Leukotriene B4 receptor 1 is differentially expressed on peripheral T cells of steroid-sensitive and -resistant asthmatics

Abstract

Background: Numbers of CD8(+) T cells expressing the leukotriene B4 (LTB4) receptor, BLT1, have been correlated with asthma severity.

Objective: To examine the activation and numbers of BLT1-expressing peripheral blood CD4(+) and CD8(+) T cells from patients with steroid-sensitive (SS) and steroid-resistant (SR) asthma.

Methods: CD4(+) and CD8(+) T cells isolated from peripheral blood of healthy human subjects and patients with SS and SR asthma were stimulated in culture with anti-CD3/anti-CD28 followed by analysis of BLT1 surface expression and cytokine production. Activation of CD8(+) T cells after ligation of BLT1 by LTB4 was monitored by changes in intracellular Ca(2+) concentrations.

Results: The number of BLT1-expressing cells was larger in patients with asthma than in controls and larger on activated CD8(+) than on CD4(+) T cells. Addition of LTB4 to activated CD8(+) T cells resulted in increases in intracellular Ca(2+) concentrations. Expansion of activated CD4(+) T cells, unlike CD8(+) T cells, was significantly decreased in the presence of corticosteroid. In patients with SS asthma, numbers of BLT1-expressing CD8(+) T cells were lower in the presence of corticosteroid, unlike in those with SR asthma in whom cell expansion was maintained. Levels of interleukin-13 were highest in cultured CD8(+) T cells, whereas interleukin-10 levels were higher in CD4(+) T cells from controls and patients with SS asthma. Interferon-γ levels were lowest in patients with SR asthma.

Conclusion: Differences in BLT1 expression, steroid sensitivity, and cytokine production were demonstrated in T lymphocytes from patients with SS and SR asthma. The LTB4-BLT1 pathway in CD8(+) cells may play an important role in asthma and serve as an important target in the treatment of patients with SR asthma.

Figure 1

BLT1 expression on stimulated CD4+ T cells. Isolated CD4+ T cells were stimulated with anti-CD3/anti-CD28 for 8 days and BLT1 expression was assessed by flow cytometry. There were no significant differences between any of the groups.

Figure 2

BLT1 expression on stimulated CD8+ T cells. Isolated CD8+ T cells were stimulated with anti-CD3/anti-CD28 for 8 days and BLT1 expression was assessed by flow cytometry. A) Representative plots are illustrated. Representative data showing isotype control staining (left panel) and BLT1 expression (center panel). The right panel shows the flow analysis of BLT1 staining and the discrimination between the FITC-isotype control and FITC-BLT1 monoclonal antibody staining. B) BLT1 expression on unstimulated and stimulated CD8+ T cells in the presence or absence of dexamethasone (Dex). #P<0.05 comparing unstimulated to stimulated group. *P<0.05 comparing steroid treated to non-treated group. +P<0.05 comparing SR to CTL and SS groups. −: no Dex, +: includes Dex.

Figure 3

Addition of LTB4 to stimulated CD8+ T cells results in increased intracellular Ca²⁺ concentrations. Isolated CD8+ T cells stimulated with anti-CD3/anti-CD28 for 8 days were incubated with LTB4 (100 or 1,000 nM) and intracellular Ca²⁺ concentrations were monitored comparing the median fluoresense ratio of indo-1 to indo-2 as described in Methods.

Figure 4

Cytokine production from cultures of CD4+ and CD8+ T cells. Isolated CD4+ and CD8+ T cells from CTL, SS, and SR patients were stimulated with anti-CD3/anti-CD28 for 48 hours and culture supernates were harvested and assayed by ELISA. *P<0.05 compared to CD4+ T cells. #P<0.05 compared to SR CD4+ T cells and CD8+ T cells. +P<0.05 comparing SR cells to CTL or SS cells.