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Chlamydia pneumoniae Affect Surfactant Trafficking and Secretion Due to Changes of Type II Cell Cytoskeleton

Clinic of Neonatology, Campus Charité-Mitte, University Children's Hospital, Humboldt-University Berlin; and Department of Internal Medicine/Infectious Diseases, Charité, Medical School of Humboldt-University, Berlin, Germany

Address correspondence to: Heide Wissel, Ph.D., Clinic of Neonatology, CCM, University Hospital Charité, Schumannstrasse 20-21, 10098 Berlin, Germany. E-mail: hwissel{at}charite.de

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Understanding the surfactant dysfunction by gram-negative bacteria pulmonary infection, the intracellular fate of Chlamydia pneumoniae (Cpn), its interaction with uptake, recycling, and secretion of surfactant and with the cytoskeleton of type II pneumocytes was investigated. Bacteria colocalized with surfactant protein (SP)-A–mediated endocytosed lipid and early endosomes (EEA1- and Rab5-positive) after 3 and 6 h of infection. No specific contact with late endosomes (Rab7- and M6PR-positive), lysosomal, or lamellar body markers (CD63, 3C9) was found after 12 h of infection. In Cpn-infected cells, SP-A–mediated lipid uptake was significantly increased. After SP-A–mediated lipid uptake followed by "re-secretion," 90% of the internalized lipid remained intracellularly. SP-A and lipid did strongly colocalize with early endosomes. Internalized SP-A cannot be resecreted rapidly to plasma membrane, and lipid is not transported toward late endosomes (Rab7- and M6PR-positive) or lamellar bodies (CD63- and 3C9-positive). These results indicate that increased surfactant internalization is caused by an inhibition in intracellular surfactant transport. Accumulation of SP-A–mediated lipid was associated with changes in ß-tubulin. Increases in surfactant secretion were associated with changes in F-actin. We postulate that Cpn infection of type II cells causes changes of the cytoskeleton, and that these effects are associated with alterations in intracellular transport and secretion of surfactant.

Abbreviations: Chlamydia pneumoniae, Cpn • inclusion-forming units, ifu • surfactant protein A, SP-A

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Chlamydia pneumoniae (Cpn) is an intracellular gram-negative bacterium, that causes serious pulmonary infections (1). Chronic infections are an important risk factor for adult-onset asthma (2) and chronic obstructive lung disease (3). After airway infection with Cpn, bronchial and alveolar epithelial cells are the first line of defense.

In bronchoalveolar lavage of patients with bacterial pneumonia, composition of surfactant phospholipid and surfactant-associated proteins is altered, comparable to changes in acute respiratory distress syndrome (ARDS) (4, 5). Alterations of the surfactant system through Cpn infection have not yet been described.

The pulmonary surfactant is synthesized and secreted by type II pneumocytes into the alveolar space via lamellar bodies, and also cleared from the alveolus. Recycling within alveolar type II cells may be an important part of surfactant metabolism. It has been shown that > 60% of surfactant is cleared from the alveoli by re-internalization in type II cells (6), a mechanism that enables the lung to cope with a variable demand for surfactant under various conditions.

We have shown in isolated type II cells that surfactant phospholipids and surfactant-associated protein A (SP-A) are internalized (7, 8) via the coated pit vesicle pathway to early endosomes (9). SP-A and lipid dissociate, and SP-A and a part of the lipid are recycled to the cell surface via Rab-4–positive recycling vesicles. Lipids are predominantly transported from early endosomes toward lamellar bodies (8, 10).

The cytoskeleton is involved in many cellular functions of eukaryotic cells, such as directed endocytosis, recycling, and secretion (11). Microtubules have been implicated in transport from early endosomes to late endosomes (12) and into apical cell membrane (13). It has been shown that microfilament (actin) is important for the early steps of endosomal distribution (14) and for the release of pulmonary surfactant from type II pneumocytes (15).

Bacterial uptake can induce rearrangement of the host cell cytoskeleton (16). Both microfilaments and microtubules influence redistribution of C. trachomatis from the host cell surface to the perinuclear region (17). However, almost nothing is known about the interaction of Cpn with the type II cell cytoskeleton and about mechanisms responsible for changes of surfactant metabolism in type II cells.

In this study we evaluated whether Cpn infection of type II pneumocytes affects regulation of surfactant uptake, intracellular pathways, or secretion, and alters the cytoskeleton of cells. We demonstrate that Cpn comes into contact with early endosomes and modifies intracellular transport and secretion of surfactant. Cpn treatment of type II cells inhibits intracellular surfactant transport in parallel with changes of the tubulin network, and increases surfactant secretion in parallel with changes of the microfilament network.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cpn strain TW183 was cultured/purified as described earlier (18). Type II cells were isolated from the lungs of adult male Wistar rats (body weight 120–140 g) according to Dobbs and coworkers (19). The cells attached to plastic dishes and glass cover slips were predominantly 87 ± 0.2% type II cells (n = 20 experiments). The viability of cells before infection was 98 ± 0.1% (n = 20 experiments). Cells were infected with 2 x 10⁶ inclusion-forming units (ifu) per 1 x 10⁶ cells.

