Introduction

Top Abstract Introduction Materials and methods Results Discussion References

Several lines of evidence suggest that destruction of interstitial matrix may result from an imbalance between proteases and antiproteases (1). Elastase and cathepsin G are thought to be two of the main causative factors of tissue damage in certain pathologic conditions, such as pulmonary emphysema (4), some immunomediated forms of glomerulonephritis (9), and experimental arthritis (12). Neutrophils are clearly implicated in the development of the extracellular matrix injury because they are the major source of these enzymes. As a consequence, neutrophil function, lysosomal enzyme content, and antiprotease screen are thought to represent key elements in the protease-antiprotease balance.

The beige mouse, a C57Bl/6J mutant with diluted pigmentation and giant lysosomes (13), has been reported to have a marked deficiency of neutrophil lysosomal proteases and thus has been widely used as a model of neutrophil cathepsin G and elastase deficiency (13). In addition, defects of neutrophil migration and degranulation have been reported for this animal (16, 17). Because of these characteristics, the beige mouse has been used for a better understanding of the role of neutrophil elastase and cathepsin G in a variety of pathologic conditions characterized by destruction of interstitial matrix (18).

However, it has been shown recently (25) that cathepsin G and a 46-kD proelastase can be detected in mature neutrophils of beige mice, and that cathepsin G is tightly bound to lysosomal membranes but is released in normal quantities in response to proinflammatory agents under in vitro (25) and in vivo conditions (26, 27). Also, in beige mice, elastase is secreted by neutrophils as a proelastase that undergoes spontaneous activation by a protease-dependent mechanism (27). Thus, these data challenge the validity of the beige mouse as a model of neutrophil elastase and cathepsin G deficiency.

To study further the role of neutrophils in beige mice, we investigated whether recruitment of neutrophils into the lungs of these mice would result in parenchymal damage and emphysema. Mice of the parent strain C57Bl/6J served as controls. This paper presents the results obtained in the course of this study.

	Materials and Methods

Top Abstract Introduction Materials and methods Results Discussion References

Source of Materials

Unless otherwise stated, all materials were obtained from Sigma Chemical Co., St. Louis, MO.

Animals

Male, 8- to 16-wk-old mice were used. Homozygous beige C57Bl/6J bg/bg and normal C57Bl/6J (+/+) mice were from our colony. The colony was started with animals originally obtained from Nossan Laboratory (Correzzana, Milan, Italy). The mice were housed in groups of two to four in macrolon cages. Room temperature was 22 to 24°C, relative humidity 40% to 50%, and food and water were supplied ad libitum.

Animal Treatment

Three groups of eight animals for each strain were used. One group received a single intratracheal instillation of 200 µg N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) in saline solution (50 µl). Another group was instilled intratracheally with the same volume of saline. All intratracheal instillations were performed under ether anesthesia. A third group was left untreated. Twenty-one days later the animals from all groups were injected with an overdose of pentobarbital sodium and exsanguinated by cutting the abdominal aorta. The lungs were then excised and processed for histologic and morphometric examination. Additionally, two animals from each group were killed 14 d after the treatment, and the lungs were processed for immunogold localization of elastase on alveolar septa.

Bronchoalveolar Lavage Cytology

To differentiate and quantitate the types of cells within the lungs after FMLP treatment, five animals of each group were killed at different time intervals. At 0, 1, 2, 3, 4, 7, and 14 d, mice were anesthetized with pentobarbital given intraperitoneally and exsanguinated by severing the abdominal aorta. The trachea was isolated and then cannulated with a 20-gauge blunt needle. With the aid of a peristaltic pump (P-1 Pharmacia, Uppsala, Sweden) the lungs were lavaged three times with 0.6 ml normal saline. The average fluid recovery was greater than 95%. When the lavage was completed, the total cell counts were performed in a hemocytometer. Differential counts of 300 cells were done on slides stained with Diff-Quik stain.

