A non-BRICHOS surfactant protein c mutation disrupts ... · maintain surfactant biosynthesis in the...

RESEARCH ARTICLE Open Access

A non-BRICHOS surfactant protein c mutationdisrupts epithelial cell function and intercellularsignalingMarkus Woischnik1, Christiane Sparr1, Sunčana Kern1, Tobias Thurm1, Andreas Hector1, Dominik Hartl1,Gerhard Liebisch2, Surafel Mulugeta3, Michael F Beers3, Gerd Schmitz2, Matthias Griese1*

Abstract

Background: Heterozygous mutations of SFTPC, the gene encoding surfactant protein C (SP-C), cause sporadic andfamilial interstitial lung disease (ILD) in children and adults. The most frequent SFTPC mutation in ILD patients leadsto a threonine for isoleucine substitution at position 73 (I73T) of the SP-C preprotein (proSP-C), however little isknown about the cellular consequences of SP-CI73T expression.

Results: To address this, we stably expressed SP-CI73T in cultured MLE-12 alveolar epithelial cells. This resulted inincreased intracellular accumulation of proSP-C processing intermediates, which matched proSP-C speciesrecovered in bronchial lavage fluid from patients with this mutation. Exposure of SP-CI73T cells to drugs currentlyused empirically in ILD therapy, cyclophosphamide, azathioprine, hydroxychloroquine or methylprednisolone,enhanced expression of the chaperones HSP90, HSP70, calreticulin and calnexin. SP-CI73T mutants had decreasedintracellular phosphatidylcholine level (PC) and increased lyso-PC level without appreciable changes of otherphospholipids. Treatment with methylprednisolone or hydroxychloroquine partially restored these lipid alterations.Furthermore, SP-CI73T cells secreted into the medium soluble factors that modulated surface expression of CCR2 orCXCR1 receptors on CD4+ lymphocytes and neutrophils, suggesting a direct paracrine influence of SP-CI73T onneighboring cells in the alveolar space.

Conclusion: We show that I73T mutation leads to impaired processing of proSP-C in alveolar type II cells, alterstheir stress tolerance and surfactant lipid composition, and activates cells of the immune system. In addition, weshow that some of the mentioned cellular aspects behind the disease can be modulated by application ofpharmaceutical drugs commonly applied in the ILD therapy.

BackgroundBiophysically active pulmonary surfactant contains amixture of lipids and hydrophobic surfactant proteins B(SP-B) and C (SP-C). A normal composition and home-ostasis of pulmonary surfactant is critical for its surface-tension-reducing properties and gas exchange in thealveolar region of the lung. SP-C is synthesized exclu-sively by alveolar type II cells (AECII) as a 21 kDa pre-protein (proSP-C). ProSP-C is further processed to the4.2 kDa mature protein through a sequence of proteoly-tic cleavages before being secreted together with lipids

and other surfactant components to the alveolar surface[1-4]. Surfactant secretion is accomplished by fusion oflamellar bodies, AECII-specific organelles for surfactantassembly and storage, with the plasma membrane [5].SNARE proteins, in particular syntaxin 2 and SNAP-23,are required for regulated surfactant secretion. Bothproteins are associated with the plasma membrane andto some degree with lamellar bodies [6,7]. In parallel tosecretion, AECII reinternalize and recycle surfactantcomponents from the alveolar surface by means ofendocytosis via clathrin-dependent and clathrin-inde-pendent pathways, which include routing to early endo-somes and multivesicular bodies [5].Interstitial lung disease (ILD) is a heterogeneous group

of diseases of known and unknown etiology [8-10].

* Correspondence: [email protected] of Pneumology, Dr. von Hauner Children’s Hospital, Ludwig-Maximilians University, Lindwurmstr. 4, Munich, 80337, GermanyFull list of author information is available at the end of the article

Woischnik et al. BMC Cell Biology 2010, 11:88http://www.biomedcentral.com/1471-2121/11/88

© 2010 Woischnik et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the CreativeCommons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly cited.

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Several histological and clinical subtypes of ILD arelinked to the SP-C protein deficiency caused by muta-tions of the corresponding SFTPC gene [3]. Many SP-Cmutations cluster within the preprotein’s BRICHOSdomain and lead to misfolding of the preprotein, aber-rant trafficking and processing [11]. To date, all affectedindividuals with BRICHOS domain mutations have beenheterozygous with no detectable mature SP-C in theirlungs, suggesting a dominant-negative effect of themutant allele (e.g. Δexon4, L188Q) [11,12]. Moreover, incell lines expressing BRICHOS domain mutations,proSP-C forms perinuclear aggregates, consistent withthe cell’s inability to clear aggregated misfolded proteinsand a toxic gain-of-function [12,13]. Various pathologicmechanisms for these mutations causing chronic accu-mulation of misfolded proSP-C have been proposed,such as induction of endoplasmic reticulum (ER) stress,cytotoxicity, and caspase 3- and caspase 4-mediatedapoptosis [14-16]. These factors might contribute toILD through cell injury and death of AECII. In additionto the BRICHOS domain mutations, a second class ofSFTPC mutations has emerged. A heterozygous mis-sense mutation, leading to a substitution of threoninefor isoleucine at position 73 of the proSP-C, is the mostfrequent SFTPC mutation [17,18]. There is a strongvariability in the phenotype of these patients, rangingfrom asymptomatic to early fatal cases [19]. I73T SP-C(SP-CI73T) is marked by mistrafficking of the preproteinto the endosomal compartment and by preserved secre-tion of both mature and aberrant proSP-C and proSP-Bforms and their intra-alveolar accumulation [17,20,21].Yet, current knowledge on SP-CI73T lacks a preciseunderstanding of the proSP-C processing abnormalities,concurrent cell stress response and cytotoxicity, as wellas perturbations of the surfactant composition andsecretion.Current treatment of the genetic interstitial lung dis-

eases in children is unfortunately empirical. Corticoster-oids are anti-inflammatory and stimulate surfactantprotein transcription [22,23]. Chloroquine and its lesstoxic derivative hydroxychloroquine [24,25] are usedand believed to act on the lysosomal function, i.e.reduce vesicle fusion, exocytosis and proteolytic degra-dation [26,27] or stimulate lamellar body biogenesis[28]. Thus, there is a need to define the cellularmechanism of the currently applied treatments. Poten-tial therapeutic targets are cell chaperones which assistin normal protein folding and are part of the cytopro-tective mechanism that keeps control over misfoldedproteins either by folding them correctly or by directingthem to the degradation pathway [29,30]. Δexon4SFTPC mutation and proSP-CΔexon4 accumulation upre-gulate the major ER chaperone BiP in an attempt tomaintain surfactant biosynthesis in the presence of ER

stress [14]. The regulation of other chaperones, likeHSP90, HSP70, calreticulin and calnexin, is unknown.Even so, without pharmacological manipulation, suchcytoprotective mechanisms may not be sufficient tomaintain production of the bioactive surfactant with anormal lipid/protein composition. In addition, AECII,stressed by aberrantly processed proteins, might signalto and activate the surrounding cells, particularly thoseof immune system, which could contribute to the SP-C-associated disease.The goal of this study was to investigate the intracel-

lular disturbances and intercellular signaling of AECIIaffected by SP-CI73T expression and the ability of thepharmaceuticals commonly used in ILD therapy tomodulate some of the cellular mechanisms behind thediseases. We demonstrate the impact of I73T mutationon proSP-C processing, AECII stress tolerance, surfac-tant lipid composition and activation of the cells of theimmune system. In addition, we investigate modulationof the disease cellular mechanisms by pharmaceuticaldrugs applied in the ILD therapy.

