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* From the Division of Pulmonology (Dr. Yong), Wonju Medical College, Yonsei University, Wonju, Korea; Carolina Respiratory Specialists (Dr. Adlakha), Charlotte, NC; and the Thoracic Diseases Research Unit (Dr. Limper), Division of Pulmonary Critical Care and Internal Medicine, Mayo Clinic, Rochester, MN.
Correspondence to: Andrew H. Limper, MD, PhD, Thoracic Diseases Research Unit, 601C Guggenheim Building, Mayo Clinic, Rochester, MN 55905; e-mail: limper.andrew{at}mayo.edu
Idiopathic pulmonary fibrosis (IPF) is characterized by patchy inflammatory infiltration of the lungs with mesenchymal cell proliferation, extracellular matrix deposition, and progressive loss of normal lung architecture. Active lesions of IPF are comprised of macrophage-rich fibrinous alveolar exudates in regions of epithelial injury. These exudates subsequently organize by fibroblast infiltration and deposition of extracellular matrix proteins including fibronectins, collagens, and proteoglycans, which generate the active fibroblastic foci typical of IPF. Prior work by our group and others has strongly implicated transforming growth factor (TGF)-ß1 in promoting fibroblastic proliferation and matrix accumulation in fibrotic lung diseases.1 2 3 Inhibition of TGF-ß has recently been proposed as a potential therapeutic avenue for the management of lung fibrosis.4
TGF-ß1 is a 25-kd homodimeric protein secreted
by numerous cells, including platelets, macrophages, epithelial cells,
and fibroblasts.5
Unlikely other peptide growth factors,
TGF-ß1 is secreted almost exclusively as a
biologically inactive or latent complex. Activation mechanisms of
TGF-ß1 are not fully understood. However,
activation can occur under a variety of conditions, including
incubation at extremes of pH, exposure to endoglycosidase F and
sialidase, proteolysis by plasmin or cathepsin D, and by interaction
with 
vß6 integrin receptors.5
6
TGF-ß1 not only stimulates the synthesis of
many extracellular matrix molecules, including fibronectin and type I
collagen and their receptors, but also decreases matrix degradation via
differential effects on the expression of proteases and their
inhibitors, strongly promoting generation of extracellular
matrix.5
7
8
Study of human IPF tissues clearly
demonstrates marked accumulation of TGF-ß1 on
regions of active matrix protein expression and fibrosis.1
Circulating TGF-ß1 levels have been shown to correlate with the development of liver and lung fibrosis after autologous bone marrow transplantation for breast cancer.9 The presence of circulating TGF-ß1 in patients with IPF has not been characterized. We therefore hypothesized that increased total and active TGF-ß circulate in plasma of patients with IPF and that TGF-ß levels are influenced by treatment of this disorder, providing a potential clinical marker for disease activity. To address this, we performed a prospective cross-sectional analysis of circulating TGF-ß in patients with IPF.
Materials and Methods
Patients had evidence of lung fibrosis for at least 3 months as reflected by clinical assessment, consisting of either diffuse pulmonary infiltrates on chest radiograph or typical findings of IPF on high-resolution CT or open-lung biopsy specimens. All 38 patients with IPF demonstrated progressive disease by clinical, radiologic, or pulmonary function assessment. Ten milliliters of blood was collected, and the plasma was frozen at - 70°C for evaluation of TGF-ß1. Plasma from six normal control subjects was obtained in an identical fashion.
Plasma TGF-ß activity was measured using the CCL-64 mink lung epithelial cell growth inhibition assay.10 In brief, CCL-64 mink lung epithelial cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum. CCL-64 cells were plated in triplicate at a cell density of 4 x 104 cells per well in 24-well tissue culture dishes and incubated for 24 h. A standard concentration of TGF-ß1 (0 to 3.0 ng/mL) was prepared and added to a series of wells. Plasma from normal control subjects and patients with IPF was diluted 1:50 in Hanks’ balanced salt solution containing 1 mg/mL of bovine serum albumin, and aliquots of each (250 L) were added to replace media on additional wells of cells in triplicate. These were incubated for an additional 22 h. The next day, wells were pulsed for 2 h with 3H thymidine (1 Ci per well) and washed with 10% trichloroacetic acid. Radionuclide incorporation in each well was quantified by scintillation counting.
Total immunoreactive TGF-ß1 in the plasma was determined using a TGF-ß1 capture enzyme-linked immunosorbent assay (ELISA [Predicta TGF-ß1 assay; Genzyme; Cambridge, MA]) as previously reported.11 Ten microliters of each plasma sample was added to siliconized tubes containing 450 µL of diluent. The diluted samples were transiently acidified with 1 N HCl to activate all latent TGF-ß1, making it accessible to the antibody used in the ELISA. Subsequently, the samples were neutralized by addition of 1 N NaOH. The activated and diluted standards and samples were plated in duplicate on test wells containing immobilized antihuman TGF-ß1 and incubated for 1 h at 37°C. Next, the wells were treated with an anti–TGF-ß1 horseradish peroxidase conjugate and incubated for an additional hour. After washing, trimethylbenzidine substrate reagent was added and incubated for 20 min at room temperature. Absorbances were read at 450 nm.
