We have recently demonstrated that primary cultured rat pneumocytes produce macrophage inflammatory protein-2 (MIP-2) in response to lipopolysaccharide (LPS) stimulation. In this study, we found that brefeldin A, by blocking anterograde transport from the endoplasmic reticulum (ER) to the Golgi apparatus, decreased LPS-induced MIP-2 in the culture medium and increased its storage in cells. This suggests that MIP-2 is secreted via a pathway from the ER to the Golgi apparatus, a process commonly regulated by microtubules. We further found that LPS induced depolymerization of microtubules as early as 1 min after LPS stimulation, and it lasted at least for 4 h. Preventing depolymerization of microtubules with paclitaxel (Taxol; 10 nM to 10 μM) partially inhibited LPS-induced MIP-2 production, whereas the microtubule-depolymerizing agents colchicine (1–10 μM) and nocodazole (1–100 μM) increased LPS-induced MIP-2 protein production without affecting MIP-2 mRNA expression. These results suggest that in pneumocytes, LPS-induced microtubule depolymerization is involved in LPS-induced MIP-2 production and that secretion of MIP-2 from pneumocytes is via the ER-Golgi pathway.
- alveolar epithelial cells
the pulmonary vasculature is the largest reservoir of polymorphonuclear neutrophils (PMNs) in the human body (42). However, the number of PMNs in normal airways and alveoli is very low. The rapid recruitment of PMNs from the lung vasculature appears to be a very important response of the host against pathogens (42). Chemokines are chemotactic cytokines for leukocyte recruitment and activation from the bloodstream to the sites of infection or tissue injury (24). PMN infiltration in the alveolar space is mainly mediated by C-X-C chemokines such as interleukin (IL)-8 in humans and its rodent homologue macrophage inflammatory protein-2 (MIP-2) (12, 24, 48). Various kinds of bacteria (15,16) or stimulants such as lipopolysaccharide (LPS) (28,39), α-quartz (13), and titanium dioxide particles (50) induce MIP-2 in the lung, which is associated with inflammation-related acute injury. Neutralization with anti-MIP-2 antibody decreases the influx of neutrophils into the lung and attenuates lung injury (13, 39, 40). Xavier et al. (49) have recently demonstrated that primary cultured rat pneumocytes produce MIP-2 in response to LPS stimulation. The amount of MIP-2 produced in these cells is at a magnitude similar to that from macrophages (49). Thus pneumocytes may be a major source of MIP-2 in the lung. LPS-induced MIP-2 production in rat pneumocytes is regulated at both the transcriptional and translational levels (49).
Intracellular MIP-2 was found mainly in the endoplasmic reticulum (ER) (49). However, like many other cytokines and chemokines, the regulation of MIP-2 secretion is largely unknown. In eukaryotic cells, most secretory proteins are transported from the ER to the Golgi apparatus. The ER network, with branching membrane tubules, extends outward along the microtubules throughout the cell, whereas the Golgi cisternae are clustered around the microtubules near the perinuclear microtubule-organizing center. Transport intermediates arising from peripheral ER sites thus often travel considerable distances to reach the Golgi complex (22). Microtubules and associated motor proteins play fundamental roles in facilitating the selective delivery of transport intermediates between the ER and the Golgi and of secretory vesicles from the Golgi to the plasma membrane (8,22). LPS is known to enhance microtubule polymerization in immune cells (2), which play an important role in mediating cytokine production from these cells in response to LPS stimulation (2, 21, 33). In contrast, Isowa et al. (19) have recently found that LPS decreases polymerization of microtubules in rat pneumocytes. Whether the change in microtubule structure is involved in cytokine production and secretion from pneumocytes is unknown. In the present study, we first found that LPS-induced MIP-2 secretion can be blocked by brefeldin A (BFA), an agent that can selectively disrupt anterograde transportation from the ER to the Golgi. We then demonstrated that LPS-induced MIP-2 production can be influenced by agents that affect the polymerization status of microtubules. We speculate that MIP-2 is secreted through the ER-Golgi pathway and that the microtubule system is involved in LPS-induced MIP-2 synthesis and secretion.
