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Originally published online as doi:10.1189/jlb.1004612 on April 14, 2005

Published online before print April 14, 2005
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(Journal of Leukocyte Biology. 2005;78:279-288.)
© 2005 by Society for Leukocyte Biology

Regulation of matrix metalloproteinase-9 release from IL-8-stimulated human neutrophils

Subhadeep Chakrabarti* and Kamala D. Patel*,{dagger},1

* Departments of Physiology and Biophysics and
{dagger} 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


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Matrix metalloproteinase-9 (MMP-9) is present in the tertiary granules of neutrophils and can be released following stimulation. We examined the signaling mechanisms that regulate interleukin-8 (IL-8)-mediated MMP-9 release from neutrophils. IL-8 activates neutrophils by interacting with two receptors: CXC chemokine receptor 1 (CXCR1) and CXCR2. Blocking CXCR1 had no effect on IL-8-mediated MMP-9 release, whereas blocking CXCR2 significantly reduced MMP-9 release. We also found that stimulating CXCR2 alone was sufficient to induce MMP-9 release. This process was independent of changes in the intracellular calcium concentration. Src-family kinases and protein kinase C (PKC) were involved in two mutually exclusive pathways regulating IL-8-mediated MMP-9 release. Inhibition of extracellular signal-regulated kinase (ERK)1/2 blocked IL-8-mediated MMP-9 release; however, inhibition of p38 mitogen-activated protein kinase had no effect on MMP-9 release. We found ERK1/2 was activated downstream of PKC, but not Src-family kinases, in this system. These data suggest that IL-8-induced MMP-9 release from neutrophils is mediated through CXCR2 and involves two distinct pathways, one involving PKC and ERK1/2 and the other involving Src-family kinases. Furthermore, our data show that the mechanisms that regulate MMP-9 release from tertiary granules are different from those that regulate primary granule release.

Key Words: signal transduction • tissue remodeling • chemokines • inflammation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Matrix metalloproteinases (MMPs) are a family of zinc-binding, calcium-dependent, proteolytic enzymes that typically degrade extracellular matrix (ECM) proteins. MMPs are crucial in normal tissue remodeling during embryogenesis, growth, and wound healing. However, excessive MMP activity is often associated with inflammatory conditions leading to destruction of normal tissue architecture. MMP-9 degrades a variety of ECM proteins such as type IV collagen, gelatin, vitronectin, and entactin [1 ]. Chemokines can also serve as substrates for MMP-9. For example, MMP-9 cleaves interleukin (IL)-8, resulting in a tenfold increase in IL-8 activity [2 ]. MMP-9 is involved in the pathogenesis of rheumatoid arthritis, septic arthritis [3 ], cryptogenic fibrosing alveolitis, and chronic obstructive pulmonary diseases (COPD) [4 , 5 ].

Neutrophils are the richest source of MMP-9 in the body. Unlike most cell types, which only express MMP-9 under proinflammatory stimuli, neutrophils constitutively produce MMP-9 and store it in their tertiary granules. Upon stimulation, neutrophils release MMP-9 rapidly from these granules. Several mediators, including formyl-Met-Leu-Phe (fMLP), tumor necrosis factor (TNF), and IL-8 [CXC chemokine ligand 8 (CXCL8)], have been shown to induce MMP-9 release from neutrophils, although the mechanisms regulating MMP-9 release remain poorly understood [6 ].

IL-8, a CXC chemokine, is a potent activator of neutrophils. Stimulation with IL-8 elicits an intracellular calcium flux and activates various signaling kinases in human neutrophils, leading to release of primary, secondary, and tertiary granule contents. IL-8 exerts its effects on neutrophils by acting at CXC chemokine receptor 1 (CXCR1) and CXCR2. Both of these receptors are highly expressed on neutrophil surfaces [7 ]. Although CXCR1 is specific for IL-8 and granulocyte chemotactic protein-2 (GCP-2; CXCL6), CXCR2 can bind to a number of CXC chemokines including growth-related oncogene {alpha} (GRO{alpha}; CXCL1), GROß (CXCL2), GRO{gamma} (CXCL3), and epithelial-derived neutrophil-activating factor-78 (ENA-78; CXCL5), in addition to IL-8 and GCP-2 [8 9 10 ].

