Evidence for early fibrosis and increased airway resistance in bone marrow transplant recipient mice deficient in MMP12

Abstract

Idiopathic pneumonia syndrome (IPS) is a significant cause of morbidity and mortality post-bone marrow transplantation (BMT) in humans. In our established murine IPS model in which lethally conditioned recipients are given allogeneic bone marrow and splenocytes, recruitment of host monocytes occurs early post-BMT, followed by donor T cells concomitant with development of severe lung dysfunction. Because matrix metalloproteinase 12 (MMP12) is important for macrophage infiltration and injury in other mouse models of lung disease such as emphysema, lethally conditioned MMP12(-/-) mice were used as allogeneic recipients to determine whether MMP12 plays a similar role in potentiating lung injury in IPS. Surprisingly, MMP12(-/-) mice developed IPS and exhibited an accelerated allogeneic T cell-dependent decrease in compliance compared with wild-type (WT) recipients. MMP12(-/-), but not WT, mice also had allogeneic T cell-dependent elevated lung resistance post-BMT. Recruitment of monocytes and T cells into the lungs was not altered on day 7 post-BMT, but the lungs of MMP12(-/-) recipients had increased collagen deposition, a feature normally not seen in our IPS model. MMP12(-/-) mice had a compensatory increase in MMP2 in the lungs post-BMT, as well as increased β6-integrin compared with WT recipients, and only in the presence of allogeneic T cells. Levels of total transforming growth factor (TGF)-β1 protein in the lungs were elevated compared with WT recipients, consistent with the profibrotic function of β6-integrin as an activator of TGF-β. These data indicate that host-derived MMP12 may be important in limiting development of IPS by allowing proper remodeling of extracellular matrix and effective repair of BMT-related injury.

Fig. 1.

Deficiency of matrix metalloproteinase 12 (MMP12)^−/− in the host does not affect the degree of cell influx. Cryosections of lung tissues harvested on day 7 post-bone marrow transplantation (BMT) were stained by hematoxylin and eosin (H&E) (top, top middle, bottom middle) or by in situ hybridization for MMP12 mRNA (bottom), as indicated (magnification ×200). No difference in perivascular or alveolar inflammation can be seen in MMP12^−/− mice compared with the wild-type (WT) mice post-BMT. However, MMP12^−/− mice receiving allogeneic bone marrow and splenocytes (BMS) had evidence of pulmonary emboli (inset). Bottom: many MMP12⁺ cells are present in WT lungs post-BMT as well as in the MMP12^−/− recipients (albeit fewer than in WT), indicating that host and donor MMP12⁺ macrophages infiltrate the lungs post-BMT of MMP12^−/− mice. Photos are representative of 6 mice per group.

Fig. 2.

MMP12^−/− recipients of BMS exhibit accelerated lung dysfunction compared with WT B6 recipients. MMP12^−/− mice also have decreased lung compliance and increased elastance on day 0 in response to pre-BMT conditioning. BM, bone marrow; MME, macrophage metalloelastase (i.e, MMP12). Results shown are representative of 3 experiments (n = 8–24 per group). A: *P < 0.05 for MMP12^−/− BMS vs. all other groups. B: *P < 0.05 for MMP12^−/− BMS vs. MMP12^−/− and B6 BM control and #P < 0.05 for B6 BMS vs. B6 BM control. C and D: +P < 0.05 for MMP12^−/− groups vs. B6 groups, *P < 0.05 for MMP12^−/− BMS vs. all other groups, #P < 0.05 for BMS groups vs. their respective BM controls. Standard deviations ranged from 4.9 to 15.2% of values.

Fig. 3.

MMP12^−/− recipients have elevated OH-proline transforming growth factor (TGF)-β1 levels, collagen deposition, and compensatory increase in MMP2 levels in the lungs compared with WT mice on day 7 post-BMT. This increase was potentiated by the addition of allogeneic splenocytes in the BM inoculum. A: OH-proline levels were determined as described in materials and methods. B: TGF-β1 protein levels were determined by ELISA. C: Masson's trichrome stains of cryosections lungs harvested on day 7 post-BMT (magnification ×100). Collagen is stained blue. D: MMP2 levels in bronchoalveolar lavage fluid (BALF) were measured by Luminex method. *Relevant comparisons with P < 0.05 are shown. n = 6/group pooled from 2 experiments.

Fig. 4.

Increased β6-integrin expression in the lungs of MMP12^−/− mice receiving allogeneic splenocytes. Lungs from day 7 post-BMT mice were evaluated for the levels of β6-integrin by qRT-PCR (A), *P < 0.05, n = 3 per group. B: Western blot, showing 3 mice per transplant group as indicated with relative quantitation. C: immunofluorescence with anti-β6-integrin antibody as described in materials and methods. MMP12^−/− recipients of BMS have significant β6 expression on bronchiolar epithelium, as well as some alveolar epithelium consistent with alveolar type II cells, compared with the BM control, and the B6 recipients. A nonimmune rabbit primary antibody control staining of a serial section of lung of MMP12^−/− recipient of BMS is also shown (magnification ×200). D: serial sections of lungs shown in C (from day 7 post-BMT mice) were stained by H&E (magnification ×100).