Departments of 1 Physiology and 2 Pediatrics, Institute for Environmental Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104
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ABSTRACT |
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We analyzed the pattern of gap junction protein (connexin) expression in vivo by indirect immunofluorescence. In normal rat lung sections, connexin (Cx)32 was expressed by type II cells, whereas Cx43 was more ubiquitously expressed and Cx46 was expressed by occasional alveolar epithelial cells. In response to bleomycin-induced lung injury, Cx46 was upregulated by alveolar epithelial cells, whereas Cx32 and Cx43 expression were largely unchanged. Given that Cx46 may form gap junction channels with either Cx43 or Cx32, we examined the ability of primary alveolar epithelial cells cultured for 6 days, which express Cx43 and Cx46, to form heterocellular gap junctions with cells expressing other connexins. Day 6 alveolar epithelial cells formed functional gap junctions with other day 6 cells or with HeLa cells transfected with Cx43 (HeLa/Cx43), but they did not communicate with HeLa/Cx32 cells. Furthermore, day 6 alveolar epithelial cells formed functional gap junction channels with freshly isolated type II cells. Taken together, these data are consistent with the notion that type I and type II alveolar epithelial cells communicate through gap junctions compatible with Cx43.
bleomycin; cell junctions; differentiation; connexin; cell-cell interactions
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INTRODUCTION |
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AT STEADY STATE, the alveolar epithelium is a heterogeneous mixture of cells, mostly type II and type I alveolar epithelial cells (12). In response to type I cell damage, type II cells are stimulated to proliferate and differentiate to replenish the type I cell population (43, 56). Although type II cells outnumber type I cells by a 2:1 ratio, the vast majority of the alveolar surface area is covered by large, flat type I cells (12). Thus most of the cell-cell interfaces consist of type I cells in direct contact with other type I cells. However, there are also numerous areas where type II cells are in contact with type I cells. Direct contact between two type II cells is rare in the normal adult lung, although it is more prominent in the developing lung and during recovery from lung injury (2, 15, 43).
Sites of cell-cell contact contain a number of different structures including gap junctions (25, 48, 53). Gap junctions contain complexes of channels that interconnect adjacent cells and enable the direct transfer of small aqueous molecules from the cytoplasm of one cell to another. Isolated primary type II alveolar epithelial cells express up to six different gap junction proteins (connexins) at the mRNA level: connexin (Cx)26, Cx30.3, Cx32, Cx37, Cx43, and Cx46 (1, 40). Of the connexins expressed by alveolar epithelial cells, we have found that three (Cx32, Cx43, and Cx46) are most prominent at the protein level (1). Freshly isolated type II cells express all three of these connexins, whereas type II cells cultured under conditions where they differentiate into a type I-like phenotype lose Cx32 mRNA expression and upregulate Cx43 and Cx46 mRNA and protein. To determine whether Cx32, Cx43, or Cx46 is differentially expressed by alveolar epithelial cells in vivo, we examined the changes in expression of these connexins after intratracheal instillation of bleomycin, which causes alveolar epithelial cell injury and induces type II cell hyperproliferation (33, 49, 61).
The role for the heterogeneity of connexin expression by alveolar
epithelial cells is not known at present. Cells that express multiple
connexins have the potential to form mixed gap junction channels
(5, 6, 20, 29, 37, 57, 58). Connexins fall into roughly
two compatibility subgroups, and
, based on dendrogram analysis
(39). With some exceptions, such as Cx40, connexins within
a given subgroup are able to form mixed gap junction channels with
other connexins in the same subgroup but not with connexins in another
subgroup (20, 58). Thus Cx43 and Cx32 are incompatible
because Cx43 is an
-connexin and Cx32 is a
-connexin. However,
although Cx46 is also an
-connexin, it is unique in that it can form
functional heteromeric gap junctions with either Cx43 or Cx32 when
expressed in Xenopus oocytes (58).
The ability of cells expressing Cx46 to couple with cells expressing Cx32 or Cx43 has not been tested in mammalian cells. We used a preloading assay (23, 37, 55) to determine whether alveolar epithelial cells expressing high levels of Cx43 and Cx46 had the capacity to communicate with HeLa cells expressing either Cx43 or Cx32. Using the same assay, we also examined the capacity of day 6 alveolar epithelial cells for heterocellular gap junctional communication with freshly isolated type II cells as a model for heterocellular communication between type I and type II cells.
