Our results suggest an entirely novel view of the sensitization to HDM allergens: the intrinsic proteolytic activity of the allergen leads to degradation of TJs in airway epithelium, thus increasing the accessibility of the sentinel dendritic antigen-presenting cells residing beneath the epithelial barrier. Specifically, the HDM allergen Der p 1 leads to cleavage of the TJ membrane protein occludin, suggesting a major role for the allergen in decreasing the effectiveness of the epithelial barrier.
That Der p 1 is a proteinase has been indicated previously (6
). We reasoned that its proteolytic effects in airway epithelium might be significant to sensitization because of the degree to which airway mucosal barrier is exposed to foreign proteins. Small amounts of HDMFP or pure Der p 1 caused TJs to break and become displaced from their normal position apical to desmosomes (11
). Whereas the effects of HDMFPs on epithelial cells were mediated by 8–10 ng of Der p 1 protein (1 fecal pellet contains ~0.2 ng of Der p 1), the proteolytic effects of Der p 1 described in other cells require microgram amounts (40
). Moreover, because Der p 1 is a cysteine proteinase susceptible to oxidation, with irreversible loss of catalytic activity, our deliberate aging of HDMFPs means that the catalytically effective amount of Der p 1 delivered to the cell surface was probably somewhat less than 8–10 ng. Thus, our findings indicate that epithelial cells could be the primary proteolytic targets of Der p 1 in vivo, because they are the cells most likely to be exposed to the highest concentrations of inhaled allergens.
Another novel aspect of our study is the use of 2PMEM to quantify TJ proteins in situ. Using 2PMEM enabled us to correlate the time-dependent decrease of both occludin and ZO-1 with the increase in epithelial permeability and with cleavage of these proteins, as shown by immunoblotting. We thus established a link between allergen proteinase activity and diminished effectiveness of the epithelial barrier.
Occludin was expressed predominantly in a 62- to 65-kDa form, with possibly phosphorylated (31
) forms at ~70–75 kDa. The 70–75-kDa band was evident in 16HBE14o–
cell extracts blotted with polyclonal antibody, but less visible in MDCK cell extracts blotted with MOC37. In 16HBE14o–
cells exposed to pure Der p 1, both high- and low-mass forms were reduced in intensity. We can conclude that the cleavage products detected all contain the COOH-terminal of occludin, because the blotting antibody recognizes a 150-residue COOH-terminal segment. Inspection of the occludin sequence and prediction of its membrane topography using the program SOSUI (44
) suggest that the 53-kDa occludin product arises by cleavage within the first external loop. The accessibility of this loop to exogenous macromolecules is demonstrated by occludin immunostaining in nonpermeabilized CaCo-2 cells (19
), using antibodies against the segment of the first extracellular loop in which we report putative cleavage sites. Furthermore, as cleavage proceeds, steric hindrance provided by the TJ itself would minimize, so that the velocity of TJ disassembly should increase progressively. In contrast to the above, the 31-kDa and 22-kDa occludin products are probably derived by intracellular processing, because their masses are consistent with the COOH-terminal domain alone.
Two general possibilities could explain the extracellular cleavage of occludin. Either Der p 1 is envisaged to cleave occludin directly or Der p 1 might activate a cell surface zymogen that then degrades occludin. Experiments favor the more direct option: (a) the immunoblotting evidence and identification of putative Der p 1 cleavage sites in a synthetic peptide strongly suggest that the first extracellular loop of occludin may be considered a substrate of Der p 1; (b) an occludin loop peptide itself acted as an inhibitor of occludin degradation in situ; (c) the failure of serine, aspartic, and metalloproteinase inhibitors to block the in situ degradation of occludin by Der p 1 argues against the existence of an extracellular proteolytic cascade involving enzymes of these families.
