1 Department of Internal Medicine, University of Iowa College of Medicine, Iowa City, Iowa 52242; and 2 Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California 94305
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ABSTRACT |
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We tested the hypothesis that histamine alters the focal apposition of endothelial cells by acting on sites of cadherin-mediated cell-cell adhesion. Focal apposition was measured as the impedance of a cell-covered electrode, which was partitioned into a cell-matrix resistance, a cell-cell resistance, and membrane capacitance. Histamine causes an immediate, short-lived decrease in the impedance of an electrode covered with human umbilical vein endothelial (HUVE) cells. ECV304 cells are a line of spontaneously transformed HUVE cells that do not express the endothelial cadherin, cadherin-5. Histamine increased ECV304 cell calcium to 600 nM. Histamine did not increase myosin light chain phosphorylation of control or transfected ECV304 cells. ECV304 cells transfected with either E-cadherin or cadherin-5 on a dexamethasone-responsive plasmid (pLKneo) increased their cell-cell resistance when stimulated with dexamethasone, whereas ECV304 cells transfected with pLKneo-lacZ did not. Histamine did not affect the impedance of ECV304 cells transfected with pLKneo-lacZ. In contrast, histamine decreased the cell-cell resistance of ECV304 cells transfected with either pLKneo-E-cadherin or pLKneo-cadherin-5. From these data, we conclude that histamine acts on sites of cadherin-mediated cell-cell apposition.
edema; permeability; cell adhesion
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INTRODUCTION |
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HISTAMINE INCREASES the permeability of postcapillary venules in many systemic vascular beds (12, 23). Early electron microscope studies indicated that histamine decreased the focal apposition of adjacent venular endothelial cells, and more recent and detailed studies from the bronchial circulation confirm the development of small (500 nm diameter) discontinuities in the apposition of the membranes of adjacent endothelial cells in response to similar peptide agonists (14). Although there is ample evidence for the development of these separations of adjacent cell membranes, the mechanisms responsible for their formation remain only partly explained.
The shape of endothelial cells is determined, at least in part, by a balance of forces, some tending to cause the cell to round up into a sphere and some causing it to flatten and spread (10). Some authors have suggested that edemagenic agonists increase a centripetally directed force and pull adjacent endothelial cells apart (3, 20, 24).
Giaever and Keese (5) reported that cells increase the impedance of a small electrode on which they are grown, and this increase in impedance reflects the apposition of the cells to each other and to their matrix. We found that histamine decreased the impedance of human umbilical vein endothelial (HUVE) cells grown on a similar electrode and that this decrease in impedance occurred without a detectable increase in centripetal tension (16). Similarly, Garcia and colleagues (4) found that ionomycin increased the permeability of cultured endothelial cell layers without increasing myosin light chain phosphorylation and, by inference, centripetal tension. In an earlier report, we found that the resting centripetal tension was both necessary and sufficient to alter endothelial cell apposition when forces of adhesion were diminished by chelating extracellular calcium, upon which many of the adhesion forces are dependent (22). Hence, there is reason to consider the hypothesis that histamine acts primarily to decrease adhesion and utilizes the resting centripetal tension to effect a change in cell shape that alters cell apposition.
ECV304 cells are a line of spontaneously transformed HUVE cells (9). They do not express the endothelial cadherin, cadherin-5 (13). When grown on a microelectrode, they develop an impedance, and the impedance does not change after addition of histamine. We hypothesized that if we transfected ECV304 cells with cDNA for E-cadherin and separately for cadherin-5, the transfected cells would respond to histamine, similar to HUVE cells. This would indicate that histamine acts primarily to alter sites of adhesion.
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METHODS |
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Materials. Fibronectin was from Collaborative Research (Bedford, MA). Tissue culture media and serum were from the Tissue Culture Core, University of Iowa. ECV304 cells and Chinese hamster ovary (CHO) cells were from ATCC (Manassas, VA). The pLKneo plasmid and the E-cadherin cDNA were a generous gift from W. James Nelson. The cadherin-5 cDNA in a pECE plasmid was a generous gift from Elizabetta Dejana. Antibody to E-cadherin (rr1, mouse monoclonal) was from the Hybridoma Studies Bank (University of Iowa). Antibody to cadherin-5 (mouse IgG, monoclonal) was from Immunotech. Secondary antibody was sheep anti-mouse IgG conjugated with horseradish peroxidase from Amersham. Fura 2 was from Molecular Probes (Junction City, OR).