Biochemical Assays
Cells were transferred on plastic dishes and preincubated with either paclitaxel (5 µM) (20), phalloidin (10 µg /ml), (21) or minimum essential medium (control) and incubated with and without Cpn. Viability (determined by trypan blue exclusion) of the infected cells (94 ± 0.2%, n = 20 experiments) was not different from uninfected cells.

For liposome internalization assay, infected and noninfected cells were incubated with 1,2-dipalmitoyl-L-3-phosphatidyl-N-[methyl-³H]choline [³H]DPPC-labeled or with 1-palmitoyl-2-[1-¹⁴C]linoleoyl-L-3-phosphatidylethanolamine ([¹⁴C]PE)-labeled liposomes ± SP-A. For determination of lipid resecretion, the samples with internalized surfactant were incubated in liposome- and SP-A–free medium. After the uptake and resecretion period, subsequently aliquots were taken to determine protein content and radioactivity as previously described (7, 8, 10). After extraction of intracellular lipid (22), the phospholipid classes were separated by two-dimensional TLC (8). For details, see online supplement. For determination of surfactant secretion, type II cells were incubated for 16 h in the presence of [³H]-palmitate. After preincubation, cells were infected with ± Cpn. Radioactivity was measured in the supernatant and the cellular fraction.

Immunocytochemistry
The cells were plated on glass coverslips, preincubated, and infected (see above). For identification of intracellular pathway of Cpn, infected and uninfected cells were fixed in paraformaldehyde and permeabilized with saponin. For staining of Cpn and intracellular endocytotic compartments, cells were subsequently incubated with primary and thereafter with secondary antibodies (detailed specification in Figure legends). To characterize intracellular surfactant pathway, cells were incubated with L--phosphatidylethanolamine-N-(lissamine rhodamine B sulfonyl)-labeled liposomes ± labeled or unlabeled SP-A. For identification of the intracellular endocytotic compartments, the cells were fixed, permeabilized, and incubated with antibodies labeling different subcellular organelles (detailed specification in Figure legends). To study the cytoskeleton, fluorescence-labeled phalloidin and anti–ß-tubulin was used (see also online supplement).

The labeled samples were analyzed with a Leica TCS NT confocal laser scanning microscope (CLSM) (Leica Lasertechnik GmbH, Heidelberg, Germany), equipped with an argon/krypton laser as described previously (10–12). Using the quantitative and statistical functions Quantify Tool-Window, Leica TCS NT Version 1.5.451 (Leica Lasertechnik GmbH), F-actin- and ß-tubulin-labeling were estimated.

Statistics
For simple paired analyses between two groups, Student's t test was chosen. For multiple comparisons, ANOVA with subsequent Fisher's protected least significant difference (PLSD) test was used. The level of significance was set at P < 0.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Distribution of Cpn in Type II Pneumocytes
After 3 h of incubation with Cpn (green), only a few bacteria had entered the periphery of type II cells (Figure 1A). Bacteria (green) colocalized with the early endosomal marker EEA1 (red) in large peripheral vesicles (yellow) (Figure 1B). Yellow indicates areas of overlap between red and green markers. Minor colocalization of bacteria (green) with SP-A–mediated internalized lipid (red) after 1 h of uptake was found (Figure 1C).

View larger version (110K): [in this window] [in a new window]   Figure 1. Determination of intracellular pathway of Cpn in type II cells with CLSM. Infected type II cells (A, 3 h; D, 6 h; G, 12 h) were fixed, permeabilized, and incubated with a mouse monoclonal anti–Chlamydia pneumoniae antibody or FITC labeled anti-mouse IgG antibodies. Bacterial uptake (green) toward the perinuclear region increased over time. (B) Labeling of Cpn after 3 h infection (bacteria, green) labeling of early endosomes with primary mouse monoclonal EEA1 antibodies and Cy3-conjugated Fab fragments goat anti-mouse IgG (red). Areas of overlapping (yellow) are representative for colocalization of bacteria with early endosomes. (C) Three-hour infected cells were incubated with rhodamine-PE–labeled lipid in the presence of unlabeled SP-A. Bacteria (green) partially colocalized with internalized lipid (red). (E) Labeling of Cpn after 6 h infection (bacteria, green) and labeling of Rab5 with primary mouse monoclonal Rab5 antibodies and Cy3-conjugated Fab fragments goat anti-mouse IgG (red). Areas of overlapping (yellow) are representative for colocalization of bacteria with early endosomes. (F) Six-hour infected cells were incubated with rhodamine-PE–labeled lipid in the presence of unlabeled SP-A. Bacteria (green) partially colocalized with internalized lipid (red). (H) Labeling of Cpn after 12 h infection (bacteria, green) and labeling of Rab7 with goat polyclonal Rab7 antibodies and anti-goat IgG secondary Alexa 594–conjugated antibodies (red). (I) Labeling of Cpn after 12 h infection (bacteria, green) and labeling of M6PR with primary mouse monoclonal M6PR antibodies and Cy3-conjugated Fab fragments goat anti-mouse IgG (red). (H and I) Bacteria were close to (but not colocalized with) late endosomes. (J) Twelve-hour infected cells were incubated with rhodamine-PE–labeled lipid in presence of unlabeled SP-A. Bacteria (green) did not colocalize with internalized lipid (red). (K) labeling of Cpn after 12 h infection (bacteria, green) and labeling of CD63 with primary mouse monoclonal CD63 antibodies and Cy3-conjugated Fab fragments goat anti-mouse IgG (red). (L) Labeling of Cpn after 12 h infection (bacteria, green) and labeling of lamellar bodies with primary mouse monoclonal 3C9 antibodies and Cy3-conjugated Fab fragments goat anti-mouse IgG (red). (K and L) Bacteria never colocalized with a component of lysosomes and lamellar bodies. Pseudo-color blue were used to highlight the contours of the cell (B, C, E, F, H–L). Bar, 10 µm. For each data point, cells from three different animals were investigated (10 cells/animal).

