PMN precursors from bone marrow
Protein disulfide isomerase
Infiltrating polymorphonuclear leukocytes (PMN) in the peritoneal cavity were found to express L-histidine decarboxylase (HDC), the rate-limiting enzyme of histamine synthesis, in a csein-induced peritonitis model. Expression of HDC was detected in the elicited PMN, but not in the peripheral blood leukocytes. The peritoneal lavage fluids in this model were found to augment histamine synthesis in PMN isolated from the bone marrow. Rapid post-translational processing of HDC was observed in PMN, and the dominant form of HDC was the mature 53-kDa form, which was found to co-localize with a granule enzyme, matrix metalloproteinase-9 (MMP-9). Treatment of PMN with the phorbol ester PMA, which stimulates the release of MMP-9, did not liberate the granular HDC. Immunofluorescence studies using an anti-HDC antibody strongly suggested that HDC is bound to the cytosolic side of the granule membranes. These observations suggest that HDC is induced upon infiltration of PMN into the mouse peritoneal cavity and that histamine is synthesized by HDC attached to the granule membranes of PMN.
Histamine has been reported to play various roles in the modulation of immune and inflammatory responses via the H1 and H2 receptors 1, such as increase invascular permeability 2, leukocyte rolling 3, cytokine production 4, 5, polarization of dendritic cells 6, and T cell and antibody responses 7, 8. These studies suggest that endogenous histamine production can modulate immune responses, although the exact source of this histamine remains to be fully determined. Many of these studies have assumed that the histamine released from mast cells and basophils via degranulation is important in such kinds of immune regulation. However, since this histamine release is generally observed at the onset of inflammatory responses, it is unlikely that only histamine released from mast cells and basophils can modulate theimmune responses, especially during the late phase responses. In an air pouch-type allergic inflammation model in rats, the release of histamine into the pouch fluid was found to be biphasic 9: in the anaphylactic phase, activated mast cells in the pouch released histamine, causing a transient increase in vascular permeability via the H1 receptors 2, andin the following chronic phase, cells of unknown identity, which were not mast cells, produced histamine, causing the inhibition of leukocyte infiltration into the pouch via the H2 receptors 10. During the chronic phase of inflammation, an intensive infiltration of polymorphonuclear leukocytes (PMN) and macrophages is usually observed. A detectable amount of histamine synthesis was found to be induced in the murine myelomonocytic cell line WEHI-3B, in parallel with their differentiation into macrophages 11. Mirossay et al. have demonstratedthe existence of trace amounts of L-histidine decarboxylase (HDC) activity and histamine content in macrophages as well as in the human promyelocytic leukemia cell line HL-60, which can be differentiated into granulocytes by all-trans-retinoic acid treatment 12. These in vitro studies suggest that both PMN and macrophages may be involved in histamine synthesis during the chronic phase of the inflammatory response. Although it still remains unknown as to which cell type is responsible for histamine synthesis in vivo, it is important to clarify the regulation of histamine synthesis by these cell types in order to appreciate the modulatory role of histamine in immune and inflammatory responses.
We previously described the post-translational processing and intracellular localization of HDC (EC 22.214.171.124), the rate-limiting enzyme in histamine synthesis in mammals, in the rat basophilic/mast cell line RBL-2H3 13. HDC was found to be translated as a 74-kDa precursor form in the cytosol, which is then processed into its mature 53-kDa form. In addition, histamine synthesis was detected in two distinct compartments, in the cytosol and in granules. On the other hand, in the preliminary study, we have found that PMN are the dominant cell type responsible for histamine synthesis in an experimental casein-induced peritonitis model 14. Granules in human PMN have been classified into three types; azurophil/primary, specific/secondary, and gelatinase/ tertiary granules 15, whereas such a classification of neutrophil granules remains to be fully determined in mice. Although the previous observation that azurophil granules in PMN share several characteristics with histamine-containing granules in mast cells 16 indicates the granular localization of HDC in PMN, the exact intracellular localization of HDC in PMN remains to be clarified. In this study, we investigated the post-translational processing and intracellular localization of HDC to elucidate the regulation of histamine synthesis in PMN.
