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PROTEIN AND VESICLE TRAFFICKING, CYTOSKELETON
Department of Anatomy and Physiology, Kansas State University, Manhattan, Kansas
Submitted 8 November 2005 ; accepted in final form 22 July 2006
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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nor interleukin-6, in the apical compartment caused a significant decrease in Rte within 8 h. These results indicate that mammary epithelium is a dynamic barrier whose cell-cell contacts are acutely modulated by cytokines and luminal electrolyte environment. Results not only demonstrate that BME-UV cells are a model system representative of mammary epithelium but also provide critical information that can be applied to other mammary model systems to improve their physiological relevance. transepithelial electrical resistance; apical cation concentration; paracellular permeability; mastitis; inflammatory cytokines; occludin
Inflammatory mediators are recruited to the mammary gland under pathological conditions, although direct effects of cytokines on mammary epithelium have not been fully delineated. During mammary infection, cytokines interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor alpha (TNF-
) are elevated in the milk within 6–8 h following bacterial inoculation of the gland (1, 25, 27, 44). It has been speculated, from work in other tissues, that these chemicals signal changes in milk composition by directly or indirectly affecting the epithelium (4, 13, 14, 19, 30). It is possible that cytokine and chemokine recruitment may directly affect the barrier function of the epithelium. Thus, it is critical to determine if these cytokines directly affect the epithelium and to determine which aspects of epithelial function are affected.
Mammary epithelial barrier function has been difficult to quantify (reviewed in Refs. 20 and 32). A previous report from this laboratory suggested that luminal composition, which is dependent on epithelial ion transport, could affect the epithelial barrier integrity as measured by transepithelial electrical resistance (Rte) (29). Nonetheless, little is known regarding the time course over which this barrier breakdown occurs, whether a paracellular pathway is affected, or the target proteins that are involved in increased epithelial permeability. Examining the effects of changes in ionic composition on the epithelia is a critical step to understanding the progression of the breakdown of cell-cell contacts, as well as illuminating subsequent options for treatment of mastitis.
Many in vitro mammary epithelial cell lines are currently being used to study the dynamics of the gland and to examine a variety of pathologies, including breast cancer (11, 15, 26, 28, 37). In most cases mammary cancer cell lines of epithelial origin are being grown on solid supports, which precludes full membrane polarization and the opportunity to assess physiological function. Also, these systems have typically utilized the same high-electrolyte medium bathing both the apical and basolateral aspects of the epithelium. Attempts are being made to grow mammary epithelial cells on permeable supports (8). However, Schmidt et al. (29) provide the only publication to date in which transformed mammary epithelial cells were cultured on permeable supports in asymmetrical media that approximates the in vivo environment and indicates a culture-dependent change in barrier function.
The primary goal of this study was to test mammary epithelial integrity following changes in apical ionic composition (to simulate changes in milk electrical conductivity) using an in vitro bovine mammary epithelial model. This attempt to determine whether luminal electrolytes affect epithelial integrity is the first to concomitantly measure Rte, permeation by uncharged particles, and the distribution of proteins associated with tight junctions directly at the level of the epithelium in an in vitro system. A secondary goal for this study was to identify and characterize effects of inflammatory cytokines (TNF-, IL-1, IL-6) on epithelial barrier function in this in vitro model. We hypothesized that exposure to increased luminal ionic concentration or inflammatory cytokines would rapidly decrease epithelial barrier function. The results indicate that mammary epithelial tight junctions are dynamic structures that can be rapidly modified by TNF- and by changes in the fluid electrolyte composition in the milk compartment. More importantly, the cytokine effects on mammary epithelial barrier function pale compared with the rapid and significant compromise of barrier function during exposure to elevated electrolytes in the luminal compartment. The decrease in barrier function is due, at least in part, to changes in localization of the tight junction protein occludin. This information brings into focus the need to reevaluate current paradigms in progression of mammary pathology.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Two sets of experiments were conducted to test for effects of changing the apical medium from, or to, H-elec medium. In both experiments, Snapwell trays were seeded with BME-UV cells in symmetrical H-elec medium. Twenty-four hours postseeding is considered "day 1" in culture. In the first experiment, the apical medium of five Snapwells in each tray was changed to L-elec medium on day 1 in culture. Beginning on day 4, 7, 10, and 13, successive monolayers were apically exposed to H-elec medium for the remaining duration of the culture period (Fig. 1A). One monolayer was exposed to H-elec apical medium throughout the entire culture period. In the second experiment, the H-elec apical medium was maintained for 1, 4, 7, 10, and 13 days before changing to apical L-elec medium on successive monolayers (Fig. 1B). In all cases, monolayers were evaluated for barrier integrity on postseeding day 15. At this time, monolayers were also fixed for assessment of tight junction-associated proteins by immunocytochemistry or harvested for Western blot analysis as described below.