In this study, we characterized the signaling mechanisms that regulate MMP-9 release following IL-8 stimulation of human neutrophils. We found that IL-8-mediated MMP-9 release was mediated largely through CXCR2 and was independent of the intracellular calcium flux. This is different from IL-8-mediated primary granule release, which depends on CXCR1 and CXCR2. We also determined roles for Src-family kinases, protein kinase C (PKC), and extracellular signal-regulated kinase (ERK)1/2 in this process.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
Hanks’ balanced salt solution (HBSS) and porcine gelatin were obtained from Sigma Chemical Co. (St. Louis, MO). Lymphoprep 1077 and Coomassie brilliant blue R-250 were from Invitrogen Life Technologies (Carlsbad, CA). Recombinant human IL-8 (72-peptide form) was purchased from Peprotech (Rocky Hill, NJ). Supersignal West Pico chemiluminescent substrate and immunopure immunoglobulin G (IgG) elution buffer were from Pierce Chemical Co. (Rockford, IL). PD 98059, SB 203580, SB 225002, PP2, and chelerythrine chloride were from Calbiochem (San Diego, CA). Fluo-3-acetoxymethyl ester (AM) and 1,2-bis(O-aminophenyl-ethane-ethane)-N,N,N',N'-tetraacetic acid (BAPTA)-AM were from Molecular Probes (Eugene, OR). Plasticware was from VWR, Canada (Toronto, Ontario). All other chemicals were from BDH Inc. (Toronto, Ontario, Canada).

Antibodies
Anti-CXCR1 (murine IgG2) and anti-CXCR2 (murine IgG1) antibodies were from BD Biosciences (Mississauga, Ontario, Canada), and control mouse IgG1 and IgG2 antibodies were from R & D Systems (Minneapolis, MN). Anti-ERK1/2 antibody was from Upstate USA (Charlottesville, VA). Antiphospho-ERK1/2 antibody was obtained from Cell Signaling Technologies (Beverly, MA). Horseradish peroxidase (HRP)-conjugated anti-rabbit IgG was from Cedarlane Laboratories Ltd. (Hornby, Ontario, Canada).

Neutrophil isolation and stimulation
Neutrophils were isolated from human blood by density centrifugation as described previously [11 ]. Neutrophils were routinely >95% pure, and eosinophils and occasional lymphocytes were the contaminating cells. Ethics approval was obtained from the University of Calgary Ethics Committee. Neutrophils were used at a concentration of 1 x 107 cells/ml in HBSS. The specified concentrations of IL-8 were added to 1 x 106 cells, and the cells were incubated at 37°C for the stated times. The cell-free supernatants were transferred to fresh microfuge tubes, and MMP-9 levels were determined by gelatin zymography as described below. In some experiments, cells were incubated with anti-CXCR1, anti-CXCR2, or control mouse antibodies for 15 min at 37°C before stimulation with IL-8. In other experiments, dimethyl sulfoxide (DMSO) alone or DMSO containing the specified concentrations of the inhibitor(s) was added to the cells for the specified duration of time before stimulation. In all cases, the minimum preincubation time that produced the maximal inhibition was used. The inhibitors used were PD 98059, SB 203580, SB 225002, PP2, chelerythrine chloride, and BAPTA. The optimum concentrations for PD 98059, SB 203580, BAPTA, and SB 225002 were determined by directly measuring the ability of the inhibitor to block its target pathway in neutrophils (see Fig. 4 and data not shown). The optimum concentrations of PP2 and chelerythrine chloride were determined by performing dose-response analysis on functions that are completely dependent on these pathways in neutrophils (data not shown).



