Abstract
Membrane-associated TNF-α cleavage is required to yield the 17.5-kD soluble product. This process is poorly understood in human cells, and no studies have related this process to the alveolar macrophage (AM). TNF-α–converting enzyme (TACE) is known to cleave TNF at the Ala-76–Val-77 site. We have evaluated the expression, regulation, and catalytic function of TACE in healthy human AMs. TACE was detected on the surface of AMs using flow cytometry. TACE protein can be upregulated by LPS (P = 0.036) and IFN-γ. LPS-induced expression is downregulated by IL-10 (P = 0.04) and TNF-α. TACE regulation was observed at the mRNA level. TACE catalytic activity as assessed by cleavage of glutathione S-transferase–proTNF fusion protein correlates significantly with TACE protein expression (P = 0.04). However, cleavage and soluble TNF-α release by AMs was inhibited by matrix metalloproteinase and serine protease inhibitors, suggesting a role for a serine protease in this process. We confirmed the presence of proteinase-3 (PR-3) on the AM surface that was functionally capable of TNF cleavage. PR-3 mRNA expression was not found in AMs. However, we determined that PR-3 from neutrophil supernatants could bind to the AM membrane, suggesting that AM-derived PR-3 is from an exogenous source, which is important in the context of inflammation.
TNF-α is a potent inflammatory cytokine that has been implicated in the pathogenesis of a number of debilitating pulmonary conditions, including acute respiratory distress syndrome (1), idiopathic pulmonary fibrosis (2), and pulmonary sarcoidosis (3, 4). Recently, it has been recognized that the bioactivity of TNF-α in biological fluids is efficiently neutralized by shed TNF receptors (5, 3), suggesting that the systemic effects of TNF-α may be restricted. Data from animal models, however, strongly suggest a pathogenic role for TNF-α in inflammatory lung diseases (6). TNF-α is initially produced in a 26-kD membrane-associated form (mTNF), which is cleaved to yield the 17.5-kD soluble product (sTNF). Much interest has centered on TNF-α converting enzyme (TACE), also known as ADAM-17, a zinc-dependent proteinase of the adamalysin family, which cleaves TNF between the amino acids Ala-76 and Val-77 (7, 8). TACE has also demonstrated the ability to cleave L-selectin, transforming growth factor-α, and amyloid precursor protein and is essential for normal mammalian development (9–11). Inhibition of TACE by broad-spectrum matrix metalloproteinase (MMP) inhibitors has demonstrated a transient increase in mTNF surface expression (12–14). The ability of mTNF to mediate proinflammatory effects in the absence of sTNF has been demonstrated in an animal model of chronic inflammatory arthritis (15), although a study by Ruuls and colleagues (16) found that expression of uncleavable TNF alone was suboptimal for the development of experimental autoimmune encephalitis. We previously demonstrated increased mTNF expression in the absence of increased sTNF in acute respiratory distress syndrome (17). These studies suggest that TACE activity is a key regulator of TNF bioavailability in the local environment. This is particularly relevant in the lung, where mTNF on the surface of macrophages may interact with a variety of cell types, including inflammatory cells and alveolar epithelial cells; the ratio of sTNF to mTNF may lead to differing outcomes.
Despite a great deal of interest in the ability of TACE to cleave TNF-α, there have been few studies exploring the regulation of TACE expression and function of human cells. In addition, the overwhelming majority of studies looking at TACE regulation have used transfected cell lines that may overexpress TACE protein.
