Effect of budesonide on fibroblast-mediated collagen gel contraction and degradation

Abstract

Background: The balance between production and degradation of extracellular matrix is crucial in maintaining normal tissue structure. This study was designed to investigate the effect of budesonide on fibroblast-mediated tissue repair and remodeling.

Methods: Using human fetal lung fibroblasts in a three-dimensional collagen gel culture system, we investigated the effect of budesonide (1-1000 nM) on collagen gel contraction and degradation in the presence or absence of Inflammatory cytokines (interleukin-1β and tumor necrosis factor α; 5 ng/mL each) and, in order to activate latent proteases, serine protease trypsin 0.25 μg/mL. The effects of budesonide on metalloproteinase production and activation were also investigated.

Results: Inflammatory cytokines significantly inhibited collagen gel contraction mediated by lung fibroblasts. Budesonide counteracted the effect of cytokines in a concentration-dependent manner (to 50%, P < 0.01). Budesonide 100 nM almost completely inhibited the release and mRNA expression of metalloproteinase-1, metalloproteinase-3, and metalloproteinase-9 induced by the cytokines (P< 0.05). Exposure to the cytokines plus trypsin increased collagen degradation and conversion of the metalloproteinases to lower molecular weight forms corresponding to their active forms. Budesonide blocked both enhanced collagen degradation (P< 0.01) and suppressed trypsin-mediated conversion of cytokine-induced metalloproteinase-9 and metalloproteinase-3 to lower molecular weight forms. Similar effects were observed with dexamethasone 1 μM, suggesting a class effect.

Conclusion: These findings demonstrate that budesonide directly modulates contraction of collagen gels and can decrease collagen degradation under Inflammatory conditions. The mechanism of this effect is through suppressing gene expression, release, and activation of metalloproteinases. By modulating the release and activity of metalloproteinases, inhaled budesonide may be able to modify airway tissue repair and remodeling.

Keywords: budesonide; metalloproteinase; tissue remodeling.

Figure 1

Effect of budesonide on collagen gel contraction in the presence or absence of cytokines. Notes: Fibroblasts (hFL-1) were cast into three-dimensional collagen gels, released into SF-DMEM with or without IL-1β and TNFα, and then incubated for 5 days with or without budesonide. Gel size was measured daily and the data presented are for gel size on day 3. The vertical axis shows gel size expressed as percent of original size (%); the horizontal axis shows budesonide concentration (nM). The square represents SF-DMEM; the triangle represents IL-1 β and TNFα (5 ng/mL for each cytokine). Each point is the mean ± standard deviation of triplicate gels from a representative experiment that was repeated three times. **P< 0.01 for treatment with medium + budesonide compared with treatment comprising cytokines and budesonide by two-way analysis of variance followed by Tukey’s test. Abbreviations: SF-DMEM, serum-free Dulbecco’s Modified Eagle’s Medium; IL-1β, interleukin-1β; TNFα, tumor necrosis factor α

Figure 2

Budesonide inhibits release of MMP-1, MMP-2, MMP-3, and MMP-9. Culture media of Figure 1 were harvested on day 5 and used for zymography or immunoblot as described in the methods. (A) Immunoblotting for MMP-1. (B) Immunoblotting for MMP-3. (C) gelatin zymography for MMP-2 and MMP-9. Notes: The clear bands indicate gelatinase activity. Data presented are representative of three separate experiments. Abbreviations: CK, cytokines (interleukin-1 β + tumor necrosis factor-α; 5 ng/mL each); Bud, budesonide; MMP, metalloproteinase.

Figure 3

Inhibition of MMP mRNA expression by budesonide. Cells were cast into three-dimensional collagen gels and maintained in serum-free Dulbecco’s Modified Eagle’s Medium with or without cytokines and/or budesonide for 3 days. Total RNA was extracted, and real-time reverse transcriptase polymerase chain reaction was performed as described in the Methods section for MMP-1 (A), MMP-3 (B), or MMP-9 (C). Notes: The vertical axis shows mRNA level expressed as fold of control (serum free Dulbecco’s Modified Eagle’s Medium condition); the horizontal axis shows treatment. Data are shown as the mean ± standard deviation for three separate experiments. *P< 0.05 by one-way analysis of variance followed by Tukey’s test for comparison of cytokines with budesonide + cytokines. Abbreviations: CK, cytokines (interleukin-1 β + tumor necrosis factor-α; 5 ng/mL each); Bud, budesonide 100 nM; MMP, metalloproteinase.