2.1 Expression of HDC in PMN
In our preliminary study, we have found that PMN are responsible for histamine synthesis in an experimental casein-induced peritonitis model 14. In this model, a drastic and transient increase in HDC enzyme activity in total peritoneal cells was observed, reaching a peak level 5 h after casein injection, at which time an intensive infiltration of PMN into the peritoneal cavity was observed (14 and data not shown). PMN were purified from the peritoneal cells 5 h after casein injection. Immunofluorescence studies revealed that the purified PMN fraction (>98% purity, confirmed by May-Grünwald-Giemsa staining) was immunoreactive to both an anti-HDC and an anti-CD11b antibody (Fig. 1A). The immunofluorescence pattern indicated a granule-like localization of HDC in the PMN. On the other hand, HDC+ cells were not found in total leukocytes from peripheral blood, both in control mice and in mice injected with casein, although a significant number of CD11b+ cells were observed in peripheral blood leukocytes from both mice (Fig. 1B). In addition, no detectable amount of HDC activity could be measured in the homogenates of peripheral blood leukocytes collected from mice treated with or without casein for 5 h (data not shown). These results indicate that HDC may be induced in response to inflammatory stimuli in the cavity. To confirm this hypothesis, we collected peripheral blood leukocytes and peritoneal lavage fluid from peritoneal cavities 5 h after casein injection. After incubation of the peripheral blood leukocytes with the lavage fluid for 6 h at 37°C, a significant number of HDC+ cells could be detected (Fig. 1C).
2.2 Induction of HDC in PMN isolated from bone marrow
Since a method for purification of PMN from murine peripheral blood leukocytes remains to be established, we isolated PMN precursors from the bone marrow (BM-PMN) by density gradient fractionation. Flow cytometric analysis demonstrated that greater than 80% of this fraction was immunoreactive to both an anti-CD11b and an anti-Gr-1 antibody (Fig. 2A). In comparison with casein-induced peritoneal PMN, similar levels of surface expression of Gr-1 and lower levels of CD11b were observed in BM-PMN. Immunofluorescence studies revealed that CD11b+ cells in BM-PMN were not immunoreactive to the anti-HDC antibody (Fig. 2B). We then investigated effects of peritoneal lavage fluids and several cytokines on the induction of histamine synthesis in BM-PMN. A drastic increase in HDC activity was observed in BM-PMN incubated for 3 h in the presence of peritoneal lavage fluids collected from mice injected with casein, but not in the presence of fluids collected from control mice (Fig. 2C). Induction of HDC by the lavage fluids was also confirmed by immunofluorescence study with the anti-HDC antibody (Fig. 2D). Among the cytokines tested, treatment with GM-CSF was found to slightly but significantly augment HDC activity in BM-PMN (Fig. 2C). IL-1β, IL-6, TNF-α, and G-CSF did not induce HDC activity in BM-PMN.
2.3 Expression and enzymatic activity of HDC in PMN
We then investigated the expression of mRNA and protein and the enzymatic activity of HDC in peritoneal PMN. Northern blot analyses demonstrated a high level of HDC mRNA expression in PMN immediately after purification, followed by a rapid decrease under culture conditions (Fig. 3A). Immunoblot analysis using the anti-HDC antibody revealed that the dominant molecular species in peritoneal PMN is the 53-kDa form (Fig. 3B). Purified PMN showed a significant level of HDC activity (∼80 pmol/min/mg protein), which gradually decreased under standard culture conditions for up to 6 h (Fig. 3C). Post-translational processing of HDC was confirmed by pulse-chase experiments (Fig. 3D, E). The 74-kDa precursor form was found to be completely converted into its mature 53-kDa form within 1 h. A band of approximately 120 kDa with strong labeling was detected, but was still detectable in the presence of excess amounts of the antigen, glutathione-S-transferase HDC fusion protein (data not shown), and hence we conclude that this band is from an unrelated protein detected in our system.