This study examined the contribution of TACE to TNF cleavage in the human lung. We have isolated alveolar macrophages (AMs) from normal human lungs by bronchoalveolar lavage to evaluate basal levels of TACE mRNA and protein expression; constitutive catalytic activity of TACE; and modification of TACE expression by the inflammatory mediators LPS, IL-10, IFN-γ, and TNF-α. We have also looked at the relationship between TACE expression and activity relative to mTNF surface expression. We report for the first time that TACE is expressed on the surface of AM, and its expression is upregulated by LPS and IFN-γ and downregulated by IL-10. The increase in LPS-induced TACE is associated with a reduction in mTNF expression. However, AM catalytic activity, as assessed by cleavage of glutathione S-transferase (GST)-proTNF fusion protein and release of soluble TNF-α, was sensitive to MMP and serine protease inhibitors. This suggested an additional role for a serine protease and proteinase-3 (PR-3) was the likely candidate because it has previously been shown to be capable of TNF cleavage (18, 19). PR-3 mRNA was not present in AMs, but incubation of AMs with neutrophil supernatant confirmed the ability of exogenous PR-3 to bind to other cells while retaining catalytic activity. Cleavage activity correlates significantly with TACE protein expression as assessed by flow cytometry, suggesting that TACE is the predominant TNF cleavage enzyme on AMs but that neutrophil-derived PR-3 may also contribute during an acute pulmonary inflammatory response.
Thirteen nonsmoking normal healthy volunteers (eight men and five women) were recruited for this study. They had a mean age of 28 yr (range 18–56 yr). None of our volunteers were smokers or ex-smokers. This study was conducted with the approval of North Bristol NHS Trust Ethics Committee.
Bronchoalveolar lavage was performed in the right middle lobe. Before the procedure, subjects were injected intramuscularly with 0.6 mg of atropine, followed by intravenous sedation with 0–2 mg alfentanil and 0–10 mg midazolam. Topical lignocaine was administered to anesthetize the upper airway. Aliquots of PBS (3 × 60 ml) were instilled and gently aspirated into a siliconized bottle kept on ice. The chilled bronchoalveolar fluid was strained through a single layer of coarse gauze to remove mucus clumps and spun at 400 × g for 5 min to recover cells. The resultant cell-free fluid was stored at −80°C until analysis. The cell pellet was resuspended in serum-free RPMI 1640 medium (Sigma, Poole, UK) supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin (Sigma) and adjusted to 1 × 106 AMs per ml, as defined by morphology. For flow cytometry, cells were cultured for 20 h in sterile Teflon wells to prevent adherence (Savillex Corp., Minnetonka, MN) in the presence or absence of LPS (Sigma), IL-10, TNF, or IFN-γ (Peprotech, London, UK). For RNA extraction, 1 × 106 AMs were plated into sterile 5-cm petri dishes (Gibco-Nunc, Paisley, UK) and incubated for 2 h with LPS, IL-10, TNF, or IFN-γ.
A recombinant pGEX-2T vector containing a GST–pro-TNF plasmid was donated by British Biotech, Oxford, UK (8). The plasmid was transfected into Escherichia coli (TOP10F; Invitrogen, Paisley, UK), and protein expression was under the control of the Taq promoter, induced by isopropylthiogalactosidase. After induction, GST-proTNF was expressed for 2.5 h before lysis by sonication. The 50-kD fusion protein was purified from the sonicates using affinity chromatography (GST purification module; Amersham Biosciences) and resuspended at 1 mg/ml. The fusion protein was stored at −80°C until needed.
Purified AMs were adhered onto 24-well tissue culture plates (1 × 106 per well) (Gibco-Nunc). Protein trafficking was arrested by preincubation for 1 h with 5 μg/ml brefeldin A in the presence or absence of 10 μM BB-3103 metalloproteinase inhibitor (courtesy of British Biotech) or 10 μM Pefabloc before addition of GST-pro-TNF substrate (10 μg per well). After 2 h, the supernatant was removed and briefly spun at 5,000 rpm to remove cell debris. Supernatants were concentrated by centrifugation at 14,000 rpm for 1.5 h at 4°C in microcon filters (YM3; Millipore, Watford, UK). Samples were run through a 15% polyacrylamide gel and transferred onto nitrocellulose membrane (Immobilon, Millipore) using a semi-dry transfer system (Bio-Rad, Hemel Hempstead, UK). Membranes were stored in 1× Tris-buffered saline before Western blotting.
