Monocyte/macrophages evoke epithelial dysfunction: indirect role of tumor necrosis factor-alpha

Mehri Zareie, Derek M. McKay, Garrett G. Kovarik, and Mary H. Perdue

Intestinal Disease Research Program, McMaster University, Hamilton, Ontario, Canada L8N 3Z5

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

We examined the ability of monocytes (MPhi ) activated by bacterial products to alter epithelial physiology. Confluent monolayers of the T84 colonic epithelial cell line were grown on filter supports and then cocultured in the presence of human MPhi with or without the activating agents bacterial lipopolysaccharide and the bacterial tripeptide formyl-methionyl-leucyl-phenylalanine. After 24 or 48 h, monolayers were mounted in Ussing chambers where parameters of epithelial function were measured. Exposure to activated MPhi resulted in a significant increase (P < 0.05) in baseline short-circuit current (250% after 48 h) that was associated with enhanced secretion of Cl-. In addition, epithelial permeability was significantly increased as shown by reduced transepithelial resistance and increased flux of 51Cr-EDTA. Activated MPhi produced substantial amounts (~3 ng/ml at 48 h) of tumor necrosis factor-alpha (TNF-alpha ). TNF-alpha was identified as a key mediator acting via an autocrine mechanism to induce epithelial pathophysiology. Our data show that MPhi , when activated by common bacterial components, are potent effector cells capable of initiating significant changes in the transport and barrier properties of a model epithelium.

epithelium; ion transport; permeability

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

MUCOSAL SURFACES, particularly the intestine, are exposed to a wide variety of commensal and potentially pathogenic bacteria. Several lines of evidence now point to a role for bacteria and/or their products in the pathogenesis of mucosal inflammatory disorders (11, 41), especially in the context of aberrant immune function (10, 18). Inflammatory bowel diseases (IBD) are often characterized by altered epithelial physiology, typically increased permeability and electrolyte secretion that can create a luminally directed driving force for water movement resulting in diarrhea. Epithelial pathophysiology may be caused by activated immune cells, since many studies have provided unequivocal evidence that the transport and barrier functions of the epithelial lining of mucosal surfaces are regulated by cells such as lymphocytes, mast cells, and neutrophils (29, 30).

Although many studies have confirmed the concept of immunomodulation of epithelial physiology, few studies have considered the ability of cells of the monocyte/macrophage (MPhi ) lineage to directly affect epithelial function. However, these cells are among the first immune cells to react on initial exposure to antigens and infective organisms. For instance, monocytes exposed to lipopolysaccharide (LPS) and the bacterial tripeptide formyl-methionyl-leucyl-phenylalanine (FMLP) respond with the production of mediators and the synthesis of cytokines/growth factors such as the interleukins (IL), IL-1, IL-6, tumor necrosis factor-alpha (TNF-alpha ), and transforming growth factor-beta (1, 8, 16, 27, 44). These factors can directly, or indirectly, affect epithelial function, altering transport and barrier characteristics (24, 25, 38, 49). In addition, processes from tissue macrophages occur close to the basement membrane of the overlying epithelium, and this spatial association may facilitate bidirectional communication between the two cell types. Finally, monocyte chemoattractant peptide-1 (MCP-1) has been immunocytochemically demonstrated in the surface epithelium of human colonic biopsies, and its expression is enhanced in tissues from patients with Crohn's disease or ulcerative colitis (35). These findings indicate a clear potential for MPhi to regulate epithelial physiology.

Resident intestinal macrophages do not normally express the LPS receptor CD14 (22). However, it was recently reported that macrophages in resected intestinal segments from patients with IBD express a unique phenotype with unusually high levels of CD14 (12, 13), presumably because of rapid recruitment of monocytes from the circulation to the gut (4, 39). Similarly, it has been shown that monocytes are recruited to the airways during an inflammatory response, and these newly recruited cells are more active in tissue damage than resident macrophages (6).

