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Published ahead of print on September 18, 2003, doi:10.1164/rccm.200210-1234OC
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American Journal of Respiratory and Critical Care Medicine Vol 169. pp. 27-33, (2004)
© 2004 American Thoracic Society

Laminin-5 {gamma}2 Chain in Cryptogenic Organizing Pneumonia and Idiopathic Pulmonary Fibrosis

Elisa Lappi-Blanco, Riitta Kaarteenaho-Wiik, Sirpa Salo, Raija Sormunen, Marko Määttä, Helena Autio-Harmainen, Ylermi Soini and Paavo Pääkkö

Departments of Pathology, Internal Medicine, and Biochemistry, University of Oulu and Oulu University Hospital, and Biocenter Oulu, Oulu, Finland

Correspondence and requests for reprints should be addressed to Elisa Lappi-Blanco, M.D., Ph.D., Department of Pathology, University of Oulu, P.O. Box 5000, FIN-90014 Oulu, Finland. E-mail: elisa.lappi-blanco{at}oulu.fi


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Insufficient reepithelialization of injured alveolar walls may be important in the pathogenesis of idiopathic pulmonary fibrosis (IPF). Laminin-5 is expressed in epithelial cells of healing wounds, promoting cell attachment and migration. In this study we have studied the extent of reepithelialization of newly formed intraluminal connective tissue, the immunohistochemical expression and ultrastructural localization of the laminin-5 {gamma}2 chain protein, and the synthesis of the laminin-5 {gamma}2 chain mRNA in regenerating epithelial cells in cryptogenic organizing pneumonia (COP) and IPF. The results show that the mean extent of reepithelialization of intraluminal connective tissue lesions was 76% (SD, ± 27%) in COP, and 54% (SD, ± 23%) in IPF (p < 0.025). The laminin-5 {gamma}2 chain was synthesized and widely expressed in regenerating epithelial cells in both diseases. Immunohistochemistry for surfactant-associated protein A suggests a pneumocyte origin for the regenerating epithelial cells in IPF. It is concluded that both in COP and IPF, regenerating epithelial cells are capable of synthesizing the laminin-5 {gamma}2 chain needed for adhesive connections to the underlying basement membrane. However, in IPF, the reepithelialization seems to be disturbed or delayed.

Key Words: alveolar epithelium • cryptogenic organizing pneumonia • idiopathic pulmonary fibrosis • laminin-5 • pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) and cryptogenic organizing pneumonia (COP) are fibrous pulmonary disorders, both of which show newly formed connective tissue in distal airspaces (1). IPF is a progressive and usually fatal lung disease with no efficient treatment (2), whereas the prognosis for COP is good, with nearly 85% of patients recovering with steroidal therapy (3). In both diseases injury to the epithelium and its basement membrane (BM) in distal air spaces leads to migration of fibroblasts and myofibroblasts into air spaces and production of extracellular matrix by these cells (4, 5). In IPF the newly formed connective tissue causes fusion of alveolar structures and interstitial remodeling (4, 5), whereas in COP the newly formed connective tissue is thought to form small collagen globules (6) or to resolve completely (7).

Laminins are a group of large heterotrimeric glycoproteins participating in cell attachment, migration, proliferation, differentiation, and apoptosis. The laminin molecule is composed of three chains: the {alpha}, ß, and {gamma} chains. Laminin-5 consists of {alpha}3, ß3, and {gamma}2 chains, of which the {gamma}2 chain is currently known to be present only in laminin-5 (8). Laminin-5 provides adhesive connections for epithelial cells (9, 10), and it also promotes migration, depending on the state of processing (11). During embryogenesis, laminin-5 is highly expressed in the epithelial cells of bronchi and alveoli (12). In normal adult lung, the laminin-5 {gamma}2 chain is localized in the BM of bronchiolar and alveolar epithelium (13). Laminin-5 is expressed by migrating keratinocytes in wounded skin when new BM is being established (14).