ResultsMLE-12 cells process proSP-CI73T differently from proSP-CWT and accumulate proSP-CI73T processing intermediatesSP-C is synthesized exclusively by AECII as a 21 kDaproSP-C which is processed to the 4.2 kDa mature pro-tein through a sequence of C-terminal and N-terminalproteolytic cleavages [2,3]. To identify potential proces-sing differences between proSP-CWT and proSP-CI73T,MLE-12 cells were transfected with plasmid vectors,allowing expression of fusion proteins of proSP-C witheither C-terminal (N1) or N-terminal (C1) EGFP tag orN-terminal HA-tag. Stable expression of the N-termin-ally HA-tagged proSP-CWT resulted in appearance of astrong protein band at ~21 kDa and weak bands at 22kDa, 17 kDa, and 14 kDa (Figure 1A, left). ProSP-CI73T

yielded the same four bands, however all at equal inten-sity in relation to each other, indicating accumulation ofproSP-CI73T forms (Figure 1A, left). The postulated pro-cessing products based on their size and the fact thatthe N-terminal HA-tag was still present are depicted inFigure 1B. Mature SP-C was never detectable because ofthe loss of the protein tag due to the final processingsteps at the N-terminus.Transient expression of N-terminal and C-terminal

EGFP fusion proteins with proSP-C were detectable 24hours post transfection (Figure 1A, middle and right).Again, the processing intermediates of the N-terminallytagged fusion proteins differed between proSP-CWT andproSP-CI73T, showing accumulation of all four proSP-CI73T bands for the mutant protein (Figure 1A, middle). Incontrast, no differences in the band pattern betweenproSP-CWT and proSP-CI73T were observed for processing


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intermediates containing the C-terminal EGFP-tag (Figure1A, right). This suggests that there is no change in thecleavage pattern regarding the truncation of proSP-C fromthe C-terminus, being the first proSP-C cleavage step [2].The lowest band (Figure 1A and 1B, #10) corresponded tothe EGFP-tag, which has a size of ~27 kDa.In summary, the expression of SP-CI73T in MLE-12

cells resulted in the intracellular accumulation of

intermediate processing products. Such processing formsare also found in the BAL fluid of patients with thismutation [17,21,31] and may reflect alterations in folding,trafficking and/or processing of proSP-CI73T. Based onthese initial experiments we considered this in vitro cel-lular system to be an appropriate model to study theeffects of SFTPC mutations on cellular physiology andstress responses.

Figure 1 Processing features of proSP-CWT or proSP-CI73T. (A) Immunoblotting of total cell lysates with tag-specific antibodies. Cell lysatesobtained from MLE-12 cells stably expressing fusion protein of proSP-C with an N-terminal HA-tag (left panel), transiently transfected cellsexpressing fusion protein of proSP-C with an N-terminal EGFP (EGFP-C1, middle panel) or a C-terminal EGFP (EGFP-N1, right panel), present withseveral bands corresponding to different proSP-C processing intermediates, in which the tag sequence is retained. (B) Based on the size of thebands, the projected corresponding intermediate species of the fusion constructs are depicted. The cleavage sites are only estimates due to thelimited resolution of the technique. EGFP-C1 (band #5) and EGFP-N1 (band #9) are expressed as a full-length product of 48 kDa, HA-SP-C (band#1) of size 22 kDa.


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ProSP-CI73T localizes to different intracellularcompartments than proSP-CWT

The intracellular localization of preprotein species, mon-itored by immunofluorescence, differed between proSP-CWT and proSP-CI73T fusion proteins in MLE-12 cellsstably expressing N-terminally HA-tagged SP-C. Again,with this approach mature SP-C was not detectedbecause of the loss of the HA-tag due to the final pro-cessing steps at the N-terminus with only proSP-Cintermediates observed. ProSP-CWT forms were foundin the lamellar body-like structures detectable asLAMP3-positive vesicles in MLE-12 cells (Figure 2A).On the other hand, the proSP-CI73T signal was less vesi-cular with a stronger cytoplasmic background and apronounced signal at the cell border, but still partiallycolocalized with the LAMP3 (Figure 2A). This indicatesthat proSP-CI73T intermediates do traffic to some extentto LAMP3-positive vesicles. None of the proSP-C forms,

WT or I73T, colocalized with the ER-specific proteincalnexin, suggesting that no proSP-C species were ERretained (Figure 2B). Surfactant secretion is dependenton the fusion of lamellar bodies with the plasma mem-brane, which requires the activity of SNARE proteins,such as syntaxin 2 and SNAP-23, both associated tosome degree with lamellar bodies [6,7]. While proSP-CWT forms colocalized well with syntaxin 2, proSP-CI73T did not (Figure 2C). In contrast, proSP-CI73T

intermediates were found partially in early endosomesdetected as EEA1-positive vesicles, while proSP-CWT

was almost not present in those compartments (Figure2D), confirming earlier data [17]. Early endosomesusually contain endocytosed material that is destined forrecycling or degradation [32]. This suggests that physio-logical proSP-CWT forms are secreted via lamellar bodyfusion with the plasma membrane, while some proSP-CI73T forms might take a different route.

Figure 2 Intracellular localization of proSP-CWT and proSP-CI73T forms in stabile MLE-12 cells. Immunofluorescent analysis with anantibody against (A) lamellar body/lysosomal protein LAMP3 (red) and (B) ER protein calnexin (red) show complete localization of proSP-CWT

(green) forms and partial localization of proSP-CI73T (green) inside of lamellar body like organelles, and no colocalization of WT or mutant formswith calnexin indicating no ER retention. While proSP-CWT localized with (C) syntaxin 2, a protein found within lamellar bodies as surfactantsecretory vesicles, and proSP-CI73T only partially, (D) proSP-CI73T was detectable in a significant amount in EEA1-positive early endosomes. Nucleiare stained with DAPI (blue). Scale bars - 10 μm.


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Expression of SP-CI73T increases susceptibility of MLE-12cells to exogenous stress imposed by pharmacologicalsubstancesIn order to determine the impairment of cells thatexpress SP-CI73T, lactate dehydrogenase (LDH) release ofstably transfected cells was determined. Expression ofSP-CI73T led to an overall slightly increased LDH release,suggesting some reduction in cell viability (Figure 3).Upon exposure of stable MLE-12 to pharmacologicalsubstances currently applied in ILD therapy, the cellsexpressing SP-CI73T showed more pronounced increasein LDH release than cells expressing SP-CWT. Azathiopr-ine treatment resulted in the most pronounced LDHrelease, while the effect of hydroxychloroquine, methyl-prednisolone and cyclophosphamide was significantlylower (Figure 3). This demonstrates that the expressionof proSP-CI73T is a stress factor that may increase cellvulnerability and susceptibility to exogenous stress. Inaddition, our data suggest that some substances used inthe ILD therapy are a potent (azathioprine) to moderate(hydroxychloroquine) or milder (methylprednisolone andcyclophosphamide) stress factor per se.