Determination of Elastase Burden by Immunoelectron Microscopy

The immunogold method (postembedding technique) was used to localize elastase in thin lung sections prepared for electron microscopy using antimouse leukocyte elastase (MLE) antibodies obtained as previously described (28). Five lung tissue blocks per animal were taken from two animals from each group. The blocks, 1 to 2 mm thick, were fixed for 3 h in 4% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2), dehydrated in acetone, and embedded in epoxy resin (Araldite) without postfixation in OsO₄. Ultrathin sections (600 Å thick) were picked up on nickel grids and pretreated with phosphate-buffered saline (PBS) containing 1% ovalbumin for 5 min. The grids were then floated on a drop of diluted anti-MLE antibodies (1:2,600) for 48 h at 4°C; the grids were thoroughly rinsed for 10 min with a mild spray of PBS and then with distilled water and transferred onto 15 µl drops of a protein A-gold particles (15 nm) (E-Y-LABS, San Mateo, CA) solution diluted 1:8 in PBS. The sections were then washed, dried, stained with uranyl acetate-lead citrate, and examined in a Philips 300 electron microscope. Ten to 12 micrographs (final magnification: ×12,000) were taken for each grid. To exclude false-positive labeling, a series of control studies (including also the use of nonimmune rabbit serum or of bovine serum albumin instead of ovalbumin) were carried out as previously described in detail (28). The density of gold particles per square micrometer of lung tissue was determined for each of the micrographs with a superimposed quadratic lattice grid. A total of 50 micrographs was thus analyzed for each animal, and the average of gold particle density on lung connective tissue of each group was calculated.

Biochemical Analysis of Lung Elastin

To quantitate lung elastin, six FMLP-treated, six saline-treated, and six untreated mice for each strain were used. Twenty-one days after the instillation, the mice were anesthetized with sodium pentobarbital and killed by severing the abdominal aorta, and the lungs were immediately removed. The lungs were weighed, immediately homogenized in ice-cold water (1:4, wt/vol) and then used for the determination of insoluble elastin. Elastin was extracted by successive extraction with 1 M NaCl, chloroform-methanol, and by hot alkali treatment at 98°C for 50 min in 0.1 N NaOH (29). The insoluble, defatted residue remaining after NaOH extraction (30) was assayed for elastin with pancreatic elastase (0.1 mg in 2 ml 0.02 M borate buffer, pH 8.8, for 3 h at 25°C) to hydrolyze the alkali-insoluble residue (operationally defined as elastin) into peptide fragments (31). After centrifugation, the supernatant peptides were determined by the method of Lowry and colleagues (32) and taken as an estimate of elastin. Elastin peptides from bovine neck ligament, obtained by elastase digestion, were used as the standard.

Lung Histology and Morphometry

The lungs from the different groups of mice were fixed intratracheally with buffered formaline (5%) at a constant pressure of 20 cm H₂O for at least 24 h. All lungs were then dehydrated, cleared in toluene, and embedded under vacuum in paraffin. Two 7-µm laterosagittal sections of each lung were cut and stained with hematoxylin and eosin. Morphometric assessment consisted of the determination of the average interalveolar distance (mean linear intercept [Lm]) (33). For each pair of lungs, 40 histologic fields were evaluated both vertically and horizontally. Examination of this number of fields meant that practically the entire lung area of each section was evaluated.

Animal Treatment with a Synthetic Serine-Proteinase Inhibitor

To assess the protease-dependence of the FMLP-induced lung lesion, a group of six animals for each strain was treated with 4-(2-aminoetyl)-benzenesulfonyl fluoride hydrochloride (Merck, Darmstadt, Germany), a potent serine proteinase inhibitor. 4-(2-Aminoetyl)-benzenesulfonyl fluoride hydrochloride, dissolved in saline at a concentration of 2.4 µg/µl, was continuously delivered at a rate of 0.5 µl/h for 2 wk by means of osmotic pumps (Alzet 2002; Alza Corp., Palo Alto, CA). The pumps were implanted subcutaneously according to the manufacturer's instructions 24 h before FMLP treatment. Twenty-one days after instillation with FMLP, the lungs were excised and processed for histologic and morphometric assessment.

Statistical Analysis

For each parameter, either measured or calculated, the values for individual animals were averaged and standard deviation (SD) was calculated. The significance of the differences between groups was calculated using one-way analysis of variance (F test) (34). A P value of less than 0.05 was considered significant.

