Surfactant Protein D Regulates Airway Function and Allergic Inflammation through Modulation of Macrophage Function

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1 Surfactant Protein D Regulates Airway Function and Allergic Inflammation through Modulation of Macrophage Function Katsuyuki Takeda, Nobuaki Miyahara, Yeong-Ho Rha, Christian Taube, Eun-Seok Yang, Anthony Joetham, Taku Kodama, Annette M. Balhorn, Azzeddine Dakhama, Catherine Duez, Amanda J. Evans, Dennis R. Voelker, and Erwin W. Gelfand Program in Cell Biology, Department of Pediatrics; and Department of Medicine, National Jewish Medical and Research Center, Denver, Colorado The lung collectin surfactant protein D (SP-D) is an important component of the innate immune response but is also believed to play a role in other regulatory aspects of immune and inflammatory responses within the lung. The role of SP-D in the development of allergeninduced airway inflammation and hyperresponsiveness (AHR) is not well defined. SP-D levels progressively increased up to 48 hours after allergen challenge of sensitized mice and then subsequently decreased. The levels of SP-D paralleled the development of airway eosinophilia and AHR. To determine if this association was functionally relevant, mice were administered rat SP-D (rsp-d) intratracheally. When given to sensitized mice before challenge, AHR and eosinophilia were reduced by rsp-d in a dose-dependent manner but not by mutant rsp-d. rsp-d administration resulted in increased levels of interleukin (IL)-10, IL-12, and IFN- in bronchoalveolar lavage fluid and reduced goblet cell hyperplasia. Culture of alveolar macrophages together with SP-D and allergen resulted in increased production of IL-10, IL-12, and IFN-. These results indicate that SP-D can (negatively) regulate the development of AHR and airway inflammation after airway challenge of sensitized mice, at least in part, by modulating the function of alveolar macrophages. Keywords: surfactant protein D; airway hyperresponsiveness; eosinophils; macrophages The incidence of allergic asthma worldwide has generated increasing alarm in terms of the economic burden imposed and the rising morbidity and mortality despite significant advances in therapeutics (1). The currently accepted theory is that the disease is the result of chronic airway inflammation, largely dependent on eosinophils that lead to airway hyperresponsiveness (AHR) and reversible airway obstruction (2, 3). With the rising incidence and therapeutic insufficiencies, it is now more apparent that asthma is first and foremost a very heterogeneous syndrome, with numerous cell types and mediators contributing to the disease phenotype. Central to the pathogenesis of the airway disease are antigen-specific, memory T cell responses and perhaps to a lesser degree, antigen-specific IgE responses (4, 5). Although antigen-specific (adaptive immune) responses are a major focus in the disease, the role of innate immunity in the regulation of airway function and inflammation may also be important (6). As an example, / T cells have been shown to exhibit both contributory and inhibitory activities in the development and expression of allergic airway disease. In a similar manner, the complement system has also attracted recent attention (Received in original form April 18, 2003; accepted in final form July 16, 2003) Correspondence and requests for reprints should be addressed to Erwin W. Gelfand, M.D., 1400 Jackson Street, Denver, CO gelfande@njc.org Am J Respir Crit Care Med Vol 168. pp , 2003 Originally Published in Press as DOI: /rccm OC on July 25, 2003 Internet address: (7). The hydrophilic surfactant proteins (SPs) SP-A and SP-D are also important components of the innate immune response (8, 9). Increasing evidence indicates that SP-A and SP-D regulate immune cell function as well as surfactant homeostasis (10). These SPs may affect allergen uptake by antigen-presenting cells or inhibit allergen-specific IgE binding to allergen, thereby inhibiting