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J Am Soc Nephrol 13:2840-2849, 2002
© 2002 American Society of Nephrology

Novel Effects of Neutrophil-Derived Proteinase 3 and Elastase on the Vascular Endothelium Involve In Vivo Cleavage of NF-{kappa}B and Proapoptotic Changes in JNK, ERK, and p38 MAPK Signaling Pathways

Gloria A. Preston*,{dagger}, Christopher S. Zarella*,{dagger}, William F. Pendergraft, III{ddagger}, Earl H. Rudolph*,{dagger}, Jia Jin Yang*,{dagger}, Stephen B. Sekura*,{dagger}, J. Charles Jennette*,{ddagger} and Ronald J. Falk*,{dagger},{ddagger}

*Department of Medicine, {dagger}Division of Nephrology and Hypertension, and {ddagger}Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina.

Correspondence to Dr. Gloria Preston, CB# 7155, 346 MacNider Bldg., Division of Nephrology and Hypertension, Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7155. Phone: 919-966-2570; Fax: 919-966-4251;E-mail: Gloria_Preston{at}med.unc.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
ABSTRACT. Leukocyte-derived proteases have long been considered simply degradative. However, emerging data raise possibilities of a complex and specific biologic role for these proteases in substrate processing and in signaling pathways within cells. This study reports that the release of neutrophilic and monocytic proteases, such as proteinase 3 (PR3) and human neutrophil elastase (HNE), can result in their entry into endothelial cells coincident with the activation of proapoptotic-signaling events through ERK, JNK, and p38 MAPK. Inhibition of JNK blocked PR3-induced apoptosis, and inhibition of p38 MAPK blocked PR3- and HNE-induced apoptosis, indicating that these pathways are required for activation of apoptosis. It is here shown that protease entry results in direct cleavage of p65 NF-{kappa}B in the N-terminal region by PR3 and in the C-terminal region by HNE. This cleavage results in diminished transcriptional activity by NF-{kappa}B as demonstrated by diminished levels of TNF-{alpha}–induced IL-8 message in the presence of PR3 or HNE. Inhibition of caspases did not block the cleavage of p65 NF-{kappa}B, and sequence analysis showed that the PR3 and HNE cleavage sites are unique with respect to reported caspase sites. The data demonstrate that PR3 and HNE have specific, fundamental roles in endothelial responses during inflammation. Upon entry, they can usurp the cell’s control of its own fate by directly intervening into caspase cascades. This provides a unique mechanism of crosstalk between leukocytes and endothelial cells at sites of inflammation that impacts both cytokine networks and cell viability.


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Acute inflammation involves the accumulation of leukocytes, especially neutrophils, at focal sites in tissue. Evolutionarily, neutrophil functions are thought to have evolved to phagocytize microorganisms and cellular debris. Vacuoles in the cytoplasm of neutrophils called azurophilic granules contain potent enzymes at millimolar concentrations, including the serine proteases studied here, proteinase 3 (PR3) and human neutrophil elastase (HNE), which are capable of digesting a variety of microbial substrates (1). These same enzymes that are beneficial when active within phagosomes are harmful to tissues when released extracellularly. For instance, PR3 and HNE are capable of digesting structural matrix proteins (2). Although protease inhibitors exist in plasma and in tissue fluid to offer some defense, protease concentrations can overwhelm the inhibitors and exist as unbound, active proteases (3). Individuals deficient in natural protease inhibitors exhibit excessive liver and lung damage (4), and even in the case of deficiency due to heterozygosity for {alpha}1-antitrypsin, associated systemic vasculitis was reported (5). Of equal importance to acute inflammation are the contributions of these proteases to chronic inflammation, as monocytes are a source of PR3 and HNE (6).

A primary aim of our research is to determine the mechanistic link between leukocyte granule proteases and endothelial cell injury. Until recently, leukocyte granule proteases were assumed to be simply degradative. This view has been challenged by data showing that particular proteases can proficiently process biologic substrates to mature forms. For example, PR3 has common functional characteristics with IL-1{beta}–converting enzyme (ICE, caspase-1) and the TNF-{alpha}–converting enzyme (TACE) in the processing of the inactive cytokine precursors to their active forms (7,8). These physiologic functions of PR3 are distinct from those of HNE, which shares many enzymatic characteristics with PR3; HNE cleaves TNF-{alpha} into fragments, rendering it inactive (9). Other PR3 substrates include immunoglobulins, IL-2 receptor, TGF-{beta}, and IL-8 (3,8,10,11).

