Published online before print November 7, 2005
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,1
* Departments of Physiology and Biophysics and
Biochemistry and Molecular Biology, Immunology Research Group, University of Calgary, Alberta, Canada
1 Correspondence: Departments of Physiology and Biophysics and Biochemistry and Molecular Biology, Immunology Research Group, University of Calgary, 3330 Hospital Dr., N.W., Calgary, Alberta T2N 4N1, Canada. E-mail: kpatel{at}ucalgary.ca
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and PKC 
in maximal MMP-9 release. These data suggest that MMP-9 release from tertiary granules uses ß2-integrin-independent signaling pathways. Furthermore, PKC isoforms play a critical role in regulating tertiary granule release.
Key Words: degranulation • signal transduction • integrins • protein kinase C • inflammation
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Neutrophils contain four different classes of granules: azurophilic (primary), specific (secondary), and gelatinase (tertiary) granules and secretory vesicles (reviewed in refs. [7 , 8 ]). These granules differ from each other in their constituent proteins, their order of appearance in the developing neutrophil, and their order of release following neutrophil stimulation [7 , 8 ]. Protein markers used to characterize the exocytosis of particular granules include myeloperoxidase (MPO) for primary granules, lactoferrin for secondary granules, and gelatinase B [matrix metalloproteinase-9 (MMP-9)] for tertiary granules [7 , 8 ]. Little is known about the signaling pathways that may underlie differences in the behavior of different classes of neutrophil granules. Some recent studies suggest profound differences in the regulation of tertiary granules versus that of primary and secondary granules in neutrophils. For example, Abdel-Latif et al. [9 ] have shown that knocking out Rac2 in murine neutrophils can inhibit the release of primary granules, and tertiary granule release remains unaffected. Takafuji et al. [10 ] have demonstrated that pretreating human neutrophils with an inhibitor of actin polymerization, which enhances primary and secondary granule release, can actually decrease tertiary granule release. Similarly, previous work in our lab has shown that interleukin (IL)-8-mediated tertiary granule release is independent of intracellular calcium changes [11 ], whereas primary and secondary granule release depends on increased intracellular calcium. Together, these data suggest that tertiary granule release is regulated differently than primary and secondary granule release.
One of the main constituents of the tertiary granules is MMP-9. Unlike most cell types, neutrophil release of MMP-9 is regulated post-transcriptionally at the level of tertiary granule release [12 ]. Several mediators have been shown to induce MMP-9 release, including formyl-Met-Leu-Phe, IL-8, and tumor necrosis factor (TNF) [13 ]; however, the mechanisms that specifically regulate MMP-9 release from stimulated neutrophils are not well understood. TNF is a multifunctional cytokine, which potently activates human neutrophils, resulting in free radical production and granule release [14 15 16 17 ]. Unlike responses to other soluble stimuli, neutrophil responses to TNF are dependent on signaling by cell-surface integrins [14 , 15 , 17 , 18 ]. In neutrophils, TNF stimulation initiates signaling events, which activate integrins (inside-out signaling), thereby leading to increased adhesiveness. Once activated and ligated, integrins themselves initiate a second cascade of signaling events (outside-in signaling). TNF-induced free radical generation as well as primary and secondary granule release rely on these secondary signaling events that occur downstream of integrin activation [14 , 15 , 17 , 18 ].
In this study, we examined the signaling pathways that regulate TNF-induced tertiary granule release from human neutrophils and found that TNF could still induce MMP-9 release in the absence of ß2-integrin signaling. Furthermore, we found that protein kinase C (PKC) isoforms were critical in regulating MMP-9 release in response to TNF stimulation.
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Antibodies
TNF receptor (TNFR) I, TNFR II, and control mouse IgG antibodies were obtained from R&D Systems. ß2-integrin antibody (Clone IB4) was obtained from Beckman Coulter Canada Inc. (Mississauga, Ontario). p38 mitogen-activated protein kinase (MAPK) antibody was from Santa Cruz Biotechnology (CA). Phospho-Src family kinase, phospho-p38 MAPK, and phospho-extracellular signal-regulated kinase (ERK)1/2 antibodies were from Cell Signaling Technologies (Beverly, MA). ERK1/2 antibody was from Upstate USA (Charlottesville, VA). Horseradish peroxidase-conjugated anti-rabbit IgG was from Cedarlane Laboratories Ltd. (Hornby, Ontario, Canada).
