Up-Regulation of Surfactant Protein Production in a Mouse Model of Secondary Pulmonary Alveolar Proteinosis Masataka Shibasaki1, Katsunori Hashimoto2, Masakazu Okamoto1, Yuta Hayashi1, Kazuyoshi Imaizumi1, Naozumi Hashimoto1, Nobuaki Ozaki3, Toyoharu Yokoi2, Kenzo Takagi2, Yoshinori Hasegawa1,4, Kaoru Shimokata1, and Tsutomu Kawabe2,4 1 Department of Respiratory Medicine, and 3Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, Nagoya, Japan; 2Department of Medical Technology, Nagoya University School of Health Science, Nagoya, Japan; and 4MEXT Innovative Research Center for Preventive Medical Engineering, Nagoya University, Nagoya, Japan

Although Pneumocystis infection might be one of the causes of secondary pulmonary alveolar proteinosis (PAP), the mechanism of its pathogenesis is uncertain. We analyzed a mouse model of secondary PAP resulting from Pneumocystis infection using mice deficient in CD40 (CD40KO), and evaluated the mechanism of the pathogenesis of secondary PAP from the viewpoint of surfactantassociated protein (SP) homeostasis, the overproduction of SP by type II alveolar epithelial cells, and the phagocytic function of alveolar macrophages (AMs). The effect of CD40 on SP production was also investigated in vitro using the H441 cell line, which has a phenotype similar to type II alveolar epithelial cells and primary alveolar epithelial cells. After long-term exposure to ovalbumin, CD40KO mice showed Pneumocystis infection and accumulation of surfactants in the alveoli (ApCD40KO). The amounts of SP production were up-regulated in ApCD40KO mice compared with wildtype mice treated using the same procedure. On the other hand, AMs from ApCD40KO mice did not show either phagocytic dysfunction or down-regulation of PU.1 expression. Furthermore, the stimulation of CD40–CD40 ligand (CD154) pathway regulated the production of SPs in H441 cells or primary alveolar epithelial cells. These results suggested that CD40KO mice could be one of the models useful for developing secondary PAP resulting from Pneumocystis infection. Surfactant accumulation was due to the overproduction in our model of secondary PAP. The CD40–CD154 interaction plays an important role in the regulation of surfactant-associated protein production. Keywords: pulmonary alveolar proteinosis; alveolar epithelial cells; surfactant; Pneumocystis; CD40

Pulmonary alveolar proteinosis (PAP) is a diffuse lung disease characterized by the accumulation of amorphous, periodic acidSchiff (PAS)-positive lipoproteinaceous material in the distal airspaces (1). This material is composed principally of the phospholipid surfactant and surfactant apoproteins, which contain four specific proteins (surfactant-associated protein [SP]-A, SP-B, SP-C, and SP-D). There are three types of PAP: acquired (or idiopathic), congenital, and secondary (1). Acquired PAP is the most common form and is likely to be an autoimmune disease. Indeed, acquired PAP is reportedly an autoimmune disease with anti–granulocyte-macrophage colony-stimulating factor (GM-CSF) autoantibodies (2). Furthermore, studies in the mouse models of PAP have revealed the critical roles of (Received in original form March 16, 2008 and in final form September 24, 2008) This study was supported in part by a Grant-in-Aid for Scientific Research from the Japan Ministry of Education, Culture, Sports, Science and Technology.

CLINICAL RELEVANCE The CD40 knockout mice developed secondary pulmonary alveolar proteinosis (PAP) resulting from Pneumocystis infection. This model revealed that surfactant protein overproduction is one of the mechanisms of secondary PAP, and that CD40 on pneumocytes in part regulates the surfactant protein overproduction.