With the discovery that a subset of vascular diseases is associated with anti-PR3 autoantibodies, termed anti-neutrophil cytoplasmic autoantibodies (ANCA), PR3-related effects on the endothelium have been of particular interest (12,13). ANCA are hypothesized to bind to PR3 expressed on the surface of neutrophils and monocytes, causing an exacerbation of the degranulation process, resulting in excessive release of proteases, thus contributing to endothelial injury and vasculitis (14,15). Do excessive levels of granule proteases explain vasculitis in ANCA patients? The concept that neutrophils and monocytes contain proteases that have proapoptotic signaling capabilities on vascular endothelia was first described by us (16). We found that apoptosis was associated with the specific internalization of PR3 by human vascular endothelial cells, whereas human lung epithelial cells did not internalize PR3 and did not undergo apoptosis (17). In this case, PR3 was proteolytically inactive, and an apoptotic function was mapped to the C-terminal domain. Thus, indications are that PR3, also known as myeloblastin, has distinct and varied functions that may or may not require proteolytic activity (18,19).

Here we focus attention on a particularly intriguing aspect of PR3 and HNE function, the mechanisms through which they activate proapoptotic signaling pathways. While searching the literature, it came to our attention that two groups had reported that endothelial cell apoptosis requires the cleavage of the transcription factor NF-{kappa}B by endogenous caspases (20,21). NF-{kappa}B is an important regulator of gene expression during immune and inflammatory responses that can protect against apoptosis (22). PR3 function has been shown to overlap with that of caspase-1 (7); we therefore posed the hypothesis that at least part of the underlying mechanism of PR3- and/or HNE-induced apoptosis involves the cleavage of NF-{kappa}B by these serine proteases. We used human umbilical vein endothelial cells (HUVEC) to examine this possibility and also to study cell signaling pathways responsive to PR3 and/or HNE stimulation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Culture
Pooled HUVEC purchased from Clonetics (San Diego, CA) were cultured for 4 to 8 doublings, or 2 to 3 wk, in EBM plus EGM BulletKit supplements (Clonetics). EA.hy926 cells were developed and donated by Dr. C. J. Edgell (Department of Pathology, University of North Carolina at Chapel Hill, NC). EA.hy926 cells were cultured in DMEM with high glucose (Invitrogen, Carlsbad, CA) with 1% penicillin/streptomycin (Invitrogen) and 10% FBS (Invitrogen).

Electron Microscopy of PR3 Internalization
Active PR3 (109 U/mg protein) was a gift from Dr. J. Wieslander (Wieslab AB, Lund, Sweden). For analysis of internalization of active PR3, HUVEC were incubated with PR3 (10 µg/mL) in medium without FBS or supplements for 10 min at 37°C and analyzed by immunogold labeling of PR3 and electron microscopy as described previously (17).

Protease and Inhibitor Treatment
Triton X-100 was removed from proteolytically active PR3 using Extracti-Gel D AffinityPak detergent-removing column (Pierce, Rockford, IL). Monolayers were washed and treated with PR3 (5 µg/mL) or HNE (Calbiochem, La Jolla, CA) (5 µg/mL) in EBM. Diisopropyl fluorophosphates (DFP; Sigma, St. Louis, MO) was added to treatment medium (5 min) to irreversibly inhibit PR3 and HNE activity, and Western analyses were conducted as described (23). Active p-JNK, p-ERK, and p-p38 MAPK were analyzed using phospho-specific Abs. Immunoblots were reprobed with Abs to detect both active and inactive forms of the proteins. For TaqMan PCR analysis of IL-8 transcripts, {alpha}1-antitrypsin was added to PR3 (30 min, RT) before addition to cell cultures (10 µg/mL). When indicated, YVAD-fmk (Calbiochem) (100 nM) was added for 2 h before and during protease treatment. Effectiveness was assessed by flow cytometric analysis of active caspase-3–positive cells after 8 h in serum-free medium with and without the inhibitor. SB203580 was used to inhibit p38 MAPK (0.75 µM) and JNK 1 and 2 (10 µM) (24) (LC Laboratories, Woburn, MA) 1 h before and during protease treatment. To inhibit PI3K, wortmanin (100 nM) was added to cell monolayers 10 min before and during PR3 or HNE treatment.

Immunofluorescence Staining
HUVEC were plated on 20-mm coverslips at 7.5 x 104 cells/slip and incubated at 37°C and 5% CO2 for 48 h before PR3 treatment (5 µg/mL). Cells were fixed with cold paraformaldehyde/PBS (2%; 30 min), permeabilized with cold methanol:acetic acid (3:1; 30 min), blocked with normal goat serum (3%; 1 h), incubated with mouse anti-NF-{kappa}B N-terminal–specific Ab (Santa Cruz Biotechnology) (3 h; RT), incubated with Alexa Fluor 488-conjugated anti-mouse IgG(H+L) (Molecular Probes, Eugene, OR) (1 h; RT), and mounted with standard anti-fade medium. All washes were with PBS.

