Introduction

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Interleukin (IL)-1 is a pleiotropic cytokine produced by activated mononuclear phagocytes, keratinocytes, and many other cell types (1). It has both immunologic and nonimmunologic activities, including the stimulation of B- and T-cells, induction of the synthesis of acute-phase proteins, activation of other proinflammatory cytokines such as IL-8, and changes in behavior, such as anorexia in animals with acute bacterial infections (1, 2). IL-1 consists of a family of three structurally related proteins, of which two are agonists (IL-1 and IL-1) and the third, the IL-1 receptor antagonist (IL- 1ra), is a competitive antagonist (3).

The two agonists IL-1 and - are the products of separate genes and have different amino-acid sequences; however, they have similar three-dimensional structures and they both bind to the IL-1 receptors I and II (IL-1RI and IL-1RII), although with different relative affinities (4). Most IL-1 remains in the cytoplasm of the cell, although some becomes associated with the cell membrane and has biologic activity that may be important in local cell-to-cell signaling. IL-1 is released from producing cells into the extracellular space and may reach the circulation. The pro-IL-1 has little biologic activity, and requires cleavage by a specific IL-1 converting enzyme (ICE) for activation (5).

The naturally occurring IL-1 receptor antagonist, IL-1ra, is produced by the same cells that produce IL-1 (3, 4). IL-1ra has a Mr of 17.5 kD; it has 30% amino-acid sequence homology to IL-1 and 19% homology to IL-1. Unlike IL-1 and IL-1, IL-1ra has a classic leader sequence and is synthesized, processed, and secreted as a 22-kD glycosylated protein (4). IL-1ra was initially isolated from mononuclear cells, but has since been identified as being produced by many cell types such as keratinocytes and respiratory epithelial cells (4). It is the natural antagonist to the activities of IL-1 and IL-1, binding principally to IL-1RI and, less avidly, to IL-1RII (the decoy receptor) without signal transduction (6).

There are many unanswered questions about the role of IL-1 in inflammatory lung diseases such as asthma, adult respiratory distress syndrome (ARDS), and cystic fibrosis (CF). Increased concentrations of IL-1 have been demonstrated in the plasma and bronchoalveolar lavage fluid (BALF) of individuals with these conditions (7). However, the presence of IL-1 alone does not establish a causal relationship between IL-1 production and the inflammatory changes in the lungs in these diseases. Moreover, increased levels of other cytokines, such as tumor necrosis factor (TNF) and IL-6, are often found in the same conditions. It would be valuable to delineate the contribution of IL-1 to the lung inflammation of these diseases, since this activity could potentially be modulated by IL-1ra therapy.

The goal of this study was to develop an animal model that would allow investigation of the relative contribution of IL-1 to the mechanism of inflammatory lung diseases. Transgenic mice containing human IL-1ra (hIL-1ra) complementary DNA (cDNA) under the transcriptional control of the 5' flanking region of the SP-C gene (SP-C promoter) were generated. Since the human SP-C promoter confers distal bronchiolar and alveolar expression to transgenes (10), its use allowed us to determine the effects of IL-1ra in the distal airway. Results from studies in which mice were challenged with IL-1 demonstrated partial protection by IL-1ra expression in the distal airway.

	Materials and Methods

Top Abstract Introduction Materials & Methods Results Discussion References

Materials

Polymerase chain reaction (PCR) primers were synthesized at the University of Cincinnati Oligonucleotide Facility. Salmon sperm DNA and agarose were purchased from Life Technologies (Gaithersburg, MD). Phenol, phenol chloroform, isoamyl alcohol (25:24:1 vol/vol), butanol, dimethylsulfoxide (DMSO), diethylpyrocarbonate (DEPC), bovine serum albumin (BSA), tetramethylbenzidine (TMB), Salmonella typhimurium lipopolysaccharide (LPS), and hexadecyltrimethyl ammonium bromide (HETAB) were obtained from Sigma Chemical Co., St. Louis, MO. The mice were of the FVB/N strain from the transgenic core facility of The Children's Hospital Research Foundation, Cincinnati, OH. Recombinant murine IL-1 was purchased from R&D Systems, Minneapolis, MN.

