Uncoordinated production of Laminin-5 chains in airways epithelium of allergic asthmatics

Asthma is a chronic inflammatory disease of the lungs that may have allergic or non-allergic causes [1‐3]. The allergic type of asthma is characterized by the accumulation of eosinophils, mast cells and lymphocytes of the Th2-type in the bronchial mucosa, whereas the non-allergic asthma has a substantial accumulation of neutrophils in addition to eosinophils and mast cells [3]. Structural changes and remodelling of the bronchial mucosa with signs of epithelial injury, subepithelial basement thickening, smooth muscle hypertrophy, increased vascularization and innervation are prominent features of the allergic type of asthma and less prominent in the non-allergic type [3].

Basement membranes (BMs) are built of cell-polymerizing networks of type IV collagens and laminins connected by nidogen/entactin [4, 5]. The major role of laminin for epithelial cells is to anchor them to BM for cell differentiation and maintenance of cell function. Laminins are heterotrimeric molecules made up by one α, one β and one γ chain. Until today we know of five α-chains, three β-chains and three γ-chains. These chains combine into at least 14 different Laminins (lns) i.e. lns 1–14. The distribution of these Laminin isoforms varies between tissues, but in most BMs more than one Laminin is present. The chains of laminins have different regions that function by binding to cellular receptor molecules among which the most abundant are integrins, dystroglycan and the recently characterized Lutheran blood group antigen [4, 6]. Several studies have shown the fundamental importance of intact Laminins in the BMs, since mutations may give rise to serious diseases such as epidermolysis bullosa in which the anchoring of the skin is grossly impaired [7]. Laminins also interact with many other cells and promote migration and angiogenesis and their functions in tumour invasion is one of the hot research topics of today [4].

The injury of the respiratory epithelium in the bronchi in allergic asthmatics may be one of the mechanisms underlying bronchial hyperresponsiveness which is one of the main features of asthma [8‐11]. The mechanisms behind the fragility of the epithelium in allergic asthmatics, i.e. the propensity of the epithelium to shed from its anchorage to the subepithelial basement membrane (SEBM) and basal cells have not been explained. Since one obligatory component in this anchoring process is mediated by Laminins, we hypothesized that uncoordinated production of Laminin chains might contribute to weaken these anchoring forces. Our aim was therefore to describe the presence of the various Laminins in the epithelium and especially SEBM of allergic asthmatics in comparison with non-allergic asthmatics and healthy non-asthmatic controls.

This study has systematically investigated the presence of most Laminin chains in the epithelium and SEBM of both allergic and non-allergic asthmatics. The primary object was to test the hypothesis that differences in Laminin chain compositions in SEBM might help explain the phenomenon of epithelial fragility and shedding as is typically seen in biopsies of allergic asthmatics [3]. We found several alterations in the Laminin chain composition in the SEBM of allergic asthmatics. Most of these differences seemed quantitative rather than qualitative. Unexpectedly we found distinct qualitative differences with respect to ln-5 chain compositions that may have a bearing on the poorer anchorage of epithelial cells to BM in allergic asthma. The finding of a close correlation between ln γ2-chain deposition and epithelial injury indeed emphasises the close relationship between laminin chain production and epithelial injury as is observed in certain subjects with asthma. However, the relationship does not tell us whether epithelial shedding is a cause of the uncoordinated laminin chain production or whether the uncoordinated production is a consequence of the repair processes induced by epithelial injury.

