Arhgef1 regulates alpha5beta1 integrin-mediated matrix metalloproteinase expression and is required for homeostatic lung immunity

Abstract

Pulmonary immunity depends on the ability of leukocytes to neutralize potentially harmful and frequent insults to the lung, and appropriate regulation of leukocyte migration and adhesion is integral to this process. Arhgef1 is a hematopoietic-restricted signaling molecule that regulates leukocyte migration and integrin-mediated adhesion. To explore a possible regulatory role for Arhgef1 in pulmonary immunity we examined the lung and its leukocytes in wild-type and Arhgef1-deficient animals. Here we report that the lungs of Arhgef1-/- mice harbored significantly more leukocytes, increased expression and activity of matrix metalloproteinases (MMPs), airspace enlargement, and decreased lung elastance compared with wild-type lungs. Transfer of Arhgef1-/- lung leukocytes to wild-type mice led to airspace enlargement and impaired lung function, indicating that loss of Arhgef1 in leukocytes was sufficient to induce pulmonary pathology. Furthermore, we showed that Arhgef1-deficient peritoneal macrophages when either injected into the lungs of wild-type mice or cultured on fibronectin significantly increased expression and activity of MMPs relative to control macrophages, and the in vitro fibronectin induction was dependent on the alpha5beta1 integrin pair. Together these data demonstrate that Arhgef1 regulates alpha5beta1-mediated MMP expression by macrophages and that loss of Arhgef1 by leukocytes leads to pulmonary pathology.

Figure 1

Arhgef1-deficient animals harbor increased numbers of lung leukocytes. A: Hematoxylin and eosin (H&E) stained lung from Arhgef1-deficient mice at the indicated ages. Top row is taken at ×10 magnification. The boxed area in the top row is shown at ×60 magnification below the respective sample. B: Leukocyte populations recovered from bronchoalveolar lavage (BAL) of 3-month-old mice. Wild-type (open bars) or Arhgef1^−/− (solid bars) mice were lavaged with 3 × 1 ml aliquots of HBSS with 5 mmol/L EDTA. Cells were enumerated with a BD Coulter counter followed by flow cytometric analysis as described in methods. Data represent mean ± SE; n = 16 for C57BL/6; n = 15 for Arhgef1^−/−. C: Leukocytes recovered from lavaged, perfused, and enzymatically digested wild-type (open bars) and mutant (solid bars) lung tissue. Cells were enumerated and defined as in B. Data represent mean ± SE; n = 8 for both Arhgef1^−/− and C57BL/6 mice. D: Number of macrophages recovered from BAL at 3 weeks, 3 months, and 6 months in wild-type (open bars, n = 4, 16 and 3, respectively) and mutant (solid bars, n = 4, 15 and 5, respectively) animals. AM indicates alveolar macrophages. *P < 0.05 Student two-tailed t test compared with C57BL/6 samples.

Figure 2

Impaired lung mechanics and airspace enlargement in the absence of Arhgef1. A: Quasi-static pressure-volume loops performed on wild-type (open boxes, dotted lines) and Arhgef1^−/− mice (solid boxes, solid lines) from 1.5 weeks to 12 months of age. Data represent mean ± SE C57BL/6 mice n = 4, 4, 11, 4, and 6; Arhgef1^−/−n = 4, 4, 12, 4, and 8 for 1.5 weeks, 3 weeks, 3 months, 6 months, and 1 year of age, respectively.B: Representative H&E stained sections of lung from C57BL/6 (left column) and Arhgef1^−/− (right column) mice from 1 day old to 1 year of age. C: Mean linear intercept (μm) of alveolar septae as measured on H & E–stained wild-type (open bars) and mutant (solid bars) inflated lungs. Data represent mean ± SE For C57BL/6 mice n = 4, 4, 7, 7, 7, and 9 for Arhgef1^−/− mice n = 3, 4, 7, 9, 7, and 18 for 1 day, 1.5 weeks, 3 weeks, 3 months, 6 months, and 1 year of age, respectively. *P < 0.05 for Student two-tailed t test compared with C57BL/6 mice.