In Vitro Cleavage of NF-{kappa}B
Human NF-{kappa}B protein was obtained by immunopurification from a human endothelial-like cell line, EA.hy926, using an {alpha}-C-terminal NF-{kappa}B–specific Ab, or immunoprecipitated in complex with I-{kappa}B{alpha}, using an {alpha}-I-{kappa}B{alpha} Ab in RIPA lysis buffer (150 mM NaCl, 1.0% NP-40, 0.5% DOC, 0.1% SDS, 50 mM Tris at pH 8.0). Peptide-competed {alpha}-NF-{kappa}B Ab was added as a control. Immune complexes were collected on protein A/G agarose beads (Pierce), washed, and resuspended in 30 µl of PBS. PR3 or HNE was added to the precipitated NF-{kappa}B complexes to a final concentration of 1 µg/mL (10 min). The reaction was stopped with hot Laemmli buffer. Sequencing of the N-terminal amino acids of the cleavage products was accomplished by Edman degradation using a Procise 492 Protein Sequencer (Applied Biosystems, Foster City, CA) at the University of North Carolina Core Protein Sequencing Facility by Dr. David Klapper. The PR3 cleavage site was determined using recombinant p65 NF-{kappa}B (residues 1 to 325) provided by Dr. Gourisankar Ghosh, Department of Chemistry & Biochemistry, UCSD, California. The HNE cleavage site was determined using immunopurified NF-{kappa}B.

Antibodies
Antibodies used were ERK 1 (K-23)-G, JNK1 (FL), I-{kappa}B{alpha} (C-21), NF-{kappa}B p65 (C-20)-G, NF-{kappa}B p65 (F-6), p38 MAPK (N-20)-G, p-JNK (G7), p-p38 MAPK (D-8) (Santa Cruz Biotechnology), phospho-p44/42 MAP kinase, and activated caspase-3 (Cell Signaling Technologies, Beverly, MA). Detection was accomplished with secondary Abs conjugated to horseradish peroxidase (Chemicon, Temecula, CA) using West Pico chemiluminescence substrate (Pierce).

Assessment of Apoptosis
Detached and attached cells were combined, fixed, and stained using the Fix and Perm kit as directed (Caltag, Burlingame, CA). Apoptotic cells were tagged using {alpha}-activated caspase-3 Ab plus FITC-conjugated goat {alpha}-rabbit secondary Ab (Dako, Carpinteria, CA), and analyzed by FACScan (Becton Dickinson Immunocytometry Systems, San Jose, CA) linked to a Cicero/Cyclops system (Cytomation, Fort Collins, Colorado). Calculations to determine rescue from apoptosis after treatment with SB203580: % rescue = [(uninhibited - inhibited) ÷ uninhibited] x 100.

Quantitative Assessment of IL-8 mRNA Levels by TaqMan PCR
Cells were exposed to PR3 or HNE (30 min) in serum-free medium before TNF-{alpha} (5 nM). RNA was isolated at 30 min and 4 h in RNA STAT-60 (Tel-Test, Friendswood, TX) as recommended. DNA was removed using RNeasy Mini Kit (Qiagen, Valencia, CA). Reverse transcription and PCR assays were performed in duplicate on a Perkin-Elmer 7700 TaqMan machine using TaqMan EZ-RT PCR kit (Applied Biosystems). Extension, annealing and amplification temperatures were used as detailed by Perkin-Elmer TaqMan. Primers and probe for IL-8 were a generous gift from Dr. Rob Silbajoris (EPA, Chapel Hill, NC). Primers and probe for cyclophilin A were purchased from Applied Biosystems. Threshold cycle (Ct) values for IL-8 genes were normalized to cyclophilin A cycle threshold values. Relative quantitation was determined by standard 2(-{Delta}{Delta}Ct) calculations setting the 0-h, nontreated control to a value of 1.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Proteolytically Active PR3 Internalization
We previously reported that uptake of inactive PR3 by HUVEC is associated with apoptosis (17). We here show that active PR3 is also capable of entry into endothelial cells. Immunogold electron micrographs show that uptake of PR3 involves internalization by endosomal-like vesicles within 10 min after exposure (Figure 1). Some staining was detected within the cytoplasm.



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  		Figure 1. Proteolytically active proteinase 3 (PR3) is internalized by human umbilical vein endothelial cells (HUVEC). Shown is a micrograph of immunogold-labeled PR3 with positive staining in endosome-like vesicles (thick arrows) and in the cytoplasm (thin arrows). Inset shows enlargement of an endosomal-like vesicle. Magnifications: x20,000 for main panel; x31,500 for inset.

