Summary
Regeneration of the mesothelium is unlike that of other epithelial-like surfaces, as healing does not occur solely by centripetal migration of cells from the wound edge. The mechanism of repair of mesothelium is controversial, but it is widely accepted, without compelling evidence, that pluripotent cells beneath the mesothelium migrate to the surface and differentiate into mesothelial cells. In this study we examined an alternative hypothesis, using in vivo cell-tracking studies, that repair involves implantation, proliferation and incorporation of free-floating mesothelial cells into the regenerating mesothelium. Cultured mesothelial cells, fibroblasts and peritoneal lavage cells were DiI- or PKH26-PCL-labelled and injected into rats immediately following mesothelial injury. Implantation of labelled cells was assessed on mesothelial imprints using confocal microscopy, and cell proliferation was determined by proliferating cell nuclear antigen immunolabelling. Incorporation of labelled cells, assessed by the formation of apical junctional complexes, was shown by confocal imaging of zonula occludens-1 protein. Labelled cultured mesothelial and peritoneal lavage cells, but not cultured fibroblasts, implanted onto the wound surface 3, 5 and 8 days after injury. These cells proliferated and incorporated into the regenerated mesothelium, as demonstrated by nuclear proliferating cell nuclear antigen staining and membrane-localised zonula occludens-1 expression, respectively. Furthermore, immunolocalisation of the mesothelial cell marker HBME-1 demonstrated that the incorporated, labelled lavage-derived cells were mesothelial cells and not macrophages as it had previously been suggested. This study has clearly shown that serosal healing involves implantation, proliferation and incorporation of free-floating mesothelial cells into the regenerating mesothelium.
Introduction
Mesothelium lines the peritoneal, pleural and pericardial cavities, with visceral and parietal surfaces covering the internal organs and body wall, respectively. Mesothelium comprises a monolayer of predominantly flattened epithelial-like cells resting on a basement membrane supported by connective tissue that contains blood vessels, lymphatics and subserosal mesenchymal cells ( Wang, 1974). Ultrastructural analysis of polarised mesothelial cells demonstrates tight junctions (zonula occludens) located towards their luminal aspect ( Baradi and Hope, 1964; Kluge and Hovig, 1967). Injury to this layer can lead to the formation of adhesions, bands of fibrous tissue that form between serosal surfaces. Adhesions occur in up to 95% of patients undergoing abdominal surgery ( Menzies and Ellis, 1990), therefore understanding the mechanisms regulating normal serosal repair may lead to novel strategies to prevent this common surgical disorder.
The mechanisms involved in mesothelial regeneration following injury are controversial. Hertzler ( Hertzler, 1919) was the first to observe that small and large peritoneal injuries healed at the same rate and concluded that the mesothelium could not regenerate solely by centripetal migration of cells at the wound edge as occurs for healing of squamous epithelia. Subsequently, several hypotheses have been proposed for the origin of the cells in regenerating mesothelium; these cells include subserosal mesenchymal precursors ( Ellis et al., 1965; Raftery, 1973; Bolen et al., 1986), bone marrow-derived precursors ( Wagner et al., 1982), free-floating macrophages ( Eskeland and Kjærheim, 1966; Ryan et al., 1973) and free-floating mesothelial cells ( Cameron et al., 1957; Watters and Buck, 1973; Whitaker and Papdimitriou, 1985). To date, the most accepted proposal is that repopulating mesothelial cells originate from a pool of pluripotent subserosal fibroblast-like cells, which migrate to the serosal surface, divide and differentiate into mesothelial cells ( Ellis et al., 1965; Raftery, 1973). However, irradiation studies have demonstrated impaired local mesothelial regeneration, which was recoverable by addition of peritoneal lavage cells ( Whitaker and Papadimitriou, 1985), suggesting that subserosal fibroblasts are not the source of regenerating mesothelial cells. In addition, studies of the kinetics of serosal repair demonstrated that subserosal cells were not essential for mesothelial healing and that the regenerating cells were likely to originate from the surrounding uninjured serosal surface ( Mutsaers et al., 2000).
