Inhibition of NF-κB Activation and Augmentation of IκBβ by Secretory Leukocyte Protease Inhibitor during Lung Inflammation

Reagents

Recombinant human SLPI and tissue inhibitor of metalloproteinases-2 (TIMP-2) were kindly provided by Dr. Thomas R. Ulich, (Amgen Inc., Thousand Oaks, CA). Rabbit polyclonal antibody to rat SLPI was prepared as described elsewhere. 1 New Zealand White rabbits were immunized with rat SLPI in incomplete Freund’s adjuvant. Immune serum (10⁻⁶ titer) was affinity purified, and the IgG administered intratracheally at a dose of 300 μg.

IgG Immune Complex-Induced Alveolitis

Pathogen-free male Long-Evans rats (275 to 300 g; Harlan Sprague-Dawley, Indianapolis, IN) were anesthetized with Ketamine HCl (150 mg/kg, intraperitoneally). A total of 1.5 mg of rabbit polyclonal IgG anti-bovine serum albumin (BSA) (ICN Biomedicals, Inc., Costa Mesa, CA) in a volume of 0.3 ml of phosphate-buffered saline (PBS) was instilled via an intratracheal catheter during inspiration. Immediately thereafter, 10 mg of BSA in 0.5 ml of PBS were injected intravenously. Negative control rats received PBS intratracheally. For analysis of pulmonary vascular permeability, trace amounts of ¹²⁵I-labeled BSA were injected intravenously. Four hours after IgG immune complex deposition, rats were exsanguinated, the pulmonary circulation was flushed with 10 ml of PBS by pulmonary artery injection and the lungs were surgically dissected. The extent of lung injury was quantified by calculating the lung permeability index (dividing the amount of radioactivity (¹²⁵I-labeled BSA) in the perfused lungs by the amount of radioactivity in 1.0 ml of blood obtained at the time of death). For analysis of NF-κB and MAPK activation, lungs were immediately frozen in liquid nitrogen after vascular perfusion with PBS.

NF-κB Activation

Nuclear extracts of whole lung tissues and alveolar macrophages were prepared as previously described 20 and analyzed by electrophoretic mobility shift assay (EMSA). Briefly, double-stranded NF-κB consensus oligonucleotide (5′-GTGAGGGGACTTTCCCAGGC-3′; Promega, Madison, WI) was end-labeled with γ[³²P]ATP (3000 Ci/mmol at 10 mCi/ml, Amersham, Arlington Heights, IL). Binding reactions containing equal amounts of nuclear protein extract (10 μg) and 35 fmols (∼50,000 cpm, Cherenkov counting) of oligonucleotide were performed for 30 minutes in binding buffer (4% glycerol, 1 mmol/L MgCl₂, 0.5 mmol/L EDTA, pH 8.0, 0.5 mmol/L dithiothreitol, 50 mmol/L NaCl, 10 mmol/L Tris, pH 7.6, 50 μg/ml poly(dI-dC); Pharmacia, Piscataway, NJ). Reaction volumes were held constant to 15 μl. Reaction products were separated in a 4% polyacrylamide gel in 0.25 × TBE buffer (25 mmol/L Tris, 22.5 mmol/L boric acid, 0.25 mmol/L EDTA) and analyzed by autoradiography. NF-κB activation was quantitated from digitized autoradiography films using image analysis software (Adobe Systems, San Jose, CA).

MAPK Activation and Western Blot Analysis

Whole lung tissue or alveolar macrophages obtained by BAL (bronchoalveolar lavage) were homogenized in lysis buffer (10 mmol/L HEPES, pH 7.9, 150 mmol/L NaCl, 1 mmol/L EDTA, 0.6% Nonidet P-40, 0.5 mmol/L phenylmethylsulfonyl fluoride, 1 μg/ml leupeptin, 1 μg/ml aprotinin, 10 μg/ml soybean trypsin inhibitor, 1 μg/ml pepstatin) on ice. Homogenates were sonicated and centrifuged at 14,000 rpm to remove cellular debris. Interfering IgG anti-BSA in homogenates was removed with GammaBind G sepharose (Pharmacia). Protein concentrations were determined as described for nuclear extracts. Samples (10 μg for MAPK analysis, 100 μg for IκB proteins) were separated in a denaturing 12.5% polyacrylamide gel and transferred to a nitrocellulose membrane. Nonspecific binding sites were blocked with Tris-buffered saline containing Tween 20 (40 mmol/L Tris, pH 7.6, 300 mmol/L NaCl, 0.1% Tween 20) containing 5% nonfat dry milk for 12 hours at 4°C. For analysis of MAPK proteins, membranes were incubated in a 1:1000 dilution of rabbit polyclonal anti-phospho p42/p44 or anti-nonphosphorlyated p42/p44 (New England Biolabs, Inc., Beverly, MA). For analysis of IκB proteins, membranes were incubated in a 1:1000 dilution of rabbit polyclonal anti-IκBα or anti-IκBβ (Santa Cruz Biotechnology, Santa Cruz, CA) in TBST. After three washes in TBST, membranes were incubated in a 1:50,000 dilution of horseradish peroxidase-conjugated donkey anti-rabbit IgG (Amersham Corp., Arlington Heights, IL). Immunoreactive proteins were detected by enhanced chemiluminescence.

