Background
Eosinophilic granulomatosis with polyangiitis (EGPA; also known as Churg–Strauss syndrome) is a rare disease characterized by the presence of allergic granulomatosis and necrotizing vasculitis. Development of the condition is preceded by peripheral blood eosinophilia and eosinophilic tissue infiltration [
1]. EGPA is classified as a vasculitis of arteries that are small or medium in diameter, although such vasculitis often is not apparent in the initial phases of the disease [
2]. Asthma is present in 96% to 100% of EGPA patients and is the cardinal feature of EGPA. Asthma may precede systemic vasculitis by approximately 8 years and, in some cases, by more than 30 years [
1],[
3],[
4]. The precise etiologies of EGPA and the asthmatic phase that precedes it, as well as the role of genetic factors, remain unknown. In our retrospective cohort study [
5], we found that most patients in the prevasculitic phase of EGPA had severe asthma requiring treatment with systemic steroids or mechanical ventilation, sinusitis, and a high percentage of eosinophils in the peripheral blood. Chronic eosinophilic pneumonia precedes systemic vasculitis in half of all patients with EGPA [
4],[
6].
Regulatory T (T
reg) cells play central roles in the maintenance of both immune homeostasis and peripheral tolerance. Naturally occurring CD4
+ T
reg (nT
reg) cells that constitutively express the forkhead box P (FOXP) 3 protein can also be identified because of their expression of cytotoxic T-lymphocyte antigen-4 (CTLA-4), CD25, and CD4 [
7]. Peripheral differentiation of inducible T
reg (iT
reg) cells form naïve CD4 T cells, which secrete IL-10, requires an elevated level of serum TGF-β and increased of IL-10 [
8]. We previously confirmed that the maintenance of T
reg cell numbers in asthma patients with chronic eosinophilic pneumonia may inhibit EGPA development via the action of cytokines, such as IL-10 and TGF-β, which are produced by CD4
+CD25
+ cells, and IL-2, which is produced by CD4
+CD25
− T cells [
9].
The mainstay of treatment for EGPA is systemic corticosteroid therapy. Additional treatment with immunosuppressive agents such as cyclophosphamide, azathioprine, methotrexate, mycophenolate mofetil, rituximab, interferon-α, anti-IgE antibodies, anti-IL-5 antibodies, anti-TNF-α (infliximab, etanercept, adalimumab), plasma exchange, and intravenous immunoglobulin therapy (IVIG) may be of benefit to some patients [
10]. The relapse rate is higher among patients with EGPA than with microscopic polyangiitis (MPA); some patients with EGPA experience frequent relapses after initial clinical remission, whereas others fail to achieve remission that lasts for an extended period [
11]. The mechanisms underlying the intractable form of EGPA remain poorly understood.
In our previous studies, frequently relapsing EGPA patients, defined as patients who relapse at least once every 2 years after an initial period of remission, had decreased numbers of T
reg cell counts, CD19
+ B cells, and a lower serum IgG concentration than those who did not experience frequent relapses. In patients with frequently relapsing EGPA, decreases in T
reg cell numbers and increased percentages of activated B cells, such as CD80
+, CD27
+, and CD95
+ B cells, may induce apoptosis of B cells [
12]. T
reg cells are involved in the etiology and mechanisms of EGPA, but their precise role is unknown, especially in relation to antigen-presenting cells such as dendritic cells (DCs).
DCs are widely recognized as the strongest antigen-presenting cells and have the unique ability to induce a primary immune response [
13]. DCs can prevent or inhibit T-cell-mediated effector responses through a variety of mechanisms, ranging from the production of pleiotropic anti-inflammatory factors that exert broadly attenuating effects to the induction of antigen-specific T cell responses that result in anergy, deletion, or instruction of T
reg cells [
14],[
15]. DCs bind to T
reg cells in the presence of the CD80/86 co-stimulatory molecule [
14]. Immature DCs induce IL-10–producing iT
reg cells, reinforcing peripheral tolerance [
16].
Conventional DCs (cDCs, also known as myeloid DCs) may play a pathophysiological role in the inflammation associated with antineutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis [
17]-[
19], granulomatosis polyangiitis–Wegener granulomatosis [
20], EGPA [
21], Kawasaki disease [
22], and giant cell arteritis (GCA) [
23]. Local increases in the number of activated myeloid DCs probably impede the maintenance of tolerance and lead to vascular autoimmune inflammation [
24].