GM-CSF and PU.1 in surfactant homeostasis and the dysfunctions of alveolar macrophages (AMs) (1). Meanwhile, secondary PAP develops in association with conditions such as some hematologic cancers, pharmacologic immunosuppression, inhalation of inorganic dust (e.g., silica) or toxic fumes, and certain infections including Pneumocystis (3–9). Although the mechanism of the pathogenesis of secondary PAP remains unclear, Pneumocystis infection is one of the causes of secondary PAP in immunocompromised hosts (10, 11). Pneumocystis infection is one of the most common opportunistic infections in patients with diseases of T cell dysfunction such as acquired immune deficiency syndrome. These conditions include Xlinked immunodeficiency with hyper-IgM caused by mutations of CD40 ligand (CD154), which is normally expressed on activated CD41 T cells (12, 13). CD40 is one of the cell surface receptors expressed on antigen-presenting cells, including AMs, and also on epithelial cells including type II alveolar epithelial cells (14–16). Mice deficient in CD40 (CD40KO) have been reported to develop fatal spontaneous Pneumocystosis infection (17), and mice deficient in CD154 (CD154KO) have been used as Pneumocystis infection model of immunodeficient mice (13). These studies suggested that the CD40–CD154 interaction is important in resistance to Pneumocystis infection (13, 18). Although the interaction between the Pneumocystis organism and SP-A and -D has been reported (19–21), the mechanisms of the pathogenesis of secondary PAP resulting from Pneumocystis infection have yet to be elucidated. We observed the accumulation of surfactant and Pneumocystis organisms in the lungs of CD40KO mice after long-term exposure to ovalbumin (OVA). In this study, we sought to assess the mechanism that underlies the accumulation of alveolar surfactant in secondary PAP resulting from Pneumocystis infection through SP homeostasis (the production of SP by type II alveolar epithelial cells and catabolism of SP by AMs).

Correspondence and requests for reprints should be addressed to Tsutomu Kawabe, M.D., Ph.D., Department of Medical Technology, Nagoya University School of Health Science, 1-1-20, Daikou-minami, Higashi-ku, Nagoya 4618673, Japan. E-mail: [email protected]

MATERIALS AND METHODS

Am J Respir Cell Mol Biol Vol 40. pp 536–542, 2009 Originally Published in Press as DOI: 10.1165/rcmb.2008-0103OC on October 17, 2008 Internet address: www.atsjournals.org

CD40KO mice on a BALB/c background were maintained as previously described (22). Wild-type (WT) BALB/c mice were purchased

Animals and Treatments

Shibasaki, Hashimoto, Okamoto, et al.: Surfactant Protein Production in Secondary PAP

from SLC (Shizuoka, Japan). All procedures were performed in accordance with the Guidelines for Animal Experimentation of Nagoya University. Female WT and CD40KO mice (8–10 wk) were sensitized on Days 1 and 14 by an intraperitoneal injection of 50 mg of OVA (Sigma, St. Louis, MO) emulsified in Al(OH)3 (Pierce Biotechnology, Rockford, IL). Fourteen days after sensitization, these mice were subjected to 30 minutes of OVA (5 mg/ml) aerosol exposures 5 days/week for 3 weeks. Twenty-four hours after the final exposure, bronchoalveolar lavage (BAL) fluid and lungs were collected for further analysis.

Isolation and Primary Culture of Murine Pulmonary Epithelial Cells Primary alveolar epithelial cells were prepared from BALB/c mice using Corti’s protocol with minor modifications (23). Dispase (Roche Diagnostics, Indianapolis, IN) was instilled into the lung via a tracheal catheter, and then the lungs were removed and incubated in a dispasecontaining solution for 45 minutes at room temperature. The parenchymal tissue was carefully teased apart in Dulbecco’s modified Eagle’s medium with 0.01% DNase I (Roche Diagnostics). The single-cell suspension was filtered through 100- and 40-mm Falcon cell strainers (BD Falcon, Franklin Lakes, NJ) and a 25-mm nylon mesh, and then centrifuged at 130 3 g at 48C. Alveolar epithelial cells were purified by incubation with biotin-conjugated antibodies against CD16/CD32 (eBioscience, San Diego, CA) and CD45 (eBioscience), followed by recovery with streptavidin-conjugated magnetic beads in a magnet separator using an LS MACS separation column (Miltenyi Biotec, Auburn, CA). Purities were determined by a FACSCalibur flow cytometer using CELLQuest (Becton Dickinson, Franklin Lakes, NJ), and typically demonstrated purities of 84–92% for both CD31 and CD45 negative cells.

Histopathology and Immunohistochemistry After measuring the wet lung weight for a calculation of the wet lungto-body weight ratio (24), the lung samples were fixed with 4% paraformaldehyde and embedded in paraffin. Lung sections were stained with hematoxylin and eosin (HE), PAS and diastase-PAS staining, or Gomori methenamine silver (GMS). Deparaffinized and rehydrated lung sections were incubated with antibodies for SP-A and pro SP-C (Chemicon, Temecula, CA) followed by horseradish peroxidase–conjugated avidin, and were detected using the DAB substrate kit (Vector Laboratories, Burlingame, CA).