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voltage/ current) was applied to the resulting current deflections to determine Rte. Transepithelial dextran permeation. Movement of large, uncharged molecules across epithelial monolayers was assessed with FITC-conjugated dextran. Methods were similar to those used by Broughman et al. (3). Briefly, BME-UV monolayers were grown on Snapwell permeable supports with changes from or to H-elec apical medium on the days postseeding as indicated. Monolayers were washed with PBScc and then bathed in Ringer solution, both apically and basolaterally. Two different sizes of FITC-conjugated dextran, 9.5 kDa and 77 kDa (Sigma-Aldrich, St. Louis, MO), were utilized to assess epithelial permeability. FITC-conjugated dextran was introduced into the apical compartment of all wells at the outset of the assay. Monolayers were incubated at 37°C in a humidified atmosphere containing 5% CO2 for 60 min after which samples of basolateral solution were obtained for analysis. Filter supports lacking cells were included to verify that the culture support did not provide a significant barrier to dextran permeation and cell monolayers exposed to Ringer solution in the absence of dextran were employed to establish that no components of the assay system contributed to the fluorescence measurements. Fluorescence in the basolateral compartment was quantified in a Fluoroskan Ascent FL plate reader (Labsystems, Helsinki, Finland). Known dilutions of each dextran stock were included in each 96-well assay plate to construct a standard curve after background subtraction.
Immunocytochemistry. After exposure to the indicated culture conditions, BME-UV cell monolayers were washed in PBScc and fixed in 4% paraformaldehyde (Fisher Scientific International, Hampton, NH), and stored less than 24 h in phosphate buffered saline for cytochemistry (PBS; composition in mM; 150 NaCl, 5 KH2PO4, 15 K2HPO4, pH 7.2). Monolayers were permeabilized with 0.1% Triton X-100 in PBS, blocked for 1 h with goat serum (Gibco-BRL, Rockville, MD; 5% vol/vol in PBS). Primary antibodies to zonula occludens-1 (rat anti-ZO-1; Chemicon, Temecula, CA), and occludin (rabbit anti-occludin; Zymed, Carlsbad, CA), diluted 1:200 with 1% goat serum in PBS were incubated with the monolayers for 2 h at room temperature. Cells were washed 3 x 10 min in PBS and subsequently incubated for 2 h at room temperature with rhodamine-conjugated goat anti-rat (for ZO-1; Vector Laboratories, Burlingame, CA) or Alexa 488-conjugated goat anti-rabbit (for occludin; Molecular Probes, Eugene OR) secondary antibody. Once again, cells were washed 3 x 10 min in PBS. Cells were further incubated with 10 µM TO-PRO3 (Invitrogen, Eugene, OR) for 1 h and cells were washed briefly with PBS immediately prior to mounting on slides with Fluormount G (SouthernBiotech, Birmingham, AL). Images were assessed by both standard (Leica Microsystems AG, Wetzlar, Germany), and confocal (Carl Zeiss MicroImaging, Thornwood, NY) microscopy using appropriate filters for each fluorophore. Identical settings were used to obtain and process images derived from paired monolayers. Negative controls included protocols with the primary antibody omitted, the secondary antibody omitted, or, when available, peptide-preadsorbed primary antibody.
Western blot analysis.