CD83 is a cell-surface marker characteristic of fully mature DCs [
25]-[
27]. In their function as antigen-presenting cells, CD83-positive DCs induce T-cell-mediated immune responses [
26]. CD83-positive DCs have been associated with activated EGPA [
21] and multiple sclerosis [
28]. Injections of soluble CD83 clearly reduce paralytic symptoms in murine experimental autoimmune encephalomyelitis [
29]. CD206 (macrophage mannose receptor, C type I) antigen is upregulated in M2-type macrophages [
30]. CD206-positive antigen-presenting cells have high phagocytic capacity. CD206 is expressed on immature DCs generated from monocytes (MoDCs), whereas CD83 is expressed on mature MoDCs [
31].
Although some reports suggest an association between DCs and T
reg cells [
18],[
32],[
33], the details are still unclear. To further explore whether DCs or T
reg cells play a role in the remission or relapse of EGPA, we examined the relationship between the maturation of DCs and the differentiation of T
reg cells in EGPA patients.
Discussion
Not only are DCs crucial for the induction of primary immune responses, but these cells may also be important for the induction of immunological tolerance and the regulation of the type of T-cell-mediated immune response [
34]. The cytokine Flt3 ligand and its receptor Flt3required for the development of plasmacytoid DCs, and cDCs [
35]. Plasmacytoid DCs develop in the bone marrow and accumulate mainly in the blood and lymphoid tissues and enter lymph nodes through the blood circulation [
36]. Like plasmacytoid DCs, pre-cDCs develop from a common DC progenitor in the bone marrow; pre-cDCs differentiate into cDCs, which migrate through the blood to lymphoid and non-lymphoid tissues [
37]. Whereas plasmacytoid DCs differentiate into immunogenic DCs that can prime T cells against viral antigens [
38], cDCs have an enhanced ability to sense and respond to tissue injury, capture environmental and cell-associated antigens, process and present phagocytosed antigens to T lymphocytes, and prime T cell responses [
37].
CD83
+ DCs have been reported to reflect disease activity in inflammatory disease [
21],[
28],[
29]. Here we have shown
in vivo that both mature CD83
+ DCs and immature CD206
+ DCs can be present in the alveoli and interstitial spaces of the lungs at the onset of EGPA. After we induced the differentiation of patient-derived MoDCs
ex vivo, we found that MoDCs that expressed CD83
+ were predominant in patients in remission, whereas MoDCs expressing CD206
+ predominated in samples from patients at relapse. Many kinds of inflammatory cells, including eosinophils, effector T cells, T
reg cells, Th1 cells, Th2 cells, and Th17 cells, are present in the peripheral blood during the active phase of EGPA [
39],[
40]. We hypothesized that the inflammatory cells present in increased numbers during the active phase might migrate into the alveoli and interstitial spaces of the lungs.
In patients with EGPA, the number and percentage of mature CD83
+ DCs were inversely correlated with those of immature CD206
+ MoDCs. In addition, the percentage of CD83
+ DCs was higher in patients with EGPA in remission than in relapse and correlated with the percentage of T
reg cells. According to previous reports [
18],[
33], CD83
+ DCs suppress the immune response, probably by inducing T
reg. Mature DCs expressing CD83
+ at remission, which were present in higher numbers after treatment with corticosteroids, immunosuppressants, and IVIG, might induce the differentiation of both iT
reg cells and nT
reg cells. In contrast, we saw few CD83
+ or CD206
+ DCs in the lung
in vivo at remission after treatment with corticosteroids, immunosuppressants, and IVIG, but this therapy did not affect the number of eosinophils, which is already high in patients with EGPA before the onset of vasculitis.
We previously reported that the percentage of CD4
+ T cells producing IL-17 and IL-22 (Th17 cells) is significantly greater in patients with active EGPA than in healthy controls or in patients with asthma, chronic eosinophilic pneumonia, or inactive EGPA [
40]. As stated earlier, eosinophilia is characteristic of EGPA, and some reports suggest that the IL-25 produced by eosinophils promotes innate adaptive immunity by enhancing Th2 cytokine production [
41]. We have demonstrated that Th17 cells are correlated with the number of CD4
+ T cells that produce IL-25 [
42]. The number of Th17 cells is higher and that of iT
reg cells is lower in patients with active EGPA than in those with inactive EGPA [
40]. The mechanism of EGPA is thought to involve the actions of Th2 cells, T
reg cells, and Th17 cells. This result may indicate again that mature DCs that express CD83
+ are associated with the differentiation of T
reg cells but with not the number of eosinophils.
