Relative corticosteroid insensitivity of peripheral blood mononuclear cells in severe asthma

Abstract

Rationale and objectives: Patients with severe asthma have a poor therapeutic response to corticosteroid therapy, and corticosteroid responsiveness cannot be easily measured in these patients. We hypothesized that this poor response is associated with a reduced effect of corticosteroids to inhibit cytokine release from activated peripheral blood mononuclear cells (PBMCs).

Methods: Patients with severe asthma were defined by American Thoracic Society criteria. We compared the suppression of LPS-induced cytokine release (monocyte chemotactic protein-1 [MCP-1], macrophage inflammatory protein [MIP] 1alpha, RANTES, tumor necrosis factor alpha, interleukin 1beta (IL-1beta), IL-8, IFN-gamma, IL-6, IL-10, and granulocyte-macrophage colony-stimulating factor [GM-CSF]) by dexamethasone from PBMCs of patients with severe asthma (n = 16), patients with nonsevere asthma (n = 19), and normal volunteers (n = 10).

Results: There was no difference in baseline spontaneous or stimulated release of these cytokines among groups. LPS-induced release of 10 cytokines was less suppressed by dexamethasone (10(-6) M) in patients with severe asthma compared with patients with nonsevere asthma, with statistical significance achieved for IL-1beta (p < 0.03), IL-8 (p < 0.03), and MIP-1alpha (p < 0.003), and borderline significance for IL-6 (p = 0.054). There was less difference between the two groups for dexamethasone at 10(-8) M. Nuclear histone deacetylase (HDAC) and histone acetyltransferase activities were reduced in patients with severe asthma compared with patients with nonsevere asthma (p < 0.01). HDAC activity reduction correlated directly to the degree of steroid insensitivity of GM-CSF (r = 0.57, p < 0.01) and IFN-gamma (r = 0.56, p < 0.05) release. Reduction in histone acetyltransferase activity related to corticosteroid use rather than asthma severity.

Conclusions: Patients with severe asthma have diminished corticosteroid sensitivity of PBMCs when compared with patients with nonsevere asthma, associated with a reduction in HDAC activity that parallels the impaired corticosteroid sensitivity.

Figure 1.

Concentrations of 10 different cytokines in cell culture supernatants of unstimulated peripheral blood mononuclear cells (PBMCs) (A) and of PBMCs stimulated with LPS (10 μg/ml) (B) from normal volunteers, patients with nonsevere asthma, and patients with severe asthma. There were no significant differences between patients with severe asthma and patients with nonsevere asthma. GM-CSF = granulocyte-macrophage colony–stimulating factor; IFN-γ = interferon-γ; IL = interleukin; MCP-1 = monocyte chemotactic protein-1; MIP = macrophage inflammatory protein; TNF = tumor necrosis factor.

Figure 2.

Effect of dexamethasone 10⁻⁶ M (A) and 10⁻⁸ M (B) on the suppression of release of 10 cytokines from PBMCs stimulated by LPS (10 μg/ml). Data are expressed as the percentage of cytokine release after exposure to LPS. *p < 0.05; **p < 0.01 between patients with severe and nonsevere asthma.

Figure 3.

Comparison of sensitivity to dexamethasone (10⁻⁶ M) between PBMCs and monocytes of a patient with severe asthma (A) and two patients with nonsevere asthma (B and C). Each panel shows the correlation between percentage cytokine release in monocytes and percentage cytokine release in PBMCs for eight different cytokines.

Figure 4.

Individual histone-deacetylase (HDAC) activities in PBMCs from normal individuals, patients with nonsevere asthma, and patients with severe asthma. The patients with nonsevere asthma are divided into those not receiving inhaled corticosteroids (ICS−) and those receiving inhaled corticosteroids (ICS+). *p < 0.05; **p < 0.01; NS = not significant.

Figure 5.

Individual histone acetyltransferase (HAT) activities in PBMCs from normal individuals, patients with nonsevere asthma, and patients with severe asthma. The patients with nonsevere asthma are divided into those not receiving inhaled corticosteroids (ICS−) and those receiving inhaled corticosteroids (ICS+). **p < 0.01.

Figure 6.

Correlation between relative steroid suppression of cytokine release for GM-CSF (A) and IFN-γ (B) and total HDAC activity. Closed circles indicate severe asthma; open circles indicate nonsevere asthma.

Figure 7.

Relationship between LPS-induced release of eight cytokines and total HAT activity in PBMCs. There was a positive correlation between the levels of cytokine release and HAT activity for seven of the cytokines, except for IFN-γ, where there was a negative correlation. Closed circles indicate severe asthma; open circles indicate nonsevere asthma.