Nitrocellulose membranes were incubated with blocking solution (5% Marvel/TBS/0.05% Tween) for 30 min before addition of biotinylated anti-GST antibody (Autogen-Bioclear, Calne, UK) (1:400 dilution in blocking solution). After 1-h incubation, membranes were washed in TBS/0.05% Tween. A 1:2,000 dilution of streptavidin-HRP (Autogen-Bioclear) in blocking solution was added for a further 1-h incubation. After four 10-min washes, ECL detection reagent (Amersham Biosciences) was added for 1 min. Membranes were immediately exposed to x-ray film (Kodak X-Omat, Sigma) and developed after 1 min. Cleavage by TACE results in a 30-kD band. The density of this band relative to the 50-kD parent molecule was quantified using Geldoc 1000 with Quantity One software (Bio-Rad).
Total cellular RNA was extracted from AMs after culture with various stimuli. Cells were washed in sterile PBS, and cellular RNA was extracted using RNAbee (AMS Biotechnology, Abingdon, UK) according to manufacturer's instructions. Cellular RNA concentration was measured using a GeneQuant II (Amersham Biosciences, Little Chalfont, UK).
RT-PCR was performed in a 20-μl one-step reaction using Reverse-IT RTase blend (ABgene, Epsom, UK) with 2 μl of total RNA as a template. RT was performed at 47°C for 30 min followed by 94°C for 2 min to inactivate the reverse transcriptase enzyme. PCR was performed with 30 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 45 s for human TACE and PR-3. GAPDH housekeeping gene was amplified for 17 cycles. Primer sequences were used as described previously (20, 21). Products were electrophoresed through a 1.5% agarose gel and visualized using ethidium bromide staining. mRNA quantity was determined by digital imaging densitometry (Geldoc 1000 with Quantity One software; Bio-Rad).
AMs were cultured for 24 h in the presence or absence of 10 μg/ml LPS, 10 μM Pefabloc, and 10 μM BB-3103. Supernatants were harvested and stored at −70°C. TNF-α levels were determined using a commercial ELISA kit (Endogen; Perbio Science, Cramlington, UK).
Blood neutrophils, isolated by density centrifugation, were preincubated for 10 min with 5 μM cytochalasin B followed by 1 μM FMLP for 30 min at 37°C and 5% CO2 to induce degranulation. The supernatants were removed, centrifuged to remove any remaining neutrophils, added to purified AMs, and incubated for 1 h in the presence of a protease inhibitor cocktail (Sigma). AMs were also incubated with purified human PR-3 (Elastin Products, Owensville, MO). PR-3 binding was detected by flow cytometry as described below.
Isolated AMs were analyzed immediately or cultured under different conditions in Teflon to prevent cell adherence before analysis. Before staining, AMs were incubated for 10 min in 40 mM citric acid buffer (pH 4.0) to remove receptor-bound proteins. Cells were washed once in PBS/0.5%BSA/0.1% sodium azide (wash buffer) before addition of 10 μg human IgG to block nonspecific binding. PE-labeled anti-human TACE antibody, FITC-labeled membrane-associated TNF antibody, or the appropriate isotype control antibodies (0.5 μg antibody/sample) (R&D Systems, Abingdon, UK) were added to the cells for 30 min at 4°C in the dark. PR-3 was detected by incubation with -PR3-ANCA Pelikine monoclonal antibody (22) (Research Diagnostics, Concord, MA), followed by rabbit anti-mouse-rPE (Dako, Ely, UK). Cells were washed twice in wash buffer and fixed in PBS/1% paraformaldehyde solution. Labeled cells (1 × 104) were acquired on an EpicsXL flow cytometer (Beckman Coulter, High Wycombe, UK) and analyzed using Expo 32 software (Beckman Coulter).
Statistical analyses were performed using GraphPad Prism (GraphPad Software Inc., San Diego, CA). The data were normally distributed as determined by the Ryan Joiner normality test. Comparisons between multiple groups were performed using one-way ANOVA, with Tukey's multiple comparison test (mct) to compare individual group differences. Paired data were compared using Student's t test. Relationships between parameters were assessed using Pearson correlation. A P value of < 0.05 was regarded as significant.
