- Top of page
- Materials and methods
Background: Matrix metalloproteinases (MMPs) digest extracellular matrix proteins and may play a role in the pathogenesis of bronchial asthma. MMP-9 levels are increased in the bronchoalveolar lavage fluid and sputum of asthmatics compared with that of controls. As exposure to cockroaches is an environmental risk factor for asthma, we sought to investigate the role of German cockroach fecal remnants (frass) on MMP-9 expression.
Methods: Human bronchial epithelial cells (16HBE14o-) and primary normal human bronchial epithelial cells were treated with cockroach frass in the absence or presence of tumor necrosis factor (TNF)α. MMP-9 mRNA, protein levels and pro-MMP-9 activity were determined using real-time polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA) and zymogram assays. Pretreatment of frass with aprotinin abolished protease activity. PD98059, a chemical inhibitor of extracellular signal regulated kinase (ERK), and SLIGKV, an activator of protease-activated receptor (PAR)-2 were also used. AP-1DNA binding was determined by electrophoretic mobility shift assay (EMSA) and ERK phosphorylation by Western blot analysis.
Results: Cockroach frass augmented TNFα-mediated MMP-9 mRNA and protein expression by a mechanism dependent on active serine proteases within frass and not on endogenous endotoxin. Frass increased ERK phosphorylation, and chemical inhibition of ERK attenuated cockroaches’ effects on MMP-9. Serine proteases are known to activate the PAR-2 receptor. We found that selective activation of PAR-2 using the peptide SLIGKV augmented TNFα-induced MMP-9 protein levels and increased ERK phosphorylation. Frass and SLIGKV each increased AP-1 translocation and DNA binding.
Conclusions: These data suggest that German cockroach frass contains active serine proteases which augment TNFα-induced MMP-9 expression by a mechanism involving PAR-2, ERK and AP-1.
Matrix metalloproteinase (MMP)-9 is a zinc- and calcium-dependent protease which can degrade collagen type IV, a major component of the epithelial basement membrane. Several observations suggest a role for MMP-9 in the pathogenesis of bronchial asthma. The level and activity of MMP-9 was increased in the sputum of severe asthmatics compared with that of patients with mild asthma or normal controls, and the level of MMP-9 was sensitive to downregulation by oral corticosteroid treatment (1). In allergic patients who underwent bronchoscopy and segmental bronchoprovocation with saline or allergen (ragweed, cat or house dust mite), MMP-9 levels were significantly increased in the bronchoalveolar lavage (BAL) fluid within 48 h (2). A second study using atopic and normal patients confirmed that allergen challenge (inhalation of house dust mite) increased MMP-9 expression in sputum from atopic patients compared with non-atopic patients (3). Together, these data suggest that MMP-9 may play a role in modulating the asthma phenotype.
Cockroach exposure has been recognized as an important cause of asthma for over 30 years. Several allergens have been cloned from German cockroach (Blattella germanica) and termed Bla g 1, Bla g 2, Bla g 4 and Bla g 5. Interestingly, while none of the cockroach allergens cloned to date are proteolytically active (4, 5), protease activity has been noted in the whole-body extract of German cockroach (5, 6). A variety of aeroallergens have been shown to exhibit protease activity including house dust mite (Dermatophagoides pteronyssinus), fungus (Aspergillus fumigatus) and cat (Felis domesticus) (7–9). This raises the question of the overall role of proteases in the development of asthma and airway hyperresponsiveness.
We have been interested in the role of German cockroach extracts in modulating human airway inflammatory responses. We have shown that serine proteases contained in cockroach whole-body extracts upregulate the expression of pro-inflammatory cytokines interleukin (IL)-8 and IL-6 in the presence of tumor necrosis factor (TNF)α (6, 10, 11). Protease-activated receptor (PAR)-2 is a G-protein-coupled receptor that is activated upon cleavage by serine or trypsin-like proteases. It has been shown that German cockroach extracts activate PAR-2 (10–13). Once PAR-2 is cleaved, a signaling cascade involving G-proteins, MEK [mitogen-activated protein (MAP) kinase/extracellular signal-regulated kinase (ERK)] and ERK is initiated (10).
It is conceivable that asthmatics would encounter cockroach-derived proteins in the context of airway inflammation (i.e. increased TNFα). As TNFα has been shown to regulate MMP-9 expression (14, 15), and since MMP-9 is increased in the BAL of asthmatics (16), we tested the hypothesis that German cockroach frass would modulate TNFα-induced MMP-9 expression in human bronchial epithelial cells.
- Top of page
- Materials and methods
The airway epithelium plays a dynamic role in the asthmatic response with its ability to synthesize important metabolically active cytokines and other proteins. Airborne proteases may come in direct contact with airway cells and may play an important role in modulating inflammation and repair processes. In this report, we find that cockroach frass contains serine proteases which modulate TNFα-induced MMP-9 expression in a mechanism dependent on PAR-2, ERK, and AP-1 translocation and DNA binding. The human MMP-9 promoter contains NF-κB, AP-1, AP-2, SP-1 and Ets binding sites (20). An absolute requirement for NF-κB in MMP-9 transcription has been previously reported (21). That frass has no effect alone but augments TNFα-induced MMP-9 expression is likely because frass does not activate NF-κB. The activation of AP-1 may not be essential for induction but may be required for maximal stimulation (21). Frass-induced activation of ERK and AP-1 further increases translocation and DNA binding of AP-1 to the MMP-9 promoter to cause an upregulation of MMP-9. Frass-induced synergy was not altered by pretreatment with inhibitors to p38, phosphatidylinositol 3-kinase or PKC. As MMP-9 plays an important role in tissue remodeling, understanding the mechanism of regulation could lend insights into therapeutic interventions.