In 1957, Cameron and colleagues proposed that mesothelial healing involved attachment of free-floating mesothelial cells to the injured surface. Peritoneal lavage fluid recovered from experimental animals following injury to the mesothelium was found to contain a significantly higher number of viable free-floating mesothelial cells two days post injury than the controls ( Whitaker and Papadimitriou, 1985). The increased free-floating cell population was thought to be caused by the proliferation of mesothelial cells adjacent to ( Johnson and Whitting, 1962; Mutsaers et al., 2000) and opposing ( Watters and Buck, 1973; Fotev et al., 1987) the serosal injury.
In this study, we have conclusively demonstrated that serosal healing involves incorporation of free-floating mesothelial cells into the regenerating mesothelium. Fluorescently labelled cell tracking confirmed implantation of cultured and peritoneal lavage-derived mesothelial cells onto the denuded wound surface in a well characterised rodent model of normal serosal repair ( Fotev et al., 1987; Mutsaers et al., 1997). Furthermore, proliferation and incorporation of fluorescently labelled mesothelial cells was demonstrated by immunolocalisation of proliferating cell nuclear antigen (PCNA) and the tight junction-associated protein, zonula occludens-1 (ZO-1), respectively.
Materials and Methods
Cell isolation and characterisation
Normal mesothelial cells were isolated from the anterior peritoneal wall of male Lewis rats (Harlan, Bicester, UK) as previously described ( Stylianou et al., 1990). Briefly, peritoneal tissue was incubated in a solution of 0.25% trypsin and 0.02% EDTA in Dulbecco's modified Eagle's medium (DMEM; Gibco, Paisley, UK) for 30 minutes at 37°C. The intact tissue was discarded and the remaining cell suspension centrifuged at 1000 rpm for 5 minutes. The cell pellet was re-suspended and the cells maintained in DMEM supplemented with 15% foetal calf serum (FCS; CSL UK Ltd, Andover, UK), 4 mM L-glutamine (Gibco, Paisley, UK), 5 ng/ml epidermal growth factor (Roche Diagnostics, Lewes, UK), 0.4μ g/ml hydrocortisone (Sigma Aldrich, Poole, UK) and antibiotics (penicillin, 100,000 units/l and streptomycin, 50 mg/l; Gibco, Paisley, UK).
Primary fibroblasts, used as a control in cell tracking studies, were isolated from the lungs of male Lewis rats. Peritoneal fibroblasts were not used owing to possible contamination with surface mesothelial cells. Diced lung parenchyma (carefully avoiding the serosal surface) was incubated in 1 mg/ml of type II collagenase (Worthington Biochemical Corp, Lakewood, New Jersey, USA) in DMEM for 2 hours at 37°C. The cell suspension was centrifuged at 1000 rpm for 5 minutes, resuspended and cells maintained in DMEM supplemented with 10% FCS, 4mM L-glutamine and antibiotics (penicillin, 100,000 units/l and streptomycin, 50 mg/l). All cells were grown in a humidified atmosphere of 10% CO2 in air at 37°C.
Mesothelial cells, which are embryologically derived from the mesoderm, share characteristics of both epithelial and mesenchymal cells ( Whitaker et al., 1982) and so were distinguished from fibroblasts using monoclonal antibodies directed against human pancytokeratin (dilution 1:20) and human vimentin (dilution 1:400; Dako Ltd, Ely, UK).
Fluorescence labelling of cultured cells
To examine the role of free-floating cell populations in vivo, cultured mesothelial cells and fibroblasts were fluorescently labelled with the celltracking probe DiI (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindo-carbacyanine perchlorate; Molecular Probes Inc., Eugene, Oregon, USA). Briefly, subconfluent cultures, up to passage three, were incubated in serum-free DMEM containing 10 μM DiI for 20 minutes under standard conditions, washed with phosphate buffered saline (PBS; pH 7.3) and then incubated in standard supplemented medium for a further 30 minutes according to the manufacturers instructions. In order to perform studies examining cell proliferation, the chloromethylbenzamido derivative of DiI, CM-DiI, was used for cell labelling because of its ability to withstand histological tissue processing ( Andrade et al., 1996).
Previous studies have demonstrated negligible transfer of DiI between adjacent membranes ( Honig and Hume, 1986), an important property for cell tracking. To confirm this finding, six replicate suspensions containing equal numbers (2.5×105) of unlabelled and DiI-labelled mesothelial cells were plated and incubated under standard conditions for 3 days. The proportion of cells labelled with DiI was then determined by direct cell counting using a Zeiss MC80 DX fluorescent microscope.