AMs expressed TACE mRNA and surface protein at a basal level in all samples. LPS stimulation for 2 h significantly increased mRNA expression to 148.6 ± 41.3% of unstimulated controls (P = 0.02) (Figure 1). By contrast, IL-10 coincubated with LPS significantly reduced expression to 24.6 ± 18.7% of unstimulated controls (P = 0.05). IFN-γ was the most potent stimulus of TACE mRNA transcription, increasing expression to 244 ± 51.1% of unstimulated controls (P = 0.008). TNF-α did not significantly affect TACE mRNA expression, although there was a trend toward reduction (56.9 ± 29.7%, P = 0.09). The changes in TACE mRNA transcripts were reflected by changes in the level of TACE surface protein expression (Figure 2). Overnight stimulation with LPS and IFN-γ increased TACE expression from 1.33 to 4.26 and 4.22, respectively (P = 0.036 and P = 0.09). LPS-stimulated TACE was significantly inhibited by addition of IL-10 to 1.44 (P = 0.04). IL-10 alone had no effect on basal levels of TACE expression (data not shown). The addition of TNF-α had no significant effect on TACE expression, although mean expression was reduced to 0.47 (P = 0.09). These results establish the presence of TACE mRNA and surface protein in healthy AMs and their potential for modulation by an inflammatory milieu.
Figure 1. Expression of TACE mRNA transcripts in AMs from normal subjects. AMs were cultured with LPS at 10 μg/ml and IL-10, TNF-α, and IFN-γ at 10 ng/ml.*Unstimulated versus LPS; P < 0.05. §LPS versus IL-10 + LPS; P = 0.05. #Unstimulated versus IFN-γ; P < 0.01, Tukey's mct. n = 13 for all conditions.
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Figure 2. Flow cytometry analysis of TACE surface protein expression expressed as a ratio of isotype control. AMs were cultured with LPS at 10 μg/ml and IL-10, TNF-α, and IFN-γ at 10 ng/ml. n = 13 for all conditions. *Unstimulated versus LPS; P < 0.05. §LPS versus IL-10 + LPS P < 0.05, Tukey's mct.
[More] [Minimize]To determine the relationship between TACE and mTNF protein expression, we compared median surface protein expression before and after LPS treatment. In unstimulated cells, median expression of TACE and mTNF was 2.96 and 18.44 as a ratio to isotype control, respectively, and we found no positive or negative association between the two proteins. However, after LPS stimulation, the median expression of TACE and mTNF was 9.0 and 26.2, respectively, and regression analysis revealed a weak negative correlation between TACE and mTNF expression (r2 = −0.33, P = 0.05) (Figure 3A). Thus, we have demonstrated that increased surface TACE is associated with increased cleavage of mTNF from the cell surface.
Figure 3. (A) Flow cytometry analysis of mTNF and TACE surface expression on AMs (n = 13) after 20-h incubation in Teflon wells with LPS (10 μg/ml), expressed as a ratio of isotype control. (B) Western blot of the in vitro cleavage of GSTproTNF by idiopathic pulmonary fibrosis AMs. Lane 1, no inhibitors. Lane 2, BB-3103. Lane 3, assay in the absence of AMs. (C) TACE expression on freshly isolated AMs determined by flow cytometry expressed as a ratio of isotype control and the corresponding cleavage of GSTproTNF determined by the in vitro cleavage assay (n = 13).
The functional catalytic activity of surface TACE on AMs was assessed by an in vitro cleavage assay, which we have developed. This assay uses a GST-proTNF fusion protein (50 kD), which is cleaved by TACE at the Ala-76–Val-77 site to yield a 30-kD GST product (Figure 3B). Using Pearson correlation, we have established a weak positive correlation between TACE surface expression and cleavage performance (Figure 3C) (r2 = 0.33, P = 0.04), leading us to conclude that TACE may be responsible for regulating the cleavage of mTNF in normal AMs.