Modulation of chaperone level in cells expressing SP-CWT

and SP-CI73T by pharmacologic substancesAfter demonstrating that SP-CI73T expression increasescell vulnerability to pharmacological stress stimuli, wefurther aimed to investigate the underlying intracellularmechanisms. Chaperone proteins are involved in the

folding of aberrantly processed proteins and producedby cells as a part of a cytoprotective mechanism to copewith increased intracellular stress and accumulation ofmisfolded proteins [29,30]. We determined a foldchange in the protein level of the two heat shock pro-teins, HSP90 and HSP70, and two ER-resident chaper-ones, calreticulin and calnexin, in MLE-12 cellsexpressing SP-CWT and SP-CI73T, before and after expo-sure to the pharmacologic substances used in ILD ther-apy: cyclophosphamide, azathioprine,methylprednisolone or hydroxychloroquine (Figure 4A-D, Table 1). The expression of HSP90 was increased sig-nificantly by all four pharmacologic substances tested inI73T cells compared to WT. Calreticulin was alsoincreased in I73T mutant after the treatment withhydroxychloroquine and HSP70 expression increasedafter treatment with cyclophosphamide compared toWT cells. Treatment with any of the four substancesdid not alter the expression of calnexin between SP-CWT and SP-CI73T-expressing cells (Figure 4). Overall,the exposure of SP-CI73T-expressing cells to selectedpharmacologic substances increased expression of somechaperones compared to SP-CWT cells, being a mechan-ism, which might enhance general cell folding capacity.

Pharmacological modulation of intracellular localizationof proSP-CWT and proSP-CI73T

Knowing that tested pharmacological substances enhancechaperone expression in cells with SP-CI73T in comparison

Figure 3 Viability of MLE12 cells expressing SP-CWT or SP-CI73T before and after treatment with drugs used in therapy. MLE-12 cellsstably expressing SP-CWT or SP-CI73T were incubated 24 hours with 10 μM of cyclophosphamide (+Cyc), azathioprine (+Aza), methylprednisolone(+Met), or hydroxychloroquine (+Hyd). LDH release, a sign of deceased cell fitness, of treated vs. untreated (-) cells is expressed as % of totalLDH. Only significant p-values are depicted.


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to those expressing SP-CWT and that proSP-CI73T formsare mislocalized to early endosomal vesicles, we investi-gated influence of two drugs used in ILD therapy, hydro-xychloroquine and methylprednisolone, on proSP-CWT

and proSP-CI73T. We applied again syntaxin 2 and EEA1as markers for correctly localized and mislocalized proSP-C respectively [7], in a quantitative immunofluorescencestudy in order to determine the percentage of proSP-C-positive vesicles that colocalized with either of the two

protein markers. As shown in Figure 2, we observed highlevel of colocalization of proSP-CWT forms with syntaxin 2and of proSP-CI73T with EEA1 (Figure 5A and 5B). Expo-sure to both pharmacological substances reduced the loca-lization of proSP-CWT to syntaxin 2-positive vesicles andmoved it toward EEA1-positive vesicles. However, evenafter the drug treatment the colocalization level of WTwith EEA1 remained significantly under the level detectedin non-treated I73T mutant (Figure 5B). Furthermore,while hydroxychloroquine did not significantly improvemislocalization defect of the proSP-CI73T forms, weobserved correctional effect of methylprednisolone onlocalization of proSP-CI73T. Namely, methylprednisoloneincreased localization of the proSP-CI73T forms to the syn-taxin 2-positive (Figure 5A) vesicles and decreased theircolocalization with EEA1 (Figure 5B). Nevertheless, evenafter the pharmacological treatment proSP-CI73T nevercompletely acquired WT localization features (Figure 5Aand 5B). Our data suggest the ability of the methylpredni-solone drug to partially correct mislocalization defect ofproSP-CI73T.

Figure 4 Modulation of chaperone level in the cells expressing SP-CWT and SP-CI73T by pharmacologic substances. Shown is the foldchange in protein level of the chaperone proteins HSP90, HSP70, calreticulin and calnexin in MLE-12 cells expressing proSP-CWT and proSP-CI73T

before and after their exposure to the drugs commonly applied in ILD therapy: (A) hydroxychloroquine, (B) methylprednisolone, (C) azathioprineand (D) cyclophosphamide. Only significant p-values are presented.

Table 1 Chaperone level in MLE-12 cells expressingmutant SP-CI73T in response to drugs used in ILDtherapy.

HSP 90 Calreticulin HSP 70 Calnexin

Cyclophosphamide + 30% * + 13% * + 41% * + 16%

Azathioprine +36% * + 8% + 7% - 13%

Hydroxychloroquine + 81% * + 75% * + 18% + 6%

Methylprednisolone + 55% * + 26% * + 8% - 9%

Presented are the results of three independent experiments and expressed as% changes of the protein amounts in SP-CI73T cells compared to SP-CWT

expressing cells; * denotes p-values < 0.05


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Alterations in the intracellular lipid composition andcomposition of secreted lipids due to expression of SP-CI73T and their response to pharmacological treatmentThe packaging and secretion of lung surfactant lipids isvery closely linked to the expression of the hydrophobicsurfactant proteins in AECII [5]. Mass spectrometriclipid analysis showed that total phospholipid amountwas not changed in transfected MLE-12 cells (Table 2).However, the phospholipid composition was significantlyaltered: phosphatidylcholine (PC) and sphingomyelinwere decreased and lyso-phosphatidylcholine (LPC) andphosphatidylethanolamine were increased in I73Tmutant cells (Table 2, Figure 6). Treatment with methyl-prednisolone or hydroxychloroquine did not correct theloss of PC in SP-CI73T expressing cells, but it did ame-liorate the LPC increase (Figure 6A). Also significantchanges in the pattern of the fatty acids molecular spe-cies of different phospholipid classes were measured(Table 2, Additional file 1: Supplemental Table S1), sug-gesting that the lipid sorting processes of the cells werealso affected substantially.The phospholipid secretion by MLE-12 cells was

assessed in the supernatant. Similar as in the intracellu-lar lipid pattern, PC was decreased by 27% and LPC wasincreased by 57% in cells expressing SP-CI73T (Figure6B), with no changes detected for other phospholipids(data not shown). Interestingly, the treatment withmethylprednisolone or hydroxychloroquine amelioratedthe reduction of PC, but had no effect on LPC. Ourdata suggest that the expression of SP-CI73T affected thelipid composition of AECII and alveolar pulmonary sur-factant profoundly. The major surfactant phospholipid

PC was reduced with a concomitant increase in LPC,suggesting increased activity of phospholipases. Treat-ment with methylprednisolone or hydroxychloroquinecorrected to some extent these alterations back towardthe WT level.

MLE-12 cells expressing SP-CI73T secrete soluble factorsthat stimulate surface expression of CCR2 and CXCR1 onCD4+ lymphocytes and CXCR1 on neutrophilsInjury of the lung epithelial cell caused by endogenousand exogenous stress may be communicated to the sur-rounding immune cells, in particular to the pulmonary

Figure 5 Influence of methylprednisolone and hydroxychloroquine on intracellular localization of SP-CWT or SP-CI73T. Stabile MLE-12cells expressing SP-CWT or SP-CI73T were incubated 24 hours with 10 μM of methylprednisolone (+Met) or hydroxychloroquine (+Hyd) andanalyzed by immunofluorescence followed by quantification of colocalization or proSP-C (HA-tag) with syntaxin 2 (marker of surfactant secretoryvesicles) and EEA1 (endosomal marker). Only significant p-values are shown.

Table 2 Phospholipid profile of transfected MLE-12 cellsstably expressing mutant SP-CI73T.

