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(American Journal of Pathology. 2000;156:1033-1039.)
© 2000 American Society for Investigative Pathology

Regular Articles

Anti-Inflammatory Effects of Mutant Forms of Secretory Leukocyte Protease Inhibitor

Michael S. Mulligan*, Alex B. Lentsch{dagger}, Markus Huber-Lang{ddagger}, Ren-Feng Guo{ddagger}, Vidya Sarma{ddagger}, Clifford D. Wright§, Thomas R. Ulich§ and Peter A. Ward{ddagger}

From the Departments of Surgery*
and Pathology,{ddagger}
University of Michigan Medical School, Ann Arbor, Michigan; the Department of Surgery,{dagger}
University of Louisville School of Medicine, Louisville, Kentucky; and Amgen, Inc.,§
Thousand Oaks, California


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The secretory leukocyte protease inhibitor (SLPI) is found in a variety of secreted fluids in mammals and is a known inhibitor of serine proteases. Wild-type (WT) SLPI has recently been shown to block nuclear factor {kappa}B (NF-{kappa}B) activation in rat lungs and to interfere with the ensuing inflammatory response and recruitment of neutrophils after an intrapulmonary deposition of IgG immune complexes. In this study, WT SLPI and SLPI mutants with various degrees of protease-inhibitory capacity (for trypsin, chymotrypsin, and elastase) were evaluated for their ability to suppress the lung-vascular leak, neutrophil accumulation, and NF-{kappa}B activation in the lung inflammatory model. The SLPI mutant with Gly72 (replacing Leu72 ) lost its ability to block in vivo activation of NF-{kappa}B, as well as its ability to suppress the lung vascular leak and neutrophil recruitment. The Phe72 and Gly20 mutants were as effective as the WT SLPI in suppressing NF-{kappa}B activation and neutrophil recruitment. The Lys72 mutant had the most suppressive effects of the lung vascular leak and for neutrophil recruitment into the lung. The in vivo suppressive effects of SLPI mutants on lung vascular permeability, neutrophil recruitment, and NF-{kappa}B activation appear to be most closely related to their trypsin-inhibiting activity. These data suggest that the suppressive effects of SLPI on the intrapulmonary activation of NF-{kappa}B and neutrophil recruitment into the lung may be linked to their antiprotease activity, directed, perhaps, at the intracellular proteases.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Although the secretory leukocyte protease inhibitor (SLPI) was originally defined as an inhibitor of serine proteases,1,2 it was eventually recognized to have other effects, such as antagonizing lipopolysaccharide-induced production of tumor necrosis factor-{alpha} (TNF-{alpha}) in stimulated phagocytic cells3 and interfering with the entry of the human immunodeficiency virus into susceptible cell lines.4,5 In rats, SLPI has since been found to inhibit inflammatory lung injury caused by an intrapulmonary deposition of IgG-immune complexes.6 This model is characterized by an intensely damaging lung-inflammatory response featuring the roles of cytokines and chemokines and the recruitment of neutrophils.7 The manner by which SLPI inhibits these inflammatory reactions and protects the lung was recently found to be related to its ability to prevent nuclear factor {kappa}B (NF-{kappa}B) activation within the lung in a manner related to preservation of the NF-{kappa}B-inhibitory protein, I{kappa}Bß.8 Further details of how this activation pathway is blocked by SLPI are not known.

In structure-function studies, mutant forms of SLPI have been evaluated in vitro, using techniques to induce site-directed mutations involving Leu72 of wild-type (WT) SLPI. Leu72 is known to be a critical site for the binding of chymotrypsin, elastase, and trypsin to SLPI.9 In the current studies, we investigated the effects of various mutants of SLPI to gain insights into the anti-inflammatory nature of this molecule. There appears to be a relationship between the protease inhibitory activity of SLPI mutants, especially correlating with the trypsin-inhibiting activity, and their ability to demonstrate in vivo protective effects through the reduction of the lung albumin leak, neutrophil accumulation, and suppression of intrapulmonary activation of NF-{kappa}B.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents

