Susceptibility of Mice Genetically Deficient in the Surfactant Protein (SP)-A or SP-D Gene to Pulmonary Hypersensitivity Induced by Antigens
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免疫学杂志 2005年第11期
Abstract
Lung surfactant protein A (SP-A) and D (SP-D) are innate immune molecules which are known to interact with allergens and immune cells and modulate cytokine and chemokine profiles during host hypersensitivity response. We have previously shown therapeutic effects of SP-A and SP-D using a murine model of lung hypersensitivity to Aspergillus fumigatus (Afu) allergens. In this study, we have examined the susceptibility of SP-A (AKO) or SP-D gene-deficient (DKO) mice to the Afu allergen challenge, as compared with the wild-type mice. Both AKO and DKO mice exhibited intrinsic hypereosinophilia and several-fold increase in levels of IL-5 and IL-13, and lowering of IFN- to IL-4 ratio in the lungs, suggesting a Th2 bias of immune response. This Th2 bias was reversible by treating AKO or DKO mice with SP-A or SP-D, respectively. The AKO and DKO mice showed distinct immune responses to Afu sensitization. DKO mice were found more susceptible than wild-type mice to pulmonary hypersensitivity induced by Afu allergens. AKO mice were found to be nearly resistant to Afu sensitization. Intranasal treatment with SP-D or rhSP-D (a recombinant fragment of human SP-D containing trimeric C-type lectin domains) was effective in rescuing the Afu-sensitized DKO mice, while SP-A-treated Afu-sensitized AKO mice showed several-fold elevated levels of IL-13 and IL-5, resulting in increased pulmonary eosinophilia and damaged lung tissue. These data reaffirm an important role for SP-A and SP-D in offering resistance to pulmonary allergenic challenge.
Introduction
Two of the hydrophilic lung surfactant proteins (SP), 3 SP-A and SP-D, are considered carbohydrate pattern recognition molecules of innate immunity which have been shown to interact with a range of pathogens, allergens, and apoptotic cells (1, 2). This interaction effects recruitment and activation of a host of immune cells, leading to differential pulmonary cytokine and chemokine profiles as a part of host response (3). The primary structure of SP-A and SP-D is organized into four regions: an N-terminal region involved in the formation of interchain disulphide bonds, a collagen region composed of Gly-X-Y repeats, a neck peptide, and a C-terminal C-type lectin domain. They are large oligomeric structures, each assembled from multiple copies of a single polypeptide chain (human SP-A has two closely-related chains). The lectin domains are spaced, in a trimeric orientation, at the end of triple-helical collagen stalks (4). Six of these trimeric subunits make up the overall structure of SP-A, while SP-D is composed of a cruciform-like structure, with four arms of equal length.
The lectin domains are usually the ligand recognition domain which are known to interact with carbohydrate structures on the surfaces of a wide range of pathogens, such as viruses, bacteria, and fungi. SP-A and SP-D are also known to interact with phagocytic cells and enhance their chemotactic, phagocytic, and oxidative properties (1, 5). Therefore, the recognition of non-self via lectin domain and subsequent engagement of collagen region with immune cells via the collectin receptor enhances killing by activated phagocytic cells (6). The interaction between the collagen region of SP-A and SP-D (when bound to ligand via lectin domain) with immune cells is generally considered to be mediated via a common collectin receptor, calreticulin/CD91 complex (7). This interaction has been shown to enhance p38 MAPK activation, NF-B activity, and production of proinflammatory cytokines/chemokines in macrophages (7). SP-A and SP-D also mediate another independent signal transduction pathway, which appears anti-inflammatory and results from direct interactions of trimeric lectin domains with specific cell surface glycoproteins (7).
SP-A and SP-D have also been shown to be involved in the modulation of pulmonary inflammatory responses and resistance to allergen-induced airway hypersensitivity (2, 8, 9, 10). Abnormal levels of SP-A and SP-D in bronchoalveolar lavage (BAL) have been reported in hypersensitivity lung diseases and asthmatics show increased amounts of SP-A and SP-D in BAL as compared with those in controls (11, 12). Serum SP-D levels for two allergic patients have been found elevated at diagnosis which decreased following corticosteroid therapy (13). The patients of birch pollen allergy and pulmonary alveolar proteinosis (PAP) showed a shift toward lower oligomeric forms of SP-A, in comparison to healthy volunteers with a possible loss or alteration of biological function (14).
SP-A and SP-D can bind via their lectin domains to allergenic extracts derived from pollens, the house dust mite, and Aspergillus fumigatus (Afu) inhibit specific IgE binding to allergens, and block allergen-induced histamine release from sensitized basophils (15, 16, 17). SP-A and SP-D can reduce the proliferation of PBMC isolated from mite-sensitive asthmatic children (18), and SP-D, in particular, has a suppressive effect on the secretions of IL-2 by PBMC (19). Using murine models of pulmonary hypersensitivity induced by allergens derived from Afu (8), the house dust mite (20), and OVA (21), it has been shown that therapeutic treatment of sensitized mice with SP-A or SP-D can reverse hypersensitivity response which involves lowering of specific IgE levels and blood and pulmonary eosinophilia, and a shift in cytokine profile from Th2 to Th1 type.
The experiments conducted using the transgenic mice, genetically deficient in SP-A or SP-D, have also emphasized a key role played by SP-A and SP-D in pulmonary immune response. The SP-A gene-deficient (AKO) mice are less effective in clearing lung pathogens (22). Concentrations of TNF-, IL-6, and IL-1 are increased in BAL fluid of AKO mice, which can increase further on adenoviral administration. Coadministration of adenovirus and purified human SP-A can ameliorate adenoviral-induced lung inflammation in AKO mice (23). Mice genetically deficient in SP-D (DKO) show chronic inflammation, foamy alveolar macrophages secreting 10-fold higher levels of hydrogen peroxide, increased activity of matrix metalloproteinases (MMP), emphysema, and fibrosis in the lungs (24).
The present study was undertaken to comparatively evaluate the effect of deficiency of SP-A or SP-D genes on eosinophilia and Th2 cytokines in view of their role in the pathogenesis of allergy and asthma. We observed that both AKO and DKO mice showed intrinsic hypereosinophilia and several-fold increase in the levels of IL-5 and IL-13, and lowering of IFN- to IL-4 ratio, suggesting a shift to a Th2 type of response in comparison to the wild-type (WT) mice. Gene expression and exogenous administration of SP-A and SP-D has been able to complement some of the defects of AKO and DKO mice (25, 26, 27, 28, 29). Therefore, we examined whether intranasal administration of native human SP-A to AKO, and SP-D or a recombinant fragment of SP-D (rhSP-D) to the DKO mice may reverse hypereosinophilia and Th2 predominance. Because both SP-A and SP-D play a role in Afu-mediated hypersensitivity, we have also examined whether AKO and DKO mice were more susceptible to Afu sensitization than WT mice and whether intranasal administration of native human SP-A, SP-D, and rhSP-D can rescue the Afu-sensitized KO mice.