Neutrophil isolation and stimulation
Neutrophils were isolated from the peripheral blood of healthy human volunteers by density centrifugation as described previously [19
]. Neutrophils were routinely >95% pure, and lymphocytes and eosinophils were the contaminating cells. We received ethics approval from the University of Calgary Ethics Committee (Alberta, Canada). Neutrophils were used at a concentration of 1 x 107 cells/ml in HBSS. TNF was added to 1 x 106 cells, and the cells were incubated in a polypropylene microfuge tube at 37°C for the stated times. Cells were gently rocked approximately once every 5–10 min to prevent them from sticking to the sides of the tube. Following centrifugation, the cell-free supernatants were diluted, and MMP-9 levels were determined by gelatin zymography and/or ELISA as described below. In some experiments, cells were incubated with anti-TNFR I (50 µg/ml), anti-TNFR II (10 µg/ml), anti-ß2-integrin (10 µg/ml), or control mouse IgG (50 or 10 µg/ml) for 15 min at 37°C prior to TNF stimulation. In other experiments, inhibitors were added to the cells for the specified times before stimulation. The appropriate solvent controls were used. The minimum preincubation time, which produced maximal inhibition, was used. The inhibitors used include PD98059, PP2, chelerythrine chloride, safingol, hispidin, rottlerin, and BAPTA-AM.
Measuring MMP-9 release by gelatin zymography
Zymography was performed as described previously [11
, 20
]. Briefly, nonreducing Laemmli’s buffer was added to the cell-free neutrophil supernatants, and the proteins were separated immediately on a 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel incorporating 1 mg/ml gelatin. After electrophoresis, the gel was washed for 15 min in a Triton X-100 containing rinse buffer and then incubated overnight in the rinse buffer at room temperature. After extensive washing with deionized water, the gel incubated for an additional 24 h at 37°C in an incubation buffer containing calcium and zinc. Staining the gel with Coomassie brilliant blue R-250 revealed clear bands, which indicate the presence of gelatinase activity. Gels were visualized using a Fluor-S-MAX MultiImager (Bio-Rad Laboratories, Hercules, CA), and the bands were quantified by densitometry using Quantity One software (Bio-Rad Laboratories). The images have been inverted to show dark bands on a clear background. Data are presented as fold increase over the unstimulated control.
Measuring MMP-9 and lactoferrin release by ELISA
Total MMP-9 was measured using ELISA according to the manufacturer’s instructions. Following stimulation, the cell-free supernatants were diluted 1:25 and assayed for total MMP-9 by ELISA. The data obtained using zymography and ELISA were equivalent. Total lactoferrin release was measured using ELISA according to the manufacturer’s instructions.
Neutrophil adhesion
Neutrophil adhesion to matrix-coated plates was performed as described previously [11
]. Briefly, neutrophils (1x106/ml) were added to 24-mm plates coated with 0.2% gelatin and were stimulated with buffer alone or buffer containing 10 ng/ml TNF. After 20 min at 37°C, the nonadherent and loosely adherent neutrophils were removed by washing, and the adherent neutrophils were lysed and quantified by measuring MPO content as described previously [11
]. Briefly, MPO activity was estimated by adding 10 µl neutrophil lysate to 100 µl one-step peroxidase substrate (Sigma Chemical Co.). After 5 min, the reaction was stopped by addition of 100 µl phosphoric acid. Absorbance at 450 nm was measured to determine the MPO content. Data are expressed as a percentage of total neutrophils added to each plate.