2.4 Co-localization of HDC with MMP-9
We investigated the intracellular localization of HDC. PMN were double-stained with the anti-HDC antibody and an antibody against matrix metalloproteinase-9 (MMP-9, gelatinase B), which is known to be a granule proteinase in PMN. In confocal microscopic observations, HDC and MMP-9 were found to co-localize in the granules of PMN (Fig. 4A), indicating that HDC is localized in the MMP-9-containing granules of PMN.
2.5 Unchanged localization of HDC upon stimulation with PMA
Since previous studies have demonstrated that MMP-9 is liberated from PMN upon treatment with phorbol 12-myristate 13-acetate (PMA) 17, extracellular release of HDC was investigated. A dose-dependent release of MMP-9 was measured as both enzyme activity and immunoreactive protein upon treatment of PMN with PMA for 30 min (Fig. 4B), whereas no release of HDC enzyme activity (data not shown) and immunoreactive protein was detected under the same conditions (Fig. 4B). The absence of MMP-9 and the granule-associated expression of HDC upon PMA treatment were also confirmed by immunofluorescence studies (Fig. 4C). Although relatively high levels of spontaneous histamine release (approximately 20%) were detected, a significant amount of stimulated release was detected upon treatment with PMA (Table 1). On the other hand, neither N-formyl-methionyl-leucyl-phenylalanine (up to 10 μM) nor leukotriene B4 (up to 1 μM) was found to induce a significant histamine release (data not shown).
|PMA (nM)||Total (ng/107 cells)||Release (ng/107 cells)||Net release (%)|
2.6 Membrane orientation of HDC
We then investigated in detail the localization of HDC in the granules of PMN using streptolysin-O (SLO), which selectively permeabilizes plasma membranes 18. Neither HDC activity nor a band immunoreactive to the anti-HDC antibody was detected in the cytosol fractions obtained after SLO treatment, whereas more than 80% of lactate dehydrogenase activity was recovered (data not shown), excluding the possibility that HDC is a cytosol-soluble protein. Immunoreactive signals were observed with the anti-HDC antibody, and not with the anti-MMP-9 antibody, in the SLO-treated PMN (Fig. 5), indicating that HDC was accessible to the antibody entering from the cytosolic side whereas MMP-9 was not. When the SLO-treated cells, after fixation, were further permeabilized with 1% Triton X-100, co-localization of HDC and MMP-9 was again confirmed.
In the previous report 14 and in the current study we demonstrated that isolated PMN express HDC and produce histamine in a casein-induced experimental peritonitis model. Expression of HDC in purified peritoneal PMN was demonstrated at both mRNA and protein levels. These results are consistent with previous findings by Shiraishi et al. 19 that infiltrating leukocytes in an air pouch-type allergic inflammation model and in a casein-induced peritonitis model in rats expressed HDC mRNA. Although their results strongly suggest that PMN are the dominant source of de novo synthesis of histamine (∼1.2 pmol/min/mg protein in infiltrating cells), they did not exclude the possibility that macrophages may produce some of this histamine. We have determined the specific activity of HDC in the peritoneal PMN (∼80 pmol/min/mg protein, Fig. 3A), which was much higher than previously reported for activated macrophages and mast cells (<10 pmol/min/mg protein) 10, 20. Furthermore, it is notable that HDC expression was induced in PMN in the peritoneal cavity, since peripheral blood leukocytes did not express HDC. It has been reported that CD11b expression in intravascular neutrophils can be enhanced by the addition of peritoneal fluid supernatant in a glycogen-induced peritonitis model 21. In the casein-induced model, we have also found that peritoneal lavage fluid has the potential to induce HDC protein in peripheral blood CD11b+ cells and in PMN isolated from the bone marrow (BM-PMN). Therefore, some humoral factors may exist in the peritoneal cavity that can induce HDC expression in PMN. The rapid decrease of HDC mRNA expression under culture conditions