Hypersensitivity pneumonitis (HP) is a group of lung diseases that result from repeated pulmonary exposure to a variety of environmental organic dusts, fungi, and molds with uncertain immunological mechanisms (1). This disorder affects the parenchyma and the small airways of the lungs and presents an acute, subacute or chronic form. Consequently, HP may lead to irreversible pulmonary damages, which cause fibrosis or emphysema, and depend on several factors such as duration of exposure and nature of the inhaled antigens (2).

Although the pathogenesis of HP is incompletely understood, it has been generally accepted that a combination of humoral and cell-mediated immune responses contributes to the development of HP (3). Several studies have suggested that the murine models of experimental HP are mediated by Th1 immune responses. In mice, adoptive transfer of Th1 clones increased cell infiltration and caused reversible HP (4). Moreover, when the Th1/Th2 balance shifts toward Th1 immune responses, the HP is more severe whereas it is attenuated by Th2-biased responses (5,6). Beside T cells, various immune cell types including neutrophils, lymphocytes, monocytes, and macrophages have been known to be involved in the development of HP (7). Upon antigen challenge, a large number of alveolar macrophages infiltrate the lungs and secrete cytokines and chemokines such as TNF-α, IL-1, IL-8, and MIP-1α, which recruit neutrophils and lymphocytes (8,9). Chemokine-induced infiltration of neutrophils is found in the alveoli and followed by an influx of activated T cells with a predominance of CD8⁺ T cells (8). In Saccharopolyspora rectivirgula (SR)-induced HP of murine models, neutrophils are most prominent in bronchoalveolar lavage fluid (BALF) immediately after antigen challenge, and they produce a large amount of IFN-γ (10). Moreover, we previously demonstrated that NKT cells suppress IFN-γ-producing Gr-1⁺ granulocytes by producing IL-4, resulting in the attenuation of SR-induced HP (11). These observations suggest that macrophages, neutrophils and NKT cells may play an important role in development of HP.

Fcγ receptors (FcγRs) for IgG are surface glycoproteins and bridge humoral and cellular immunity by directing the interaction of antibodies with effector cells. These receptors are expressed on effector cells of the immune system and mediate phagocytosis, antibody-dependent cell-mediated cytotoxicity (ADCC), and activation of inflammatory cells (12,13). To date, four different classes of FcγRs (FcγRI, FcγRIIB, FcγRIII and FcγRIV) have been recognized in mice (14,15). FcγRIIB provides inhibitory signals, whereas FcγRI, FcγRIII, and FcγRIV act as activating receptors in immune cells (16). In mice, monocytes and macrophages express all activating and inhibitory FcγRs (FcγRI-FcγRIV), while neutrophils express the inhibitory FcγRIIB and the activating FcγRIII and FcγRIV. In addition, the expression of FcγRI, FcγRIIB, and FcγRIII dominates on dendritic cell (DC)s (17). In rodents, cell surface expression and signaling capacity of all activating FcγRs (FcγRI, FcγRIII, and FcγRIV) are dependent on the γ-chain. Therefore, genetic deletion of this subunit leads to loss of cell surface expression of FcγRs, resulting in functional deficiency of these activating FcγRs. Therefore, γ-chain of Fc receptor-knockout animals showed dramatically impaired antibody mediated effector cell responses (14,18-21).

During HP, immune complexes formed by soluble antigens and IgG antibodies trigger the complement cascade, resulting in activation of alveolar macrophage, in sequence, recruitment of neutrophils and lymphocytes (2). Furthermore, prominent humoral responses have been reported in pigeon fancier's disease, one of the most common HP in human (22), as revealed by the elevated levels of IgGs in BALF and the infiltration of B lymphocytes into interstitial and alveolar spaces (23,24). Based on these observations, we postulate that immune complexes of exposed antigens and IgGs activate neutrophils and macrophages via FcγRs, which may contribute to initiation of pulmonary inflammation upon SR antigen challenge. Therefore, in this study, we investigated the functional roles of FcγRs in the development of murine SR-induced HP using Fcγ-chain knockout mice.

During the development of HP, the inhaled antigen enters the alveolar spaces, crosses the alveolar endothelium, and binds to circulating antibody leading to immune complexes deposition within the lung (30). Moreover, these immune complexes activate various immune cells via engagement of IgG Fc receptors containing an activation motif (ITAM), resulting in initiation and propagation of an inflammatory response (31). Therefore, it has been suggested that FcγR play a critical role in the development of HP. Consistent with this suggestion, our experiments demonstrated that FcγR^-/- mice exhibited an attenuated phenotype of HP in terms of histologic alterations compared with B6 mice, indicating that activating FcγRs contribute to the development of SR-induced HP. The activating FcγRs (FcγRI, III, and IV) are usually expressed on innate cells such as monocytes, macrophages, dendritic cells, whereas B cells express inhibitory FcγRIIB and none of FcγRs is detected on T cells (17). Thus, this expression pattern of FcγR on immune cells enables us to hypothesize that innate immune cells might be involved in FcγR-mediated immune responses of SR-induced HP rather than adaptive immune cells. Consistent with this hypothesis, the cellular compositions of BALF were strikingly different between FcγR^-/- and B6 mice in SR-induced HP. Macrophages and neutrophils were significantly reduced in BALF of FcγR^-/- mice compared to B6 mice, whereas T and B lymphocytes were increased. These findings indicate that FcγRs contribute to activation and recruitment of macrophages or neutrophils via immune complexes, resulting in attenuation of histopathology of lung parenchyma in FcγR^-/- mice during SR-induced HP. HP has been considered to involve two pathologic mechanisms: immune complex-mediated immune response and T cell-mediated immune response (32). These mechanisms occur either simultaneously and/or independently at different stages of the disease. The former mechanism usually initiates and mediates the acute HP, while the latter mediates the subacute and chronic HP. Therefore, it is more likely that FcγR-mediated activation and recruitment of innate immune cells might be an early event of HP rather than occur at chronic phase of disease.

