Roxithromycin enhances lymphocyte apoptosis in Dermatophagoides-sensitive childhood asthma

Noma, Takeshi; Ogawa, Norifumi

« Previous Next »The Journal of Allergy and Clinical Immunology
Volume 111, Issue 3 , Pages 646-647, March 2003

Roxithromycin enhances lymphocyte apoptosis in Dermatophagoides-sensitive childhood asthma

Department of Pediatrics Kitasato University School of Medicine Kanagawa, Japan

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To the Editor:

Roxithromycin (RXM), a new macrolide, might be an effective treatment for asthma.¹ The unbalanced cytokine profile of patients with mite antigen-induced asthma might be corrected by RXM, which might reproduce the changes of the immune system seen in patients in remission who are tolerant of Dermatophagoides farinae (Df) antigen.² Apoptosis, or programmed cell death, appears to play an important role in the thymic selection of T cells and in the development of peripheral tolerance. Dysregulation of apoptosis might lead to the development of autoimmune diseases and allergic diseases.³ Our data show the antiallergic properties of RXM, focusing on its effect on Df-induced apoptosis in patients with asthma.

PBMCs were obtained from 13 patients with Df-sensitive childhood asthma (age range, 9-15 years; M:F, 5:8) and 8 nonallergic healthy volunteers. The diagnostic criteria for asthma were those established by the American Thoracic Society. The total serum IgE concentration and the IgE score for mite antigen, determined by radioallergosorbent testing, were 926 ± 213 IU/mL and 4 to 6, respectively. At the time of the study, no patients had acute exacerbations and none were taking oral corticosteroids or antiallergy agents.

To verify the induction of apoptosis and exclude necrosis in the late phase of apoptosis, the proportion of apoptotic PBMCs was determined either by annexin V staining⁴ through use of a MEBCYTO Apoptosis Kit (MBL Co, Nagoya, Japan) or by fragmented nuclei with propidium iodide (PI) staining.⁵ Stained cells were analyzed by means of flow cytometry. Serial dilutions of RXM dissolved in RPMI 1640 medium adjusted to pH 7.4 were added to the PBMCs (1 × 10⁶/mL). Data are expressed as means ± SEs. Data were analyzed through use of ANOVA with Bonferonni correction. Any difference with a P value of < .05 was considered statistically significant.

RXM induced the early phase of apoptosis (annexin-positive, PI-negative cells) in activated cells, after which the cells proceeded to the late phase of apoptosis (annexin-positive, PI-positive cells), because low concentrations of RXM (1 to 500 ng/mL) augmented the early phase, but not the late phase, of apoptosis in Df-stimulated PBMCs. However, a higher concentration of RXM (1 μg/mL), the maximum clinical level of which is approximately 6 μg/mL, augmented both the early and late phases of apoptosis in Df-stimulated cells. In unstimulated cells, RXM did not significantly affect apoptosis. In contrast, RXM did not induce apoptosis in normal subjects. Thus, RXM can induce apoptosis when lymphocytes are activated by a relevant antigen in patients with asthma.

Apoptosis induced by Df was decreased in lymphocytes from patients with mite-sensitive asthma, and spontaneous apoptosis as well as Df-stimulated apoptosis was markedly increased among patients with asthma in remission.³ Clearance of CD8⁺ T cells by increased apoptosis is likely to be involved in an individual's outgrowing asthma.³ Df stimulation induced apoptosis of activated cells in normal individuals rather than in patients with active asthma. RXM-induced apoptosis of Df-stimulated cells mimics the increase of apoptosis in patients with remission who are tolerant of mite antigen.

This increase of apoptosis was not induced by other antibiotics, including cefazolin, which indicates that RXM was specifically involved in the augmentation of apoptosis.

Fas ligand expression by Df-stimulated cells, but not by unstimulated or PHA-stimulated cells, was significantly increased after treatment with 1 μg/mL RXM in comparison with no treatment (Table I). These results clearly show that RXM is involved in Fas ligand expression and Df-induced apoptosis. In contrast, Fas receptor expression was not changed by simulation with Df or PHA. Bcl-2 expression was reduced by treatment with RXM in the case of untstimulated as well as Df-stimulated cells (Table I). These results suggest that the impaired apoptotic response to Df stimulation in patients with mite-sensitive asthma was reversed by treatment with RXM. Induction of Fas/Fas ligand and reduced Bcl-2 expression might contribute to this increased apoptosis.

Table I. Augmented expression of Fas ligand and Bcl-2 by lymphocytes from patients with asthma

Early plus late phases of apoptosis (%) (as measured by annexin V and PI staining)	No treatment	1 μg/mL RXM
Unstimulated	13.1 ± 0.8*	11.0 ± 4.8
1 μg/mL Df	11.5 ± 0.7	15.9 ± 3.1†
Unstimulated	11.6 ± 1.1*	8.9 ± 2.6‡
1 μg/mL Df	12.1 ± 1.0	8.4 ± 3.6‡
Fas ligand (%)
Unstimulated	4.4 ± 2.6*	0.3 ± 0.2
1 μg/mL Df	1.4 ± 1.3	9.7 ± 4.9†
BcL-2 (mean intensity)§
Unstimulated	393.5 ± 23.6*	372.1± 64.0†
1 μg/mL Df	261.6 ± 42.9	253.5 ± 76.1†
*Data from 5 independent experiments were pooled. †P < .05 (in comparison with untreated). ‡Stimulation with 2 g/mL cefazolin instead of RXM. §Intracellular expression of Bcl-2 was determined through use of CellQuest software. Results are represented as means ± SEs of the fluorescence channel intensity in Bcl-2⁺ cells.

Supported by a grant from the Ministry of Education, Culture and Science, Japan.

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