Specific enzyme inhibitors were added to the cleavage assay to determine the relative contribution of TACE to the cleavage process. The mean inhibition in response to the MMP inhibitor BB-3103 was 23.6 ± 6.89% as a percentage of AMs alone (Figure 4). Treatment with the serine protease inhibitor (Pefabloc) alone reduced cleavage to 58.6 ± 24.6% as a percentage of AMs alone (P < 0.05). Furthermore, Pefabloc in addition to BB-3103, increased the level of inhibition to 14.8 ± 8.6% as a percentage of AMs alone (P < 0.01). These data suggest that a serine protease may make a small contribution, additional to TACE, to cleavage of mTNF by normal AMs.
Figure 4. Effect of inhibitors on GSTproTNF cleavage by AMs in culture (n = 13 for each condition). Gray bars, AMs alone. Diagonal bars, 10 μM BB3103. Horizontal bars, 10 μM pefabloc. Hatched bars, pefabloc + BB3103. **P < 0.005 BB3103 versus normal subject AMs alone; *P < 0.05 pefabloc versus AMs alone; §P < 0.01 BB-3103 + pefabloc versus AMs alone. Student's paired t test.
[More] [Minimize]We explored the effect of TACE and serine protease inhibition on the release of soluble TNF-α from AM cultures stimulated with 10 μg/ml LPS. We found that BB-3103 alone was able to significantly reduce LPS-induced expression from 2.10 ± 0.12 ng/ml to 0.47 ± 0.06 ng/ml (P < 0.0001). Serine protease inhibition by Pefabloc reduced LPS-induced TNF-α release to 1.24 ± 0.31 ng/ml (P < 0.05), and when combined with BB-3103, TNF-α release was reduced to 0.088 0.026 ng/ml (P < 0.0001) (Figure 5). These data provide more direct support for a contribution of serine proteases to TNF-α cleavage.
Figure 5. Effect of inhibitors on soluble TNF-α release by AMs stimulated by 10 μg/ml LPS in culture (n = 6 for each condition). Gray bars, LPS alone. Diagonal bars, 10 μM BB3103. Horizontal bars, 10 μM pefabloc. Hatched bars, pefabloc + BB3103. ***P < 0.0001 BB3103 versus LPS alone; *P < 0.05 pefabloc versus LPS alone; §P < 0.0001 BB-3103 + pefabloc versus LPS alone. Student's paired t test.
[More] [Minimize]Because PR-3 has been suggested previously as a candidate for TNF-α cleavage, we performed experiments to investigate whether PR-3 protein was present on AMs. Flow cytometry revealed staining for PR-3 on the surface of AMs (Figure 6A). However, we found no evidence of de novo synthesis of PR-3 mRNA in AMs, using THP-1 mRNA as a positive control (Figure 6B). This suggests that the PR-3 may be derived from an exogenous source.
Figure 6. (A) Representative flow cytometry of PR-3 on the AM surface detected with cANCA antibody. White indicates isotype control; gray indicates normal subject AMs. (B) PR-3 mRNA expression in AMs determined by RT-PCR. Lane 1, 100-bp ladder. Lanes 2–5, AMs (PR-3). Lanes 6 and 7, THP-1–positive controls. Lane 8, water control. Lanes 9–14, corresponding GAPDH expression.
[More] [Minimize]Experiments were performed to evaluate whether PR-3 could bind to the AM surface from an exogenous source. AMs from normal subjects had a mean PR-3 expression of 1.57 ± 0.26 relative to isotype control (Figure 7). This was significantly increased after incubation with purified human PR-3 (7.79 ± 1.21, P < 0.01; paired t test). Incubation with untreated neutrophil supernatant had no effect on PR-3 expression (1.81 ± 0.36). However, when AMs were incubated with supernatant from neutrophils pretreated with FMLP and cytochalasin B, this resulted in increased PR-3 binding to the AM surface (3.52 ± 0.91) relative to AMs alone (P < 0.05). Thus, PR-3 from activated neutrophils is able to bind to the surface of AMs, suggesting that these cells are the likely source of AM-bound PR-3.
Figure 7. Flow cytometry analysis of PR-3 binding to the AM surface after incubation with neutrophil supernatant (unstimulated or treated with 1 μM FMLP and 5 μM cytochalasin B [Cyto B]) or exogenous PR-3 (10 ng/ml). §P < 0.01 versus AMs blank; *P < 0.05 versus AMs blank. Student's paired t test (n = 8).