Cell cultures. Cultured HUVE cells were prepared by collagenase treatment of freshly obtained human umbilical veins and cultured. Cultures were identified as endothelial by their characteristic uniform morphology, uptake of acetylated low-density lipoprotein, and indirect immunofluorescent staining for factor VIII (16). Madin-Darby canine kidney (MDCK) cells were from a clone we have previously reported on (21), originally obtained from Dr. B. Gumbiner.
MDCK cells were cultured in MEM with 10% FBS, basal medium Eagle vitamins and amino acids, glucose (5 mM), glutamine (2 mM), penicillin (100 µg/ml), and streptomycin (100 µg/ml).
ECV304 cells were grown in medium 199 (M199) supplemented with 10% FBS, basal medium Eagle vitamins and amino acids, glucose (5 mM), glutamine (2 mM), penicillin (100 µg/ml), and streptomycin (100 µg/ml).
Plasmid preparation and transfections. The pLKneo vector was prepared by cutting with EcoR I (in the Bam-Hind configuration) and phosphatase treated with calf intestinal phosphatase. The cadherin-5 cDNA insert was cut from the pECE plasmid with EcoR I and ligated in the prepared vector with T4 DNA ligase. The correct orientation was confirmed by sequencing. The ampicillin and G418 resistance of pLKneo are in a sequence driven by an SV40 promoter that is separate from the dexamethasone-responsive LTR promoter-driven segment into which the cadherin cDNA was ligated (8). SCS110 competent cells were transformed with 1 ng of the ligation product, and colonies were selected on LB-ampicillin plates. Colonies were picked, grown overnight, resuspended in Tris (pH 8), EDTA, and glucose buffer, and lysed in NaOH-SDS. After phenol-chloroform extraction, DNA was precipitated from the supernatant with ethanol and dried after centrifugation. The isolated DNA was digested with EcoR I, and the digest was separated on a 1% agarose gel.
Colonies with the appropriate size digest products were grown on a large scale. DNA was isolated from these cultures and then sequenced (University of Iowa DNA Core). DNA from colonies with a sequence identical to the published sequence for human cadherin-5 was used for transfection. ECV304 cells were transfected with this DNA using LipofectAMINE (Life Technologies; as per the manufacturer's protocol). The pLKneo plasmid containing cDNA for E-cadherin was similarly amplified, and LipofectAMINE was used to transfect additional ECV304 cells (1, 8).
Transfected cells were selected with G418 (1 mg/ml) in M199 with 10% FBS. Surviving cells were isolated, and clones were expanded in the presence of G418. Clones were tested for expression of cadherins after stimulation with dexamethasone for 18 h using either Western blotting of proteins solubilized with SDS sample buffer or fluorescence-activated cell sorting (FACS; University of Iowa Core FACS Facility) of cells prepared to examine surface expression of the cadherins.
Analysis of cadherin expression. Protein was solubilized from transfected cells with SDS sample buffer, and equal masses of cell proteins were separated on 8% PAGE and transferred to polyvinylidene difluoride membranes in 25 mM Tris, 192 mM glycine, 15% methanol, and 0.02% SDS buffer for 18 h at 4°C and 18 volts. The membranes were blocked with 5% nonfat milk, 1% Tween 20 in 10 mM Tris, and 150 mM NaCl buffer. Blocked membranes were incubated with primary antibody (mouse monoclonal) in blocking buffer for 2 h at 27°C, washed three times with blocking buffer, and then incubated with the secondary antibody (sheep anti-mouse IgG). Blots were examined with enhanced chemiluminescence (Amersham).