BME-UV cell lysates were prepared in the 1.13 cm2 Snapwell culture substrates using Phosphosafe lysis buffer (Novagen, San Diego, CA) including 1% protease inhibitor cocktail (Sigma-Aldrich) and maintained in a frost-free freezer at –20°C until assay. Total protein content was determined using a bicinchoninic assay (Pierce, Rockford, IL) and 20 µg of total protein was loaded in each well of a 10–20% SDS-PAGE prepoured gel (Bio-Rad, Hercules, CA) for electrophoresis. Gels were run at 160 V for 45 min, then transferred to Millipore PVF-Immobilon membranes for 8.5 h at 90 mV. Transfer was confirmed by staining gels with Gelcode Blue (Pierce). Membranes were blocked in SuperBlock Blocking Buffer (Pierce) and probed with rat anti-ZO-1 and rabbit anti-occludin diluted in SuperBlock Blocking Buffer. Protein was visualized by enhanced chemiluminescence with Pico-chemiluminescence substrate (Pierce), using a peroxidase-conjugated secondary antibody (1:5,000). Membranes were stripped with Restore Western Blot Stripping Buffer (Pierce), and stripping was confirmed with visualization using Femto-chemiluminescence substrate (Pierce), a more sensitive chemiluminescence substrate to preclude any residual labeling. Membranes were reprobed with mouse monoclonal primary antibody for either GAPDH (Abcam, Cambridge, MA ) or -actin (Sigma-Aldrich) as a loading control for densitometric analysis. Membranes were exposed on CL-Xposure film (Pierce) and analyzed using a Kodak RP X-OMAT (model M7B) film analyzer. Imaging and densitometric analysis were performed on a Fluor Chem 8900 Alpha Innotech Imaging System with Alpha Ease FC StandAlone Software (Alpha Innotech, San Leandro, CA).
Amiloride/EIPA-exposure experiments. BME-UV cells were seeded onto Snapwell permeable supports (in groups of six) as described above. The apical medium of three Snapwells in each tray was changed to L-elec medium on day 1, and additional wells were exposed apically to H-elec medium conditions for the duration of the 15-day culture period. Beginning on day 13, one L-elec and one H-elec medium monolayer were cultured in the presence of amiloride (10 µM; a selective blocker of the epithelial Na+ channel, ENaC), and one L-elec and one H-elec medium monolayer were cultured in the presence of 5-(N-ethyl-N-isopropyl)-amiloride (EIPA, 1 µM; an amiloride analog that preferentially inhibits the Na+/H+ exchanger, NHE) in the apical culture media for 24 h prior to evaluation of Rte in the Ussing chamber.
Alternative solutions for Ussing chamber experiments. Reduced Na+ and/or Cl– solutions were employed for certain experiments by equimolar replacement of 120 mM NaCl with N-methyl-D-glucosamine·HCl (NMDG-Cl), Na-gluconate, or NMDG-gluconate. Alternatively, in some experiments 60 mM NaCl was replaced by 120 mM lactose to test for effects of electrolyte reduction.
Cytokine treatments.
BME-UV cell monolayers, cultured in the presence of L-elec apical medium, were exposed to cytokines that have been associated with mammary inflammation (25, 27, 44; reviewed in Ref. 1), including TNF- (0.5 µg/ml), IL-1 (0.1 µg/ml), or IL-6 (1 µg/ml) in the apical medium for 8 or 12 h prior to assessment of Rte in modified Ussing chambers.
Data analysis. Quantitative results were analyzed via ANOVA using SAS (SAS Institute, Cary, NC). Data are presented as means ± SE. Differences are considered statistically significant when the probability of a type I error is <0.05.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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·cm2). In fact, within 1 h of continuous exposure to H-elec medium, barrier function, as measured by Rte, began to decline (data not shown). Four-day exposure to H-elec medium showed further incremental reduction in Rte to less than 30% of the L-elec medium value. Although a trend toward further reduction in Rte was observed with additional exposure to H-elec medium, the incremental changes did not achieve statistical significance. These results demonstrate that short-term apical exposure to H-elec medium compromises the mammary epithelial barrier function, as assessed by electrical means.