Cell Count of Type II Alveolar Epithelial Cells The lung sections were stained with anti–TTF-1 mAb (DAKO, Carpinteria, CA) followed by fluorescein-conjugated goat anti-mouse antibody (Vector Laboratories) and vectashield mounting medium containing propidium iodide (PI) for nuclei staining (Vector Laboratories). The number of type II alveolar epithelial cells was examined and compared by counting TTF-1/PI double-positive cells. Two independent physicians and one pathologist read more than 10 fields from four independent experimental groups, and compared the ratio of the number of type II alveolar epithelial cells per total cells.

Quantitative Real-Time PCR Reverse transcription and real-time PCR were performed as described previously (25). Probes and primers to detect murine SP-A (Sftpa1), B (Sftpb), C (Sftpc), D (Sftpd), GM-CSF (Csf2), glyceraldehyde-3phosphate dehydrogenase (Gapdh), and human SP-A (SFTPA1) were purchased from Applied Biosystems (Foster City, CA). Those of human CD40 (CD40) and human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were from NIPPON EGT (Toyama, Japan). SPA, -B, -C, -D, and GM-CSF mRNAs were indexed to the GAPDH mRNA. All experiments were repeated more than three times.

Phagocytosis Assay AMs obtained by BAL were incubated with fluorescein isothiocyanate (FITC)-labeled latex beads (Polysciences, Warrington, PA) in culture medium for 2 hours at 378C, and washed gently with 0.025% Tween 20 in PBS. Cells were stained with phycoerythrin (PE)-labeled anti-I-Ad and biotin-conjugated anti-CD11b mAbs, followed by streptavidin-

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CyChrome (PharMingen, San Diego, CA). The stained cells were evaluated on a FACSCalibur flow cytometer using CELLQuest. AMs were gated using the forward scatter-side scatter, and I-Ad and CD11b properties. The fluorescence intensity associated with the gated cells was regarded as phagocytic activity of the AMs. All experiments were done in triplicate.

Immunoblotting Protein extraction and immunoblotting using antibodies for PU.1 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), SP-A, pro–SP-C, and SP-D (Chemicon International, Temecula, CA) of BAL cells, BAL fluids concentrated 10-fold using CENTORICON (Millipore, Billerica, MA), and lungs were performed using a standard technique as described previously (24). The intensities were estimated by the public domain NIH image 1.61 program.

Measurement of mRNA Levels of SPs in Alveolar Epithelial Cells The lung adenocarcinoma cell line NCI-H441 was purchased from the American Type Culture Collection (Rockville, MD). To evaluate the effects induced by CD40 on SPs mRNA expression, H441 cells were incubated in the presence of recombinant human CD154 (1.5, 3, and 6 mg/ml; R&D Systems, Minneapolis, MN) and murine primary alveolar epithelial cells were incubated with anti-CD40 mAb (1C10; R&D Systems) for 3 days, then harvested, after which total RNA was isolated. H441 cells were also treated with phorbol 12-myristate 13acetate (PMA; Sigma) for positive control.

Statistical Analysis The results were analyzed by Mann-Whitney U test for a comparison between the two groups. Data are expressed as mean 6 SD, and were considered statistically significant at P , 0.05. All analyses were performed using the Stat View 5 statistical package for Windows (SAS Institute Inc., Cary, NC).

RESULTS Histologic Evaluation of Lungs in Mice after Repeated OVA Challenge

Although the lungs of WT mice after repeated OVA challenge showed no significant histologic findings within the alveoli (Figure 1A), lungs of all CD40KO mice after repeated OVA challenge showed accumulations of granular and eosinophilic materials within the alveoli, which were PAS-positive and diastase-resistant (Figures 1D, 1C, and 1F), and the normal alveolar architecture was preserved, though the alveolar septa were slightly thickened. The appearance of contiguous alveoli containing these materials in OVA-challenged CD40KO mice resembled that of alveolar proteinosis (Figures 1D, 1C, and 1F). Since Pneumocystis infection was one of the causes of secondary PAP, and because CD40KO and CD154KO mice were reported to readily develop Pneumocystis infection (10, 17, 26), we suspected such an infection and included GMS staining in our models. Although there were few GMS-stained organisms in the lungs of OVA-challenged WT mice (Figure 1B), they were detected in the lungs of all OVA-challenged CD40KO mice examined (Figure 1E). The more eosinophilic materials we observed within the alveoli, the more GMS-stained organisms were detected in the lungs of OVA-challenged CD40KO mice. The lung tissues of aged mice were examined to assess whether the Pneumocystis infection and the accumulation of eosinophilic materials occurred naturally in the alveoli of CD40KO mice. Pneumocystis and the accumulation of eosinophilic materials have occasionally been observed in the alveoli of 60- to 80week-old CD40KO mice, although none were seen in the alveoli of WT mice of the same age group (data not shown). We also examined the lungs of OVA-challenged CD40KO mice