Western blotting
Following stimulation, neutrophils were rapidly chilled by the addition of ice-cold HBSS. Neutrophils were pelleted, the supernatants were removed, and the cell pellets were lysed by the addition of hot, reducing Laemmli’s buffer containing protease inhibitors (5 mM sodium vanadate, 5 mM EDTA, 2 mM phenylmethylsulfonyl fluoride, 0.1 U/ml aprotinin, 20 µg/ml leupeptin). Cellular proteins were resolved using SDS-PAGE and transferred to polyvinylidine difluoride membranes, which were subsequently probed with specific antibodies. Antibody binding was detected using chemiluminescence and was visualized using a Fluor-S-MAX MultiImager.
Statistics
All experiments were performed between three and five times using different blood donors. Densitometry data are shown as mean ± SEM. Statistical differences among experimental groups were evaluated using a one-way ANOVA with an appropriate post-test. P values of less than 0.05 were considered significant. Data were compared against the unstimulated control and the TNF-stimulated samples. No differences were observed between TNF alone and TNF containing nonimmune IgG or dimethyl sulfoxide (DMSO).
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450 ng/ml MMP-9 per 107 cells (data not shown).
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Figure 1. TNF induces the rapid release of MMP-9 from human neutrophils through interactions with TNFR I. Neutrophils were treated with (A) various concentrations of TNF for 30 min at 37°C or (B) 10 ng/ml TNF for the specified times at 37°C. MMP-9 release was determined by gelatin zymography of cell-free supernatants followed by densitometry as described in Materials and Methods. (C and D) Neutrophils were left untreated or were pretreated with anti-TNFR I (50 µg/ml) antibody, anti-TNFR II (10 µg/ml) antibody, or both for 15 min prior to stimulation with 10 ng/ml TNF. After 30 min at 37°C, cell-free supernatants were collected and analyzed for MMP-9 content by (C) gelatin zymography followed by densitometry or (D) ELISA as described in Materials and Methods. Representative zymograms are shown. Data represent the mean ± SEM of between three and five experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001, compared with control; and , P < 0.01; , P < 0.001, compared with TNF. ns, Not significant; mAb, monoclonal antibody.
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ERK1/2 is not critical for TNF-induced MMP-9 release from neutrophils
The MAPK mediate diverse functions such as cell division and degranulation in various cell types. TNF stimulation of neutrophils activates all three MAPK: ERK1/2, Jun N-terminal kinase, and p38 MAPK [24
]. p38 MAPK and ERK1/2 have been shown to be involved in the degranulation of granulocytes [25
, 26
]; however, we have previously demonstrated that inhibiting p38 with SB203580 has no effect on TNF-induced MMP-9 release [20
]. In contrast, ERK1/2 can regulate IL-8-mediated MMP-9 release from neutrophils [11
]. Based on these findings, we examined the role of ERK1/2 in TNF-mediated MMP-9 release from neutrophils, which were pretreated with the MAPK kinase inhibitor PD98059 prior to stimulation with TNF. PD98059 (10 µM) was selected based on its ability to block TNF-induced ERK1/2 phosphorylation (ref. [11
] and data not shown). We found that PD98059 had no effect on MMP-9 release (MMP-9-fold increase: TNF alone, 4.92±0.46; TNF+PD98059, 4.75±0.99). Together, these data suggest that ERK1/2 and p38 MAPK are not critical for TNF-induced MMP-9 release.
TNF-induced MMP-9 release is partially independent of ß2-integrin signaling
Several groups have demonstrated that TNF-induced free radical production and degranulation are dependent on ß2-integrin adhesion and subsequent outside-in signaling through the Src family kinases [17
, 27
28
29
]. These studies defined degranulation as the release of lactoferrin from secondary granules but did not measure the tertiary granule marker MMP-9 [17
, 28
]. We used a function-blocking antibody directed against ß2-integrins (Clone IB4) to determine if TNF-induced MMP-9 release from tertiary granules also required ß2-intetgrin-mediated adhesion. Neutrophils were treated with 10 µg/ml IB4 prior to stimulation with TNF. We found that blocking ß2-integrins only partially attenuated MMP-9 release (Fig. 2A
and 2B
). The ability of TNF-stimulated neutrophils to release MMP-9 in the presence of a ß2-integrin antibody occurred when cells were stimulated in suspension and when cells were stimulated in culture dishes (Fig. 2C)
. Under identical conditions, this antibody totally prevented TNF-induced adhesion to gelatin (Fig. 2D)
, demonstrating that we blocked ß2-integrin-mediated adhesion effectively. As release of lactoferrin from secondary granules has been reported to be reduced to control levels following ß2-integrin blockade [17
], we examined the release of lactoferrin from secondary granules in TNF-stimulated neutrophils in the presence of a ß2-integrin antibody. Consistent with previous findings, we found that blocking ß2-integrins abolished the release of lactoferrin from the secondary granules (Fig. 2E)
under the same conditions in which MMP-9 release was only partially attenuated (Fig. 2A
and 2B)
.