WT I73T P(Anova)

Total phospholipids (nmol/mgprotein)

152.80 ±9.6

153.10 ±10.0

ns

Phospholipid classes (% of totalPL)

Phosphatidylcholine 57.80 ±1.0

53.70 ± 0.8 < 0.001

Lyso-Phosphatidylcholine 0.60 ± 0.1 1.00 ± 0.8 < 0.001

Phosphatidylglycerol 0.30 ± 0.0 0.20 ± 0.1 ns

Sphingomyelin 6.20 ± 0.3 5.50 ± 0.2 < 0.05

Ceramide 1.80 ± 0.1 1.80 ± 0.1 ns

Glucosyl-Ceramide 0.10 ± 0.0 0.10 ± 0.0 ns

Phosphatidylethanolamine 11.20 ±0.5

14.70 ± 0.8 < 0.001

Phosphatidylserine 6.50 ± 0.1 6.60 ± 0.3 ns

Data are means of three independent experiments each performed induplicate. P-values are given.


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leukocytes, leading to inflammation and cell remodeling.Chemokine receptors recruit leukocytes to the alveolarsite of inflammation and orchestrate local immuneresponses [33,34]. In the previous studies we demon-strated that, among a plethora of chemokine receptorsinvolved in this network, specifically CCR2 on lympho-cytes and CXCR1 on neutrophils, modulate pulmonaryimmunity in human inflammatory lung diseases [35,36].Therefore, we examined whether cells expressing SP-CI73T stimulated the expression of CCR2 on lymphocytes

and CXCR1 on neutrophils by incubating isolated neu-trophils or lymphocytes with 7-fold concentrated super-natants of MLE-12 cells expressing WT or I73T SP-C. Asa result, CD8+ lymphocytes did not show a differencebetween WT and I73T mutant, however CD4+ lympho-cytes showed an increased level of surface receptor CCR2expression in response to the supernatant of proSP-CI73T

expressing cells (Figure 7A). We observed the same pat-tern with CXCR1, which was increased on CD4+ lym-phocytes after incubation with the mutant cell

Figure 6 Intracellular lipid content and lipid secretion of MLE-12 cells expressing SP-CWT or SP-CI73T. (A) Intracellular lipid content of cellsstably expressing WT SP-C or I73T mutant were quantified by mass spectrometry. Untreated cells (-) or cells treated with 10 μMmethylprednisolone (+Met) or hydroxychloroquine (+Hyd) for 24 hours prior to sample isolation. Values were calculated as % of the mean of theuntreated WT values. (B) after removal of detached cells, the lipids in the cell supernatant were analysed and presented as in (A). The graphsshow relative amounts of phosphatidylcholine and lyso-phosphatidylcholine. Only significant p-values are depicted.


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supernatant, but was unaltered on CD8+ lymphocytes(Figure 7B). We further analyzed the surface receptorexpression on neutrophils. The supernatant of SP-CI73T

expressing cells increased the level of CXCR1 expressionon neutrophils, but did not affect CD11b levels (Figure7C). Non-concentrated supernatants gave the sameresults, although less pronounced and a clear concentra-tion dependency of the effects was observed (data notshown). This suggests that SP-CI73T-expressing MLE-12cells were able to modulate the surface receptor expres-sion on the cells of immune system through the secretionof soluble factors into the medium.

DiscussionMutations in the SFTPC gene are a known cause of sur-factant deficiency and very variable genetic ILD in chil-dren and adults. We investigated the intracellulardisturbances and intercellular signaling of MLE-12 cellsexpressing SP-CI73T and the ability of pharmaceuticaldrugs used in ILD therapy to modulate some of the cel-lular consequences of SP-C deficiency caused by thismutation. MLE-12 cells were chosen as a model systemsince they contain structures, which resemble lamellarbodies seen in AECII [37]. The presence of lamellarbody-like structures in the cells used was confirmed byelectron microscopy (data not shown). Here we namedthe organelles detectable as LAMP3-positive vesicles,lamellar body-like structures.A potential limitation of the study is that our system

corresponds rather to a homozygous than to a heterozy-gous SFTPC mutation where one WT copy is still pre-sent. Although endogenous SP-C is expressed in theMLE-12 cells [38], expression of exogenous SP-C fromthe CMV promoter present on the plasmid vector islikely higher. However, all known patients with SP-Cmutations are heterozygous, expressing one copy of thewild type gene. Thus, the experimental model reflectsthe in vivo condition. In addition, in contrast to SP-CΔexon4 or SP-CL188Q, I73T is a non-BRICHOS domainmutation and does not lead to the conformational dis-ease observed for BRICHOS domain mutations wherethe mutant allele acts in a dominant-negative way [13].Consequently, I73T still allows for the production ofmature SP-C in vivo [17].Stable transfection of MLE-12 cells with SP-CWT or

SP-CI73T led to the intracellular accumulation of proSP-CI73T processing intermediates which were not found incells with proSP-CWT, but corresponded well to speciesin the BAL fluid of patients with this mutation [17] (Fig-ure 1). The first step in proSP-C processing is a cleavageat the C-terminal end [2]. Using an EGFP-tag fused tothe C-terminus of proSP-C showed no difference in pro-cessing intermediates of proSP-CWT and proSP-CI73T

(Figure 1, right). This means that (a) the first cleavage

Figure 7 Surface receptor expression on human lymphocytesand neutrophils. Neutrophils and lymphocytes were isolated fromthe whole blood of different human donors and incubated with 7-fold concentrated supernatants obtained from MLE-12 cellsexpressing SP-CWT or SP-CI73T prior to flow cytometry analysis. Non-concentrated supernatants gave the same results, although lesspronounced with a clear concentration dependency of the effects(data not shown). The receptor levels on the surface of lymphocytesafter incubation with antibodies directed against (A) CCR2 or (B)CXCR1 are shown and expressed as mean fluorescence intensity(MFI). Another second marker-specific antibody was applied todistinguish between CD4+ and CD8+ lymphocytes. (C) The levels ofCXCR1 and CD11b on isolated neutrophils. Significant changes aredepicted with the corresponding p-values.


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step happening at C-terminus is not influenced by thismutation and (b) the mutation does not interfere withthe export from the ER and Golgi, because this cleavageoccurs after trafficking through these compartments[2,3]. In addition, immunofluorescence assays showedneither proSP-CWT nor proSP-CI73T retention in the ERcompartment (Figure 2), supporting the conclusionsmade from the immunoblots. To examine the proces-sing following the first C-terminal cleavage, we appliedN-terminal protein tags. Dominant proSP-CWT inter-mediates, that were also identified for proSP-CI73T, werethe species after the first C-terminal cleavage (Figure 1,bands #2 and #6), and the species before the first N-terminal cleavage (Figure 1, band #8). The primary full-length translation product (Figure 1, bands #1 and #5)was only faintly detectable for proSP-CWT. Expressionof proSP-C in this model is under control of a CMV-promoter, not the native SP-C promoter. It is thereforeunlikely that a feedback mechanism is responsible for ahigher expression of proSP-CI73T intermediates. It ismore likely that the I73T mutation slows down the pro-cessing and/or trafficking of the mutant proSP-C, lead-ing to accumulation of incompletely processed proSP-C.It is not known how this mutation affects the folding ofproSP-C, but subtle changes in conformation may alsobe responsible for the abundance of another processingintermediate, of size ~17 kDa (Figure 1, band #3 and#7). This intermediate can be found in the BAL fluid ofpatients with the I73T mutation, suggesting that thisproSP-C form is being secreted from AECII along withthe mature SP-C that is produced by AECII regardlessof the presence of the I73T mutation [17].Immunofluorescence assay of stably transfected MLE-