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  		Figure 4. Chelation of intracellular calcium has no effect on IL-8-mediated MMP-9 release. Neutrophils were treated with the intracellular calcium chelator BAPTA-AM (10 µM) for 10 min. (A) Neutrophils were stimulated with 10–7 M IL-8 for 30 min at 37°C. The cell-free supernatants were collected, and MMP-9 release was quantified by gelatin zymography followed by densitometry, as described in Materials and Methods. Data in (A) represent the mean ± SEM of five experiments. (B) Neutrophils were loaded with the calcium-sensitive dye Fluo-3 (2 µM) for 30 min followed by treatment with BAPTA-AM (10 µM) for 10 min. Neutrophils were then stimulated with 10–7 M IL-8, and changes in intracellular calcium were measured by flow cytometry. Data in (B) represent three independent experiments.

 
Measuring MMP-9 release by gelatin zymography
Nonreducing Laemmli’s buffer was added to the cell-free neutrophil supernatants, and the proteins were separated on a 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) containing 1 mg/ml gelatin. Following electrophoresis, this gel was washed once in rinse buffer (2.5% Triton X-100, 50 mM Tris-HCl, 10 mM calcium chloride, 1 mM zinc chloride) for 15 min and then incubated in rinse buffer overnight at room temperature. The gel was then rinsed with deionized water for 5 min before overnight incubation in reaction buffer (50 mM Tris-HCl, 10 mM calcium chloride, 1 mM zinc chloride) at 37°C. Finally, the gel was stained with Coomassie brilliant blue R-250 for 1 h and then destained until clear bands were visible on a blue background. The clear bands indicated gelatinolytic activity was present at the expected molecular weight for MMP-9 and was identical to those reported by many other groups [1 ]. The identity of the gelatinolytic band as MMP-9 was determined by comigration with a MMP-9 standard and side-by-side dose-response curves using zymography and enzyme-linked immunosorbent assay (ELISA), as described previously for eosinophil MMP-9 [12 ]. Gels were scanned using a Fluor-S-MAX multi-imager (Bio-Rad, Hercules, CA), and the bands were quantified by densitometry using Quantity One software (Bio-Rad). Data are presented as fold increase above the unstimulated control. Data obtained by zymography were equivalent to data obtained using a MMP-9 ELISA kit (R&D Systems).

Static adhesion assay
Neutrophil adhesion was performed as described previously [13 , 14 ]. Briefly, tissue culture plates were coated with 0.2% gelatin for at least 1 h at 37°C. Plates were then washed with HBSS, and freshly isolated neutrophils (106/mL) were added to each well. Cells were stimulated with IL-8 and allowed to adhere for 20 min at 37°C. The nonadherent and loosely adherent neutrophils were removed by washing. Neutrophil adherence was determined by lysing the adherent neutrophils in hexadecyltrimethylammonium bromide (HTAB)-containing buffer and measuring peroxidase content as described [14 ]. Data were correlated with microscopic examination (data not shown). In some experiments, neutrophils were treated with antibodies for 15 min prior to addition to gelatin-coated plates.

Myeloperoxidase (MPO) release
Primary granule release was determined by measuring the release of MPO. Neutrophils were stimulated as described, and the cell-free supernatant was collected and transferred to a fresh microfuge tube. The cell-free supernatant was diluted in HTAB buffer, and MPO content/activity was measured using a colometric assay. Diluted sample (10 µl) was added to 100 µl one-step peroxidase substrate (Sigma Chemical Co.). After 5 min, the reaction was stopped by the addition of 100 µl phosphoric acid. Absorbance at 450 nm was then measured. MPO release was expressed as a percentage of the total MPO present in unstimulated, lysed neutrophils.

Western blotting
Triton X-100-soluble lysates were separated by SDS-PAGE, and proteins were transferred to polyvinylidene difluoride membranes. Membranes were blocked with 5% bovine serum albumin in Tris-buffered saline (TBS) containing 0.2% Tween 20 (TBST) for 1 h at room temperature and then probed with 1 µg/mL of a phospho-specific, anti-ERK1/2 antibody overnight at 4°C. Membranes were washed four times with TBST and probed with a HPR-conjugated secondary antibody for 1 h at room temperature. The membranes were washed with TBS, and Supersignal West Pico chemiluminescent substrate was then used to visualize the bands, which were detected using a Flour-S-Max multi-imager and quantified by densitometry using Quantity One software. The data were normalized by stripping the membranes with Immunopure IgG elution buffer, according to the manufacturer’s instructions, and then reprobing the membranes with 1 µg/mL of an antibody directed against total ERK1/2, which was then detected as described above.