AKO and DKO mice showed a distinct immune response to Afu sensitization. Although DKO showed a cytokine profile similar to that of WT mice on Afu sensitization, the magnitude of the effect was higher suggesting that the DKO mice are more susceptible than the WT mice. AKO mice showed a different trend in the cytokines in comparison to WT mice on Afu sensitization. However, the magnitude of change was not significant suggesting that AKO may be resistant to Afu sensitization. SP-D and rhSP-D were effective in rescuing the Afu-sensitized DKO mice while SP-A administered Afu-sensitized AKO mice showed manyfold elevated levels of IL-5 and IL-13, resulting in severe pulmonary eosinophilia and damaged lung tissue.
Results
Comparative evaluation of eosinophilia and cytokine profile of WT, AKO, and DKO control mice on day 0
AKO mice showed elevated peripheral eosinophilia (1.85-fold) and EPO activity (1.29-fold) than WT mice (Table II), consistent with increased eosinophil infiltrations seen in the lung sections (Fig. 1). AKO mice showed an increase in IL-13 (13.1-fold), IL-5 (3.93-fold), and IL-2 (3.43-fold) and a 1.92-fold decrease in IFN- than WT mice (Fig. 2; Table III). The ratio of IFN- to IL-4 was 1.525-fold less in AKO than WT mice, suggesting that AKO mice have a Th2 bias, as opposed to the predominantly Th1 profile of the WT C57BL/6 mice (Table II).
SP-D treatment to WT-Ag mice led to increase in all the cytokines, with significant increase in IL-5 (3.66-fold), IL-2 (6.75-fold) except IL-4 (1.5-fold decrease) on day 4, followed by a decrease in IFN- (3.29-fold) and a further increase in IL-5 (6-fold) (Fig. 7). The IFN- to IL-4 ratio increased on day 4 and decreased on day 10 (from 6.522 on day 0 to 13.33, i.e., 2.04-fold increase and 1.63 i.e., 4-fold decrease) on days 4 and 10, respectively (Table VI). Administration of rhSP-D to WT-Ag mice led to an increase in IL-2 (4.5-fold), and a decrease in IL-4 (1.71-fold decrease), IL-12 (1.88-fold) and IFN- (2.15-fold) on day 4 followed by a decrease in IL-13 (1.87-fold), IL-2 (2.0-fold), IL-4 (8.4-fold), and IFN- (9.33-fold) (Fig. 8). The IFN- to IL-4 ratio decreased from 6.522 on day 0 to 3.9 (1.67-fold) and 4.5 (1.45-fold) on days 4 and 10, respectively (Table VI).
Administration of BSA to sensitized AKO-Ag and DKO-Ag mice led to decrease in peripheral eosinophilic count (2.08- and 3.12-fold, respectively) and EPO activity (1.69- and 1.14-fold, respectively) (Table VI). Lung histopathology showed decreased eosinophilic infiltrations in BSA-treated AKO-Ag and DKO-Ag mice. No significant increase in levels of anti-BSA IgG or IgE Ab was observed in these mice. BSA treatment to AKO-Ag mice showed a decrease in all the cytokines on day 4: IL-13 (8.28-fold), IL-5 (3.46-fold), IL-4 (2.15-fold), IL-2 (2.38-fold) IL-10 (2.63-fold) and IL-12 (3.1-fold). The levels of IL-13 (11.6-fold), IL-5 (3.46-fold), IL-4 (4.75-fold), IL-2 (31-fold), IL-10 (2.16-fold), IL-12 (2.04-fold), IFN- (2.4-fold) and TNF- (5.47-fold) further decreased on day 10. IFN- to IL-4 ratio did not change significantly (Table VI). BSA treatment to DKO-Ag mice showed a significant increase in IL-4 (6.75-fold) and IFN- (3-fold) on day 4 and IL-13 (2.64-fold), IL-4 (6.75-fold), and IFN- (3.75-fold) on day 10. IFN- to IL-4 ratio decreased from 2.298 on day 0 to 1.4 (1.64-fold) and 1.85 (1.24-fold) on day 4 and 10, respectively (Table VI).
Administration of SP-D or rhSP-D has therapeutic effects on Afu-sensitized DKO mice
Administration of SP-D or rhSP-D to DKO-Ag mice led to decrease in peripheral eosinophilic count (1.61- and 2.5-fold, respectively), EPO activity (1.6- and 2.04-fold, respectively), while a decrease in Afu IgE (0.94-, 0.7-fold) (Table VI). Lung sections of SP-D or rhSP-D-treated DKO-Ag mice showed reduced eosinophilic infiltrations on day 10 in comparison to untreated DKO-Ag mice on day 0 and rhSP-D administration was more effective in reducing eosinophilia than SP-D (Fig. 4).
DKO-Ag-SP-D mice showed a decrease in IL-13 (3.4-fold) and IL-5 (3.37-fold) while an increase in IL-4 (8-fold), and IL-2 (2.44-fold) (Fig. 7). The IFN- to IL-4 ratio decreased on day 4 and increased on day 10 (from 2.298 on day 0 to 0.625 (3.67-fold) and 3.75 (2.23-fold) on days 4 and 10, respectively (Table VI). rhSP-D administration to DKO-Ag led to an increase in all the cytokines, with significant increases in IL-4 (10.8-fold), IL-10 (2.13-fold), IL-2 (2.4-fold), TNF- (2.53-fold), IL-12 (2.58-fold), and IFN- (20.5-fold) on day 4. On day 10, however, cytokine levels decreased: IL-13 (2.7-fold), IL-5 (2.31-fold), IL-10 (2.17-fold), and IL-2 (2.22-fold) except IFN-, which increased by 9-fold (Fig. 8). The IFN- to IL-4 ratio decreased followed by an increase from 2.298 on day 0 to 1.8 (1.27-fold) and 3.0 (2.23-fold) on days 4 and 10, respectively (Table VI).
Administration of SP-A to AKO-Ag mice led to decrease in peripheral eosinophilic count (2.27-fold) on day 4 followed by further decrease on day 10 (4.16-fold) (Table VI). SP-A treatment led to an increase in levels of IL-13 (2.9-fold) on day 4 and a decrease in levels of IL-4 (2.14-fold), IL-2 (5.16-fold), IFN- (2.4-fold), and TNF- (3.45-fold) on day 10 (Fig. 6). The IFN- to IL-4 ratio did not change significantly (Table VI). Lung sections showed significantly increased eosinophilic infiltrations on days 4 and 10 in comparison to AKO-Ag mice on day 0 and showed collapse of the alveolar structure (Fig. 4). It is important to note here that although the peripheral eosinophil count decreased with SP-A administration to Afu-sensitized AKO mice, the pulmonary eosinophilia worsened.