Development of Transgenic Mice

Transgenic mice were developed containing human interleukin-1 receptor antagonist protein (IRAP) cDNA under the transcriptional control of the 3.7-kb 5' flanking region of the human SP-C gene. The IRAP cDNA was cloned by reverse transcription-PCR (RT-PCR) amplication of total RNA from the human monocyte cell line U937. Sequence analysis was used to confirm that the cDNA was intact, and the cDNA was cloned into a vector containing the 3.7-kb SP-C 5' flanking region. A polyadenylation signal was present immediately downstream from the IRAP cDNA. This construct was injected into the male pronuclei of mouse zygotes, which were then introduced into the uteri of pseudopregnant mice. Transgenic mice were identified by Southern blot analysis. Lung expression of the IRAP gene was confirmed by Northern blot analysis, using human IRAP cDNA as a probe. The mice were maintained in a specific-pathogen-free (SPF) environment.

Histology

Whole lungs from mice 8 to 12 wk old were fixed in 4% paraformaldehyde as previously described (11). After dehydration in a series of ethanol solutions, the tissues were embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E). All sections were evaluated by a blinded observer.

Quantification of IL-1ra by Enzyme Immunoassay

The presence of human IRAP was detected in BALF by enzyme immunoassay (EIA). EIA plates (Corning Costar, Cambridge, MA) were coated with 100 µl of 5 µg/ml goat antihuman IL-1ra neutralizing antibody (R&D Systems) diluted in 0.1 M carbonate buffer (pH 9.6), and were incubated at 4°C for 16 h. The plates were then washed three times with washing buffer (1× phosphate-buffered saline [PBS], pH 7.4; 0.5% Tween-20). After washing of the plates, they were incubated with blocking buffer (1× PBS, pH 7.4; 0.5% Tween-20; 2% BSA) at 37°C for 2 h. To prepare a standard curve, serial dilutions of a standard, purified recombinant human IL-1ra (R&D Systems) were prepared in blocking buffer and added to the plates. BALF samples (100 µl) or standards were added to the plates in duplicate, followed by incubation for 2 h at 37°C, washing three times, and blocking for 15 min at room temperature. Plates were then incubated with 100 µl of 10 µg/ml rabbit antihuman IL-1ra (Genzyme, Cambridge, MA) at 37°C for 2 h. After incubation, the plates were washed three times with washing buffer and incubated with 100 µl of 2 µg/ml goat antirabbit IgG (whole molecule) horseradish peroxidase-conjugated antibody (CalBiochem, San Diego, CA) at 37°C for 2 h. Plates were washed three times with washing buffer, incubated with 150 µl K Blue Substrate (Neogen, Lexington, KY), and color developed for 20 min at room temperature. Following color development, the plates were read with an automated microplate reader (Molecular Devices, Menlo Park, CA) at a wavelength of 650 nm. The EIA used for IL-1ra is a modification of a standard method (12); the range of sensitivity is from 10 pg/ml to 10 ng/ml.

Intratracheal Administration of Murine IL-1

For the study of the IL-1ra transgenic mice, we adapted a previously characterized rat model of IL-1-induced acute lung inflammation (13, 14). Briefly, FVB/N mice at least 8 wk of age were anesthetized with isoflurane. Intratracheal injections were given with a 30-gauge needle after blunt dissection of the soft tissues of the neck to expose the trachea. One-hundred microliters of vehicle (sterile PBS with 0.1% BSA) or 100 µl of vehicle containing 100 ng murine IL-1 in sterile PBS with 0.1% BSA were instilled intratracheally. The dose of murine IL-1 was chosen on the basis of the dose-response curve in rats (16). The incisions were closed with Nexaband adhesive (UPL, Phoenix, AZ) and the mice were allowed to recover. These experiments were approved by the Institutional Animal Use Committee of the University of Cincinnati.

Bronchoalveolar Lavage

In order to quantify bronchial and alveolar inflammation following IL-1 instillation, bronchoalveolar lavage (BAL) was performed. Animals were killed with sodium pentobarbital (Nembutal) (10 mg), and the trachea was isolated by blunt dissection. The upper portion of the trachea was incised and then cannulated with S/P medical grade silicone tubing (0.047 OD) attached to a blunt needle; BAL was performed with 1 ml saline, which was then withdrawn. The BAL supernatant was stored at 80°C until EIAs could be performed; cell pellets were resuspended in 650 µl PBS (50 µl was used for total cell counts, and the rest of the cells were used to make cytospin preparations with a Cytospin 2 centrifuge [Shandon, Pittsburgh, PA]). The slides were stained with Giemsa and allowed to dry in air before a differential cell count was performed.

