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Trafficking of lymphocytes to lung is a critical component of pulmonary immune defense and surveillance. Selectins, expressed on vascular endothelium, regulate T lymphocyte emigration into tissues, such as skin, but the role of the selectins in trafficking of T cells to lung has not been well characterized. Here, we used a model of lung inflammation induced by adoptive transfer of alloreactive Th1 cells to analyze the role of P- and E-selectin in Th1 cell trafficking to lung in vivo. We found that both P- and E-selectin play an important role in Th1 lymphocyte migration to lung. We confirmed that the Th1 cells express P-selectin glycoprotein ligand-1, which was functional in binding to P- and E-selectin in vitro. However, our studies reveal that a ligand distinct from P-selectin glycoprotein-1 also binds these selectins in vitro and appears to play a physiologic role in in vivo emigration of Th1 lymphocytes into the lung.

Tissue inhibitors of metalloproteinases (TIMPs) are multifunctional proteins that have the capacity to modify cellular activities and to modulate matrix turnover. We demonstrate that TIMP-1 messenger RNA (mRNA) and protein expression are selectively and markedly increased in a murine model of bleomycin-induced pulmonary fibrosis. Northern analysis showed that lung steady-state TIMP-1 mRNA levels increased 14-fold after bleomycin administration compared with control mice. Expression of the genes for TIMP-2, TIMP-3, and interstitial collagenase (matrix metalloproteinase-13) was unaltered in the injured lung. In situ hybridization demonstrated that TIMP-1 gene induction was spatially restricted to areas of lung injury. Metalloproteinase inhibitory activity of relative molecular mass of ~ 21 to 28 kD, corresponding to the molecular weights for TIMP-1 and TIMP-2, was identified in lung extracts of bleomycin-injured mice by reverse zymography. Western analysis demonstrated that TIMP-1 protein levels in bronchoalveolar lavage fluid (BALF) of bleomycin-treated mice increased 220- and 151-fold at Days 4 and 28, respectively, compared with control mice. TIMP-2 immunoreactive protein in the BALF increased 20- and 103-fold relative to controls at Days 4 and 28, respectively. These results demonstrate that TIMP-1 gene expression is selectively increased, and that the expression of TIMP-1 and TIMP-2 is differentially regulated in bleomycin-induced pulmonary fibrosis. The profound and durable increase in TIMP-1 and TIMP-2 proteins suggests an important regulatory role for these antiproteases in the inflammatory and fibrotic responses to bleomycin-induced lung injury.

Despite evidence that implicates transforming growth factor-alpha (TGF-alpha) in the pathogenesis of acute lung injury, the contribution of TGF-alpha to the fibroproliferative response is unknown. To determine whether the development of pulmonary fibrosis depends on TGF-alpha, we induced lung injury with bleomycin in TGF-alpha null-mutation transgenic mice and wild-type mice. Lung hydroxyproline content was 1.3, 1.2, and 1.6 times greater in wild-genotype mice than in TGF-alpha-deficient animals at Days 10, 21, and 28, respectively, after a single intratracheal injection of bleomycin. At Days 7 and 10 after bleomycin treatment, lung total RNA content was 1.5 times greater in wild-genotype mice than in TGF-alpha-deficient animals. There was no significant difference between mice of the two genotypes in lung total DNA content or nuclear labeling indices after bleomycin administration. Wild-genotype mice had significantly higher lung fibrosis scores at Days 7 and 14 after bleomycin treatment than did TGF-alpha-deficient animals. There was no significant difference between TGF-alpha-deficient mice and wild-genotype mice in lung inflammation scores after bleomycin administration. To determine whether expression of other members of the epidermal growth factor (EGF) family is increased after bleomycin-induced injury, we measured lung EGF and heparin-binding- epidermal growth factor (HB-EGF) mRNA levels. Steady-state HB-EGF mRNA levels were 321% and 478% of control values in bleomycin-treated lungs at Days 7 and 10, respectively, but were not significantly different in TGF-alpha-deficient and in wild-genotype mice. EGF mRNA was not detected in normal or bleomycin-treated lungs of mice of either genotype. These results show that TGF-alpha contributes significantly to the pathogenesis of pulmonary fibrosis after bleomycin-induced injury, and that compensatory increases in other EGF family members do not occur in TGF-alpha-deficient mice.