In this report, we used cockroach frass to determine the effects on MMP-9 expression. Frass is a likely source of allergen exposure as desiccated fecal remnants may easily be incorporated in house dust. It has been shown that after disturbance, dust particles containing cockroach proteins >10 μm in diameter may be deposited in the airways (22). Once in the airways, proteins elute from these particles and may reach high local concentrations. In addition, we have preliminary data to suggest that proteases remain active in dust collected from carpeting in homes (K. Page and B. Lanphear, unpubl. obs.). This suggests that the proteases may continue to be active even after lying dormant for a period of time.
MMP-9 protein levels were increased following treatment with TNFα and augmented with frass and TNFα treatment; however, these treatments did not significantly affect TIMP-1 levels. TIMP-1 is a natural inhibitor of MMP-9 and is thought to bind to MMP-9 in a 1:1 ratio. There was a significant excess of TIMP-1 compared with MMP-9 in these cells. This is likely why we were only able to detect the latent pro-MMP-9 (92 kDa) form of MMP-9 and not the active (88 kDa) form by zymogram. It is conceivable that the ratio of MMP-9 and TIMP-1 synthesized from different types of cells would be different. We have data to suggest that in neutrophils, which can degranulate and release MMP-9, the secretion of MMP-9 is in excess over TIMP-1 (K. Page and V.S. Hughes, unpublished data). The excess of TIMP-1 secretion from airway bronchial epithelial cells could be a mechanism to prevent disruption of the type IV collagen basement membrane. In a study comparing sputum samples from non-asthmatics, stable asthmatics and acute asthmatics, it was shown that pro-MMP-9 (92 kDa) activities were higher in asthmatic patients (1). They were unable to detect the active form of MMP-9 (88 kDa) in any patient, but rationalized that the concentration of active MMP-9 was small compared with the pro-MMP-9 concentration. Their data suggested that airway inflammation following an asthma exacerbation correlates with overproduction of MMP-9.
The consequence of increased MMP-9 expression following inhalation of cockroach proteases could be to aid in the migration of inflammatory cells. MMP-9 has been shown to be important for human polymorphonuclear neutrophil (PMN) and eosinophil migration across Matrigel-coated micropore membranes (23, 24). The transmigration of dendritic cells, an important antigen-presenting cell in asthma, was impaired in a MMP-9 knockout mouse. In addition, MMP-9 deficiency inhibited allergic airway inflammation characterized by a decrease in PMN and eosinophilic infiltration (25). In another study using MMP-9 knockout mice, lymphocyte and PMN infiltration was decreased following exposure to allergen (26). However, at least one study showed that neutrophil migration is not altered in MMP-9 null mice (27). Recent evidence suggests that pores exist within the basement membranes, and matrix degradation may not be as important as previously expected (28, 29). Additional studies are needed to clarify the importance of MMP-9 in cellular migration.
The MMP-9 could also play a role in the repair response in asthmatics. Some growth factors (such as platelet-derived growth factor, basic fibroblast growth factor and transforming growth factor) are stored in the extracellular matrix by being bound to proteoglycans (30, 31). It has been shown in the endobronchial biopsies of patients with asthma that MMP-9 immunoreactivity is localized in the extracellular matrix of the bronchial submucosa (32). In support of this, it has been shown that MMP-2, -3 and -7 have been shown to degrade decorin, a proteoglycan found in the extracellular matrix (33). It is possible that MMP-9-induced degradation of the collagen type IV in the basement membranes would release growth factors which could stimulate airway smooth muscle cell proliferation, a feature found in the asthmatic airway.
Our previous work has shown that serine proteases in cockroach whole body extract synergistically increased TNFα-induced IL-8 and IL-6 expression (6). IL-8 is a potent neutrophil chemoattractant, and can cause degranulation of recruited neutrophils. Van den Steen (20) has shown that MMP-9 derived from primary human neutrophils cleaves the amino terminus of IL-8 to increase its potency by more than 10-fold. It is conceivable that airway epithelial cells which encounter both cockroach proteases and a pro-inflammatory mediator such as TNFα. TNFα binds to a receptor and causes an intracellular signaling cascade which results in the increase of NF-κB activation. Cockroach proteases bind and cleave the PAR-2 receptor, which triggers its own intracellular signaling cascade resulting in activation of G-coupled protein activation, Ras, MEK (10), ERK and AP-1 (11). The end result is the increase in IL-8, IL-6 and MMP-9 secretion from human bronchial epithelial cells. Neutrophils will be attracted to the airways by the secretion of IL-8 and may also degranulate and release more MMP-9 into the airway. The amount of MMP-9 secretion from the human bronchial epithelial cell is minimal compared to what a neutrophil could secrete, but may play an important role in mediating the early stages of airway inflammation or repair.
Overall, our data suggest that the active serine proteases in cockroach frass play an important role modulating airway responses in the presence of an inflammatory stimulus, like TNFα. Inhalation of active proteases from cockroach frass, or from other sources, may elicit local inflammatory events in the airways, thus causing asthma exacerbations. Further understanding of the role of proteases in modulating airway remodeling and inflammation could result in novel therapeutic interventions for the treatment of asthma.