Fluorescence labelling of peritoneal lavage cells
To collect peritoneal lavage cells, male Lewis rats received a widespread abrasion injury to the anterior peritoneal wall with a sterile gauze swab. Animals were sacrificed 2 days post injury, when the free-floating mesothelial cell population was maximal ( Whitaker and Papadimitriou, 1985), and the peritoneal cavity was lavaged with 20 ml serum-free DMEM. The lavage fluid was centrifuged at 1000 rpm for 5 minutes, the cell pellet re-suspended in serum-free DMEM containing 10 μM DiI and incubated for 30 minutes at 37°C, before washing with PBS and resuspending the labelled cells in serum-free DMEM.
Peritoneal free-floating macrophages have been suggested as a potential source of regenerating mesothelial cells ( Eskeland and Kjærheim, 1966; Ryan et al., 1973). Therefore, peritoneal free-floating cells were labelled using the red fluorescent dye, PKH26-PCL (Sigma Aldrich, Poole, UK), which is specifically taken up by phagocytic cells and remains within the cells for more than 21 days in vivo ( Melnicoff et al., 1989). Rats were injected intraperitoneally (i.p.) with 0.5μ M PKH26-PCL 2 days post injury, killed 2 hours later and the peritoneal cavity lavaged with 20 ml serum-free DMEM to retrieve labelled lavage cells.
Mesothelial healing model
7 to 9 week old male Lewis rats (Harlan, Bicester, UK) weighing 160-170 g were used throughout this study (n=3 for each experimental treatment). Animals were housed in groups of five and fed on a commercial diet and water ad libitum. A testicular thermal injury model ( Fotev et al., 1987; Mutsaers et al., 1997) was used to examine normal serosal healing. Briefly, a metal probe, consisting of a mica-coated brass rod with a 1 cm diameter tip heated to 60°C, was applied to a standard site on both testicular serosal surfaces for 3 seconds. The tunica vaginalis and scrotal skin were closed using 4-0 silk sutures.
In vivo cell tracking
An equal number of labelled and unlabelled cells, suspended in serum-free DMEM at a concentration of 1×106 cells/ml were used for all in vivo cell tracking studies. The inclusion of unlabelled cells allowed clear distinction of DiI-labelled cells. Aliquots (1 ml) containing labelled and unlabelled, cultured or lavage-derived cells were injected i.p. immediately following serosal injury. To assess whether mesothelial cell implantation was restricted to the wound site, an additional set of uninjured animals was injected i.p. with DiI-labelled cultured mesothelial cells.
At 3, 5 and 8 days post injury, animals were sacrificed and both testes excised. In order to assess cell implantation and expression of the tight-junction-associated protein, ZO-1, and the mesothelial cell surface marker HBME-1, the testes were washed with PBS, the serosal surface dried with compressed air and mesothelial monolayer imprints obtained on 5% gelatin coated microscope slides ( Mutsaers et al., 1997). This technique removed almost all cells from the regenerating surface, although occasional cells failed to adhere to the gelatin. Whole testes from animals injected with CM-DiI labelled cultured mesothelial cells were removed 4 days post injury, when there is maximal mitotic activity on the wound surface ( Watters and Buck, 1973; Mutsaers et al., 2000), and processed for histology and PCNA immunohistochemistry.
Cell proliferation
Paraffin-wax-embedded sections (3 μm) of whole testes were microwaved in 10 mM citrate buffer, pH 6.4, for 10 minutes to allow nuclear antigen retrieval. Endogenous peroxidase activity and non-specific binding sites were blocked by incubating sections in 1% hydrogen peroxide and 1.5% normal rabbit serum, respectively. Sections were incubated with a monoclonal antibody directed against PCNA (dilution 1:75; Dako Ltd, Ely, UK) for 2 hours in a humidified chamber at room temperature. Negative controls were treated with isotype-specific mouse IgG2a antibody (PharMingen, San Diego, California, USA). Sections were then incubated with biotinylated rabbit anti-mouse antisera (dilution 1:100; Dako Ltd) for 1 hour followed by streptavidin-HRP (dilution 1:200; Dako Ltd) for 30 minutes with subsequent detection using the chromogenic substrate 3,3′-diaminobenzidine (DAB; Sigma Aldrich, Poole, UK). Sections were mounted with DPX (BDH, Poole, UK) and consecutive sections were examined by confocal laser scanning microscopy for the presence of CM-DiI labelled cells.
