Effects of macrolides on antigen presentation and cytokine production by dendritic cells and T lymphocytes

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Summary

Macrolides are effective therapeutic agents for chronic respiratory tract diseases, such as chronic sinusitis, sinobronchial syndrome and diffuse panbronchiolitis. Although only limited information is available about their mechanisms, suppression of various inflammatory cytokines (IL-8, etc.) and some transcription factors has been reported to be involved. Non-typeable Haemophilus influenzae (NTHI) is one of the most important pathogens of the respiratory tract. P6 is one of the outer membrane proteins of NTHI and the target antigen of protective antibodies. To analyze the influence of macrolides on human dendritic cells (DCs), we treated DCs with macrolides and used them as antigen-presenting cells (APCs). Clarithromycin, roxithromycin and prednisolone suppressed the in vitro proliferative response of CD4+ T cells to P6 and also the production of cytokines. As a control, we also cultured DCs alone and exposed them to the medicament, while conversely culturing T cells without adding any drugs to the cultures. The results showed similar tendencies for suppression of immune responses. These findings suggest that macrolides suppress the antigen-specific immune responses of DCs in vitro.

Introduction

It is thought that a vicious circle prevails in chronic respiratory tract inflammatory diseases such as chronic sinusitis and diffuses panbronchiolitis, involving infection by pathogenic microbes on the one hand and the body's immune response on the other. Pathogens disrupt the local defense mechanisms and thus become established locally, and this induces an immune response on the part of the host. Especially in the case of inflammation having IL-8 and neutrophils as key elements, the local defense mechanisms are further disrupted by esterases and active enzymes, the clearance of the pathogens is interfered with, and a vicious circle results [1].

Fourteen-member lactone-ring macrolide antimicrobial agents are used as immunomodulators in the treatment of such chronic respiratory tract inflammatory diseases as chronic sinusitis, sinobronchial syndrome and diffuse panbronchiolitis, etc., and they have been reported to be useful in this application [2], [3], [4], [5], [6]. It is said that low-dose long-term chemotherapy of macrolides for these chronic respiratory tract inflammatory diseases stops the excessive immune response of the host and makes it possible to eliminate the vicious circle. There have been multiple reports that the underlying mechanism of macrolides in this role is that they inhibit the production of IL-8 and various other inflammatory cytokines [7], [8], [9], [10], [11], while it has also been reported that macrolides show inhibitory effects at the levels of mRNA and NF-κB and AP-1 transcription factors [12], [13], [14], [15], [16], [17]. Iino et al. demonstrated that the expression of a costimulator of antigen-presenting cells was inhibited by macrolides [18], [19]. In addition, Asano et al. showed that macrolides specifically inhibit Th2 cytokines [20], [21]. However, there remain many facets of the action mechanism underlying the anti-inflammatory effects of macrolides that are not fully understood.

Dendritic cells (DCs) are distributed in many tissues of the body, and it is known that they play an important role in inducing immune responses by carrying out antigen presentation to T cells. In addition, in recent years it has been shown that DCs are also involved in immunologic tolerance, and attention has thus been given to the aspect of DCs as immunoregulatory cells. It has also recently been shown that culture of CD14+ human peripheral blood monocytes in the presence of GM-SF and IL-4 for about 7 days is capable of inducing differentiation of DCs, and research on DCs themselves has become vigorous [22]. Nevertheless, there have still been few reports of studies of the effects of macrolides on human DCs.

Haemophilus influenzae is an important bacterial causative organism of chronic airway inflammatory diseases. One of the outer membrane proteins of this bacterium is the P6 protein, which is a commonly shared antigen of all H. influenzae strains. P6 is a target antigen for induction of infection-preventing antibodies, and it has also received much attention as the target of vaccine therapy [23], [24], [25], [26].

The objective of the present study was to investigate the effects of macrolides on such antigen-specific immune responses of CD4+ T cells as their proliferative response and their cytokines production (IL-8 and Th1/2 cytokines). These investigations were carried out using DCs generated from peripheral blood and the P6 outer membrane protein of H. influenzae.

Section snippets

Methods

P6 was purified from NTHi 1479 strain by the previously described method with some modifications [27], [28], [29]. Endotoxin was not detected by TOXICOLOR® (SEIKAGAKU CORPORATION, Tokyo, Japan) in the purified P6 preparation.

DCs were used as antigen-presenting cells (APCs), which were generated from CD14-positive monocytes as previously reported [22]. PBMCs were isolated from the peripheral blood of five healthy male donors by gradient centrifugation (Ficoll Paque Plus®; Amersham Pharmacia

Results

Fig. 1 presents the bar graphs of the results for the measurements of the proliferative response. The mean CPM resulting from stimulation with P6 protein alone was 20230.8. In comparison with when no macrolide was added to the cell (lymphocytes + DCs) cultures, both roxithromycin and clarithromycin showed concentration-dependent inhibition of the proliferative response. Each of these macrolides showed statistically significant inhibition of the response when present at a concentration of 1.0 μg/mL

Discussion

Fourteen-member lactone-ring macrolide antimicrobial agents are employed in the treatment of chronic respiratory tract inflammatory diseases since they show immunomodulating activity, and their usefulness in that application has been reported [2], [3], [4], [5], [6]. With regard to the underlying mechanism of macrolides in this role, they have been reported to inhibit the production of mRNA for IL-8 and various other inflammatory cytokines [7], [8], [9], [10], [11], and there have also been

Conclusions

Studies were carried out to elucidate the immune responses of DCs and CD4+ T cells originating from healthy adult volunteers to the P6 protein when the cells were exposed to macrolides. Both roxithromycin and clarithromycin showed inhibition of the proliferative response of the CD4+ T cells to P6 protein that was similar to the inhibition shown by prednisolone. Each of the tested drugs showed concentration-dependent inhibition of the productions of IFN-γ, IL-5 and IL-8, but production of IL-4

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