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·cm2). Monolayers exposed to H-elec medium for 13, 10, and 7 days before an apical transition to L-elec medium for 1, 4, and 7 days, respectively, exhibited incrementally greater Rte. Differences in Rte were not associated with longer periods (i.e., up to 14 days) of apical exposure to L-elec medium. These results suggest that a reduction in apical electrolyte concentration, to a value that more closely approximates that of typical (i.e., healthy) milk, rapidly promotes the enhancement of the mammary epithelial barrier and an electrically tight monolayer. Apical H-elec medium increases paracellular permeability. Reductions in Rte might reflect an increase in transcellular permeability, paracellular permeability, or some combination of these two permeation pathways. Thus, gradient-driven permeation of 9.5 and 77 kDa dextran was employed to assess changes in the paracellular pathway resulting from differences in the apical solution. As shown in Fig. 3A, monolayers exposed apically to L-elec medium for the entire culture period exhibited the lowest permeation rates (i.e., most substantial barrier) to diffusion compared with other time points for both 9.5 and 77 kDa dextran. Apical exposure to H-elec medium for as little as 1 day, following 13 days of L-elec medium, was associated with significantly greater permeation of both sizes of dextran. Incrementally greater permeation was observed with 7 days exposure to apical H-elec medium. The greatest permeation was observed when monolayers were exposed apically to H-elec medium throughout the entire culture period. These observations demonstrate that short-term exposure to a serum-like medium on the apical face of bovine mammary epithelial cells promotes a loss of the barrier integrity that is associated with the opening of the paracellular pathway to allow the permeation of high molecular weight solutes; an observation that is consistent with the reduction in Rte (Fig. 2A).
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Occludin, but not ZO-1, distribution is altered by apical medium. To test whether reduced integrity of the epithelial barrier was related to changes in junctional proteins, and to rule out the possibility that apical H-elec medium was causing a portion of the cells to "release" from the culture substrate and/or the epithelium, experiments were conducted to visually evaluate the epithelium and to assess the distribution of the tight junction proteins ZO-1 and occludin. Examination of H-elec medium-exposed BME-UV cell monolayers revealed no evidence for the loss of cells. In every case, a survey of at least five objective fields per filter revealed a confluent monolayer with a cobblestone appearance throughout. Monolayers exposed to L-elec medium for 14 days were colabeled with anti-occludin primary antibody that was visualized using an Alexa 488-conjugated secondary antibody, and with anti-ZO-1 primary antibody that was visualized using either Alexa 594- (confocal microscopy) or rhodamine- (light microscopy; data not shown) conjugated secondary antibody. TO-PRO3 staining of nuclear material was included to provide an additional reference point within each cell. Occludin (Fig. 4A) and ZO-1 (Fig. 4B) immunoreactivities were identified in the apical-lateral portions of all cells in the epithelial monolayer. Figure 4 provides the image at a single focal plane that is near the apical aspect of the cells and was selected from a stack of images through the BME-UV cell monolayer. In the BME-UV cell monolayer, each cell is fully circumscribed by immunoreactivity for each of the epitopes. The combined image (Fig. 4D) clearly shows that the distribution of these two tight junction-associated proteins is virtually identical in these conditions.
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-actin controls for normalization of total protein loading. Two clear bands were evident for occludin protein. Although it is possible that these multiple mobilities are due to different phosphorylation states for the occludin protein, experiments were not performed to test this possibility. No significant differences in immunoreactive occludin protein were identified in monolayers exposed to different apical conditions (Fig. 8). These observations indicate that occludin redistribution, rather than expression, is one of the defining factors for loss of barrier function across this epithelium.
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decreases BME-UV monolayer barrier function.