HBME-1 and ZO-1 localisation
Immunolocalisation of the mesothelial cell surface marker HBME-1 and the tight-junction-associated protein ZO-1 on mesothelial imprints was performed to demonstrate incorporation of labelled mesothelial cells into the reconstituted serosal surface. Imprints were fixed with 4% (w/v) paraformaldehyde, pH 7.4, for 5 minutes and permeabilised for 5 minutes in PBS, pH 7.0, containing 20 mM HEPES (N-2-hydroxyethylpiperazine-N′-2-ethane sulfonic acid), 300 mM sucrose, 50 mM sodium chloride, 3 mM magnesium chloride and 0.5% Triton X-100. Non-specific staining was blocked with 5% newborn calf serum before incubating the imprints with monoclonal antibodies directed against ZO-1 (dilution 1:25; Zymed Laboratories Inc, San Francisco, California, USA) or HBME-1 (dilution 1:100; Dako Ltd, Ely, UK) for 1 hour at room temperature in a humidified chamber. Negative controls were treated with isotype-specific antisera (Dako Ltd). Imprints were then incubated with rabbit anti-mouse fluorescein isothiocyanate (FITC)-conjugated antisera (dilution 1:40; Dako Ltd) for 1 hour at room temperature in a humidified chamber before being washed, mounted in Immu-mount (Shandon, Runcorn, UK) and examined using confocal laser scanning microscopy.
Microscopy and imaging
Gelatin imprints and tissue sections of regenerating mesothelium were examined by confocal laser scanning microscopy using the Leica TCS NT system. Photomultiplier tube voltage thresholds for confocal microscopy were set to gate out background fluorescence produced by isotype-specific negative controls. Fluorescent images were sequentially collected through regenerating mesothelial imprints and tissue sections for FITC and TRITC (tetrarhodamine isothiocyanate) fluorochromes at 488 and 568 nm emission wavelengths, respectively. Tissue sections stained for PCNA were examined using an Olympus BX40 light microscope, and images were captured using KS300 image analysis software.
Results
Cell culture and characterisation
Cultured peritoneal mesothelial cells were bipolar or multipolar in appearance but at confluence they adopted a polygonal configuration and became increasingly fibroblast-like with repeated passage. Cultured fibroblasts were more elongated and formed a whorl-like pattern at confluence. Both mesothelial and fibroblast cell cultures expressed the mesenchymal intermediate filament, vimentin, in a diffuse filamentous distribution, whereas only mesothelial cells expressed the epithelial intermediate filament, cytokeratin, confirming the isolation of pure cultures of mesothelial cells (data not shown).
Transfer of DiI between cultured cells
Equal numbers of unlabelled and DiI-labelled mesothelial cells were cultured for 3 days, and the proportion of DiI-labelled cells were determined. Cell counts demonstrated no significant change in the proportion of DiI-labelled mesothelial cells between the time of plating (50%) and 3 days (45.8±2.7%; Student's two-tailed t-test: p > 0.05).
Implantation of DiI- and PKH26 PCL-labelled cells onto a denuded serosal surface
DiI-labelled cultured mesothelial cells, which demonstrated red fluorescence localised to both the plasma membrane and vesicle-like structures within the cell cytoplasm ( Fig. 1A; inset), were injected i.p. into injured rats to determine whether these cells were capable of implanting onto a denuded serosal surface. 5 days post injury, imprints comprised islands of predominantly rounded cells, but after 8 days the wound surface was completely covered with cells, which had assumed a more polygonal configuration. Imprints of regenerating mesothelium at 5 and 8 days post injury demonstrated the presence of DiI-labelled cells ( Fig. 1A), which were most numerous at the wound centre and least in number at the periphery. However, labelled cells were absent on imprints following transplantation of DiI-labelled fibroblasts at all time points examined ( Fig. 1B). In addition, imprints of uninjured mesothelium, taken from animals injected with DiI-labelled cultured mesothelial cells, did not demonstrate any incorporation of DiI-labelled cells (data not shown).