the triggering of allergic responses (11). Alveolar macrophages, which are major resident cells in the airways may be playing a role in the SP-D regulatory effects in allergic inflammation (12 14). SP-A and SP-D have been shown to play protective roles in an allergic bronchopulmonary aspergillosis model in mice (15). It has been reported that mite allergen induced airway inflammation leads to decreased levels of SP-A and SP-D in the bronchoalveolar lavage fluid (BALF) from sensitized mice (16). In contrast, elevated levels of SP-A and SP-D have also been found in BALF from sensitized and challenged mice (17) and in the BALF of patients with asthma (18). The purpose of the present study was to determine if SP-D levels are linked to allergic inflammation and AHR, whether administration of SP-D could attenuate airway inflammation, and normalize airway responsiveness, and to define potential mechanisms for such effects. METHODS Animals Female BALB/c mice from 6 to 8 weeks of age were obtained from Jackson Laboratories (Bar Harbor, ME). The animals were maintained on an ovalbumin (OVA)-free diet. Experiments were conducted under a protocol approved by the Institutional Animal Care and Use Committee of the National Jewish Medical and Research Center. Sensitization and Airway Challenge Mice were sensitized by intraperitoneal injection of 20 g of ovalbumin (OVA, Grade V; Sigma Chemical Co., St. Louis, MO) emulsified in 2.25 mg alum (AlumImuject; Pierce, Rockford, IL) in a total volume of 100 l on Days 1 and 14. Mice were challenged via the airways with OVA (1% in saline) for 20 minutes on Days 28, 29, and 30 using an ultrasonic nebulizer (model NE-07; Omron Healthcare, Vernon Hills, IL). The control groups of mice were challenged alone, that is, received three challenges but no sensitization. In all groups, assays were performed 48 hours after the last challenge. Determination of Airway Responsiveness Airway responsiveness was assessed as a change in airway function after challenge with aerosolized methacholine (Sigma). Mice were anesthetized, tracheostomized, mechanically ventilated, and lung function was assessed as described previously (19). Briefly, the mice were anesthetized with an intraperitoneal injection of pentobarbital sodium (100 mg/kg). When an appropriate level of anesthesia was achieved, a stainless steel 18G tube was inserted as a tracheostomy cannula and tied into place. The tracheostomy tube was passed through a hole in the whole body plethysmograph. A four-way connector was attached to

2 784 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL Figure 1. (A) Changes in airway resistance (RL) after sensitization and challenge at various time points. RL values were obtained in response to increasing concentrations of inhaled methacholine (MCh). Values are expressed as mean ( SEM) (n 8). Open squares, challenge alone; open triangles, 0 hours after the last antigen challenge; closed squares, 24 hours after; closed diamonds, 48 hours after; closed triangles, 1 week after; open diamonds, 2 weeks after. *Significant differences (p 0.05) occurred between the groups of challenge alone, 0 hours, and 2 weeks versus 24 hours, 48 hours, and 1 week after challenge. (B) Numbers of eosinophils in bronchoalveolar lavage fluid (BALF) at different time points after the last antigen challenge. *After antigen challenge, mice developed significantly higher number of eosinophils (p 0.05) when compared with the challenged-alone group. (C) surfactant protein D (SP-D) levels in BALF at different time points after antigen challenge. Significant differences (*p 0.05) occurred between 24 hours, 48 hours, or 1 week after and challenge alone. n 8 in each group. the tracheostomy tube, with two ports connected to the inspiratory and expiratory sides of a ventilator (SN-480-7; Shinano, Tokyo, Japan). Ventilation was achieved at 160 breaths per minute and a Vt of 0.16 ml with a positive end-expiratory pressure of 2 to 4 cm H 2 O. Changes in lung volume were measured by detecting