Chemicals derived from inflammatory cells, including the cytokines TNF-, IL-1, and IL-6, are increased under pathological conditions in the bovine mammary gland and may directly or indirectly affect epithelial cell function. Experiments were conducted to test for effects of these inflammatory cytokines on mammary epithelial integrity via analysis of Rte. Exposure to TNF- for 8 h, a time point at which these inflammatory cytokines have been detected to be elevated in vivo, was associated with a 30% decrease in Rte (Fig. 11). Twelve hour exposure to TNF- was associated with more variable results that were not significantly different from the control (data not shown). No significant effect on Rte was noted after exposure to IL-1 or IL-6 (Fig. 11). Likewise, increased time of exposure, at 24 and 30 h, demonstrated no effect on Rte (data not shown). These observations demonstrate the importance of examining each of these effects both separately and together to determine the overall effect of challenge to the gland within the optimal physiological context.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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BME-UV cells provide the basis for an in vitro system to characterize mammary epithelial cell function. The original description included the claim that these immortalized cells exhibited many characteristics of mammary epithelia, including a cobblestone appearance when grown on solid supports, cytokeratin production, and synthesis of -lactalbumin and s1-casein (41). It has since been reported that BME-UV cells respond to a variety of hormones and pharmacological agents with changes in cell signaling, cellular metabolism, proliferation, and apoptosis (6, 12, 39, 40, 42). It has also been shown that BME-UV cells grow on a permeable support system to form a confluent, polarized monolayer that is capable of regulated ion transport (29). The fact that these cells form a polarized and electrically tight epithelial monolayer provides opportunities to study both barrier function and ion transport across the mammary epithelium in a way that is not possible in the native gland, with freshly isolated cells, or in a 3-dimensional culture system.
Leaders in the field have noted that the mammary epithelium is a structure with complex geometry and, therefore, have suggested that an in vitro model such as this would be amenable to study with powerful techniques such as the Ussing chamber (32). The BME-UV cell system is amenable to tightly controlled systematic experimentation. Optimization of the system by Schmidt et. al. (29) demonstrated the benefit of a milk-like apical medium. The current report utilizes the polarized monolayer configuration and demonstrates that BME-UV cells exhibit functional responses to changes in electrolyte composition and to cytokines that would be predicted based upon in vivo observations. The time course of these changes is important to understanding the progression of epithelial barrier modulation within the mammary gland.
The results indicate that mammary epithelial integrity is modulated by changes in the electrolyte or carbohydrate concentration in the apical medium. Three separate and complementary techniques were employed to document differences between the treatments. First, Rte was employed as a rapid and sensitive indication of the ease with which small charged solutes, especially monovalent ions, move through the epithelium. Since the lactating mammary gland secretes a high volume (30–35 kg/day in dairy cows) of isosmotic solution, one might expect the epithelium to exhibit a relatively low resistance. However, the composition of milk is distinctly different from serum, which might suggest the presence of a "tight" epithelium. Furthermore, it was reported that cultures of freshly isolated mammary epithelial cells exhibited resistances of >1,000 ·cm2 (34), although the contribution of the substrates were not reported. Thus, the previous report (29) that BME-UV achieved a maximal Rte of <200 ·cm2 was somewhat surprising and suggested that optimal culture conditions had not been fully defined. The hypothesis that a "milk-like" medium on the apical face of the monolayer would enhance resistance was tested and determined to be correct. The goal was to create a medium that was low in total electrolytes, relatively high in K+, relatively high in lactose, and isosmotic with the basolateral medium. There were, however, certain constraints that guided the apical medium construction. First, one cannot remove a solute and maintain osmolarity. Hence, there is not an experiment to test for effects of changing electrolyte concentrations without a concomitant change in carbohydrate to maintain osmolarity. A previous report shows that similar results are obtained when either mannitol or lactose are used as the compensating osmolyte when electrolytes are reduced (29). Additionally, the optimal medium defined by Zavizion et al. (41), included 15% serum, which includes a substantial amount of Na+ and Cl–. It was determined that a systematic evaluation of serum withdrawal was beyond the scope of the current studies. Rather, the current study focused on the time course over which medium-induced differences in Rte could be observed.
In every experimental block, recent duration of L-elec medium exposure is associated with the greatest electrical resistance. Although block to block and experiment to experiment variation in the maximal resistance was observed, continuous exposure to H-elec medium is associated with the lowest Rte in every block. It was previously reported that BME-UV cell monolayers reach a maximal plateau in Rte between 7 and 10 days of culture that is maintained consistently to at least day 14 (29). Thus, the observations that the transition from apical L-elec to H-elec medium for as little as one day at the end of the 14 day culture period results in a >25% reduction in Rte is striking. The results suggest that high electrolyte/low carbohydrate at the apical membrane causes a change in the epithelium that permits gradient-driven flux of small charged solutes, either through transcellular or paracellular routes. Similarly, a one day transition to L-elec medium can result in a doubling of Rte. The pathway(s) that are affected by the change in apical composition can be readily modulated in either direction. Furthermore, the response appears to be complete or nearly complete within four days. There is a fourfold reduction in Rte with four days of apical H-elec medium exposure and there is a fourfold increase in Rte with four days of apical L-elec medium exposure. This time frame for changes in barrier function is important because it parallels the period over which substantial changes are seen in milk composition and in mammary function at the initiation of lactation, at the initiation of involution, and in the course of mastitis.