Statistics
All experiments were performed between three and six times using different individuals as blood donors. All densitometry data are presented as mean ± SEM. Statistical differences among experimental groups were calculated by using a Student’s t-test (for two groups) or one-way ANOVA (for more than two groups). A P value of less than 0.05 was taken as statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
IL-8 induces the rapid release of MMP-9 from neutrophils
Previous studies have shown that IL-8 can induce the release of MMP-9 from human neutrophils after 30–60 min of stimulation [15 ]. Also, IL-8 has been shown to induce MMP-9 release from whole blood within 10 min of stimulation [6 ]. We first examined the minimum time required to induce significant release of IL-8 from freshly isolated human neutrophils, which were stimulated for 1, 5, 10, 15, or 30 min with 10–7 M IL-8. The cell-free supernatants were then collected and assayed for the presence of MMP-9 by gelatin zymography and ELISA. We found that IL-8 induced significant release of MMP-9 as early as 5 min (Fig. 1A and 1B ). MMP-9 release was maximal by 15–30 min (Fig. 1A and 1B , and data not shown); thus, we treated neutrophils with 10–7 M IL-8 for 30 min in our subsequent experiments.



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  		Figure 1. MMP-9 is released rapidly from IL-8-stimulated neutrophils, which in suspension, were treated with 10–7 M IL-8 for the specified times at 37°C. The cell-free supernatants were collected, and MMP-9 release was quantified by gelatin zymography followed by densitometry as described in Materials and Methods. (A) Data represent the mean ± SEM of five experiments, and a representative zymogram is shown (B). *, P < 0.05.

 
CXCR2, but not CXCR1, is required for IL-8-mediated MMP-9 release
IL-8 can bind to and signal through two different receptors: CXCR1 and CXCR2. Both are G-protein-coupled receptors expressed on neutrophil surfaces [8 ]. Receptor-specific blocking antibodies are available for CXCR1 and CXCR2. In addition, CXCR2 can be blocked with a nonpeptide receptor antagonist. Using these tools, we examined the role of each receptor in IL-8-mediated MMP-9 release. We found that blocking CXCR1 alone had no effect on IL-8-induced MMP-9 release (Fig. 2A and 2B ), whereas blocking CXCR2 with an antibody or a nonpeptide receptor antagonist inhibited most MMP-9 release (Fig. 2A 2B 2C) . Using anti-CXCR1 and anti-CXCR2 antibodies together inhibited MMP-9 release to the same extent as blocking with the anti-CXCR2 antibody alone, suggesting that CXCR1 was not required for MMP-9 release (Fig. 2A and 2B) . This was surprising, as most IL-8 responses in human neutrophils are dependent on CXCR1 and CXCR2. To address this, we examined IL-8-induced, primary granule release and IL-8-induced adhesion. Both of these activities require CXCR1 and CXCR2. We found that an anti-CXCR1 antibody partially blocked IL-8-induced static adhesion, an anti-CXCR2 antibody had no effect on static adhesion, and using both antibodies together decreased adhesion greater than using either antibody alone (Fig. 2D) . When we examined MPO release from primary granules, we found that blocking CXCR1 or CXCR2 alone attenuated MPO release, and blocking both receptors together completely inhibited MPO release (Fig. 2E) . These data validate the blocking abilities of our CXCR1 antibody and suggest that CXCR1 is not required for mediating MMP-9 release from IL-8-stimulated neutrophils. Together, these data suggest that CXCR2, but not CXCR1, regulates IL-8-mediated MMP-9 release from human neutrophils.