Pulmonary surfactant is responsible for lowering the surface tension at the air–liquid interface in the alveoli. Pulmonary surfactant is synthesized by Type II pneumocytes. It is a mixture of phospholipids and proteins, including four distinct surfactant proteins (SPs): SP-A, SP-B, SP-C, and SP-D (15). Serum levels of SP-A and SP-D have been shown to be elevated in IPF (16, 17).

It is presumed that in IPF, a failure of reepithelialization of fibroblast foci maintains fibroblast/myofibroblast activity and extracellular matrix synthesis (18, 19). Here we have studied the extent of reepithelialization of newly formed intraluminal connective tissue lesions, and the expression and synthesis of the laminin-5 {gamma}2 chain in regenerating epithelial cells in COP and in IPF. Surfactant-associated protein A was used to characterize the regenerating epithelial cells. Some of the results have been previously reported in the form of an abstract (20).


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
All open or thoracoscopic lung biopsies from the files of the Department of Pathology, Oulu University Hospital (Oulu, Finland), from 1977 to 2001 were evaluated. Additional cases were collected from two other Finnish university hospitals and one central hospital. After reevaluating the histologic slides, 29 patients (8 women and 21 men) were selected, 14 fulfilling the histologic and clinical criteria for COP and 15 showing IPF with fibroblast foci in distal air spaces (2). The study protocol was accepted by the ethics committee of Oulu University Hospital and the Finnish Social and Health Ministry.

For immunoelectron microscopy, tissue samples from remodeled areas of peripheral lung were available from three patients with IPF, and a tissue sample of a normal bronchus was available from one patient who had undergone an operation for a pleural scar. Uninvolved peripheral lung tissue, used as a control, was obtained from four patients operated on for a malignant lung tumor.

Immunohistochemistry for Laminin-5 {gamma}2 Chain, Cytokeratin, and Surfactant-associated Protein A
A polyclonal (anti-LAM{gamma}2-III) anti-human laminin-5 {gamma}2 chain antibody recognizing amino acid residues 391–567 of the human laminin-5 {gamma}2 chain was used. A monoclonal mouse anti-human cytokeratin antibody (clone MNF-116) recognizing a number of discrete 40- to 58-kD keratin polypeptides, including keratins 5, 6, 8, 17, and probably 19 (DakoCytomation, Glostrup, Denmark), was used to recognize epithelial cells of human alveoli and terminal bronchioli (21). SP-A (N-19) is a goat polyclonal antibody against a peptide mapping near the amino terminus of pulmonary surfactant protein A (Santa Cruz Biotechnology, Santa Cruz, CA).

Immunohistochemistry for paraffin sections was performed as previously described (22). An overnight incubation (14 µg/ml) at 4°C was used for the anti-LAM{gamma}2-III antibody and a 1-hour incubation (dilution, 1:100) at room temperature was used for MNF-116. For SP-A, citrate buffer (pH 6) treatment in a microwave oven (2 minutes at 850 W and 10 minutes at 150 W) was used for antigen retrieval, and this was followed by a 1-hour incubation with the antibody (dilution, 1:150) at room temperature. Sections incubated with phosphate-buffered saline instead of the primary antibody were used as negative controls.

For the analysis, intraluminal fibromyxoid connective tissue lesions were evaluated in cases with COP, and fibroblast foci with loose, myxoid stroma in IPF. The extent of reepithelialization is given as a percentage of regenerating cytokeratin-positive cells on surface area of intraluminal connective tissue lesions and fibroblast foci. The proportion of laminin-5 {gamma}2–positive epithelial cells was identified in the same lesions from adjacent paraffin sections, and was similarly evaluated and given as a percentage of the cytokeratin-positive regenerating epithelial cells in COP and IPF. In COP, 275 intraluminal fibromyxoid lesions were evaluated, and 134 fibroblast foci were evaluated in IPF. As a control, part of the material was evaluated with an ocular micrometer. Evaluation of the extent of reepithelialization based on ocular micrometer measurements and routine microscopic evaluations gave results that were surprisingly repeatable, the Pearson correlation coefficient being 0.9 (p < 0.01).

