Our results show that patients with severe asthma, defined according to ATS criteria, have diminished corticosteroid sensitivity of their circulating PBMCs when compared with patients with nonsevere asthma. We used LPS as a stimulator of these cells and measured the release of 10 cytokines. We showed that at the higher dose of 10−6 M of dexamethasone, there was less reduction of cytokine release in cells from patients with severe asthma compared that from patients with nonsevere asthma; however, this difference was not observed at the concentration of 10−8 M of dexamethasone. In addition, we found that the total activities of the two enzymes that determine histone acetylation status, HDAC and HAT, were reduced in patients with severe asthma compared with patients with nonsevere asthma, indicating potential involvement of these enzymes in determining the relative corticosteroid insensitivity in PBMCs from patients with severe asthma.
The lack of inhibition of a monocyte-derived neutrophil activating factor and of cytokines such as TNF-α, IL-1β, and GM-CSF from blood monocytes by corticosteroids of patients with corticosteroid-resistant asthma as compared with patients with corticosteroid-sensitive asthma has been described (7
). TNF-α–induced GM-CSF release from PBMCs was also found to be less suppressible by exogenous dexamethasone in a group of patients with corticosteroid-resistant and corticosteroid-dependent asthma (9
). In these studies, the severity of the patients with asthma was not clearly defined. Our study is the first to indicate that there is an impairment of corticosteroid suppression of cytokine release measured in PBMCs from patients with severe asthma. The corticosteroid sensitivity of different cytokines released from PBMCs compared with that from monocytes in three patients with asthma indicates that the monocyte within the PBMC population is likely to be the cell being tested for corticosteroid sensitivity. This makes sense because LPS is known to activate monocytes to cause release of the cytokines that we have measured, but this does not exclude potential interactions between monocytes and lymphocytes in the response of PBMCs to LPS. A reduction in the inhibitory effects of corticosteroids on the proliferation of blood lymphocytes from patients with corticosteroid-resistant asthma has been reported (4
Monocytes and macrophages are sensitive to LPS, which induces an array of cytokines through the transmembrane signaling receptor Toll-like receptor 4, which activates several intracellular signaling pathways, such as nuclear factor-κB (NF-κB) and the mitogen-activated protein kinase pathways (10
). For example, the transcriptional induction of GM-CSF, IL-8, TNF-α, or RANTES is dependent on the activation of IκB kinase NF-κB, which binds to and activates coactivator molecules through the acetylation of core histones, leading to increased cytokine gene transcription (12
). Corticosteroids are likely to inhibit the expression of these cytokines through the reversal of histone acetylation at the site of the cytokine gene expression by direct binding of the activated corticosteroid receptor to NF-κB–associated coactivators or by recruitment of HDACs to the activated transcription complex (15
). There was no difference in the spontaneous release or in the LPS-stimulated release of these cytokines between patients with severe and patients with nonsevere asthma, indicating that the differences in steroid sensitivity observed were not related to a greater expression of cytokines, probably reflecting no differences in intracellular signaling induced by LPS between the two groups. We found that in terms of differences between severe and nonsevere asthma, there was less suppression of at least 7 of the 10 cytokines assayed in PBMCs of severe asthma, with significant differences for IL-8, MIP-1α, and IL-1β (and with borderline significance for IL-6). Differences were less obvious for the release of IL-10, MCP-1, and RANTES. One potential explanation for these different sensitivities may be the differential contribution of corticosteroid-sensitive intracellular pathways, such as NF-κB or mitogen-activated protein kinase activation, induced by LPS.
HAT and HDAC are families of enzymes that regulate the structure of chromatin which ultimately modulates the gene expression of inflammatory genes (17
). Acetylation of histones by coactivator proteins, such as cAMP response element binding (CREB)-binding protein p300 and TAFII
250, which possess HAT activity, leads to DNA unwinding, which allows transcription factors and RNA polymerase II to initiate gene transcription (18
). On the other hand, deacetylation of histones leads to the repression of transcription (19
). It has therefore been proposed that the balance of histone acetylation and of histone deacetylation may determine the inflammatory state. Glucocorticoids have been shown to suppress inflammatory gene expression, and this effect may involve the recruitment of HDACs to the transcriptional machinery complex by the activated glucocorticoid receptor (20
Changes in HDAC and HAT activity have been previously described in bronchial biopsies of patients with nonsevere asthma (21
). Compared with normal subjects, these subjects had a reduction in HDAC activity and a reciprocal increase in HAT activity. In patients treated with inhaled corticosteroids, the reduction in HDAC and the increase in HAT activity were attenuated (21
). Because repression of HAT activity and recruitment of HDAC activity is caused by corticosteroids in vitro
), these results were interpreted as reflecting the effect of corticosteroids in inhibiting HAT and recruiting HDAC activities. In our study, HDAC and HAT activities were significantly reduced in PBMCs from patients with severe asthma compared with patients with nonsevere asthma, indicating that changes in chromatin modification in severe asthma are more complex than the reciprocal change in the activities of HAT and HDAC described in nonsevere asthma.