supports this hypothesis. We found an increase in HDC activity in BM-PMN incubated in the presence of GM-CSF. G-CSF and IL-6 demonstrated neither additive nor synergistic effects on GM-CSF-mediated induction of HDC, although these cytokines are also involved in regulation of PMN functions. Since the potential of the peritoneal lavage fluids to augment histamine synthesis was much greater than that of GM-CSF, it is possible that the peritoneal lavage fluids contain other inducing factors for HDC. TNF-α is one of the possible candidates, since Endo has previously reported that a systemic injection of TNF-α was able to induce histamine synthesis in mouse bone marrow cells and spleen cells 22 and since a significant amount of TNF-α release from mast cells has been detected during the anaphylactic phase of a mouse peritonitis model 23. However, TNF-α may not be involved in the induction of HDC in our model, as TNF-α did not augment HDC activity in BM-PMN and an intraperitoneal injection of an anti-TNF-α neutralizing antibody did not inhibit the induction of HDC (data not shown). Another proinflammatory cytokine, IL-1β, was also found to have no inducible effects on HDC expression in PMN. Further studies are surely required to determine the factors responsible for induction of HDC.
The enzymatic activity of HDC in peritoneal PMN was found to rapidly decrease under culture conditions, before the decrease in HDC mRNA and protein. This result indicates the occurrence of enzymatical inactivation of HDC in peritoneal PMN in addition to degradation of its mRNA and protein, although such inactivation has not been previously reported. Rapid suppression of histamine synthesis in peritoneal PMN may contribute to the prevention of exacerbated inflammatory responses, which could be induced by prolonged histamine release.
The immunofluorescence studies using the anti-HDC antibody revealed a granular localization of HDC in PMN. We previously demonstrated that HDC was translated in the cytosol and targeted to the endoplasmatic reticulum (ER), where post-translational processing occurred in the rat mast cell line RBL-2H3 13. We also observed that histamine was produced in both the cytosol and the granule fractions of the cells, mediated by the 74-kDa form and the 53-kDa form, respectively. Compared with the intracellular localization of HDC in the mast cell line, which is mainly in the ER, the current study demonstrated that HDC is co-localized with MMP-9 in PMN, indicating the granular localization of HDC. These results indicate that HDC may be efficiently processed into the mature 53-kDa form and transported to the gelatinase-containing granules in PMN. Indeed, the pulse-chase experiments showed the rapid and complete processing of HDC as only the 53-kDa form was detected upon immunoblot analyses using the anti-HDC antibody.
We previously demonstrated that the 53-kDa form of HDC was not accessible to the anti-HDC antibody in SLO-treated RBL-2H3 cells 13, which strongly suggests that the 53-kDa form of HDC is localized in the luminal compartments of the cells. Indeed, the 53-kDa form was found to be resistant to trypsin digestion in SLO-treated RBL-2H3 cells. On the contrary, the 53-kDa form was accessible to the anti-HDC antibody in SLO-treated PMN. Since HDC was not released upon SLO-treatment, HDC may be bound to the cytosolic side of granule membranes in PMN. We have no experimental evidence giving a clear explanation for this inconsistency in the localization of HDC between mast cells and PMN. Membrane orientation and targeting of HDC remains largely unknown. Using the in vitro translation system with rabbit reticulocyte lysates, we previously demonstrated that targeting of the 74-kDa form to the microsomal membranes occurs post-translationally 24, although the exact mechanism remains to be determined. Since the 74-kDa form of HDC lacks the amino-terminal signal sequence, which can bind to the signal recognition particle, the membrane targeting of HDC may be mediated by an unknown mechanism. Further analyses are required to clarify this mechanism.