Adhesion of cells to components of the extracellular matrix has been shown to be critical in normal lung development, particularly during the pseudoglandular stage, when conducting airways are forming through a process of branching morphogenesis. Expression of factors that inhibit cellular adhesion might also modulate branching morphogenesis. SPARC is a secreted glycoprotein that exhibits antiadhesive effects on cultured cells and is widely expressed in embryonic tissues. In this report, we examine the distribution of SPARC in fetal rat lung during development and its effect on the process of branching morphogenesis. Immunohistochemistry and in situ hybridization studies revealed that SPARC was present in the airway epithelial cells during the pseudoglandular stage of lung development, and in blood vessels and smooth muscle cells associated with airways during the canalicular and saccular stages of development. We used an in vitro model of rat lung branching morphogenesis to examine airway branching in the presence of: a) a neutralizing anti-SPARC antibody; or b) a synthetic peptide from a region of SPARC that, like the native protein, perturbs cell adhesion and diminishes the synthesis of fibronectin and thrombospondin 1. Lungs cultured in the presence of either reagent exhibited diminished branching and an abnormal morphology that was characterized in part by dilated airways. These findings implicate SPARC in the development of the airways.

To investigate the potential role of transforming growth factor-alpha (TGF-alpha) and the epidermal growth factor receptor (EGF-R) in the fibroproliferative response to acute lung injury, we determined lung steady-state TGF-alpha and EGF-R mRNA levels, TGF-alpha protein levels, and the distribution of TGF-alpha and EGF-R immunoreactive protein of bleomycin-injured and control rat lungs. At 2 and 4 days after a single intratracheal injection of bleomycin, TGF-alpha mRNA levels increased to 159% and 184% of control values, respectively. EGF-R mRNA levels increased to 163%, 314%, and 170% of control values at 1, 7, and 14 days after bleomycin instillation. TGF-alpha protein levels in whole lung extracts increased to 230% of control values at 4 days after bleomycin administration. TGF-alpha and EGF-R immunoreactivity was detected in macrophages, alveolar septal cells, and airway epithelium of control and bleomycin-injured animals with an apparent increase in the intensity and number of specifically immunostained cells following lung injury. TGF-alpha and EGF-R immunoreactive proteins were detected in foci of cellular proliferation and in areas of intraalveolar fibrosis. We conclude that TGF-alpha and the EGF-R are present in normal and bleomycin-injured rat lung and that the expression of this growth factor and its receptor are up-regulated following lung injury. These results suggest that increased expression of TGF-alpha and the EGF-R may be an important mechanism that modulates the fibroproliferative response to acute lung injury.

Transforming growth factor-alpha (TGF-alpha), a member of the epidermal growth factor (EGF) family, is a potent mitogen for several cell types. To investigate the possible role of TGF-alpha in the development of midgestation human fetal lung, we studied its distribution with immunohistochemistry and determined levels of steady-state TGF-alpha mRNA by Northern analysis of cellular RNA isolates from lung. Lung was obtained from fetuses at 10 to 22 wk of gestation (n = 14) and immunostained for TGF-alpha. TGF-alpha was localized in epithelial cells at all gestational ages examined. Immunostaining was particularly prominent in bronchiolar epithelial cells. TGF-alpha immunoreactivity was also associated with arterial smooth muscle cells, as well as with nerves. Occasional chondrocytes were also associated with TGF-alpha immunoreactivity. Total cellular RNA was isolated from lung tissue obtained from additional fetuses at gestational ages 10 to 24 wk (n = 22). TGF-alpha mRNA was present in RNA extracts of all fetal lungs studied. We conclude that TGF-alpha is probably produced in human fetal lung during mid-gestation. The prominent immunostaining of bronchiolar epithelial cells for TGF-alpha is consistent with its playing a role in distal airway formation.