For FACS analysis, transfected and nontransfected ECV304 cells were grown in M199 and 10% FBS with dexamethasone (1 µM) for 18 h, cultured in M199 and 10% FBS without dexamethasone for 24 h, washed in PBS, and released from the plates in 137 mM NaCl, 4.2 mM NaHCO3, 5.4 mM KCl, 5.6 mM glucose, and 0.5 mM EDTA (lifting solution). The cells were suspended in PBS, centrifuged at 150 g for 5 min, resuspended, and rotated in PBS containing primary antibody for 1 h at 27°C; next, the cells were washed two times with PBS and resuspended in PBS containing sheep anti-mouse IgG conjugated to fluorescein isothiocyanate (FITC). Cells were rotated for 1 h at 27°C, resuspended in PBS with propidium iodide, and then analyzed for surface expression of E-cadherin or cadherin-5 by FACS.
Monolayers of cells were fixed with paraformaldehyde, permeabilized with Triton X-100 (1%) in PBS, and stained with primary antibody to E-cadherin or cadherin-5 and secondary anti-mouse IgG conjugated with rhodamine (Molecular Probes). Stained cells were visualized in the confocal microscope, and digitized images were recorded to document the distribution of the particular cadherin in the cells.
Cell impedance measurements. Cell apposition was assayed by measuring the impedance of a cell-covered electrode and comparing it with the impedance of a cell-free electrode as we have described (16). The total impedance was partitioned into its three components, the cell-cell resistance, the cell-matrix resistance, and the membrane capacitance as described by Giaever and Keese (5). To do this, we measured the impedance of the cell-covered electrode at three different frequencies (1, 4, and 16 kHz) every second. For each of these time points, we used the downhill simplex method (18) to determine the set of parameters (cell-cell resistance, cell-matrix resistance, and membrane capacitance) that best fit the observed data. The software for data acquisition and analysis was written using LabVIEW (National Instruments, Austin, TX).
Cells were plated on the electrode at 1.5 × 105 cells/cm2. Five hours after being plated, control ECV304 cells and cells transfected with the pLKneo constructs were exposed to dexamethasone for 18 h followed by an additional 24 h in the absence of dexamethasone before being studied. All other cells were simply cultured for 24 h on the electrode before being studied.
Measurement of cell calcium and myosin light chain phosphorylation. Cell calcium was measured with the fluorescent probe fura 2 as previously described (2). Phosphorylation of myosin light chain in ECV304 cells was measured with two-dimensional electrophoresis as described (16).
Statistical analysis. Peak changes in total, cell-cell, and cell-matrix resistances were compared by ANOVA, and individual group comparisons were done using a Tukey honestly significant difference test for post hoc comparisons of means. Differences were considered significant at the P < 0.05 level.
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RESULTS |
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Expression of cadherins in cells.
Nontransfected ECV304 cells and CHO cells do not express cadherin-5 or
E-cadherin. Western blotting confirmed expression of cadherin-5 in CHO
cells transfected with cadherin-5 in the pECE or pLXSN plasmids (Fig.
1). However, these cells did not develop an
impedance on the electrode that was greater than that of nontransfected
cells (data not shown), and FACS analysis indicated a low level of
surface expression (Fig. 2). In contrast,
when ECV304 cells were transfected with E-cadherin or cadherin-5 in the
pLKneo plasmid and stimulated with dexamethasone, there was a high
level of protein expression, and surface expression approached that of
control HUVE or MDCK cells (Figs. 1 and 2). Only 5% of nontransfected
ECV304 cells gated at 1 log of FITC fluorescence for either
cadherin-5 or E-cadherin expression. Seventy-three percent of both
control HUVE cells and ECV-pLKneo-cadherin-5 transfectants gated at
1
log for cadherin-5 expression. Eighty-eight percent of MDCK cells gated
at
1 log, and 78% of pLKneo-E-cadherin transfectants gated at that
level for E-cadherin expression. Hence, the pLKneo plasmids offered a
high level of surface expression of cadherin-5 and E-cadherin. Nontransfected ECV304 cells did not express the cadherins when exposed to dexamethasone.
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The expression of the transfected cadherins was largely dependent on
stimulation of the cells with dexamethasone, and the cadherin proteins
persisted for >72 h after dexamethasone was removed from the culture
medium (Fig. 3). The results for the E-cadherin transfectants were essentially identical with those for
cadherin-5 (data not shown).