pressure changes in the plethysmographic chamber through a port in the connecting tube with a differential pressure transducer and then referenced to a second copper gauze filled 1.0-L glass bottle. Flow was measured by digital differentiation of the volume signal. Lung resistance was continuously computed (Labview; National Instruments, Austin, TX) by fitting flow, volume, and pressure to an equation of motion, using a recessive least squares algorithm. The aerosolized methacholine was administered through bypass tubing via an ultrasonic nebulizer (model 5500D; DeVilbiss, Somerset, PA) placed between the expiratory port of the ventilator and the fourway connector. Aerosolized methacholine was administered for 8 seconds with a Vt of 0.45 ml and frequency of 60 breaths per minute by another ventilator (model 683; Harvard Apparatus, South Natwick, MA). The data of lung resistance was continuously collected for up to 3 minutes. Maximum values of lung resistance were taken to express changes in airway function. BAL Immediately after assessment of AHR, lungs were lavaged via the tracheal tube with Hank s balanced solution (1 1mlat37 C). The volume of collected BALF was measured in each sample and the number of leukocytes was counted (Coulter Counter; Coulter Corporation, Hialeah, FL). Differential cell counts were performed by counting at least 300 cells on cytocentrifuged preparations (Cytospin 3; Shandon Ltd., Runcorn, Cheshire, UK). Slides were stained with modified Wright-Giemsa (Hema 3; Protocol, Swedesboro, NJ) and differentiated by standard hematologic procedures. Administration of Rat SP-D and Mutant Rat SP-D To determine the direct effects of SP-D on the airway, we administered rat SP-D (rsp-d), which shares a high degree of homology with mouse SP-D (identities 91% and positives 94%). A mutated form of rsp-d with altered carbohydrate binding was used as a negative control [E321Q, N323D]. rsp-d and the [E321Q-N323D] mutant were isolated from culture medium of CHO-K1 cells expressing the recombinant protein by the method described previously (20). Briefly, the harvested medium containing rsp-d was centrifuged at 3,000 g at 4 C for 15 minutes to obtain cell debris free solution, and the resultant supernatant was applied to a mannose-sepharose affinity matrix in the presence of 5 mm calcium. For purification of mutant SP-D, multilamellar liposomes containing phosphatidylinositol, phosphatidylserine, and cholesterol (3:4:3) were mixed with the protein and subsequently separated by centrifugation. The proteins were subsequently eluted from affinity columns or liposomes with either 2 or 15 mm ethylenediaminetetraacetic acid, respectively. The ethylenediaminetetraacetic acid was subsequently removed by gel filtration. The purified SP-D contained both dodecamers and larger molecular aggregates of the proteins. The proteins were dialyzed against Tris-buffered saline (5 mm tris(hydroxymethyl)aminomethane hydrochloride, 150 mm sodium chloride, ph 7.4) at 4 C and stored at 20 C until use. Endotoxin levels in the purified SP preparations were determined using a kit (QCL-1000; Biowhittaker, Inc., Wakersville, MD) and the levels were under 0.25 pg for each sample. At these levels, there are no effects on airway inflammation (neutrophils) or AHR (data not shown). Before administration of wild-type or mutant rsp-d, the mice were anesthetized with avertin (400 mg/kg) and 2 g of the recombinant proteins in 50 l or the same volume of vehicle per mouse was administered as an aerosol using the intratracheal aerosolizer (Microsprayer; PennCentury, Philadelphia, PA). Agents were given 1 day before and 5 hours before the first allergen challenge. To examine the dose-dependent effects of rsp-d, various doses of the protein (0.02, 0.2, 2, and 10 g in50 l) were instilled into the trachea. Measurements of SP-D Levels in BALF Mouse SP-D levels in lavage were quantified by ELISA (21). Recombinant mouse SP-D used as a standard was purified from culture supernatants of CHO-K1 cells expressing the protein using methods similar to rsp-d as described previously. Horseradish peroxidase conjugated rabbit anti-mouse SP-D IgG was used for antigen detection. 2,2 -azi-