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Figure 1. Histologic evaluation of lungs in ovalbumin (OVA)challenged wild-type (WT) and CD40KO Mice. Light photomicrographs of paraffin-embedded sections of lung tissues stained with hematoxylin and eosin and Gomori methenamine silver (GMS). (A and B) OVA-challenged WT mice. (D and E) OVA-challenged CD40KO mice. Lung sections of OVAchallenged CD40KO mice were stained with periodic acid Schiff (PAS), and diastase-PAS (C and F). Sections shown are of one representative mouse from a group of six. All experiments were independently repeated five times, and PAS-positive materials were revealed in alveoli of all OVA-challenged CD40KO mice. Scale bars 5 100 mm. (G) Changes in wet lung-to-body weight ratio of challenged WT (open bar) and CD40KO (solid bar) mice. (H) Immunoblots of surfactant protein (SP)-A and SP-D in bronchoalveolar lavage fluids from OVA-challenged WT and CD40KO mice. Data represent mean 6 SEM (*P , 0.05).

without systemic sensitization by an intraperitoneal injection of OVA. Although we observed only minor accumulations of eosinophilic materials, GMS-stained organisms were recognized in the lungs of OVA-challenged CD40KO mice without intraperitoneal OVA sensitization (data not shown). To exclude the possibility of the colonization of Pneumocystis in the lungs of CD40KO mice, we investigated the presence of GMS-stained organisms in the lungs of naı¨ve and PBS control (OVA priming followed by PBS aerosol challenge) CD40KO mice. However, few GMS-stained organisms were observed in those lungs (data not shown). We estimated the wet lung-to-body weight ratios, which were correlated with the severity of lung injury (24). Those ratios in OVA-challenged CD40KO mice were significantly higher than in OVA-challenged WT mice (0.024 6 0.007, 0.014 6 0.003, respectively, P , 0.01) (Figure 1G). The protein levels of both SP-A and SP-D were increased approximately 4fold in BAL fluids from OVA-challenged CD40KO mice compared with those from OVA-challenged WT mice (Figure 1H). These results suggested that CD40KO mice were prone to develop Pneumocystis pneumonia, resulting in a disease that resembled alveolar proteinosis (ApCD40KO mice). Overproduction of SPs in Lungs of ApCD40KO Mice

Because ApCD40KO mice developed accumulations of eosinophilic materials in the alveoli such as are seen in human alveolar proteinosis, we examined the mRNA levels of SPs in total lungs by quantitative real-time PCR. The mRNA levels of SP-A, SP-B, SP-C, and SP-D were significantly increased in total lungs of ApCD40KO mice, compared with those in challenged WT mice (Figure 2A). Furthermore, we evaluated the protein levels of SPs by immunoblotting. Although the protein levels of b-actin decreased somewhat relatively in the total lungs of ApCD40KO mice compared with those in challenged WT mice, the protein levels of SPs increased significantly in ApCD40KO mice. The ratios of protein levels of SP-A, SP-C, and SP-D against those of b-actin were up-regulated approximately 2-, 13-, and 7-fold, respectively, in total lungs of ApCD40KO mice compared with those in challenged WT mice (Figures 2B and 2C). Next, we examined the immunohistochemical distribution of the SP-A and SP-C; both were more

expressed in ApCD40KO mice than in challenged WT mice (Figures 3A–3D). Furthermore, we investigated the number of type II alveolar epithelial cells as TTF-1–positive cells. The number of the type II cells (yellow) per the total cells (red plus yellow) in the field was 1.22- 6 0.12-fold increased in ApCD40KO compared with OVA-challenged mice (Figures 3E and 3F). These results suggested that the accumulation of eosinophilic materials in the alveoli of ApCD40KO mice might be due to an up-regulation of SPs. Phenotypic Analysis of AMs in BAL Cells