Unless otherwise indicated, all reagents were purchased from Sigma Chemical Co. (St. Louis, MO). Recombinant human SLPI and SLPI mutants were used in all studies. The SLPI mutants were prepared by site-specific mutagenesis using the Mutagene in vitro mutagenesis kit purchased from Bio-Rad (Hercules, CA). The plaques were screened initially by hybridization using the mutagenic oligonucleotide as the probe, and those clones that were positive by hybridization were plaque-purified and sequenced using modified T7 DNA polymerase (U.S. Biochemical Corp., Cleveland, OH). Mutants having the desired sequence were grown in JM109 cells and M13 RF DNA was prepared as described elsewhere.9

IgG Immune Complex-Induced Alveolitis

Pathogen-free male Long-Evans rats (275–300 g; Harlan Sprague-Dawley, Indianapolis, IN) were anesthetized with ketamine HC1 (150 mg/kg, i.p.). The rats received an intratracheal administration of phosphate-buffered saline, pH 7.4, or 2 mg of rabbit IgG antibody to bovine serum albumin (BSA) from ICN Biomedicals (Costa Mesa, CA) in a total volume of 300 µl, followed by an intravenous infusion of 10 µg of BSA. When the permeability index was measured,125I-BSA was also injected intravenously.8 When SLPI proteins were used, 400 µg were added to the anti-BSA preparation. This amount has been shown to be suppressive of the lung inflammatory response.8 When {alpha}1-antiproteinase ({alpha}1-PI) was used, 1.0 mg was added to the anti-BSA preparation before the intratracheal instillation. Four hours after the IgG-immune complex deposition in the lung, the rats were exsanguinated. For the measurement of lung vascular permeability, the pulmonary artery was perfused with 10 ml of phosphate-buffered saline to remove any residual blood in the pulmonary vasculature. The total radioactivity of the lungs was measured and compared with the amount of radioactivity present in 1.0 ml of blood obtained from the inferior vena cava at the time of sacrifice. This ratio was the computed permeability index.8 For the measurement of pulmonary neutrophil accumulation, bronchioalveolar lavage (BAL) was performed with three repetitive washes with 5 ml of sterile saline. The BAL-fluid neutrophil counts were determined by microcytometry. For each experimental group, n = 7.

Assessment of NF-{kappa}B Activation by Electrophoretic Mobility Shift Assay

Nuclear extracts of whole-lung tissues were prepared by the method of Deryckere and Gannon.10 Protein concentrations were determined by a bicinchoninic-acid assay with TCA precipitation using BSA as a reference standard (Pierce, Rockford, IL).The NF-{kappa}B consensus oligonucleotide (5'-AGTGAGGGGACTTTCCCAGGC-3'; Promega, Madison, WI) was end-labeled with [{gamma}-32P]ATP (3000 Ci/nmol/L at 10 mCi/ml; Amersham, Arlington Heights, IL). The binding reactions containing equal amounts of protein (10 µg) and 35 fmol (~50,000 cpm, Cherenkov counting) of oligonucleotide were performed for 30 minutes in binding buffer (4% glycerol, 1 mmol/L MgC12, 0.5 mmol/L ethylenediaminetetraacetic acid, pH 8.0, 0.5 mmol/L dithiothreitol, 50 mmol/L NaC1, 10 mmol/L Tris, pH 7.6, 50 µg/ml poly (dI·dC); Pharmacia, Piscataway, NJ). For supershift assays, 1 µl of polyclonal antibody to p50, p52, p65, p68, or p75 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was added to the binding reactions. The reaction volumes were held constant to 15 µl. The reaction products were separated in a 4% polyacrylamide gel and analyzed by autoradiography. The NF-{kappa}B activation was quantitated from digitized autoradiography films using image analysis software (Adobe Systems, Inc., San Jose, CA).

Assessment of Inhibitory Activities of SLPI and {alpha}1-PI

Serine proteases were assayed using specific chromogenic peptide-p-nitroanilide (pNA) substrates in a 96-well microtiter plate format. Each protease was incubated with various concentrations of human WT SLPI or {alpha}1-PI for 15 minutes at 37°C in specific assay buffer. The residual protease activity was measured after the addition of the appropriate substrate. The pNA product of proteolysis was quantified at 405 nm on a SpectraMAX 340 plate reader (Molecular Devices, Sunnyvale, CA). The human neutrophil elastase (Calbiochem-Novabiochem International, San Diego, CA) was assayed using pyroGlu-Pro-Val-pNA (Pharmacia Hepar Inc., Franklin, OH) in 100 mmol/L Tris-HCl, pH 8.3, 0.96 mmol/L NaCl, 1% BSA.11 Bovine pancreatic trypsin (-1-Tosylamide-2-phenylethyl chloromethyl ketone-treated; Sigma) was assayed using N-{alpha}-benzoyl-L-Arg-pNA (Boehringer Mannheim Corp., Indianapolis, IN) in 50 mmol/L Tris-HCl, pH 8.2, 20 mmol/L CaCl2.12 Bovine pancreatic chymotrypsin (Boehringer Mannheim) was assayed using N-Suc-Ala-Ala-Pro-Phe-pNA (Sigma) in 100 mmol/L Tris-HCl, pH 7.8, 10 mmol/L CaCl2.13 The dissociation constants (Ki) of human WT SLPI and {alpha}1-PI against each proteolytic enzyme were determined as previously described.14

