Supplementary Online Material
Materials and Methods
Reagents
Cathepsin H from human liver was obtained from Calbiochem (San Diego, CA, USA). Primary antibodies for immunoblots were a polyclonal rabbit anti-cathepsin H (Fitzgerald, Concord, MA, USA) and a monoclonal mouse anti-human napsin A (IBL, Hamburg, Germany). Dr. Steinhilber (Nycomed, Konstanz) kindly provided rabbit polyclonal antibodies against mature surfactant proteins B and C, and recombinant SP-B and SP-C. A rabbit polyclonal antibody against proSP-B, and recombinant proSP-B was a gift from S. Guttentag (Philadelphia, PA, USA).
BAL sample preparation, isolation of large and small aggregate surfactant
Bronchoscopy and BAL (4 x 1 ml of 0.9% NaCl per kg body weight) were performed as described previously [1]. In the patients with pulmonary alveolar proteinosis, the therapeutic lavages were done with pre-warmed normal saline. Lavage aliquots were filtered through two layers of sterile gauze, cells and debris were removed by centrifugation at 300 xg for 10 minutes and the supernatant was stored at -80° C until used for analysis.
From the cell pellets total numbers of cells were determined and differential cell counts were read from May-Grünwald-Giemsa stained cytospins. Viability of cells was determined by erythrosin dye exclusion test (Supplementary table 1). Total protein content of cell-free BAL fluid was determined with Bradford reagent (Biorad, Hercules, CA, USA), using bovine serum albumin as standard. Mean protein concentrations were 271 22 g/ml for control group, 1043 128 g/ml for juvenile PAP, and 1054 237 g/ml for adult PAP patients, respectively (means SE).
For the isolation of large and small aggregate surfactant, aliquots of the BAL fluid were centrifuged at 40,000 xg for 30 minutes. The supernatant, containing small surfactant aggregates (SAs), was separated from the large surfactant aggregates (LAs) containing pellet.
Immunoblot analysis of human BAL fluid, LAs, and SAs
Aliquots of human BAL fluid, LAs, SAs, and cathepsin H were separated under reducing conditions using 10% NuPage Bis-Tris gels with MES running buffer and transferred to Immobilon-P PVDF membrane (Millipore, Concord, MA, USA) according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA). Membranes were blocked with 5% bovine serum albumin (BSA) in phosphate buffered saline (PBS) for one hour, followed by incubation with the primary antibody in PBS over night at 4°C. Blots were washed three times with PBS-T (PBS - 0,05% Tween-20), and incubated for one hour at room temperature with peroxidase-coupled secondary antibody; the blots were washed again three times, and developed with enhanced chemiluminescence detection kit ECL (GE Healthcare, Chalfont St. Giles, UK). Bands corresponding to cathepsin H and napsin A were densitometrically quantified with QuantityOne Software (Biorad, Hercules, CA, USA).
For the quantification of surfactant protein B, -C, and proSP-B, aliquots of human BAL fluid, recombinant SP-B, SP-C, or proSP-B were separated, detected and quantified as described above, using specific polyclonal antibodies against SP-B, SP-C, and proSP-B for detection.
Quantification of cystatin C in BAL fluid by ELISA
Cystatin C levels in human BAL fluid samples were quantified in duplicate by sandwich ELISA according to the manufacturer’s instructions (Human cystatin C ELISA, BioVendor, Heidelberg, Germany). Concentrations were calculated from standard curves. The lower limit of detection was 0.25ng/ml; the upper limit of the assay was 25 ng/ml.
Cathepsin H activity assay
Cathepsin H activity was determined by a modified protocol of Schwartz and Barrett [2], using the specific substrate H-Arginine 7-Amino-4-methylcoumarin hydrochloride (H-Arg-AMC 2 HCl, Bachem, Weil am Rhein, Germany). Samples (20 µl) were pre-incubated for 5 minutes at 37°C with 80 µl assay buffer (100 mM 2-Morpholino-ethanesulfonic acid pH 6.8, 1 mM ethylenediamine-tetra acetic acid (EDTA), 1 mM dithiothreitol (DTT)), before adding H-Arg-AMC substrate in assay buffer to a final concentration of 10 µM. Liberated 7-Amino-4-methylcoumarin was determined by measuring the fluorescence (excitation at 355 nm, emission at 460 nm) over 20 minutes at 37°C in a fluorometer (GENios, Tecan, Munich, Germany). Enzyme activity was calculated as amount cathepsin H per volume sample by comparison of the slopes of the kinetic curves with a standard curve, using mature cathepsin H as reference substance. The lower limit of detection was 0.25g/ml; the assay was linear to 5g/ml. To document the specificity of the enzymatic activity, the assay was also performed in the presence of 14 µM cysteine protease inhibitor trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E-64, Sigma, Germany), which blocked the substrate cleavage completely. Although the majority of BAL fluid samples analyzed were aliquots and not repetitively thawed and frozen, we verified that cathepsin H activity was not affected by repeated freeze-thaw cycles of the BAL fluid samples (data not shown).