In Situ Hybridization for Laminin-5 {gamma}2 Chain mRNA
For the laminin-5 {gamma}2 chain, a PstI–EcoRI fragment of clone L15 (bases 2995–3840) was subcloned into pSP64 and pSP65 vectors in the sense and antisense orientations. The probe in the sense orientation served as a control for nonspecific hybridization (22). The hybridized tissue sections of lung biopsies were examined by light microscopy, and the number of grains over the cells was evaluated in general, and especially in locations where laminin-5 {gamma}2 chain immunoreactivity was located. Cells hybridized with the 35S-labeled antisense laminin-5 {gamma}2 probe were considered positive if they contained more grains than corresponding cells that had been hybridized with the 35S-labeled sense laminin-5 {gamma}2 chain probe.

Immunoelectron Microscopy of Laminin-5 {gamma}2 Chain
Immunoelectron microscopy was performed as previously described (23). Sections were incubated with polyclonal antibody to human laminin-5 {gamma}2 chain (14 µg/ml) for 60 min followed by protein A–gold complex (size, 10 nm) for 30 minutes, made according to Slot and Geuze (24). The controls were prepared by carrying out the labeling procedure without primary antibody. The sections were examined in a Philips CM100 transmission electron microscope (FEI, Hillsboro, OR).


   RESULTS
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 RESULTS
DISCUSSION
 REFERENCES
 
Immunohistochemical Features of Reepithelialization
Cytokeratin staining was strongly positive in all epithelial cells; that is, in normal Type I and Type II pneumocytes and bronchiolar epithelial cells, regenerating epithelial cells in COP and IPF, and in metaplastic squamous-type and bronchiolar-type epithelium seen in IPF. On the basis of the amount of cytokeratin-positive cells covering the intraluminal connective tissue lesions, the extent of reepithelialization was significantly higher in COP (76%; SD, ± 27%) when compared with IPF (54%; SD, ± 23%; p < 0.025).

In COP, regenerating epithelial cells were usually uniform, sometimes having a flat shape reminiscent of Type I pneumocytes (Figures 1A and 1B) . Disarray of epithelial cells was slight or nonexistent in COP (Figures 1A–1D). Exfoliation of epithelial cells positive for both cytokeratin and laminin-5 {gamma}2 chain was seen in 2 of 15 COP cases (Table 1) . In IPF, reepithelialization was more heterogeneous, poorly and well epithelialized intraluminal lesions appearing simultaneously (Figure 1E). In IPF, there was morphologic variation of epithelial cells characterized by variation in cell size and shape as well as in the morphology of nuclei. Some of the epithelial cells appeared swollen with vacuolated cytoplasm suggestive of degenerative changes (Figure 1F). The epithelium showed disordered layering, and piling of epithelial cells was sometimes seen at the leading edge of the migrating epithelium. Exfoliation of laminin-5 {gamma}2 chain–positive epithelial cells into the alveolar lumen was seen in 11 of 15 IPF cases both in fibroblast foci and outside them (Figures 1G and 1H, and Table 1). Statistically, exfoliation of epithelial cells was seen significantly less frequently in COP compared with IPF (p < 0.001 by Fisher's exact probability test). The mural-incorporating lesions were widely or totally reepithelialized.



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  		Figure 1. The high extent of reepithelialization and regular histology of regenerating pneumocytes in COP (A–D) contrasted with the variable histology seen in IPF (E–H). In COP, the regenerating Type II pneumocytes were layered in an orderly fashion and often adapted a flat shape reminiscent of Type I pneumocytes. Staining against cytokeratin (MNF-116); original magnification, x100 (A) and x210 (B). A well-epithelialized intraluminal connective tissue lesion covered by cells positive both for cytokeratin (C) and laminin-5 {gamma}2 chain (D) in COP. Original magnification, x50 (C and D). Low extent of reepithelialization and disordered organization of morphologically variable epithelial cells was a usual finding in IPF. Immunohistochemical staining against cytokeratin (MNF-116); original magnification, x100 (E and F). Exfoliating cells positive for cytokeratin (G) and laminin-5 {gamma}2 chain (H) in IPF. Original magnification, x100 (G and H).