Measurement of Lung Myeloperoxidase Activity

Lung neutrophil content was assessed through myeloperoxidase (MPO) activity as an indirect measurement. Lung MPO content was measured colorimetrically with a modification of the microtiter assay system previously described by Stark (15) and Remick (16). Briefly, whole lungs were removed and washed in sterile saline, blotted dry, and weighed to determine a wet weight. The lungs were then homogenized in 3 ml of 100 mM sodium acetate, pH 6.0; 0.5% HETAB; and 5 mM EDTA. The homogenate was sonicated and then centrifuged at 13,000 × g for 15 min. The supernatant was mixed at a ratio of 1:7.5 in assay buffer (3.2 mM TMB and 1.0 mM H₂O₂) in a microtiter plate. The plate was read immediately at 650 nm over a period of 4 min. MPO units were calculated as the change in absorbance over time/whole-lung wet weight.

RT-PCR Analysis of Mouse Lung RNA Cytokine Expression

The following primer pairs were used:

-actin:

  Top 5'-GTGGGCCGCTCTAGGCACCAA-3'

  Bottom 5'-CTCTTTGATGTCACGCACGATTTC-3'

MIP-1:

  Top 5'-ACTGCCCTTGCTGTTCTTCTCT-3'

  Bottom 5'-AGGCATTCAGTTCCAGGTCAGT-3'

MIP-2:

  Top 5'-TGCTGGCCACCAACCAGG-3'

  Bottom 5'-TCAGTTAGCCTTGCCTTTGTT-3'

TNF-:

  Top 5'-CCAGACCCTCACACTCAGAT-3'

  Bottom 5'-AACACCCATTCCCTTCACAG-3'

IL-1:

  Top 5'-GTTCTGCCATTGACCATCTCTC-3'

  Bottom 5'-CCAGAAGAAAATGAGGTCGGTC-3'.

cDNA were synthesized from whole-lung RNA obtained with the Phase Lock Gel system (5 Prime-3 Prime, La Jolla, CA). Two micrograms of RNA were added to 10 U ribonuclease (RNAse) inhibitor (Boehringer Mannheim, Indianapolis, IN), 0.5 µg of oligo-deoxythymidine (oligo-dT) (Life Technologies). This mixture was incubated for 10 min at 70°C and then chilled immediately on ice. After this incubation, 0.5 mm deoxynucleotide triphosphate (dNTP), 1 mm dithiothreitol (DTT), and first-strand buffer were added to a total volume of 20 µl and incubated for 2 min at 42°C. One microliter of Superscript II reverse transcriptase (Life Technologies) was added, and the reaction was incubated for 50 min at 42°C. The reaction was inactivated at 70°C for 15 min. For PCR, 2 µl of the cDNA mixture was added to 0.1 mM dNTP, 0.5 µg each of the top and bottom primers, and 1 µl Taq DNA polymerase (10 U) in PCR buffer in a total volume of 100 µl. This mixture was covered with mineral oil and incubated at 94°C for 5 min. The mixture was then put through cycles consisting of 95°C for 30 s, annealing at 59°C for 30 s, and extending at 72°C for 30 s for a total of 30 cycles, followed by a final extending cycle for 7 min at 72°C. PCR products were assessed by gel electrophoresis through a 2% agarose gel containing ethidium bromide in TAE electrode buffer (0.04 M Tris-acetate and 0.002 M EDTA). Gels were photographed under UV light and the bands quantitated with the Digital Imaging System IS-1000 (Alpha Innotech Corp., San Leandro, CA).

Statistics

A one-way analysis of variance (ANOVA) test (Quattro Pro; Borland International, Scotts Valley, CA) was used to test for significant differences between the experimental groups overall, and comparisons between any two groups were made with Student's t test.

	Results

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Identification and Analysis of Transgenic Mice

Three founder lines were obtained, and their hemizygous offspring were identified for the presence of the human IL-1ra sequence by Southern analysis. These lines were maintained, and one of them was chosen for further study based on consistent high-level expression of IL-1ra mRNA by Northern analysis (Figure 1) and enzyme-linked immunosorbent assay (ELISA) of BAL. The concentration of hIL-1ra in BALF from these mice was 2.45 ng/ml ± 0.28 (SD) (n = 12), and the mean serum concentration was 6.6 pg/ml (range: 0 to 18 pg/ml in five animals); no IL-1ra could be detected in the BALF or serum from nontransgenic animals. Four controls and four transgenic mice were examined histologically by a blinded veterinary pathologist. There were no significant differences in lung histology between the transgenic mice and the nontransgenic littermates.