Napsin A activity assay
Napsin A activity was determined by measuring proteolytic cleavage of the substrate MCA-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Lys(Dnp)-D-Arg-NH2 (Bachem, Weil am Rhein, Germany). Samples (20 µl) were pre-incubated for 5 minutes at 37°C with or without the aspartic acid protease inhibitor pepstatin (10 µM), followed by the addition of substrate in 100 µl assay buffer (100 mM sodiumacetate pH 4.7, 20 mM ethylenediamine-tetra acetic acid (EDTA)) to a final concentration of 5 µM. Proteolysis was monitored by measuring the fluorescence (excitation at 355 nm, emission at 460 nm) over 20 minutes at 37°C in a fluorometer (GENios, Tecan, Munich, Germany). Enzyme activity was expressed as relative activity, using the same unrelated patient sample as internal reference in each experiment. The specificity of the enzymatic activity was verified by use of the aspartic acid protease inhibitor pepstatin, which blocked substrate cleavage completely (data not shown). As with cathepsin H, repeated freeze-thaw cycles of the BAL fluid samples did not result in a significant change in proteolytic activity (data not shown).
Lactate dehydrogenase (LDH) assay
LDH activity in BAL fluid was determined by the method of Decker and Lohmann-Matthes [3]. Briefly, 100 μl of BAL fluid were mixed with 30 μl dye solution (18 mg/ml L-Lactate, 1 mg/ml Iodonitrotetrazolium (INT) in PBS). After addition of 15 μl catalyst (3 mg/ml NAD+, 2.3 mg/ml diaphorase, 0.03% BSA, 1.2% sucrose in PBS), absorbance at 492 nm was determined at one minute intervals for 15 minutes at 37°C. Absolute LDH activity was calculated from a standard curve, using purified LDH (Sigma, Germany). The lower limit of detection was 20 Units/L; the assay was linear to 2500 Units/L.
References
1.Ratjen F, Bredendiek M, Brendel M, Meltzer J, Costabel U. Differential cytology of bronchoalveolar lavage fluid in normal children. Eur Respir J 1994; 7: 1865-1870.
2.Schwartz WN, Barrett AJ. Human cathepsin H. Biochem J 1980; 191: 487-497.
3.Decker T, Lohmann-Matthes ML. A quick and simple method for the quantitation of lactate dehydrogenase release in measurements of cellular cytotoxicity and tumor necrosis factor (TNF) activity. J Immunol Methods 1988; 115: 61-69.
Table legends
Supplementary Table 1: Cellular characteristics of the BAL fluids in the patient groups investigated
Figure legends
Supplementary Fig. 1: Correlation of protease amounts with mature surfactant proteins.The correlation ofcathepsin H amounts with amounts of mature SP-B (8 kDa form), and with amounts of mature SP-C (4.3 kDa form) is shown in panel A, and panel B, respectively. The same SP-B and SP-C values are correlated with napsin A amounts in panel C, and panel D, respectively. Correlation coefficients and p-values are shown in the diagrams.
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Supplementary Table 1.Cellular characteristics of the BAL fluids in the patient groups investigated
Patient Group / n / Total cell count[x103 ml-1] / Macrophages
[%] / Lymphocytes
[%] / Neutrophiles
[%] / Eosinophiles
[%] / Vitality
[%] / Recovery
[%]
Control / 16 / 210.9 30.6 / 72.8 3.0 / 13.8 2.0 / 12.3 3.2 / 0.7 0.3 / 88.9 1.9 / 64.2 3.4
PAP juvenile / 20 / 349.6 81.0 / 63.7 7.1 / 9.2 2.6 / 25.9 16.1 / 0.02 0.02 / 88.7 2.5 / 61.3 2.3
PAP adult / 13 / 177.5 22.5 / 53.0 21.2 / 20.7 8.9 / 24.1 6.1 / 2.1 1.7 / 59.0 9.0 / n.d.
Data are means standard error (SE); n.d.: not determined; PAP: pulmonary alveolar proteinosis
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