Maximal internalization of bacteria (green) was prominent after 12 h of infection, when Cpn were found in the perinuclear region of cells (Figure 1G). At this time point, bacteria were close to (but not colocalized with) antibodies against Rab-7 (red) (Figure 1H) and M6PR (red) (Figure 1J), components of late endosomes. Most of the internalized lipids (red) was found in vesicles distinct from bacteria (green) (Figure 1I).

Bacteria (green) never colocalized with antibodies against CD63 (red) (Figure 1K), a component of lysosomes and lamellar bodies, or 3C9 (red) (Figure 1L), an antibody recognizing a component of the limiting membrane of classic lamellar bodies in type II cells.

Influence of Cpn Infection on Lipid Uptake
SP-A–mediated lipid uptake was significantly increased 1.9-fold after 3 h (P = 0.0008), 1.5-fold after 6 h (P < 0.0001), and 1.3-fold after 12 h (P = 0.0154) of infection when compared with uninfected cells. Lipid uptake in infected cells (3, 6, 12 h) was significantly lower in the absence of SP-A (P < 0.001) when compared with lipid uptake in the presence of SP-A. No significant differences in intracellular amount of lipid were found between infected and noninfected cells in the absence of SP-A (Figure 2).

View larger version (28K): [in this window] [in a new window]   Figure 2. [3H]DPPC-labeled lipid uptake in 3, 6, and 12 h Cpn-infected type II cells compared with noninfected cells. Vertically striped bars indicate liposomal uptake in absence of SP-A in uninfected cells. Open bars represent lipid uptake in presence of SP-A in uninfected cells. Filled bars indicate lipid uptake without SP-A in infected cells. Horizontally striped bars represent lipid uptake with SP-A in infected cells. Values are expressed as nmol DPPC/106 cells, means ± SE, n = 3 experiments. In the presence of SP-A the lipid uptake was significantly higher in 3 h (+P = 0.0008), 6 h (*P < 0.0001), and 12 h (#P = 0.0154) Cpn-infected type II cells when compared with noninfected cells.

Influence of Cpn Infection on the Surfactant Recycling Pathway
Table 1 shows results in 3 h infected and noninfected cells after 1 h lipid uptake in the presence of SP-A and 30 min subsequent incubation in lipid- and SP-A–free medium ("resecretion" period). In noninfected cells, 67.5 ± 3.8% of the internalized lipid remained in the cells (P = 0.0044 versus uptake). In infected type II cells, 90 ± 6% of the intracellular lipid label remained intracellular (resecretion versus uptake not significantly different). In those assays ± Cpn run without SP-A, differences between lipid uptake and the intracellular amount of lipid after "resecretion" period were not significant.

Influence of Cpn Infection on Intracellular Surfactant Pathway
To understand the influence of Cpn upon the surfactant endocytosis and recycling, the intracellular transport of surfactant along the endocytotic compartments was studied using CLSM.

Early Endosomal Compartments
In 3 h infected cells, 1 h after the start of internalization, FITC-labeled SP-A– (green) colocalized with rhodamine–PE-labeled lipid (red) in large vesicles (yellow) (Figure 3A). At this time point, labeled lipid (red) in the presence of unlabeled SP-A did strongly colocalize with the early endosomal marker EEA1 (green) (Figure 3C). After internalization followed by resecretion, the extent of colocalization of SP-A and lipid did not decrease (results not shown). In comparison with uninfected cells, less internalized labeled SP-A (green) and labeled lipid (red) was found (Figure 3B). In addition, only minor colocalization of lipid (red) with EEA1 (green) was present at this time point (Figure 3D). FITC–SP-A (green) in the presence of unlabeled liposomes in infected cells did strongly colocalize with Rab4-positive (red) compartments (Figure 3E). SP-A (green) in uninfected cells partially colocalized with Rab4-positive (red) compartments (Figure 3F). After internalization followed by resecretion in uninfected cells, SP-A and lipid label colocalized in a markedly smaller fraction of the cells (results not shown).