12 showed that proSP-CI73T colocalized often withEEA1 positive vesicles (Figure 2A), confirming our pre-vious report (12). Early endosomes generally containmaterial that is taken up by endocytosis and is eitherrecycled or routed for degradation [32]. Up to 80% ofused lung surfactant is known to be reinternalized byAECII from alveolar space [5]. Although immunofluor-escence does not allow the distinction between differentEGFP-positive species depicted in Figure 1B, we specu-late that the proSP-CI73T species in the EEA1 positivecompartment might be primarily the additional prepro-tein species accumulating in the I73T mutant. They aresecreted only by the AECII with the I73T mutation andmight be reinternalized as well. On the other hand,proSP-CWT, but rarely proSP-CI73T, colocalized withsyntaxin 2, a SNARE protein involved in the secretionof lung surfactant, found in the plasma membrane andlamellar bodies of AECII (Figure 2B). Interestingly, ourdata propose the influence of hydroxychloroquine andmethylprednisolone on localization and routing ofproSP-CWT moving it toward early endosomal vesicles.

On the other hand, methylprednisolone showed thecapacity to partially correct the mislocalization/routingdefect of proSP-CI73T (Figure 5).The expression of mutated proteins frequently results

in elevated cell stress. This has been shown for the BRI-CHOS domain SP-C mutations L188Q and Δexon4[14,15]. We found that the constitutive expression ofSP-CI73T moderately increased cell lethality under nor-mal growth conditions (Figure 3A), maybe as a result ofthe ability of the cellular system to adapt to the pre-sence of stress, as reported in [39]. The additional exo-genous stress, imposed in our experiments by exposureto pharmaceuticals used in ILD therapy, might shift thisbalance out of the tolerable range. Treatment of thecells with azathioprine drug almost doubled the numberof dying I73T mutant cells compared to WT. Thisaggravation was much less pronounced in the presenceof methylprednisolone, hydroxychloroquine orcyclophosphamide.Intracellular stress is in part handled by endogenous

chaperones. Still without pharmacological boost, suchcytoprotective mechanisms may not always be sufficientto normalize the cell function and maintain productionof the bioactive surfactant with a normal lipid/proteincomposition. We determined the change in expressionof the four important chaperones under the influence ofthe same ILD drugs. We found that the influence ofazathioprine on the chaperones was almost the same inproSP-CWT-and proSP-CI73T-expressing cells, leaving noprotection for additional stress, being a potent stressfactor per se (Figure 4A-D). In contrast, hydroxychloro-quine treatment led to an 81% increase in HSP90, and75% increase in calreticulin expression in I73T mutantcells over WT cells (Table 1), thereby possibly protect-ing the cells against the additional stress and enhancingthe ER folding capacity. HSP90 seemed to be targetedby all tested pharmaceuticals, while calnexin levels wererefractory to stimulation (Figure 4). Treatment with thefour drugs did not change the pattern of the proSP-Cprocessing bands observed in the immunoblots in Figure1A (data not shown).The lipid composition of the stable MLE-12 cells was

similar to that previously described in human foetalAECII, especially with regard to PC composition [40]. Inthe SP-CI73T expressing cells we found a pronounceddrop of total cellular PC, whereas LPC was increased(Figure 6A, additional file 1: supplemental Table S1). Itis known that PC is degraded to LPC by an intrinsicphospholipase A2-like activity, and that LPC is toxic tovarious cells [41]. Increased LPC may therefore be aresult of increased phospholipase activity due to the pre-sence of mutated SP-C. SP-C dysfunction may also leadto a diminished activity of acyltransferases which reace-tylate LPC. LPC is a known inhibitor of the lung


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surfactant activity and has the ability to penetratedirectly into interfacial films to impair lowering of thealveolar surface tension during dynamic compression[42,43]. Elevated LPC levels in the SP-CI73T-expressingcells could also explain the heightened sensitivitytowards exogenous stress described above. Generationof LPC cannot account for the decrease of PC mass inSP-CI73T expressing cells, but additional factors, whichdirectly interfere with the synthesis and packaging ofPC, must also be responsible. This is in line with theobserved grossly altered pattern of the fatty acid speciesof the different phospholipid classes, including PC inSP-CI73T cells (Table 2, additional file 1: supplementalTable S1).AECII secrete the surfactant phospholipids into the

alveolar space where it lowers surface tension. Amongphospholipids secreted by the I73T mutants PC wasagain decreased by 27% and LPC was increased by 57%,compatible with a reduced surfactant function [40,43].Treatment with methylprednisolone or hydroxychloro-quine ameliorated the increase in intracellular andsecreted LPC and decrease in secreted PC, but did notcompletely correct it (Figure 6A, B). The capacity of thetreatment with methylprednisolone and hydroxychloro-quine to correct the lipid disturbances caused by I73Tmutation (Figure 6B) represent one of the mechanismsby which these treatments are empirically helpful insome patients with I73T mutations (own unpublishedresults, [19]).Lastly, the index patient with the I73T mutation in

our previous study displayed a mild interstitial chronicinflammation and most of the infiltrated leukocyteswere CD3+ and CD4+ T-lymphocytes [17]. We foundthat cells with the I73T mutation released soluble fac-tors into the medium that increase surface expression ofCCR2 and CXCR1 on CD4+ lymphocytes and CXCR1on neutrophiles (Figure 7A-C). When activated, the highaffinity IL-8 receptor CXCR1 mediates antibacterial kill-ing capacity [36,44]. Increases in surface expressionlevels of CCR2 and CXCR1, respectively, might have thepotential to modulate the pulmonary immune responsewith regard to antibacterial (CXCR1) and profibrotic(CCR2) responses [36,45]. However, the soluble factorsinvolved in the induction of chemokine receptor expres-sion as well as the functional consequences of this phe-nomenon remain to be addressed in future studies.

ConclusionsWe showed impaired proSP-C processing, altered cellu-lar stress tolerance and unfavorable changes of the sur-factant lipid composition in a murine AECII model cellline. Some of the demonstrated cellular aspects behindthe disease could be modulated with drugs used in thetherapy of ILD patients, thereby giving insight into their

potential therapeutic mechanism on a cellular level. Wealso demonstrated that AECII with I73T mutation couldsignal to the surrounding cells of the immune systemthrough secretion of soluble factors. Therefore, ourstudy adds to understanding of the effects that SFTPCmutations impose on (pro)SP-C and AECII cell biologyand pave the way for a more precise pharmacologicaltargeting in patients with SP-C deficiency.

MethodsPlasmid vectorsEukaryotic expression vectors containing the full humanSFTPC gene fused to either EGFP-tag (pEGFP-N1/hSP-C1-197 and pEGFP-C1/hSP-C1-197 to obtain proSP-C withEGFP fused to the C- or N-terminus respectively) orhemagglutinin (HA)-tag (proSP-C with N-terminal HA-tag) were obtained as previously described [12]. I73Tpoint mutation was introduced into the wild-type (WT)SFTPC gene in these vectors using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, USA) andthe following primers: I73T_forward: 5’-GGT TCT GGAGAT GAG CAC TGG GGC GC-3’, I73T_reverse:5’-GCG CCC CAG TGC TCA TCT CCA GAA CC-3’,following the recommended protocol. The successfulmutagenesis was confirmed by DNA sequencing.