Results
A total of 38 patients with IPF and 6 normal control subjects were enrolled in this study. The mean duration of symptoms was 2.7 ± 2.0 years prior to evaluation. Pulmonary function testing of the IPF group revealed a mean FVC of 62.4% predicted and a mean diffusion capacity of the lung for carbon monoxide of 55.1% predicted, indicating moderate restrictive lung disease. Total immunoreactive TGF-ß1 was significantly higher in patients with IPF compared to normal control plasma (Fig 1 ). The mean (± SD) total plasma TGF-ß1 in patients with IPF was 33.2 ± 24.6 ng/mL compared to 8.2 ± 4.0 ng/mL in the control group (p = 0.0001). We further investigated whether TGF-ß activity was also increased in the plasma of patients with IPF compared to normal subjects. Functional TGF-ß activity was evaluated using the CCL-64 cell growth inhibition assay, without activation of latent TGF-ß. In a similar manner, active TGF-ß in the plasma was increased in patients with IPF compared to control subjects (Fig 1) . Mean TGF-ß activity in plasma of patients with IPF was 2.7 ± 3.5 ng/mL compared to 0.3 ± 0.2 ng/mL in control subjects (p = 0.03).
|
20
mg/d of prednisone at our initial evaluation. Patients who received
prior corticosteroid treatment exhibited no difference in total plasma
TGF-ß1 compared to previously untreated
patients. In contrast, plasma TGF-ß activity in the previously
treated group was significantly lower than in untreated patients
(1.2 ± 0.5 ng/mL vs 2.8 ± 1.4 ng/mL; p = 0.03). We further prospectively investigated six previously untreated patients with active IPF who began therapy after enrollment. Plasma was collected before and 3 months after treatment was initiated.12 Before treatment, total TGF-ß1 level was 41.2 ± 17.2 ng/mL. After 3 months of treatment, total TGF-ß1 level was significantly lower (24.9 ± 17.8 ng/mL; p = 0.02). Active TGF-ß was also significantly decreased by treatment (from 2.8 ± 1.4 to 1.2 ± 0.5 ng/mL; p = 0.03). Notably, posttreatment levels of total and active TGF-ß1 were still significantly higher than in normal control subjects (p = 0.04 and p = 0.006, respectively).
Discussion
Our data indicate that TGF-ß1, a potent profibrotic cytokine that strongly drives the deposition of extracellular matrix proteins in lung, also circulates at elevated levels in the plasma of patients with IPF, and may be decreased during treatment of this disorder. This observation is highly complementary to prior studies1 2 that have revealed augmented local tissue expression of TGF-ß1 in lung biopsy specimens and elevated TGF-ß1 level in the BAL fluid of the patients with IPF.
Recent work utilizing immunologic and molecular approaches has suggested that antagonism of TGF-ß activity may alleviate fibrosis following lung injury. Transient gene transfer of Smad 7 to inhibit TGF-mediated cell signaling was recently found to prevent bleomycin-induced lung fibrosis in mice.4 Previous studies using anti–TGF-ß neutralizing antibodies or TGF inhibitory proteins have revealed similar findings in preventing fibrosis in bleomycin-treated animals. Such studies provide additional strong evidence supporting TGF-ß antagonism as a desirable goal in designing effective therapies for IPF.
Our current study demonstrates persistently higher levels of circulating plasma TGF-ß1 in patients with active IPF compared to normal control subjects, even following conventional treatment. We demonstrated that prior treatment with corticosteroid is associated with reduced TGF-ß levels, compared to untreated patients. Nevertheless, the posttreatment TGF-ß levels were still significantly higher than in untreated control subjects. We further observed that prospective treatment with colchicine (0.6 mg bid) also significantly suppressed circulating levels of TGF-ß activity, although this suppression again did not reduce the level to that of untreated control subjects. Clinically, neither corticosteroids nor colchicine arrests progressive lung fibrosis in a majority of patients with IPF.12 This disappointing lack of efficacy might be explained by the inability of these current agents to completely control the exuberant tissue expression of TGF-ß that promotes the accumulation of extracellular matrix proteins. Serial monitoring of circulating total and active TGF-ß may represent a useful parameter in assessing disease activity and monitoring response to therapy during IPF. We further postulate that therapies directed at suppressing TGF-ß activity to normal levels will have optimal efficacy in treating IPF.
Footnotes
Abbreviations: ELISA = enzyme-linked immunosorbent assay; IPF = idiopathic pulmonary fibrosis; TGF = transforming growth factor
This research was supported by an American Heart Association research grant to Dr. Limper. Dr. Yong was supported by funds from Wonju Medical College, Yonsei University.
References
v ß 6 binds and activates latent TGF ß 1: a mechanism for regulating pulmonary inflammation and fibrosis. Cell 96,319-328[CrossRef][ISI][Medline]
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  K. A. Keogh, J. Standing, G. C. Kane, A. Terzic, and A. H. Limper Angiotensin II antagonism fails to ameliorate bleomycin-induced pulmonary fibrosis in mice Eur. Respir. J., April 1, 2005; 25(4): 708 - 714. [Abstract] [Full Text] [PDF]
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