IFN-γ produced by T cells and neutrophils in the lungs contributes to the development of murine HP, categorized as a Th1 disease (5,33). Moreover, IFN-γ-producing neutrophils contribute to granuloma formation in the absence of IFN-γ-producing T cells during SR-induced HP (10). These findings indicate that innate immune cells more contribute to IFN-γ production and development of HP than adaptive immune cells do. In our experiments, the subset analysis of BALF revealed that neutrophils were remarkably reduced in BALF of FcγR^-/- mice during SR-induced HP, which led us to hypothesize that small numbers of neutrohpils in the lungs of FcγR^-/- mice produce small amount of IFN-γ, resulting in the marked reduction of IFN-γ production in the lungs. However, we did not detect statistically significant reduction of IFN-γ production in the lungs of FcγR^-/- mice compared to B6 mice. Here, there are several lines of evidences to explain this discrepancy in IFN-γ production of FcγR^-/- mice during SR-induced HP. First, in contrast to neutrophils, CD4⁺ T cells, known to be another main source of IFN-γ in the lungs during SR-induced HP, significantly increased in BALF of FcγR^-/- mice upon SR antigen inoculation. These findings suggest that IFN-γ produced by CD4⁺ T cells might compensate reduced amount of IFN-γ produced by neutrophils during SR-induced HP. Second, we recently reported that IL-4-producing NKT cells play a protective role in SR-induced HP by suppressing IFN-γ-producing neutrophils (11). Moreover, NKT cells express an activating receptor FcγRIII that provides strong activating signals for activation of these cells (34-36). Our experiments in antibody-induced joint inflammation demonstrated that the engagement of FcγRIII via immune complexes activates NKT cells, resulting in modulation of joint inflammation (37). Thus, there is a possibility that signals via FcγRIII also contribute to activation of NKT cells during SR-induced HP, which suppresses IFN-γ-producing neutrophils in the lungs. Based on this possibility, we postulate that loss of activating signals via FcγRIII on NKT cells in FcγR^-/- mice causes the reduction of suppressive effects on IFN-γ-producing neutrophils, resulting in enhancement of IFN-γ production by neutrophils during SR-induced HP. Combined, it is suggested that increased number of IFN-γ-producing CD4⁺ and CD8⁺ T cells in the lungs and loss of NKT cell-mediated suppression of IFN-γ-producing neutrophils in FcγR^-/- mice compensate reduced IFN-γ production by FcγR-deficient neurtophils.

Considering clinical aspects of HP, the presence of serum precipitating antibodies or cell-mediated hypersensitivity against presumptive antigens may be helpful for the diagnosis of patients with HP (2,28,29). Moreover, in several studies using the SR-induced murine HP model, SR-spedific serum IgG or IL-2 production of BAL cells was measured to evaluate the adaptive immune responses in SR-induced HP (38,39). Therefore, to examine adaptive immune responses, we measured the SR-specific immune cell proliferation and the IgG levels both in sera and BALF. In contrast to histological alteration and cellular composition of BALF, there was no difference in adaptive immune responses between B6 and FcγR^-/- mice upon SR-antigen exposure. However, our experiments demonstrated that the number of CD4⁺ and CD8⁺ T, and B cells in BALF of FcγR^-/- mice increased compared to that of B6 mice. These findings suggest that adaptive immune responses induced by T and B cells against SR antigen are not affected in FcγR^-/- mice although T and B cells more infiltrated into the lungs of FcγR^-/- mice compared to B6 mice. Very recently, we reported that CD4⁺CD25⁺ regulatory T (Treg) cells attenuate SR-induced HP by suppressing IFN-γ-producing CD4⁺ and CD8⁺ T cells in the lungs via both direct cell-to-cell contact and IL-10 dependent manners (40). Even though the exact number and function of Treg cells needs to be determined in FcγR^-/- and B6 mice, it is hypothesized that increased numbers or function of Treg cells upon SR angtigen challenge contributes to more suppression of CD4⁺ and CD8⁺ T cells in FcγR^-/- mice upon SR antigen exposure, resulting in similar adaptive immune responses between FcγR^-/- and B6 mice.

Avoidance of the causative antigens and steroid treatment are well-known treatment of patients with HP. However, some patients develop advanced pulmonary fibrosis despite of treatment (2). Therefore, alternative therapeutic modalities must be developed. Here, we propose a therapeutic approach using blockage of activating FcγRs based on our current research (11). However, it is likely that the blockage of FcγRs might attenuate the protective function of FcγRIII-expressing NKT cells on HP, which provides adverse effects on the treatment of HP. Therefore, a comprehensive approach should be considered to optimize therapeutic effects on HP.

In summary, our results demonstrate that activating signals through Fcγ receptors play an important role in the development of HP by provoking innate immune cells to mobilize and to produce cytokines in particular Th1 inducing cytokines. These results provide new insights into the complex immunological mechanisms underlying HP, and lead to the development of more effective therapeutic approaches for HP.