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METHODS |
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Bleomycin-induced lung injury. Animal protocols were reviewed and authorized by the Institutional Animal Care and Use Committees of both the University of Pennsylvania and The Children's Hospital of Philadelphia (Philadelphia, PA). Sprague-Dawley rats were anesthetized, and bleomycin (8 U/kg) in 0.25 ml of a normal saline solution was intratracheally instilled through a 1-cm incision in the anterior neck as previously described (51). Control animals received saline vehicle alone or were untreated. The incision was then sutured, and the animals were allowed to recover. Seven days after treatment, the animals were euthanized, and the lungs were harvested and then processed for either histochemical or mRNA analysis.
Immunofluorescence.
Polyclonal anti-Cx43 and anti-Cx46 antibodies were produced as
previously described (11, 38). Anti-Cx32 antibody was from Zymed (South San Francisco, CA). Lungs obtained from untreated, saline-treated, and bleomycin-treated rats were inflated, fixed by
perfusion with 2% paraformaldehyde, and then sequentially incubated in
5 mM NH4Cl, 10% sucrose, 20% sucrose, and 30% sucrose in
PBS. The samples were chopped into ~3-mm2 pieces and then
frozen into embedding medium (Tissue-Tek). The samples were sliced with
a microtome to 4-µm thickness and mounted onto slides. Tissue
sections were incubated for 30 min at room temperature with 1 M glycine
in PBS to reduce nonspecific cross-linking and autofluorescence, washed
with PBS, incubated for 10 min at room temperature with 1 mg/ml of
NaBH4 in PBS to further reduce autofluorescence, and then
washed with PBS plus 0.5% Triton X-100 (PBS+TX) and PBS plus 0.5%
Triton X-100 plus 2% goat serum (PBS+TX+GS). The sections were
incubated overnight at 4°C with primary antiserum diluted with
PBS+TX+GS, washed, and then incubated with indocarbocyanine-conjugated goat anti-rabbit IgG (Roche) for 2 h at room temperature. To
preferentially label type II cells, FITC-conjugated mouse anti-pan
cytokeratin clone C-11 (Sigma, St. Louis, MO) was used
(52). We chose to use anti-pan cytokeratin as a marker
because the cytokeratins recognized by this antibody (cytokeratins 8 and 18) remain strongly expressed by alveolar type II cells during the
course of bleomycin injury (31, 60) and in development
(52, 59) as opposed to other markers such as surfactant
proteins (SPs) (13, 16, 50), lamellar bodies
(50), or T1 (36) that can have variable or
reduced expression patterns in bleomycin-treated animals. Macrophages were labeled with FITC conjugated to an antibody raised to a macrophage antigen (ED-1, Labgen Immunochemicals, Frankfurt, Germany). For isolated cells plated onto glass coverslips, the cells were washed with
PBS, fixed, and permeabilized with 1:1 methanol-acetone for 2 min;
washed in PBS, PBS+TX, and PBS+TX+GS; and then immunostained as
described above except that the incubation with primary antiserum was
for 1 h at room temperature. The slides were mounted in MOWIOL and
visualized with fluorescence microscopy with an Olympus IX-70 microscope. Images were obtained with a Hamamatsu Orca-1 and Image Pro
image analysis software.
RT-PCR. PCR primers corresponding to Cx32, Cx43, and Cx46 (1) and SP-C (45) were obtained from Operon (Alameda, CA) as previously described. Semiquantitative RT-PCR was performed as follows. RNA was isolated from untreated, saline-treated, and bleomycin-treated lungs with TRIzol reagent (Life Technologies). RNA was treated with DNase (Promega, Madison, WI) to remove contaminating genomic DNA and then converted to cDNA with reverse transcriptase and random hexamer primers (Clontech, Palo Alto, CA). This was used as the source material for the PCR that used primers specific for a given connexin construct. We used QuantumRNA 18S internal standards (Ambion, Austin, TX) to normalize for input cDNA. The size and amount of the PCR products generated were confirmed by agarose gel electrophoresis in the presence of ethidium bromide and analyzed with the Kodak EDAS 1-d analysis package. Different starting amounts of cDNA and ratios of cDNA to 18S standards were assessed to ensure that we were in a linear range for product formation. Data were from triplicate determinations, and significance was determined with Student's t-test.