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Figure 2. TNF-induced MMP-9 release has ß2-integrin-dependent and -independent components. Neutrophils were left untreated or were pretreated with 10 µg/ml anti-ß2-integrin antibody (IB4) or nonimmune IgG (NI-IgG) for 15 min. (A and B) MMP-9 release was measured after 30 min stimulation in suspension with buffer alone or 10 ng/ml TNF. (C) Alternatively, MMP-9 release was measured after 20 min stimulation of cells added to culture plates with buffer alone or 10 ng/ml TNF. Supernatants were analyzed for total MMP-9 levels by (A and C) gelatin zymography followed by densitometry or (B) ELISA, as described in Materials and Methods. (D) Adhesion to plates coated with 0.2% gelatin was measured after 20 min of stimulation buffer alone or 10 ng/ml TNF. (E) Lactoferrin release into the cell-free supernatant was measured by ELISA after 30 min of stimulation with buffer alone or 10 ng/ml TNF. The data represent the mean ± SEM of five experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001, as compared with control; and , P < 0.01; , P < 0.001, as compared with TNF.
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Figure 3. The role of Src family kinases in TNF-induced MMP-9 release. (A) Neutrophils stimulated for the specified times with 10 ng/ml TNF were lysed directly in reducing Laemmli’s buffer, and Western blotting was performed using a pan-specific phospho-Src (p-src) family kinase antibody. All lanes were loaded with equal amounts of total protein as demonstrated by total ERK1/2 binding (data not shown). A representative Western blot is shown. (B) Neutrophils were pretreated for 5 min with DMSO alone or with the specified concentrations of the Src family kinase inhibitor PP2 prior to 30 min of stimulation with 10 ng/ml TNF at 37°C. Supernatants were analyzed for MMP-9 levels by gelatin zymography followed by densitometry as described in Materials and Methods. (C) Neutrophils were pretreated for 5 min with DMSO alone or with 10 µM PP2 prior to 30 min of stimulation with 10 ng/ml TNF at 37°C. Lactoferrin release into the cell-free supernatant was measured by ELISA. The data represent mean ± SEM of between three and five experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001, as compared with control; and , P < 0.001, as compared with TNF.
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Figure 4. Src family kinases and ß2-integrins together only attenuate TNF-induced MMP-9 release. (A and B) Neutrophils were pretreated for 5 min with DMSO alone or for 5 min with 10 µM PP2 prior to 30 min of stimulation with 10 ng/ml TNF or 500 µM Mn2+ at 37°C. (C and D) Neutrophils were pretreated with 10 µM PP2, 10 µg/ml of an anti-ß2-integrin antibody (IB4), or both prior to 30 min of stimulation with 10 ng/ml TNF at 37°C. Supernatants were analyzed for total MMP-9 levels by (A and C) gelatin zymography followed by densitometry or (B and D) ELISA as described in Materials and Methods. The data represent mean ± SEM of between three and five experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001, as compared with control; and , P < 0.05; , P < 0.01, as compared with TNF.