3 Takeda, Miyahara, Rha, et al.: Surfactant Protein D and Airway Hyperresponsiveness 785 Figure 2. A) Airway responsiveness to methacholine (MCh) and (B) eosinophil numbers in bronchoalveolar lavage fluid (BALF) after administration of different doses of rat surfactant protein D (rsp-d). Airway responsiveness is expressed as the MCh concentration required to induce 200% change over saline-treated mice (PC200). Significant differences (p 0.05) * compared with vehicle treatment; # compared with SP-D 0.02 g treatment (n 8). nobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS, Sigma) was used as the substrate for the peroxidase reaction. Antibody binding to the proteins was measured by absorbance at 410 nm, and protein levels in BALF were expressed as nanograms per ml (ng/ml). Effects of SP-D on Cytokine Production from Alveolar Macrophages To elucidate the direct effects of SP-D on cytokine production from alveolar macrophages, the cells were purified from BAL and cultured with SP-D and/or OVA. Briefly, mice were anesthetized with avertin as described previously, lungs of sensitized but nonchallenged mice were lavaged via the tracheal tube with Hank s balanced solution (5 1ml37 C). Cells were washed with Hank s balanced solution and cells were cultured in 200 l of RPMI 1640 medium with l-glutamine (Mediatech, Herndon, VA) supplemented with 10% fetal bovine serum and 10 g/ml polymyxin B sulfate (Sigma). The cells were confirmed to be more than 99% macrophages on stained cytocentrifuged slides. To obtain sufficient macrophages for these experiments, BALF was pooled from 2 to 3 mice. Cells were incubated with OVA, and various concentrations of rsp-d and the supernatants were harvested after 24 hours. The optimal OVA concentration was determined to be 10 g/ ml in preliminary experiments. Measurement of Cytokines Cytokine levels in the BALF or supernates of cultured airway macrophages were measured by ELISA as described previously (22). Briefly, measurements of interleukin (IL)-10, IL-12, IFN-, IL-4, and IL-5 were performed using OptEIA sets (BD PharMingen, San Diego, CA) with 96-well plates (Immulon 2; Dynatech, Chantilly, VA) according to the manufacturer s protocol. The limits of detection were 4 pg/ml for IL-4 and IL-5, and 10 pg/ml for IL-12, IL-10, and IFN-. For measuring IFN- levels from cultured macrophages, OptEIA set for small amounts (AN-18; BD PharMingen) was also used and the limit of detection was 1.5 pg/ml. Histologic and Immunohistochemistry Studies Lungs were inflated through the trachea with 1 ml of 10% formalin and fixed in 10% formalin by immersion. Blocks of lung tissue were cut around the main bronchus and embedded in paraffin. For detection of mucus-containing cells in formalin-fixed airway tissue, sections (6 m) were cut and stained with periodic acid Schiff and counterstained with hematoxylin. Goblet cell numbers were counted in sections by measuring the length of epithelium and the luminal area using an National Institutes of Health image analysis system (23, 24). Statistical Analysis All results were expressed as the mean and SEM. Analysis of variance was used to determine the levels of difference between all groups. Pairs of groups were compared by unpaired two-tailed Student s t test. The p values for significance were set at Figure 3. (A) Airway response to inhaled methacholine (MCh) after rat surfactant protein D (rsp-d), mutant SP-D, or vehicle treatment before ovalbumin (OVA) challenge after sensitization. Values are expressed as mean ( SEM) (n 8). Open squares, vehicle treatment; open triangles, mutant SP-D treatment; closed squares, rsp-d treatment. (B) Numbers of eosinophils in bronchoalveolar lavage fluid (BALF) from same groups of mice. Significant differences (p 0.05) between *, rsp-d versus vehicle treatment, # rsp-d versus mutant SP-D.