View larger version (46K): [in this window] [in a new window]   Figure 1.   Representative autoradiograph of the IL-1ra and -actin signals from Northern analysis of six transgenic mice (Q2-C10) compared with two wild-type controls (R5-Q1), demonstrating expression of human IL-1ra in all the transgenic mice.

Evaluation of Lung Protection by Overexpression of IL-1ra

To determine the biologic efficacy of the overexpression of IL-1ra, mice were challenged by intratracheal administration of recombinant murine IL-1. They were injected with 100 ng murine IL-1, allowed to recover, and killed at 6 h. Differential cell counts on BAL samples showed a significant reduction in the number of polymorphonuclear neutrophils (PMN) at 6 h in the transgenic mice (14.8% ± 4.2) as compared with controls (58.3% ± 7.6; n = 10; P = 0.0005) (Figure 2).

View larger version (24K): [in this window] [in a new window]   Figure 2.   Neutrophil count following lung challenge with mouse recombinant IL-1. The percentages of neutrophils in BALF are shown for transgenic mice with lung-specific expression of human IL-1ra (hIL-1ra), compared with transgene-negative (wild-type) littermate controls (Wt) 6 h after challenge with 100 ng intratracheal murine IL-1 or vehicle (sterile PBS with 0.1% BSA). (n) = number of animals per group. *P < 0.001 versus wild-type controls that received vehicle. **P < 0.001 versus wild-type mice that received IL-1.

Visual examination of the lung specimens showed occasional, focal, perivascular accumulations of neutrophils in the lungs of the IL-1-treated mice, but there was no significant difference, by standard histologic evaluation, between the IL-1-treated wild-type mice and those that received saline (data not shown).

Evaluation of the wet weights of the lungs as a measure of lung leak showed that the lungs of the IL-1-treated wild-type mice weighed significantly more than those of the saline-treated wild-type controls (mean: 0.175 ± 0.017 g [SD] versus 0.151 ± 0.020 g; P < 0.01 by Student's t test, n = 10 per group), but there was no significant difference between the weights of the IL-1-treated wild-type mice and the IL-1-treated transgenic mice (0.175 ± 0.017 g versus 0.167 ± 0.024 g, n = 10 per group).

To measure PMN accumulation in the lungs, MPO assays were performed on whole-lung homogenate. The results showed an increase in MPO activity (reflective of increased neutrophil content) following IL-1 administration, which was attenuated significantly in the transgenic mice (Figure 3).

View larger version (33K): [in this window] [in a new window]   Figure 3.   Neutrophil content of lungs following intratracheal challenge with IL-1, as assessed by MPO activity. The MPO activity of whole-lung homogenate was determined for hIL-1ra transgenic mice (IL-1ra) as compared with transgene-negative littermate controls (Wt) at 6 h after challenge with 100 ng intratracheal murine IL-1 or vehicle (sterile PBS with 0.1% BSA). (n) = number of animals per group. *P < 0.001 versus wild-type controls that received vehicle. **P < 0.001 versus wild-type mice that received IL-1.

Cytokine Expression following IL-1 Administration

RT-PCR analysis was performed to assess the degree of activation of the cytokines IL-1, MIP-1, MIP-2, and TNF-. The results showed a significant increase in the mRNA level for MIP-1 and MIP-2 at 6 h after intratracheal IL-1 administration, with a significant attenuation in the transgenic mice (Figure 4). IL-1 mRNA was found to be expressed constitutively, and did not change following IL-1 administration; there was no detectable TNF- mRNA in this assay.

View larger version (23K): [in this window] [in a new window]   View larger version (21K): [in this window] [in a new window]   Figure 4.   MIP-1 and MIP-2 mRNA levels following intratracheal challenge with mouse recombinant IL-1. mRNA/-actin ratios were determined by RT-PCR analysis of whole-lung mRNA from hIL-1ra transgenic mice, as compared with transgene-negative littermate controls, at 6 h after challenge with 100 ng intratracheal murine IL-1 as compared with vehicle (sterile PBS with 0.1% BSA). Numbers of animals are shown in parentheses. (a) MIP-1: *P < 0.00005 versus wild-type controls that received vehicle (sterile PBS with 0.1% BSA). (b) MIP-2: *P < 0.000005 versus wild-type controls that received vehicle (sterile PBS with 0.1% BSA). **P < 0.0005 versus wild-type control mice that received IL-1.