Isolation of type II cells. Sprague-Dawley rat alveolar type II cells were isolated from lavaged, perfused lungs by elastase digestion with the method of Dobbs et al. (18) with modifications (1). Cells were biopanned with IgG-coated culture dishes to remove alveolar macrophages and other Fc receptor-expressing cells. To obtain type II cells with high purity, we used a second round of immunodepletion by incubating the cells with Biomag beads coated with rabbit IgG (PerSeptive Biosystems, Framingham, MA). Using this approach, we were able to routinely get preparations that were 90-95% type II cells (1). To allow the cells to progress to a type I-like phenotype (4, 14), the cells were cultured on treated standard tissue culture plastic dishes in Earle's minimal essential medium (Life Technologies, Rockville, MD) containing 2 mM glutamine, 100 U/ml of penicillin, 100 µg/ml of streptomycin (MEM), and 10% fetal bovine serum.
Preloading assay for dye transfer. The preloading assay was done as previously described (23, 37, 55), with some modifications. Donor cells on 60-mm tissue culture dishes were washed and then incubated for 30 min at 37°C in MEM containing 1 mg/ml of Texas Red dextran (mol wt 10,000) and 10 µM calcein-AM (Molecular Probes). Texas Red dextran is internalized by fluid-phase endocytosis, whereas calcein-AM permeates the plasma membrane and is then hydrolyzed to free calcein in the cytosol to act as a marker for gap junctional intercellular communication. The donor cells were then washed and resuspended (0.25% trypsin-EDTA for 5 min at 37°C). In experiments with freshly isolated type II cells as the donor cells, the cells were labeled in suspension and washed by centrifugation at 500 g for 10 min. The donor cells were cocultured with unlabeled acceptor cells for 5 h at 37°C in MEM plus 10% FBS and imaged by fluorescence and phase-contrast microscopy. For quantitation, donor or acceptor cell interfaces were manually identified and then examined for the specific transfer of calcein. Data are expressed as the fraction of calcein-positive acceptor cells as calculated from at least 50 interfaces/experiment done in triplicate.
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RESULTS |
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Connexin expression by intact and injured alveolar epithelia.
Cx32, Cx43, and Cx46 undergo the most dramatic changes in expression
when isolated type II alveolar epithelial cells differentiate in
culture (1, 40). We used immunofluorescence to determine whether these connexins are also expressed by alveolar epithelial cells
in situ (Fig. 1). The immunofluorescence
profile for untreated control lungs was virtually identical to that for
saline-treated lungs (data not shown). Each of the anti-connexin
antisera had distinct labeling in rats 7 days after treatment with
saline vehicle alone. Cx32 had a limited distribution, and virtually
every Cx32-positive cell was also labeled with anti-pan cytokeratin
(31), consistent with Cx32 expression being limited to
type II cells. In contrast, nearly all of the epithelial cells had Cx43
immunostaining. Cx46 had a somewhat intermediate pattern of expression
in which some Cx46-positive cells showed expression of the type II cell
marker, whereas others did not.
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Gap junctional communication by type I-like cells. Isolated type II alveolar epithelial cells cultured under conditions where they assume a type I-like phenotype increase the expression of Cx46 (1). Because Cx46 was also upregulated by alveolar epithelial cells in response to bleomycin and Cx46 has the potential to form functional gap junction channels with either Cx32 or Cx43 (58), we examined the ability of primary alveolar type II cells cultured for 6 days (day 6 cells) to form gap junctions with other types of cells.
A preloading assay was used to measure the ability of cells to form gap junctions. In this assay, gap junction channel formation was determined by observing the transfer of calcein from a labeled donor cell to an unlabeled acceptor cell. Donor cells were identified with endocytosed 10-kDa molecular mass Texas Red dextran as a nontransferable marker. As shown in Fig. 4, day 6 alveolar epithelial cells showed extensive transfer of calcein from double-labeled donor cells to acceptor cells with the preloading assay, indicating that they formed functional gap junctions. Consistent with this observation, these cells showed both Cx43 and Cx46 localized to the cell surface by immunofluorescence microscopy (Fig. 5).
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Specificity of gap junction formation. It has been previously shown with transfected oocytes that Cx46 is able to form heterotypic gap junctions with Cx32 (58). Cx43 and Cx32 are not compatible to form functional gap junction channels. Because Cx32 is also expressed by type II cells, we postulated that one possible role for Cx46 expression was to enable gap junctional communication through heteromeric Cx46/Cx32 gap junction channels. To test this possibility, we substituted HeLa cells expressing either Cx43 or Cx32 for day 6 alveolar epithelial cells as a partner for the preloading assay.