 

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  		TABLE 1. Extent of reepithelialization and expression of LAMININ-5 {gamma}2 chain in regenerating epithelial cells, number of positive signals in LAMININ-5 {gamma}2 chain mRNA in situ hybridization in intraluminal connective tissue lesions, and exfoliation of epithelial cells in cop and ipf*

 
Immunohistochemistry for Laminin-5 {gamma}2 Chain
Wide positivity for the laminin-5 {gamma}2 chain was found in regenerating epithelial cells in paraffin sections (anti–LAM{gamma}2-III) in both COP and IPF. The staining pattern was intracytoplasmic, with occasional positive staining along BMs. The epithelial location of laminin-5 {gamma}2 chain immunohistochemistry was defined with adjacent sections stained with cytokeratin (Figures 1C and 1D). Staining intensity was mostly moderate or strong, and the staining pattern was diffuse or slightly granular.

In COP, 73% (SD, ± 32%) of regenerating epithelial cells covering intraluminal connective tissue lesions were positive for the laminin-5 {gamma}2 chain. Correspondingly, in IPF laminin-5 {gamma}2 chain positivity was seen in 90% (SD, ± 13%) of regenerating cells. No significant difference was seen between COP and IPF in the percentage of positively stained epithelial cells.

In both diseases, intracytoplasmic positivity for the laminin-5 {gamma}2 chain was also seen in some alveolar walls covered by cuboidal cells. In IPF, wide intracytoplasmic laminin-5 {gamma}2 chain positivity was found in remodeled air spaces lined by regenerating cuboidal or metaplastic squamous-type epithelium. In IPF, bronchiolar-type epithelium close to distal bronchioli and alveolar ducts showed usually a faint stringlike staining pattern along BMs like normal bronchiolar and alveolar epithelial cells. In COP, there was positive intracytoplasmic staining in some basal cells of terminal bronchioli containing loose connective tissue.

Immunohistochemistry for SP-A
In COP, SP-A was clearly positive in normal alveolar Type I and Type II pneumocytes and in regenerating cuboidal cells on alveolar walls close to distal bronchioli and alveolar ducts. However, the regenerating cells covering intraluminal fibromyxoid connective tissue lesions were mostly negative for SP-A (Figure 2A) . In IPF, SP-A was consistently positive in regenerating cells covering fibroblast foci (Figure 2B). SP-A was also positive in cuboidal or metaplastic squamous-type epithelium covering remodeled alveolar spaces. Bronchiolar-type epithelium covering distorted air spaces close to distal bronchioli and alveolar ducts remained SPA negative.



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  		Figure 2. Immunohistochemical staining against SP-A was mostly negative in regenerating epithelial cells in COP (A), whereas in IPF the regenerating epithelial cells were positive against SP-A (B). Original magnification, x100 (A and B).

 
In Situ Hybridization for Laminin-5 {gamma}2 Chain mRNA
Positive signals for the laminin-5 {gamma}2 chain mRNA were observed in 12 cases of 13 of COP and in 10 of 15 cases of IPF. The positive signals were located at the leading edge of the migrating epithelium on the surfaces of the intraluminal connective tissue lesions (Figure 3) . There were usually only a few positive cells in an intraluminal lesion. In COP, positive signals were seen only in intraluminal connective tissue lesions. In IPF, a few positive signals were also found in cuboidal or squamous-type epithelium covering remodeled air spaces.



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  		Figure 3. In laminin-5 {gamma}2 chain mRNA in situ hybridization, positive signals were located at the leading edge of the migrating epithelium. Antisense laminin-5 {gamma}2 probe (A), sense laminin-5 {gamma}2 probe (B), and staining against cytokeratin (MNF-116) (C). Original magnification, x225 (A–C).