Integrating these themes, this study examined the specific hypothesis that human MPhi activated by the bacterial products LPS and FMLP can influence epithelial electrolyte transport and barrier functions. Here, we used the human colonic T84 cell line as a model epithelium (9) and a coculture approach analogous to that used to define the ability of lymphocytes and polymorphonuclear cells (PMN) to regulate epithelial physiology (19, 24). Our data show that purified MPhi (in the absence of other classes of immune cells) significantly increased luminally directed Cl- secretion and disrupted epithelial barrier function. These changes in epithelial function were inhibited by inclusion in the coculture system of a neutralizing antibody against TNF-alpha , implicating this cytokine as a critical mediator in gut pathophysiology. Further studies indicated an important autocrine mechanism of action for TNF-alpha on MPhi . Thus MPhi have been identified as being capable of directly modulating epithelial function. We speculate that given appropriate environmental conditions, activation of MPhi could be a precipitating event in the onset of pathophysiology leading to chronic secretory or inflammatory disease in the intestine.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cell Culture

Epithelial cells. T84 cells (passage 45-65) were seeded onto tissue culture-treated semipermeable filter supports (0.4-µm pore size, 1.0-cm2 surface area; Costar, Cambridge, MA) at a concentration of 106 cells/ml and grown in culture media consisting of equal volumes of DMEM and F-12 medium, supplemented with 1.5% (vol/vol) HEPES, 2% (vol/vol) penicillin-streptomycin, and 10% newborn calf serum (all from GIBCO Laboratories, Grand Island, NY) (24). After culture for 7 days, confluent T84 monolayers consistently displayed electrical resistances >= 1,000 Omega  · cm2.

Immune cells. Human peripheral blood mononuclear cells (PBM) from healthy volunteers (male and female, ages 23-45 yr) were isolated by one-step density centrifugation of whole blood over Ficoll-Hypaque (Pharmacia Biotech, Uppsala, Sweden) and resuspended in fresh media at 106 cells/ml. The MPhi population was obtained by plastic plating of PBM (4 h at 37°C) and subsequent removal of nonadherent T and B cells. Fresh media were added to the adherent cells, which were then incubated for 18 h at 37°C before use in coculture studies. Assessment of T cells and monocytes in the adherent cell population was carried out by two-color flow cytometry analysis [fluorescence-activated cell sorter (FACS)] after staining the cells with FITC-conjugated anti-CD3 (OKT3, Coulter Immunology, Hialeah, FL) and phycoerythrin-conjugated anti-CD14 (Caltag Laboratories, San Francisco, CA), respectively. Analysis was performed using FACScan (Becton Dickinson, Mississauga, Ontario, Canada) followed by data analysis using PC-Lysys II computer software (Becton Dickinson).

Immune Cell Activation

MPhi were activated by addition of Salmonella minnesota LPS (10 µg/ml) and the Escherichia coli-derived tripeptide FMLP (0.1 µM) (both from Sigma Chemical, St. Louis, MO) to the culture media before coculture. Activation was indicated by production of TNF-alpha measured in conditioned medium (CM) by an ELISA (Biotrack, Oakville, Ontario, Canada). The sensitivity of the assay was 4 pg/ml. IL-2 (a marker of T cell contamination) was assessed in the same samples by ELISA (Advanced Magnetics, Cambridge, MA).

Coculture Studies

Confluent T84 monolayers were cocultured for 24 or 48 h with LPS/FMLP-activated MPhi (25,000-200,000 cells/well; unless stated otherwise 2 × 105 MPhi were used) placed in the basal compartment of the coculture wells. Control groups included 1) T84 monolayers, 2) T84 monolayers cultured with LPS/FMLP, and 3) T84 monolayers cultured with nonactivated MPhi . Some experiments were conducted with naive T84 monolayers exposed to 50% CM for 48 h (prepared by culture of MPhi with LPS/FMLP for 24 h).