 
PR3- and HNE-Induced Apoptosis Involves NF-{kappa}B Cleavage
We hypothesized that the mechanism underlying PR3- and/or HNE-induced apoptosis involves the cleavage of transcription factor NF-{kappa}B. We tested whether protease treatment of HUVEC resulted in p65 NF-{kappa}B cleavage. PR3 exposure resulted in a cleavage fragment of approximately 56 kD produced by the removal of N-terminal amino acids, as determined by loss of epitope recognition by a N-terminal–specific Ab (Figure 2A). Of particular importance, only when time for internalization of PR3 was allowed, 30 s (no internalization) versus 30 min (internalization), did we see the cleavage product (Figure 2B). We then asked if NF-{kappa}B localization was affected after PR3 exposure. As detected by immunofluorescence, positive staining was observed in the cytoplasm of mock-treated cells using an anti-NF-{kappa}B Ab (Figure 2C). In contrast, PR3 treatment produced some nuclear staining, with particulates in the cytoplasm that could be cleaved protein. PR3-induced p65 NF-{kappa}B cleavage was specific and distinct from HNE-induced cleavage, which resulted in the removal of the C-terminus generating an approximately 48-kD fragment (Figure 2D). We confirmed the activation of apoptosis in the protease-treated cells after 6 h as compared with mock-treated controls (Figure 2E).



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  		Figure 2. In vivo cleavage of p65 NF-{kappa}B after protease exposure. (A) Western blot analysis of HUVEC treated with PR3 or human neutrophil elastase (HNE) revealed a NF-{kappa}B N-terminally truncated fragment (approximately 56 kD) in the PR3-treated groups, as detected by an {alpha}-NF-{kappa}B C-terminus-specific Ab. (B) NF-{kappa}B cleavage occurs intracellularly and not during the cell lysis procedure. The cleavage product was seen in PR3-treated cells (P) only when time for internalization (30 min) was permitted, whereas the 30-s time point showed no cleavage product. (C) Immunofluorescence analysis of p65 NF-{kappa}B using a C-terminal–specific {alpha}-NF-{kappa}B Ab. Mock-treated HUVEC show cytoplasmic staining (left), wheras PR3 treatment results in some nuclear staining with some particulate staining in the cytoplasm (right) (magnification, x40). (D) Reprobe of blot with an N-terminus–specific {alpha}-NF-{kappa}B Ab revealed a C-terminally truncated product (approximately 48 kD) in the HNE-treated group. (E) NF-{kappa}B cleavage is associated with PR3- and HNE-induced apoptosis, as detected by flow cytometric analysis of activated caspase-3–positive cells.

 
PR3 and HNE Cleave NF-{kappa}B In Vitro
Further clarification was need as to whether the observed p65 NF-{kappa}B cleavage was a direct or indirect effect of PR3 and HNE activities. To test this, we used purified PR3, HNE, and p65 NF-{kappa}B to determine direct cleavage in vitro. p65 NF-{kappa}B was immunopurified from nontreated logarithmically growing human cells by using an {alpha}-p65 NF-{kappa}B–specific Ab (Figure 3A). To ensure specificity and for comparison, p65 NF-{kappa}B was also purified using {alpha}-I-{kappa}B{alpha} Ab (Figure 3, B and C). Addition of PR3 to p65 NF-{kappa}B in vitro resulted in cleavage and detection of an approximately 56-kD fragment with the C-terminal–specific {alpha}-NF-{kappa}B Ab, as was seen in vivo (Figure 3, A and B). Protein sequence analysis of the fragment mapped the cleavage site to the VGKDC95-R96 motif of p65 NF-{kappa}B, two amino acids upstream of the reported caspase-3 site (21). This site would be available even when p65 NF-{kappa}B is complexed with p50 NF-{kappa}B and I{kappa}B, according to the published crystal structure (25) (Figure 3D). HNE cleaved NF-{kappa}B in the C-terminal domain, generating an approximately 48-kD fragment detected by the N-terminal-specific {alpha}-NF-{kappa}B Ab (Figure 3C). The small C-terminal HNE cleavage product of approximately 22 kD was detected using the C-terminal-specific {alpha}-NF-{kappa}B Ab (Figure 3, A and B). Protein sequence analysis of the approximately 22-kD fragment mapped HNE cleavage to the SRSSA339-S340 motif of p65 NF-{kappa}B. These data confirm that PR3 and HNE cleave p65 NF-{kappa}B directly. For comparison, a schematic representation of these cleavage sites, as well as those reported caspases-3 and -6, are shown in Figure 3D (20,21).