[More] [Minimize]TACE catalysis of mTNF is a critical event regulating the bioavailability of soluble versus membrane-associated TNF-α. Although TNF-α production is an essential protective component of the host-defense response, inappropriate regulation can lead to pathologic effects, such as increased permeability, cytotoxicity, and fibrosis, directly or indirectly as a result of induction of other proinflammatory mediators (23). As a consequence, there have been many studies investigating the production and activity TNF-α in the context of inflammation. However, there have been few studies looking at its processing at the cell surface, and our study is the first to investigate TACE activity relative to the human lung and to recognize the contribution of PR-3 in this context. The AM was the cell of choice because it is the sentinel cell of the host-defense system in the normal lung and the main source of TNF-α during an inflammatory response (24).
We have shown for the first time that basal expression of TACE on the AM can be upregulated by IFN-γ and LPS. IFN-γ is known to induce TNF-α production by macrophages (25), and the concomitant increase in TACE may contribute to the increased availability of soluble TNF-α during an inflammatory response. LPS is the primary bacterial trigger of this response and can upregulate an array of proinflammatory cytokines, including IL-1β and IL-8 (26). Although LPS can increase TNF-α production per se, the ability of LPS to increase surface TACE expression may contribute to the increase in soluble TNF-α observed in animal models of endotoxemia (27) and macrophage culture supernatants after LPS stimulation. We selected a 20-h time point because kinetic studies in our laboratory have shown that TACE increases gradually in response to LPS, peaking at 16–20 h before gradually declining. The peak mRNA response at 2 h suggests that de novo protein synthesis is occurring. Our findings are in contrast to a study by Robertshaw and Brennan (28) who demonstrated in blood monocytes that LPS stimulation decreases TACE surface protein expression. However, they did show a 14-fold increase in TACE activity in response to LPS, peaking at 2 h before declining. Differences in response between the two studies may reflect the phenotypic differences between monocytes and macrophages and the different sources and doses of LPS used. We have also shown that the anti-inflammatory cytokine IL-10 can inhibit the LPS-induced increase in TACE expression. This is in accord with the ability of IL-10 to downregulate LPS-induced TNF-α release by AMs and may be part of the protective effects of IL-10 during an inflammatory response (29).
The relationship between regulation of TACE production and surface TACE expression is contentious. We have shown that the alterations in surface TACE on AMs are reflected at the mRNA level, suggesting that TACE is subjected to transcriptional regulation during an inflammatory response. However, it is also known that TACE, in common with other adamalysins, is produced in a pro-form that is cleaved by a furin protease in the trans-Golgi network (30). It has also been reported that the majority of TACE is located in the perinuclear compartment (30), suggesting that there may be a large pool of inactive enzyme that is processed intracellularly. However, another study demonstrated that any processing of TACE is tightly coupled to transport to the cell membrane (31), suggesting that the majority of processed TACE rapidly appears on the cell surface.
The measurement of GST-proTNF provides a useful model for the investigation of cleavage activity on the surface of cells. We used GST detection in the Western blots to prevent interference from AM-derived TNF-α. However, we cannot be certain that the product we are measuring is being cleaved in an identical fashion to native mTNF. We have confirmed the specificity of the cleavage by Western blotting and demonstrated a 17.5-kD band using TNF-α–specific antibodies (data not shown). Our assumption is supported by the observed significant correlation between TACE expression and GST-proTNF cleavage and the reduction of cleavage in the presence of the MMP inhibitor BB-3103, which has previously been demonstrated to inhibit TACE catalytic activity in vitro (32). These findings suggest that TACE function on the cell membrane plays an important role in the cleavage of mTNF on the surface of the normal AMs. BB-3103 is not specific for TACE alone, having been shown to inhibit a broad spectrum of MMPs, such as MMP-7, which can also cleave TNF-α. Tissue inhibitor of metalloproteinases (TIMPs) could have been used to discriminate between TACE (inhibited only by TIMP-3) and MMP activity (inhibited by TIMP-1, -2, -3, and -4), but the ability to perform multiple analyses such as these is limited by the relatively small number of AMs that can be obtained from an individual subject for ex vivo experimentation. The correlation observed between TACE expression and GST-proTNF cleavage supports the suggestion that the BB-3103 effects demonstrated are primarily TACE dependent.
