Human T cells stimulate fibroblast-mediated degradation of extracellular matrix in vitro

Abstract

Several chronic diseases are characterized by inflammation, T cell recruitment and tissue remodelling. We hypothesized that activated T cells may stimulate remodelling of extracellular matrix (ECM) in vitro. Total T cells (CD3+) as well as CD4+ and CD8+ subsets were isolated from peripheral blood and stimulated, after which conditioned media (CM) were obtained. CM was added to human lung fibroblasts in three-dimensional collagen gels and the area of gels was measured daily. Hydroxyproline was determined as a measure of collagen degradation in the gels. Matrix metalloproteinase (MMP) activity in the culture media was analysed by gelatine zymography. Cytokine secretion of stimulated CD4+ and CD8+ T cells was analysed. CD3+ CM augmented collagen gel contraction in a time- and dose-dependent manner (P < 0.0001). CD4+ T cell CM was more potent than CD8+ T cell CM (P < 0.001). CD3+ CM and CD4+ T cell CM, but not CD8+ T cell CM, stimulated fibroblast-mediated collagen degradation and MMP-9 activity. A broad-spectrum MMP-inhibitor added to the culture system inhibited both gel contraction and MMP activity. Activated CD4+ T cells secreted significantly more tumour necrosis factor (TNF) and interleukin (IL)-6 compared to CD8+ T cells. CD3+ CM from patients with chronic obstructive pulmonary disease stimulated fibroblast-mediated collagen gel contraction to the same magnitude as CD3+ CM from healthy controls. In conclusion, activated CD4+ T cells can stimulate fibroblast-mediated degradation of ECM in vitro. This could be a mechanism by which activated T cells stimulate degradation of lung tissue leading to pulmonary emphysema.

Fig. 1

T cell conditioned media (CM) stimulate fibroblast-mediated gel contraction. Fibroblasts [3 × 10⁵ human fetal lung fibroblasts (HFL)] were cast into collagen gels in a 24-well culture plate. After gelation the gels were released into a 60 mm tissue culture dish containing serum-free Dulbecco's modified essential medium (DMEM) with or without T cell CM. The area of the floating gels was measured daily with an image analyser. (a) Photograph of collagen gels after 4 days of culture. (i) Control gels with fibroblasts. (ii) Gels with fibroblasts stimulated with 1/5 CD3⁺ CM. (b) Time- and concentration-dependent augmentation of fibroblast-mediated collagen gel contraction by various concentrations of CD3⁺ CM (1/40, 1/20, 1/10 and 1/5). Vertical axis: the percentage of initial gel area. Horizontal axis: time in days. Data are presented as mean of triplicate gels ± standard error of the mean (s.e.m.) from one representative experiment. (c) CD4⁺ and CD8⁺ T cell stimulation of fibroblast-mediated collagen gel contraction. CD4⁺ and CD8⁺ T cell CM were diluted 1/5 in serum-free DMEM. Data presented as in (b).

Fig. 2

T cell conditioned media (CM) stimulate fibroblasts to release active matrix metalloproteinase (MMP)-9 in three-dimensional cultures. Media from gel cultures were collected and concentrated. Proteins were separated by electrophoresis on an acrylamide gel containing gelatine. MMP-2 and MMP-9 activity were detected after proteolysis. Densitometry was used to determine the intensity of MMP-9 activity. (a) MMP-2 and MMP-9 activity in media stimulated with CD3⁺ CM (lanes 3, 4 and 6) from three separate experiments were compared to control samples [human fetal lung fibroblasts (HFL) cultured in Dulbecco's modified essential medium (DMEM) alone, lanes 2 and 5]. The first lane contained supernatants from HT1080 cells (positive control). (b) MMP-2 and MMP-9 activity after stimulation with CD4⁺ T cell CM and CD8⁺ T cells CM separately from four different experiments (CD4-1/CD8-1, CD4-2/CD8-2, CD4-3/CD8-3 and CD4-4/CD8-4). (c) The relative MMP-9 activity as densitometric value in media from gel cultures stimulated with CD4⁺ and CD8⁺ T cell CM (P < 0·05, Mann–Whitney, n = 4), n.d. = not detected.

Fig. 3

Matrix metalloproteinase (MMP) inhibitor GM6001 attenuates CD3⁺ conditioned media (CM) mediated collagen gel contraction. Fibroblasts [human fetal lung fibroblasts (HFL)-1] were cast in collagen gels and released into serum-free Dulbecco's modified essential medium (DMEM) with or without CD3⁺ CM and the broad-spectrum MMP-inhibitor GM6001. After 4 days of culture, the area of the gels was measured with an image analyser. Vertical axis: percentage of initial gel area. Horizontal axis: condition. Data shown as mean ± standard error of the mean of triplicate gels from three separate experiments. (**P < 0·01, ***P < 0·001, analysis of variance followed by Tukey's).

Fig. 4

T cell conditioned media (CM) stimulate collagen degradation. Collagen gels from day 4 were prepared and assayed for hydroxyproline as a measure of collagen content. (a) Hydroxyproline content in triplicates of gels from four separate experiments with control gels [human fetal lung fibroblasts (HFL)-1] and gels incubated with CD3⁺ CM. Vertical axis: μg hydroxyproline. Horizontal axis: samples (*P < 0·05, Mann–Whitney). (b) Hydroxyproline content in triplicate gels stimulated with CD4⁺ T cell CM and CD8⁺ T cell CM from four separate experiments. Vertical axis: hydroxyproline (% of control, HFL-1). Horizontal axis: samples (*P < 0·05, Mann–Whitney).