MLE-12 cell lines and transfectionThe mouse MLE-12 lung epithelial cell line (CRL-2119)[38] was obtained from the American Type Culture Col-lection (ATCC) and maintained in RPMI medium sup-plemented with 10% FBS. Cells were transfected usingFuGene 6 (Roche, Penzberg, Germany) according to themanufacturer’s protocol. Stable transfection of MLE-12cells with pcDNA3/HA-hSP-C1-197 and pcDNA3/HA-hSP-CI73T vectors was obtained by selecting transfectedcells in the presence of 600 μg/ml G418 in RPMI med-ium for four weeks. For drug exposure experimentsstable cells were grown 24 hours in the presence of 10μM of cyclophosphamide, azathioprine, methylpredniso-lone or hydroxychloroquine.

ImmunoblottingTotal cell proteins were obtained by lysing the cells inlysis buffer (PBS, 20 mM EDTA, 1% v/v Elugent (Cal-biochem, Bad Soden, Germany), protease inhibitor(Complete; Roche, Manheim, Germany). For immuno-blotting 30 μg protein were separated under reducingconditions using 10% NuPage Bis-Tris (Invitrogen,Karlsruhe, Germany) and transferred to a PVDF mem-brane. The following primary antibodies were used:monoclonal rat anti-HA-tag (1:1000; Roche), monoclo-nal mouse anti-GFP (1:500; Clontech, Heidelberg, Ger-many) and polyclonal goat anti-calnexin (1:500),polyclonal goat anti-calreticulin (1:500), monoclonal


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mouse anti-HSP90a/b, polyclonal goat anti-HSP70(1:1000) and monoclonal anti-b-actin HRP conjugate(1:10000) (all from Santa Cruz Biotechnology, SantaCruz, CA). Signal was detected using chemiluminiscentlabeling with Amersham ECL Detection Reagents (GEHealthcare), densitometrically quantified and normalizedto the b-actin signal.

Immunofluorescence24 hours after transfection cells grown on coverslips werefixed with 4% paraformaldehyde, permeabilised with 10%Triton X-100, blocked 30 min in PBS with 5% FBS. Thefollowing primary antibodies were used and all in 1:200dilution: polyclonal rabbit anti-mouse LAMP3 (SantaCruz), monoclonal mouse anti-human CD63/LAMP3(Chemicon, Schwalbach, Germany), polyclonal rabbitanti-EEA1 (Acris Antibodies, Herford, Germany), mono-clonal mouse anti-ubiquitin (Biomol, Hamburg, Ger-many) and polyclonal rabbit anti-syntaxin 2 (SynapticSystems, Berlin, Germany). Species specific Alexa Fluor488 or Alexa Fluor 555 secondary antibodies (Invitrogen)were used at 1:200. Samples were mounted and AlexaFluor or GFP fluorescence was examined with Axiovert135 fluorescent microscope and evaluated with AxioVi-sion 4.7.1 software (Carl Zeiss, Jena, Germany). For semi-quantitative assessment of colocalization, high magnifica-tion confocal microscope images were used. On 14 to 27different coverslips at least 100 vesicles stained for SP-Cand/or syntaxin 2 were counted in a blinded fashion andthe percentage of vesicles showing staining for both mar-kers was calculated. Similarly, the percentage of vesiclesstained for SP-C and EEA-1 was calculated.

Lactate dehydrogenase (LDH) assayLDH activity in cell lysates and culture supernatants wasdetermined by the method of Decker and Lohmann-Matthes [46]. Briefly, 100 μl of samples were mixed with30 μl dye solution (18 mg/ml L-lactate, 1 mg/ml iodoni-trotetrazolium in PBS). After adding 15 μl of the catalyst(3 mg/ml NAD+, 2.3 mg/ml diaphorase, 0.03% BSA,1.2% sucrose in PBS), absorbance at 492 nm was deter-mined at one minute intervals for 15 minutes at 37°C.Absolute LDH activity was calculated from a standardcurve, using purified LDH (Sigma, Munich, Germany).The lower limit of detection was 20 Units/L; the assaywas linear to 2500 Units/L.

Mass spectrometric lipid analysisFor lipid analysis cells grown in Petri dishes were har-vested by scraping off in 2 mL PBS supplemented withprotease inhibitor (Complete, Roche). The cell suspen-sion was sonicated (four strokes, 10 seconds; BransonDigital Sonifier S450D). Lipid classes and subspecieswere determined by electrospray ionization tandem mass

spectrometry (ESI-MS/MS) using direct flow injectionanalysis, as described previously [47,48]. Cells wereextracted according to the Bligh and Dyer method in thepresence of non-naturally occurring lipid species used asinternal standards [49]. A precursor ion scan of m/z 184specific for phosphocholine containing lipids was usedfor phosphatidylcholine (PC), sphingomyelin (SPM) [48]and lysophosphatidylcholine (LPC) [47]. Neutral lossscans of m/z 141 and m/z 185 were used for phosphati-dylethanolamine (PE) and phosphatidylserine (PS),respectively [50]. Phosphatidylglycerol (PG) was analyzedusing a neutral loss scan of m/z 189 of ammoniumadduct ions [51]. Ceramide and glucosylceramide wereanalyzed as previously described [52] using N-heptadeca-noyl-sphingosine as internal standard. Quantification wasachieved by calibration lines generated by addition ofnaturally occurring lipid species to pooled cell homoge-nate. All lipid classes were quantified with internal stan-dards belonging to the same lipid class, except SM (PCinternal standards). Each lipid class was calibrated with avariety of species covering chain lengths and number ofdouble bonds of naturally occurring species. Correctionof isotopic overlap of lipid species and data analysis wasperformed by self-programmed Excel macros for all lipidclasses according to the described principles [48].

Flow cytometryHuman lymphocytes and neutrophils were isolated fromwhole blood using LeucoSep (Greiner Bio-One, Solin-gen-Wald, Germany) and Ficoll-Isopaque gradient den-sity isolation method (GE Healthcare) according to themanufacturer’s instructions. Cells were incubated for 6hours (neutrophils) or 24 hours (lymphocytes) at 37°Cwith supernatants of MLE-12 cells expressing wild-typeor mutant proSP-C. Cell-free supernatants were col-lected after 48 hours of growth and concentrated 7-fold,using Microsep 1 k centrifugal concentrators (Millipore,Schwalbach, Germany). Cells were analyzed by four-col-our flow cytometry (FACSCalibur; BD Pharmingen, Hei-delberg, Germany) as described previously [35]. Thefollowing antibodies were used: PE-conjugated mouseanti-human CCR2-B (R&D Systems, Minneapolis, USA),FITC labeled anti-human CD8, FITC labeled anti-human CD4, PE-conjugated mouse anti-human CD11b/Mac-1, PE-conjugated mouse anti-human CD181(CXCR1, IL-8RA) (all BD Pharmingen). Results are pre-sented as mean fluorescence intensity (MFI) after sub-tracting background binding provided by non-specificisotypes. Calculations were performed with CellQuestanalysis software (BD Pharmingen).

Statistical methodsSince the data was distributed non-Gaussian, non-para-metric tests were used for comparison of two unpaired


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groups (Mann Whitney test). The results are given asmeans ± standard error (SE) of the individual number ofdifferent subjects, each individual value representing themean of 3-4 determinations or as indicated. For lipid analy-sis the results are presented as means with standard devia-tion and comparisons were made by ANOVA followed byTukey’s post hoc multi comparisons test. For correlations,Spearman’s non-parametric test was used. P-values of lessthan 0.05 were considered statistically significant.