 
The results of the immunohistochemical and mRNA in situ hybridization analyses are summarized in Table 1.

Immunoelectron Microscopy of Laminin-5 {gamma}2 Chain
In basal cells of a normal bronchus, labeling for the laminin-5 {gamma}2 chain was seen at the outer zone of the lamina densa without any intracytoplasmic staining (Figure 4A) . In IPF, remodeled alveoli were covered mostly by Type II pneumocytes recognized by lamellar bodies. Occasional Type I pneumocytes were also seen. Labeling for laminin-5 {gamma}2 chain was seen both at the inner and outer zones of the lamina densa in Type I and II pneumocytes (Figures 4B and 4C). The labeling was somewhat stronger in BMs of Type I pneumocytes when compared with BMs of regenerating Type II pneumocytes. No definite intracytoplasmic labeling was observed in alveolar epithelial cells.



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  		Figure 4. Immunoelectron microscopy showed laminin-5 {gamma}2 chain both at the inner and outer zones of lamina densa of basal membrane (arrows). BM = basal membrane (arrowheads; A–C); br = basal cells of a normal bronchus (A); pnI = Type I pneumocyte (B); pnII = Type II pneumocyte (C). Scale bars: 500 nm.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our study shows that the laminin-5 {gamma}2 chain is synthesized and widely expressed in regenerating epithelial cells in both COP and IPF. The intracytoplasmic staining pattern can be seen as a sign of regeneration, contrasting with the stringlike staining pattern along BMs in normal alveolar and bronchiolar epithelial cells. The proportional number of laminin-5 {gamma}2 chain–positive epithelial cells as well as the number of cells synthesizing laminin-5 {gamma}2 chain mRNA showed no significant difference in these diseases. This finding indicates that in both diseases, the regenerating epithelial cells are able to synthesize the laminin-5 {gamma}2 chain, and thus there is an attempt to stabilize the adhesions between the reepithelializing cells and underlying stroma. The importance of laminin-5 for epithelial cell adhesions has been shown in several previous studies (9, 10, 25). Moreover, it has been shown that the short arm of the laminin-5 {gamma}2 chain plays a pivotal role in the incorporation of laminin-5 into the extracellular matrix as well as in cell adhesion (26).

The extent of reepithelialization demonstrated by cytokeratin immunohistochemistry was significantly wider in COP when compared with that in IPF, suggesting disturbed or delayed reepithelialization in IPF. Our results support the new concept of abnormal alveolar wall healing as an important part of the pathogenesis of idiopathic IPF (27). The results are also in line with our previous results of a low level of angiogenesis and diminished apoptotic activity in the newly formed intraluminal fibromyxoid lesions in IPF (28, 29).

An interesting finding was that the regenerating cells were mostly negative for SP-A in COP, whereas they were constantly SP-A positive in IPF. This finding indicates that the regenerating cells are of different phenotypes and may be of different origins in these diseases, which may also contribute to the extent of reepithelialization. On the basis of SP-A positivity and the immunoelectron microscopy findings of our study, the regenerating cells are Type II pneumocytes in IPF. However, the origin of the regenerating cells in COP remains to be studied. Our findings are in agreement with the findings of Myers and Katzenstein (30). In an ultrastructural study they observed that in idiopathic bronchiolitis obliterans organizing pneumonia (i.e., COP) the regenerating cells contained only occasional lamellar bodies characteristic of Type II pneumocytes. However, no further characterization of the regenerating cells was achieved.