View larger version (170K): [in this window] [in a new window]   Figure 3. Identification of intracellular surfactant pathway in Cpn infected type II cells with CLSM. Three-hour infected cells (A) and uninfected cells (B) were incubated with FITC-labeled SPA and with rhodamine-PE–labeled lipid. (A) SP-A (green) and lipid (red) did strongly colocalize in large vesicles (yellow). (B) Internalization of SP-A and lipid in uninfected cells was lower versus infected cells. SP-A partially colocalized with lipid. Three-hour infected cells (C) and uninfected cells (D) were incubated with rhodamine-PE–labeled lipid (red) in presence of unlabeled SP-A and then labeled with early endosomal marker EEA1/Alexa 488-conjugated anti-mouse IgG (green). (C) Lipid did strongly colocalize with EEA1. (D) Lipid partially colocalized with EEA1. Three-hour infected cells (E) and uninfected cells (F) were incubated with FITC-labeled SP-A (green) in presence of unlabeled lipid and then labeled with polyclonal goat Rab4 antibodies and anti-goat IgG secondary Alexa 594–conjugated antibodies (red). (E) SP-A did strongly colocalize with Rab4. (F) SP-A partially colocalized with Rab4. Twelve-hour infected cells (G) and uninfected cells (H) were incubated with rhodamine-PE–labeled lipid (red) in presence of unlabeled SP-A and then labeled with EEA1/Alexa 488–conjugated anti-mouse IgG (green). (G) Lipid did strongly colocalize with EEA1. (H) Lipid partially colocalized with EEA1. Twelve-hour infected cells (I) and uninfected cells (J) were incubated with rhodamine-PE–labeled lipid (red) in presence of unlabeled SP-A and then labeled with polyclonal goat Rab7 antibodies and anti-goat IgG secondary Alexa 488–conjugated antibodies (green). (I) Lipid did not colocalize with Rab7. (J) Lipid colocalized with Rab7. Twelve-hour infected cells (K) and uninfected cells (L) were incubated with rhodamine-PE–labeled lipid (red) in presence of unlabeled SP-A and then labeled with primary mouse monoclonal M6PR antibodies and secondary antibodies Alexa 488–conjugated goat anti-mouse IgG (green). (K) Lipid did not colocalize with M6PR. (L) Lipid colocalized with M6PR. Twelve-hour infected cells (M) and uninfected cells (N) were incubated with rhodamine-PE–labeled lipid (red) in presence of unlabeled SP-A and then labeled with monoclonal CD63 antibodies and secondary antibodies Alexa 488–labeled anti-mouse IgG (green). (M) Lipid did not colocalize with CD63. (N) Lipid colocalized with CD63. Twelve-hour infected cells (O) and uninfected cells (P) were incubated with rhodamine-PE–labeled lipid (red) in presence of unlabeled SP-A and then labeled with monoclonal 3C9 antibodies and secondary antibodies Alexa 488–labeled anti-mouse IgG (green). (O) Lipid did not colocalize with 3C9. (P) Lipid and 3C9 did strongly colocalize. Pseudo-color blue were used to highlight the contours of the cells. Bar, 10 µm. For each data point, cells from three different animals were investigated (10 cells/animal).

Late Endosomal Compartments
In 12 h infected cells (1 h after the start of lipid internalization), in most cells, labeled lipid (red), taken up in the presence of unlabeled SP-A, did not colocalize with Rab7- (Figure 3I), M6PR- (Figure 3K), CD63- (Figure 3M), and 3C9-positive (green) compartments (Figure 3O). In uninfected cells, internalized labeled lipid (red) in presence of unlabeled SP-A did colocalize with Rab7- (Figure 3J), M6PR- (Figure 3L), CD63- (Figure 3N), and 3C9 (Figure 3P)-positive (green) compartments.

Similar results were found in 3 and 6 h infected cells, no colocalization with SP-A–mediated lipid and Rab-7, M6PR-, CD63-, and 3C9-positive compartments (results not shown).

Cpn Infection Induced Depolymerization of Microfilaments and Microtubules by Type II Cells
We investigated the effect of Cpn infection on microtubulin and microfilaments using fluorescent staining and CLSM. Uninfected cells and cells after 3 h of infection were stained with mouse monoclonal anti–ß-tubulin antibody followed by Alexa 594-labeled anti-mouse IgG (red) and for F-actin by using Alexa 488–labeled phalloidin (green) under the same conditions. Figure 4 shows labeling of ß-tubulin (red) and F-actin (green).