Surfactant is cleared by its uptake into type II alveolar epithelial cells and AMs. AMs are specially important to internalize and catabolize the remaining surfactant pool (1). We investigated the phagocytic function as a representative function of AMs. To quantify the phagocytic capacity of AMs, FITC-labeled latex beads were incubated with cells from the BAL fluid of challenged WT and ApCD40KO mice. After a 2-hour incubation, fluorescence intensity of the cells was assessed with flow cytometry (FACS) analysis. Our analysis of AMs by FACS revealed that the level of the phagocytic capacity of AMs in ApCD40KO mice was not impaired compared with that of challenged WT mice (Figures 4A and 4B). Furthermore, we evaluated the mRNA levels of GM-CSF in total lungs and the protein levels of PU.1 in BAL cells. PU.1 is the transcriptional activator that is apparently critical in GM-CSF–initiated signaling in AMs (27, 28). The mRNA levels of GM-CSF in total lungs and the protein levels of PU.1 in BAL cells of ApCD40KO mice were not inferior to those in challenged WT mice (Figures 5A and 5B). These data indicated that there were no significant differences in the properties of AMs between ApCD40KO and challenged WT mice. Stimulation of CD40 Signaling Inhibits mRNA Levels of SPs in Pulmonary Alveolar Epithelial Cells

The relation between CD40 and the production of SPs was investigated by quantitative real-time PCR in an in vitro experiment using the H441 cell lines, because they were considered to have phenotypes similar to type II alveolar epithelial cells and to produce surfactants (29, 30). CD40 is

Shibasaki, Hashimoto, Okamoto, et al.: Surfactant Protein Production in Secondary PAP

Figure 2. Increased SPs in ApCD40KO mice. Production of surfactant proteins was evaluated at mRNA and protein levels. (A) mRNA levels of SPs were evaluated by quantitative real-time PCR in challenged WT mice lung tissues (open circles, n 5 7) and ApCD40KO mice lung tissues (solid circles, n 5 7) as a fold of the GAPDH gene expression. (B) Immunoblotting of SP-A, pro–SP-C, SP-D, and corresponding b-actin in total lungs from challenged WT mice (left three lanes) and ApCD40KO mice (right three lanes). Results were representative of three independent experiments (n 5 3–7 per group). (C) Ratios of the protein levels of SP-A, pro–SP-C, and SP-D against those of b-actin. Ratios of SP-A, pro–SP-C, and SP-D against those of b-actin in ApCD40KO mice (solid bars) and challenged WT mice (open bars), assuming figures of challenged WT mice as one. Result represents mean 6 SD (*P , 0.05).

expressed on lung parenchymal cells, including alveolar epithelial cells (16, 31). First, we examined and confirmed the expression of CD40 on H441 cells by FACS (Figure 6A). We examined whether or not the stimulation of CD40 signaling with recombinant soluble CD154 would inhibit the mRNA levels of SP-A in H441 cells, using PMA as a positive control; PMA is reported to inhibit those levels dependently on the concentration (32). In the presence of recombinant soluble CD154 (3 or 6 mg/ml), the mRNA levels of SP-A were significantly reduced in a dose-dependent manner in H441 cells compared with those in the absence of CD154 (Figure 6B). However, the mRNA levels of CD40 were unchanged in both the presence and absence of CD154 (Figure 6C). Based on these results using H441 cells, we examined whether or not the stimulation of CD40 signaling would inhibit the mRNA levels of SPs in primary murine alveolar epithelial cells. In the presence of anti-CD40 mAb (10 mg/ml), the mRNA levels of SP-A, SP-C, and SP-D were reduced compared with those in the absence of anti-CD40 mAb (Figure 6D). These results indicated that the stimulation of CD40 signaling inhibited the mRNA levels of SPs in alveolar epithelial cells.