Northern Blot Analysis

Total RNA from whole lung tissue was extracted using a guanidinium-isothiocyanate method as described previously. 23 Samples (20 μg of RNA) were fractionated electrophoretically in a 1% formaldehyde gel and transferred to a nylon membrane (MSI, Westboro, MA). cDNA for rat IκBβ was generated by reverse transcriptase-polymerase chain reaction using the following oligonucleotide primers based on the murine sequence: 24 5′ primer: 5′-CATGTAGCTGTCATCCACA-3′ and 3′ primer: 5′-TGTGCACGGAGGAGGCG-3′. The polymerase chain reaction product was sequenced for verification, and the rat IκBβ cDNA probe was end labeled with [³²P]dCTP (NEN-DuPont, Boston, MA) using Random Prime (Amersham Corp.). Northern blots were hybridized with the cDNA probe in QuikHyb^® hybridization solution (Stratagene Cloning Systems, La Jolla, CA) at 68°C for 4 hours and an autoradiogram developed on Kodak BioMax-MR film (Rochester, NY). Equal loading of RNA samples was confirmed by probing Northern blots with cDNA to β-actin (Stratagene Cloning Systems) end-labeled with [³²P]dCTP.

BAL Fluid Analyses

BAL fluids were collected by instilling and withdrawing 5 ml of sterile PBS three times from the lungs via an intratracheal cannula. BAL content of TNF-α was measured using a standard WEHI cell cytotoxicity assay as previously reported. 25 Measurement of macrophage inflammatory protein-2 (MIP-2), cytokine-induced neutrophil chemoattractant (CINC), and C5a in BAL fluids were by enzyme-linked immunosorbent assay as described elsewhere. 26,27

Lung Vascular Expression of ICAM-1

Rats were injected intravenously with 1.5 μCi of ¹²⁵I-labeled anti-ICAM-1 (clone 1A29; PharMingen, Inc. San Diego, CA) 3.75 hours after induction of lung injury. The specific activity of the anti-ICAM-1 was 11.3 μCi/μg. Fifteen minutes later (4 hours after induction of lung injury), rats were sacrificed, and the lung vasculature was flushed with 10 ml of PBS. Lung vascular ICAM-1 expression (binding index) was calculated by dividing the amount of radioactivity (¹²⁵I-labeled antibody) in lungs by the amount of radioactivity in 1.0 ml of blood. To control for nonspecific binding and potential accumulation of anti-ICAM-1 antibody in lung parenchyma caused by injury, 1.5 μCi of ¹²⁵I-labeled nonspecific isotype-matched mouse IgG (clone MOPC-21, ICN Biomedicals, Inc.) were administered in a separate set of rats. The specific activity of the irrelevant IgG antibody was 13.0 μCi/μg.

Lung Myeloperoxidase (MPO) Content

Whole lung MPO activity was quantitated as described previously. 28 Briefly, whole lungs homogenates were diluted in 50 mmol/L potassium phosphate buffer containing 0.5% hexadecyltrimethylammonium bromide, pH 6.0. After sonication and two freeze-thaw cycles, samples were centrifuged at 4000 × g for 30 minutes. The supernatants were reacted with H₂O₂ (0.3 mmol/L) in the presence of tetramethylbenzidine (1.6 mmol/L). MPO activity was assessed by measuring the change in absorbance at 655 nm.