Ussing Chamber Studies

Epithelial ion transport. After coculture, T84 monolayers were mounted in Ussing chambers as previously described (24). Epithelial monolayers were bathed in oxygenated Krebs buffer (37°C) containing 10 mM glucose as an energy source in the serosal buffer, which was osmotically balanced by 10 mM mannitol in the mucosal buffer. The epithelial spontaneous potential difference was maintained at 0 V by the continuous injection of an external current by an automated voltage clamp (World Precision Instruments, Sarasota, FL). This short-circuit current (Isc, in µA/cm2) reflects net active ion transport across the preparation. Baseline Isc was recorded after a 15-min equilibration period. Stimulated ion secretion was measured by addition of the cholinergic agonist carbachol (10-4 M) or the adenylate cyclase-activating agent forskolin (10-5 M) (both from Sigma Chemical) to the serosal side of the T84 monolayers and recording the maximum increase in Isc (24).

The mucosal-to-serosal (Mright-arrowS), serosal-to-mucosal (Sright-arrowM) and net fluxes of 22Na and 36Cl were determined using standard methodologies. Briefly, after T84 monolayers had established a stable baseline Isc, 22Na and 36Cl were added to either the serosal or mucosal buffer at final concentrations of 4 µCi/ml for 22Na and 2 µCi/ml for 36Cl. After a 20-min equilibration period, samples (50 µl) were taken from the "hot" buffers for calculation of the tracer specific activity. One-milliliter samples were obtained from the cold buffer at 15-min intervals and replaced with appropriate Krebs buffer. Radioactivity in each sample was measured in a gamma -counter and a scintillation counter, and flux rates were calculated using standard formulas (42).

Epithelial permeability. Electrical resistance is a measure of the barrier property of the epithelium to passive ion movement. At intervals during each experiment, potential difference across the monolayer was clamped at 1.0 mV (differential pulse method, 1 pulse/30 s), and the resulting change in current was used to calculate the transepithelial ion resistance (R, in Omega  · cm2) according to Ohm's law (33). As an indication of epithelial permeability to larger molecules, the Mright-arrowS movement of the inert probe 51Cr-EDTA (362.3 Da, diameter 1.15 nm) was measured. 51Cr-EDTA (Radiopharmacy, McMaster-Chedoke Hospital, Hamilton, Ontario, Canada) was added to the mucosal buffer at a final concentration of 6.5 µCi/ml. Nonradioactive Cr-EDTA was added to the serosal buffer to maintain the osmotic balance. Fluxes were determined using 30-min flux periods (14).

Cell Viability

T84 monolayer viability was assessed by measuring release of lactate dehydrogenase (LDH) (20). After coculture, T84 monolayers were removed and rinsed three times in fresh PBS. Epithelial monolayers were lysed by immersing each filter in 0.1% (vol/vol) Triton X-100 (Sigma Chemical)/PBS for 30 min at room temperature followed by vigorous manual pipetting. The lysate was centrifuged at 500 rpm for 5 min, and the supernatant was analyzed for LDH activity using an automated multiple point rate test (Kodak, Rochester, NY).

Studies to Determine the Role of TNF-alpha

The effect of 48-h exposure to human recombinant TNF-alpha (3 or 6 ng/ml; Centocor) on T84 function was assessed. These concentrations were chosen based on the detected levels of TNF-alpha in CM at 24 or 48 h. Additional T84 monolayers were cocultured with MPhi activated with human recombinant TNF-alpha (>= 6 ng/ml; Centocor).

The role of TNF-alpha in the MPhi modulation of epithelial transport and barrier functions was assessed by inclusion of a neutralizing antibody to TNF-alpha , cA2 (1 µg/ml, >100-fold excess of the TNF-alpha measured in the CM) (Centocor). An irrelevant isotype-matched antibody (anti-hepatoma IgG1, AF20; Centocor) was used as control. Additional studies examined the effect of cA2 in CM added to T84 cells or to MPhi .

