Background
Pulmonary alveolar proteinosis (PAP) syndrome is thought to occur due to multiple causes including genetic defects [
1‐
3], immune deficiencies [
4], malignancies [
5,
6] and infection [
7]. Patients with PAP have milky alveolar infiltrates that often contain excess surfactant material and large numbers of white blood cells. PAP is usually treated by whole lung lavage (WLL) [
8‐
12] or with GM-CSF administration [
13‐
16]. GM-CSF is a hematopoietic growth factor known to stimulate stem cells to proliferate into granulocytes or monocytes [
17], promote differentiation of monocytes into alveolar macrophages [
18‐
20], and increase the catabolism within alveolar macrophages [
21,
22], and increase the innate immune potential of neutrophils [
23]. Therefore, GM-CSF is an important cytokine that could regulate PAP at multiple levels.
PAP-like condition is also reported in surfactant protein D (SP-D) deficient mice [
24‐
26]. SP-D is an innate immune collectin that is present in lungs and other mucosal surfaces [
27]. It opsonizes pathogens and dying cells, and enhances their uptake by the alveolar macrophages [
28,
29]. SP-D deficient mice also accumulate lipid-laden foamy macrophages [
25,
26] and apoptotic cells in their lungs [
30]. Moreover, transgenic over-expression of SP-D [
31] or administration of recombinant fragments of SP-D [
30] has been shown to enhance immune function in the lungs in mice. Therefore, SP-D and GM-CSF could significantly enhance the innate immune functions of alveolar macrophages.
PAP is one of the deadliest phenotypes seen in Lysinuric protein intolerance (LPI) [
32‐
34]. LPI is an autosomal recessive disorder characterized by mutations in the SLC7A7 (solute carrier family 7, member 7) gene, which encodes a dibasic cationic amino acid transporter, y+LAT1 [
35,
36]. Mutant SLC7A7 proteins cause defective transport of the cationic amino acids arginine, lysine, and ornithine [
35‐
38]. PAP develops in some LPI patients and appears to be different from idiopathic PAP [
32]. PAP in LPI presents with large numbers of cholesterol crystals and granulomas [
32,
33,
39]. The precise cause or appropriate treatment options for the clinical presentation of PAP in LPI is currently unknown. Since LPI is not a well-characterized disease, and has multiple symptoms [
37], it may have also been misdiagnosed as other conditions, and hence, the prevalence of this disease may be higher than the reported numbers [
37]. Therefore, it is important to study the disease phenotypes and treatment options. We hypothesized that defective clearance of materials present in the airways contributes to the presentation of PAP in LPI patients, and treating the LPI cells with SP-D and GM-CSF could enhance the innate immune potential of these cells.
Consistent with our hypothesis, we found that SP-D and GM-CSF increase the uptake of proteins and dying cells by LPI AMs. Unexpectedly, we found that the LPI AMs spontaneously formed granulomas, ex vivo. Moreover, while control cells remained unchanged, the addition of GM-CSF to the LPI AM cultures resulted in a marked increase in the number of granulomatous structures. Notably, SP-D counteracted the negative effect of GM-CSF. In conclusion, although GM-CSF may have therapeutic advantage in certain types of PAPs, it may not be suitable for treating PAP of the LPI patients.
Materials and methods
Mutation analysis
SLC7A7 gene mutation analysis was performed by Dr. Ginafranco Sebastio in Naples, Italy. ABCA3, SP-B and SP-C genes were sequenced and analyzed by Ambrey Genetics.
Reagents
All buffer salts and reagents were obtained from Sigma unless otherwise stated. SP-D was isolated from BALF of an adult PAP patient as described previously [
40,
41]. Recombinant GM-CSF expressed in yeast (Leukine) was produced by Berlex (Seattle, WA). All cell culture media were obtained from Invitrogen (Carlsbad, CA).