Statistical Analyses

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

Effects of SLPI on IgG Immune Complex-Induced Lung Injury

Previous studies demonstrated that when administered intratracheally, 1 mg of SLPI suppressed IgG immune complex-induced lung injury. 21 To determine the lowest effective dose of intratracheally administered SLPI, dose response experiments were performed with the endpoint of extravascular leakage of ¹²⁵I-albumin. As shown in Figure 1 ▶ , neither 5 nor 25 μg of SLPI (given intratracheally along with the anti-BSA) caused any significant reduction in IgG immune complex-induced lung vascular permeability. However, 100 μg of SLPI reduced lung vascular permeability by 45% (P = 0.006). TIMP-2 had similar effects, reducing albumin leak by 40% (Figure 1) ▶ . The effects of 100 μg SLPI on IgG immune complex-induced increases in proinflammatory mediators in BAL fluids were assessed. As anticipated, intrapulmonary deposition of IgG immune complexes caused significant increases in BAL content of TNF-α, MIP-2, CINC, and C5a (Table 1) ▶ . Co-treatment with 100 μg of SLPI did not significantly alter BAL levels of any of these proinflammatory mediators.

Because of a key role for vascular ICAM-1 in this lung inflammatory model, experiments were designed to determine whether SLPI had any effect on the expression of ICAM-1 in the pulmonary vasculature. Intrapulmonary deposition of IgG immune complexes caused the expected increase in expression of lung vascular ICAM-1 (Figure 2) ▶ . The increase in binding index for the anti-ICAM-1 antibody could not be attributed to nonspecific binding or sequestration from lung injury as the binding index for the irrelevant IgG control antibody was only 0.091 ± 0.011 in inflamed lungs (versus 0.065 ± 0.015 in noninflamed lungs). In the presence of 100 μg of SLPI, there was a 67% reduction (P = 0.041) in ICAM-1 expression in the pulmonary vasculature. Because SLPI caused a substantial decrease in lung vascular ICAM-1 expression, we examined whether this effect was associated with decreased lung recruitment of neutrophils. Pulmonary accumulation of neutrophils was quantitated by lung content of MPO. As expected, intrapulmonary deposition of IgG immune complexes caused a greater than sevenfold increase in lung MPO content (Figure 3) ▶ . Co-treatment with SLPI reduced IgG immune complex-induced lung MPO content by 39% (P < 0.001).

Effects of SLPI on Intrapulmonary Activation of NF-κB and MAPK

To investigate whether the protective effects of SLPI might be related to inhibition of pulmonary NF-κB activation (defined as nuclear translocation), nuclear extracts from whole lungs harvested 4 hours after IgG immune complex deposition were analyzed by EMSA. As expected, little NF-κB was present in lung nuclei from rats treated intratracheally with PBS (Figure 4) ▶ . Treatment with IgG immune complexes resulted in a significant increase in nuclear translocation of NF-κB. In the presence of 100 μg of SLPI, NF-κB activation was greatly reduced. Image analysis of digitized EMSA blots demonstrated that the presence of SLPI reduced nuclear localization of lung NF-κB by 64% (P = 0.005). Interestingly, 100 μg of TIMP-2 had no effect on lung NF-κB activation.

Effects of SLPI on lung MAPK activation in the lung injury model were assessed by Western blot analysis of lung homogenates (Figure 5) ▶ . Four hours after intrapulmonary deposition of IgG immune complexes, phosphorylation (a measure of activation) of MAPK was evident. No phosphorylation of p42 or p44 was found in extracts from lungs instilled with PBS, in striking contrast to phosphorylation of p42 and p44 in inflamed lungs (Figure 5 ▶ , upper frame). Phosphorylation was primarily associated with the p44 form of MAPK (slower migrating band) as contrasted to the p42 form. It also seems likely that in this model of inflammation there is induction of p44 because the Western blot bands were more intense in lungs receiving IgG immune complexes (Figure 5 ▶ , lower frame, second lane versus third and fourth lanes). Quite clearly, co-treatment with SLPI did not seem to interfere with MAPK activation in lung.

We have previously shown that NF-κB activation in alveolar macrophages in vivo is increased as early as 30 minutes after deposition of IgG immune complexes. 29 Furthermore, in a macrophage cell line transgenically over-expressing SLPI, LPS-induced NF-κB activation was attenuated. 5 Therefore, we assessed whether 100 μg of SLPI administered intratracheally reduced NF-κB activation in alveolar macrophages following intrapulmonary deposition of IgG immune complexes. Treatment with SLPI had no effect on NF-κB activation in alveolar macrophages retrieved by BAL 30 minutes after initiation of IgG immune complex deposition (Figure 6) ▶ . Similarly, NF-κB activation in alveolar macrophages stimulated in vitro with 100 μg/ml IgG-BSA immune complexes or LPS (50 ng/ml) was unaffected by co-treatment with 10 μg/ml SLPI (data not shown). Intrapulmonary instillation of SLPI failed to inhibit MAPK activation in alveolar macrophages retrieved by BAL 30 minutes after IgG immune complex deposition (data not shown). Thus, it would appear that the effects of SLPI in lung are not directed toward alveolar macrophages.