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  		Figure 2. CXCR2 is required for IL-8-mediated MMP-9 release. (A, B, D, and E) Neutrophils were pretreated with 10 µg/mL anti-CXCR1, 10 µg/mL anti-CXCR2, both, or isotype-matched control antibodies for 15 min prior to stimulation with 10–7 M IL-8 at 37°C. (C) Alternatively, neutrophils were pretreated with the specified concentrations of the CXCR2 inhibitor SB 225002 for 5 min prior to stimulation with 10–7 M IL-8 at 37°C. (A–C) The cell-free supernatants were collected and analyzed for MMP-9 content by gelatin zymography followed by densitometry of the zymogram or ELISA. (D) Following preincubation with antibodies, neutrophil adhesion in response to 10–7 M IL-8 was determined as described in Materials and Methods. (E) To measure primary granule release, the cell-free supernatants were collected and analyzed for MPO content as a measure of primary granule release, as described in Materials and Methods. Data are the mean ± SEM of five experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant.

 
Stimulation of CXCR2 alone is sufficient to induce MMP-9 release from neutrophils
We next determined if stimulation of CXCR2 alone was sufficient to induce MMP-9 release. Although CXCR1 can bind only to IL-8 and GCP-2, CXCR2, in addition to binding these chemokines, can also bind to GRO{alpha}, -ß, and -{gamma}, as well as ENA-78 and neutrophil-activating peptide-2 (NAP-2). As GRO{alpha} can be used as a selective agonist of CXCR2 [10 ], we stimulated neutrophils with GRO{alpha} to determine if selective activation of CXCR2 was sufficient to induce MMP-9 release. We found that GRO{alpha} induced a significant increase in MMP-9 release from neutrophils (Fig. 3 ). GRO{alpha}-induced MMP-9 release was completely abolished by an anti-CXCR2 blocking antibody, and the control IgG1 antibody had no effect (Fig. 3A and 3B) . A similar level of inhibition was obtained by blocking CXCR2 with SB 225002 (Fig. 3C) . These data validate our CXCR2 blocking reagents and further suggest that CXCR2 is necessary for IL-8-induced MMP-9 release and alone, is sufficient to induce MMP-9 release from neutrophils.



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  		Figure 3. GRO{alpha} acts at CXCR2 to induce MMP-9 release from neutrophils, which in suspension, were pretreated with (A and B) 10 µg/mL anti-CXCR2 or nonimmune control antibody for 15 min or with (C) the specified concentration of the CXCR2 antagonist SB 225002 for 5 min. Neutrophils were then stimulated for 45 min with 50 ng/ml GRO{alpha} at 37°C. The cell-free supernatants were collected, and MMP-9 release was quantified by (A and C) gelatin zymography followed by densitometry, as described in Materials and Methods, or (B) by ELISA. Data represent mean ± SEM of five experiments. **, P < 0.01.

 
Intracellular calcium influx is not necessary for IL-8-mediated MMP-9 release from neutrophils
IL-8 induces a robust influx of calcium ions in human neutrophils. This calcium influx leads to activation of calcium-sensitive pathways and regulates various functions in IL-8-stimulated neutrophils [16 ]. We blocked the effects of this intracellular calcium influx by treating neutrophils with the intracellular calcium chelator BAPTA-AM prior to stimulating with IL-8. Chelating intracellular calcium had no effect on IL-8-mediated MMP-9 release (Fig. 4A ). We used flow cytometry to measure intracellular calcium in neutrophils pretreated with BAPTA to verify that the concentrations of BAPTA used in these experiments were sufficient to quench the intracellular calcium flux induced by IL-8. We found that BAPTA-AM completely abolished the IL-8-induced intracellular calcium influx in our system (Fig. 4B) . These data suggest that increased intracellular calcium is not required for IL-8-induced MMP-9 release.