Dextran permeation across the BME-UV monolayer indicates flux through the paracellular pathway. The electrical parameters described above are blind to transcellular vs. paracellular movement and are insensitive to the movement of any nonionic solute. Numerous mechanisms, both transcellular and paracellular, have been described to account for solute movement across mammary epithelium (3). Thus, a protocol was conducted to assess flux through the paracellular pathway. The experimental design tested for changes in paracellular permeation that paralleled the changes observed in electrical measurements and assessed the approximate size of the solutes that could readily traverse the epithelium. The results show that the BME-UV cells can be a formidable barrier to the diffusional movement of large solutes, with basal permeation rates that are similar to a canine kidney epithelial cell line (MDCK), and to T84 (colonic origin) and Calu-3 (airway origin; Ref. 3 and unpublished observations). The experimental results provide compelling evidence that changes in the apical medium affect the paracellular pathway with a time course that is similar to that observed for the effects on Rte. One day of exposure to H-elec medium is associated with a doubling in the rate of permeation by both the small (9.5 kDa) and large (77 kDa) dextran molecules. Paralleling the changes observed for Rte, dextran permeation was incrementally greater after longer periods of H-elec medium exposure. The results with 7 or more days exposure to L-elec medium are consistent with the Rte measurements in that 7 days exposure to L-elec medium produces a significantly greater barrier to solute flux and that, within these experiments, no additional incremental changes are associated with additional exposure to L-elec medium. Dextran of 77 kDa was selected because of its similarity in size to serum albumin (
66 kDa). Thus, one can infer from these results that changes in the paracellular pathway that are caused by the modified apical medium are sufficient to allow for the permeation of albumin into the milk. Both the appearance of albumin and the elevation in electrolytes have been used as clinical measures of mastitis (9, 18, 23). The fact that milk conductivity is used as an early indicator of mastitis suggests, but does not demonstrate, that changes in milk electrolyte composition precede changes in the epithelial barrier integrity. Thus the current results provide impetus to conduct systematic measurements in milk electrolyte concentration and serum albumin content at the onset of mastitis.
Immunocytochemistry revealed that occludin, but not ZO-1, was absent from the expected apical lateral location following exposure to H-elec medium. Systematic metabolic assays were not conducted, so no inferences can be drawn regarding the underlying mechanisms that are affected. However, there appears to be a consistent amount of overall occludin immunoreactivity in the epithelial cells following H-elec medium exposure, when examined via Western blot analysis, but less localization of occludin at the level of the tight junctions. The possibility that the H-elec medium treatment caused a general redistribution of proteins or modified overall cell structure is ruled out by the observation that ZO-1 distribution and intensity were unaffected by the H-elec medium treatment, whereas specific redistribution of occludin protein was apparent. It is intriguing that a similar kinetic profile is observed for the decrease in resistance, increase in permeability, and decrease in occludin immunoreactivity. The results clearly show that these modifications in epithelial function occur concurrently during exposure to H-elec medium. Additionally, it is shown that transition to L-elec medium affects all three parameters (Rte, dextran permeation, occludin distribution) in the opposite direction of that seen with H-elec medium and with similar time course.
The magnitude and duration of exposure to altered apical medium composition required to cause a change in epithelial integrity or occludin distribution remain to be determined. The current results show that an abrupt and substantial change in apical medium composition causes a change in barrier Rte, dextran permeation and occludin distribution that can be readily observed within one day. However, one would not necessarily expect to observe such abrupt changes in milk composition in a physiological or clinical setting, although mammary saline infusion has been employed in a research setting (32). Furthermore, ion transport mechanisms that are present in mammary epithelium and account for electrolyte movement into or out of the milk compartment remain to be defined, along with their associated regulatory cascades. Knowledge of these mechanisms is required to establish pharmacological targets that can be used to modulate milk electrolyte composition in vivo. Evidence has been provided for the anion channel that is mutated in cystic fibrosis (CFTR) and for ENaC (2, 29) in mammary epithelial cells, along with regulation by both hormones and neurotransmitters. The magnitude or rate by which milk electrolyte composition can change, however, has not been determined. Clearly, additional experiments are required to test for effects of more subtle changes in apical composition on epithelial function.