Statistics

Results are presented as means ± SE. Because of variability in absolute values between different batches of T84 cells, data were normalized to control values in each experiment (expressed as percentage of control); n values represent the number of experiments (different blood donors) in which two to four monolayers were examined for each condition. Data were analyzed using one-way ANOVA followed by Newman-Keuls comparison. Student's t-test was used where appropriate for individual comparisons. Statistically significant differences were accepted at P < 0.05.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Immune Cells

FACS analysis showed that >95% of the adherent immune cell population expressed CD14 and were the appropriate size for monocytes (n = 6). Less than 5% of cells expressed CD3, indicating that virtually no T cells were present. MPhi stimulated by LPS/FMLP secreted substantial amounts of TNF-alpha after 24 h compared with nonactivated MPhi (5.58 ± 1.52 vs. 1.15 ± 0.51 ng/ml; n = 3) and 48 h (3.09 ± 1.26 vs. 0.71 ± 0.31 ng/ml; n = 8). IL-2 was not detected (<4 pg/ml). Viability was >96% as measured by trypan blue exclusion after recovery of cells after plastic adherence.

Epithelial Physiology After Coculture With Monocytes

T84 monolayers cultured with LPS/FMLP in the absence of MPhi displayed transport characteristics that were not significantly different from control T84 monolayers cultured in media alone (data not shown). Therefore, T84 cells with no additions were used as controls in subsequent experiments.

Epithelial Ion Transport

Baseline Isc of T84 monolayers was unaltered after 24-48 h of coculture with nonactivated MPhi (Fig. 1A). In contrast, LPS/FMLP-activated MPhi evoked a significant increase (P < 0.05) in baseline Isc after 24 and 48 h of coculture to 174 ± 14 and 251 ± 16% of control values, respectively (Fig. 1A). As few as 5 × 104 cells/well caused a significant elevation (P < 0.05) in baseline Isc after 48 h of coculture (146 ± 21% of control values) (Fig. 1B). Absolute values from one representative experiment were 0.8 ± 0.1 and 3.8 ± 0.3 µA/cm2 for control and T84 monolayers cocultured with activated MPhi for 48 h, respectively (n = 4 replicates). Because Isc values are relatively insensitive in the low range (<10 µA), we measured bidirectional transepithelial fluxes of Na+ and Cl-. After 48 h of coculture, there was a significant increase in apically directed flux of Cl- across T84 monolayers compared with control values as well as an increased net flux of Cl- (Table 1). Under these experimental conditions, there was no significant difference in the transepithelial movement of Na+.


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Fig. 1.   Percent change from control values (T84 cells alone) of epithelial baseline short-circuit current (Isc) after 24 or 48 h of coculture with nonactivated monocytes (MPhi ) or lipopolysaccharide (LPS)/formyl-methionyl-leucyl-phenylalanine (FMLP)-activated monocytes (A-MPhi ) (n = 12 experiments) (A) and after 48 h of coculture with various numbers of A-MPhi (n = 4 experiments) (B). Values represent means ± SE with 2-4 monolayers per experiment. * P < 0.05 compared with control (100%).

                              
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Table 1.   Transepithelial fluxes of Na+ and Cl-

The Isc increase evoked by forskolin (88.0 ± 9.3 µA/cm2) was unaltered by coculture with nonactivated MPhi (24-48 h) or by exposure to activated MPhi for 24 h. However, after 48 h of culture with activated MPhi , there was a significantly diminished response (Fig. 2). In contrast, the stimulated Isc evoked by carbachol was unaffected by coculture (24 or 48 h) with MPhi or activated MPhi (119 ± 24 and 95 ± 21% of control values, respectively).


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Fig. 2.   Percent changes from control values (T84 cells alone) of epithelial secretory responses to forskolin (Fsk; 10-5 M) after 24 or 48 h of coculture with nonactivated monocytes (MPhi ) or LPS/FMLP-activated monocytes (A-MPhi ) (n = 12 experiments). Values represent means ± SE with 2-4 monolayers per experiment. * P < 0.05 compared with control (100%).

Epithelial Permeability

Coculture with nonactivated MPhi resulted in a small drop in transepithelial resistance (from 1,865 ± 130 to 1,492 ± 112 Omega  · cm2). This change in the barrier function of the epithelium was considerably enhanced by activation of MPhi . Thus, after 24 h of coculture with activated MPhi , T84 resistance was reduced to 50 ± 4% of that of time-matched controls. T84 resistance was consistently <40% of control values after 48 h of coculture with activated MPhi (Fig. 3A). The reduction in resistance was dependent on the number of MPhi present in the culture (Fig. 3B), with as few as 2.5 × 104 cells/well causing a significant decrease (P < 0.05) to 60 ± 16% of control values after 48 h of coculture. The decrease in resistance was maximal at 2 × 105 cells (30 ± 8% of control values).