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  		Figure 3. Purified PR3 and HNE cleave NF-{kappa}B in vitro. NF-{kappa}B was immunopurified (IP) using (A) {alpha}-NF-{kappa}B Ab or (B and C) {alpha}-I-{kappa}B{alpha} Ab. Antigenic peptide-competed {alpha}-NF-{kappa}B Ab, labeled C is shown in lane 1. (A) PR3 (P) cleavage resulted in an approximately 56-kD N-terminally-truncated fragment, as detected with a C-terminus–specific {alpha}-NF-{kappa}B Ab versus untreated (U). HNE (E) cleavage resulted in an approximately 22-kD N-terminally-truncated fragment. The bands at 55 kD and 25 kD are {alpha}-goat Ab heavy and light chain cross-reactivity. (B) Fragmentation of NF-{kappa}B by PR3 was shown also when NF-{kappa}B was purified using {alpha}-I-{kappa}B{alpha} Ab. (C) Western immunoblots (IB) were reprobed with {alpha}-NF-{kappa}B N-terminus–specific Ab. The remaining N-terminal 48-kD fragment of NF-{kappa}B generated by HNE was visualized. (D) The PR3 cleavage site on p65 NF-{kappa}B is indicated (closed arrow) in a model of the published crystal structure (28), thus indicating that this site is accessible when p65 NF-{kappa}B (green) is in complex with p50 NF-{kappa}B (purple) and I{kappa}B (red). The caspase cleavage site is indicated with an open arrow. (E) Schematic comparisons of fragments generated by PR3 and HNE, as determined by protein sequence analyses, compared to those reported for caspase-3 and caspase-6 (23–24).

 
PR3- and HNE-Mediated Cleavage of NF-{kappa}B Are Caspase-Independent
Although our in vitro data confirmed that PR3 and HNE could directly cleave p65 NF-{kappa}B, for thoroughness, we examined whether intracellular cleavage could occur in the presence of the broad-spectrum caspase inhibitor YVAD-fmk. This would conclusively determine whether caspases are contributing to p65 NF-{kappa}B cleavage by PR3 and HNE in vivo. We first proved that this caspase inhibitor is effective in HUVEC by showing that it blocks caspase-3 activation, as measured by flow cytometry (Figure 4A). However, we found that caspase inhibition did not block generation of the respective NF-{kappa}B fragments (Figure 4, B and C), confirming that both PR3- and HNE-induced cleavage is direct and caspase-independent.



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  		Figure 4. NF-{kappa}B cleavage by PR3 and HNE is caspase-independent. (A) Flow cytometric analysis determined that activation of caspase-3 is completely eliminated in the presence of the YVAD-fmk inhibitor. (B and C) Western blot analyses of HUVEC incubated with or without caspase inhibitor 2 h before and during protease treatments showed that in vivo NF-{kappa}B cleavage by PR3 (B) and HNE (C) occurs in the presence of caspase inhibitor.

 
NF-{kappa}B Inactivation Alone Is Not Sufficient to Induce Apoptosis in Endothelial Cells
Is inactivation of p65 NF-{kappa}B sufficient for PR3 or HNE to cause apoptosis, or is the mechanism more complex, requiring additional cellular signaling? If inactivation alone is sufficient, then inhibition of p65 NF-{kappa}B function by the exogenously expressed super repressor I{kappa}B{alpha}-SR should cause an apoptotic event. To test this, we infected HUVEC with the dominant negative inhibitor of NF-{kappa}B, I{kappa}B{alpha}-SR (26), or with the {beta}-galactosidase gene as a control. Percent death 24 h post-infection with I{kappa}B{alpha}-SR was 7.5%, compared with 5.6% in cells infected with {beta}-galactosidase. At 48 h, I-{kappa}B{alpha}-SR-expressing cells showed no increase above background at 6.3% death. Blocking NF-{kappa}B function by expression of I-{kappa}B{alpha}-SR did not induce apoptosis in HUVEC, indicating that additional events are required for apoptosis to occur.

PR3 and HNE Affect Signal Transduction Pathways
It is generally accepted that cleavage of NF-{kappa}B is required but not sufficient for successful apoptosis; therefore, additional signaling would be necessary. On the basis of reports that sustained activation of c-Jun N-terminal kinase (JNK) contributes to endothelial cell apoptosis (27,28), we reasoned that PR3 and/or HNE may be activating the JNK pathways as part of the mechanism to induce apoptosis. We compared the activation status of three stress-related pathways, including JNK, extracellular signal-regulated protein kinase (ERK), and p38 mitogen-activated protein kinase (p38 MAPK) (Figure 5A), using phospho-specific Abs. The major differences observed after protease treatment were in the JNK and ERK pathways. Specifically, PR3 treatment caused an increase in JNK 2, whereas HNE caused a decrease in JNK 1 compared with mock-treated control cells (Figure 5B). Both proteases completely downregulated ERK 1 and 2; in controls, the only change was a slight decrease in ERK 1. Moreover, active ERK in control cells caused an increase in the level of c-Fos, an ERK-responsive gene. The only remarkable change in p38 MAPK activity was in the HNE-treated cells, which showed a slight decrease. We wanted to examine further the mechanism involved in PR3 signaling that would result in sustained JNK 2 activation. Because PI3K activity is linked to JNK and/or p38 MAPK activity (29), we asked if we could block PR3-induced changes in JNK 2 activity by inhibiting PI3K with the drug wortmanin. The data show that inhibition of PI3K blocked PR3-induced JNK 2 activation (Figure 5C).