4 786 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL Figure 4. Cytokine levels in bronchoalveolar lavage fluid (BALF) after rat surfactant protein D (rsp-d) or vehicle treatment. Animals were treated with 2 g SP-D, 1 day and 5 hours before antigen challenge. Lung lavage was performed 2 days after antigen challenge. *Significant differences (p 0.05) occurred between rsp-d and vehicle. n 8 in each group. RESULTS Kinetics of AHR, Eosinophil, and SP-D Responses Sensitization followed by challenge to allergen-induced significant AHR to inhaled methacholine and resulted in increased numbers of eosinophils in the BALF. Naive mice, that were sensitized but not challenged, and mice challenged alone, showed lower levels of SP-D when compared with sensitized and challenged mice (data not shown). To assess any association between SP-D, AHR, and eosinophilic infiltration into the airways, these parameters were examined at different time points after antigen challenge of sensitized and challenged mice. AHR was detected 24 hours after the third allergen challenge and was sustained for 1 week. By 2 weeks after challenge, airway function returned to near baseline levels (Figure 1A). Investigation of the number of eosinophils in the BALF revealed a similar time course: eosinophils began to increase earlier than airway responsiveness; after 2 weeks, eosinophil numbers were still significantly elevated when compared with nonsensitized animals (Figure 1B). The kinetics of the elevations in SP-D in the BALF revealed a similar pattern to airway responsiveness or eosinophil numbers in BALF (Figure 1C). Increased levels of SP-D were detected 24 hours after OVA challenge and returned to baseline levels 2 weeks after the last challenge. All three parameters, AHR, eosinophil numbers, and BAL SP-D levels peaked 48 hours after the last of the three OVA challenges. Effects of SP-D Administration Despite the association of elevated SP-D levels, AHR and eosinophilic infiltration in the lungs, the role played by SP-D in these Figure 5. Goblet cell hyperplasia was quantified in periodic acid-schiff (PAS) stained sections and expressed per millimeter of basement membrane. Values are expressed as mean ( SEM). *Significant differences (p 0.05) occurred between groups (n 8) receiving rat surfactant protein D (rsp-d) treatment or vehicle. responses remained to be defined. To directly address this issue, we administered exogenous rsp-d intratracheally and determined whether this treatment improved or exacerbated AHR and eosinophilic airway inflammation. rsp-d was administered at different doses as shown in Figure 2; AHR and eosinophil numbers in BAL were reduced in a dose-dependent manner. After preliminary experiments to determine the most effective dose, 2 g of rsp-d was administered through the trachea before antigen challenge. As a control, the [E321Q, N323D] mutant rsp-d was administered in the same manner as rsp-d. As shown in Figure 3, only rsp-d treatment significantly decreased AHR and eosinophil numbers in BALF, whereas the mutant protein was without effect, implying a requirement for wild-type carbohydrate recognition specificity for the response (it should be noted that the instillation of any substance, even saline, results in lower eosinophil numbers [Figures 2 and 3] than seen without any manipulation [Figure 1]). Levels of cytokines in BALF and goblet cell hyperplasia were also assessed after administration of rsp-d. Intratracheal instillation of rsp-d before challenge was associated with increased levels of IL-10, IL-12, and IFN-, as shown in Figure 4. The levels of IL-4 or IL-5 were not significantly altered in response to rsp-d treatment (data not shown). There was also a small decrease in goblet cell numbers after rsp-d treatment (Figure 5). Effects of SP-D on Cytokine Production by Alveolar Macrophages On the basis of these results, we investigated the effect of SP-D on in vitro cytokine production by alveolar macrophages obtained from mice after sensitization to OVA. As shown in Figure 6, culture of the macrophages with either allergen alone or rsp-d alone resulted in small increases in cytokine production. In contrast, in vitro production of IFN-, IL-12, and IL-10 was significantly upregulated when cells were cultured together with allergen and rsp-d, which is consistent with the increase in levels of cytokines after in vivo rsp-d treatment. IL-4 and IL-5 were not detected under these conditions (data not shown). DISCUSSION SP-D belongs to the collectin protein family that also includes SP-A. Both of these bronchoalveolar proteins are considered to be important regulatory molecules of the pulmonary innate immune system, involved in the first-line defense of the lung against microbial or allergen challenge (15, 25 27). In addition to