View larger version (104K): [in this window] [in a new window]   Figure 4. Characterization of Cpn infection on cytoskeleton of type II cells by CLSM. Three-hour uninfected cells (A–D) and infected cells (E–H) were stained with phalloidin/Alexa 488–labeled (green) and monoclonal anti–ß-tubulin antibody and secondary antibodies Alexa 594–labeled anti-mouse IgG (red) under the same conditions. (A and E) Containing only ß-tubulin label. (B and F) Containing only F-actin label, corresponding fluorescence images to C and G. (C and D) Uninfected cells containing both labels. F-actin (green) is typically present at the basal portion of the cell, around the cell periphery, as a dense lattice of short branching irregular filaments organized into bundles, stress fibers (C), and as punctuate deposits or very short filaments (D). ß-Tubulin (red) is distributed diffusely throughout the cytoplasm, which form a relatively dense network of microtubules in irregularly shaped regions. ß-Tubulin is perinuclearly concentrated. (G andH) Infected cells containing both labels. ß-Tubulin and F-actin are colocalized (yellow) and distributed to a perinuclear local aggregate. F-actin was localized deeper within the cell cytoplasm, ß-tubulin is closer to the plasma membrane. Three-hour infected cells (I and J) and uninfected cells (K and L) were incubated with rhodamine-PE–labeled lipid (red) in presence of unlabeled SP-A, fixed, permeabilized, and stained with monoclonal anti–ß-tubulin antibody and secondary antibodies Alexa 488–labeled anti-mouse IgG (green). (I and J) Internalized lipid (red) in infected cells colocalize with the ß-tubulin marker (green). (K and L) In uninfected cells lipid label (red) and ß-tubulin label (green) are in part separately distributed. Pseudo-color blue were used to highlight the contours of the cell (D, H, I–L). Bar, 10 µm. For each data point, cells from three different animals were investigated (10 cells/animal).

In addition, to verify changes in the pattern of cell arrangement, ß-tubulin and F-actin labeling was quantified in 3, 6, and 12 h infected cells and compared with uninfected cells using the xy-intensity histogram function. Intensity distribution of the gray values of image within marked areas of cells from three different animals were investigated (20 cells/animal). The fluorescence intensity of ß-tubulin staining in 3 h infected cells was 49 ± 3 (1.6-fold smaller) versus 76 ± 4 of the uninfected cells. The fluorescence intensity in the 6 h and 12 h infected cells was 35 ± 3 (2.1 fold smaller) versus 73 ± 4 of the uninfected cells. Differences were significant (P < 0.0001). In uninfected cells no significant difference in ß-tubulin label was found over the time period studied.

The fluorescence intensity of staining for F-actin in 3 h infected cells was 42 ± 2 (1.8-fold smaller) versus 76 ± 4 of the uninfected cells. Values in the 6 h infected cells were 36 ± 2 (2.1-fold smaller) versus 77 ± 4 of the uninfected cells. The staining for F-actin in 12 h infected cells was 34 ± 3 (2.3-fold smaller) versus 79 ± 3 of the uninfected cells. Differences were significant (P < 0.0001). The labeling of F-actin in control cells was not significantly affected over the time period studied.

Role of Cytoskeleton Changes in the Surfactant Endocytosis and Recycling of Cpn-Infected Type II Cells
We first compared the effects of Cpn infection (3 h) on the subcellular localization of SP-A–mediated internalized lipid (red) and ß-tubulin (green). In infected cells lipid and tubulin were colocalized (Figures 4I and 4J). In uninfected cells, labels were relative separately distributed and only partially colocalized in a punctuate vesicular pattern throughout the cell (Figures 4K and 4L).

To explore the effect of changes in cellular microtubuli involved in lipid endocytosis and recycling, we have measured the effect of paclitaxel on SP-A–mediated lipid uptake by 3 h infected cells and noninfected cells (Figure 5). Treatment of cells with paclitaxel promotes assembly of microtubules and inhibits tubulin disassembly, causing a stabilization of microtubules (20). In 3 h infected cells, pretreatment with paclitaxel (5 µM) for 30 min at 37°C was associated with a significantly decreased lipid uptake of control level (P < 0.0001). In uninfected cells, pretreatment with paclitaxel did not affect SP-A–mediated lipid uptake. Treatment with the actin filament stabilizer, phalloidin (21), had no effect on lipid uptake, neither in infected nor in uninfected cells (data not shown).

View larger version (29K): [in this window] [in a new window]   Figure 5. Effect of paclitaxel on SP-A–mediated lipid uptake in Cpn-infected type II cells. Type II cells were ± paclitaxel preincubated and then infected with Cpn for 3 h or not infected. Thereafter, the cells were incubated with [3H]DPPC-labeled lipid in the presence of SP-A for 1 h. Vertically striped bars indicate liposomal uptake in absence of paclitaxel in uninfected cells. Open bars represented lipid uptake in presence of paclitaxel in uninfected cells. Horizontally striped bars represented lipid uptake in absence of paclitaxel in infected cells. Filled bars indicate lipid uptake in presence of paclitaxel in infected cells. Values are expressed as nmol DPPC/106 cells (means ± SE, n = 3 experiments). #P < 0.0001 versus uninfected cells. *P < 0.0001 versus infected cells in absence of paclitaxel.

We could not detect differences in the distribution of SP-A and lipid on intracellular pathway due to phalloidin or paclitaxel with CLSM (results not shown).

As an index for cell toxicity, the release of lactate dehydrogenase (LDH) was measured. The measurements were as follows: 3.1 ± 0.1% (after 3 h infection), 2.7 ± 0.2% (after pretreatment of cells with paclitaxel), 3.4 ± 0.4% (after pretreatment of cells with phalloidin), and 3.7 ± 0.2% (in noninfected cells) of LDH were found extracellularly (n = 3 experiments). Differences were not statistically significant.