We confirmed the specificity of the preloading assay using transfected HeLa cells (Fig. 6). HeLa/Cx43 cells were able to form gap junctions with other HeLa/Cx43 cells, with a coupling index of 0.55 ± 0.09 (n = 3 experiments). Communication between HeLa/Cx32 cells had a similar coupling index of 0.52 ± 0.16 (n = 3 experiments). However, we were unable to detect intercellular communication between HeLa/Cx32 and HeLa/Cx43 cells using the preloading assay regardless of which cell type was the donor and which was the acceptor.
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Communication between day 6 and freshly isolated alveolar
epithelial cells.
Because Cx32 is one of the most prominent connexins expressed by type
II cells, we examined the ability of freshly isolated type II cells to
communicate with day 6 alveolar epithelial cells and vice
versa (Fig. 8). Type II donor cells
communicated with day 6 alveolar epithelial acceptor cells,
with a coupling index of 0.64 ± 0.03 (n = 3 experiments). When day 6 alveolar epithelial cells were the
donors, the coupling index was 0.28 ± 0.08 (n = 3 experiments). Again, the coupling index correlated with the ability of
the acceptor cells to attach and spread because freshly isolated type
II cells typically require >5 h to adhere and spread poorly.
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DISCUSSION |
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In response to alveolar cell injury, type II cells are induced to proliferate. Based on DNA labeling kinetics, acute lung injury results in the appearance of a kinetically distinct subpopulation of alveolar epithelial cells before labeled type I cells appear (21). The precise phenotype of these cells is not known; however, our results raise the possibility that Cx46 may be a specific marker for a potential intermediate alveolar epithelial phenotype between the type II and type I end points. Consistent with this possibility, Cx46 was found to be clustered in focal areas of hyperplasia in the lung, and these cells showed little expression of a type II cell marker.
Bleomycin also appeared to increase the extent of Cx43 expression at the level of immunofluorescence. Consistent with our observations, Kasper et al. (32) found that Cx43 protein expression was increased during the course of radiation-induced pulmonary fibrosis. Although Kasper et al. did not examine Cx43 mRNA expression, we were unable to detect a change in net Cx43 mRNA in response to bleomycin. However, the overall level of Cx43 expression in the lung is quite high, so an increase in Cx43 expression may be due to the increase in cell number due to bleomycin injury as opposed to a change in Cx43 gene expression.
The differentiation of alveolar epithelial cells in culture is reminiscent of the differentiation of type II cells in response to type I cell injury (30, 56). This suggests that the increase in Cx46 expression due to bleomycin may be the in vivo equivalent to the transient increase in Cx46 observed when isolated type II cells differentiate into a type I-like phenotype in culture (1). Underscoring a likely role for Cx46 in the injury response, Cx46 expression is also upregulated in response to peripheral nerve injury (8). However, the function of Cx46 in an injury-response cascade in either tissue remains unclear at present.
Nearly all the studies on Cx46 function (19, 26, 29, 35) have been done with lens epithelium where Cx46 and Cx50 form mixed gap junction channels. Consistent with a function for Cx46 in lens, Cx46-deficient mice develop premature cataracts (24, 42). However, unchallenged Cx46-deficient mice have no obvious pulmonary phenotype and are viable. Future studies examining the effect of bleomycin on Cx46-deficient mice are needed to elucidate the potential roles for Cx46 in recovery from acute lung injury.
Alveolar epithelial cells do not express Cx50 (1), so it is likely that the Cx46 expressed by alveolar epithelial cells may either form independent gap junction channels or intermix with Cx43. Based on an in vitro assembly study (22) and observations from other cell types (Das Sarma J and Koval M, unpublished observations), Cx43 and Cx46 coexpressed in the same cell have the capacity to form heteromeric gap junction channels. Day 6 alveolar epithelial cells show both Cx43 and Cx46 at the plasma membrane (Fig. 5) (1), consistent with this possibility. However, the ability of Cx43 and Cx46 to interact is also likely to be a regulated process because both alveolar epithelial cells and osteoblasts can maintain separate pools where Cx43 is localized to the plasma membrane and Cx46 is retained in the trans-Golgi network (1, 38).
The formation of mixed gap junction channels with Cx43 and Cx46 may enable alveolar epithelial cells to regulate gap junctional communication, perhaps by altering channel permeability, gating, or open frequency. The electrophysiological properties of Cx43/Cx46 heteromeric gap junction channels have not been characterized; however, heteromeric gap junction channels containing Cx43/Cx45 (46) or Cx43/Cx37 (5) show intermediate gating characteristics compared with a channel composed of a single connexin. Consistent with the possibility that Cx46 may help regulate gap junctional communication, the extent of coupling between alveolar epithelial cells increases with increasing time in culture (1, 40), in parallel with increasing Cx46 expression at the plasma membrane (1).