Discussion
In view of the important roles of SP-A and SP-D in pulmonary immune response, we had earlier examined the effect of SP-A, SP-D, and rhSP-D in a murine model of Afu-induced pulmonary hypersensitivity (8). Afu is the fungus most commonly implicated in causing both IgE-mediated and non-IgE-mediated hypersensitivity in humans leading to development of ABPA, which is characterized by activated Th2 cells and asthma. Intranasal administration of SP-A, SP-D, or rhSP-D (three doses on consecutive days) significantly lowered eosinophilia and specific Ab levels in ABPA mice (8). Lung sections of the ABPA mice showed extensive infiltration of lymphocytes and eosinophils, which were considerably reduced following treatment (8). The levels of IL-2, IL-4, and IL-5 were decreased, while that of IFN- was raised in supernatants of the cultured spleen cells, indicating a marked Th2Th1 shift (8). This study highlighted a central role for SP-A and SP-D in regulation of pulmonary hypersensitivity (8). As a logical next step, we wished to examine the nature of immune response in AKO and DKO mice when challenged with Afu allergens to validate whether deficiency of these proteins made mice more susceptible to pulmonary hypersensitivity.
AKO and DKO show intrinsic hypereosinophilia
Both AKO and DKO mice showed elevated peripheral and pulmonary eosinophilia and a significant increase in EPO activity in comparison to the WT mice. A significant monocytic infiltration has been reported in the peribronchiolar and perivascular regions of the lungs in DKO mice (24). In addition, an increased accumulation of alveolar macrophages and lymphocytes was observed in DKO mice (32, 36). Because treatment with SP-A, SP-D or rhSP-D has been shown to lower IL-5, peripheral and pulmonary eosinophilia in the Afu-sensitized WT BALB/c mice (8), an alteration in the peripheral and pulmonary eosinophil counts in the AKO and DKO mice, was not surprising. A significantly raised level of IL-5 and IL-13 in both AKO and DKO mice may be one of the mechanisms causing hypereosinophilia (37, 38). SP-A can inhibit IL-8 expression and production from eosinophils, thus probably preventing the autocrine cycle for recruitment of human eosinophils by inhibiting IL-8, a chemotactic cytokine (39). Eosinophils are the important effector cells for the pathogenesis of allergic inflammation via the secretion of highly cytotoxic granular proteins and Th2 type of cytokines. Blood and tissue eosinophilia is a common manifestation of late-phase allergic inflammation causing tissue damage. Hypereosinophilia exhibited by both AKO and DKO mice suggests that SP-A and SP-D have a role in regulating the eosinophil infiltration and modulation in the lung in response to environmental stimuli.
Genetic deficiency of SP-A or SP-D shifts the cytokine profile of C57BL/6 mice toward Th2 type
The cytokine profile of both AKO and DKO mice suggested a Th2 bias (elevated IL-13 and IL-5 levels) and down-regulation of Th1 cytokine, IFN- (more pronounced in DKO than AKO mice). IL-13 and IL-5 have important roles in allergen induced asthma and airway hyperresponsiveness (AHR). Overexpression of IL-13 in mice leads to 70-fold increase in SP-D, 3-fold increase in SP-A, and 6-fold increase in the phospholipid pool (38). Remarkably similar to DKO mice, IL-13 overexpressing mice have characteristic foamy macrophages, type II cell hypertrophy, fibrosis, massive inflammation involving eosinophilia, protease-dependent acquired emphysema, and AHR (38). IL-13, produced in the airway by a variety of cells (T cells, eosinophils, and mast cells), mediates mucus production and AHR through its combined actions on epithelial cells and smooth muscle cells independently of IL-5 and eotaxin (40, 41). IL-13 also directly promotes eosinophil survival, activation, and recruitment (42, 43, 44). Alveolar macrophages of DKO mice show increased expression of reactive oxygen species (ROS), hydrogen peroxide, MMP-9, MMP-12, and NF-B (45). Because IL-13 has been reported to inhibit the production of proinflammatory mediators by monocytes and macrophages, including ROS, through a mechanism that probably involves NF-B, it appears that the increased levels of IL-13 are produced in DKO mice to regulate their increased oxidative state (46, 47, 48, 49, 50). However, IL-13 and SP-D have also been described as potent stimulators of MMP in the lung (37, 51). It is likely that certain physiological effects and hypereosinophilia observed in AKO and DKO mice arise due to overexpression of IL-13, although AKO mice do not show abnormalities, such as foamy macrophages, type II cell hypertrophy, and fibrosis similar to DKO mice. However, sequential targeting of both SP-A and SP-D genes (double knockout) show exaggerated alveolar proteinosis and emphysema compared with DKO mice, suggesting that SP-A deficiency may contribute to physiological abnormalities in the lungs (52).
Transgenic mice overexpressing IL-5 also exhibit intrinsic AHR (even in the absence of any antigenic stimuli) and increased numbers of eosinophils and lymphocytes in the lung tissue (53). The observation that AKO and DKO mice have elevated IL-5 levels, which is lowered by therapeutic delivery of SP-A or SP-D/rhSP-D, appears to suggest that SP-A and SP-D inhibit allergen mediated eosinophilia in the lungs through down-regulation of IL-5. It is worth noting that mice genetically deficient in GM-CSF also show pulmonary alveolar proteinosis associated with a marked increase in phospholipid pool similar to DKO mice. GM-CSF-deficient mice showed 50-fold increase in SP-D, while only a 3-fold increase in SP-A, similar to IL-13 overexpressing mice. It has been proposed that GM-CSF mediates some of the physiological changes seen in DKO mice as ablation of GM-CSF in DKO mice leads to alleviation of macrophage proliferation and type II cell hypertrophy (54). It is also possible that the actions of GM-CSF and SP-D leading to similar pathophysiological changes are distinct (55). It is to be noted that IL-5, IL-13, and GM-CSF genes are situated on the same chromosomal location (5q31). Furthermore, GM-CSF, along with IL-5, is known to regulate IL-13 secretion from human eosinophils (56).
Distinct immune response to BSA by WT, AKO, and DKO
Intranasal administration of BSA on days 1–3 was included in the study as a control protein similar to our earlier studies. However, C57BL/6 mice responded to the BSA administered as a short-term allergen challenge with a characteristic Th2 response with increased peripheral eosinophil count and pulmonary eosinophilia, which has also been reported earlier (57). WT mice, however, showed no significant increase in anti-BSA IgG or IgE Abs. Interestingly, BSA-specific IgG and IgE Abs were observed in both BSA-treated AKO and DKO mice with AKO mice also showing a significant down-regulation of IFN- to IL-4 ratio (shift to Th2 response) with an increase in peripheral eosinophil count and pulmonary eosinophilia while DKO mice showed only an increase in pulmonary eosinophilia. These observations suggest that both AKO and DKO mice show a different pulmonary immune response to short-term sensitization than the WT mice and both SP-A and SP-D have important roles in regulation of humoral immune response to short-term allergen sensitization in the lung.
