Allergy Asthma Immunol Res.  2012 Jul;4(4):231-239. 10.4168/aair.2012.4.4.231.

Expression and Roles of MMP-2, MMP-9, MMP-13, TIMP-1, and TIMP-2 in Allergic Nasal Mucosa

• Mori S
• Pawankar R
• Ozu C
• Nonaka M
• Yagi T
• Okubo K

• KMID: 1970712
• DOI: http://doi.org/10.4168/aair.2012.4.4.231

Abstract

PURPOSE
Allergic rhinitis (AR) and asthma share many characteristics, but structural changes are observed far less often in AR. Matrix metalloproteinases (MMPs) constitute a family of Zn-dependent endopeptidases that can decompose the extracellular matrix and basement membrane, and regulate cell infiltration. We analyzed the expression of MMPs and their inhibitors, tissue inhibitors of metalloproteinases (TIMPs), in allergic nasal mucosa after nasal allergen challenge (NAC) and determined their relationship to inflammatory cells.
METHODS
Nasal mucosa specimens were obtained at surgery performed for hypertrophied turbinates. We performed NAC with house dust mite (HDM) allergen disks and control disks, and took biopsies at 30 minutes, 6 hours, and 12 hours after NAC. Cells expressing MMP-2, MMP-9, MMP-13, TIMP-1, and TIMP-2, as well as eosinophils and mast cells, were analyzed immunohistochemically. The MMPs and TIMPs in allergic nasal mucosa were quantified using enzyme-linked immunosorbent assays.
RESULTS
At 30 minutes post-NAC, HDM-exposed nasal mucosa exhibited significantly more MMP-2+, MMP-9+, MMP-13+, TIMP-1+, and TIMP-2+ cells compared with control mucosa, and the numbers of MMP-9+ and TIMP-1+ cells correlated strongly with the number of mast cells. At 6 hours post-NAC, the numbers of MMP+ and TIMP+ cells did not differ significantly between HDM-exposed mucosa and control mucosa, but the ratios of MMP+ cells to TIMP+ cells were higher in HDM-exposed mucosa. At 12 hours post-NAC, the number of MMP-13+ cells tended to be higher in HDM-exposed mucosa and was strongly correlated with the number of eosinophils. Quantitatively, the levels of MMP-2 and MMP-13 were significantly higher than the MMP-9 level, and the TIMP-2 level was significantly higher than the TIMP-1 level in allergic nasal mucosa.
CONCLUSIONS
We demonstrated increased expression of MMP-2, MMP-9, and MMP-13 in allergic nasal mucosa, high MMPs-to-TIMP-1 ratios, and a strong correlation between MMP-9 and mast cells and between MMP-13 and eosinophils. The imbalance between MMPs and TIMPs may contribute to the migration of inflammatory cells such as eosinophils and mast cells to the nasal mucosa of AR patients, suggesting a possible active role of MMPs in AR.

Keyword

Allergic rhinitis; MMP; TIMP; cell infiltration; mast cells; eosinophils

MeSH Terms

Asthma
Basement Membrane
Biopsy
Endopeptidases
Eosinophils
Extracellular Matrix
Humans
Mast Cells
Matrix Metalloproteinases
Metalloproteases
Mucous Membrane
Nasal Mucosa
Pyroglyphidae
Rhinitis
Rhinitis, Allergic, Perennial
Tissue Inhibitor of Metalloproteinase-1
Tissue Inhibitor of Metalloproteinase-2
Turbinates
Endopeptidases
Matrix Metalloproteinases
Metalloproteases
Tissue Inhibitor of Metalloproteinase-1
Tissue Inhibitor of Metalloproteinase-2

Figure

• 

  Fig. 1 Immunoreactivity and localization of MMP-2, MMP-9, MMP-13, TIMP-1, and TIMP-2 in the nasal mucosa of PAR patients. Immunohistochemistry using the APAAP method was performed as described in the Materials and Methods section. (A) MMP-2 was expressed mainly in inflammatory cells, but also in fibroblasts, epithelial cells, and endothelial cells. (B) MMP-9 was expressed mainly in inflammatory cells, but also in fibroblasts, epithelial cells, and acinar cells. (C) MMP-13 was expressed mainly in inflammatory cells, but also in fibroblasts, endothelial cells, and acinar cells. (D) TIMP-1 was expressed mainly in inflammatory cells, but also in fibroblasts, epithelial cells, and endothelial cells. (E) TIMP-2 was expressed mainly in inflammatory cells, but also in fibroblasts and endothelial cells. (F) The negative control showed no immunoreactivity (magnification, ×200-400 HPF).