Collection of WLL and BALF
Therapeutic WLL was performed in the LPI patient or idiopathic PAP patient, and the BALF samples were colleted in 100 ml vials. Diagnostic BALF sample was also obtained from another airway disease patient. BALF was filtered with nylon mesh and centrifuged at 200 × g for 10 min at 4°C. The pellets containing the cells were then washed twice with Hank's Buffered Salt Solution (HBSS). The supernatant was centrifuged again at 10,000 × g for 40 min at 4°C to collect surfactant lipid pellets, which were stored at -80°C until further analyses.
Analysis of the BALF
BCA
® Protein Assay kit from Pierce (Rockford, IL) was used for measuring the protein concentrations in the pre-filtered BALF from each collection vial according to the manufacturer's instructions. SP-D concentration levels and degradation were assessed in the BALF from the LPI patient by Western blotting using anti-human SP-D rabbit sera as described previously [
42]. An aliquot of the BALF from each collectin tube was immediately mixed with 0.2% (w/v) Trypan Blue solution and counted using a Hemocytometer at 20× or 40× normal magnification. The amount of cholesterol present in the BALF samples were assessed using the Cholesterol Assay Kit (Cayman Chemical Co., Ann Arbor, MI).
Histological staining
Cytospin preparations were made using 50 and 100 μl BALF, cells were air dried and subjected to Hemacolor® staining (EMD Chemicals, Inc, Gibbstown, NJ) for histological analysis. Other cytospin preparations were fixed with 2% (v/v) paraformaldehyde (PFA) in PBS for 1 h at 4°C, and were subjected to staining with Oil Red O (Polysciences, Inc, Washington, PA) for lipid analysis of foamy macrophage cells. Oil Red O stock solution was prepared by dissolving 0.5% (w/v) Oil Red O powder in isopropanol at 37°C. This stock solution was then diluted to 60% (v/v) with dH2O to make the working solution. Cytospin preparation of cells were rinsed with 60% (v/v) isopropanol, and stained with freshly prepared Oil Red O working solution for 15 min and counterstained with Hemacolor Solution 3 for nuclei. Cells were imaged using Leitz Laborlux D microscope with Leica IM 50 image manager software.
Electron microscopy
Cell pellets and surfactant lipid pellets obtained from 200 × g and 10,000 × g centrifugation of BALF, respectively, were fixed for at least 18 h at 4°C with 2% (v/v) paraformaldehyde (PFA) and 4% (v/v) glutaraldehyde (Electron Microscopy Sciences, Fort Washington, PA) in a buffer containing 50 mM HEPES (pH 7.4), 150 mM NaCl, and 1 mM CaCl2. Samples were then processed, stained with 2% (w/v) uranyl acetate and analyzed by transmission electron microscopy (JEOL TEM 1011) with AMT Image Capture Engine software at 2,000-40,000 times normal magnification.
Cell culture
Cell pellets obtained from 200 × g centrifugation of the BALF were immediately re-suspended in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% (v/v) heat-inactivated Fetal Bovine Serum (FBS) with 100 U/ml penicillin, 100 μg/ml streptomycin, and 2.5 μg/ml Fungezone. Cells were then allowed to adhere to chamber slides or 24- or 48-well culture plates for 1 h, and non-adherent cells were washed. Cells were cultured in the same media, and subsequently, the media was changed to Macrophage Serum-Free Media (MSFM), a specific media available for culturing macrophage cells (Invitrogen), and does not require supplementation with serum, prior to the experiments. The Jurkat T cell line was maintained in RPMI containing 10% (v/v) FBS.
Induction of apoptosis
Jurkat T cells were first labelled with 2 μl/ml DiI membrane dye (Invitrogen) in MSFM for 4 min at 37°C, followed by three washes with HBSS. Apoptosis was induced by UVC irradiation for 30 s using UV Stratalinker 2400 (120 J/cm2). Cells were then placed in the humidified incubator at 37°C for 2 h in the absence or presence of SP-D (5 μg/ml).
