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Overlapping and independent contributions of MMP2 and MMP9 to lung allergic inflammatory cell egression through decreased CC chemokines¹

DAVID B. CORRY^*,,, ATTILA KISS^*, LI-ZHEN SONG^*, LING SONG^*, JIE XU^*, SEUNG-HYO LEE^*,, ZENA WERB and FARRAH KHERADMAND^*,,2

Departments of
^* Medicine and
Immunology, and
Section of Pulmonary and Critical Care Medicine, Baylor College of Medicine, Houston, Texas, USA; and
Department of Anatomy, University of California, San Francisco, California, USA

²Correspondence: Baylor College of Medicine, One Baylor Plaza, Suite 520B, Houston, TX 77030, USA. E-mail: farrahk{at}bcm.tmc.edu

SPECIFIC AIMS

Mechanisms that initiate allergic lung inflammation are relevant to expression of diseases such as asthma, but the factors underlying resolution of inflammation are equally important. Immune cells recruited to sites of allergic inflammation express MMP9, but the physiological role of this enzyme in allergic lung disease remains unclear. We hypothesized that, much like MMP2, the closely related protease MMP9 may play an important role in facilitating the clearance of preapoptotic inflammatory cells in the allergic lung.

PRINCIPAL FINDINGS

1. MMP9 is expressed by recruited leukocytes during allergic lung inflammation
To understand the lung expression and distribution of MMP9 in mice that exhibit the asthma phenotype, we assessed whole lung mRNA in complete Aspergillus allergen (CAA) challenge. After airway immunization with CAA, MMP9 expression was dramatically enhanced in the peri-bronchovascular regions of the lung where allergic inflammatory cells typically concentrate. The constitutive lung tissues, especially the airway epithelium, did not express MMP9. We found intense MMP9 mRNA expression in macrophages and other recruited inflammatory cells that spared entirely the epithelium and mesenchymal cells, i.e., fibroblasts and endothelium. Although the epithelium does not express MMP2, the MMP9 expression pattern is otherwise in striking contrast to MMP2 expression, which, in mice exhibiting the identical asthma phenotype, is strongly expressed in mesenchymal cells. Thus, although both MMP2 and MMP9 are up-regulated under similar conditions, different cell types differentially express them under naive and inflammatory conditions.

2. Recruitment of allergic inflammatory cells and AHR are independent of MMP9 and MMP2
To understand the role of MMP9 and the potential synergistic effects of MMP9 and MMP2 in allergic lung disease, we assessed the asthma phenotype in wild-type (WT) and MMP-deficient mice using CAA. Similar to MMP2-deficient mice, WT, MMP9^–/–, and MMP9/MMP2 double knockout (dko) mice immunized with CAA showed characteristic allergic and obstructive features, including similar degrees of airway hyperresponsiveness, mucin, and glycoprotein hypersecretion and elevated serum IgE levels. In contrast, total bronchoalveolar lavage (BAL) cells, notably increased in allergen-challenged WT mice, were significantly reduced in both MMP9 and dko mice. Differential cell count of the BAL cells revealed a significant reduction in total number of eosinophils and neutrophils in the lavage fluid of MMP9^–/– and dko mice. Thus, several features of the asthma phenotype were preserved in the absence of MMP2 and MMP9, but lack of either or both enzymes disrupted trafficking of inflammatory cells into the airway. Although decrease in BAL inflammatory cells was also observed in MMP2^–/– mice, it was entirely accounted for by fewer eosinophils. Our observed decrease in neutrophils and eosinophils in the BAL of mice deficient in MMP9 reveals similar but non-overlapping specificity of MMP9 in comparison with MMP2.