Implantation of fluorescence-labelled cultured mesothelial cells and peritoneal lavage-derived cells on representative areas of serosal injury. DiI-labelled cultured mesothelial cells, characterised by intense, punctate, red fluorescence in cell suspension (inset A), were present on imprints of regenerating mesothelium 5 days post injury (A), whereas DiI-labelled cultured fibroblasts were absent at the same time point (B). DiI-labelled peritoneal lavage cells were also present 8 days post injury (C). DiI-labelled cells (arrowed) were clearly distinguished from adjacent unlabelled cells (arrowheads). PKH26-PCL-labelled phagocytic cells were characterised by intense cytoplasmic red fluorescence in peritoneal lavage fluid obtained 2 days after peritoneal abrasion injury (inset D). Labelled macrophages (arrowed) were implanted onto the wound surface at 3 and 5 days post injury (D and E, respectively) but were absent by 8 days (F). Bars, 10 μm.
When animals were injected with DiI-labelled peritoneal lavage cells, labelled cells were found on imprints of regenerating mesothelium at both 5 and 8 days post injury ( Fig. 1C) and displayed a similar distribution to that found using cultured mesothelial cells. However, although PKH26-PCL-labelled peritoneal lavage phagocytes ( Fig. 1D inset) were present on imprints 3 and 5 days post injury ( Fig. 1D, E), they were completely absent by 8 days ( Fig. 1F).
Proliferation of implanted DiI-labelled cultured mesothelial cells on the regenerating serosal surface
Four days post injury, transverse sections of healing serosa, immunostained for the proliferation marker PCNA, demonstrated positive cells within the seminiferous tubules ( Fig. 2A) and the regenerating mesothelium ( Fig. 2B). Isotype-specific negative controls did not reveal any non-specific staining (data not shown). Adjacent sections revealed multiple layers of CM-DiI-labelled cultured mesothelial cells at the wound site ( Fig. 2C), which corresponds to the position of PCNA-positive cells.
Proliferation of CM-DiI-labelled cultured mesothelial cells in regenerating mesothelium. Immunolocalisation of PCNA 4 days post injury shows proliferating cells in seminiferous tubules (A) and regenerating mesothelium (boxed area and B). The adjacent tissue section shows CM-DiI-labelled mesothelial cells in multiple layers at the wound site (C), some corresponding to PCNA-positive cells (arrowed). Bar, 10 μm.
Incorporation of fluorescence-labelled cells into the regenerating mesothelium
To confirm the identity of implanted DiI-labelled peritoneal lavage cells, imprints were examined for expression of the mesothelial cell surface marker HBME-1. Normal mesothelium showed a plasma membrane distribution of HBME-1 expression ( Fig. 3A), and this was not present on isotype-specific-treated negative controls (data not shown). Dual colocalisation of DiI and HBME-1 demonstrated that implanted DiI-labelled lavage cells expressed the mesothelial cell surface marker at both 5 and 8 days post injury ( Fig. 3B).
Identification of mesothelial cells by HBME-1 immunostaining. HBME-1 expression in normal mesothelium (A). Dual localisation of DiI and HBME-1 on gelatin imprints of regenerating mesothelium, 8 days following injection of DiI-labelled peritoneal lavage cells, demonstrating membranous HBME-1 expression on the surface of implanted DiI-labelled cells (B, arrowed). Bar, 10 μm.
The incorporation of DiI-labelled cultured mesothelial and peritoneal lavage cells into regenerating mesothelium was examined by immunolocalisation of the tight-junction-associated protein ZO-1 on mesothelial imprints. In normal mesothelium, ZO-1 immunoreactivity was detected at the plasma membrane with weak staining observed within the cell cytoplasm ( Fig. 4A). In regenerating mesothelium, cells on the wound surface 5 days post injury demonstrated ZO-1 staining localised towards the plasma membrane and displayed a punctate distribution at sites of cell-to-cell contact ( Fig. 4B), which was increased at day 8 ( Fig. 4C). Confocal overlay images revealed cell membrane localised expression of ZO-1 in implanted DiI-labelled cultured mesothelial cells ( Fig. 4B) and lavage-derived cells ( Fig. 4C) at both 5 and 8 days post injury. Weak cytoplasmic expression of ZO-1 was also observed in regenerating mesothelium at all time points examined in both DiI-labelled and unlabelled cells. Negative controls treated with specific isotypes demonstrated negligible FITC staining (data not shown).