MATERIALS AND METHODS
LPS (Escherichia coli), BFA, paclitaxel (Taxol), colchicine, nocodazole, and rat IgG were purchased from Sigma (St. Louis, MO). DMEM, fetal bovine serum (FBS), and gentamicin were purchased from GIBCO BRL (Mississauga, ON). Porcine pancreatic elastase was purchased from Worthington Biochemical (Freehold, NJ). Pentobarbital sodium was purchased from Bimeda-MTC Pharmaceuticals (Cambridge, ON). Rat anti-mammalian α-tubulin antibody and FITC-conjugated rabbit anti-rat IgG were purchased from Serotec (Kidlington, UK).
Rat pneumocyte isolation and culture.
Alveolar type II cells were obtained with the method of Dobbs (10) as previously described (19, 49). Briefly, male adult Sprague-Dawley rats (Harlan, Indianapolis, IN) weighing ∼250 g were anesthetized with pentobarbital sodium (100 mg/kg body wt ip) and killed. Pneumocytes were separated from the alveolar basement membrane by incubation of the isolated lung tissue with porcine pancreatic elastase. Alveolar macrophages were removed by differential adherence to rat IgG-precoated petri dishes.
The cells were cultured in DMEM containing 10% (vol/vol) FBS and 12.5 μg/ml of gentamicin. To further reduce contaminating alveolar macrophages in the primary culture, the culture medium was changed daily for 2 days before LPS treatment. As Isowa et al. (19) and Xavier et al (49) have previously reported, this maneuver effectively reduces the number of macrophages to an undetectable level as determined by cell surface ectoenzyme-alkaline phosphatase staining or by immunofluorescent staining with anti-CD45 antibody. The purity of pneumocytes in the culture system was assessed and confirmed by phase-contrast microscopy and immunofluorescent staining with anti-cytokeratin and anti-surfactant proprotein C antibodies (specific markers for epithelial cells and type II pneumocytes, respectively).
Immunocytofluorescent staining for microtubules and confocal microscopy.
One milliliter of freshly isolated lung cell suspension (5 × 105 cells/ml) was seeded in each well of four-well Lab-Tek chamber slides (NUNC, Naperville, IL), and the culture medium was changed daily for 2 days before LPS treatment. On the day of the experiment, the culture medium was replaced with 10% FBS-DMEM with and without LPS (10 μg/ml) for 1 min, 15 min, or 4 h. The cells were immunostained for α-tubulin as previously described (19). Because tubulin exists free in the cytosol as well as polymerized in the microtubules, the cells were washed after permeabilization to remove free tubulin before fixation (2). Briefly, after being washed, fixed, and permeabilized, the cells were stained with rat anti-mammalian α-tubulin antibody (1:100 in distilled water) for 30 min at room temperature. After being washed with PBS, the cells were incubated with FITC-conjugated rabbit anti-rat IgG (1:100 in distilled water) for 30 min at room temperature in the dark. After being washed with PBS, the slides were mounted with an antifading reagent (SlowFade, Molecular Probes, Eugene, OR). To determine the specificity of staining, the first antibody was replaced with nonspecific rabbit IgG (Sigma) or was omitted from the staining procedure. Confocal laser scanning was performed with a confocal microscope (MRC-600, Bio-Rad, Mississauga, ON) equipped with a krypton or argon laser.
LPS stimulation and drug treatments.
Freshly isolated rat lung cells (1 × 106 cells/ml) were plated on 24-well culture plates (1 ml/well; Corning Glass Works, Corning, NY) in 10% FBS-DMEM and maintained at 37°C with 5% CO2. Two days after inoculation, the cells were treated with various concentrations of BFA, paclitaxel, colchicine, or nocodazole for 2 h followed by stimulation with LPS for 4 h in the presence of the corresponding drugs in 10% FBS-DMEM. Cell culture medium or cell lysates were collected and analyzed for MIP-2 with ELISA.
The cytotoxic effects of LPS, BFA, paclitaxel, colchicine, and nocodazole were examined by simultaneous double staining with fluorescein diacetate and propidium iodide (20) as previously reported (19, 49). Briefly, after treatment with all agents, the cells were washed twice with Dulbecco's phosphate-buffered saline. Freshly diluted fluorescein diacetate-propidium iodide solution was added to each well for 3 min at room temperature. Viability of the cells was examined with a fluorescent microscope with 520- and 590-nm filters. Viable cells fluoresced bright green, whereas nonviable cells were bright red. The viability of cells in all groups was found to be comparable to that of the untreated control groups. No cytotoxic effect on rat pneumocytes was found for these compounds with the concentrations used in the present study.