Compared with mast cells, histamine release from PMN is characterized by high levels of spontaneous release, indicating the rapid turnover of nascent histamine in PMN. On the other hand, the observation that relatively small amounts of histamine were released upon PMA treatment indicates the existence of heterogeneous pools of granule histamine. Granule-associated localization of HDC may contribute to uptake of nascent histamine into granules, and spontaneous release of granule contents may occur in PMN infiltrated into the peritoneal cavity. The transport system for histamine across the plasma membrane and vesicular membranes remains largely unknown. There has been no experimental evidence that excludes the possibility that histamine produced in the cytosol may be directly released via a transporter in the plasma membrane. Indeed, we have recently reported that histamine is transported into the cytosol of mouse macrophages in a Na+-independent manner and that cytosolic histamine is liberated from the cells after depletion of extracellular histamine, although the molecular identity of the transporter remains to be determined 25. We surely need further experimental evidence to address this problem.
It has been demonstrated that large amounts of histamine are transiently released from mast cell granules in the anaphylactic phase of inflammatory responses, whereas continuous histamine release is usually observed in the late phase, which is accompanied by an increase in histamine synthesis 9. These distinct characteristics of histamine release may reflect the difference in the fashion of histamine synthesis and storage between mast cells and PMN; histamine is accumulated and stored in the granules of mast cells, whereas it is rapidly released from PMN.
In summary, we demonstrated in this study that HDC is induced in PMN infiltrated into the peritoneal cavity and that the dominant 53-kDa form, which is converted by a rapid post-translational processing, is localized on the cytosolic side of gelatinase-containing granules in PMN. PMN may be one of the important sources of histamine in inflammatory responses.
4 Materials and methods
An anti-HDC antibody was prepared as described 26. This antibody was raised against the partial amino acid sequence (residues 1–210) of mouse HDC and can recognize both the precursor 74-kDa form and the mature 53-kDa form of HDC 13. The following materials were purchased from the sources indicated: an anti-MMP-9 antibody from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); an anti-protein disulfide isomerase (PDI) antibody from StressGen (Victoria, BC); a FITC-conjugated anti-CD11b antibody and a phycoerythrin-conjugated anti-Gr-1 antibody from PharMingen (San Diego, CA); a FITC-conjugated anti-rabbit IgG antibody and a rhodamine-conjugated anti-mouse IgG antibody from Leinco Technologies, Inc. (Ballwin, MO); a rhodamine-conjugated donkey anti-goat IgG antibody and a FITC-conjugated donkey anti-rabbit IgG antibody from CHEMICON (Temecula, CA); an Alexa594-conjugated anti-rabbit IgG antibody from Molecular Probes, Inc. (Eugene, OR); polyvinylidene difluoride (PVDF) membranes from Millipore (Tokyo, Japan); a horseradish peroxidase-conjugated anti-rabbit IgG antibody from Dako (Glostrup, Denmark); ISOGEN from Nippon Gene (Tokyo, Japan); Percoll, ECL Western blot detection reagent and protein A Sepharose CL-4B from Amersham Pharmacia (Uppsala, Sweden); Biodyne A from Pall BioSupport Corporation (East Hills, NY); SLO, N-formyl-methionyl-leucyl-phenylalanine, and leukotriene B4 from Sigma Aldrich (St. Lois, MO); [35S]methionine (1,000 Ci/mmol) and [α-32P]dCTP (3,000 Ci/mmol) from Du Pont-New England Nuclear (Boston, MA). All other chemicals were commercial products of reagent grade.
Female BALB/c mice (7–8 weeks old) were obtained from Shimizu Experimental Animal Lab, Co. Ltd. (Kyoto, Japan). All experiments were performed according to the Guideline for Animal Experimentsof Kyoto University.