The unexpected findings were both related to the ln γ2-chain staining. According to the present knowledge the γ2-chain is only found as part of ln-5 (α3β3γ2) [4, 18]. In our biopsies we found exclusive staining with the antibodies against the γ2-chain in the epithelium, with no sign of simultaneous staining with antibodies against the α3-chain. The epithelium staining could be a reflection of the fact that airways epithelium is a producer of the ln γ2-chain and that the staining reflects the deposition of non-secreted protein. It was of particular interest that we found accumulation of immunoreactivity in the apical part of intact columnar epithelium in allergic asthmatics, but not in the other two groups, whereas staining of the basal cells were seen in all three groups in areas of epithelial destruction. This staining was observed without any concomitant staining of the complementary ln-5 α3 chain, which suggests an uncoordinated production of the γ2 chain in the epithelium of allergic asthmatics resulting in the intracellular accumulation of the γ2 chain, since the extracellular secretion probably requires the assembly of the heterotrimeric molecule [18]. The staining pattern could also indicate the presence of a hitherto unrecognised Laminin or alternatively that the α3 chain had been proteolytically modified with the loss of the particular epitope recognized by our monoclonal antibodies [19].

The intense staining of the cytoplasm of basal cells in areas of epithelial injury suggests that the basal cells are producers of the ln γ2 chain and probably also of the whole heterotrimeric complex of ln-5, although no staining of the other two chains was observed. Indeed, sole expression of ln γ2 chain has been shown in invading tumour cells[4, 20], which shows that uncoordinated production of the three ln-5 chains may take place under certain conditions. It is also of interest that Lappi-Blanco et al. in a recent report found ln γ2 chain expression to be increased in regenerating epithelial cells and also found γ2 chain in basal cells of normal bronchus [21]. These results suggest that our findings of intense staining seen in the basal cells at areas of tissue injury may be a sign of re-epithelialization and repair.

The SEBM showed the presence of mainly three Laminins i.e. ln-5 (α3β3γ2), ln-10 (α5β1γ1) and ln-2 (α2β1γ1). The two former were expected based on earlier findings [4, 22], whereas the presence of ln-2 mostly is associated with BMs surrounding tissues such as muscles and nerves [23]. In a previous report we indicated the wide presence of α1-chains in SEBM, which is seemingly contrasted by the present results[24]. Those results, however, were based on the false assumption that the monoclonal antibody 4C7 specifically recognizes α1-chains, which is not the case. The 4C7 antibody only recognizes the α5-chains [14, 15]. As was previously found the thickness of the Laminin layer in the SEBM was increased in allergic asthmatics as compared to both non-allergic asthmatics or healthy controls [3]. This difference was most obvious for ln-10, since also the thickness found in non-allergic asthmatics was increased as compared to healthy non-asthmatic controls. This was contrasted by the increased thickness of ln-5 and -2, which was only seen in allergic asthmatics. These differences, therefore suggest qualitative differences in the production of various chains in allergic and non-allergic asthmatics, which may relate to the differences in the inflammatory processes going on in these two diseases. The allergic asthma being eosinophil-mast cell-Th2 driven and the non-allergic asthma being more neutrophil-mast cell driven, although eosinophils are also present at increased amounts in the non-allergic asthma [3].

As mentioned above the primary aim of this work was to test the hypothesis that an imbalance in Laminin chain production might explain the observed epithelial cell loss in allergic asthmatics. This hypothesis is seemingly refuted by our data, since others have shown that ln-5 induces the formation of hemidesmosomes [25], which actually promote stable cell:matrix adhesion. Another interesting property of ln-5 is the biological activity of the proteolytically modified fragments, which might modify cellular behaviours [26]. However, it should also be noted that mutations or modifications of any of the chains of ln-5 are associated with severe disease due to separation of epithelia from the underlying basement membrane [7]. Thus, we cannot exclude any processing of ln-5 in the inflamed tissue of allergic asthma as an explanation of poor anchorage of the epithelial cells in the bronchi to the underlying basement membrane.

We conclude that Laminin chain deposition in the epithelium and SEBM of allergic and non-allergic asthmatics differs in qualitative and quantitative terms and that there is a close relationship between ln γ2-chain deposition and epithelial injury. The uncoordinated production of the chains of ln-5 in the epithelium of allergic asthmatics may be of particular interest, since ln-5 promotes the formation of hemidesmosomes, which promote stable cell matrix adhesion.