Measurement of MIP-2.
After treatment with each agent, the culture medium was collected. The cells were washed twice with cold PBS and harvested with cell lysis buffer containing 10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, and 0.6% (octylphenoxy)polyethoxyethanol (Nonidet P-40) (26). Cells were sonicated and centrifuged at 14,000 rpm for 3 min. The supernatants were measured for MIP-2 and protein content. MIP-2 concentrations in the culture medium and cell lysates were measured in duplicate or triplicate with a commercial ELISA kit (Biosource, Camarillo, CA). The optical density (OD) of each well was read at 450 nm with a NM600 microplate reader (Dynatech Laboratories, Chantilly, VA). The detection range of the MIP-2 kit is 10–640 pg/ml. The protein content was determined by the Bradford (6) method. The MIP-2 value of cell lysates was standardized to the amount of protein tested.
RNA extraction and semiquantitative reverse transcriptase-polymerase chain reaction.
Cells were cultured in six-well plates (4 × 106cells/well) in 10% FBS-DMEM. Forty-eight hours after cell isolation, the cells were treated with and without paclitaxel, colchicine, or nocodazole for 2 h followed by stimulation with LPS (10 μg/ml) in 10% FBS-DMEM for 4 h with and without the agents tested. The medium was removed, and the cells were washed twice with ice-cold PBS. RNA extraction and reverse transcriptase-polymerase chain reaction (RT-PCR) were performed as previously described (19, 49). Two micrograms of RNA were used for the reverse transcription reaction, and RT products from 0.2 μg RNA were used for PCR. PCR primers for β-actin and MIP-2 were synthesized by ACGT Corporation (Toronto, ON). The sequences of primers for β-actin and MIP-2 have been previously described (49). Ten microliters of PCR product were electrophoresed on 1% agarose gels, with ethidium bromide staining for visualization, and the gels were photographed and quantified with a gel documentation system (Gel Doc 1000, Bio-Rad, Hercules, CA). To ensure comparability, RT-PCR was performed simultaneously on all samples collected from each experiment. PCR products were analyzed on the same gel. The OD of the PCR product bands was quantified with integrated image analysis software (Molecular Analyst version 1.5, Bio-Rad). With optimized PCR conditions, all data were collected without saturation or missing bands. The background OD reading for each band was subtracted locally. RT-PCR was conducted at least two times for each sample to ensure reproducibility.
All experiments were carried out with materials collected from at least two to three separate cell cultures in duplicate or triplicate. All data are expressed as means ± SE from separate measurements and were analyzed with a personal computer with SigmaStat for Windows version 1.0 (Jandel, San Rafael, CA). Comparison of groups was carried out with one-way or two-way analysis of variance followed by Student-Newman-Keuls test, with significance defined asP < 0.05.
BFA inhibits MIP-2 secretion from alveolar epithelial cells.
Xavier et al. (49) have recently demonstrated that incubation with LPS (10 μg/ml) for 4 h significantly stimulated MIP-2 production in rat pneumocytes. To study the secretory mechanism of MIP-2, pneumocytes were pretreated with various concentrations of BFA for 2 h and then treated with LPS (10 μg/ml) in the presence of BFA for 4 h. BFA is known to block vesicular traffic from the ER to the Golgi apparatus (11), which inhibited the amount of MIP-2 in the culture medium in a dose-dependent fashion (Fig.1). BFA had no cytotoxic effects on the cells at any concentration examined (data not shown). To further confirm that the decrease in MIP-2 was due to a blocking effect of BFA on MIP-2 secretion, the cells were treated with BFA (500 ng/ml), and the amount of MIP-2 in the cell lysates and the culture medium was measured separately. The basal level of MIP-2 in the cell lysates was 311 ± 85 ng/well, representing 29.6 ± 5.2% of total MIP-2. After LPS stimulation, it increased to 3,507 ± 104 ng/well (P < 0.05), representing only 8.4 ± 0.4% of total MIP-2 (P < 0.05). Therefore, LPS stimulated not only MIP-2 production but also its secretion. BFA inhibited both basal and LPS-induced MIP-2 accumulation in the culture medium (Fig.2 A), and significantly increased MIP-2 protein in the cell lysates (Fig. 2 B). These results indicate that MIP-2 secretion is through the ER-Golgi pathway.