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  		Figure 5. Stress-related signal transduction pathways are required for PR3- and HNE-induced apoptosis. (A) Schematic of kinases analyzed for activity (circles) and the major pathways associated with these kinases (B). Phosphorylated/active p-JNK, p-ERK, and p-p38 MAPK were analyzed using phospho-specific Abs. Immunoblots were reprobed with Abs to detect both active and inactive forms. (C) Western blot analyses of the effects of the PI3K inhibitor, wortmanin (100 nM), on protease-induced JNK phosphorylation/activation using a phospho-specific Ab. Wortmanin inhibits PR3-induced JNK 2 phosphorylation/activation. In summary, PR3 blocks the downregulation of JNK 2 through a PI3K-dependent mechanism, whereas JNK 1 and p38 MAPK remain active and ERK is downregulated. In comparison, HNE signaling involves the activity of p38 MAPK, whereas ERK and JNK 1 are downregulated. (D) Cells are rescued from apoptosis if signaling pathways are inhibited. The percent rescue from apoptosis, due to inhibition of p38 MAPK (0.75 µM) and JNK 1 and 2 (10 µM) with SB203580.

 
To test whether p38 MAPK and JNK activation are functionally coupled to the downstream signals of apoptosis induced by these proteases, the drug SB203580 was used to inhibit p38 MAPK (0.75 µM) and JNK 1 and 2 (10 µM) (24). Inhibition of p38 MAPK resulted in a 75% rescue from PR3-induced apoptosis and a 57% rescue from HNE-induced apoptosis (Figure 5D). Inhibition of JNK resulted in a 65% rescue from PR3-induced apoptosis; however, only 14% rescue was observed in the HNE-treated group. The data indicate that unique signaling pathways are functionally coupled to the apoptotic effect of each of these proteases and that JNK activation is required for PR3-induced apoptosis.

Proposed Model of PR3-Induced Effects on NF-{kappa}B Function
The primary purpose of this work is to explore PR3-induced effects to delineate mechanisms of endothelial damage incurred at sites of excessive neutrophil and monocyte degranulation. Because NF-{kappa}B is an important regulator of gene expression during immune and inflammatory responses (30,31), we asked what the consequences of NF-{kappa}B cleavage by PR3 would be in the setting of an inflammatory response. Does NF-{kappa}B cleavage by PR3 result in inactivation, as occurs after caspase cleavage (20)? Our working model is shown in Figure 6. During inflammation, cytokines, such as TNF-{alpha}, trigger the activation of NF-{kappa}B, which leads to transcription of chemokines, IL-8 for example (32). Increased IL-8 would result in neutrophil infiltration and eventual release of granule constituents, including PR3. Endothelial cells then internalize PR3, NF-{kappa}B is cleaved, and IL-8 production is decreased. Neutrophil infiltration would then decrease favoring the resolution of inflammation. This scenario provides a feedback mechanism to block signals for perpetuation of the response, thus favoring the resolution of inflammation. To test this model, we monitored the influence of PR3- on TNF-{alpha}–induced changes in IL-8 mRNA levels by TaqMan PCR. In response to TNF-{alpha}, IL-8 levels in HUVEC steadily increased over a 4-h period, as compared with mock-treated controls (Table 1). The net increase was 2.2-fold between the 30 min and 4 h time points. Addition of PR3 to TNF-{alpha}–treated cells reduced the levels to 1.3-fold. This reduction was due to the proteolytic activity of PR3, because addition of {alpha}-1-anti-trypsin restored IL-8 levels to 1.8-fold. HNE effectively reduced TNF-{alpha}–induced IL-8 levels by 50% (Table 1). This trend was observed consistently with both PR3 and HNE in eight different TaqMan reactions representative of three separate experiments.