Until recently (18
), occludin was the only known transmembrane protein of TJs. Despite the proposal that claudin-1 and -2 may be more significant in TJ structure (20
), functional evidence indicates that disruption of occludin causes epithelial barriers to fail. Peptides mimicking extracellular loops of occludin open TJs (45
) and prevent the gain of adhesiveness in occludin-null cell lines transfected with occludin (19
). These results would not be expected if occludin were of secondary importance in TJ structure and function. Our data provide clear evidence that Der p 1 increases epithelial permeability and disrupts TJs. The inescapable conclusion is that occludin and all other TJ adhesion proteins that regulate paracellular permeability must be affected in this process, and that initiation of the response ultimately depends upon the proteolytic activity of Der p 1.
Potential Der p 1 cleavage sites exist in both extracellular loops of claudin-1. In 64
cleavage of LL was facile (as seen in occludin). This suggests that like papain (46
), Der p 1 exhibits a P1
′ preference for hydrophobic residues. Other cleavages, especially of YG in occludin and in loop 2 of claudin-1, were less facile. Interestingly, the facile LL cleavage site of claudin-1 is replicated in 5 of the 8 known claudins (47
), whereas in claudins 4, 5, and 7 the equivalents of P1 69
L are conservative substitutions (M,V,V) (47
). Collectively, these observations suggest that occludin and claudins are all targets of Der p 1 and that there may be a preference for small, uncharged polar residues at P2
in its natural mammalian substrates.
In addition to cleavage of occludin, ZO-1 (22
) was degraded following exposure of cells to HDMFPs or Der p 1. Because ZO-1 is intracellular (9
), and therefore unlikely to have been directly degraded by Der p 1, we propose that its breakdown is a consequence of TJ disassembly. A similar explanation has been invoked to explain intracellular cleavage of the transmembrane protein N-cadherin following loss of intercellular contacts (48
The possible modes of action of Der p 1 on TJs are shown schematically in Figure . To summarize, our data are consistent with direct extracellular cleavage of occludin, and possibly claudins (mechanism in cell A). This may trigger intracellular processing, or, alternatively, intracellular cleavage could arise through a receptor and transduction pathway that is decoupled from TJs (the cell surface receptor and intracellular pathway in cell B). However, it seems less likely that TJs are degraded by activation of a cell surface zymogen that then attacks TJs (extracellular pathway in cell B). Whatever the mechanism of intracellular proteolysis, the effect is to open the epithelial barrier (cell C).
Figure 11 Schematic summary of how Der p 1 might open TJs in lung epithelium. In the simple case, cell A, Der p 1 acts upon extracellular loops of occludin, and possibly claudin-1. Extracellular cleavage of TJs initiates intracellular processing of junctional constituents. (more ...)
TJ cleavage evoked by HDMFP or Der p 1 differs from other exogenous proteinases that cause aberrant TJ-like strands to be formed (49
). However, aberrant TJ strands increase the plasticity of TJs because paracellular permeability increases during their formation (50
). This difference in behavior between Der p 1 and certain other proteinases might explain why Der p 1 is an insidious allergen capable of promoting a high level of reactivity in individuals with a constitutional predisposition to allergy.
Our results indicate that epithelial permeability is changed nonselectively by HDMFPs and pure Der p 1. This disruption was demonstrated using mannitol, which permeates the epithelial barrier only paracellularly (53
), providing a sensitive index of TJ function (29
). Furthermore, and of significance for allergen presentation, the increased permeability following TJ cleavage facilitated the transepithelial permeation of Der p 1.The nonselectivity of the increase implies that the transepithelial permeation of all proteins would be enhanced. This would lead to an increased probability of any inhaled allergen being able to encounter antigen-presenting cells of the airway’s immune system at sites where epithelial permeability was increased. We note that allergy to Der p 1 is frequently associated with reactivity to unrelated allergens (54
), as would be anticipated from our proposed mechanism.
Our observations suggest that allergic sensitization may be promoted by any environmental proteinase that attacks, directly or indirectly, the integrity of the epithelial barrier. We therefore suggest that prevention of TJ disruption (by inhibiting the environmental proteinase or even promoting TJ reassembly/assembly) could provide an entirely new approach to the treatment of asthma by limiting exposure of the immune system to allergens.