Incorporation of labelled cells into regenerating mesothelium. ZO-1 expression in normal mesothelium is localised to the plasma membrane (A). Dual localisation of DiI and ZO-1 on imprints of regenerating mesothelium, 5 days following injection of DiI-labelled cultured mesothelial cells (B, arrowed) and 8 days following injection of DiI-labelled peritoneal lavage cells (C, arrowed), demonstrating relocation of ZO-1 to the plasma membrane at points of cell-to-cell contact. Bar, 10 μm.
Discussion
This study clearly demonstrates for the first time that cultured and lavage-derived mesothelial cells attach to injured serosal surfaces, proliferate and incorporate into the regenerating mesothelium. These studies were performed using the lipophilic cell tracking dye, DiI, to determine the fate of mesothelial cells transplanted into the peritoneal cavity following mesothelial injury. DiI has been used for long-term cell tracking studies in vivo and its presence in cell membranes does not affect cell viability, proliferation or other physiological processes ( Honig and Hume, 1986; Kuffler, 1990). Furthermore, it was confirmed in this study that DiI is not transferred between neighbouring cells. Using this technique, DiI-labelled cells were clearly demonstrated on the injured serosal surface and within the reconstituted mesothelium. PKH26-PCL-labelled peritoneal macrophages also attached to the wound surface but did not incorporate into the regenerated mesothelium; however DiI-labelled fibroblasts failed to attach, demonstrating the selective implantation of certain cell types. Furthermore, labelled cultured mesothelial cells failed to attach to uninjured mesothelium, suggesting that mesothelial cells adhere to exposed extracellular matrix (ECM) substrates following injury ( Tietze et al., 1999; Leavesley et al., 1999).
It has previously been shown that there is an increase in the number of free-floating mesothelial cells in peritoneal fluid following serosal injury ( Whitaker and Papadimitriou, 1985). Peritoneal lavage fluid also contains inflammatory exudate cells, predominantly macrophages, and it has been proposed that macrophages can transform into mesothelial cells to reconstitute the mesothelium ( Eskeland and Kjærheim, 1966; Ryan et al., 1973). To assess whether macrophages are a source of mesothelial cells, free-floating phagocytic cells were labelled with PKH26-PCL and transplanted into the peritoneal cavity following injury. Labelled cells were present on the wound surface at 3 and 5 days but were absent at 8 days, demonstrating that macrophage transformation into mesothelial cells does not occur. This supports previous studies in which peritoneal macrophages labelled with trypan blue ( Ellis et al., 1965) or loaded with polystyrene spheres ( Raftery, 1973) were not identified within healed mesothelium.
Although new mesothelial cells do not originate from inflammatory cells, previous studies have suggested that macrophages secrete mitogens, which stimulate mesothelial proliferation and initiate healing ( Fotev et al., 1987; Rodgers and diZerega, 1992). Mesothelial cells surrounding a serosal lesion proliferate between 24 and 48 hours after injury, when collections of macrophages are present on the wounded area. Maximal cell proliferation at the centre of the wound occurs 4 days post injury following attachment of free-floating mesothelial cells and migration of cells at the edge of the lesion towards the wound centre ( Whitaker and Papadimitriou, 1985; Watters and Buck, 1973; Mutsaers et al., 2000). Fotev and coworkers ( Fotev et al., 1987) demonstrated mitogenic activity for mesothelial cells in wound lavages from injured serosal tissue and conditioned medium from macrophage cultures. Subsequently, Mutsaers et al. ( Mutsaers et al., 1997) identified fibroblast growth factor-2, tumour necrosis factor-α and platelet-derived growth factor as cytokines with significant mesothelial cell mitogenic potency in vivo. Our studies demonstrated numerous CM-DiI-labelled mesothelial cells and corresponding PCNA-positive nuclei on the healing serosal surface 4 days post-injury, confirming proliferation of free-floating mesothelial cells once implanted onto the wound. It is likely that these cells proliferate in response to mediators secreted by macrophages present on the wound surface early in the process of regeneration.