Association of Changes of Cytoskeleton and Influence of Surfactant Secretion
To investigate whether Cpn infection affects basal surfactant secretion and alters the F-actin network, infected and uninfected type II cells were prelabeled with [³H]-palmitate, thereafter preincubated with and without phalloidin (10 µg/ml). Figure 6 shows that after 3 h of infection, secretion of surfactant was significantly increased (P < 0.0001 versus uninfected cells). Phalloidin inhibited surfactant secretion by infected cells significantly (P < 0.0001 versus infected cells in absence of phalloidin). No phalloidin effect on basal surfactant secretion was found in uninfected cells. Paclitaxel had no effect on surfactant secretion, neither in infected nor in uninfected cells (results not shown).

View larger version (39K): [in this window] [in a new window]   Figure 6. Effect of phalloidin on Cpn-induced surfactant secretion of type II cells. Type II cells were incubated for 16 h with [3H]-palmitate. Preincubation without and with phalloidin was performed for 30 min and cells were infected or not infected thereafter for 3 h with Cpn. Vertically striped bars indicate basal secretion of uninfected cells in absence of phalloidin. Open bars represented basal secretion in presence of phalloidin in uninfected cells. Horizontally striped bars represented surfactant secretion in absence of phalloidin in infected cells. Filled bars indicate surfactant secretion in presence of phalloidin in infected cells. Results are expressed as % secreted tracer in supernatant in relation to the total amount of tracer in supernatant and in cells (means ± SE, n = 3 experiments). *P < 0.0001 versus uninfected cells. #P < 0.0001 versus infected cells in absence of phalloidin.

In paclitaxel- or phalloidin-pretreated cells and for 3 h infected cells, bacteria (green) did not colocalize with SP-A–mediated internalized lipid (red) after 1 h of uptake (Figure 7A), nor with EEA1-positive compartments (Figure 7B). After 12 h of infection in cells pretreated with paclitaxel or phalloidin, bacteria were colocalized with Rab7- (Figure 7C), M6PR- (Figure 7D), and CD63-positive compartments (Figure 7E). Minor colocalization with 3C9 antibody against limiting membrane of lamellar bodies was found (Figure 7F).

View larger version (113K): [in this window] [in a new window]   Figure 7. Effect of phalloidin and paclitaxel of intracellular pathway of Cpn in type II cells. (A) With paclitaxel preincubated and for 3 h with Cpn-infected type II cells were incubated with rhodamine-PE–labeled lipid in presence of unlabeled SP-A and then labeled with a mouse monoclonal anti–Chlamydia pneumoniae antibody and Alexa 488–conjugated anti-mouse IgG. Bacteria (green) but not colocalized with internalized lipid (red). (B) With paclitaxel pre-incubated and for 3 h with Cpn infected type II cells were labeled of Cpn with a mouse monoclonal anti–Chlamydia pneumoniae antibody and Alexa 488–conjugated anti-mouse IgG and of early endosomes with primary mouse monoclonal EEA1 antibodies and Cy3-conjugated Fab fragments goat anti-mouse IgG. Bacteria (green) not colocalized with early endosomes (red). (C) With paclitaxel preincubated type II cells and for 12 h with Cpn infected were labeled of Cpn and of Rab7 with goat polyclonal Rab7 antibodies and anti-goat IgG secondary Alexa 594–conjugated antibodies. (D) With phalloidin preincubated type II cells and for 12 h with Cpn infected were labeled with Cpn and then labeled with primary mouse monoclonal M6PR antibodies and Cy3-conjugated Fab fragments goat anti-mouse IgG (red). (E) With paclitaxel preincubated type II cells and for 12 h with Cpn infected were labeled with Cpn and labeled with primary mouse monoclonal CD63 antibodies and Cy3-conjugated Fab fragments goat anti-mouse IgG. (F) Labeled with primary mouse monoclonal 3C9 antibodies and Cy3-conjugated Fab fragments goat anti-mouse IgG. Bacteria (green) colocalized with components of late endosomes (red) (C, D) and lysosomes (red) (E), and partially with lamellar bodies (red) (F). Pseudo-color blue were used to highlight the contours of the cells. Bar, 10 µm. For each data point, cells from three different animals were investigated (10 cells/animal).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

In the current work, we demonstrate that Cpn are internalized into type II pneumocytes. We have examined the intracellular pathway of bacteria after 3, 6, and 12 h of infection (Figure 1). We did not study later time points because isolated type II cells, have been shown to express type I phenotypic markers and to cease type II–specific functions, within 2–5 d on plastic culture (25).

The CLSM results indicate that bacteria interact with small fractions of early endosomes (EEA1- and Rab5-positive) and with small fraction of internalized surfactant lipid after 3 and 6 h infection. Our results are in accordance with studies published for C. trachomatis, C. psittaci, and C. pneumoniae, suggesting that bacteria selectively accumulate in early endosomal compartments (26, 27). The interaction of early endosomes with bacteria and with endocytosed SP-A–mediated lipid at 3 h and 6 h after infection indicate that bacteria and surfactant in the early phase of infection come into contact. After prolonged infection (12 h), bacteria in cells were transported to a perinuclear location near the late endosomes (Rab7- and M6PR-positive); bacteria did not, however, enter late endosomes. A similar result has been obtained for C. trachomatis and C. pneumoniae, which also do not reside in the late endosomes (26, 27). In the present study, neither lysosomal nor lamellar body markers were close to the bacteria. Intracellular pathogens have developed several different strategies for survival within eukaryotic host cell. Survival of chlamydiae depends upon prevention of fusion with lysosomal compartments (28).