Alternate or additional proteases may contribute to cleavage. When we added the specific serine protease inhibitor Pefabloc to assess any inhibitory effect on the cleavage process, we found a small effect that was additional to TACE. Furthermore, addition of the serine protease inhibitor reduced soluble TNF-α release in response to LPS by over 30% and showed an additive effect to the inhibition of TACE activity by BB-3103. Although there has been a great deal of interest in the ability of TACE to cleave TNF-α, it was demonstrated several years earlier that mTNF could also be cleaved by a serine protease (33). This was later to be identified as PR-3 (34), and the cleavage site is located at Arg-Val (18), which is immediately adjacent to the Ala-Val cleavage site of TACE, both yielding a 17.5-kD product. Apart from cleavage of cytokines, PR-3 is able to degrade extracellular matrix, and its contribution to inflammatory disease has been demonstrated by the induction of emphysema in hamsters after intratracheal installation (35). PR-3 is also increased in the sputum of patients with cystic fibrosis, where it correlates with disease severity (36). PR-3 is the target antigen for cANCA autoantibody in Wegener's granulomatosis, but whether it contributes to pathology or is an epiphenomenon is not clear (37). As a homolog of neutrophil elastase and cathepsin G, PR-3 is produced by cells of myelomonocytic origin, and the mature enzyme is stored in azurophilic granules of neutrophils that release it upon degranulation (38). However, it is also known to be present on the surface of monocytes (39), endothelial cells (40), and AMs (41). Whether this represents production of PR-3 by these cells or reflects insertion of exogenous hydrophobic PR-3 into the cell membranes (42) has been matter of contention. We found no evidence for de novo synthesis of PR-3 in our AMs, but we have demonstrated that purified PR-3 and to a lesser extent PR-3 present in activated neutrophil supernatants are able to bind to AMs in vitro. This finding is supported by another study that showed that PR-3 was able to bind to the outer plasma membrane of human umbilical vein endothelial cells (40). We have also demonstrated the presence of PR-3 on the surface AMs in normal subjects. This may reflect the situation in vivo, although it is possible that the bronchoscopy and lavage procedures lead to increased infiltration of alveolar neutrophils (43), resulting in artifactual binding of PR-3 to resident AMs.
The inhibition of cleavage by BB-3103 relative to Pefabloc in normal AMs supports a dominant role for TACE in the cleavage of mTNF from the AM surface in the healthy lung. This is further supported by the significant correlation between mTNF and TACE expression after LPS stimulation. However, it has been suggested that TACE is able to cleave its substrates in intracellular vesicles; this is supported by the observation that TACE can cleave amyloid precursor protein (44). Other studies have reported that mTNF processing occurs predominantly at the cell surface (12–14). The exact site of mTNF processing by TACE in AMs is under investigation in our laboratory. The ability of increased TACE on the cell surface to increase mTNF cleavage does suggest that pharmacologic inhibition of TACE by broad-spectrum MMP inhibitors may lead to an accumulation of mTNF on the cell surface. In inflammatory disorders, these inhibitors could have potentially pathological consequences due to the ability of mTNF to mediate proinflammatory and cytotoxic effects on adjacent cells. However, our data suggest that in an inflammatory situation, there may be changes in TACE and in other proteases, such as neutrophil-derived PR-3, which may contribute to mTNF cleavage.
These results suggest that the regulation of TNF bioavailability is highly controlled, as is appropriate for such a key mediator of the inflammatory response. At least two enzymes are involved in this process, and both could be considered for pharmacologic targeting in inflammatory disorders.
The authors thank Professor Caroline Savage, University of Birmingham, UK, for advice regarding the PR-3 investigations. The authors acknowledge Action Medical Research for supporting Dr. Lynne Armstrong and British Biotech for supplying the GSTproTNF vector and the BB-3103 inhibitor.
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