It is well accepted that there is an increase in the number of fibroblasts in the airways of patients with asthma that correlates with thickness of lamina reticularis and disease severity. Moreover, fibroblast activation and differentiation to myofibroblasts are also evident.1–4
In the present study, we aimed to investigate the in vitro effect of glucocorticosteroids and short-acting β2
-agonists widely used as first-line antiasthmatic drugs on human lung fibroblast proliferation and IL-6 production. We specifically choose to evaluate fibroblast proliferation because this is the first hallmark of fibrosis taking place. IL-6 was selected among a plethora of proinflammatory profibrotic cytokines produced by the fibroblast22
that mainly influences the inflammatory response.23,24
We found that dexamethasone and salbutamol alone and in combination increase both human fetal lung and human bronchial fibroblast proliferation. Moreover, we demonstrate for the first time that when the fibroblasts are preincubated with either drug and then with an activator such as mast cell sonicate or eotaxin, fibroblast proliferation is further increased. In contrast, the production of the proinflammatory and profibrotic cytokine IL-6 by confluent fibroblast monolayers was found to be decreased by dexamethasone, while it was increased in the presence of salbutamol. When the 2 drugs were added together, dexamethasone was still able to slightly decrease IL-6 production.
We have previously shown that mast cells enhance proliferation of human lung fibroblasts, an effect also seen with histamine and to a lesser extent with tryptase.7
In a more recent study, we have also defined the profibrotic properties of eotaxin/CCL11,8
a central mediator of asthma that can also be produced by mast cells.25
In that study, we did not find significant differences between the human fetal lung fibroblast line and human bronchial fibroblasts regarding their proliferative responses to eotaxin/CCL11. This was explained by the fact that they were found to express similar levels of surface CCR3. Similarly, in the present study, we did not observe significant differences in the response of the fetal lung and the bronchial fibroblasts toward the proliferative effects of either the drugs or the stimulants. This is an interesting observation because fibroblasts from cell lines and primary cultures might behave differently. The fact that we did not detect major differences between these 2 sources of fibroblasts might be due to the fact that primary cells did not need to be cultured and subcultured for extensive time and when used were not senescent.
Many studies have been carried out to investigate the effects of glucorticosteroids on several sources of fibroblasts such as human fetal lines, or primary human lung, asthmatics endobronchial biopsies, human nasal polyps derived or mouse 3T3 and rat lung and even adipose tissue derived providing sometimes similar and sometimes contrasting results. We opted for the most commonly used source of human lung fibroblasts, the cell line MRC-5 and primary human bronchial fibroblasts.
Regarding our data, it is interesting to note that generally in the absence of the mast cell sonicate, dexamethasone effect was always greater than that of salbutamol. In this work, we showed for the first time that salbutamol caused a further increase in fibroblast proliferation in a dose-dependent fashion in fibroblasts treated with eotaxin. This proliferative event is probably carried out by the interaction of eotaxin with its receptor and by salbutamol with β2-receptors.
Glucocorticosteroids in general and specifically dexamethasone are known to decrease gene expression of proinflammatory cytokines. Several studies have found that dexamethasone decreases cytokine secretion by fibroblasts, among them IL-6.26
The results of our study correlate with these findings. But also this decreasing effect is not universally detected and depends on the exposure duration and the cytokine under study.27
Studies that tested the effect of β2
-agonists on IL-6 secretion on different cell types found that these drugs enhanced IL-6 secretion.28
As far as we know, the effect of β2
-agonists on IL-6 secretion from lung fibroblasts has not yet been studied. We found that salbutamol increased slightly IL-6 production. Cyclic adenosine monophosphate (cAMP) is known as an IL-6 secretion enhancer (airway smooth muscle cells, 3T3 fibroblasts, and gingival fibroblasts), even though it has been reported that lung fibroblast cell line had an inverse relationship between the amount of cAMP and the amount of IL-6 secreted from the cell.29
In our study, salbutamol and forskolin that increase cAMP production, both increased IL-6 secretion from the fibroblasts. These findings strengthen the assumption that the mechanism through which salbutamol affects the increase in IL-6 secretion is cAMP dependent. Interestingly, when dexamethasone and salbutamol were added together, the decrease in IL-6 production was smaller than the one obtained in the presence of dexamethasone alone.
Many mediators, including growth factors, IL-6, and other cytokines, are involved in fibroblast proliferation regulation. In a previous study, we found that when added to fetal lung fibroblasts, IL-6 increased their proliferation up to 140% (data not published). Therefore, we might speculate that salbutamol can influence lung fibroblast proliferation by causing IL-6 production. The effect of β2
-agonists in general and specifically of salbutamol on fibroblast proliferation has almost not been studied. Agarwal and Glasel described the effect of treatment with opioid and β-adrenergic agonists on cell proliferation.30
They found that although both receptors increase cAMP production, opioid agonists lowered the level of proliferation, while a β2
-agonist (procaterol) increased cell proliferation. Silvestri et al20
found that salmeterol decreased fetal lung line fibroblast proliferation induced by bFGF. The source of the cells is indeed similar to the cells in our study, but salmeterol is a long-acting β2
-agonist as opposed to salbutamol.20
Another important difference is that the study by Silvestri et al,20
and other studies, tested the drug's effect on proliferation of cells that were incubated with a growth factor (in this case basic fibroblast growth factor). It is difficult to determine whether the drug's effect is independent of the tested growth factor and whether this can be compared with our research in which salbutamol's effect on fibroblasts was assessed either without additional treatment or in the presence of mast cells or eotaxin.
It is difficult to speculate how the dexamethasone carries out its profibrogenic response. Several mechanisms have been suggested regarding the fashion in which the glucocorticosteroids enhance fibroblast proliferation. For example, they can either enhance or decrease the activity of different growth factors (among them, insulin growth factor-1 and fibroblast growth factor) and their receptors (ie, platelet derived growth factor11
) or their production (ie, connective tissue growth factor31
). The time of drug addition to the cultures can make a difference. When it is added after the cells reached confluence, it causes enhancement of proliferation, while drug administration during the exponential growth phase usually causes a decrease.32
In our protocol, we added dexamethasone to subconfluent monolayer and still it had a profibrogenic effect.
It is interesting to point out that the literature on the effect of drug combination on fibroblast proliferation presents a complex picture. Many researches describe an additive and a synergistic effect of glucocorticosteroids and β2
-agonists. On the other hand, other studies have found classes of the two drugs to have antagonistic effects. Peters et al33
reported that β2
-agonists, among them salbutamol, have the ability to lower the steroid effect on gene transcription by inhibiting the binding to the glucocorticosteroid responsive element on the DNA. On the other hand, salbutamol and salmeterol have been described as having the ability to activate the receptor by a mechanism related to cAMP increase.34
Our findings obtained in vitro show that whatever the underlying mechanism(s) is, the drugs have a direct fibrogenic effect and a synergistic effect with central players, such as mast cell–derived mediators in the asthmatic process. If these in vitro observations are found to be true also in the in vivo complexity of the asthmatic patient with airway remodeling and fixed airway obstruction, it is then possible to speculate that this may not only be part of the natural course of the disease but a complication of the accepted drug treatment and a product of the drugs' interactions with the inflammation mediators. Therefore, if the profibrogenic effect of glucocorticosteroids and β2-agonists is reconfirmed in vivo, the combination of the remodeling and the drugs' effect can be dangerous. In conclusion, more in vivo studies are warranted to clarify what the risks and benefits are of monotherapy versus combination of inhaled glucocorticosteroids and short- and long-acting β2-agonists on airway remodeling in asthma.