To show that implanted mesothelial cells become incorporated into the reconstituted mesothelium, we examined the formation of apical junctional complexes between mesothelial cells in the healing monolayer. Mesothelial cells form a number of junctional complexes including tight junctions ( Baradi and Hope, 1964; Kluge and Hovig, 1967). ZO-1, a plaque protein associated with apical junctions, links the cadherin-catenin complex with the actin-based cytoskeleton ( Itoh et al., 1997). In uninjured mesothelium, ZO-1 expression localised towards the plasma membrane at sites of cell-to-cell contact. During mesothelial regeneration, ZO-1 expression was predominantly cytoplasmic, owing to the loss of intercellular communicating junctions. By 5 days, however, ZO-1 was detected towards sites of cell-to-cell contact, implying the reformation of apical junctional complexes. Mobilisation of ZO-1 from the cytoplasm has previously been demonstrated in Madin-Darby canine kidney cells in which low calcium levels prevented apical junction formation, whereas switching to normal calcium levels resulted in a re-distribution of ZO-1 to the cell surface ( Rajasekaran et al., 1996). The extent and intensity of ZO-1 staining at the cell membrane appeared to have increased by 8 days after injury, which coincided with the reestablishment of an intact mesothelial monolayer. Whether the intensity of staining was due to an upregulation of ZO-1 was not determined.
To show that mesothelial cell implantation and incorporation occurs on other serosal surfaces in different models of injury, we repeated these studies using an abrasion injury to the peritoneal wall. All results were consistent with the testicular injury model (data not shown), suggesting that free-floating mesothelial cells are a source of new mesothelium on all serosal surfaces.
In summary, the following model is proposed. Mesothelial regeneration requires recruitment of inflammatory cells to the wound surface and release of mitogenic cytokines to activate and stimulate mesothelial cell proliferation surrounding the wound ( Fotev et al., 1987; Mutsaers et al., 1997). Activated mesothelial cells break their cell-to-cell contacts and migrate onto the wound surface ( Whitaker and Papadimitriou, 1985). Recent evidence suggests that this may be induced by hepatocyte growth factor, which is secreted by mesothelial cells ( Warn et al., 2001) and surrounding fibroblasts ( Yashiro et al., 1996). Additional mesothelial cells detach and become free-floating ( Whitaker and Papadimitriou, 1985), accounting for a 12-fold increase in peritoneal lavage mesothelial cell counts 2.5 days after serosal injury ( Fotev et al., 1987). However the mechanisms by which these cells become detached from the basement membrane and remain viable in the serosal fluid is not known. Free-floating mesothelial cells move down chemotactic gradients, attach to ECM components exposed beneath the mesothelium or are deposited from the serosal fluid, then proliferate and reconstitute an intact mesothelial monolayer.
Our findings complement the studies of Bertram et al. ( Bertram et al., 1999) who demonstrated a significant reduction in adhesion formation following the intraperitoneal transplantation of autologous mesothelial cells, suggesting that the implanted cells may enhance serosal healing. This may be of therapeutic significance to certain subgroups of patients at high risk of peritoneal adhesion formation, for example those receiving continuous ambulatory peritoneal dialysis as they have ready access to peritoneal lavage fluid. In conclusion, we have shown that cultured and lavage-derived mesothelial cells, but not cultured lung fibroblasts or macrophages, implant onto areas of serosal injury, proliferate and become incorporated into the reconstituted mesothelium, conclusively demonstrating that free-floating mesothelial cells are an origin of the regenerating mesothelium.
Acknowledgements
This work was supported by the Middlesex Hospital Special Trustees, the Wellcome Trust (061566) and Johnson and Johnson Medical/COSAT. We are indebted to Geoffrey Bellingan for his advice during the study and Michael Horton for access to the confocal laser scanning microscope facilities. We also appreciate the critical comments from Grenham Ireland (University of Manchester, Manchester, UK) in the preparation of this manuscript.
- Accepted January 8, 2002.
- © The Company of Biologists Limited 2002
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