Figure 3

Loss of Arhgef1 leads to increased metalloproteinase expression and activity in the lung. A: MMP2 (top), MMP9 (middle), and MMP12 (bottom) expression measured by qPCR in whole lung at day 1, adult lavaged lung tissue, adult BAL leukocytes, adult liver, bone marrow neutrophils (BMN), or peritoneal elicited macrophages (PEM). Arhgef1^−/− (solid bars, n = 8) MMP expression is shown as fold relative to control samples (open bars n = 8) with all samples normalized to 18s RNA. B: Gelatin zymography of BAL supernatant from wild-type (+/+) and homozygous (−/−) mutants. Molecular weight standards and respective enzymatic activity of MMPs are shown. Shown below zymogram is the quantitation of MMP activity as determined by densitometric analysis. MMP activity is shown as fold relative to control samples wild-type (open bars) n = 4 and Arhgef1^−/− (solid bars) n = 6 from two independent experiments. C: Gelatin zymography of BAL supernatant from Arhgef1^−/− mice adsorbed with anti-MMP2 or anti-MMP9. Molecular weight standards and respective enzymatic activity of MMPs are noted. Shown below zymogram is the quantitation of MMP as determined by densitometric analysis. MMP activity is shown as fold relative to BSA adsorbed. Open bars represent BSA adsorbed sample, gray bars represent anti-MMP2 adsorbed sample, and solid bars represent anti-MMP9 adsorbed samples. Data represent mean ± SE. *P < 0.05 Student two-tailed t test compared with C57BL/6 samples. ND indicates not detected.

Figure 4

Transfer of Arhgef1-deficient BAL cells causes pathology in adult lungs. Eight-week-old wild-type mice received intratracheal instillations of BAL cells recovered from either C57BL/6 mice or Arhgef1-deficient mice once a week for 4 weeks. A: Quasi-static pressure-volume loops performed on wild-type mice that received either wild-type BAL cells (open boxes, dotted lines, n = 5) or Arhgef1^−/− BAL cells (solid boxes, solid lines, n = 9). Data represent mean ± SE. B: Mean linear intercept (μm) of alveolar septae as measured on H & E–stained inflated lungs from wild-type mice that received either wild-type BAL cells (open bar, n = 5) or Arhgef1^−/− BAL cells (solid bar, n = 9). C: MMP2, MMP9, and MMP12 expression measured by qPCR in BAL cells recovered 48 hours after the transfer of peritoneal macrophages from wild-type (n = 4) and Arhgef1^−/− (n = 7) into wild-type recipient mice. D: Representative gelatin zymography of BAL supernatant recovered from mice described in C. Whole cell lysate of BAL cells from LPS-treated wild-type mouse is included as positive control for pro-MMP9. E: MMP activity in BAL supernatant from mice described in C as determined by densitometric analysis. MMP activity is shown as fold relative to control samples. Data represent mean ± SE. *P < 0.05 for Student two-tailed t test comparing mice that received wild-type cells to mice that received Arhgef1^−/− cells.

Figure 5

Arhgef1-deficient peritoneal elicited macrophages exhibit increased integrin-mediated MMP expression. A: Adhesion of wild-type (open bars) and Arhgef1^−/− (solid bars) PEMs to BSA, laminin, ICAM-1, VCAM-1, and fibronectin after 48 hours of incubation. Wild-type (n = 6) and Arhgef1^−/− (n = 6) from 3 independent experiments. B: MMP2 (top), MMP9 (middle), and MMP12 (bottom) expression measured by qPCR of wild-type (open bars) and Arhgef1^−/− (solid bars) PEMs cultured for 48 hours on the indicated substrates. MMP expression is shown as fold relative to wild-type samples cultured on BSA with all samples normalized to 18s RNA. Wild-type (n = 8) and Arhgef1^−/− (n = 12) from 4 independent experiments. C: MMP activity as quantified by densitometric analysis of conditioned media from macrophages cultured on fibronectin, wild-type (open bars; n = 6), and Arhgef1^−/− (solid bars; n = 6). Data represent mean ± SE. *P < 0.05 Student two-tailed t test compared with cells on BSA. **P < 0.05 Student two-tailed t test comparing wild-type to Arhgef1^−/− cells.

Figure 6

Induction of MMPs on fibronectin is dependent on α5β1 integrin signaling. MMP2 expression (A) and MMP9 expression (B) as measured by qPCR of wild-type (open bars, n = 6) and Arhgef1^−/− (solid bars, n = 6) PEMs cultured for 24 hours on fibronectin with either anti-CD29 (2 μg/ml) or anti-CD49E (50 μg/ml) antibodies. MMP expression is shown as percentage of untreated fibronectin plated PEMs. Data represent mean ± SE from two independent experiments. *P < 0.05 Student two-tailed t test comparing untreated samples to respective antibody treatments.