Decreased allergic lung inflammatory cell egression and increased susceptibility to asphyxiation in MMP2-deficiency

Abstract

Clearance of recruited immune cells is necessary to resolve inflammatory reactions. We show here that matrix metalloproteinase 2 (MMP2), as part of an interleukin 13 (IL-13)-dependent regulatory loop, dampens inflammation by promoting the egress of inflammatory cells into the airway lumen. MMP2-/- mice showed a robust asthma phenotype and increased susceptibility to asphyxiation induced by allergens. However, whereas the lack of MMP2 reduced the influx of cells into bronchoalveolar lavage (BAL), numerous inflammatory cells accumulated in the lung parenchyma. BAL of MMP2-/- mice lacked normal chemotactic activity, whereas lung inflammatory cells from the same mice showed appropriate chemotactic responses. Thus, MMP2 establishes the chemotactic gradient required for egression of lung inflammatory cells and prevention of lethal asphyxiation.

Figure 1. OVA challenge of BALB/c mice induces a robust asthma phenotype and increased MMP2 activity in BAL

Mice were immunized intraperitoneally with OVA-alum and subsequently challenged intranasally with either saline (OVA-saline) or OVA (OVA-OVA). (a) AHR, assessed as PC₂₀₀. (b) Total number of cells recovered from BAL. (c) The abundance of eosinophils (Eosin), macrophages (Mac), neutrophils (Neut) and lymphocytes (Lymph) in BAL, as assessed by modified Giemsa staining. (d) Serum OVA-specific IgE, as determined by ELISA. (e) IFN-γ, IL-4 and IL-5 concentrations in BAL fluid, as determined by ELISA. (f) Detection of BAL MMP activity by zymography. (Right) More active MMP2 (68 kD) and MMP9 (98 kD) were expressed in OVA-OVA–treated mice (n = 2 mice for each condition). The higher molecular weight bands indicated the presence of relatively inactive (pro-MMP) zymogens (left). Protease activity was neutralized by the addition of 1,10-phenanthroline, a zinc chelator, which confirmed the identity of the MMPs.

Figure 2. Lung MMP2 is expressed in the presence of allergic inflammation and IL-13

(a) Zymogram showing expression of active MMP2 and pro-MMP2 in BAL in response to i.n. challenge of mice with either saline (−) or rIL-13 (+). (b) Northern blot analysis of MMP2 expression. Total RNA from the lungs of BALB/c mice that were challenged with saline or were immunized and then challenged with either OVA (OVA-OVA) or rIL-13 was probed for MMP2 expression. The MMP2 band (3 kb) and relative loading of total RNA (as indicated by the 18S ribosomal RNA subunit band) are shown. (c–f) Lung MMP2 in situ hybridization (ISH). Airway tissue from (c,d) OVA-saline– or (e,f) OVA-OVA–challenged mice were fixed with formalin and stained with H&E; representative samples (n=3) are shown. MMP2 mRNA was detected in contiguous sections with a radiolabeled antisense MMP2-specific probe. Arrowheads indicate bright ISH signals, which appear as dark grains in H&E-stained sections (c,e) or white grains in the darkfield views (d,f). Significant amounts of MMP2 mRNA were detected only in lung mesenchyme (arrowheads) that showed active allergic inflammation (e,f); it was largely absent from airway epithelium (arrows). Bar, 100 μm.

Figure 3. Effect of MMP inhibition on AHR and BAL cell egression

BALB/c mice were immunized with OVA-alum and intranasally challenged with saline or OVA as in Fig. 1. A third immunized group was given the MMP inhibitor GM6001 (150 mg/kg) 1 h before i.n. challenge with OVA (OVA-OVA GM6001). (a) AHR, assessed as PC₂₀₀. (b) Inhibition of cell egression into BAL relative to OVA-OVA–challenged mice (see Methods). At doses of 250 mg/kg, GM6001 inhibited BAL inflammatory cell recruitment by 65%. *P≤0. 05 relative to (a) saline or (b) 50 mg/kg of GM6001.

Figure 4. Comparison of GM6001 treatment to MMP2 deficiency

Wild-type and syngeneic MMP2^−/− mice were challenged intranasally with saline or CAA and responses were compared to those of allergen-challenged WT mice treated with GM6001. (a) AHR, assessed as PC₂₀₀. (b) Total serum IgE, measured by ELISA. (c) Total BAL mucin. (d) Total BAL cells. (e) BAL cell differential, which indicates the total numbers of individual inflammatory cells. (f–i) Photomicrographs of representative lung bronchovascular bundles stained with H&E. The insets at the upper right depict higher magnification images of the airway epithelium stained with periodic acid-Schiff. (g–i) Abundant mucus-producing goblet cells (pink cytoplasm) are seen lining the airway epithelium of all mice receiving the CAA allergen. (h,i) Relative to the mice challenged with saline, peri-bronchovascular inflammatory cells were visible in the lungs of allergen-challenged mice but were more prominent in MMP2^−/− and GM6001-treated animals. (j) This was confirmed by counting the cells within 40,000 μm² areas. *P≤0.05 compared to WT saline. **P≤0.05 compared to WT CAA. Bar, 100μm.

Figure 5. MMP2^−/− mice aberrantly accumulate T_H2 cytokine mRNA in the lung and show increased susceptibility to lethal asphyxiation

(a) Total RNA was obtained from lungs of mice treated as in Fig. 4. Equal quantities of RNA (10 ng) were then analyzed for the indicated T_H2 cytokines and housekeeping genes (L32 and GAPDH) by RPA. (b) Specific mRNA species are indicated by dark bands that are shifted slightly downward in the gels relative to the gene probes. Relative to wild-type, all mRNA species were over-represented in CAA-challenged MMP2^−/− mice, but IL-4 and IL-13 were disproportionately affected. This was confirmed by quantitative real-time PCR which indicates an increase in expression of IL-4 and IL-13 in the absence of MMP2. Mice were given i.n. CAA every 4 days in the (c) absence (n=10) or (d) presence (n=20) of 100% supplemental oxygen (given for 10 min before and after CAA challenge). Survival was assessed within 30 min of each antigen challenge. tRNA, transfer RNA (used as negative control). *P≤0.05 compared to MMP2^−/−.

Figure 6. Aberrant chemotaxis of lung inflammatory cells in response to MMP2^−/− BAL

(a) The migratory responses of lung inflammatory cells from CAA-challenged wild-type and MMP2^−/− mice in response to RPMI (media), BAL from CAA-challenged MMP2^−/− (BAL MMP2^−/−) or wild-type (BAL WT) mice and recombinant MCP-3 were determined by transfilter assays. The total number of cells that migrated into the chamber filters are shown. Note the normal chemotactic response of MMP2^−/− cells in response to wild-type BAL. *P≤0.05 compared to the media control. **P≤0.05 compared to MMP2^−/− BAL. (b) Concentrations of CCL11, CCL7 and CCL17 in BAL from wild-type and MMP2^−/− mice challenged with CAA, as measured by ELISA. *P≤0.05 compared to wild-type mice.