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PKC is critical for TNF-mediated MMP-9 release
Binding of TNF to TNFR I activates a variety of protein kinases in neutrophils, including PKC [33
]. We initially used chelerythrine chloride, a pan-specific PKC inhibitor, to investigate the role of PKC in TNF-induced MMP-9 release. Neutrophils were pretreated with various concentrations of chelerythrine chloride for 5 min prior to stimulation with TNF. We found that chelerythrine chloride significantly reduced TNF-mediated MMP-9 release, suggesting a role for PKC in this process (Fig. 5A
and 5B
). Human neutrophils express the , ßI, ßII, , and 
isoforms of PKC [34
35
36
37
]. PKC , ßI, and ßII are classical isoforms, which are activated by diacylglycerol (DAG) and calcium. PKC is a novel isoform, which is activated by DAG but is independent of calcium. PKC is an atypical isoform, which is independent of DAG and calcium. Using inhibitors directed against specific PKC isoforms, we found that blocking any single PKC isoform had no effect on MMP-9 release; however, blocking PKC and PKC together completely blocked MMP-9 release (Fig. 5C) . PKC is calcium-dependent, whereas PKC is calcium-independent; thus, chelating intracellular calcium with BAPTA can block the activation of PKC but not PKC . Similar to blocking PKC alone, chelating intracellular calcium with BAPTA alone had no effect on MMP-9 release (Fig. 5D)
. BAPTA used in combination with a PKC inhibitor blocked MMP-9 release (Fig. 5D)
. These data were consistent with the data using PKC and PKC inhibitors together (Fig. 5C)
, and together, these data support a role for PKC and PKC in TNF-induced MMP-9 release.
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Figure 5. PKC and PKC regulate TNF-mediated MMP-9 release. (A) Neutrophils in suspension were pretreated for 5 min with DMSO alone or various concentrations of the pan-PKC inhibitor chelerythrine chloride. (B) Neutrophils in suspension were pretreated for 5 min with DMSO alone or with 20 µM chelerythrine chloride (Chel-Cl). (C) Neutrophils were pretreated 5 min with DMSO alone or were with inhibitors directed against specific PKC isoforms used including 20 µM chelerythrine chloride (All), 20 µM Safingol (), 10 µM Hispidin (ß), 10 µM Rottlerin (), or combinations of these inhibitors. (D) Neutrophils were pretreated 5 min with DMSO alone or 10 µM BAPTA-AM, 10 µM Rottlerin (PKC ), or both. (A–C) Cells were then stimulated for 30 min with 10 ng/ml TNF at 37°C. Supernatants were analyzed for MMP-9 levels by (A, C, and D) gelatin zymography followed by densitometry or (B) ELISA, as described in Materials and Methods. The data represent mean ± SEM of between three and five experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001, as compared with control; , P < 0.05; , P < 0.001, as compared with TNF.
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Using MMP-9 as a marker for tertiary granule release, we found that TNF induced significant MMP-9 release when ß2-integrin-mediated adhesion was blocked using an inhibitory antibody (Fig. 2) . Similarly, TNF-induced MMP-9 release was only partially attenuated by inhibition of the Src family kinases (Fig. 3) . Blocking ß2-integrins and Src family kinases together did not have an additive effect on blocking MMP-9 release, suggesting that a significant portion of tertiary granule release can occur independently of ß2-integrin-mediated adhesion and Src family kinase signaling. ß2-integrin signaling and Src family kinases did participate in a portion of TNF-induced MMP-9 release. As neutrophils were stimulated in suspension in these experiments, it is likely that the adhesive signal was a result of cell–cell aggregation, which occurred during the experiment. There are several other pathways activated downstream of integrins, including phosphorylation and activation of the focal adhesion kinase Pyk2 and Syk [16 , 31 , 32 ]. Han et al. [31 ] demonstrated that blocking Pyk2 activity prevented TNF-induced Syk phosphorylation in human neutrophils and decreased superoxide production by 80% yet had no effect on secondary granule release. Similar to these findings, we found that Syk inhibition had no effect on MMP-9 release (data not shown).