Our key finding was that HDAC activity in PBMCs from patients with severe asthma was reduced when compared with patients with nonsevere asthma. Furthermore, HDAC activity in patients with nonsevere asthma was not influenced by inhaled corticosteroid use, suggesting that inhaled steroids do not contribute to changes in HDAC activity in PBMCs. The reduction in HDAC activity in PBMCs from patients with severe asthma correlated with the impairment of steroid responsiveness of GM-CSF and IFN-γ release, supporting a link between diminished HDAC activity and the development of steroid insensitivity in severe asthma. By contrast, the reduction in HAT activity in PBMCs from patients with severe asthma is probably due to the use of high-dose inhaled (and oral) corticosteroids rather than asthma severity because HAT activity in patients with severe asthma was not significantly different from the subgroup of patients with nonsevere asthma taking inhaled corticosteroids. Furthermore, HAT activity was reduced in patients with nonsevere asthma on inhaled corticosteroids when compared with those not on this therapy. These findings are consistent with the in vitro
inhibition of histone acetylation by glucocorticoids found previously (17
). We also found that among patients with asthma, HAT activity was reduced in parallel with the reduction of inflammatory gene expression by dexamethasone for 7 of the 10 cytokines (except IFN-γ) assayed, indicating that the reduction in HAT activity is related to corticosteroid use but dissociated from asthma severity. We do not have an adequate explanation for the inverse relationship obtained for IFN-γ.
Prior treatment with corticosteroids was unlikely to have influenced PBMC responsiveness because we found no difference in corticosteroid sensitivity among the patients with severe asthma who took regular oral prednisolone in comparison to those that did not take oral corticosteroids. The effect of oral prednisolone therapy on corticosteroid responsiveness is unknown, but a 10-d course of prednisolone at a dose of 40 mg/d had no effect on the number or affinity of corticosteroid receptors on PBMCs (23
). The reduced sensitivity of PBMCs to corticosteroid in patients with corticosteroid-dependent asthma has been reproduced by incubating PBMCs from nonasthmatic volunteers with a combination of IL-2 and IL-4, and this was associated with a reduction in affinity of the glucocorticoid receptor (GR) on PBMCs, including T cells (23
). In this study of steroid-dependent severe asthma, the binding affinity of dexamethasone to the glucocorticoid receptor was threefold higher than that of patients with corticosteroid-dependent asthma, which could account for the differences in cytokine inhibition by corticosteroids.
Our data show that PBMCs of patients with severe asthma express relative corticosteroid insensitivity. It is not known whether this abnormality would be reflected in lung macrophages, such as bronchoalveolar lavage macrophages. A recent study has demonstrated that nuclear translocation of the glucocorticoid receptor α by dexamethasone is impaired in alveolar macrophages of patients with glucocorticoid-insensitive asthma, indicating that this abnormality could occur in alveolar macrophages of patients with severe asthma (25
). Whether this is an intrinsic defect of PBMCs or an acquired defect (e.g., secondary to the effect of circulating mediators) is not known. In addition, it is not known whether the degree of corticosteroid sensitivity measured in the PBMCs reflects disease severity, although our corticosteroid sensitivity does not seem to correlate with various markers of severity of asthma.
In summary, severe asthma is characterized by a greater degree of steroid insensitivity measured in PBMCs than nonsevere asthma, along with a reduction in HDAC and HAT activities. Our results indicate that diminished HDAC activity may be linked to the presence of relative steroid insensitivity in severe asthma, whereas reduced HAT activity probably relates to corticosteroid use rather than disease severity. Determining the mechanisms underlying impaired steroid responsiveness may yield new therapeutic targets for severe asthma.