for the secretion of TNF-α.

FIGURE 4.

Peritoneal and serum cytokine levels. Levels of TNF-α, IL-1β, IL-6, and MIP1α were measured at the times indicated in the peritoneum lavage 2 h after the i.p. injection of vehicle/PBS (WT PBS) or C5a (a) or in serum 2 h after the i.v. injection of vehicle/PBS or C5a (b) in WT (WT C5a), VAMP8+/− (VAMP8+/− C5a), and VAMP8−/− (VAMP8−/− C5a) mice. Data shown as means ± SD (n = 5). *, p < 0.05 and **, p< 0.01; Student’s t test p values compared with five C5a-induced WT mice.

FIGURE 4.

Peritoneal and serum cytokine levels. Levels of TNF-α, IL-1β, IL-6, and MIP1α were measured at the times indicated in the peritoneum lavage 2 h after the i.p. injection of vehicle/PBS (WT PBS) or C5a (a) or in serum 2 h after the i.v. injection of vehicle/PBS or C5a (b) in WT (WT C5a), VAMP8+/− (VAMP8+/− C5a), and VAMP8−/− (VAMP8−/− C5a) mice. Data shown as means ± SD (n = 5). *, p < 0.05 and **, p< 0.01; Student’s t test p values compared with five C5a-induced WT mice.

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FIGURE 5.

C5a-induced cytokine release and degranulation in macrophages. a, Levels of TNF-α, IL-1β, IL-6, and MIP1α were measured in supernatant of macrophages 24 h after the addition of vehicle/PBS (WT PBS) or C5a to macrophages cultured from WT (WT C5a), VAMP8+/− (VAMP8+/− C5a), and VAMP8−/− (VAMP8−/− C5a) mice. b, Levels of β-hexosaminidase were measured in the supernatant of peritoneal macrophages 1 h after the addition of vehicle/PBS (WT PBS) or C5a to macrophages cultured from WT (WT C5a), VAMP8 +/− (VAMP8+/− C5a), and VAMP8−/− (VAMP8−/− C5a) mice. Data shown as means ± SD (n = 3). *, p < 0.05 and **, p < 0.01; Student’s t test p values compared with C5a-induced WT mice. c and d, Confocal microscopy of WT peritoneal macrophages stained for intracellular TNF-α and VAMP8 and merged (c) or stained for intracellular β-hexosaminidase (β-hexos) and VAMP8 and merged (d). Scale white bar equals 25 μm. Data shown is representative of at least three separate experiments.

FIGURE 5.

C5a-induced cytokine release and degranulation in macrophages. a, Levels of TNF-α, IL-1β, IL-6, and MIP1α were measured in supernatant of macrophages 24 h after the addition of vehicle/PBS (WT PBS) or C5a to macrophages cultured from WT (WT C5a), VAMP8+/− (VAMP8+/− C5a), and VAMP8−/− (VAMP8−/− C5a) mice. b, Levels of β-hexosaminidase were measured in the supernatant of peritoneal macrophages 1 h after the addition of vehicle/PBS (WT PBS) or C5a to macrophages cultured from WT (WT C5a), VAMP8 +/− (VAMP8+/− C5a), and VAMP8−/− (VAMP8−/− C5a) mice. Data shown as means ± SD (n = 3). *, p < 0.05 and **, p < 0.01; Student’s t test p values compared with C5a-induced WT mice. c and d, Confocal microscopy of WT peritoneal macrophages stained for intracellular TNF-α and VAMP8 and merged (c) or stained for intracellular β-hexosaminidase (β-hexos) and VAMP8 and merged (d). Scale white bar equals 25 μm. Data shown is representative of at least three separate experiments.

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Another important function of anaphylatoxin is the triggering of phagocyte degranulation, an important effector function of phagocytic cells. Thus, we investigated whether VAMP8 plays any role in C5a-triggered degranulation. C5a-stimulated degranulation was strongly inhibited in VAMP8-deficient macrophages (Fig. 5,b). Moreover, confocal microscopy reveals that in WT macrophages, VAMP8, at least in part, colocalizes with TNF-α (Fig. 5,c) and with vesicles containing β-hexosaminidase, a marker for macrophage granules (Fig. 5 d).