Cx46 is unusual because it has the potential in transfected
Xenopus oocytes to form heterotypic gap junction channels
with either Cx43, an -connexin, or Cx32, a
-connexin
(58). Because type II cells express Cx32, this raised the
possibility that a transient increase in Cx46 expression by
differentiating alveolar epithelial cells might enable cells to
communicate through Cx32-compatible channels. Although Cx32 and Cx46
formed functional gap junction channels with transfected
Xenopus oocytes (58), our results indicate that
this is not the case for alveolar epithelial cells expressing
endogenous connexins intermixed with HeLa cells transfected with Cx32.
Thus Cx46 expression by alveolar epithelial cells was not sufficient to
enable intercellular communication through Cx32-compatible gap junction
channels. One possibility is that Cx46 expressed by alveolar epithelial
cells forms mixed Cx43/Cx46 gap junction channels that, in turn, are
incompatible with Cx32. Alternatively, Cx46 expressed by
Xenopus oocytes may be in a context that enables more
promiscuous gap junction protein interactions than the native mammalian
cell environment. For instance, heterocellular communication between
mammalian cells may require other cofactors, such as cell adhesion
molecules (27, 47), that may not be required for communication between transfected oocytes.
Secretory epithelial cells frequently express Cx32 (44). For instance, Cx32 expression is upregulated by mammary epithelium during lactation (41). Pancreatic acini are also coupled through Cx32, which is required for proper regulation of amylase secretion (9, 10). However, the functional significance of Cx32 expression by type II cells is unclear because our results indicate that gap junctional communication between type I and type II cells was mediated through Cx43-compatible gap junction channels, which are not compatible with Cx32.
One possibility is that Cx32 is expressed to maintain selective gap junctional communication between type II stem cells and daughter cells as a putative second channel for intercellular communication. Consistent with this notion, we observed some adjacent type II cells that expressed Cx32 in response to bleomycin injury (Fig. 2, A-C). Interestingly, Cx32 function by intestinal epithelium is important for protection from ischemia-reperfusion injury in intestinal epithelium, presumably by enabling cells to share protective metabolites such as glutathione (28). Whether type II cells use Cx32-compatible channels as a protective mechanism is not known; however, if this is the case, then we predict that disruption of Cx32 expression by type II cells may increase their sensitivity to injury.
The signals that are propagated through gap junctions to regulate epithelial function are largely unknown at present. One possibility is that gap junctions coordinate intercellular transmission of transient increases in intracellular calcium. For instance, coupling between pancreatic acinar cells controls the frequency of calcium oscillations that, in turn, modulates amylase secretion (10, 54). Consistent with these observations and a role for heterocellular gap junctions in the regulation of surfactant secretion induced by mechanical stress, Ashino et al. (3) found that gap junctional communication from type I to type II cells in situ was required for both the propagation of calcium transients and stimulated surfactant secretion. Whether coupling between type I and type II alveolar epithelial cells mediates the direct transmission of calcium or whether this involves another signaling molecule, such as inositol trisphosphate, is not known at present. Using strategies based on dominant negative connexins that we have developed to alter gap junctional permeability in other systems (37), studies are underway to better define the classes of signaling molecules transmitted between type I and type II cells.
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Note added in proof |
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Consistent with our results, Guo et al. used the transfer of microinjected Lucifer yellow to show that freshly isolated type II cells and day 3 alveolar epithelial cells in coculture were coupled by gap junctions (Guo U, Martinez-Williams C, Yellowley CE, Donahue HJ, and Rannels DE. Connexin expression by alveolar epithelial cells is regulated by extracelluar matrix. Am J Physiol Lung Cell Mol Physiol 280: L191-L202, 2001).
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ACKNOWLEDGEMENTS |
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We thank Jayasri Das Sarma and Fushan Wang for helpful comments on the manuscript.
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FOOTNOTES |
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This work was supported by grants from the American Heart Association, Arthritis Foundation, and March of Dimes Foundation; National Institute of General Medical Sciences Grant GM-61012 (to M. Koval); and National Heart, Lung, and Blood Institute Grant HL-62472 (to R. C. Savani).
Address for reprint requests and other correspondence: M. Koval, Institute of Environmental Medicine, Univ. of Pennsylvania Medical School, 1 John Morgan Bldg., 3620 Hamilton Walk, Philadelphia, PA 19104-6068 (E-mail: mkoval{at}mail.med.upenn.edu).
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.
Received 8 August 2000; accepted in final form 19 October 2000.
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