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Fig. 3.   Percent change from control values (T84 cells alone) of epithelial resistance after 24 or 48 h of coculture with nonactivated monocytes (MPhi ) or LPS/FMLP-activated monocytes (A-MPhi ) (n = 12 experiments) (A) and after 48 h of coculture with various numbers of A-MPhi (n = 4 experiments) (B). Values represent means ± SE with 2-4 monolayers per experiment. * P < 0.05 compared with control (100%). # P < 0.05 compared with MPhi . ** P < 0.05 compared with control and 25,000 MPhi .

The degree of the epithelial barrier defect was further assessed by determination of the flux of the radiolabeled probe 51Cr-EDTA. After 48 h of coculture with activated MPhi (but not nonactivated MPhi ), the Mright-arrowS movement of 51Cr-EDTA across the T84 monolayers was significantly increased compared with control monolayers (3.12 ± 1.49 vs. 0.76 ± 0.66 nmol · h-1 · cm-2, respectively) (Fig. 4).


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Fig. 4.   Changes in mucosal-to-serosal movement of 51Cr-EDTA across T84 monolayers after 48 h of coculture with nonactivated monocytes (MPhi ), LPS/FMLP-activated monocytes (A- MPhi ), or A-MPhi with cA2 (1 µg/ml) (n = 6 experiments). Values represent means ± SE with 2-4 monolayers per experiment. * P < 0.05 compared with control (100%). # P < 0.05 compared with A-MPhi .

Epithelial Viability

After 48 h, there was no significant difference in LDH released from T84 epithelial cells cultured in media only or cocultured with activated MPhi (1,497 ± 71 vs. 1,560 ± 88 U/l).

Epithelial Physiology After Culture With CM

The altered epithelial ion transport properties (elevation of baseline Isc and reduced responsiveness to forskolin) were also observed after 48-h culture with CM from activated MPhi (Table 2). In addition, 48-h culture with CM caused a significant drop in resistance of T84 monolayers compared with control values (Table 2).

                              
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Table 2.   Effect of A-MPhi or conditioned medium on T84 monolayer physiology

Role of TNF-alpha

Addition of cA2 (anti-TNF-alpha antibody) to the coculture system completely prevented the increase in T84 baseline Isc that had previously been observed after 48 h of coculture with activated MPhi (Fig. 5). This amelioration of the increased ion transport was accompanied by a return of the active Sright-arrowM Cl- flux to control levels (0.52 ± 0.05 compared with 0.97 ± 0.11 µeq · h-1 · cm-2 for activated MPhi  + anti-TNF-alpha vs. activated MPhi , respectively). In addition, cA2 restored the diminished secretory response to forskolin (92 ± 8 vs. 72 ± 6% for activated MPhi  + anti-TNF-alpha vs. activated MPhi , respectively) and ameliorated the resistance change and flux of 51Cr-EDTA (Figs. 4 and 5). Inclusion of the irrelevant antibody had no effect on epithelial barrier and secretory defects.


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Fig. 5.   Percent change from control values (T84 cells alone) of epithelial baseline Isc and transepithelial resistance of T84 monolayers after 48 h of coculture with LPS/FMLP-activated monocytes (A-MPhi ) or A-MPhi in presence of cA2 (1 µg/ml) (n = 8 experiments). Values represent means ± SE with 2-4 monolayers per experiment. * P < 0.05 compared with A-MPhi .