LPS depolymerizes microtubules in alveolar epithelial cells.
Microtubules are very important for transport between the ER and the Golgi (23, 37). Isowa et al. (19) have recently reported that the intensity of microtubule immunofluorescent staining in pneumocytes was decreased by incubation with LPS for 15 min. To determine whether LPS-induced microtubule depolymerization is related to MIP-2 production, in this study we examined microtubule structure of the cells treated with LPS (10 μg/ml) for up to 4 h with immunofluorescent staining and confocal microscopy. The intensity of microtubule staining of the LPS-stimulated cells decreased compared with that of control cells at all periods examined (Fig.3). The reduced polymerization of microtubules was observed as early as 1 min after LPS stimulation and lasted at least for 4 h (Fig. 3).
Paclitaxel inhibits LPS-induced MIP-2 release from pneumocytes.
Paclitaxel is a plant derivative that induces and stabilizes the bundling of microtubules (38). To determine whether the effect of LPS on the depolymerization of microtubules is related to LPS-induced MIP-2 production in pneumocytes, we treated cells with paclitaxel before and during LPS stimulation. Paclitaxel (10 nM to 10 μM) partially but significantly inhibited LPS-induced MIP-2 release from pneumocytes in a dose-dependent manner (Fig.4). Paclitaxel had no effects on MIP-2 release from the cells without LPS stimulation (data not shown).
Colchicine or nocodazole increases LPS-induced MIP-2 release from pneumocytes.
We then examined whether depolymerizing microtubules can further affect LPS-induced MIP-2 release from pneumocytes. Colchicine is a plant alkaloid that irreversibly binds to the protein subunit of microtubules (1). Nocodazole is a synthetic compound that binds reversibly to tubulin (17). Both colchicine and nocodazole are commonly used as microtubule-disrupting agents. Treatment with colchicine (1–10 μM) or nocodazole (1–100 μM) before and during LPS stimulation significantly increased MIP-2 release from alveolar epithelial cells (Fig. 5). In the absence of LPS, neither colchicine nor nocodazole affected MIP-2 in the culture medium (Fig. 5) or in the cell lysates (data not shown). To study the synergistic effects between colchicine or nocodazole and LPS on MIP-2 release, we treated cells with colchicine (1 μM) or nocodazole (10 μM) and varying concentrations (10 pg/ml to 10 μg/ml) of LPS. MIP-2 release was increased in a LPS dose-dependent manner as found previously by Xavier et al. (49). With the higher concentrations of LPS (0.1–10 μg/ml), both colchicine and nocodazole significantly increased MIP-2 release from alveolar epithelial cells (P < 0.05). However, neither colchicine nor nocodazole showed any effects on MIP-2 release when cells were treated with the lower concentrations of LPS (Fig.6).
Paclitaxel, colchicine, or nocodazole does not affect LPS-induced MIP-2 mRNA expression in pneumocytes.
In a human bronchial epithelial cell line, depolymerization of microtubules with colchicine induced IL-8 gene expression, which was inhibited by polymerization of microtubules with paclitaxel pretreatment (41). Xavier et al. (49) previously demonstrated that MIP-2 mRNA in pneumocytes was induced by LPS stimulation. Therefore, we examined whether polymerization or depolymerization of microtubules affects the LPS-induced elevation in MIP-2 mRNA. Cells were incubated with paclitaxel (10 μM), colchicine (1 μM), or nocodazole (10 μM) before and during LPS (10 μg/ml) stimulation. Total RNA was extracted, and steady-state levels of MIP-2 mRNA were determined with semiquantitative RT-PCR. Because β-actin gene transcription was not affected by either LPS or any agents tested, the ratio of densitometry units between MIP-2 and β-actin mRNAs was used to represent the steady-state levels of MIP-2 mRNA. Similar to previous observations by Xavier et al., LPS induced an increase in MIP-2 mRNA. However, it was not affected by colchicine, nocodazole, or paclitaxel (Fig. 7). To confirm that this result was not due to saturated cDNA products resulting from an excess number of PCR cycles, we also examined mRNA expression using different PCR cycles (24, 27, and 30 cycles). The results were the same (data not shown).