Additional material

Additional file 1: Table S1. Phospholipid classes and molecularspecies profile of transfected MLE-12 cells stably expressing SFTPCI73T mutation. Data are means and standard deviation of threeindependent experiments, each performed in duplicate. The mutant(I73T) and wild type (WT) were compared by ANOVA followed by Tukeysmultiple comparison test. P-values are shown.

List of abbreviationsSP-C: surfactant protein C; SFTPC: surfactant protein C gen; SP-B: surfactantprotein B; ILD: interstitial lung disease; BAL: bronchial lavage; AECII: alveolartype II cells; SNARE: SNAP receptors; SNAP-23: soluble NSF attachmentprotein 23; EGFP: enhanced green fluorescent protein; HA: hemagglutinin;LAMP3: lysomal-associated membrane protein 3; EEA1: early endosomalantigen 1; HRP: horseradish peroxidase; LDH: lactate dehydrogenase; ER:endoplasmic reticulum; HSP70/90: heath shock protein 70/90; PC:phosphatidylcholine; LPC: lysophosphatidylcholine; PE1:phosphatidylethanolamine; PS: phosphatidylserine; SPM: sphingomyelin; PG:phosphatidylglycerol; CXCR1: CXC chemokine receptor; CCR2: chemokine C-C motif receptor 2; FITC: fluorescein isothiocyanate; PE2: phycoerythrin; MFI:mean fluorescence intensity; CYC: cyclophosphamide; AZA: azathioprine;MET: methylprednisolone; HYD: hydroxychloroquine.

Authors’ contributionsMG, MW, CS and SK conceived and designed the study as well as analyzedand interpreted the data. CS, MW, SK, TT performed the experiments withexception of MS lipid analyses, performed by GL and GS, and flowcytometry analyses, performed by AH and DH. MW, SK and MG wrote thepaper. SM and MFB generated and provided the necessary wild type vectorsand participated in drafting and reviewing the manuscript. All authors readand approved the final manuscript.

Authors’ informationThis paper contains parts of the doctoral thesis of Christiane Sparr. Until2009 Sunčana Kern published as Sunčana Moslavac.

AcknowledgementsThis study was supported by Deutsche Forschungsgemeinschaft (DFG)Grants GR970-7.2 and GR970-7.3 to M.G. and National Institute of HealthGrant HL-19737 to M.F.B. We thank to Eva Kaltenborn for critical reading ofthe text and Stefanie Gruschka for excellent technical assistance.

Author details1Department of Pneumology, Dr. von Hauner Children’s Hospital, Ludwig-Maximilians University, Lindwurmstr. 4, Munich, 80337, Germany. 2Institutefor Clinical Chemistry and Laboratory Medicine, University of Regensburg,Franz-Josef-Strauss-Allee 11, Regensburg, 93053, Germany. 3Pulmonary andCritical Care Division, University of Pennsylvania School of Medicine, 380 S.University Avenue, Philadelphia, PA 19104, USA.

Received: 7 May 2010 Accepted: 20 November 2010Published: 20 November 2010

References1. Beers MF, Kim CY, Dodia C, Fisher AB: Localization, synthesis, and

processing of surfactant protein SP-C in rat lung analyzed by epitope-specific antipeptide antibodies. J Biol Chem 1994, 269:20318-20328.

2. Brasch F, Ten Brinke A, Johnen G, Ochs M, Kapp N, Muller KM, et al:Involvement of cathepsin H in the processing of the hydrophobicsurfactant-associated protein C in type II pneumocytes. Am J Respir CellMol Biol 2002, 26:659-670.

3. Beers MF, Mulugeta S: Surfactant protein C biosynthesis and its emergingrole in conformational lung disease. Annu Rev Physiol 2005, 67:663-696.

4. Hamvas A, Cole FS, Nogee LM: Genetic disorders of surfactant proteins.Neonatology 2007, 91:311-317.

5. Andreeva AV, Kutuzov MA, Voyno-Yasenetskaya TA: Regulation ofsurfactant secretion in alveolar type II cells. Am J Physiol Lung Cell MolPhysiol 2007, 293:L259-L271.

6. Abonyo BO, Wang P, Narasaraju TA, Rowan WH, McMillan DH,Zimmerman UJ, et al: Characterization of alpha-soluble N-ethylmaleimide-sensitive fusion attachment protein in alveolar type IIcells: implications in lung surfactant secretion. Am J Respir Cell Mol Biol2003, 29:273-282.

7. Abonyo BO, Gou D, Wang P, Narasaraju T, Wang Z, Liu L: Syntaxin 2 andSNAP-23 are required for regulated surfactant secretion. Biochemistry2004, 43:3499-3506.

8. Coultas DB, Zumwalt RE, Black WC, Sobonya RE: The epidemiology ofinterstitial lung diseases. Am J Respir Crit Care Med 1994, 150:967-972.

9. Clement A, Eber E: Interstitial lung diseases in infants and children. EurRespir J 2008, 31:658-666.

10. Hartl D, Griese M: Interstitial lung disease in children – geneticbackground and associated phenotypes. Respir Res 2005, 6:32.

11. Nogee LM: Alterations in SP-B and SP-C expression in neonatal lungdisease. Annu Rev Physiol 2004, 66:601-623.

12. Wang WJ, Mulugeta S, Russo SJ, Beers MF: Deletion of exon 4 fromhuman surfactant protein C results in aggresome formation andgeneration of a dominant negative. J Cell Sci 2003, 116:683-692.

13. Nerelius C, Martin E, Peng S, Gustafsson M, Nordling K, Weaver T, et al:Mutations linked to interstitial lung disease can abrogate anti-amyloidfunction of prosurfactant protein C. Biochem J 2008, 416:201-209.

14. Mulugeta S, Nguyen V, Russo SJ, Muniswamy M, Beers MF: A surfactantprotein C precursor protein BRICHOS domain mutation causesendoplasmic reticulum stress, proteasome dysfunction, and caspase 3activation. Am J Respir Cell Mol Biol 2005, 32:521-530.

15. Mulugeta S, Maguire JA, Newitt JL, Russo SJ, Kotorashvili A, Beers MF:Misfolded BRICHOS SP-C mutant proteins induce apoptosis via caspase-4- and cytochrome c-related mechanisms. Am J Physiol Lung Cell MolPhysiol 2007, 293:L720-L729.

16. Lawson WE, Crossno PF, Polosukhin VV, Roldan J, Cheng DS, Lane KB, et al:Endoplasmic reticulum stress in alveolar epithelial cells is prominent inIPF: association with altered surfactant protein processing andherpesvirus infection. Am J Physiol Lung Cell Mol Physiol 2008, 294:L1119-L1126.

17. Brasch F, Griese M, Tredano M, Johnen G, Ochs M, Rieger C, et al:Interstitial lung disease in a baby with a de novo mutation in the SFTPCgene. Eur Respir J 2004, 24:30-39.

18. Cameron HS, Somaschini M, Carrera P, Hamvas A, Whitsett JA, Wert SE,et al: A common mutation in the surfactant protein C gene associatedwith lung disease. J Pediatr 2005, 146:370-375.

19. Abou TR, Jaubert F, Emond S, Le Bourgeois M, Epaud R, Karila C, et al:Familial interstitial disease with I73T mutation: A mid- and long-termstudy. Pediatr Pulmonol 2009, 44:167-175.