In IPF, poorly and well reepithelialized intraluminal lesions occurred simultaneously. This gives rise to a question concerning whether the low extent of reepithelialization reflects only temporal heterogeneity of IPF. However, the clear morphologic and structural abnormality of regenerating epithelial cells favors the theory of disturbed or delayed reepithelialization in IPF. In COP, the regenerating epithelium was uniform and it usually showed appropriate layering on the surface of intraluminal connective tissue lesions. This regular histology contrasted with the histology of IPF, where disordered layering of regenerating epithelial cells, morphologic variety of the cells, and exfoliation of laminin-5 {gamma}2 chain–positive epithelial cells into alveolar spaces were seen. The morphologic difference between IPF and COP suggests that despite the synthesis of the laminin-5 {gamma}2 chain, the attachment of the cells to the underlying stroma is disturbed in IPF. This may be due to a plethora of factors, such as altered processing of the {gamma}2 chain or dysfunction of other laminin-5 chains, laminin receptors, or other BM components. Loss of processed laminin-5 in the extracellular matrix may also expose epithelial cells to increased apoptosis (31), which may be of great importance in the pathogenesis of usual interstitial pneumonia (19).

Laminin-5 promotes both cell adhesion and migration in remodeling tissues, depending on the stage of processing (11, 32). Whereas unprocessed laminin-5 is a biologically active adhesion ligand, the specific cleavage of laminin-5 {gamma}2 chain by matrix metalloprotease-2 induces migration of epithelial cells (11). On the other hand, the unprocessed {alpha}3 chain induces motility of the epithelial cells, but conversion of the {alpha}3 chain by tissue-type plasminogen activator and plasminogen triggers adhesion of cells (32). These results indicate that tight regulation of enzymatic activity is needed for reepithelialization in normal wound healing. Previous studies suggest an imbalance between matrix metalloproteinases and their tissue inhibitors in IPF (33–35). However, more detailed studies are needed to clarify the role of matrix metalloproteases in reepithelialization and tissue remodeling in IPF.

Localization of positive signals in laminin-5 {gamma}2 chain mRNA in situ hybridization correlated with the immunohistochemical expression of the laminin-5 {gamma}2 chain protein, showing that laminin-5 {gamma}2 chain is synthesized in epithelial cells with no detectable synthesis by stromal cells. The small amount of positive signals in in situ hybridization corresponds to the normal wound healing model, in which laminin-5 {gamma}2 mRNA has been observed only at the leading edge of the migrating epithelium (14). In the present study, the small amount of laminin-5 {gamma}2 chain mRNA is in contrast with the wide immunohistochemical expression of the protein. Obviously, in nonneoplastic cells the synthesis of laminin-5 {gamma}2 chain is strictly regulated and momentary.

The immunoelectron microcopy findings correlate well with the results of laminin-5 {gamma}2 chain immunohistochemistry. Positive labeling for the laminin-5 {gamma}2 chain was seen at BMs of normal Type I pneumocytes and regenerating Type II pneumocytes of remodeled alveoli in IPF, as well as in basal cells of a normal bronchus. No intracytoplasmic labeling was seen in regenerating cells, but this may be due to sampling error.

In conclusion, our results show that in both COP and IPF regenerating epithelial cells are capable of synthesizing the laminin-5 {gamma}2 chain needed for stabilized epithelial cell adhesions. In COP, reepithelialization and synthesis of the laminin-5 {gamma}2 chain apparently parallel normal wound healing, which requires spatially and temporally regulated synthesis and modulation of different proteins. The stem cell for regenerating epithelial cells in COP remains to be characterized. In IPF, regenerating cells are Type II pneumocytes, but reepithelialization seems to be disturbed or delayed, which may contribute to ongoing fibroproliferation. The disturbed reepithelialization in IPF may be caused by several different factors, the identification of which calls for further study.


   FOOTNOTES
 
Supported in part by the Finnish Anti-Tuberculosis Association Foundation and the Paulo Foundation, Finland.

Conflict of Interest Statement: E.L-B. has no declared conflict of interest; R.K-W. has no declared conflict of interest; S.S. has no declared conflict of interest; R.S. has no declared conflict of interest; M.M. has no declared conflict of interest; H.A-H. has no declared conflict of interest; Y.S. has no declared conflict of interest; P.P. has no declared conflict of interest.

Received in original form October 29, 2002; accepted in final form September 13, 2003


   REFERENCES
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 DISCUSSION
 REFERENCES
 

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