PKC and Src-family kinases participate in IL-8-mediated MMP-9 release from neutrophils
Stimulation of neutrophils by IL-8 activates a variety of protein kinases including isoforms of PKC [17 ]. We used chelerythrine chloride, a pan-specific PKC inhibitor, to address the role of PKC in IL-8-mediated MMP-9 release from neutrophils. We found that blocking PKC attenuated IL-8-induced MMP-9 release (Fig. 5A ). Chelerythrine chloride at identical concentrations is able to abolish TNF-induced MMP-9 release completely, indicating that chelerythrine is able to inhibit PKC completely in this system (data not shown). IL-8 binding also activates the Src-family kinases in neutrophils [18 ]. We used the Src-family kinase inhibitor PP2 to determine the role of this pathway in IL-8-mediated MMP-9 release. We found that PP2 produced only a partial inhibition in MMP-9 release in response to IL-8 (Fig. 5A) . Partial blockade of MMP-9 release was not a result of the incomplete inhibition of Src-family kinases by PP2, as longer incubation periods and higher concentrations of PP2 did not cause greater inhibition of MMP-9 release (data not shown). Inhibition of PKC and Src-family kinases by using chelerythrine chloride and PP2 together caused greater inhibition of MMP-9 release than either compound used alone (Fig. 5A) and reduced MMP-9 release to control levels. This suggests that mutually exclusive pathways, one dependent on PKC and the other involving Src-family kinases, may regulate IL-8-mediated MMP-9 release independently from neutrophils.



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  		Figure 5. Src-family kinases, PKC, and ERK1/2 mitogen-activated protein kinase (MAPK) participate in IL-8-mediated MMP-9 release from neutrophils, which (A) were pretreated for 5 min with the Src-family kinase inhibitor PP2 (10 µM), the PKC inhibitor chelerythrine chloride (20 µM), both inhibitors, or DMSO prior to stimulation with 10–7 M IL-8 for 30 min at 37°C. (B) Neutrophils were pretreated for 5 min with the MAPK kinase (MEK)-1,2-inhibitor (which blocks phosphorylation of ERK1/2) PD 98059 (10 µM), p38 MAPK inhibitor SB 203580 (1 µM), or DMSO alone before stimulation with 10–7 M IL-8 for 30 min at 37°C. The cell-free supernatants were collected, and MMP-9 release was quantified by gelatin zymography followed by densitometry, as described in Materials and Methods. Equivalent results were obtained with ELISA (data not shown). Data represent mean ± SEM of five experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

 
ERK1/2 is required for IL-8-mediated MMP-9 release from neutrophils
The MAPKs mediate diverse functions such as cell division and degranulation in many cell types [19 20 21 ]. Human neutrophils express ERK1/2, Jun N-terminal kinase, and p38 MAPKs [22 , 23 ], and ERK1/2 and p38 MAPKs are activated following stimulation with IL-8 [24 ]. Activation of ERK1/2 and p38 regulates various effects in neutrophils and other granulocytes including release of granule contents by degranulation [25 , 26 ]. We investigated effects of these MAPKs in IL-8-mediated MMP-9 release by using specific pharmacological inhibitors. PD 98059 and SB 203580 were used to inhibit the ERK1/2 and p38 MAPK pathways, respectively. We found that inhibition of the ERK1/2 pathway using PD 98059 attenuated MMP-9 release, whereas blocking the p38 pathway with SB 203580 had no effect on MMP-9 release (Fig. 5B) . We found that these concentrations of PD 98059 and SB 203580 completely blocked agonist-induced phosphorylation of ERK1/2 and MAPKAP-2, respectively, in neutrophils (data not shown).

PKC regulates IL-8-induced phosphorylation of ERK1/2 in neutrophils
We have found roles for PKC, Src-family kinases, and ERK1/2 in regulating MMP-9 release from IL-8-stimulated neutrophils. In various cells, Src-family kinases and PKC have been described as being upstream to the activation (by phosphorylation) of MAPKs including ERK1/2 [27 28 29 30 ]. We investigated whether PKC or Src-family kinases were upstream to ERK1/2 in our system. Neutrophils were pretreated with PKC and/or Src-family kinase inhibitors prior to stimulation with IL-8. Cells were lysed, and the lysates were analyzed by Western blotting using antibodies against phosphorylated and total ERK1/2. Data were expressed as the ratio of phosphorylated to total protein. We found that inhibition of PKC abolished the phosphorylation of ERK1/2 in response to IL-8 (Fig. 6A 6B 6C ). In contrast, blocking Src-family kinases had no effect on ERK1/2 phosphorylation (Fig. 6A 6B 6C) . Combining both inhibitors did not have any effect greater than that shown by the PKC inhibitor alone (Fig. 6A 6B 6C) . Based on these data, we next examined IL-8-induced MMP-9 release when the Src-family kinases and the ERK1/2 pathways were blocked. We found that inhibiting the Src-family kinases in combination with ERK1/2 led to a greater decrease in MMP-9 release than blocking either pathway alone in four out of five experiments (Fig. 6D) . These data suggest that at least two different pathways are involved in IL-8-mediated MMP-9 release from human neutrophils, one involving the PKC and ERK1/2 and the other involving Src-family kinases.