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  		Figure 6. Working model of PR3 effects at sites of inflammation. Once elevated, cytokines (e.g., TNF-{alpha}) induce NF-{kappa}B activation, leading to increased transcription of chemokines (e.g., IL-8) followed by increased secretion of protein. Neutrophils migrate to the site and release granule constituents, including PR3. When a threshold concentration is reached, PR3 binds and is internalized by the endothelial cell. PR3 uptake results in NF-{kappa}B cleavage and a decrease in IL-8 production. Eventually, neutrophil infiltration would subside, and the cell that internalized PR3 would be eliminated from the site by apoptosis.

 

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  		Table 1. Fold-change in IL-8 levels as detected by TaqMan PCR
 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Neutrophil accumulation in tissue and loss of vessel wall integrity has long been implicated in the development of diverse cardiovascular and inflammatory disorders. Accordingly, considerable interest has been directed toward understanding the mechanistic link between secreted neutrophil granule proteases and endothelial cell damage. Here we provide evidence that the serine proteases PR3 and HNE, released at sites of neutrophil and/or monocyte infiltration, can enter an endothelial cell and, in the process, push the balance of cellular signaling pathways toward a proapoptotic event. Concomitantly, PR3 and HNE recognize p65 NF-{kappa}B as a natural substrate, thus debilitating its transcriptional activity. PR3 removes the DNA-binding domain in the amino terminus of p65 NF-{kappa}B by cleavage at a site in the vicinity of a caspase-3 cleavage site, leaving an N-terminally truncated form that contains two potent activation domains and the cytosolic/nuclear localization domain. We think that cleavage releases NF-{kappa}B from retention in the cytoplasm, allowing nuclear localization of the dysfunctional fragment, although the mechanism through which this might occur is yet to be determined. Nuclear localization of the fragment would be anticipated because the nuclear localization site remains intact, and indeed p65 NF-{kappa}B fragments have been implicated as having dominant-negative effects (20). HNE removes the carboxy-terminus of p65 NF-{kappa}B in the vicinity of a site predominantly cleaved by caspase-6, leaving a C-terminally truncated form that retains the ability to bind DNA but lacks the ability to initiate transcription.

One issue for discussion is whether the effects of PR3 on NF-{kappa}B are functionally linked to the ability of PR3 to induce apoptosis. Indeed, we reported earlier that proteolytically inactive PR3 can also induce apoptosis. To explore this, we asked if inactivation of NF-{kappa}B alone in endothelial cells was sufficient to induce apoptosis, and we found that overexpression of a dominant negative I{kappa}B super repressor did not induce apoptosis. This finding is in agreement with other reports that NF-{kappa}B cleavage is required but not sufficient to induce an apoptotic event (20). Cleavage of NF-{kappa}B is proposed to prevent transcription of anti-apoptotic genes, thereby insuring efficient execution of the apoptotic process. So how are these events achieved in the case of proteolytically inactive PR3? One obvious difference between inactive and active PR3-induced death is the time component. Inactive PR3-induced apoptosis requires 24 h (17), whereas a similar response with active PR3 requires only 6 to 8 h. We propose that in the case of inactive PR3, the apoptotic process requires the involvement of caspases to cleave and inactivate NF-{kappa}B, whereas active PR3 can intervene in this pathway and accelerate the process by directly cleaving NF-{kappa}B. In teleologic terms, this capability would be especially important in a situation where caspase-dependent death is blocked by invading organisms that carry a caspase inhibitor.

Even though both PR3 and HNE are serine proteases and similar in many respects, there is a series of examples delineating their unique specificity of substrates (33). In particular, the thrombin receptor is a natural substrate for both proteases, but the cleavage sites are different (34); in the case of the 28-kD heat shock protein (hsp28), PR3 cleaves it into discrete fragments, but HNE does not cleave it at all (35).

We do not know at this point if uptake is necessarily linked with PR3-induced changes in signal transduction pathways. Several lines of evidence suggest that a dynamic equilibrium among three major pathways, ERK, JNK, and p38 MAPK, determines whether a cell will survive or die (36). Our data indicate that both p38 MAPK and JNK pathways are responsive to protease treatment and that activation is probably mediated through the PI3K pathway. We are actively addressing the question of how PR3 and/or HNE are internalized by endothelial cells, and we hope to identify PR3 and/or HNE receptor/s. To date, efforts to find a PR3 receptor have revealed candidates including a 111-kD membrane molecule on HUVEC (37), GM-CSF receptor (18), and the soluble protein C receptor (38).