Statistical Analyses

All values are expressed as mean ± SEM. Data were analyzed with a one-way analysis of variance and subsequent Student-Newman-Keuls test. The differences were considered significant when P < 0.05. For calculations of the percent change, negative control values were subtracted from the positive control and treatment group values.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of SLPI Mutants on the Lung Permeability Index

The ability of WT SLPI and SLPI mutants to affect the permeability index 4 hours after the initiation of the lung inflammatory injury is shown in Figure 1 . As expected, the permeability index values for the negative and positive controls were different by a factor of more than threefold. The presence of WT SLPI reduced the permeability index by nearly 47% (P < 0.001). The Gly20 mutant was similarly protective (P < 0.001). The Gly72 mutant had no protective effects (P not significant). The Phe72 mutant was statistically as protective as the WT SLPI (P = 0.002), whereas the Lys72 mutant was the most protective of all the forms of SLPI (P = 0.001). In addition, the Lys72 mutant was the only form of SLPI that possessed protective activities that exceeded those of the WT SLPI (P = 0.02; Figure 1 ).



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  		Figure 1. Effects of mutant forms of SLPI in reducing the lung vascular permeability index in rats after intrapulmonary deposition of IgG immune complexes. When used, WT SLPI and SLPI mutants (400 µg) were instilled into the airways together with 2 mg of IgG anti-BSA. P values were compared with the positive control group not otherwise treated and to the WT SLPI group. Values represent mean ± SEM with n = 7 for each group.

 
Effects of SLPI Mutants on Lung Recruitment of Neutrophils

The SLPI and its mutant forms were evaluated for their effects on the neutrophil accumulation in BAL fluids from rat lungs 4 hours after the intrapulmonary deposition of the IgG immune complexes. The neutrophils are largely responsible for the damage occurring in this model.15 As expected, when the BAL content of neutrophils was compared in the negative and positive control groups, there was a nearly 20-fold increase in the number of neutrophils recruited into inflamed lungs (Figure 1) . In the presence of 400 µg WT SLPI, there was a 48% reduction (P = 0.001) in neutrophil accumulation in the inflamed lungs. The Gly20 mutant had inhibitory effects similar to WT SLPI, decreasing the BAL neutrophil counts by 50% (P < 0.001). In the presence of the Gly72 mutant of SLPI, the inhibitory effects were lost, compared with the positive control group (P, not significant). The Phe72 mutant of SLPI had inhibitory effects (40% decrease, P = 0.001) that were indistinguishable from those of WT SLPI. The Lys72 mutant of SLPI caused the greatest reduction in BAL neutrophils compared with the positive control group (82% decrease, P < 0.001). When compared with WT SLPI for the effect on the BAL accumulation of neutrophils, Lys72 had significantly greater inhibitory properties (P = 0.001).