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  		Figure 6. PKC regulates IL-8-induced ERK1/2 phosphorylation in neutrophils, which were pretreated for 5 min with the Src-family kinase inhibitor PP2 (10 µM), the PKC inhibitor chelerythrine chloride (20 µM), both inhibitors, or DMSO alone prior to 1 min of stimulation with 10–7 M IL-8 at 37°C. The neutrophils were lysed, and Western blotting was performed with an antiphospho-ERK1/2 antibody. All gels were normalized by reprobing with an anti-ERK1/2 antibody, as described in Materials and Methods. Blots were analyzed by densitometry, and the ratio of (A) phospho-ERK1 (P-Erk1) to total ERK1 or (B) phospho-ERK2 to total ERK2 was calculated and expressed as fold increase over unstimulated control. (C) A representative blot is shown. (D) Neutrophils were pretreated for 5 min with the MEK-1,2-inhibitor PD 98059 (10 µM), Src-family kinase inhibitor PP2 (10 µM), both, or DMSO alone before stimulation with 10–7 M IL-8 for 30 min at 37°C. The cell-free supernatants were collected, and MMP-9 release was quantified by gelatin zymography followed by densitometry, as described in Materials and Methods. (A, B, and D) Data represent the mean ± SEM of five experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we investigated the mechanisms of IL-8-mediated MMP-9 release from human neutrophils. IL-8 mediates its biological responses by binding to the G-protein-coupled chemokine receptors CXCR1 and CXCR2 [31 32 33 ], which share 77% amino acid sequence homology [34 ]. They differ in their N-terminal extracellular domains, their C-terminal intracellular domains, and in their ligand specificity [10 , 35 ]. Neutrophils express both of these receptors on their surface [7 ]. Most IL-8-mediated effects on human neutrophils are mediated through CXCR1 and CXCR2 [31 32 33 ], and IL-8-induced priming of neutrophils for the respiratory burst has been found to be mediated exclusively through CXCR1 [36 , 37 ]. We now show that IL-8-mediated tertiary granule release is regulated differently than other IL-8-mediated responses such as primary granule release and adhesion. Our data show that CXCR2 and not CXCR1 is critical in IL-8-mediated MMP-9 release from neutrophils.

We found that inhibition of CXCR2 by blocking antibodies or the CXCR2-specific pharmacological inhibitor SB 225002 can significantly attenuate MMP-9 release from IL-8-stimulated neutrophils (Fig. 2) . Furthermore, we stimulated neutrophils with the CXCR2-specific ligand GRO{alpha} and found that GRO{alpha} induced significant release of MMP-9, which was completely dependent on activation of CXCR2 (Fig. 3) . Few IL-8-mediated neutrophil functions in humans are ascribed readily to CXCR2 in vitro; however, clinical data suggest that CXCR2 may have a role in several inflammatory diseases. Patients suffering from inflammatory lung diseases, such as cryptogenic fibrogenic alveolitis, express selective up-regulation of CXCR2 but not CXCR1 on their neutrophils [38 ]. Similarly, patients suffering exacerbations in COPD have much higher levels of CXCR2 on their neutrophils compared with those with more stable COPD or controls [39 ]. In both of these conditions, there is infiltration of neutrophils as well as tissue damage and remodeling that may be influenced by MMP-9. The up-regulation of CXCR2 may make the tissue neutrophils in these conditions more sensitive to the effects of IL-8, leading to greater MMP-9 release and more severe tissue injury. CXCR2-specific chemokines such as GRO{alpha}, NAP-2, and ENA-78 are also up-regulated in inflammatory disorders including arthritis, COPD, and pulmonary fibrosis [40 41 42 ]. All of these are conditions characterized by neutrophil infiltration and tissue remodeling involving MMP-9. CXCR2 may play a previously unappreciated role in regulating MMP-9 release from the neutrophils in these conditions; hence, targeting CXCR2 could be potentially beneficial in these inflammatory conditions.