Afu sensitization provokes distinct immunological responses in AKO and DKO mice
Following Afu sensitization, C57BL/6 mice, which were used as a control for AKO and DKO mice (both in C57BL/6 background) showed no change in Afu-IgE, and an increase in Afu-IgG, peripheral and pulmonary eosinophilia. Allergen challenge led to a decrease in IFN- and IL-4 in lung suspensions (hence an increase in IFN- to IL-4 ratio). IL-2, IL-5, and IL-13 levels decreased significantly in the lung and spleen suspensions, suggesting the Th1 predominance in the mouse strain. This is consistent with the observation that response to allergenic challenge varies in different strains of mice and C57BL/6 mice show a predominantly Th1 response to a high dose of allergen sensitization (57).
Both AKO and DKO mice showed comparable increase in Afu-IgG and peripheral eosinophil count, and DKO mice showed more severe pulmonary eosinophilia than AKO mice, following Afu sensitization. AKO mice showed a corresponding increase in Afu-IgE level as well. The EPO activity was down in DKO mice, while in AKO mice, it remained unchanged. AKO mice showed an increase in all the cytokines in lung suspension (<2-fold) except IL-13 and IL-5. Increased levels of Th2 cytokines in Afu-sensitized AKO mice than WT and DKO mice suggests that the phenotype of these mice is more complex than previously reported. However, AKO-Ag mice consistently showed Th1 predominance in BAL as well as in lung and spleen suspensions. DKO mice showed a decrease in all cytokine levels in lung suspension, similar to WT mice, but in a more pronounced manner. Both lung and spleen suspensions of DKO-Ag mice showed a Th1 response; however, BAL had an increase in IL-13, IL-4, and IL-2 levels, similar to WT-Ag mice. A recent study showed similar results wherein, following OVA sensitization and challenge in vivo, SP-D–/– mice expressed higher BAL eosinophils, IL-10 and IL-13 concentrations and lower IFN- expression at early time points compared with WT mice (58). It is evident that AKO mice are almost nonresponsive to the Afu sensitization, while DKO mice show a pronounced response. Afu sensitization led to a similar increase in the ratio of IFN- and IL-4 in both WT and DKO mice (no significant increase in AKO mice). Significant down-regulation of Afu-IgE Ab was specific to DKO mice. AKO mice, in contrast, showed a significant increase in Afu-IgE Ab. This differential responsiveness to Afu sensitization in AKO and DKO mice may be accounted for by a 50% decrease in SP-A levels in DKO mice and a 7-fold increase in SP-D levels in AKO mice (59). It appears that both SP-A and SP-D contribute to the homeostasis to the allergenic challenge in an interdependent manner and absence of any one of them disturbs this balance.
Administration of SP-A, SP-D, or rhSP-D to the Afu-sensitized WT and KO mice can partially rescue them
As previously reported (8), Afu-IgE, and Afu-IgG, peripheral and pulmonary eosinophilia in WT-Ag were down-regulated by SP-A, SP-D, or rhSP-D treatment (the increased IFN- to IL-4 ratio was also reversed). SP-D was able to restore IL-5 and IL-2 levels increased by allergen challenge. Afu sensitization led to a decrease in IL-13, while an increase in IL-2, IL-4, IL-10, IL-12, IFN-, and TNF- in AKO mice. SP-A treatment containing the peripheral eosinophil count, however, showed increased pulmonary eosinophilia with extensive tissue damage, possibly caused by increased levels of IL-13 and IL-5 in AKO mice. SP-A was also not able to bring down increased Afu-IgG and Afu-IgE levels, suggesting that SP-A treatment (3 μg per mice for 3 consecutive days) is not leading to complete alleviation of the Afu-induced changes in AKO mice.
SP-D treatment to DKO-Ag mice restored IL-2, IL-4, IL-12, and TNF- levels. IL-13 and IL-5 levels showed a further decrease with treatment, the levels being significantly lower than the DKO-C mice. IL-13, IL-5, and IL-2 levels in rhSP-D-treated DKO-Ag mice were comparable to WT-C mice, while IL-10 and IL-12 went down significantly compared with all other groups. Thus, SP-D and rhSP-D were more effective than SP-A in rescuing the respective gene-deficient mice from the effects of Afu sensitization. Previously, coadministration of SP-D has been shown to normalize viral clearance and cytokine response in DKO mice challenged with influenza A virus (IAV) (60). Expression of SP-D/conglutinin chimeric protein in epithelial cells of DKO mice substantially corrected the increased lung phospholipids and increased the clearance of IAV but could not ameliorate the ongoing lung inflammation, enhanced metalloproteinase expression, and alveolar destruction (28).
It appears likely that SP-A and SP-D influence the lung immunity by directly or indirectly modulating the nuclear factors. DKO mice have been shown to have elevated levels of transcripts for NF-B and AP-1 (45). NFAT1 (NF regulating expression of many genes encoding immunoregulatory cytokines)-deficient mice also show increased levels of IL-4, IL-5, and IL-13 as well as enhanced eosinophilia, similar to AKO and DKO mice. It is possible that NFAT1 is involved in the downstream signaling of SP-A and/or SP-D (61). In conclusion, the present study reports that both SP-A and SP-D have important roles in the regulation of cytokine milieu and eosinophilia in the lungs, and their absence leading to inherent hypersensitivity in mice highlights their essential role in host defense against allergic airway challenges. Thus, both these versatile macromolecules enable the lung to achieve homeostasis probably through distinct mechanisms. It is important to note here that both AKO and DKO mice are different and the anatomical and functional abnormalities reported only in DKO mice, and not in AKO mice, may be underlying issues for their behavior and susceptibility to Afu sensitization.
Acknowledgments
The SP-A and SP-D gene knockout mice were kindly provided by Dr. S. Hawgood (Cardiovascular Research Institute and Department of Pediatrics, University of California San Francisco, CA). We are grateful to Dr. Howard Clark for his technical help with breeding knockout mice.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by the Council for Scientific and Industrial Research (to T.M., M.S., and P.U.S.), the Medical Research Council (to K.B.M.R.) and the European Commission (to K.B.M.R. and U.K.).
2 Address correspondence and reprint requests to Dr. P. Usha Sarma, Institute of Genomics and Integrative Biology, Delhi University Campus, Mall Road, Delhi-110007, India. E-mail address: u_sarma{at}hotmail.com
3 Abbreviations used in this paper: SP-A, human surfactant protein A; SP-D, human surfactant protein D; BAL, bronchoalveolar lavage; PAP, pulmonary alveolar proteinosis; Afu, Aspergillus fumigatus; AKO, SP-A gene deficient; DKO, SP-D gene deficient; MMP, matrix metalloproteinase; rhSP-D, a recombinant fragment of human surfactant protein D, composed of homotrimeric neck and C-type lectin domains; WT, wild type; 3wcf, three week culture filtrate; ABPA, allergic bronchopulmonary aspergillosis; EPO, eosinophil peroxidase; ROS, reactive oxygen species; IAV, influenza A virus.