Alveolar epithelial cells play an important role in host defense by regulating the recruitment and activation of immune cells such as macrophages and neutrophils through the secretion of cytokines and chemokines in responding to various kinds of stimulation (29, 43,44). Neutrophil infiltration in the alveolar space is one of the most important events in acute lung injury (45), which is mainly mediated by C-X-C chemokines (39). Xavier et al. (49) have recently found that LPS-stimulated rat pneumocytes produced MIP-2, an important member of the C-X-C family. We investigated the role of the microtubule system in LPS-induced MIP-2 production in this study.
Most secretory proteins endowed with a signal sequence of hydrophobic amino acids (46) are first translocated across the membrane of the ER and then transported through the Golgi apparatus to the plasma membrane (30). Many cytokines such as tumor necrosis factor-α (TNF-α) and IL-6 (36) contain such a signal sequence. TNF-α secretion from human monocytes (32,36) and rat mast cells (3) or IL-6 secretion from human monocytes (36) was inhibited by BFA treatment. BFA is an agent that not only blocks anterograde transport from the ER to the Golgi but also induces retrograde transport from the Golgi to the ER (23). Some cytokines, such as IL-1β, do not contain the signal peptide and are released via alternative pathways. For example, IL-1β is contained, in part, within intracellular vesicles in activated human monocytes, and its secretion is not inhibited by BFA (36). A 31-residue signal peptide was noted from rat MIP-2 (27). We expected that MIP-2 secretion is mediated through the ER-Golgi pathway. Indeed, BFA blocked both the basal and LPS-induced MIP-2 secretion from pneumocytes. A 22-residue signal peptide (25) was also noted from human IL-8. Therefore, the ER-Golgi pathway could be a common route shared by several cytokines and chemokines. There are two ways that proteins exit the Golgi toward the cytoplasmic membrane: regulated and constitutive secretion. Proteins that exit through regulated secretion are usually stored as secretory vesicles and are released when stimulatory signals are exerted on the cells. Constitutive secretion means that the newly synthesized protein is released continuously after synthesis. BFA has been shown to reversibly block early but not late protein transport steps in the yeast secretory pathway (14). Thus the effective blockade of MIP-2 secretion by BFA also suggests that the post-Golgi storage of MIP-2 in pneumocytes is transient and limited, supporting a constitutive secretion of MIP-2.
Isowa et al. (19) have previously shown that incubation with LPS for 15 min decreased polymerization of microtubules in pneumocytes. With postembedding immunolabeling techniques and electron microscopy, it has been shown that LPS can enter the cytoplasm of rat type II pneumocytes and colocalize with microtubules (34). With biochemical and electron microscopy analyses, it has been shown that LPS can bind to microtubules and inhibit the polymerization of microtubules in vitro in a LPS concentration-dependent process (35). At lower concentrations, LPS selectively displaced the microtubule-associated proteins from the polymerized microtubules, whereas at higher concentrations, LPS inhibited the polymerization of microtubules (5). In the present study, LPS exposure reduced microtubule polymerization rapidly, within 1 min (Fig. 3,A and B), and this effect lasted throughout the 4-h LPS treatment (Fig. 3, C–F). The function of this rapid microtubule depolymerization needs to be elucidated. The prolonged effect of LPS on microtubule depolymerization is concurrent with the increased MIP-2 production. Furthermore, blocking depolymerization of microtubules by pretreatment with paclitaxel partially inhibited LPS-induced MIP-2 (Fig. 4), whereas colchicine or nocodazole, microtubule-depolymerizing agents, enhanced LPS-induced MIP-2 (Fig. 5) from pneumocytes. Taken together, these results suggest that the LPS-induced microtubule depolymerization is involved in MIP-2 production. We further found that LPS-induced MIP-2 mRNA expression was not affected by these microtubule-polymerizing or -depolymerizing agents (Fig. 7). Thus the effects of these agents on MIP-2 production from LPS-stimulated pneumocytes should be mainly at the posttranscriptional level.