4.3 Isolation of elicited peritoneal PMN
Female BALB/c mice were used for all experiments. Casein in saline (5%, w/v, 2 ml/cavity) was injected intraperitoneally. Peritoneal cell types were determined by microscopic observation afterMay-Grünwald-Giemsa staining. Cells in the peritoneal cavity were harvested 5 h after injection by lavage of the cavities with 3 ml sterile PBS. Lavage fluids were centrifuged at 200×g for 5 min at 4°C, and the pellet was resuspended in 2 ml PBS. PMN were purified by centrifugation on discontinuous Percoll gradients 27. Briefly, 2 ml cell suspension were carefully layered on top of the discontinuous Percoll gradient prepared with 2 ml solution B (density 1.090 g/ml) and 2 ml solution A (density 1.070 g/ml). After centrifugation at 500×g for 20 min at 4°C, two distinct leukocytic layers were obtained. The lower band was collected as the PMN fraction and washed twice in PBS. Determination of the cell population by May-Grünwald-Giemsa staining indicated that more than 98% of the cells obtained were neutrophils, whereas the rest were mononuclear cells.
4.4 Isolation of PMN from bone marrow
Bone marrow cells were harvested from the femurs of female BALB/c mice and washed in Hanks' balanced salt solution. PMN in bone marrow were isolated according to the procedure described 28 with minor modifications. Briefly, bone marrow cells were resuspended in PBS (5×107–7×107 cells/ml) and carefully layered on top of the discontinuousPercoll gradient consisting of three layers 29 (3 ml each; densities 1.082, 1.075, 1.053 g/ml). After centrifugation at 500×g for 30 min at 4°C, the bottom fraction (1 ml) was collected as the PMN fraction (BM-PMN) and washed twice in PBS. Determination of the cell population was performed by May-Grünwald-Giemsa staining and by flow cytometry.
4.5 Culture of peritoneal and bone marrow PMN
Purified PMN or bone marrow PMN were cultured in RPMI 1640 medium containing 50 μM 2-mercaptoethanol, 1 mM sodium pyruvate, 100 U/ml penicillin, 0.1 mg/ml streptomycin and 10% heat-inactivated fetal calf serum in a fully humidified 5%-CO2 atmosphere (standard culture conditions) for the indicated times. The viability of greater than 98% of the cells was confirmed by trypan blue exclusion.
4.6 Flow cytometric analysis
Flow cytometric analyses were performed with a FACSCalibur (Becton Dickinson) using a FITC-conjugated anti-CD11b antibody and a phycoerythrin-conjugated anti-Gr-1 antibody to determine the neutrophil populations. The population of positive cells was determined by comparison to cells stained with FITC-or phycoerythrin-conjugated isotype-matched immunoglobulin.
4.7 Immunofluorescence studies
PMN were centrifuged onto round cover glasses (▪=18.0 mm), which were then placed in 12-well culture plates. Immunofluorescence studies were performed as described 13. An anti-HDC antibody (1:500), an anti-MMP-9 antibody (1:500), an anti-PDI antibody (1:500) and a FITC-conjugated anti-CD11b antibody (1:1,000) were used as primary antibodies. Alexa594-conjugated goat anti-rabbit IgG antibody (1:1,000) was used for HDC and CD11b double-staining. A rhodamine-conjugated donkey anti-goat IgG antibody (1:100) and a FITC-conjugated donkey anti-rabbit IgG antibody (1:100) were used for MMP-9 and HDC double-staining, respectively. Stained cells were observed by confocal microscopy (MRC-1024, Bio-Rad Laboratories, Hercules, CA).
4.8 Northern blot analyses
Total RNA was extracted from purified PMN using ISOGEN according to the manufacturer's instructions, separated (3 μg/lane) by electrophoresis on a 1.5% agarose/formaldehyde gel and transferred onto a Biodyne A membrane in 20× SSC (1× SSC is composed of 0.15 M NaCl and 0.015 M sodium citrate) by capillary blotting. Hybridization was performed with a 32P-labeledcDNA fragment specific for murine HDC (Pvu II-digested fragment) in hybridizing solution (6× SSC, 5× Denhardt's solution, 0.5% SDS, and 100 μg/ml salmon sperm DNA) at 68°C overnight 30. The filter was rinsed twice in 2× SSC containing 1% SDS at room temperature and twice in 0.2× SSC containing 0.1% SDS at 60°C. A 32P-labeled probe specific for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used in rehybridization for the loading control. The filter was then analyzed using a Fujix BAS 2000 Bio-Imaging Analyzer.