Received for publication July 23, 2004. Accepted for publication March 18, 2005.
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Lung surfactant protein A (SP-A) and D (SP-D) are innate immune molecules which are known to interact with allergens and immune cells and modulate cytokine and chemokine profiles during host hypersensitivity response. We have previously shown therapeutic effects of SP-A and SP-D using a murine model of lung hypersensitivity to Aspergillus fumigatus (Afu) allergens. In this study, we have examined the susceptibility of SP-A (AKO) or SP-D gene-deficient (DKO) mice to the Afu allergen challenge, as compared with the wild-type mice. Both AKO and DKO mice exhibited intrinsic hypereosinophilia and several-fold increase in levels of IL-5 and IL-13, and lowering of IFN- to IL-4 ratio in the lungs, suggesting a Th2 bias of immune response. This Th2 bias was reversible by treating AKO or DKO mice with SP-A or SP-D, respectively. The AKO and DKO mice showed distinct immune responses to Afu sensitization. DKO mice were found more susceptible than wild-type mice to pulmonary hypersensitivity induced by Afu allergens. AKO mice were found to be nearly resistant to Afu sensitization. Intranasal treatment with SP-D or rhSP-D (a recombinant fragment of human SP-D containing trimeric C-type lectin domains) was effective in rescuing the Afu-sensitized DKO mice, while SP-A-treated Afu-sensitized AKO mice showed several-fold elevated levels of IL-13 and IL-5, resulting in increased pulmonary eosinophilia and damaged lung tissue. These data reaffirm an important role for SP-A and SP-D in offering resistance to pulmonary allergenic challenge.
Introduction
Two of the hydrophilic lung surfactant proteins (SP), 3 SP-A and SP-D, are considered carbohydrate pattern recognition molecules of innate immunity which have been shown to interact with a range of pathogens, allergens, and apoptotic cells (1, 2). This interaction effects recruitment and activation of a host of immune cells, leading to differential pulmonary cytokine and chemokine profiles as a part of host response (3). The primary structure of SP-A and SP-D is organized into four regions: an N-terminal region involved in the formation of interchain disulphide bonds, a collagen region composed of Gly-X-Y repeats, a neck peptide, and a C-terminal C-type lectin domain. They are large oligomeric structures, each assembled from multiple copies of a single polypeptide chain (human SP-A has two closely-related chains). The lectin domains are spaced, in a trimeric orientation, at the end of triple-helical collagen stalks (4). Six of these trimeric subunits make up the overall structure of SP-A, while SP-D is composed of a cruciform-like structure, with four arms of equal length.
The lectin domains are usually the ligand recognition domain which are known to interact with carbohydrate structures on the surfaces of a wide range of pathogens, such as viruses, bacteria, and fungi. SP-A and SP-D are also known to interact with phagocytic cells and enhance their chemotactic, phagocytic, and oxidative properties (1, 5). Therefore, the recognition of non-self via lectin domain and subsequent engagement of collagen region with immune cells via the collectin receptor enhances killing by activated phagocytic cells (6). The interaction between the collagen region of SP-A and SP-D (when bound to ligand via lectin domain) with immune cells is generally considered to be mediated via a common collectin receptor, calreticulin/CD91 complex (7). This interaction has been shown to enhance p38 MAPK activation, NF-B activity, and production of proinflammatory cytokines/chemokines in macrophages (7). SP-A and SP-D also mediate another independent signal transduction pathway, which appears anti-inflammatory and results from direct interactions of trimeric lectin domains with specific cell surface glycoproteins (7).
SP-A and SP-D have also been shown to be involved in the modulation of pulmonary inflammatory responses and resistance to allergen-induced airway hypersensitivity (2, 8, 9, 10). Abnormal levels of SP-A and SP-D in bronchoalveolar lavage (BAL) have been reported in hypersensitivity lung diseases and asthmatics show increased amounts of SP-A and SP-D in BAL as compared with those in controls (11, 12). Serum SP-D levels for two allergic patients have been found elevated at diagnosis which decreased following corticosteroid therapy (13). The patients of birch pollen allergy and pulmonary alveolar proteinosis (PAP) showed a shift toward lower oligomeric forms of SP-A, in comparison to healthy volunteers with a possible loss or alteration of biological function (14).
SP-A and SP-D can bind via their lectin domains to allergenic extracts derived from pollens, the house dust mite, and Aspergillus fumigatus (Afu) inhibit specific IgE binding to allergens, and block allergen-induced histamine release from sensitized basophils (15, 16, 17). SP-A and SP-D can reduce the proliferation of PBMC isolated from mite-sensitive asthmatic children (18), and SP-D, in particular, has a suppressive effect on the secretions of IL-2 by PBMC (19). Using murine models of pulmonary hypersensitivity induced by allergens derived from Afu (8), the house dust mite (20), and OVA (21), it has been shown that therapeutic treatment of sensitized mice with SP-A or SP-D can reverse hypersensitivity response which involves lowering of specific IgE levels and blood and pulmonary eosinophilia, and a shift in cytokine profile from Th2 to Th1 type.
The experiments conducted using the transgenic mice, genetically deficient in SP-A or SP-D, have also emphasized a key role played by SP-A and SP-D in pulmonary immune response. The SP-A gene-deficient (AKO) mice are less effective in clearing lung pathogens (22). Concentrations of TNF-, IL-6, and IL-1 are increased in BAL fluid of AKO mice, which can increase further on adenoviral administration. Coadministration of adenovirus and purified human SP-A can ameliorate adenoviral-induced lung inflammation in AKO mice (23). Mice genetically deficient in SP-D (DKO) show chronic inflammation, foamy alveolar macrophages secreting 10-fold higher levels of hydrogen peroxide, increased activity of matrix metalloproteinases (MMP), emphysema, and fibrosis in the lungs (24).
The present study was undertaken to comparatively evaluate the effect of deficiency of SP-A or SP-D genes on eosinophilia and Th2 cytokines in view of their role in the pathogenesis of allergy and asthma. We observed that both AKO and DKO mice showed intrinsic hypereosinophilia and several-fold increase in the levels of IL-5 and IL-13, and lowering of IFN- to IL-4 ratio, suggesting a shift to a Th2 type of response in comparison to the wild-type (WT) mice. Gene expression and exogenous administration of SP-A and SP-D has been able to complement some of the defects of AKO and DKO mice (25, 26, 27, 28, 29). Therefore, we examined whether intranasal administration of native human SP-A to AKO, and SP-D or a recombinant fragment of SP-D (rhSP-D) to the DKO mice may reverse hypereosinophilia and Th2 predominance. Because both SP-A and SP-D play a role in Afu-mediated hypersensitivity, we have also examined whether AKO and DKO mice were more susceptible to Afu sensitization than WT mice and whether intranasal administration of native human SP-A, SP-D, and rhSP-D can rescue the Afu-sensitized KO mice.