5 Takeda, Miyahara, Rha, et al.: Surfactant Protein D and Airway Hyperresponsiveness 787 Figure 6. Cytokine production from airway macrophages cultured together with allergen and/or surfactant protein D (SP-D). The cells were cultured in the presence of ovalbumin (OVA) or SP-D as indicated for 24 hours. The cell-free medium was analyzed for cytokines by ELISA. The levels of (A) IL-10, (B) IFN-, (C) IL-12 are expressed as mean ( SEM) (n 8). Significant differences (p 0.05) are indicated by * when treatments are compared with medium. Significant differences (p 0.05) are also indicated by # when treatments are compared with OVA or rsp-d alone. promoting clearance of infectious pathogens, SP-D can modulate cellular immune responses in response to allergen challenge. Here, we examined the interrelationship between SP-D and allergen-induced AHR and airway inflammation by first determining if there was an association between these processes after allergen challenge of sensitized mice. Allergen challenge of sensitized mice resulted in significant increases in the levels of SP-D in the BALF. The kinetics of the SP-D response (increases) appeared to closely follow those of airway eosinophilia and AHR to inhaled methacholine. Moreover, administration of wild-type rsp-d, but not a variant with altered carbohydrate recognition before allergen challenge, reversed the consequences of allergen exposure by reducing airway eosinophilia, inducing IL-10 and Th1 cytokines (IL-12, IFN- ), and normalizing airway function. The ability of allergen challenge in sensitized, but not in nonsensitized, mice to trigger an increase in the levels of SP-D, implicates this protein in the response to allergen. SP-D is a multivalent homooligomeric protein that binds carbohydrate, lipids, and protein ligands with high affinity (9). SP-D binds to oligosaccharides on the surface of a variety of pathogenic microorganisms as well as directly to cells such as alveolar macrophages (28). SP-D also enhances chemotaxis of phagocytic cells (29) and exhibits immunomodulatory function, inhibiting T cell proliferation and IL-2 production (30). SP-D is also capable of inhibiting specific IgE binding to allergen and preventing allergen-induced histamine release from human basophils (31). The mechanism underlying the increase in SP-D levels in BALF in this model is not known. The response appears specific as it was only seen in mice challenged with the original sensitizing allergen. The levels of BALF SP-D appeared to parallel the inflammatory response in the lung, especially BAL eosinophil numbers. Similar results have been demonstrated in human asthma and other animal models of allergic inflammation (17, 18, 32, 33). Serum levels of SP-D also have served as a biomarker of inflammation in interstitial lung disease (34). Although SP-D may serve as a marker of acute airway inflammation, such as in acute asthma exacerbation, increased levels of SP-D in the airway may also be a consequence of epithelial cell damage. SP-D could play a role in limiting the allergic inflammatory response, limiting proliferation of various inflammatory cells by binding to the allergen and interfering with its recognition by antigen-presenting cells (29, 30) or by modifying the production and secretion of various inflammatory cytokines (35 37). Thus, endogenous SP-D production after allergen challenge of sensitized mice may play a role in limiting the extent and persistence of the inflammatory changes, for example, by curtailing the eosinophilic inflammatory response. To directly assess the influence of SP-D on allergic inflammation, we administered rsp-d after sensitization but before allergen challenge. A microsprayer capable of delivering small particles was used to deliver the protein to ensure a diffuse distribution to the airways. When administered in this way, we observed a marked reduction in the eosinophilic inflammatory response; eosinophil numbers in the BAL were reduced to roughly 50% by rsp-d but not by a mutant form of the protein or vehicle. The effects of rsp-d were dose-dependent. These results suggest that SP-D is playing a role through its carbohydrate recognition domain, similar to that shown previously in an RSV infection model (38). When BAL cytokine levels were examined, increased levels of the three critical disease-modifying cytokines (IL-10, IFN-, IL-12) were demonstrated, suggesting that the effects of SP-D were not likely due to interference with allergen recognition or antigen-presenting cell function. After sensitization and challenge, the levels of these three cytokines typically decrease in BALF. IL-10 has been shown to play a complex role in allergic inflammation. Tournoy and coworkers