Indoleamine 2,3-dioxygenase (IDO) can be induced in many human cell types
in vitro by stimulation with interferons, lipopolysaccharide, tumor-necrosis factor-α (TNF-α), Toll-like receptor (TLR) ligands, or FcεRI. IDO is an interferon-inducible enzyme that suppresses adaptive T-cell immunity by catabolizing the essential amino acid tryptophan in the cellular microenvironment [
43]. IDO expression in monocytes from EGPA patients is positively correlated with the percentage of CD4
+CD25
+ T
reg cells producing IL-10 and inversely correlated with the percentage of Th17 cells [
42]. Therefore, EGPA relapse may be linked to elevated levels of IL-25–producing CD4
+ T cells, which promote Th2 inflammation and decrease iT
reg cell subpopulations, as does decreased IDO expression in monocytes. Therefore, the percentages of Th17 cells and of CD4
+CD25
+ T
reg cells producing IL-10 both reflect the disease activity of EGPA [
42].
Together with co-stimulatory molecules such as CD80 and CD86, CD83 is strongly upregulated during inflammation [
44]. Ligation of CTLA-4 to CD80 or CD86 or both may trigger the IDO pathway in DCs, in turn activating the transcription factor FOXP3 (which regulates immune functioning) and inhibiting cytokine production by DCs [
45]. We demonstrated that patients with EGPA who experience frequent relapses after initial clinical remission have decreased T
reg cell counts; increased percentages of B cells positive for CD80, CD27, or CD95; lower CD19
+ B cell counts; and lower serum IgG than do those who maintain remission [
12]. In contrast, other patients never achieve clinical remission [
12]. These results suggest that an interaction between T
reg cells and B cells via the overexpression of costimulatory molecules such as CD80 is related to EGPA disease severity.
TLRs constitute a family of transmembrane proteins that are expressed by various cell types, including antigen-presenting cells. TLRs function to discriminate pathogens and initiate inflammatory signaling pathways [
46]. TLR4, TLR5, TLR7, and TLR9 may play a role in vasculitis [
47],[
48]. DCs localized at the adventitia–media border of medium-sized arteries in patients with GCA express a series of TLR receptors, produce chemokines, and activate T cells [
49]. LPS-induced triggering of TLR4 activates DCs, suggesting a key role for TLR4 in the induction of the inflammatory processes in GCA [
50]. These researches may help to elucidate the etiology of vasculitis.
The remission rate after initial treatment is reported to be 81% to 91% in EGPA patients, compared with 30% to 93% in patients with GPA and 75% to 89% in those with MPA [
11]. However, the relapse rate during the first 2 years after diagnosis is higher in EGPA patients (35%) than in MPA patients (8%) [
11]. The relapse rate at 5 years or more after disease onset is greater than 60% in patients with EGPA, compared with 20% in MPA patients [
51]. Why the relapse rate of EGPA is so high is unknown, but vasculitis relapse might occur when unknown events in the peripheral blood or blood vessels lead to an interaction between DCs and T
reg cells, thereby causing an immune response.
Competing interests
No author has any conflict of interest to disclose. The work was not funded by a grant or any other external source of financial support. N. Tsurikisawa, H. Saito, C. Oshikata, T. Tsuburai, M. Ishiyama, H. Mitomi, K. Akiyama.
Authors’ contributions
NT examined the patients, analyzed data and statistics, and was the main contributor to manuscript preparation. HS assayed the levels of FOXP3+CD4+ T cells; CD4+ T cells producing IL-10, TGF-β, and IL-2; and DCs derived from CD14+ monocytes. CO and TT examined the patients and contributed to discussions about the patients. HM and MI performed immunohistochemistry on CD83+ DCs and CD206+ DCs derived from CD14+ monocytes in vitro and in lung biopsy tissues. KA is the president of the hospital and contributed to discussions about the manuscript. All authors read and approved the final manuscript.