DISCUSSION In this study, we showed that all the ApCD40KO mice we examined developed Pneumocystis infections and, consequently, accumulations of surfactant. ApCD40KO mice seem

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Figure 3. Immunohistochemical analysis of SP-A and SP-C expression in lungs of challenged WT and ApCD40KO mice. Representative immunohistochemistry for SPs in lungs of challenged WT mice (A and C) and those of ApCD40KO mice (B and D). Both SP-A (A and B) and SP-C (C and D) were more strongly expressed in ApCD40KO mice than in challenged WT mice. Representative immunofluorescence staining of challenged WT mice and ApCD40KO mice with anti–TTF-1 mAb and PI (E and F). Nuclei of type II alveolar epithelial cells were stained with TTF1/PI (yellow) and other cells (red). Scale bars 5 100 mm.

to be one of the excellent animal models of secondary PAP resulting from Pneumocystis infection. We investigated the mechanism of the pathogenesis of secondary PAP using this model, and showed that surfactant accumulations were due to an overproduction rather than to a decrease of clearance in our model of secondary PAP. We also investigated the relationship between SP production and CD40 signaling, and found that the stimulation of CD40 signaling regulated the production of SPs in alveolar epithelial cells. Since both CD40KO and CD154KO mice were reported to be highly susceptible to opportunistic infection, including Pneumocystis (13, 17, 26), we thought that ApCD40KO mice were not able to immunologically recover from the Pneumocystis infection. In the naive and PBS control CD40KO mice, Pneumocystis was seldom observed and the accumulation of surfactants was low. The severity of Pneumocystis infection and the consequent accumulation of surfactant depended on OVA priming. Furthermore, Pneumocystis and the accumulation of surfactants were only occasionally observed in the lungs of aged CD40KO mice. It has been reported that chronic OVA exposure followed by OVA priming causes chronic T-helper type 2 (Th2) airway inflammation (33, 34). Using a mouse model for a Leishmania major infection, Kamanaka and coworkers reported that the CD40–CD154 interaction plays an important role in the generation of a T-helper type 1 (Th1) response (35). Our results suggested that ApCD40KO mice, which were shifted toward Th2-type airway inflammations, developed Pneumocystis infection, resulting in the accumulation of surfactant. We examined the specific mechanism of the pathogenesis of secondary PAP resulting from Pneumocystis infection in ApCD40KO mice. To clarify whether or not secondary PAP

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Figure 5. Evaluation of mRNA levels of GM-CSF in total lungs, and protein levels of PU.1 in AMs. (A) mRNA levels of GM-CSF evaluated in total lungs of challenged WT mice (open circles), and ApCD40KO mice (solid circles) (P 5 0.063). (B) Protein levels of PU.1 evaluated by immunoblotting in AMs of naı¨ve WT (lane 1), naı¨ve CD40KO (lane 2), challenged WT (lane 3), and ApCD40KO (lane 4). Results were representative of three independent experiments. (C) Protein levels of PU.1 relative to corresponding b-actin (naı¨ve WT [vertically striped bar], naı¨ve CD40KO [horizontally striped bar], challenged WT mice [open bar], and ApCD40KO mice [solid bar]). Results from five to seven mice per group.

Figure 4. Phagocytic capacity of alveolar macrophages (AMs). Fluorescence-activated cell sorter analysis was done to assess phagocytic capacity of AMs. AMs (1 3 106 cells) from challenged WT and ApCD40KO mice were incubated with fluorescein isothiocyanate– labeled 0.1-mm latex beads/ml (100 particles/cell) in culture medium for 2 hours at 378C (black line). Control cells were incubated with supernatants of the beads solution (gray line). (A) Representative flowcytometry histograms of phagocytosis by AMs from three independent experiments. Each successive peak represents number of cells with an increased number of beads per cell. Level of phagocytic capacity of AMs in ApCD40KO mice (bottom panel) was not impaired compared with that in challenged WT mice (top panel). (B) Proportion of macrophages was shown according to number of ingested beads per cell. Data were shown as mean 6 SD from three independent experiments. No significant differences were detected between challenged WT (open bars) and ApCD40KO (solid bars) mice.

with Pneumocystis infection was caused by the up-regulation of SPs, we evaluated the expression levels of SPs in both ApCD40KO and challenged WT mice. We observed that the mRNA and protein levels of SPs increased in total lungs of ApCD40KO mice, compared with those in challenged WT mice. In addition, immunohistochemical expressions of SP-A and SP-C supported their overproduction in the lungs of ApCD40KO mice. Although an increased number of type II epithelial cells in the lungs of ApCD40KO mice was one potential cause of the increased amount of surfactant proteins there, the increase in the number of type II alveolar epithelial cells was not so much as that in the amount of surfactant proteins. Based on these results, we concluded that the up-regulation of SP productions was involved in the pathogenesis of secondary PAP resulting from infection with Pneumocystis. Most studies in mouse models of acquired PAP revealed the critical roles of GM-CSF and PU.1 in surfactant homeostasis. It has been reported that the accumulation of surfactants was due to a defect in the surfactant clearance by AMs, rather than to a production increase in acquired PAP (1). We investigated the phagocytic function of AMs, the mRNA levels of GM-CSF in lungs, and the protein levels of PU.1 in AMs, and we observed no dysfunction of AMs in ApCD40KO mice, unlike that in