BSA/Apopotic cell uptake assay
Cells were incubated first in MSFM overnight prior to the experiment. Cells were then treated with SP-D (1 μg/ml) for 4 h, and GM-CSF (10 ng/ml) for 30 min prior to the experiment. Alexa-647 conjugated BSA was added to the culture for 30 min, at which point the cells were fixed with 2% (v/v) PFA for 10 min at room temperature, followed by fresh 2% (v/v) PFA for 1 h at 4°C. DiI-labelled apoptotic Jurkat T cell uptake assay was performed in the same manner as BSA using the Jurkat cells 2 h after the induction of apoptosis (1:5 of apoptotic cells:macrophages).
Fluorescence microscopy and image analysis
Images for the uptake assays, and granuloma assay were captured using Zeiss Axiovert 200 M fluorescence microscope with Volocity software. Measurement of cell area, color (label-specific gray levels) and size was performed using Volocity software.
Cells were incubated in the presence or absence of SP-D (1 μg/ml) and/or GM-CSF (10 ng/ml) for 29 days ex vivo. Culture media was replaced with a fresh media with the addition of appropriate reagents (SP-D and/or GM-CSF) every 3-5 days. After 28 days, the granulomas started to dislodge from the substratum.
Statistical analysis
A two-tailed student's t-test was performed using Microsoft Excel on Oil Red O positive cell counting and apoptotic Jurkat cell uptake analysis. Protein uptake assay, and cell shape and size were analyzed using Dunnett's multiple mean comparison tests using JMP statistical analysis software (Version 5.0; SAS Institute Inc.). A p-value was set at 0.05 for statistical significance. Non-linear regression analysis and comparisons of regression lines by ANOVA and F-test was performed using GraphPad PRISM statistical analysis software (Version 4.0; GraphPad Software).
Discussion
PAP in LPI is often untreatable and results in death. In this study, we carefully characterized BALF and tissue samples obtained from an LPI patient. The data revealed elevated levels of protein and dying cells in the airways. In addition, large cholesterol crystals and abnormal tubular myelin structures were found. Our primary cell culture assays using Alexa-647 conjugated BSA and apoptotic Jurkat T cells showed that pre-incubation of the LPI BAL cells with SP-D and GM-CSF increased their innate immune functions. Surprisingly, many of these LPI cells, but not the control cells, became elongated and stopped internalizing foreign material; eventually they formed granulomas ex vivo. Notably, treating these cells with GM-CSF ex vivo dramatically increased granuloma formation. However, treating the cells with SP-D reduced GM-CSF-mediated granuloma formation. Therefore, these findings may provide important clues for devising better treatment of PAP in LPI patients.
Although various SLC7A7 mutations can cause LPI, the importance of different mutations in the disease phenotype is not clearly established [
45]. LPI is relatively frequent in Finland and Italy [
46,
47], and in Japan, 1:119 individuals are heterozygous carriers for this LPI gene mutations with estimated frequency of 1:50000 individuals living with LPI [
48]. LPI has also been reported in other countries where variable penetrance of specific founder mutations was shown to be responsible for the disease [
37,
49]; hence, many LPI individuals with SLC7A7 mutations may be misdiagnosed.
This is the first report that shows LPI in a patient with Dutch ancestry. Consistent with the definition of PAP [
1,
12,
27], this patient's BALF had PAS positive material (data not shown) and >100-fold protein levels compared to control, as well as increased numbers of dead cells (Figure
1). High amount of cholesterol and several cholesterol crystals were also seen (Figure.
2). These pathological indices are consistent with reduced lung function seen in these patients.