Endogenous SLPI Regulates Lung NF-κB Activation

Because treatment with SLPI almost completely ameliorated IgG immune complex-induced NF-κB activation in whole lung tissues, we investigated whether endogenous SLPI regulated activation of lung NF-κB. For these experiments, a reduced intratracheal dose of anti-BSA (250 μg) was used to produce minimal activation of NF-κB. Endogenous SLPI was blocked by intratracheal administration of 300 μg of anti-rat SLPI together with the anti-BSA. This dose of anti-SLPI was shown to effectively neutralize endogenous SLPI in this model of lung inflammation. 1 Positive controls received 300 μg of preimmune rabbit IgG. Four hours after intrapulmonary deposition of IgG immune complexes there was a detectable increase in lung NF-κB activation (Figure 7) ▶ . In the presence of anti-SLPI there was significantly increased NF-κB activation in lung. Image analysis of digitized EMSA blots dem- onstrated that anti-SLPI increased nuclear localization of lung NF-κB by 141% (P = 0.006). Blockade of endogenous SLPI had no effect on lung MAPK activation (data not shown).

Effects of SLPI on IκBα and IκBβ

As SLPI inhibited NF-κB activation in whole lungs (Figure 4) ▶ , we designed experiments to determine whether the inhibitory effects of SLPI during lung inflammation might be related to effects on the cytoplasmic NF-κB regulatory proteins, IκBα and IκBβ. Western blot analysis of whole lung homogenates obtained 4 hours after initiation of lung injury confirmed decreases in the amounts of detectable IκBα protein after intrapulmonary deposition of IgG immune complexes as compared with intrapulmonary instillation of PBS (Figure 8A ▶ , upper panel). Neither co-treatment with SLPI nor TIMP-2 had any measurable effect on decreases in IκBα protein in the inflamed lung. In striking contrast, IgG immune complex deposition appeared to cause an increase in the amount of detectable IκBβ protein compared with treatment with PBS (Figure 8A ▶ , lower panel). Image analysis of digitized Western blots is shown in Figure 8B ▶ . Western blots of samples from lungs treated with IgG immune complexes showed a second, faint, and more rapidly migrating IκBβ band. Other studies have identified this band to be a dephosphorylated form of IκBβ, which is an intermediate during IκBβ degradation. 30 Co-treatment with SLPI resulted in a marked increase in the slower migrating, presumably hyperphosphorylated, form of IκBβ when compared with treatment with IgG immune complexes. Co-treatment with TIMP-2 had no effect on IgG immune complex-induced changes in IκBβ protein (Figure 8A ▶ , lower panel).

To assess whether SLPI might be directly augmenting the production of IκBβ and/or suppressing IκBβ degradation, rats received lung instillation of PBS, SLPI, or TIMP-2 (each at 100 μg) in the absence of IgG immune complexes. Neither SLPI nor TIMP-2 had any detectable effect on IκBα protein expression compared with treatment with PBS (Figure 9 ▶ , upper panel). However, treatment with SLPI, but not with TIMP-2, resulted in a marked increase in IκBβ protein expression compared with treatment with PBS (Figure 9 ▶ , lower panel). In these experiments only the slower migrating IκBβ band was observed, suggesting that under these conditions degraded IκBβ could not be detected.

To determine whether SLPI-induced increases in IκBβ protein expression were related to enhanced IκBβ gene activation, Northern blot analysis of IκBβ mRNA in whole lung extracts was performed. Lung extracts from rats undergoing 4 hours of injury were analyzed. As shown in Figure 10 ▶ , intrapulmonary deposition of IgG immune complexes caused a marked increase in IκBβ mRNA expression compared with PBS controls. Co-treatment with SLPI failed to provide any clear evidence for IgG immune complex-induced increases in IκBβ mRNA expression. Finally, lung instillation of SLPI in otherwise unmanipulated rats did not induce expression of mRNA for IκBβ (Figure 10) ▶ .