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When ECV304 cells transfected with the pLKneo plasmids were fixed and
stained with primary antibody to cadherin-5 or E-cadherin and with a
rhodamine-tagged secondary antibody, both cadherins localized to the
cell cortex at sites of cell-cell apposition (Fig.
4).
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Effects of transfectants on impedance.
The pLKneo-cadherin-5 and pLKneo-E-cadherin transfectants did increase
the impedance of the cell-covered electrode after stimulation with
dexamethasone. The increase in impedance was due to an increase in the
cell-cell resistance (Fig. 5).
Dexamethasone did not alter the impedance of cells transfected with
pLKneo alone (Fig. 5) or cells transfected with a pLKneo-lacZ plasmid
(data not shown).
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Response of ECV cell calcium to
histamine. When fura 2-loaded ECV304 cells were exposed
to histamine, there was an increase in cell calcium to ~600 nM (Fig.
6). This was less than the response in HUVE
cells in which the peak exceeds 1 µM (2).
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Histamine increased myosin light chain phosphorylation in HUVE cells
(15). Histamine did not increase myosin light chain phosphorylation in
either control, pLKneo-lacZ-transfected, or pLKneo-cadherin-5-transfected ECV304 cells after exposure to
dexamethasone [change in myosin light chain phosphorylation (mol
phosphate/mol myosin light chain) = 0.052 ± 0.075 in control ECV
cells, 0.010 ± 0.093 in pLKneo-lacZ- transfected cells, and
0.021 ± 0.055 in pLKneo-cadherin-5-transfected cells].
Response of resistances of ECV304 cells to
histamine. The resistances of nontransfected ECV304
cells did not change when the cells were exposed to histamine (Fig.
7A). The
resistances of cells transfected with pLKneo alone or with pLKneo-lacZ
also did not change after exposure to histamine (data not shown). In
contrast, the resistances of ECV304 cells transfected with either
pLKneo-E-cadherin or pLKneo-cadherin-5 promptly fell and then recovered
after exposure to histamine (Fig. 7, B
and C). Although the response of the
transfected cells was a little less than that of control HUVE cells
(Fig. 7D), the general pattern was
very similar, and in each type of cell, most of the change was in the
cell-cell resistance. In nontransfected ECV304 cells, total resistance
remained at 98.4 ± 0.4% of basal total resistance, and cell-cell
resistance remained at 97.9 ± 1.9% of basal levels after histamine
(n = 19, 8 of which were treated with
dexamethasone and showed no difference from the
nondexamethasone-treated cells). In the ECV-pLKneo-E-cadherin cells,
histamine decreased total resistance to 82.0 ± 4.9% of basal total
resistance and decreased cell-cell resistance to 63.5 ± 12.1% of
basal levels (n = 9). In the
ECV-pLKneo-cadherin-5 cells, histamine decreased total resistance to
77.4 ± 2.2% of basal total resistance and decreased cell-cell
resistance to 65.8 ± 6.6% of the basal level (n = 11). In HUVE cells, histamine
decreased total resistance to 55.8 ± 8.2% of basal total
resistance and decreased cell-cell resistance to 33.3 ± 4.0% of
basal levels (n = 14).
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DISCUSSION |
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Inflammatory edema is an important physiological response that facilitates the delivery of immunocompetent proteins and cells from the vascular space to the extravascular tissues. Agonists such as histamine, bradykinin, and some of the neuropeptides bind to receptors and activate signaling pathways that result in the development of transitory, small focal discontinuities in the apposition of adjacent endothelial cells to each other and potentially to substrate (12, 14, 23). These changes in cell shape represent, at least in part, a change in the balance of the forces that maintain normal cell shape.