4.9 Immunoblot analyses
PMN were homogenized in 50 mM HEPES-NaOH pH 7.3, containing 0.2 mM dithiothreitol, 0.01 mM pyridoxal 5′-phosphate, 2% polyethylene glycol 300, 0.2 mM PMSF and 0.1% TritonX-100 and centrifuged at 15,000×g for 30 min at 4°C. The resultant supernatant (50 μg/lane) was subjected to SDS-PAGE (10% slab gel), and the separated proteins were transferred electrophoretically to a PVDF membrane. Immunoblot analysis was performed as described 13. An anti-HDC antibody (1:500) or an anti-MMP-9 antibody (1:500) was used as the primary antibody, and a horseradish peroxidase-conjugated anti-rabbit IgG antibody (1:3,000) or a horseradish peroxidase-conjugated anti-goat IgG antibody (1:3,000) was used as the secondary antibody. The membranes were stained using an ECL kit according to the manufacturer's instructions.
4.10 Histidine decarboxylase assay
PMN were rinsed with PBS followed by centrifugation, and the cell pellet was lysed (1×107 cells/ml) with 50 mM HEPES-NaOH pH 7.3 containing 0.2 mM dithiothreitol, 0.01 mM pyridoxal 5′-phosphate, 2% polyethylene glycol 300, 0.2 mM PMSF and 0.1% Triton X-100 on ice for 30 min. The cells were centrifuged at 100,000×g for 1 h at 4°C, and the supernatant was used for the measurement of histidine decarboxylase activity as described 13. The precipitate fraction exhibited no detectable enzymatic activity. The histamine formed was separated on a cation exchange column, WCX-1 (Shimadzu, Kyoto, Japan), by HPLC and then measured by the o-phtalaldehyde method 31.
PMN were starved for 30 min in methionine-free RPMI 1640 medium supplemented with 10% dialyzed fetal calf serum and then pulse-labeled with [35S]methionine (10 μCi/ml) for the indicated periods. In the chase experiments, 1 mM cold methionine was added to the cells after pulse labeling and incubated for the indicated periods at 37°C. Immunoprecipitation with an anti-HDC antibody was performed as described previously 13. The dried gel was analyzed using a Fujix BAS 2000 Bio-Imaging Analyzer.
4.12 Gelatin zymography
Gelatin zymography was performed according to the procedure described 32. Briefly, SDS-PAGE was performed with 10% acrylamide slab gels containing 0.28% gelatin. After electrophoresis, gels were incubated twice in 2.5% Triton X-100 at room temperature for 30 min, and the in-gel gelatinase reaction was performed in 100 mM Tris-HCl pH 7.5 containing 10 mM CaCl2 at 37°C for 12 h. Gelatin digestion was confirmed by Coomassie brilliant blue staining.
4.13 Streptolysin-O treatment
PMN were incubated with SLO (12,500 U/ml; preactivated by incubation for 15 min on ice with PBS containing 10 mM dithiothreitol) in PBS at 4°C for 10 min. After this binding step, cells were rinsed twice with PBS and then incubated at 37°C for 3 min to cause permeabilization. More than 80% of lactate dehydrogenase activity was recovered in the leaked fraction under these conditions. No immunoreactive band was detected in the leaked fraction upon immunoblot analyses with the anti-PDI antibody, indicating that the ER membrane was intact. Detergent-free buffer was used in the immunofluorescence experiments for analysis of cells after selective permeabilization of the plasma membrane.
This study was supported by grants-in-aid for Scientific Research from the Ministry of Education, Culture, Science, Sports and Technology of Japan. We thankMs. A. Popiel for her help in preparation of the manuscript.