Apart from the Src family kinases, we found that two PKC isoforms, PKC and PKC , also participated in MMP-9 release (Fig. 5)
. Only when both isoforms were blocked was MMP-9 release inhibited. The dependence on two PKC isoforms may reflect the ability of both PKC isoforms to phosphorylate similar targets required for MMP-9 release, such that if one isoform is inhibited, the other can still phosphorylate the target. Only when both isoforms are blocked will phosphorylation of the target be blocked and MMP-9 release inhibited. This would also explain why chelation of intracellular calcium alone, which would only block PKC and not PKC , had no effect on MMP-9 release, but chelation of calcium combined with blocking PKC inhibited MMP-9 release (Fig. 5)
. Potential targets for PKC and are the soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) proteins, synaptosome-associated protein 23 (SNAP-23) and syntaxin-4. SNARE proteins mediate vesicle fusion in many cell types [39
] and have been shown to participate in secondary and tertiary granule release in neutrophils [40
, 41
]. The activity of SNAREs can be regulated by phosphorylation. This has been examined carefully in platelets but not in neutrophils. In platelets, SNAP-23 and syntaxin-4 become rapidly phosphorylated following stimulation with thrombin [42
, 43
], and phosphorylation parallels degranulation [42
, 43
]. PKC is central to this process, as a PKC inhibitor blocks thrombin-induced SNAP-23 and syntaxin-4 phosphorylation. In vitro, PKC isoforms can phosphorylate SNAP-23 and syntaxin-4; this phosphorylation alters interactions between SNAP-23 and syntaxin-4, suggesting that phosphorylation may regulate vesicle fusion. Examining the role of PKC in SNARE phosphorylation in TNF-stimulated neutrophils is warranted.
MMP-9 has been implicated in the pathogenesis of inflammatory lung diseases such as COPD [44 , 45 ], asthma [46 , 47 ], and ARDS [48 ]. Relative to control individuals, COPD and asthma patients show elevated MMP-9 levels in lung biopsy specimens [49 , 50 ], bronchoalveolar lavage fluid [50 ], and induced sputum [2 ]. MMP-9 is proposed to regulate the tissue remodeling and inflammation that characterize these diseases. Infiltrated neutrophils are an important source of lung MMP-9 in these inflammatory diseases. In COPD and asthma patients, MMP-9 localizes to neutrophils in lung biopsy tissue [50 , 51 ], and MMP-9 levels in induced sputum correlate with sputum neutrophil levels [2 , 52 ]. Our data suggest that TNF-stimulated neutrophils may contribute a significant amount of MMP-9 at an inflamed site. In addition, our data suggest that TNF can induce MMP-9 release from nonadherent neutrophils. This may be relevant in patients with diseases such as sepsis, where elevated levels of TNF and MMP-9 are found in the peripheral blood [13 ]. In patients with varicose veins, MMP-9 levels, but not primary or secondary granule proteins, are elevated in the peripheral blood [53 ], a finding that may suggest selective release of the tertiary granule contents from circulating neutrophils.
In this study, we identified specific mechanisms that regulate TNF-induced MMP-9 release from human neutrophils. By understanding the mechanisms that uniquely regulate specific activation responses in neutrophils, it might be possible to selectively modulate the release of tissue-degrading factors such as MMP-9 without compromising the bactericidal functions mediated by the generation of free radicals and the release of primary and secondary granule proteins from the neutrophil.
Received June 30, 2005; revised September 5, 2005; accepted September 6, 2005.
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}L{ß}2 J. Leukoc. Biol. 72,718-726 induced neutrophil apoptosis Microbiol. Immunol. 40,463-465[Medline] in the induction of human polymorphonuclear neutrophil chemiluminescence Immunol. Lett. 53,45-50[CrossRef][Medline] Blood 93,341-349-stimulated production of O2- by human neutrophils adherent to fibrinogen Inflammation 23,167-178[CrossRef][Medline] and PI 3-kinase in TNF--mediated antiapoptotic signaling in the human neutrophil Am. J. Physiol. Cell Physiol. 283,C48-C57 in human neutrophils FEBS Lett. 349,338-342[CrossRef][Medline] in human neutrophils J. Immunol. 157,4641-4647[Abstract]This article has been cited by other articles:
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