3. MMP9^–/– and MMP2/MMP9 double knockout mice show increased inflammatory cells in lung parenchyma
Our previous studies with MMP2^–/– mice showed that this enzyme is required for neither recruitment to the lung of inflammatory cells nor their localization to bronchovascular bundles. We therefore reasoned that because much of the asthma phenotype was preserved in MMP9^–/– and dko mice, allergic inflammatory cell recruitment to lung would not be reduced. Pathologic examination of lungs indicated that, relative to saline-challenged mice, lack of MMP9 or MMP9 and MMP2 did not impair lung recruitment of inflammatory cells or their ability to localize to the peri-bronchovascular space after allergen challenge (Fig. 1 a). We noted larger and more organized aggregates of inflammatory cells clustering tightly around bronchioles and arterioles of MMP-deficient animals, suggesting that either cellular recruitment was increased or egression of cells into the lavage is diminished (arrowheads, Fig. 1a ). We noted a second abnormality unique to MMP9^–/– and dko mice: alveolar inflammation, present diffusely in WT mice, was nearly 3-fold higher and confined more to the perivascular bundle in these mice (Fig. 1a, b ). In the absence of MMP9, expression of IL-4 and IL-13 mRNA was 2- and 3-fold higher, respectively, by real-time polymerase chain reaction (PCR) relative to WT mice after allergen challenge (Fig. 1d ), in keeping with the larger number of inflammatory cells. These findings indicate that in MMP9^–/– and dko mice, the excess inflammatory cells that accumulate abnormally around bronchovascular bundles in the lung parenchyma produce IL-4 and IL-13.

View larger version (69K): [in this window] [in a new window]   Figure 1. Histopathology and cytokine production in MMP9 and MMP2;MMP9 null lungs. a) Photomicrographs of representative bronchovascular bundles stained with H&E. Arrows indicate alveolar inflammation prominent in WT mice; arrowheads show interstitial, peri-bronchovascular inflammation, which was more prominent in MMP-deficient animals. Relative size is indicated by 200 µm bar. b) Relative to WT CAA-challenged mice, 3- to 4-fold more peri-bronchovascular inflammatory cells are visible in lungs of allergen-challenged MMP9 and dko mice as confirmed by enumeration of cells within 40,000 µm2 areas. *P < 0.05 relative to CAA-challenged WT mice. c) Fold difference in IL-4 and IL-13 mRNA production from lungs of the same mice by real-time PCR. *P < 0.05 relative to saline-challenged WT. **P < 0.05 relative to CAA-challenged WT.

4. Excess lung apoptotic cells in the absence of MMP9 and MMP2
An essential aspect of any inflammatory response is the prompt disposition of effete immune cells. Unnecessary inflammatory cells most likely prolong potentially deleterious immune responses; lysed or apoptotic cells are capable of inciting additional tissue injury and inflammation. There were virtually no apoptotic cells in the BAL or parenchyma of saline-challenged animals. Compared with WT mice, MMP9-deficient mice challenged with CAA showed a reduction in the absolute number of apoptotic cells in their BAL as determined by TUNEL assay. In contrast, there was massive accumulation of apoptotic cells in the lungs of allergen-challenged MMP9^–/– mice compared with lungs of allergen-challenged WT mice.

5. Decreased CCL11, CCL17, and CCL7 concentrations in BAL but not lung tissue of MMP9^–/– and dko mice
The excess accumulation of lung parenchymal inflammatory cells combined with diminished cellularity of the bronchoalveolar lavage seen in MMP9^–/– and dko mice suggested that the normal trafficking of recruited lung cells into the airway lumen had been disrupted, favoring their accumulation in parenchyma. Only CCL11 (eotaxin) was affected by lack of MMP2, suggesting an important, but limited, contribution of this metalloproteinase to cellular egression. To understand the contribution of MMP9 and MMP9/MMP2 to transepithelial chemokine gradient formation, we quantified CCL11, CCL7, CCL17, CXCL10, and CXCL3 in BAL of WT, MMP9^–/–, and dko mice challenged with allergen. In contrast to MMP2^–/– mice, concentrations of all three CC chemokines were 30–70% less than those of the corresponding chemokines of WT mice (Fig. 2 a–c). These alterations in CC chemokine concentration were confined to the airway since total lung protein levels of these chemokines did not differ significantly between WT and MMP9 allergen-instilled animals. Because we noted a decrease in transepithelial migration of neutrophils in MMP9 and dko mice, we measured CXCL3 (KC) and CXCL10 (IP-10). We did not detect KC or IP-10 proteins in the BAL of the naive or allergen-challenged WT, MMP9, or dko mice.