acquired PAP. Based on these results, we considered that in secondary PAP resulting from Pneumocystis infection, surfactant accumulation was due to an overproduction rather than to a reduced clearance. An obvious and important question is the relation between OVA priming and the accumulation of surfactants in secondary PAP resulting from Pneumocystis infection. Reports regarding the models of PAP by an increased SP expression have been confined to animal studies with transgenic mice for IL-4 and IL-13 (36, 37). Airway inflammation being more inclined to Th2-type might lead to a situation in which the increased abundance of SPs is more likely to occur in ApCD40KO mice. As CD40 was reported being expressed on lung parenchymal cells, including alveolar epithelial cells (16, 31), we examined the effect of CD40–CD154 interaction on the regulation of SP production. The stimulation of CD40 signaling in alveolar epithelial cells showed that the CD40 signaling inhibited the mRNA levels of SPs. On the other hand, this stimulation did not affect the mRNA levels of CD40. These results indicated that the CD40–CD154 interaction regulated type II alveolar epithelial cells to produce SPs. Pulmonary surfactant was initially identified as a lipoprotein complex that reduced surface tension at the air–liquid interface of the lung (38, 39). Furthermore, the host-defense functions of surfactant are primarily mediated by SP-A and SP-D, which are members of the collectin family of proteins (40). The lung is exposed to inhaled particles and pathogens that are cleared by the actions of an innate host defense system including the pulmonary collectins (40, 41). Although there were several prior studies reporting that the pulmonary collectins (SP-A and SP-D) interacted with Pneumocystis infection (42–44), it has not been elucidated whether or not type II alveolar epithelial cells overproduce SPs when infected with Pneumocystis. Without the interaction between CD40-expressing type II alveolar epithelial cells and CD154-expressing cells, increased abundance of SPs were observed in Pneumocystis infection. Immunocompetent individuals have a regulation system of innate immunity. It might be possible that once acquired immunity is developed, activated T cells expressing CD154 could migrate to the alveoli and regulate the production of surfactant in immunocompetent individuals. A lack of the CD40–CD154 interaction is reported to un-

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nism of secondary PAP was an overproduction of SPs, suggesting that their production could be regulated by the CD40 signaling. Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript. Acknowledgments: The authors sincerely thank Ms. Kumi Kawase for her technical assistance.

References

Figure 6. Inhibition of SP-A mRNA levels in H441cells by stimulation of CD40. (A) Surface expression of CD40 in H441cells (CD40, black line; isotype control, gray line). (B) mRNA levels of SP-A in H441 coincubated with recombinant CD154 (0, 1.5, 3, 6 mg/ml) and PMA (0–1.2 nM) as a control. (C) mRNA levels of CD40 in H441 coincubated with recombinant CD154 (solid bar), vehicle (open bar), and PMA as a control (gray bars). Data shown as mean 6 SED from three independent experiments (*P , 0.05). (D) mRNA levels of SP-A, SP-C, and SP-D in murine primary alveolar epithelial cells co-incubated with anti-CD40 mAb (10 mg/ml) (solid bars) or without (open bars). Results were representative of three independent experiments.

dermine resistance to Pneumocystis infection by inhibiting the interaction of T cells with antigen-presenting cells, rapidly leading to Pneumocystis infection (13). As for ApCD40KO mice, when CD40KO mice were infected with Pneumocystis, the production of SPs was not regulated because they lacked the regulatory effect that was triggered by the CD40 signaling, and consequently surfactants accumulated. The administration of soluble CD154 or CD40 antibody would be one of the potential therapeutic options of secondary PAP associated with Pneumocystis infection. Further investigations are necessary to verify the critical role of the CD40-CD154 system in secondary PAP associated with immunocompromised subjects. In summary, we found that ApCD40KO mice were one of the good models of secondary PAP resulting from Pneumocystis infection. Furthermore, we showed that one potential mecha-

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