To find out whether VAMP8 is uniquely used by the C5a receptor, we investigated the role of VAMP8 following fMLP stimulation. Our results show that, similar to C5a, the fMLP-triggered TNF-α secretion (Fig. 6,a) and degranulation is inhibited in macrophages from VAMP8−/− mice (Fig. 6,b). Furthermore, neutrophil and monocyte infiltration into the peritoneal cavity of mice i.p. injected with fMLP is also inhibited in VAMP8−/− mice (Fig. 6, c and d). This suggests that VAMP8 may regulate TNF-α secretion and degranulation from macrophages activated by different stimuli.

FIGURE 6.

fMLP-induced cytokine release and degranulation and peritonitis. a, Levels of TNF-α, IL-1β, IL-6, and MIP1α were measured in supernatant of macrophages 24 h after the addition of vehicle/PBS (WT PBS) or fMLP to macrophages cultured from WT (WT fMLP) or VAMP8−/− (VAMP8−/− fMLP) mice. b, Levels of β-hexosaminidase were measured in the supernatant of peritoneal macrophages 1 h after the addition of vehicle/PBS (WT PBS) or fMLP to macrophages cultured from WT and VAMP8−/− mice. c, Neutrophil infiltration. Neutrophil numbers in the peritoneum lavage, following the i.p. injection of vehicle/PBS (WT PBS) or fMLP, at the times indicated in WT and VAMP8−/− mice. d, Monocyte infiltration. Monocyte numbers in the peritoneum lavage, following the i.p. injection of vehicle/PBS (WT PBS) or fMLP, at the times indicated in WT and VAMP8−/− mice. Data shown as means ± SD (n = 5). **, p< 0.01; Student’s t test p values compared with fMLP-induced samples from WT mice.

FIGURE 6.

fMLP-induced cytokine release and degranulation and peritonitis. a, Levels of TNF-α, IL-1β, IL-6, and MIP1α were measured in supernatant of macrophages 24 h after the addition of vehicle/PBS (WT PBS) or fMLP to macrophages cultured from WT (WT fMLP) or VAMP8−/− (VAMP8−/− fMLP) mice. b, Levels of β-hexosaminidase were measured in the supernatant of peritoneal macrophages 1 h after the addition of vehicle/PBS (WT PBS) or fMLP to macrophages cultured from WT and VAMP8−/− mice. c, Neutrophil infiltration. Neutrophil numbers in the peritoneum lavage, following the i.p. injection of vehicle/PBS (WT PBS) or fMLP, at the times indicated in WT and VAMP8−/− mice. d, Monocyte infiltration. Monocyte numbers in the peritoneum lavage, following the i.p. injection of vehicle/PBS (WT PBS) or fMLP, at the times indicated in WT and VAMP8−/− mice. Data shown as means ± SD (n = 5). **, p< 0.01; Student’s t test p values compared with fMLP-induced samples from WT mice.