Effects of SLPI Mutants on NF-{kappa}B Activation in Lung

Using this lung-inflammatory model, we have recently shown that the anti-inflammatory effects of WT SLPI are associated with the inhibition of the intrapulmonary activation of NF-{kappa}B through the preservation of I{kappa}Bß.8 To determine how mutant forms of SLPI compare to the effects of WT SLPI on pulmonary NF-{kappa}B activation, nuclear extracts from whole lungs that were harvested 4 hours after IgG-immune complex deposition (in the absence or presence of SLPI proteins) were analyzed by electrophoretic mobility shift assay (EMSA). As expected, there was little evidence of NF-{kappa}B activation in the negative control lungs (Figure 3 , upper frame, first lane, arrow). The intrapulmonary deposition of IgG immune complexes resulted in a significant increase in the nuclear translocation of NF-{kappa}B (second lane). In the presence of 400 µg of WT SLPI, NF-{kappa}B activation was greatly reduced (third lane). Image analysis of digitized EMSA autoradiograms indicated that WT SLPI reduced NF-{kappa}B activation by 55% (P = 0.002) when compared with positive controls in the absence of SLPI (Figure 3 , lower frame). Treatment with the Gly20 mutant of SLPI also had suppressive effects, reducing NF-{kappa}B activation by 47% (P = 0.020; fourth lane). The effects of the Gly72 mutant of SLPI (fifth lane) were indistinguishable from the positive control group. The effects of the Phe72 mutant of SLPI on the lung NF-{kappa}B activation were similar to WT SLPI, decreasing IgG-immune complex-induced NF-{kappa}B activation by 51% (P = 0.006; sixth lane). The Lys72 mutant had the most suppressive effects, virtually abolishing evidence of NF-{kappa}B activation (P = 0.003; seventh lane). Accordingly, there appears to be a correlation between SLPI effects on NF-{kappa}B activation (Figure 3) , reductions of the permeability index (Figure 1) , and the diminished recruitment of neutrophils (Figure 2) .



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  		Figure 3. Effects of W. T. SLPI and SLPI mutants on IgG immune complex-induced lung NF-{kappa}B activation as detected by EMSA. A: The main NF-{kappa}B activation band (arrow) is shown in whole-lung tissues harvested 4 hours after intratracheal administration of 2 mg of IgG anti-BSA (IgG-IC) followed by intravenous infusion of 10 mg of BSA. WT SLPI and SLPI mutants (400 µg) were administered intratracheally with the IgG anti-BSA. B: Quantitation of EMSA blots by image analysis.

 


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  		Figure 2. Effects of WT SLPI and SLPI mutants on accumulation of neutrophils in bronchoalveolar (BAL) fluids obtained from lungs of rats 4 hours after intrapulmonary deposition of IgG immune complexes. When used, WT SLPI and SLPI mutants (400 µg) were instilled into the airways together with 2 mg of IgG anti-BSA. Values represent mean ± SEM with n = 7 for each group.

 
We have previously shown that the NF-{kappa}B complex activated by the IgG-immune complexes in the lung consists of p50 and p65 subunits.16 In the present studies it was observed that treatment with WT SLPI and, to a lesser extent, Gly20 and Phe72 mutants altered the NF-{kappa}B banding patterns in the EMSA’s (Figure 3) . Specifically, a prominent, slower-migrating NF-{kappa}B band was observed in these treatment groups. To identify the components of these EMSA bands, supershift assays were performed on nuclear extracts of inflamed lungs in the presence of 400 µg WT SLPI. The results are shown in Figure 4 . Indicated at the left side of the figure are the positions of the primary NF-{kappa}B band (solid arrow), the slower migrating band (solid arrowhead), and the supershift bands (open arrow). In the presence of the antibody to p50, some supershift of NF-{kappa}B was found (second lane). Supershifts were not observed in the presence of the antibody to p52, p68, or p75. In the presence of the antibody to p65, there was some supershift of the primary NF-{kappa}B band and a complete supershift of the slower-migrating NF-{kappa}B band. These data suggest that the primary NF-{kappa}B band (solid arrow) is composed largely of p50/p65 heterodimers, and that the slower-migrating NF-{kappa}B band observed (solid arrowhead) in SLPI treatment groups is composed of p65/p65 homodimers.



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  		Figure 4. Supershift analysis of SLPI-induced NF-{kappa}B components. DNA-binding reactions were done with nuclear extracts from whole lungs harvested 4 hours after intratracheal administration of IgG immune complexes, using the 32P-labeled NF-{kappa}B consensus oligonucleotide in the absence or presence of antibodies to the NF-{kappa}B proteins indicated (p50, p52, p65, p68, p75). Supershifts of p50 and p65 are indicated by the open arrow. The solid arrowhead indicates the position of the higher molecular weight NF-{kappa}B complex induced by SLPI. The solid arrow indicates the position of the primary NF-{kappa}B band.