These data indicate that at early phase (3 and 6 h) of infection, Cpn are found in close apposition to the compartments containing internalized surfactant. However, the later pathway of Cpn (12 h of infection) differs from that of endocytosed surfactant in type II cells.

Repeated cycling of surfactant components between type II cells and alveolar liquid plays a substantial role in the host defense system of the lung (29). Surfactant is secreted into the alveolar space via lamellar bodies, and is also cleared from the alveolus, partly via reinternalization by type II cells (6). In vitro assays suggest that in the presence of SP-A, surfactant lipids are not degraded via the pathway to lamellar bodies (30). We have recently shown that in type II cells, endocytosed SP-A and lipid are transported toward a common early endosomal compartment. Thereafter, SP-A and a part of surfactant lipid is rapidly recycled to the cell surface via Rab4-associated recycling vesicles, and a large part of surfactant lipid is transported toward lamellar bodies (7, 8, 10), as schematically depicted in Figure 8A.

View larger version (34K): [in this window] [in a new window]   Figure 8. Model of SP-A and lipid metabolism in type II cells in the absence (A) and presence (B) of Cpn infection. (A) In uninfected cells the SP-A–lipid complex accumulates at clathrin-coated pits of the plasma membrane, which bud off to yield clathrin-coated vesicles at the plasma membrane. SP-A–mediated lipid endocytosis is typically initiated by the formation of clathrin-coated vesicles at the plasma membrane. The vesicles lose their coats, which facilitates fusion with early endosomes, which results in the delivery of SP-A–lipid complex to early endosomes (array of tubules and vesicles, EEA1+, Rab5+, Rab4+). Here, a part of SP-A–lipid complex dissociates, thus resulting in the return of SP-A and lipid to plasma membrane in recycling vesicles (Rab4+). Dissociated lipid is transferred from early to late endosomes (Rab7+, M6PR+). A main part of lipid is takes an the formation of lamellar bodies (3C9+, Rab7+, CD63+); a small part is transferred to lysosomes (CD63+), where digestable content is degraded. (B) In infected cells lipid endocytosis is typically initiated by the formation of clathrin-coated vesicles at the plasma membrane, which results in the delivery of SP-A and lipid complexes to early endosomes. In this array of tubules and vesicles SP-A and lipid accumulated in early endosomes. The recycling of SP-A and lipid to plasma membrane and the recycling to late endosomes or to the lamellar bodies is inhibited. The results suggested that the inhibition of lipid recycling inhibits the formation of lamellar bodies inhibited.

To clarify the intracellular transport routes of SP-A and lipid in infected cells in comparison to uninfected cells, we used the specific localization of intracellular compartments with specific fluorescence-labeled antibodies. Pathways of fluorescence-marked surfactant components were tracked with CLSM-based techniques (Figure 3). The microscopy of Cpn-infected cells suggests that SP-A and lipid accumulate in early endosomes. The finding that SP-A strongly colocalized with Rab4-positive recycling vesicles could mean that recycling from early endosomes to the plasma membrane is inhibited. In addition, surfactant lipid colocalized with early endosomes and was not identified in late endosomes or lamellar bodies, suggesting that transport of lipid from early endosomes to lamellar bodies is blocked. Biochemical assays, as well as confocal microscopy experiments, demonstrate that surfactant recycling back to the plasma membrane and to the lamellar bodies is inhibited by Cpn infection of type II cells as schematically depicted in Figure 8B.

Thus, the findings that surfactant recycling is inhibited, and that Cpn causes an increase of basal surfactant secretion (Figure 6), would mean that the Cpn infection leads to a reorganization of cell compartments.

We also aimed to study the role of the cytoskeleton in Cpn-infected type II cells, and its subsequent effect of endocytosis to recycling and secretion of surfactant. The cytoskeleton is vital to the function of all eukaryotic cells, and is involved in processes ranging from maintenance of cell shape and endocytosis to recycling and secretion (11, 13).

The host cell cytoskeleton is known to play a vital role in the life cycles of several microorganisms. Studies of cellular entry by C. trachomatis showed that both microtubules or microfilaments are involved in the chlamydial migration from the host cell surface to a perinuclear region (17). Invasion of several bacteria is accompanied by rearrangement of the F-actin filaments within host cells, in which microtubules have been observed to act as anchoring structures for F-actin (31). It is known that the process of nonphagocytic cell invasion in many pathogenic bacteria involves triggering of host cell signal transduction pathways. The final result of this process is the rearrangement of the cytoskeleton (32). It has been proposed that C. trachomatis produces cytoskeletal changes in epithelial cells (33). The roles of cytoskeleton in host cells in Cpn infections is still unclear. In the present study we could demonstrate that infection of Cpn by type II cells changed the pattern of cell arrangement of ß-tubulin and F-actin (Figure 4). This observation suggests that the early infection of bacteria (after 3 h) alters the pattern of cell arrangement by type II cells. The Cpn interaction with type II cells (namely attaching and invasion, triggering of host cell signal transduction pathways, cytoskeletal rearrangement) may be different manifestations of the same process.