A mechanistic link between apical medium composition and tight junction integrity has not been ascertained. The initial supposition was that either Na+ or Cl– in the apical medium might affect membrane electrical potential or, by movement through channels or transporters, cytosol composition to precipitate a change in cell function. This hypothesis was tested both pharmacologically and by substitution with impermeant ions. The results, however, were not particularly instructive except to indicate that these hypotheses were inadequate. Likewise, an osmotic effect is ruled out by meticulous care to insure that all media were isosmotic (290 mosmol/kgH2O). The possibility that a high apical lactose concentration is required to enhance barrier integrity is ruled out by a previous report, which showed that similar results were obtained with mannitol as the compensating osmolyte (29). That a mechanism is not currently revealed does not diminish the impact of the observations that changes in apical composition affect epithelial integrity. Rather, these observations provide additional impetus to identify players in the regulatory cascade.
Times when luminal electrolyte composition would be expected to change, including parturition and involution, are associated with increased incidence of infection, inflammation, and cytokine recruitment (1, 5, 7, 14, 16, 21). Cytokines, including TNF-, IL-1, IL-6, and IL-8, have been reported to modulate the mammary epithelial barrier in vivo (44). Intramammary TNF- infusion is associated with the recruitment of neutrophils, decreased milk proteins, and the appearance of serum proteins in milk (38). TNF- has also been shown to downregulate occludin expression in other cell systems (36), and to alter Na+ and Cl– movement across epithelial barriers (19). Whether the effects are direct or indirect, however, is still open to question. The results presented in this manuscript indicate that TNF- has a direct effect on mammary epithelial cells to cause a breakdown in the barrier function, with the same time course that has been observed in vivo. Alternatively, the results failed to support a direct role for IL-1 or IL-6 in modulation of Rte in mammary epithelium. It is important to note that the conditions used in the present assays were not selected to mimic any particular clinical study. Thus, the discordance may reflect differences in the health status of the mammary gland that was used relative to the in vitro cell system. The results demonstrate that the BME-UV cell system is sensitive to selected cytokines and will provide an excellent system to delineate the cellular mechanisms that are involved in the response.
The current results provide a new factor, luminal electrolyte composition, that must be considered as one investigates mammary epithelial function. Changes in epithelial function can occur rapidly and do not require ion permeability into the cells. Rather, mammary epithelial monolayers are extremely sensitive to changes in luminal fluid composition, which is one of the earliest measurable indicators of mastitis. This evidence is complimented by observations that lowering the apical ion concentration leads to an increase in Rte, indicating enhanced barrier function. These results identify new factors that contribute to the progression of mastitis damage in the mammary gland and offer hope for new targets to circumvent (prevent) or treat inflammatory disease within the mammary gland. Reducing direct damage to the epithelium via altering cytokine effects, decreasing electrolytes, or modulating ion transport mechanisms may provide new options for investigation and ultimately for intervention. Work toward understanding the mechanism(s) of action that leads to changes in luminal ion concentration, cytokines, and epithelial remodeling will provide novel targets for prevention and treatment of mastitis at the earliest stages of the disease.
In summary, the current results demonstrate, with an in vitro model, that cytokines and apical electrolytes rapidly affect the epithelium that separates milk from blood. The epithelial barrier function is directly modulated by the local environment and some cytokines. These data provide information for those working with other in vitro mammary models to improve the quality of those systems by more closely representing the in vivo environment. Most importantly, the data indicate that there can be a feedback mechanism whereby the composition of milk that is secreted by the epithelium can affect barrier integrity. This observation provides impetus to define the cellular mechanisms that account for the low electrolyte content of milk and to seek interventions to therapeutically target the activity of these mechanisms.
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ACKNOWLEDGMENTS |
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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. Section 1734 solely to indicate this fact.
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