However, exposure of naive T84 monolayers to 3 or 6 ng/ml of recombinant TNF-alpha (amount measured in CM from activated MPhi after 24 and 48 h, respectively) for 48 h had no significant effect on baseline secretory properties and transepithelial resistance of the epithelial cells (data not shown). In addition, cA2 in CM from activated MPhi added to T84 cells did not prove beneficial in correcting the altered epithelial ion secretion (baseline Isc of 276 ± 66 vs. 263 ± 57% of control values) or barrier properties (resistance of 49 ± 5.6 vs. 48 ± 10% of control values) for CM vs. CM + cA2, respectively. This suggested an indirect effect of TNF-alpha , perhaps via autocrine action on MPhi themselves. To examine this possiblity, T84 monolayers were exposed to the CM that was prepared in the presence of cA2 antibody. Under these conditions, the increased baseline Isc was completely returned to control levels, and the reduced T84 resistance was significantly corrected (Table 2). This hypothesis was confirmed, since T84 monolayers cocultured with MPhi activated with human recombinant TNF-alpha for 48 h consistently showed elevated baseline Isc, diminished responses to forskolin, and lowered epithelial resistance that were significantly different from control values (Fig. 6). These changes in epithelial function were very similar to those observed in coculture with LPS/FMLP-activated MPhi .


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Fig. 6.   Percent change from control values (T84 cells alone) in epithelial baseline Isc (A), secretory response to forskolin (Fsk; 10-5 M) (B), and transepithelial resistance (C) of T84 monolayers after 48 h of coculture with LPS/FMLP-activated monocytes (MPhi  + LPS/FMLP) or monocytes activated with human recombinant tumor necrosis factor-alpha (MPhi  + TNF-alpha ; 10 ng/ml) (n = 4 experiments). Values represent means ± SE with 2-4 monolayers per experiment. * P < 0.05 compared with control (100%).

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

We have used an in vitro coculture model system to demonstrate that MPhi activated with common bacterial products, in the absence of other immune cell types, can alter epithelial ion transport and permeability. Our results showed that after 48 h of coculture, activated MPhi stimulated epithelial Cl- secretion and increased permeability leading to impaired epithelial barrier function. We also demonstrated that MPhi -derived TNF-alpha was a key factor in mediating these abnormalities as addition of a neutralizing antibody against TNF-alpha in the coculture system inhibited the epithelial defects.

Monocytes/macrophages play a central role in immune and inflammatory events in the intestinal mucosa. Resident macrophages are located close to the basal membrane of the intestinal epithelium and represent the first line of defense by immune cells. Additional monocytes may be attracted to the intestinal mucosa during inflammation by locally produced MCP-1 (35). Recent studies have demonstrated the appearance of monocyte subpopulations with a different phenotype in IBD mucosa (22, 39) that unlike resident macrophages in normal intestine express high levels of the LPS receptor CD14 (4, 12, 39). The newly recruited cells are more easily activated, resulting in the production of excessive amounts of potent inflammatory mediators (2, 39). Isolated mononuclear cells from the colonic mucosa of IBD patients have an increased ability to undergo respiratory burst and to stimulate immunoglobulin secretion and an enhanced antigen-presenting activity (5). Furthermore, it has been recently reported that the CD14+ subset of macrophages from IBD mucosa have a different cytokine profile compared with the resident macrophages and are primed for the production of TNF-alpha , IL-1, and IL-6, all of which can directly or indirectly affect epithelial function (40). Despite these data, the ability of MPhi to directly regulate epithelial physiology has not been examined. Additionally, LPS is present in large quantities in the intestinal lumen, and when exposed to LPS, MPhi synthesize a plethora of proinflammatory mediators (8, 16, 27, 36) that are capable of inducing local tissue damage through their interactions with T cells, leukocytes, and endothelial cells (28). The bacterial tripeptide FMLP (43) induces monocyte chemotaxis and adherence as well as the production of oxygen radicals and proinflammatory eicosanoids (26). Because LPS and FMLP are usually present simultaneously in the gut lumen, we chose to add both agents directly to MPhi (mimicking events after their uptake from the lumen) and then determine the consequence of this MPhi activation on epithelial ion transport and permeability.