The assumption that in infected cells the intracellular accumulation of internalized SP-A and lipid and the increase basal surfactant secretion are directly related to the rearrangement of ß-tubulin and F-actin is based on the following:

1. Paclitaxel, a drug which stabilizes microtubuli (20), did significantly reduce cellular accumulation of lipid in infected cells (Figure 5, Table 3). Phalloidin, which stabilizes F-actin (21), had no effect on cellular accumulation of surfactant. The data indicate that the microtubulin network is involved in the intracellular transport of endocytosed surfactant. These findings are compatible with the observation that the pattern of ß-tubulin arrangement and the distribution of arrested lipid are changed in infected cells.
   
2. In infected cells, SP-A and lipid accumulated in early endosomes. SP-A did strongly colocalize with Rab4-positive recycling vesicles. The fact that 90% of SP-A–mediated lipid remained associated intracellularly suggests that rapid recycling to cell surface was inhibited. In addition, surfactant lipid was not transported to late endosomes or lamellar bodies. The data suggest a role for microtubulin. It is known that the host cell microtubular cytoskeleton plays an important role in dissociation of endocytosed molecules in early endosomes. A part of the material from early endosomes is transferred via Rab4-positive recycling vesicles to plasma membrane; the other part is transported to late endosomes (34). Recycling is mediated by a series of protein mediators of vesicle formation, docking, and fusion, requiring a coordinate interaction between endosomes and cytoskeleton (34). Experiments with microtubuli-depolymerizing agents prevents the dissociation of internalized molecules from early endosomes to plasma membrane and to late endosomes (35, 13).
   
3. The basal surfactant secretion is increased in 3 h infected type II cells. Phalloidin inhibited basal surfactant secretion significantly, indicating that surfactant secretion is directly related to changes in F-actin (Figure 6). Paclitaxel had no effect of surfactant secretion. Previous reports demonstrated that exposure of type II cells to secretagogues and mechanical stretch involves the depolymerization of the actin microfilament network with a subsequent effect on surfactant secretion. The enhancement of baseline secretion was directly related to the depolymerization of F-actin by C2 toxin exposure of type II cells (15).
   
4. Our results show that paclitaxel and phalloidin changed the trafficking of Cpn in type II cells (Figure 7). In paclitaxel- or phalloidin-treated cells internalized lipid and early endosomes did not come into contact with bacteria. Cpn interact with late endosomal or lysosomal compartments. In non–paclitaxel- or non–phalloidin-treated cells, Cpn come into contact with internalized lipid and early endosomes but avoid contact with late endosomal or lysosomal compartments. Our results support the hypothesis that an undisturbed cytoskeleton is required to protect Cpn fusion with lysosomes. Stabilization of cytoskeleton with paclitaxel or phalloidin channels Cpn into the lysosomal compartment.
   

In summary, this study shows that Cpn at an early infection time point (3 and 6 h) are in contact with early endosomes and internalized SP-A–mediated lipid in type II cells. At a later infection time point (12 h), a pathway of bacteria can be recognized, that is separate from the endocytosed surfactant transport pathway. The infection of type II cells suggests that the interaction of cells with bacteria alters the regulation of surfactant mechanisms and the cell arrangement. After infection, lipid internalized via the SP-A–mediated pathway, accumulates in type II cells, due to blocking of intracellular transport. The influence of ß-tubulin is linked with inhibition of intracellular surfactant transport to the cell surface and to lamellar bodies. The change of F-actin is linked with increased basal surfactant secretion.

We postulate that changes in cytoskeleton are associated with changes in the metabolism of surfactant by type II cells and leads to alterations in surfactant homeostasis. Understanding the mechanisms of impairment of surfactant recycling and secretion by type II cells in Cpn infection may be important for understanding the surfactant dysfunction by specific gram-negative bacteria. Future work will explore signaling pathways that mediate the effect of Cpn to improve our understanding of the effect on surfactant metabolism.

	Acknowledgments

 
The authors thank Helga Kemmer (Clinic of Neonatology, Campus Charité-Mitte, University Children's Hospital, Humboldt-University Berlin, Germany) for their technical assistance in cell preparation, and Petra Klein (Institute of Biology, Department of Membrane Physiology, Humboldt-University Berlin, Germany) for help with the CLSM. This work was supported by Grants Ste 459/4-3 and 4-4 and Wi 2074/1-1 from the Deutsche Forschungsgemeinschaft, and by research advancement (387-02) from the Charité, Humboldt-University Berlin.

	Footnotes

 
This article has an online data supplement, which is accessible from this issue's table of contents online at www.atsjournals.org

Received in original form November 12, 2002

Received in final form March 26, 2003

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