Figure 4

Budesonide inhibits activation of MMPs by trypsin. Fibroblasts were cast into collagen gels and maintained in serum free Dulbecco’s Modified Eagle’s Medium with or without cytokines, trypsin, and budesonide. After 3 days, the media were harvested and used to assess MMP release. Immunoblottings for MMP-1 (A) and MMP-3 (B). (C) gelatin zymography for MMP-2 and MMP-9. Notes: The clear bands indicate gelatinase activity. The labels indicate the expected positions for the latent and active forms of the MMPs. The left-most lane in Panels A and B shows the molecular weight standards. The data presented are one representative of three separate experiments. Abbreviations: Bud, budesonide 100 nM; CK, cytokines (interleukin-1 β + tumor necrosis factor-α; 5 ng/mL for each); Tryp, trypsin 0.25 μg/mL; MMP, metalloproteinase.

Figure 5

Effect of budesonide on collagen gel degradation induced by cytokines plus trypsin. Fibroblasts were cast into three-dimensional collagen gels and maintained in floating culture for 5 days in the presence or absence of cytokines, trypsin, and budesonide. (A) Collagen gel contraction assay. Vertical axis shows gel size expressed as percent of initial size (%); horizontal axis shows time (day). (B) hydroxyproline assay. In replicated experiments, gels were harvested on day 3 and hydroxyproline amount was determined. Vertical axis shows hydroxyproline amount expressed as μg/gel; horizontal axis shows media with or without cytokines. Notes: The data presented in both panels are the mean ± standard deviation of three separate experiments, each of which included triplicate gels for each condition. **P< 0.01 by one-way analysis of variance followed by Tukey’s test for the comparison of trypsin with trypsin + budesonide in the presence of cytokines. Abbreviations: CK, cytokines (interleukin-1β + tumor necrosis factor-α; 5 ng/mL each); Bud, budesonide 100 nM; Tryp, trypsin 0.25 μg/mL; hoop, hydroxyproline.

Figure 6

Effect of dexamethasone on MMP production and activation. gels were cultured in various conditions (as indicated in the lanes) for 5 days. Media were harvested for gelatin zymography and immunoblotting. (A) Immunoblottings for MMP-1 and (B) MMP-3. (C) gelatin zymography for MMP-2 and MMP-9. Notes: Arrows indicate expected positions of latent and active forms of the MMPs. Data presented are one representative of three separate experiments. Abbreviations: CK, cytokines (interleukin-1β + tumor necrosis factor-α; 5 ng/mL each); Dex, dexamethasone 1 μM; Tryp, trypsin 0.25 μg/mL; MMP, metalloproteinase.

Figure 7

Inhibition of MMP mRNA expression by dexamethasone. Cells were cast into three-dimensional collagen gels and maintained in serum-free Dulbecco’s Modified Eagle’s Medium with or without cytokines and/or dexamethasone for 3 days. Total RNA was extracted and real-time reverse transcriptase polymerase chain reaction was performed as described in the Methods section for MMP-1 (A), MMP-3 (B), and MMP-9 (C). Notes: The vertical axis shows mRNA level expressed as fold of control (serum-free Dulbecco’s Modified Eagle’s Medium condition); the horizontal axis shows treatment. Data are presented as the mean ± standard deviation for three separate experiments. *P< 0.05 by one-way analysis of variance followed by Tukey’s test for the comparison of cytokines with dexamethasone + cytokines. Abbreviations: CK, cytokines (IL-1β + TNFα; 5 ng/mL each); Dex, dexamethasone 1 μM; MMP, metalloproteinase.