AKO and DKO mice showed a distinct immune response to Afu sensitization. Although DKO showed a cytokine profile similar to that of WT mice on Afu sensitization, the magnitude of the effect was higher suggesting that the DKO mice are more susceptible than the WT mice. AKO mice showed a different trend in the cytokines in comparison to WT mice on Afu sensitization. However, the magnitude of change was not significant suggesting that AKO may be resistant to Afu sensitization. SP-D and rhSP-D were effective in rescuing the Afu-sensitized DKO mice while SP-A administered Afu-sensitized AKO mice showed manyfold elevated levels of IL-5 and IL-13, resulting in severe pulmonary eosinophilia and damaged lung tissue.
Results
Comparative evaluation of eosinophilia and cytokine profile of WT, AKO, and DKO control mice on day 0
AKO mice showed elevated peripheral eosinophilia (1.85-fold) and EPO activity (1.29-fold) than WT mice (Table II), consistent with increased eosinophil infiltrations seen in the lung sections (Fig. 1). AKO mice showed an increase in IL-13 (13.1-fold), IL-5 (3.93-fold), and IL-2 (3.43-fold) and a 1.92-fold decrease in IFN- than WT mice (Fig. 2; Table III). The ratio of IFN- to IL-4 was 1.525-fold less in AKO than WT mice, suggesting that AKO mice have a Th2 bias, as opposed to the predominantly Th1 profile of the WT C57BL/6 mice (Table II).
SP-D treatment to WT-Ag mice led to increase in all the cytokines, with significant increase in IL-5 (3.66-fold), IL-2 (6.75-fold) except IL-4 (1.5-fold decrease) on day 4, followed by a decrease in IFN- (3.29-fold) and a further increase in IL-5 (6-fold) (Fig. 7). The IFN- to IL-4 ratio increased on day 4 and decreased on day 10 (from 6.522 on day 0 to 13.33, i.e., 2.04-fold increase and 1.63 i.e., 4-fold decrease) on days 4 and 10, respectively (Table VI). Administration of rhSP-D to WT-Ag mice led to an increase in IL-2 (4.5-fold), and a decrease in IL-4 (1.71-fold decrease), IL-12 (1.88-fold) and IFN- (2.15-fold) on day 4 followed by a decrease in IL-13 (1.87-fold), IL-2 (2.0-fold), IL-4 (8.4-fold), and IFN- (9.33-fold) (Fig. 8). The IFN- to IL-4 ratio decreased from 6.522 on day 0 to 3.9 (1.67-fold) and 4.5 (1.45-fold) on days 4 and 10, respectively (Table VI).
Administration of BSA to sensitized AKO-Ag and DKO-Ag mice led to decrease in peripheral eosinophilic count (2.08- and 3.12-fold, respectively) and EPO activity (1.69- and 1.14-fold, respectively) (Table VI). Lung histopathology showed decreased eosinophilic infiltrations in BSA-treated AKO-Ag and DKO-Ag mice. No significant increase in levels of anti-BSA IgG or IgE Ab was observed in these mice. BSA treatment to AKO-Ag mice showed a decrease in all the cytokines on day 4: IL-13 (8.28-fold), IL-5 (3.46-fold), IL-4 (2.15-fold), IL-2 (2.38-fold) IL-10 (2.63-fold) and IL-12 (3.1-fold). The levels of IL-13 (11.6-fold), IL-5 (3.46-fold), IL-4 (4.75-fold), IL-2 (31-fold), IL-10 (2.16-fold), IL-12 (2.04-fold), IFN- (2.4-fold) and TNF- (5.47-fold) further decreased on day 10. IFN- to IL-4 ratio did not change significantly (Table VI). BSA treatment to DKO-Ag mice showed a significant increase in IL-4 (6.75-fold) and IFN- (3-fold) on day 4 and IL-13 (2.64-fold), IL-4 (6.75-fold), and IFN- (3.75-fold) on day 10. IFN- to IL-4 ratio decreased from 2.298 on day 0 to 1.4 (1.64-fold) and 1.85 (1.24-fold) on day 4 and 10, respectively (Table VI).
Administration of SP-D or rhSP-D has therapeutic effects on Afu-sensitized DKO mice
Administration of SP-D or rhSP-D to DKO-Ag mice led to decrease in peripheral eosinophilic count (1.61- and 2.5-fold, respectively), EPO activity (1.6- and 2.04-fold, respectively), while a decrease in Afu IgE (0.94-, 0.7-fold) (Table VI). Lung sections of SP-D or rhSP-D-treated DKO-Ag mice showed reduced eosinophilic infiltrations on day 10 in comparison to untreated DKO-Ag mice on day 0 and rhSP-D administration was more effective in reducing eosinophilia than SP-D (Fig. 4).
DKO-Ag-SP-D mice showed a decrease in IL-13 (3.4-fold) and IL-5 (3.37-fold) while an increase in IL-4 (8-fold), and IL-2 (2.44-fold) (Fig. 7). The IFN- to IL-4 ratio decreased on day 4 and increased on day 10 (from 2.298 on day 0 to 0.625 (3.67-fold) and 3.75 (2.23-fold) on days 4 and 10, respectively (Table VI). rhSP-D administration to DKO-Ag led to an increase in all the cytokines, with significant increases in IL-4 (10.8-fold), IL-10 (2.13-fold), IL-2 (2.4-fold), TNF- (2.53-fold), IL-12 (2.58-fold), and IFN- (20.5-fold) on day 4. On day 10, however, cytokine levels decreased: IL-13 (2.7-fold), IL-5 (2.31-fold), IL-10 (2.17-fold), and IL-2 (2.22-fold) except IFN-, which increased by 9-fold (Fig. 8). The IFN- to IL-4 ratio decreased followed by an increase from 2.298 on day 0 to 1.8 (1.27-fold) and 3.0 (2.23-fold) on days 4 and 10, respectively (Table VI).
Administration of SP-A to AKO-Ag mice led to decrease in peripheral eosinophilic count (2.27-fold) on day 4 followed by further decrease on day 10 (4.16-fold) (Table VI). SP-A treatment led to an increase in levels of IL-13 (2.9-fold) on day 4 and a decrease in levels of IL-4 (2.14-fold), IL-2 (5.16-fold), IFN- (2.4-fold), and TNF- (3.45-fold) on day 10 (Fig. 6). The IFN- to IL-4 ratio did not change significantly (Table VI). Lung sections showed significantly increased eosinophilic infiltrations on days 4 and 10 in comparison to AKO-Ag mice on day 0 and showed collapse of the alveolar structure (Fig. 4). It is important to note here that although the peripheral eosinophil count decreased with SP-A administration to Afu-sensitized AKO mice, the pulmonary eosinophilia worsened.