6 788 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL showed that endogenous IL-10 can suppress airway allergic inflammation, including numbers of BAL eosinophils as well as airway responsiveness (39), whereas van Scott and coworkers showed that IL-10 can increase AHR (40). Both IL-12 and IFN- have also exhibited similar negative influences on eosinophilic airway inflammation and AHR (41, 42). Of interest, levels of IL-4 and IL-5 were unaffected by administration of SP-D under these conditions. Overexpression of IL-4 in Clara cells was associated with marked increases in SP-D levels in the lung (26). In a model of allergic bronchopulmomary aspergillosis, administration of SP-D did decrease IL-4 and IL-5 while increasing IFN- levels, when spleen cells from the treated mice were cultured in vitro (15). Because of its widespread effects, there are a number of different ways that SP-D may have functioned to reduce airway inflammation and normalize airway function. Because rsp-d but not mutant SP-D is capable of binding mannose glucose (20), a mannosylated glycoprotein such as OVA may be captured and cleared from the airway more efficiently after administration of rsp-d. This could contribute to some of the effects of rsp-d, but would not explain the increases in IL-10, IFN-, or IL-12 levels. Alternatively, rsp-d coupled with OVA may be a more effective activator of macrophages, accounting for the increases in these cytokine levels both in vivo and in vitro. IL-10, IFN-, and IL-12 may be produced by non-t cells, epithelial cells, dendritic cells, and macrophages. SP-D has previously been shown to induce IL-10 and IL-12 production in macrophages (36). We investigated the effects of culturing airway macrophages together with rsp-d and/or allergen to determine whether SP-D could modify cytokine production from these cells in vitro. Together with allergen, rsp-d increased macrophage production of IFN-, IL-10, and IL-12 in a dose-dependent fashion. Because airway macrophages are the dominant cells in the airways of sensitized but not challenged mice, these data suggest that one of the mechanisms underlying the antiinflammatory effects of SP-D on airway allergic inflammation is by upregulating Th1 type cytokine production from airway macrophages and potentially other cell types. SP-D may do so by enhancing antigen presentation by antigen-presenting cells (43). Furthermore, SP-D is reported to decrease metalloproteinase activity and oxidant production in the lung (44), effects that may also result in normalization of airway function. SP-D may regulate tissue metalloproteinase activity by increasing tissue inhibitor of metalloproteinase activity production from alveolar macrophages via increasing levels of IL-10 (45). Finally, SP-D may play a significant role in the pulmonary clearance of apoptotic cells by alveolar macrophages, contributing to the resolution or inhibition of inflammation (27, 46). In summary, SP-D exhibits a number of potentially important activities in the lung during the inflammatory response elicited by allergen challenge of sensitized mice. These effects of SP-D may be expressed on cells of both the innate and adaptive immune systems and appear dependent on the integrity of the carbohydrate recognition domain. For these reasons, SP-D may be of benefit in the reduction or resolution of allergen-induced AHR and airway inflammation. Conflict of Interest Statement : K.T. has no declared conflict of interest; N.M. has no declared conflict of interest; Y-H.R. has no declared conflict of interest; C.T. has no declared conflict of interest; E-S.Y. has no declared conflict of interest; A.J. has no declared conflict of interest; T.K. has no declared conflict of interest; A.B. has no declared conflict of interest; A.D. has no declared conflict of interest; C.D. has no declared conflict of interest; A.E. has no declared conflict of interest; D.R.V. has no declared conflict of interest; E.W.G. has no declared conflict of interest. References 1. Sly RM. Decreases in asthma mortality in the United States. Ann Allergy Asthma Immunol 2000;85: Lacy P, Moqbel R. Immune effector functions of eosinophils in allergic airway inflammation. Curr Opin Allergy Clin Immunol 2001;1: Tattersfield AE, Knox AJ, Britton JR, Hall IP. Asthma. Lancet 2000;360: Oshiba A, Hamelmann E, Haczku A, Takeda K, Conrad DH, Kikutani H, Gelfand EW. 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