20. Tredano M, Griese M, Brasch F, Schumacher S, de Blic J, Marque S, et al:Mutation of SFTPC in infantile pulmonary alveolar proteinosis with orwithout fibrosing lung disease. Am J Med Genet A 2004, 126A:18-26.

21. Galetskiy D, Woischnik M, Ripper J, Griese M, Przybylski M: Aberrantprocessing forms of lung surfactant proteins SP-B and SP-C revealed byhigh-resolution mass spectrometry. Eur J Mass Spectrom (Chichester, Eng)2008, 14:379-390.

22. Fisher JH, McCormack F, Park SS, Stelzner T, Shannon JM, Hofmann T: Invivo regulation of surfactant proteins by glucocorticoids. Am J Respir CellMol Biol 1991, 5:63-70.


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http://www.biomedcentral.com/content/supplementary/1471-2121-11-88-S1.PDF

23. Solarin KO, Ballard PL, Guttentag SH, Lomax CA, Beers MF: Expression andglucocorticoid regulation of surfactant protein C in human fetal lung.Pediatr Res 1997, 42:356-364.

24. Avital A, Godfrey S, Maayan C, Diamant Y, Springer C: Chloroquinetreatment of interstitial lung disease in children. Pediatr Pulmonol 1994,18:356-360.

25. Barbato A, Panizzolo C: Chronic interstitial lung disease in children.Paediatr Respir Rev 2000, 1:172-178.

26. Meshnick SR: Chloroquine as intercalator: a hypothesis revived. ParasitolToday 1990, 6:77-79.

27. Kalina M, Socher R: Endocytosis in cultured rat alveolar type II cells: effectof lysosomotropic weak bases on the processes. J Histochem Cytochem1991, 39:1337-1348.

28. Reasor MJ: A review of the biology and toxicologic implications of theinduction of lysosomal lamellar bodies by drugs. Toxicol Appl Pharmacol1989, 97:47-56.

29. Buck TM, Wright CM, Brodsky JL: The activities and function of molecularchaperones in the endoplasmic reticulum. Semin Cell Dev Biol 2007,18:751-761.

30. Yoshida H: ER stress and diseases. FEBS J 2007, 274:630-658.31. Bai Y, Galetskiy D, Damoc E, Ripper J, Woischnik M, Griese M, et al: Lung

alveolar proteomics of bronchoalveolar lavage from a pulmonaryalveolar proteinosis patient using high-resolution FTICR massspectrometry. Anal Bioanal Chem 2007, 389:1075-1085.

32. Spang A: On the fate of early endosomes. Biol Chem 2009, 390:753-759.33. Luster AD: Chemokines–chemotactic cytokines that mediate

inflammation. N Engl J Med 1998, 338:436-445.34. Luster AD: The role of chemokines in linking innate and adaptive

immunity. Curr Opin Immunol 2002, 14:129-135.35. Hartl D, Griese M, Nicolai T, Zissel G, Prell C, Konstantopoulos N, et al:

Pulmonary chemokines and their receptors differentiate children withasthma and chronic cough. J Allergy Clin Immunol 2005, 115:728-736.

36. Hartl D, Latzin P, Hordijk P, Marcos V, Rudolph C, Woischnik M, et al:Cleavage of CXCR1 on neutrophils disables bacterial killing in cysticfibrosis lung disease. Nat Med 2007, 13:1423-1430.

37. Sorokina EM, Feinstein SI, Milovanova TN, Fisher AB: Identification of theamino acid sequence that targets peroxiredoxin 6 to lysosome-likestructures of lung epithelial cells. Am J Physiol Lung Cell Mol Physiol 2009,297:L871-L880.

38. Wikenheiser KA, Vorbroker DK, Rice WR, Clark JC, Bachurski CJ, Oie HK, et al:Production of immortalized distal respiratory epithelial cell lines fromsurfactant protein C/simian virus 40 large tumor antigen transgenicmice. Proc Natl Acad Sci USA 1993, 90:11029-11033.

39. Bridges JP, Xu Y, Na CL, Wong HR, Weaver TE: Adaptation and increasedsusceptibility to infection associated with constitutive expression ofmisfolded SP-C. J Cell Biol 2006, 172:395-407.

40. Postle AD, Gonzales LW, Bernhard W, Clark GT, Godinez MH, Godinez RI,et al: Lipidomics of cellular and secreted phospholipids fromdifferentiated human fetal type II alveolar epithelial cells. J Lipid Res2006, 47:1322-1331.

41. Hite RD, Seeds MC, Safta AM, Jacinto RB, Gyves JI, Bass DA, et al:Lysophospholipid generation and phosphatidylglycerol depletion inphospholipase A(2)-mediated surfactant dysfunction. Am J Physiol LungCell Mol Physiol 2005, 288:L618-L624.

42. Wang Z, Notter RH: Additivity of protein and nonprotein inhibitors oflung surfactant activity. Am J Respir Crit Care Med 1998, 158:28-35.

43. Holm BA, Wang Z, Notter RH: Multiple mechanisms of lung surfactantinhibition. Pediatr Res 1999, 46:85-93.

44. Nathan C: Neutrophils and immunity: challenges and opportunities. NatRev Immunol 2006, 6:173-182.

45. Hogaboam CM, Bone-Larson CL, Lipinski S, Lukacs NW, Chensue SW,Strieter RM, et al: Differential monocyte chemoattractant protein-1 andchemokine receptor 2 expression by murine lung fibroblasts derivedfrom Th1- and Th2-type pulmonary granuloma models. J Immunol 1999,163:2193-2201.

46. Decker T, Lohmann-Matthes ML: A quick and simple method for thequantitation of lactate dehydrogenase release in measurements ofcellular cytotoxicity and tumor necrosis factor (TNF) activity. J ImmunolMethods 1988, 115:61-69.

47. Liebisch G, Drobnik W, Lieser B, Schmitz G: High-throughput quantificationof lysophosphatidylcholine by electrospray ionization tandem massspectrometry. Clin Chem 2002, 48:2217-2224.

48. Liebisch G, Lieser B, Rathenberg J, Drobnik W, Schmitz G: High-throughputquantification of phosphatidylcholine and sphingomyelin byelectrospray ionization tandem mass spectrometry coupled with isotopecorrection algorithm. Biochim Biophys Acta 2004, 1686:108-117.

49. Bligh EG, Dyer WJ: A rapid method of total lipid extraction andpurification. Can J Biochem Physiol 1959, 37:911-917.

50. Brugger B, Erben G, Sandhoff R, Wieland FT, Lehmann WD: Quantitativeanalysis of biological membrane lipids at the low picomole level bynano-electrospray ionization tandem mass spectrometry. Proc Natl AcadSci USA 1997, 94:2339-2344.

51. Schwudke D, Liebisch G, Herzog R, Schmitz G, Shevchenko A: Shotgunlipidomics by tandem mass spectrometry under data-dependentacquisition control. Methods Enzymol 2007, 433:175-191.

52. Liebisch G, Drobnik W, Reil M, Trumbach B, Arnecke R, Olgemoller B, et al:Quantitative measurement of different ceramide species from crudecellular extracts by electrospray ionization tandem mass spectrometry(ESI-MS/MS). J Lipid Res 1999, 40:1539-1546.

doi:10.1186/1471-2121-11-88Cite this article as: Woischnik et al.: A non-BRICHOS surfactant protein cmutation disrupts epithelial cell function and intercellular signaling.BMC Cell Biology 2010 11:88.

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