Discussion
In view of the important roles of SP-A and SP-D in pulmonary immune response, we had earlier examined the effect of SP-A, SP-D, and rhSP-D in a murine model of Afu-induced pulmonary hypersensitivity (8). Afu is the fungus most commonly implicated in causing both IgE-mediated and non-IgE-mediated hypersensitivity in humans leading to development of ABPA, which is characterized by activated Th2 cells and asthma. Intranasal administration of SP-A, SP-D, or rhSP-D (three doses on consecutive days) significantly lowered eosinophilia and specific Ab levels in ABPA mice (8). Lung sections of the ABPA mice showed extensive infiltration of lymphocytes and eosinophils, which were considerably reduced following treatment (8). The levels of IL-2, IL-4, and IL-5 were decreased, while that of IFN- was raised in supernatants of the cultured spleen cells, indicating a marked Th2Th1 shift (8). This study highlighted a central role for SP-A and SP-D in regulation of pulmonary hypersensitivity (8). As a logical next step, we wished to examine the nature of immune response in AKO and DKO mice when challenged with Afu allergens to validate whether deficiency of these proteins made mice more susceptible to pulmonary hypersensitivity.
AKO and DKO show intrinsic hypereosinophilia
Both AKO and DKO mice showed elevated peripheral and pulmonary eosinophilia and a significant increase in EPO activity in comparison to the WT mice. A significant monocytic infiltration has been reported in the peribronchiolar and perivascular regions of the lungs in DKO mice (24). In addition, an increased accumulation of alveolar macrophages and lymphocytes was observed in DKO mice (32, 36). Because treatment with SP-A, SP-D or rhSP-D has been shown to lower IL-5, peripheral and pulmonary eosinophilia in the Afu-sensitized WT BALB/c mice (8), an alteration in the peripheral and pulmonary eosinophil counts in the AKO and DKO mice, was not surprising. A significantly raised level of IL-5 and IL-13 in both AKO and DKO mice may be one of the mechanisms causing hypereosinophilia (37, 38). SP-A can inhibit IL-8 expression and production from eosinophils, thus probably preventing the autocrine cycle for recruitment of human eosinophils by inhibiting IL-8, a chemotactic cytokine (39). Eosinophils are the important effector cells for the pathogenesis of allergic inflammation via the secretion of highly cytotoxic granular proteins and Th2 type of cytokines. Blood and tissue eosinophilia is a common manifestation of late-phase allergic inflammation causing tissue damage. Hypereosinophilia exhibited by both AKO and DKO mice suggests that SP-A and SP-D have a role in regulating the eosinophil infiltration and modulation in the lung in response to environmental stimuli.
Genetic deficiency of SP-A or SP-D shifts the cytokine profile of C57BL/6 mice toward Th2 type
The cytokine profile of both AKO and DKO mice suggested a Th2 bias (elevated IL-13 and IL-5 levels) and down-regulation of Th1 cytokine, IFN- (more pronounced in DKO than AKO mice). IL-13 and IL-5 have important roles in allergen induced asthma and airway hyperresponsiveness (AHR). Overexpression of IL-13 in mice leads to 70-fold increase in SP-D, 3-fold increase in SP-A, and 6-fold increase in the phospholipid pool (38). Remarkably similar to DKO mice, IL-13 overexpressing mice have characteristic foamy macrophages, type II cell hypertrophy, fibrosis, massive inflammation involving eosinophilia, protease-dependent acquired emphysema, and AHR (38). IL-13, produced in the airway by a variety of cells (T cells, eosinophils, and mast cells), mediates mucus production and AHR through its combined actions on epithelial cells and smooth muscle cells independently of IL-5 and eotaxin (40, 41). IL-13 also directly promotes eosinophil survival, activation, and recruitment (42, 43, 44). Alveolar macrophages of DKO mice show increased expression of reactive oxygen species (ROS), hydrogen peroxide, MMP-9, MMP-12, and NF-B (45). Because IL-13 has been reported to inhibit the production of proinflammatory mediators by monocytes and macrophages, including ROS, through a mechanism that probably involves NF-B, it appears that the increased levels of IL-13 are produced in DKO mice to regulate their increased oxidative state (46, 47, 48, 49, 50). However, IL-13 and SP-D have also been described as potent stimulators of MMP in the lung (37, 51). It is likely that certain physiological effects and hypereosinophilia observed in AKO and DKO mice arise due to overexpression of IL-13, although AKO mice do not show abnormalities, such as foamy macrophages, type II cell hypertrophy, and fibrosis similar to DKO mice. However, sequential targeting of both SP-A and SP-D genes (double knockout) show exaggerated alveolar proteinosis and emphysema compared with DKO mice, suggesting that SP-A deficiency may contribute to physiological abnormalities in the lungs (52).
Transgenic mice overexpressing IL-5 also exhibit intrinsic AHR (even in the absence of any antigenic stimuli) and increased numbers of eosinophils and lymphocytes in the lung tissue (53). The observation that AKO and DKO mice have elevated IL-5 levels, which is lowered by therapeutic delivery of SP-A or SP-D/rhSP-D, appears to suggest that SP-A and SP-D inhibit allergen mediated eosinophilia in the lungs through down-regulation of IL-5. It is worth noting that mice genetically deficient in GM-CSF also show pulmonary alveolar proteinosis associated with a marked increase in phospholipid pool similar to DKO mice. GM-CSF-deficient mice showed 50-fold increase in SP-D, while only a 3-fold increase in SP-A, similar to IL-13 overexpressing mice. It has been proposed that GM-CSF mediates some of the physiological changes seen in DKO mice as ablation of GM-CSF in DKO mice leads to alleviation of macrophage proliferation and type II cell hypertrophy (54). It is also possible that the actions of GM-CSF and SP-D leading to similar pathophysiological changes are distinct (55). It is to be noted that IL-5, IL-13, and GM-CSF genes are situated on the same chromosomal location (5q31). Furthermore, GM-CSF, along with IL-5, is known to regulate IL-13 secretion from human eosinophils (56).
Distinct immune response to BSA by WT, AKO, and DKO
Intranasal administration of BSA on days 1–3 was included in the study as a control protein similar to our earlier studies. However, C57BL/6 mice responded to the BSA administered as a short-term allergen challenge with a characteristic Th2 response with increased peripheral eosinophil count and pulmonary eosinophilia, which has also been reported earlier (57). WT mice, however, showed no significant increase in anti-BSA IgG or IgE Abs. Interestingly, BSA-specific IgG and IgE Abs were observed in both BSA-treated AKO and DKO mice with AKO mice also showing a significant down-regulation of IFN- to IL-4 ratio (shift to Th2 response) with an increase in peripheral eosinophil count and pulmonary eosinophilia while DKO mice showed only an increase in pulmonary eosinophilia. These observations suggest that both AKO and DKO mice show a different pulmonary immune response to short-term sensitization than the WT mice and both SP-A and SP-D have important roles in regulation of humoral immune response to short-term allergen sensitization in the lung.