View larger version (38K): [in this window] [in a new window]   Figure 2. Chemokine concentration of and chemotactic response to bronchoalveolar lavage fluid in MMP-deficient mice. Wild-type (WT) mice were challenged with saline or allergen (CAA) and compared with MMP9–/– and dko mice for concentrations of a) eotaxin, CCL11, b) MARC, CCL7, and c) TARC, CCL17 in the bronchoalveolar lavage. *P < 0.05 relative to saline-challenged WT. **P < 0.05 relative to CAA-challenged WT. d) Migratory responses of lung inflammatory cells from CAA-challenged WT cells and MMP9–/– mice (MMP9–/– cells) in response to media (RPMI), BAL from CAA-challenged MMP9–/– (BAL MMP9–/–), or BAL WT mice were determined in transfilter assays. Data are expressed as the total number of cells migrating into the chamber filters per low-power field. Note the normal chemotactic response of MMP9–/– cells in response to wild-type BAL. *Significantly different from MMP9–/– BAL.

6. Normal chemotaxis of MMP9-deficient inflammatory cells
Migration and chemotaxis of neutrophils and eosinophils in MMP9-deficient mice may be affected by the loss of this enzyme. Therefore, we assessed the chemotactic ability of inflammatory cells obtained from lungs of allergen-challenged WT and MMP9-deficient mice in response to BAL from the same mice. We found that lung inflammatory cells showed similar chemotaxis in response to WT BAL regardless of genotype (Fig. 2d ). However, a significant reduction in chemotaxis was observed when the same cells were tested against the BAL of MMP9^–/– mice, indicating that chemotactic activity is deficient in MMP9^–/– BAL (Fig. 2d ). These findings are consistent with the hypothesis that, like MMP2, MMP9 controls the formation of transepithelial CC chemokine gradients that control the egression (chemotaxis) of inflammatory cells.

CONCLUSIONS AND SIGNIFICANCE

We show here that leukocyte MMP9 is the dominant airway MMP controlling inflammatory cell egression. The allergic lung phenotype of MMP9^–/– mice was similar to WT and not altered by concomitant deletion of the MMP2 gene (dko). Inflammatory cells accumulated aberrantly in the lungs of allergen-challenged MMP9^–/– and dko mice; fewer eosinophils and neutrophils were present in bronchoalveolar lavage. These aberrant cellular trafficking patterns were explained by disruption of transepithelial chemokine gradients, in MMP2^–/– mice affecting only eotaxin (CCL11) but in MMP9^–/– and dko mice involving eotaxin, CCL7, and CCL17. Thus, by establishing multiple transepithelial chemokine gradients, MMP9 is broadly implicated in the resolution of allergic inflammation, an essential protective mechanism that overlaps with a more limited role played by MMP2 (Fig. 3 ).

View larger version (41K): [in this window] [in a new window]   Figure 3. Schematic diagram depicting the migration (extravasation, intraparenchymal homing and transepithelial egression) of allergic inflammatory cells recruited to the lungs. Cellular migration is shown progressing downwards, with recently extravasated cells (including T cells, monocytes, eosinophils, and mast cells) traversing the pulmonary interstitium and the airway epithelium to enter the airway lumen, where they are cleared. Interstitial inflammatory cells are recruited to the lumen by establishing a transepithelial chemotactic gradient in which chemokines are strongly expressed on the apical surface of epithelial cells relative to the interstitium. Lack of MMP9 and MMP2 disrupts formation of this chemokine gradient and impairs migration of cells at points marked X.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-1412fje;

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