 
The suppressive effects of SLPI proteins on NF-{kappa}B activation appeared to correlate most closely with the published trypsin-inhibitory effects of these proteins (Table 1) . In view of this possibility, the effects of {alpha}1-PI on pulmonary NF-{kappa}B activation were also investigated, because {alpha}1-PI is a potent inhibitor of trypsin and other serine proteases, including chymotrypsin and elastase.17,18 The administration of 1 mg of {alpha}1-PI in rat lungs also failed to reduce lung NF-{kappa}B activation, which is induced by IgG-immune complexes (Figure 5) . It should be noted, however, that the amount of {alpha}1-PI administered was, on a molar basis, less than the amount of SLPI (60 mmol/L versus 110 mmol/L, respectively). The addition of {alpha}1-PI in the airways of the rats also failed to inhibit either the permeability index (positive control values in the absence or presence of 1.0 mg {alpha}1-PI were 0.537 ± 0.049 and 0.53 ± 0.024, respectively) or the numbers of neutrophils (in millions) in the BAL fluids (positive control values in the absence or presence of 1.0 mg {alpha}1-PI were 5.32 ± 0.582 and 5.70 ± 0.483, respectively). Thus, although {alpha}1-PI is a broad inhibitor of serine proteases, in this experimental model it does not suppress NF-{kappa}B activation, the permeability index, or the number of neutrophils recruited into the lung.


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  		Table 1. Relative Protease Inhibitory Effects of SLPI Mutants and {alpha}1-PI

 


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  		Figure 5. Effects of {alpha}1-proteinase inhibitor ({alpha}1-PI) on IgG immune complex (IgG-IC)-induced lung NF-{kappa}B activation (arrow). NF-{kappa}B activation was evaluated in whole-lung tissues harvested 4 hours after intratracheal administration of 2 mg of IgG anti-BSA followed by intravenous infusion of 10 mg BSA. {alpha}1-PI (1 mg) was administered intratracheally with the IgG anti-BSA.

 
Inhibitory Activities of SLPI and {alpha}1-PI

Because WT SLPI, but not {alpha}1-PI, blocked NF-{kappa}B activation in the lung (Figures 3 and 5) , the kinetic dissociation constants (Ki) of the two protease inhibitors were determined. As shown in Table 2 , the protease-inhibitory activity of SLPI revealed a descending rank order of chymotrypsin = elastase >> trypsin. In the case of {alpha}1-PI, the descending order of inhibition was chymotrypsin > elastase > trypsin. A comparison of the Ki values for WT SLPI and {alpha}1-PI revealed that the protease inhibitory activities of SLPI and {alpha}1-PI are similar in the case of trypsin and chymotrypsin, whereas {alpha}1-PI is about 1/10 as effective as SLPI for inhibition of elastase.


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  		Table 2. Comparison of the Anti-Protease Activities of SLPI and {alpha}1-PI

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
SLPI contains two molecular domains (1 and 2) and a total of eight Cys-Cys groups, resulting in a very stable, oxidant-resistant molecule. The substitution of Leu in position 72 significantly affects the protease inhibitory capacity of SLPI.9 In the N-terminal domain 1, although it was speculated that Arg20 might be a binding site for trypsin and other serine proteases, substituting Gly for Arg in this position failed to affect the protease-inhibitory capacity of SLPI, implying that this is not a binding site for serine proteases.9 Similarly, the Gly20 mutant of SLPI retained the full in vivo anti-inflammatory effects when compared with WT SLPI (Figures 1, 2) .

The C-terminal domain 2 of WT SLPI contains Leu72, which is known to be critical for the binding of serine proteases to SLPI.9 The availability of mutant forms of SLPI has allowed us to assess how replacements of Leu72 alter the anti-inflammatory activities of SLPI, in turn perhaps providing information on whether the anti-inflammatory effects of SLPI are correlated with its protease inhibitory activity. Such studies could suggest whether proteases are required for the full development of a lung inflammatory injury. In this report, we have evaluated the effects of several substitutions of Leu72 SLPI with Gly72, Phe72 ,or Lys72 and have determined to what extent these mutants demonstrate altered ability to reduce lung-vascular permeability, suppress neutrophil accumulation, and inhibit NF-{kappa}B activation in vivo. Substitutions of Leu72 are known to affect positively or negatively the anti-protease activities of SLPI (reference 9; Table 1 ). The replacement of Leu72 with Gly72 dramatically reduced the binding activity of mutant SLPI for all three proteases (Table 1) . Gly72 SLPI also lost its in- vivo protective effects in the inflammatory model, having no effect on lung-vascular permeability (Figure 1) , neutrophil accumulation (Figure 2) , or NF-{kappa}B activation (Figure 3) . The replacement of Leu72 with Phe72 increased binding interactions of mutant SLPI with chymotrypsin (by 40-fold) and for trypsin (by 10-fold), but the binding for elastase was decreased (by 2.5-fold). This mutant was similar to WT SLPI in terms of the suppression of neutrophil recruitment and intrapulmonary NF-{kappa}B activation. Perhaps the most dramatic effects were found with Lys72 SLPI, in which the binding interactions for chymotrypsin and elastase are known to be reduced by 100-fold and 2000-fold, respectively, but the binding interactions for trypsin were increased by more than 1000-fold (Table 1) . This mutant form of SLPI was most effective in reducing lung-vascular permeability, in suppressing neutrophil accumulation, and in inhibiting NF-{kappa}B activation in vivo.