We also demonstrated that IL-8-mediated MMP-9 release was calcium-independent. IL-8 stimulation of neutrophils elicits a rapid and transient increase in intracellular calcium. This transient influx of calcium ions activates numerous signaling cascades that play major roles in neutrophil functions. Previous data have shown that agonist-mediated primary and secondary granule release and superoxide generation in neutrophils can be regulated by increases in intracellular calcium [43 44 45 46 ]. Similarly, increasing intracellular calcium with an ionophore elicits primary, secondary, and tertiary granule release from neutrophils [47 ]. Our findings show that increased intracellular calcium is not required for IL-8-mediated tertiary granule release. We found that chelation of intracellular calcium by BAPTA had no effect on MMP-9 release (Fig. 4A) , although it was sufficient to abolish the calcium flux generated by IL-8 stimulation (Fig. 4B) .

Others have also shown that neutrophil granule release can be regulated differentially. Recently, Abdel-Latif et al. [48 ] demonstrate that fMLP-stimulated degranulation of primary granules is dependent on Rac2, whereas neither secondary nor tertiary granule release required Rac2. Similarly, neutrophils from an individual with a mutation in Rac2 show defective primary granule release but normal secondary granule release in response to the same stimuli [49 ]. Given these findings, we explored some of the signaling pathway that regulates tertiary granule release.

Of the many signaling pathways activated in IL-8-stimulated neutrophils, we found that Src-family kinases, ERK1/2, MAPK, and PKC, participated in MMP-9 release. However, blockade of any pathway alone produced only a partial decrease in MMP-9 release, and blocking these together had an additive effect (Figs. 5A and 6D) , suggesting existence of mutually exclusive pathways leading to degranulation of tertiary granules. We showed that ERK1/2 phosphorylation was dependent on PKC; however, blocking Src-family kinases in combination with PKC led to a greater decrease in MMP-9 release than blocking Src-family kinases in combination with the ERK1/2 pathway. This suggests that additional targets of PKC are likely involved in MMP-9 release. Possible targets of PKC might be the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. SNARE proteins regulate vesicle fusion in various cell types and have been shown to mediate secondary and tertiary granule release in neutrophils [50 , 51 ]. A role for PKC in phosphorylation (and hence, activation) of SNARE proteins in platelets has been shown [52 , 53 ]. The involvement of the Src-family kinases is likely a result of their signaling downstream of ß2-integrins [54 , 55 ]. Once neutrophils become adherent, outside-in signaling occurs through the integrins and Src-family kinases [56 ]; thus, the Src-family kinases may be representative of the integrin-dependent component of MMP-9 release, and PKC may be participating in several integrin-independent components.

These data show that the mechanisms that regulate tertiary granule release from IL-8-stimulated neutrophils are different from those that regulate other neutrophil responses, in that MMP-9 release was dependent on CXCR2 alone. As neutrophil-derived MMP-9 has been implicated in many inflammatory disorders, identifying mechanisms that disrupt MMP-9 release from tertiary granules while leaving other neutrophil functions intact may offer unique opportunities for therapeutic intervention in these diseases.


   ACKNOWLEDGEMENTS
 
The Alberta Lung Association and the Canadian Institutes of Health Research (CIHR) funded this work. K. D. P. is a Canada Research Chair and an Alberta Heritage Foundation for Medical Research Senior Scholar. We thank Evelyn Lailey for her excellent technical assistance, Dr. Victoria Stubbs for her critical reading of this manuscript, Ching Yee Fung for her assistance with key experiments, and the Canadian Institutes of Health Research Group Grant in Leukocyte Trafficking for technical assistance.

Received October 22, 2004; revised February 8, 2005; accepted February 23, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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