Although Western blot analyses showed that SP-D was present in the BALF, some SP-D was modified or tightly bound to other components. Furthermore, a considerable amount of SP-D was partially cleaved (Figure
1D). These conditions would render SP-D less functional [
50,
51]. Furthermore, electron microscopy analysis of the surfactant lipid pellets showed alterations in tubular myelin structures; notably, the tubular myelin had circular lattice structures, as opposed to the SP-A-mediated square lattice-like structures that are typically seen in healthy humans (Figure
1E and
1F) [
43,
52]. Tubular myelin with circular lattice structures is not reported in normal human lungs, but could be generated with SP-D and phosphatidylinositol,
in vitro [
43]. Since these lipid structures could trap SP-D, together with the fact that there was degraded SP-D, we suggest that the bioavailability of free SP-D is limited in the airways of PAP lungs.
Electron microscopy analysis further revealed the presence of large numbers of cholesterol crystals in the surfactant consistent with previous reports on LPI with PAP [
32,
33,
39]. Since the cholesterol level is elevated in airways and serum of idiopathic PAP patients [
53], cholesterol may be one of the important contributors of the pathology in all types of PAP. High cholesterol levels in the lung surfactant increases surface tension [
54,
55], and thus may reduce overall lung function. Interestingly, lovastatin, a cholesterol-reducing drug, has been shown to increase apoptotic cell clearance [
56]. Therefore, we suggest that it would be worthwhile to explore the possibility of lowering cholesterol in the lungs while increasing dead cell clearance. This may be an alternative treatment option for reducing pulmonary complications in LPI patients.
Since GM-CSF [
2,
3,
22,
23,
57‐
62] and SP-D deficiency [
24‐
26,
42] cause a PAP-like phenotype, we examined their potential therapeutic role
ex vivo. Although both GM-CSF and SP-D are usually present in the lungs, it is important to determine whether the BAL cells isolated from the patient respond to these proteins. We were able to show a positive effect of GM-CSF and SP-D in the uptake of protein and apoptotic cells (Figure
3). These results are consistent with the roles of GM-CSF and SP-D suggested by
in vivo mouse experiments [
30,
63,
64]. Therefore, our results show that, similar to wild type cells, "healthy" LPI cells can respond to SP-D and GM-CSF effectively.
These healthy viable LPI cells however also formed granulomatous structure
ex vivo (Figure.
5). Within about one week, many of these cells become elongated and large. They stopped internalizing foreign materials regardless of the presence or absence of SP-D or GM-CSF (Figure
5). Some of the cells formed elongated fibers and connected with each other whereas others migrated to a common location and formed granulomatous structures. It is logical to consider that defective surfactant homeostasis [
1‐
7,
22‐
26,
42,
57‐
62,
65‐
68], accumulation of dead/dying cells [
30] and foamy macrophages [
21,
25,
26,
69], and increased granulomas [
39] would reduce lung function in the patients with PAP and LPI. Therefore, alternative strategies are necessary to treat PAP in these patients.
Aerosol [
70,
71] and subcutaneous [
14,
72] GM-CSF therapy have been successful in treating some patients with PAP [
14,
70‐
72]. Overexpression of GM-CSF in mouse [
20,
73] and rat [
74,
75] lungs has been shown to increase the recruitment of monocytes [
20,
75] and proliferation [
73] and differentiation of alveolar macrophages [
20]. Under certain conditions, GM-CSF also increases granuloma formation [
75] and fibrosis [
74] in the lung. Subcutaneous GM-CSF therapy (6 μg/kg per day) has been tried for treating PAP in another child with LPI, but was unsuccessful because the administration of GM-CSF had side effects and lead to excessive leukocytosis [
34]. A recent report described a successful therapeutic WLL on a 10-year-old Italian boy with LPI who presented with PAP [
46]. The pulmonary condition in the patient described in our report, however, did not improve after WLL, and the patient eventually died of pulmonary insufficiency.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
DND performed experiments and drafted the manuscript. NF performed experiments in this manuscript. SD performed BAL procedure and participated in manuscript preparation. HG participated in data interpretation and manuscript preparation. NP devised the study and participated in the interpretation of data and manuscript preparation. All authors read and approved the final manuscript.