• 

  Fig. 2 The numbers of MMP-2+, MMP-9+, MMP-13+, TIMP-1+, and TIMP-2+ cells in the nasal mucosa of PAR patients at 30 min after NAC with HDM allergen disks versus control disks. Immunohistochemistry using the APAAP method was performed as described in the Materials and Methods section. Positively stained cells in an area of 0.202 mm2 were counted at ×400 HPF. Box plots represent the median values with 25 and 75% interquartiles. Error bars represent the 10th and 90th percentiles; the squares indicate the means. (A) At 30 min post-NAC, the numbers of MMP-2+, MMP-9+, and MMP-13+ cells in the HDM-exposed mucosa were significantly higher than those in the control-exposed mucosa (n=10). (B) At 30 min post-NAC, the numbers of TIMP-1+ and TIMP-2+ cells in the HDM-exposed mucosa were significantly higher than those in the control-exposed mucosa (n=10). *P<0.05, **P<0.02, by Wilcoxon signed-rank test.

• 

  Fig. 3 The numbers of MMP-2+, MMP-9+, MMP-13+, TIMP-1+, and TIMP-2+ cells in the nasal mucosa of PAR patients at 6 hr after NAC with HDM allergen disks versus control disks. Immunohistochemistry using the APAAP method was performed as described in the Materials and Methods section. Positively stained cells in an area of 0.202 mm2 were counted at ×400 HPF. Box plots represent the median values with 25 and 75% interquartiles. Error bars represent the 10th and 90th percentiles; the squares indicate the means. (A) At 6 hr post-NAC, there was no significant difference in the number of MMP-2+, MMP-9+, or MMP-13+ cells between HDM-exposed and control-exposed nasal mucosa (n=10). (B) At 6 hr post-NAC, there was no significant difference in the number of TIMP-1+ or TIMP-2+ cells between HDM-exposed and control-exposed nasal mucosa (n=10).

• 

  Fig. 4 The numbers of MMP-2+, MMP-9+, MMP-13+, TIMP-1+, and TIMP-2+ cells in the nasal mucosa of PAR patients at 12 hr after NAC with HDM allergen disks versus control disks. Immunohistochemistry using the APAAP method was performed as described in the Materials and Methods section. Positively stained cells in an area of 0.202 mm2 were counted at ×400 HPF. Box plots represent the median values with 25 and 75% interquartiles. Error bars represent the 10th and 90th percentiles; the squares indicate the means. (A) At 12 hr post-NAC, there was no difference in the number of MMP-2+, MMP-9+, or MMP-13+ cells between HDM-exposed and control-exposed nasal mucosa (n=10). (B) At 12 hr post-NAC, there was no difference in the number of TIMP-1+ or TIMP-2+ cells between HDM-exposed and control-exposed nasal mucosa (n=10).

• 

  Fig. 5 Correlation of MMP-9+ and TIMP-1+ cells with mast cells in the nasal mucosa of PAR patients. (A) There was a strong correlation between the number of mast cells and the number of MMP-9+ cells at 30 min after NAC with HDM allergen disks. (r=0.8, P<0.02, by Spearman's rank correlation coefficient; n=10.) (B) There was a strong correlation between the number of mast cells and the number of TIMP-1+ cells at 30 min after NAC with HDM allergen disks. (r=0.8, P<0.05, by Spearman's rank correlation coefficient; n=10.)

• 

  Fig. 6 Correlation between MMP-13+ cells and eosinophils in the nasal mucosa of PAR patients. The number of eosinophils was strongly correlated with the number of MMP-13+ cells at 12 hr after NAC with HDM allergen disks (r=0.75, P<0.05, by Spearman's rank correlation coefficient; n=10).

• 

  Fig. 7 The levels of MMP-2, MMP-9, MMP-13, TIMP-1, and TIMP-2, as determined by specific ELISA, in the nasal mucosa of PAR patients. MMP-2 and MMP-13 levels were higher than the MMP-9 level, and the TIMP-2 level was higher than the TIMP-1 level in allergic nasal mucosa. *P<0.05, **P<0.01, Wilcoxon signed-rank test; n=6.

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