Kolodney and Wysolmerski (11) and more recently Moy et al. (16) observed that thrombin caused a myosin light chain kinase-dependent increase in a centripetally directed tension in HUVE cells. These data supported the possibility that edemagenic agonists might act by initiating a contraction that pulled the cells apart. However, using the same technology, we found that histamine did not increase the centripetal tension of HUVE cells. Moreover, when myosin light chain kinase was inhibited and tension development was prevented, thrombin still caused a loss of focal apposition of HUVE cells (16). These data suggested that agonists such as histamine and thrombin decreased focal apposition by directly altering adhesion, independent of increased tension. Increased tension might increase the magnitude and/or duration of the loss of focal apposition, but the tension was not necessary to initiate the loss of focal apposition. This implied that histamine-initiated signaling directly affected sites of adhesion.
The observations in this manuscript support the concept that histamine-initiated signaling primarily affects sites of adhesion. Histamine did not affect the resistances of dexamethasone-treated, nontransfected ECV304 cells or dexamethasone-treated ECV304 cells transfected with pLKneo alone. These cells had readily measured resistances reflecting cell-cell and cell-matrix apposition. If histamine increased a centripetal tension in these cells that tore the cells from each other, it should have decreased the resistance of these nontransfected cells. This did not happen.
Histamine did not increase myosin light chain phosphorylation in control or transfected ECV304 cells. The reason for this is uncertain, although the attenuated increase in cell calcium may contribute. The lack of an increase in myosin light chain phosphorylation makes it unlikely that histamine would increase tension in the cells.
When the ECV304 cells were transfected with either E-cadherin or cadherin-5, the transfected proteins increased the cell-cell resistance of the cells. Histamine caused a prompt fall in the resistance of the cells expressing these cadherins, similar to the effect of histamine on HUVE cells. This indicates that histamine signaling primarily affects endothelial cell apposition through sites of cadherin-mediated cell adhesion.
Although the decrease in resistance of the transfected cells was smaller than the decrease seen in HUVE cells, it followed a similar pattern and time course. The smaller magnitude may reflect the attenuated increase in cell calcium or potentially the lack of an increase in myosin light chain phosphorylation.
Sandoval et al. (19) reported in an abstract that thrombin also did not affect the resistance of ECV304 cells. Whether their observations reflect a lack of cadherin responsiveness or other defects in thrombin signaling is uncertain.
When blots of extracts of ECV304 cells were probed with antibody to the conserved cytoplasmic tail of the cadherins, two cadherins were present in nontransfected cells (data not shown). HUVE cells express N- and P-cadherin in addition to cadherin-5, and the two cadherins present in the nontransfected ECV304 cells are most likely N- and P-cadherin. Our experiments suggest that there may be specificity in the histamine response in that histamine did not affect the resistance created by P- and N-cadherin but only that created by E-cadherin or cadherin-5. This is of interest as Navarro and colleagues (17) have reported that N-cadherin can compete with cadherin-5 for binding to the cytoskeleton. However, it is also possible that the nontransfected ECV304 cells have an inadequate mass of cadherin to respond and that overexpression of cadherin-5 or E-cadherin established a critical mass of cadherin that could respond to histamine.
Our data suggest that histamine acts primarily to alter sites of cadherin-mediated cell-cell adhesion. This reinforces our prior observations that the initial loss of cell apposition with histamine occurs independent of an increase in centripetal tension (16). Moore and colleagues (15) have hypothesized and supported the hypothesis that calcium-mediated changes in cAMP concentrations alter apposition of endothelial cells. Histamine could affect cadherin-mediated cell adhesion by this mechanism, although the specific biochemical events are not presently known.
In summary, histamine decreased cell-cell apposition of adjacent ECV304 cells only when the cells expressed E-cadherin or cadherin-5. We conclude that histamine acts primarily to decrease cell apposition at sites of cadherin-mediated cell adhesion and that it does not act by increasing a tension that tears the cells apart.
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ACKNOWLEDGEMENTS |
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M. Winter was supported by National Heart, Lung, and Blood Institute (NHLBI) Grant HL-07344. This work was supported by NHLBI Grant HL-33540.
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FOOTNOTES |
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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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: M. Winter, Dept. of Internal Medicine, Univ. of Iowa College of Medicine, B191 ML, Iowa City, IA 52242 (E-mail: michael-winter{at}uiowa.edu).
Received 8 December 1998; accepted in final form 4 June 1999.
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