	Results

Top Abstract Introduction Materials and methods Results Discussion References

Bronchoalveolar Lavage Cytology

The total and differential cell counts in bronchoalveolar lavage fluid (BALF) of the beige and control C57Bl/6J mice after FMLP instillation are reported in Table 1. As can be seen, no differences at various times have been observed between beige and C57Bl/6J mice belonging to the same experimental group. An average of 2.7 ± 0.2 × 10⁵ cells/animal was observed in untreated animals. At 24 and 48 h after FMLP treatment, the total BALF cell number increased more than fourfold above control levels. At 72 h the total number of the cells was still about twofold above control levels. The change in total BALF cell population after FMLP exposure was characterized by a relative increase in the number of neutrophils (approximately 100-fold) that far exceeded in absolute value the number of macrophages (10 × 10⁵ cells versus 1.5 × 10⁵ after 24 h). Similar changes in the cytologic profile of BALF were previously observed in C57Bl/6J mice and in other strains of mice after FMLP treatment (27). The values of the total and differential cell counts in BALF of the beige and control C57Bl/6J mice returned to control levels at Day 4 after FMLP challenge, remaining unaltered until Day 14. 

Elastase Burden

The number of colloidal gold particles found after immunoreaction with anti-MLE antibodies within the alveolar walls from the various groups for each strain is reported in Table 2. Similar results were observed in beige and C57Bl/ 6J mice belonging to the same experimental group. Very few colloidal gold particles were found in association with the connective tissue of the alveolar walls of untreated and saline-treated mice 14 d after saline instillation (Figure 1). However, 14 d after FMLP treatment a significant increase in gold particle density was observed on lung interstitium and was associated with the amorphous component of elastic fibers (Figure 2).

View larger version (143K): [in this window] [in a new window]   Figure 1.   Electron micrograph of an alveolar wall from a saline-injected control mouse after immunostaining with anti-MLE IgG. Few or no colloidal particles are found on interstitium. Original magnification: ×34,000.

View larger version (151K): [in this window] [in a new window]   Figure 2.   Pulmonary parenchyma from an FMLP-treated beige mouse after immunolocalization with anti-MLE IgG. Colloidal particles are evident on alveolar interstitium in association with the amorphous component of elastic fibers. Original magnification: ×34,000.

Lung Elastin

The results of the biochemical analysis of lung elastin expressed as micrograms per lung in the different groups for each strain are presented in Table 2. Untreated and saline-treated mice held very similar values. However, after FMLP treatment both beige and C57Bl/6J mice had significantly lower values (18% and 17%, respectively) than after saline treatment.

Histology and Morphometry

Twenty-one days after FMLP instillation, the lungs from beige mice showed diffuse areas of enlargement of the air spaces with destruction of alveolar septa (Figure 3a). These destructive changes were not present in lungs from uninjected and saline-injected mice (Figure 3b). It should be mentioned, however, that the control beige mice, but not the C57Bl/6J mice, have a form of air space enlargement that is due to an inborn defect of alveolar formation (21). Following FMLP challange, changes similar to those detected in beige mice were also observed in the C57Bl/6J mice.

View larger version (113K): [in this window] [in a new window]   View larger version (126K): [in this window] [in a new window]   View larger version (129K): [in this window] [in a new window]   Figure 3.   Representative histologic sections from lungs of C57Bl/ 6J beige mice 21 d after (a) FMLP, (b) saline, and (c) 4-(2-aminoetyl)-benzenesulfonyl fluoride hydrochloride and FMLP treatment. Hematoxylin and eosin stain. Original magnification: ×100.

Table 2 shows the Lm values obtained for the different groups. As can be seen, a significant increase of Lm values was found in beige and C57Bl/6J FMLP-treated mice (+30% and +28%, respectively) in comparison with the respective saline-treated groups. No significant difference was observed between saline-injected and uninjected mice of both strains.

Effect of a Serum-Proteinase Inhibitor Treatment on FMLP-Induced Lesions

The treatment of animals with 4-(2-aminoetyl)-benzenesulfonyl fluoride hydrochloride, a potent serine-proteinase inhibitor, significantly prevented the FMLP-induced lesions in beige mice (Figure 3c). The Lm value in these mice (50.66 ± 0.96 µm) is not significantly different from those observed in untreated and saline-treated control mice (Table 2).