Afu sensitization provokes distinct immunological responses in AKO and DKO mice
Following Afu sensitization, C57BL/6 mice, which were used as a control for AKO and DKO mice (both in C57BL/6 background) showed no change in Afu-IgE, and an increase in Afu-IgG, peripheral and pulmonary eosinophilia. Allergen challenge led to a decrease in IFN- and IL-4 in lung suspensions (hence an increase in IFN- to IL-4 ratio). IL-2, IL-5, and IL-13 levels decreased significantly in the lung and spleen suspensions, suggesting the Th1 predominance in the mouse strain. This is consistent with the observation that response to allergenic challenge varies in different strains of mice and C57BL/6 mice show a predominantly Th1 response to a high dose of allergen sensitization (57).
Both AKO and DKO mice showed comparable increase in Afu-IgG and peripheral eosinophil count, and DKO mice showed more severe pulmonary eosinophilia than AKO mice, following Afu sensitization. AKO mice showed a corresponding increase in Afu-IgE level as well. The EPO activity was down in DKO mice, while in AKO mice, it remained unchanged. AKO mice showed an increase in all the cytokines in lung suspension (<2-fold) except IL-13 and IL-5. Increased levels of Th2 cytokines in Afu-sensitized AKO mice than WT and DKO mice suggests that the phenotype of these mice is more complex than previously reported. However, AKO-Ag mice consistently showed Th1 predominance in BAL as well as in lung and spleen suspensions. DKO mice showed a decrease in all cytokine levels in lung suspension, similar to WT mice, but in a more pronounced manner. Both lung and spleen suspensions of DKO-Ag mice showed a Th1 response; however, BAL had an increase in IL-13, IL-4, and IL-2 levels, similar to WT-Ag mice. A recent study showed similar results wherein, following OVA sensitization and challenge in vivo, SP-D–/– mice expressed higher BAL eosinophils, IL-10 and IL-13 concentrations and lower IFN- expression at early time points compared with WT mice (58). It is evident that AKO mice are almost nonresponsive to the Afu sensitization, while DKO mice show a pronounced response. Afu sensitization led to a similar increase in the ratio of IFN- and IL-4 in both WT and DKO mice (no significant increase in AKO mice). Significant down-regulation of Afu-IgE Ab was specific to DKO mice. AKO mice, in contrast, showed a significant increase in Afu-IgE Ab. This differential responsiveness to Afu sensitization in AKO and DKO mice may be accounted for by a 50% decrease in SP-A levels in DKO mice and a 7-fold increase in SP-D levels in AKO mice (59). It appears that both SP-A and SP-D contribute to the homeostasis to the allergenic challenge in an interdependent manner and absence of any one of them disturbs this balance.
Administration of SP-A, SP-D, or rhSP-D to the Afu-sensitized WT and KO mice can partially rescue them
As previously reported (8), Afu-IgE, and Afu-IgG, peripheral and pulmonary eosinophilia in WT-Ag were down-regulated by SP-A, SP-D, or rhSP-D treatment (the increased IFN- to IL-4 ratio was also reversed). SP-D was able to restore IL-5 and IL-2 levels increased by allergen challenge. Afu sensitization led to a decrease in IL-13, while an increase in IL-2, IL-4, IL-10, IL-12, IFN-, and TNF- in AKO mice. SP-A treatment containing the peripheral eosinophil count, however, showed increased pulmonary eosinophilia with extensive tissue damage, possibly caused by increased levels of IL-13 and IL-5 in AKO mice. SP-A was also not able to bring down increased Afu-IgG and Afu-IgE levels, suggesting that SP-A treatment (3 μg per mice for 3 consecutive days) is not leading to complete alleviation of the Afu-induced changes in AKO mice.
SP-D treatment to DKO-Ag mice restored IL-2, IL-4, IL-12, and TNF- levels. IL-13 and IL-5 levels showed a further decrease with treatment, the levels being significantly lower than the DKO-C mice. IL-13, IL-5, and IL-2 levels in rhSP-D-treated DKO-Ag mice were comparable to WT-C mice, while IL-10 and IL-12 went down significantly compared with all other groups. Thus, SP-D and rhSP-D were more effective than SP-A in rescuing the respective gene-deficient mice from the effects of Afu sensitization. Previously, coadministration of SP-D has been shown to normalize viral clearance and cytokine response in DKO mice challenged with influenza A virus (IAV) (60). Expression of SP-D/conglutinin chimeric protein in epithelial cells of DKO mice substantially corrected the increased lung phospholipids and increased the clearance of IAV but could not ameliorate the ongoing lung inflammation, enhanced metalloproteinase expression, and alveolar destruction (28).
It appears likely that SP-A and SP-D influence the lung immunity by directly or indirectly modulating the nuclear factors. DKO mice have been shown to have elevated levels of transcripts for NF-B and AP-1 (45). NFAT1 (NF regulating expression of many genes encoding immunoregulatory cytokines)-deficient mice also show increased levels of IL-4, IL-5, and IL-13 as well as enhanced eosinophilia, similar to AKO and DKO mice. It is possible that NFAT1 is involved in the downstream signaling of SP-A and/or SP-D (61). In conclusion, the present study reports that both SP-A and SP-D have important roles in the regulation of cytokine milieu and eosinophilia in the lungs, and their absence leading to inherent hypersensitivity in mice highlights their essential role in host defense against allergic airway challenges. Thus, both these versatile macromolecules enable the lung to achieve homeostasis probably through distinct mechanisms. It is important to note here that both AKO and DKO mice are different and the anatomical and functional abnormalities reported only in DKO mice, and not in AKO mice, may be underlying issues for their behavior and susceptibility to Afu sensitization.
Acknowledgments
The SP-A and SP-D gene knockout mice were kindly provided by Dr. S. Hawgood (Cardiovascular Research Institute and Department of Pediatrics, University of California San Francisco, CA). We are grateful to Dr. Howard Clark for his technical help with breeding knockout mice.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by the Council for Scientific and Industrial Research (to T.M., M.S., and P.U.S.), the Medical Research Council (to K.B.M.R.) and the European Commission (to K.B.M.R. and U.K.).
2 Address correspondence and reprint requests to Dr. P. Usha Sarma, Institute of Genomics and Integrative Biology, Delhi University Campus, Mall Road, Delhi-110007, India. E-mail address: u_sarma{at}hotmail.com
3 Abbreviations used in this paper: SP-A, human surfactant protein A; SP-D, human surfactant protein D; BAL, bronchoalveolar lavage; PAP, pulmonary alveolar proteinosis; Afu, Aspergillus fumigatus; AKO, SP-A gene deficient; DKO, SP-D gene deficient; MMP, matrix metalloproteinase; rhSP-D, a recombinant fragment of human surfactant protein D, composed of homotrimeric neck and C-type lectin domains; WT, wild type; 3wcf, three week culture filtrate; ABPA, allergic bronchopulmonary aspergillosis; EPO, eosinophil peroxidase; ROS, reactive oxygen species; IAV, influenza A virus.
Received for publication July 23, 2004. Accepted for publication March 18, 2005.
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