The redundancy of substrates among caspases and leukocyte proteases suggests evolutionarily that duplication of function provides a fail-safe method to activate apoptosis when necessary. Here we show that PR3 and HNE mimic caspase-3 and caspase-6 in their ability to cleave p65 NF-{kappa}B. PR3 mimics caspase-2 and caspase-3 by cleaving the Sp1 transcription factor (39) and also mimics caspase-1 in its ability to process IL-1{beta} (8). In addition to the caspase-like functions of PR3 and HNE, the T cell proteases (i.e., granzymes) also have substrate specificity that overlaps with caspases. Granzyme B can mimic proapoptotic caspase-8 and caspase-9 and can cleave and activate proforms of caspase-3 and caspase-7 in vitro (40). It has been suggested that such redundancy evolved to protect cells from viruses that produce caspase inhibitors, such as crmA expressed by cowpox virus (41). It is important to note that although neutrophils are generally associated with defense against bacterial invasion, they do play an important role in viral infection (42–45).

A point for discussion is a previous report that indicated that PR3 potently upregulates IL-8 in endothelial cells independently of proteolytic activity (46), which appears to be contradictory to our findings that the proteolytic activity of PR3 is associated with an inhibitory effect on IL-8 transcription. Comparisons of the reported experiments indicate that Berger et al. (46) treated endothelial cells in the presence of serum over a 24-h period and measured IL-8 in the culture medium, whereas our experiments were performed in serum-free medium to circumvent the complexities of contaminating inhibitors. They discuss the possibility that the serum added contained protease inhibitors; indeed, when they added additional {alpha}-1-antitrypsin to the cultures, no difference in IL-8 production was observed, although this was not the case when examining the effects of HNE. Therefore, they concluded that loss of proteolytic activity was not a factor in their studies; however, they did not test PR3 activity in the presence of their serum-containing medium. Moreover, our experiments were designed to address a different question, "What effect does PR3 have on TNF-{alpha}–induced cytokine production?" We suggest here that TNF-{alpha}–induced IL-8 production by endothelial cells is blunted by the proteolytic activity of PR3. However, our data indicate that activation of IL-8 transcription by PR3 alone may involve PR3-induced JNK activation and/or other signal transduction pathways, and we did find that PR3 alone could induce a twofold increase in IL-8 message by 2 h, which corresponds with the report by Berger et al. (46). The subsequent inactivation of NF-{kappa}B may be part of a negative feedback loop that requires the proteolytic activity of PR3. Our observations are not in total opposition to those of Berger and colleagues, but they indeed provide a broader picture of what may occur within the inflammatory microenvironment, where both active and inactive PR3 may contribute to the balance between the perpetuation and the resolution of inflammation.

Pathophysiologic consequences of secreted proteases take on another dimension of complexity when considering the proteolytic modifications of NF-{kappa}B function reported here. This observation, combined with published reports that PR3 and HNE directly process cytokines at focal sites of inflammation (reviewed in reference 7), indicates that they are involved also in controlling cytokine concentrations. In turn, cytokine concentrations would influence the balance of factors that favor perpetuation versus resolution of inflammation (47). The specific and restricted effects of PR3 and HNE on NF-{kappa}B as well as defined effects on signal transduction pathways clearly show that PR3 and HNE are indeed more than indiscriminately destructive and strongly support the hypothesis that these enzymes play specific regulatory roles in inflammatory processes.

How would these functions of PR3 and HNE contribute to vascular injury in the ANCA setting? Some of these autoantibodies are PR3-specific, and binding of the antibody to PR3 can have an inhibitory effect (48). Interestingly, an increased titer of these autoantibodies correlated with increased vasculitic disease activity in patients with Wegener’s granulomatosis. Although this may seem counterintuitive, our data imply that PR3 inhibition may not necessarily be protective in some microenvironments.

We feel that one of the most intriguing aspects of the data presented here is that neutrophils and monocytes carry proteases that can act like caspases, except they move into the cell from outside and thereby usurp the cell’s control of its own fate. This provides a unique mechanism of crosstalk between these leukocytes and endothelial cells at sites of inflammation that affects both cytokine networks and cell viability. The awareness of the specific functional capabilities of these secreted proteases should lend to the development of methods that can manipulate the inflammatory process.


   Acknowledgments
 
We wish to thank Dr. Albert Baldwin, Jr., University of North Carolina–Chapel Hill, for his scientific direction, for providing reagents, and for his critique of the manuscript. We wish to thank Drs. Julian Preston, EPA, and David Alcorta, University of North Carolina–Chapel Hill, for their helpful discussions and for their critique of the manuscript. We would also like to thank Jorgan Weislander for his generous gift of purified PR3. We thank Dr. Gourisankar Ghosh, Department of Chemistry & Biochemistry at UCSD, for the recombinant NF-{kappa}B used for determining the PR3 cleavage site. We wish to acknowledge the UNC-CH Microscopy and Protein Sequencing Core Facilities for their expert contributions to this work. This work was supported by NIHDK-58335-01.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received for publication May 22, 2002. Accepted for publication August 4, 2002.




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