	Discussion

Top Abstract Introduction Materials & Methods Results Discussion References

IL-1 is an important proinflammatory mediator that, like TNF, increases early in the time course of diseases such as sepsis and endotoxemia, whereas IL-6 and IL-8 increase later. In the lung, it is likely that IL-1 has an important role in the pathophysiology of ARDS (9), asthma (17), interstitial pulmonary fibrosis (18), CF (7, 19, 20), and chronic lung disease of prematurity (21). We therefore developed an animal with protection against the proinflammatory effects of IL-1 that would allow the role of IL-1 in these diseases to be defined.

The transgenic mice developed for this study, with lung-specific production and secretion of hIL-1ra by the respiratory epithelium, are healthy and fertile, indicating that inhibition of IL-1 activity was not toxic or embryonically lethal. hIL-1ra mRNA transcription was detected by Northern analysis of whole-lung mRNA. Previous studies demonstrated that the human 3.7-kb SP-C promoter directs transgene expression in the murine lung in bronchial and alveolar respiratory epithelial cells by 10 to 11 days of gestation (22).

The IL-1ra expressed in these animals was the 17.5-kD human protein, which shares 77% amino-acid homology with the murine protein (23). We have not demonstrated that the human protein binds directly to murine IL-1 receptors, although other investigators have shown this in vitro for both type I and type II IL-1 receptors on murine cell lines (24, 25). Human IL-1ra has also been shown to protect mice against the lethal effects of endotoxin, presumably by blocking the murine IL-1 receptor in vivo (26).

The hypothesis that the transgenic mice used in the study have partial protection against the actions of IL-1 was tested by intratracheal injection of murine recombinant IL-1 in a modified rodent model of IL-1-induced lung injury (27). IL-1 was used rather than IL-1 on the basis of earllier studies (27, 28). Initial experiments, in which human IL-1 was used, did not lead to any changes in the cell differential count or the histologic appearance of the murine lung, unlike the reported experience with the rat model (27). When recombinant murine IL-1 was used, there was a marked increase in the percentage of neutrophils in BALF at 6 h, which correlated with an increase in the MPO content of whole-lung homogenate. By contrast, the transgenic mice with respiratory epithelial expression of hIL-1ra showed a reduction in the percentage of neutrophils and in the MPO activity of the whole-lung homogenate. These results support earlier studies of rats given intratracheal IL-1, in which treatment with hIL-1ra before and after insult decreased lung leak, lavage leukocytes, and lavage neutrophils, and in which treatment before insult decreased lung MPO activity (28). It is interesting to note that these rats were given 100 mg/kg hIL-1ra subcutaneously, and achieved plasma concentrations of 10,000 to 15,000 ng/ml, which were much greater than were demonstrated in BALF from our animals (2.45 ng/ ml), even accounting for the dilution that is involved in the sampling of BALF. By having apically directed release of hIL-1ra, we were able to achieve similar protective effects against intratracheal IL-1 with a much smaller quantity of hIL-1ra than in the earlier study (28).

IL-1-induced neutrophil immigration into the lung is potentially mediated by activation of several chemokines. RT-PCR was used as a semiquantitative method to investigate the relative degrees of activation of IL-1, TNF-, MIP-1, and MIP-2. Other chemokines are potentially important in this model, such as KC and Gro (29), but they were outside the scope of the study. The reduction in ratio of MIP-1 and MIP-2 mRNA/-actin associated with a reduction in the accumulation of neutrophils in the lung provides evidence for the role of these two chemokines in neutrophilic inflammation in the murine lung (30, 31), and indicates that the inflammatory response to intratracheal IL-1 may be partly mediated by the activation of these chemokines. Although we did not ascertain the cellular sources of the chemokines, our data are consistent with other studies that have shown that IL-1 induces expression of MIP-1 by monocytes and macrophages in vitro (32), and increased expression of MIP-2 in airway mononuclear cells and airway epithelium in rats treated with intratracheal IL-1 4 h previously (33).

In conclusion, we have generated a transgenic mouse in which respiratory epithelial cells express hIL-1ra as shown by ELISA assay and Northern analysis. These mice have a normal phenotype and are partly protected against the proinflammatory effects of intratracheal IL-1 administration. They will be useful in elucidating the role of IL-1 in inflammatory disorders of the lungs. The effects of hIL-1ra in this study also suggest that therapeutic administration of hIL-1ra to the airway could be beneficial in the treatment of diseases such as ARDS, since higher local levels of the receptor antagonist achieved by airway administration should be more effective at reducing pulmonary inflammation than intravenous administration.