In general, interrupting microtubule polymerization blocks secretion (4), but there are also reports that secretion is unimpaired in cells depleted of microtubules (4). The role of microtubules in intracellular transport may depend on cell type, molecules of interest, involvement of microtubule-related motor proteins, and other confounding factors. Microtubules are well known to be involved in the regulation of intracellular trafficking of secretion vesicles (7, 23, 31) by regulating the transport efficiency and guiding the appropriate destination as “highways” (4). A “recycling” mechanism has been proposed, which consists of retrograde transport from the Golgi to the ER and anterograde transport from the ER to the Golgi, both of which are associated with microtubules (7, 23). With proteins tagged with a green fluorescent protein, ER-to-Golgi trafficking (31,37) and microtubule-dependent transport of secretory vesicles (47) have been visualized in real time. Microtubule-disrupting agents (i.e., nocodazole) inhibit retrograde movement, causing Golgi proteins to accumulate in the intermediate compartment (23). Similarly, microtubule depolymerization, either induced by LPS alone or further enhanced by colchicine or nocodazole, may selectively reduce the retrograde transport from the Golgi to the ER and hence increase the secretion of MIP-2 from rat pneumocytes. MIP-2 produced from pneumocytes was enhanced by colchicine or nocodazole only when the cells were treated with relatively higher concentrations of LPS (100 ng/ml to 10 μg/ml; Fig. 6). It is possible that a relatively small amount of MIP-2 induced by lower concentrations of LPS could be secreted efficiently. Higher concentrations of LPS induce microtubule depolymerization, which may facilitate the secretion of an excess amount of MIP-2 produced by lung alveolar epithelial cells. Isowa et al. (19) have recently found that LPS-induced TNF-α secretion from pneumocytes was not enhanced by microtubule depolymerization with colchicine. The amount of TNF-α secreted from these cells is only several hundred picograms per milliliter; thus the microtubule-dependent mechanism may not be necessary for TNF-α release from pneumocytes.
Microtubules are also very important in maintaining the intracellular localization of the ER and the Golgi complex, which are essential in determining the pathways for secretory proteins. When cells are treated with nocodazole, depolymerization of microtubules leads to redistribution of the ER and the Golgi (7, 9, 23, 47). LPS-induced depolymerization of microtubules, especially in the presence of nocodazole or colchicine, may change the distribution of the ER and the Golgi in alveolar epithelial cells, which may lead to microtubule-independent secretion from the ER to the plasma membrane. As we found, paclitaxel only partially inhibited LPS-induced MIP-2 secretion (Fig. 4). We (18) have recently found that LPS-induced depolymerization of microfilaments of pneumocytes also contributes to LPS-induced MIP-2 secretion. Thus LPS-induced MIP-2 production from rat pneumocytes is mediated by both microtubule-dependent and -independent mechanisms.
In summary, in this study, we found that both basal and LPS-induced MIP-2 secretion occur through the ER-Golgi pathway in a constitutive fashion. In addition to activating MIP-2 gene expression and protein synthesis, LPS also induces microtubule depolymerization that may enhance MIP-2 production. Secretion of other chemokines and cytokines from pneumocytes may be regulated by similar mechanisms.
We acknowledge the technical assistance of Ewa Dziak, Xiao-Hui Bai, Xiao-Ming Zhang, Dr. Michiharu Suga, and Dr. Stefan Fisher.
This research was supported by operating grants from the Medical Research Council of Canada (MT-13270), James H. Cummings Foundation, Canadian Cystic Fibrosis Foundation, and Ontario Thoracic Society.
N. Isowa is a recipient of a fellowship from Department of Surgery and Faculty of Medicine, University of Toronto (Toronto, ON). M. Liu is a Scholar of the Medical Research Council of Canada.
Address for reprint requests and other correspondence: M. Liu, Thoracic Surgery Research Laboratory, Toronto General Hospital, Univ. Health Network, Room CCRW 1-816, 200 Elizabeth St., Toronto, Ontario, Canada M5G 2C4 (E-mail:).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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