There are several possible explanations for these observations. Proteases can directly activate phagocytic cells, resulting in the enhanced generation of oxidants and cytokines/chemokines.19 Trypsin and related proteases (elastase and cathepsin G) are known to be able to cleave the fifth component of the complement (C5), releasing the powerful anaphylatoxin, C5a,20 which is a potent agonist for phagocytic cells.21-23 The recent demonstration that a powerful synthetic inhibitor of elastase is protective in the same model of lung injury24 provides additional evidence that the serine proteases are somehow involved in the processes leading to injury in this inflammatory model. It is also possible that SLPI proteins gain access to the intracellular environment of cells, such as lung macrophages, which are known to be essential for the chain of events leading to lung damage25 and blocks intracellular proteases involved in signal transduction and NF-{kappa}B activation events. The cellular internalization of SLPI has been suggested in a report demonstrating that SLPI and SLPI mutants bind with a high affinity to a 55-kd receptor on the surface of monocytes.5 Surface binding was followed by a rapid decrease in the ability to detect SLPI on the cell surface or in supernatants.5

The activation of the transcription factor, NF-{kappa}B, has been shown to be a required event for the development of lung-inflammatory injury induced by IgG immune complexes16,26 and by systemic administration of LPS.27 Under normal conditions, NF-{kappa}B is retained in the cytoplasm complexed to inhibitory proteins of the I{kappa}B family. In response to inflammatory stimuli, I{kappa}B proteins are phosphorylated, ubiquitinated, and degraded by the multicatalytic proteinase complex (MCP).28 Inhibitors of the MCP have been shown to attenuate the activation of NF-{kappa}B.29 The MCP possesses chymotrypsin and trypsin-like activities,30 which may suggest potential points of action for the inhibitory effects of SLPI proteins. If SLPI is able to bind to a cell-surface receptor and be internalized, as has been suggested elsewhere,5 it could be processed or complexed, possibly with the MCP. The data contained in the current report suggests that SLPI blocks activation of NF-{kappa}B in association with its protease inhibitory activities. The inability of another serine protease inhibitor, {alpha}1-PI, to block intrapulmonary activation of NF-{kappa}B suggests that a secreted extracellular serine protease may not be involved in the NF-{kappa}B activation process. Evidence supporting the internalization of uncomplexed {alpha}1-PI has not been reported. Alternatively, the relatively large size of {alpha}1-PI (53 kd) compared with SLPI (12 kd), may also prevent this molecule from gaining access to the intracellular environment.

In summary, mutant forms of SLPI with various degrees of antiprotease capacity have been applied to a model of inflammatory lung injury in rats and compared with the effects of WT SLPI, which we have recently shown to have potent anti-inflammatory activity. The protective effects of mutant SLPI forms appear to correlate with their antiprotease activity, especially with a trypsin inhibiting capacity. It is suggested that the anti-inflammatory effects of SLPI may be linked to intracellular protease activity that is required for activation of NF-{kappa}B.


   Acknowledgements
 
We thank Beverly Schumann for her secretarial assistance.


   Footnotes
 
Address reprint requests to Peter A. Ward, MD, Department of Pathology, The University of Michigan Medical School, M5240 Medical Science I, Box 0602, 1301 Catherine Road, Ann Arbor, MI 48109-0602. E-mail: pward{at}umich.edu

Supported by grants from the National Heart Lung and Blood Institute (PO1-HL-31963) and National Institute of General Medical Sciences (NIH-GM-29507).

Accepted for publication November 28, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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