	Discussion

Top Abstract Introduction Materials and methods Results Discussion References

The beige mouse is thought to be the mouse counterpart of the human Chediak-Higashi syndrome (CHS) (13, 35, 36). The beige gene localizes at the centromeric end of chromosome 13 (37), and this region is highly conserved on human Chr 1q42-q44 (38). This mouse has been widely used as a model of neutrophil elastase and cathepsin G deficiency to demonstrate or exclude the role of these proteases in certain pathologic conditions (18).

However, although several phenotypic features of the human CHS are present in the beige mouse (13, 16, 35) and some homology has been recently demonstrated between beige and Chediak-Higashi genes (37), characteristics that are important by a pathogenic point of view in the human syndrome (i.e., impaired migration and defective degranulation of neutrophils) are not present in beige mouse (27).

Also, data from our and other laboratories indicate that the beige mouse cannot be used as a model of cathepsin G deficiency because this enzyme is present in giant lysosomes of beige neutrophils and can be released in normal quantities on exocytosis under in vitro and in vivo conditions (25).

With regard to neutrophil elastase, the results reported in our previous papers (25, 27) demonstrate that a latent 46-kD form of elastase is present in lysosomes of mature beige neutrophils and that this proenzyme can undergo spontaneous activation by a proteolytic mechanism when released in the extracellular milieu. However, the pathogenic potential of this enzyme in matrix degradation has not been previously ascertained.

In this study we report the results of a study carried out in beige mice to examine the possibility that elastolytic proteases from neutrophils recruited into the lungs have the capability to damage the alveolar structures. The intratracheal instillation of FMLP in beige mice results in (1) marked influx of neutrophils in BALF, (2) an increase in lung elastase burden, (3) a decrease in lung elastin content, (4) an increase of mean linear intercept, and (5) the development of morphologic emphysema. These results indicate that the beige mouse's responses to FMLP do not differ substantially from those of C57Bl/6J, a strain characterized by normal neutrophil function and a deficiency in serum ₁-proteinase inhibitor (8). Recent data from our laboratory (unpublished results) have shown that similar to the parent C57Bl/6J strain, the beige mutant has low elastase inhibitory capacity levels in serum and in BALF. This may contribute to development of the lung elastolytic lesions in mice with a C57Bl/6J background. In the present study, after FMLP challenge in both C57Bl/6J and beige strains, neutrophils in the BALF peaked between 24 and 48 h and returned to control values at 96 h. However, significant neutrophil elastase burden was found in the septa of beige and C57Bl/6J mice 14 d after FMLP challenge. These data indicate that neutrophil elastase is long lived in the septa. This is consistent with data previously reported by Stone and coworkers (40), who found that after a single injection of ³H-methylated elastase to hamsters this enzyme could be detected in alveolar septa for several months after the instillation. Alternatively, we cannot exclude that after FMLP there is a continuous recruitment of neutrophils into the septa that is not reflected by an increase in the number of neutrophils in BALF. A time-course investigation of the septa by means of transmission electron microscopy would not answer this question because it is well known that neutrophils in the septa are very short lived (41). The protease dependency of the lung injury following the intratracheal instillation of FMLP in the beige mouse has been further demonstrated by the protective effect of 4-(2-aminoetyl)-benzenesulfonyl fluoride hydrochloride, a protease inhibitor active toward serine proteinases, and in particular of leukocyte elastase (42).

It has been previously reported that neutrophil recruitment into the lungs, induced by intratracheal injection of endotoxin, did not produce emphysema in beige mice (21). In that study, however, control beige mice had a particularly severe form of inborn air space enlargement. Thus the difference of the results obtained between the present and the previously mentioned studies may also depend on the severity of the preexisting lung changes.

In conclusion, the data reported in this paper suggest that the beige C57Bl/6 mutant mouse cannot be used as a model of neutrophil function impairment (CHS), and in particular of elastase deficiency. The presence of an activatable proelastase in beige lysosomes also explains why under several experimental conditions the beige mouse does develop (even if with a delay) lesions that have been ascribed to the proteolytic activity of neutrophil elastase (18, 19, 21, 43). In our opinion, many of the conclusions drawn from studies in which this strain has been used as a model of elastase deficiency (18) should be reconsidered in light of the data reported here.