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Anaphylatoxins, and more specifically C5a, are known to play key roles in the inflammatory cascades that lead to inflammation in various diseases, including autoimmune diseases, traumas, infections, and septic endotoxic shock triggered by bacteria-derived products, where activation of the serum complement system leads to a substantial elevation of circulating C5a (2, 3). Our data shows that the systemic (i.v.) injection of recombinant C5a into mice triggered a neutropenic response, however, in VAMP8−/− mice, neutropenia was substantially reduced. It is known that neutrophil, macrophage, and endothelial cells respond robustly to C5a to up-regulate cell adhesion molecule expression (27). This process leads to the rapid adhesion of neutrophils to the vascular endothelium and the rapid decrease in levels of circulating neutrophils. This decrease in circulating leukocytes is normally due to leukocyte rolling movement along the vessel wall and the subsequent solid adhesion to the endothelial cells and migration into tissues (28). Thus, we investigated the effect of VAMP8 in the recruitment and activation of acute inflammatory leukocytes in vivo in C5a-induced systemic and localized inflammatory models, and showed that, following C5a i.v. administration, the migration of leukocytes into the lungs was induced and peritoneal administration of C5a induced a rapid recruitment of neutrophils into the peritoneal cavity. Moreover, this process was accompanied by vascular permeability and up-regulation of cell adhesion molecule expression. It is well established that P-selectin mediates the rolling of PMNs, whereas ICAM-1 contributes to the firm adhesion and emigration of PMN. It has been shown that P-selectin-independent, as well as ICAM-1-independent, adhesive mechanisms are required for proper migration, and when one of these molecules is deficient there is a reduction in PMN migration (29). In line with these observations, our data shows that in VAMP8-deficient mice there is a substantial reduction in immune cell infiltration and an inhibition in the vascular levels of ICAM-1, VCAM-1, and P-selectin, suggesting that VAMP8 may contribute to leukocyte migration by transporting CAMs to the plasma membrane. However, as cell adhesion molecule expression in the vasculature is also dependent on the activity of various proinflammatory cytokines released early during inflammation, we investigated the role of VAMP8 in proinflammatory cytokine secretion. C5a administration triggered the secretion to the proinflammatory cytokines IL-1β, IL-6, and TNF-α and the chemokine MIP1α. Interestingly, in VAMP8-deficient mice the serum and peritoneal levels of TNF-α were substantially reduced, whereas the levels of IL-1β, IL-6, and MIP1α were unaffected, indicating a selective role for VAMP8 in the production and/or secretion of the important cytokine TNF-α. This is a key observation, as the secretion of TNF-α is one of the most important proinflammatory landmarks of inflammation (30). Paradoxically, in mast cells it was shown that the FcεRI-mediated release of cytokines, including TNF-α, was VAMP8-independent (23). Thus, to further investigate this issue, we performed in vitro studies in primary mouse macrophages. Our data showed that, in vitro, in WT murine macrophages C5a triggers the release of IL-1β, IL-6, TNF-α, and MIP1α, and furthermore, in VAMP8-deficient macrophages, similar to the VAMP8−/− in vivo data, TNF-α release was substantially reduced, whereas the secretion of IL-1β, IL-6, and MIP1α was unaffected. Moreover, in WT macrophages, TNF-α colocalizes with VAMP8-containing vesicles. Interestingly, similar to FcεRI-mediated degranulation in mast cells (23), C5a-triggered degranulation in macrophages is inhibited in VAMP8-deficient mice, and moreover in WT macrophages, VAMP8 colocalizes with β-hexosaminidase containing granules.

In conclusion, our study presents evidence that VAMP8 plays an important role in neutrophil and monocyte recruitment and phagocyte and vascular activation during acute inflammation, also showing a key role for VAMP8 in macrophage degranulation and in the secretion of TNF-α, a previously unidentified phenomenon with profound implications in the inflammatory disease field. These findings on the molecular function of VAMP8 in C5a-mediated inflammatory response expand our knowledge of the physiological roles for VAMP8 in cellular responses and identify a novel mechanism by which C5a contributes to the overall activation of the inflammatory parameters, potentially including those in sepsis, where C5a has been shown to play a very detrimental role (30, 31). Moreover, the effects of VAMP8-delition are not restricted to C5a alone; we also show that similar to C5a, fMLP-triggered TNF-α secretion and degranulation is inhibited in macrophages from VAMP8−/− mice, and that neutrophil and monocyte infiltration into the peritoneal cavity of mice injected with fMLP is inhibited in VAMP8−/− mice. Thus, our findings indicate a new functional role for VAMP8 and strongly suggest that the VAMP8-mediated fusion process represents a novel therapeutic target to selectively interfere with excessive inflammatory responses.

This work was supported by a National University of Singapore Academic Research Fund Grant No. R-185–000-109–112 and a University of Glasgow Start-up Fund. We thank A.-K. Fraser-Andrews for proofreading the manuscript.

The authors have no financial conflict of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

2

Abbreviations used in this paper: SNARE, soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor; CAM, cell adhesion molecule; hC5a, human C5a; PMN, polymorphonuclear neutrophil; WT, wild type.

1
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