Our study clearly shows that MPhi activated by LPS/FMLP cause significant changes in epithelial ion transport and barrier functions. Coculture of T84 cells with activated MPhi elicited a significant increase in epithelial ion secretion as shown by the elevation of T84 baseline Isc. The elevated baseline secretion was most pronounced 48 h after coculture and was associated with increased Cl- secretion. However, the Isc increase was less than would be expected based on Cl- secretion alone, suggesting that there is another transport event that is decreasing the measured Isc that is not accounted for. From the data presented in Table 1, it appears that altered Na+ is not responsible. These observations complement findings documenting that LPS-stimulated alveolar macrophages altered ion transport in isolated rat lung epithelial cells (6). In comparison with our findings, T84 monolayers cocultured with PMN also display an increase in baseline Isc (19).

Activated MPhi increased epithelial ionic permeability as illustrated by a significant reduction in transepithelial resistance of the monolayer after 48 h of coculture. Concomitant with the reduced resistance was an increased transepithelial flux of the inert probe 51Cr-EDTA, which is suggestive of increased paracellular permeability (34, 42). These changes in epithelial permeability were evoked by remarkably few cells (2.5-5 × 104 MPhi ). Because epithelial cells were plated at an original density of 106 T84 cells/filter, a significant increase in epithelial permeability was observed at a ratio of >= 40:1 of epithelial cells to MPhi . Similar changes in epithelial physiology have been documented at a ratio of 20:1 of epithelial cells to PMN (15, 19). Thus our data indicate that MPhi have a potent ability to alter epithelial ion transport and permeability. It is clear that MPhi can be added to the growing list of immune cells (T cells, neutrophils, and eosinophils; Refs. 19, 23, 24, 37) that regulate epithelial physiology.

Increased permeability of the epithelial monolayers could potentially be because of epithelial cell cytotoxicity. However, the effects of activated MPhi on T84 cells were not simply the result of decreased epithelial viability, since release of LDH from MPhi cocultured epithelial monolayers was not different from control monolayers. A number of previous studies have shown similar increases in permeability of T84 epithelial monolayers cocultured with other immune cells (20, 24) or infected with enteropathogenic E. coli (31) without any significant epithelial cytotoxicity. Moreover, after exposure to activated MPhi , the epithelial monolayer was still capable of substantial vectorial ion secretion as indicated by the elevated baseline Isc of the monolayer and significant cAMP- and normal Ca2+-mediated Cl- secretion. These events indicate a functionally intact monolayer.

Cell-free CM from activated MPhi was equally effective in producing the epithelial abnormalities observed after coculture with activated MPhi . Stimulation of MPhi by LPS and FMLP leads to the production of an array of proinflammatory cytokines (8, 16, 27), and among these, TNF-alpha presents itself as a clear candidate for the mediation of the altered epithelial physiology. Several studies have reported increased TNF-alpha protein and mRNA levels in biopsies from IBD patients, particularly in Crohn's disease (17, 32). Significantly increased concentrations of TNF-alpha have been reported in stools of children with active chronic IBD (3). Other studies have also reported a rise in circulating TNF-alpha and the soluble TNF-alpha receptor (p55) in patients with active IBD that were significantly correlated with the clinical and/or laboratory measures of disease activity (11, 17). Furthermore, in a recent multicenter, placebo-controlled trial, anti-TNF-alpha antibody (cA2 antibody; same antibody used in this study) treatment resulted in prolonged clinical improvement in some patients with Crohn's disease (46). Therefore, having demonstrated significant TNF-alpha production by MPhi to our cocktail of LPS/FMLP, we proceeded to examine the role of TNF-alpha in MPhi modulation of epithelial function. Neutralization of TNF-alpha in the coculture model by addition of an anti-TNF-alpha antibody reduced the T84 baseline Isc response to control levels and significantly improved the T84 transepithelial resistance. In addition, anti-TNF-alpha treatment inhibited the increased epithelial permeability to 51Cr-EDTA evoked by culture with activated MPhi . The correction of the abnormal epithelial function by anti-TNF-alpha suggested that TNF-alpha directly affected T84 physiology, alone or in concert with other immune mediators, or that the TNF-alpha effect was one of autocrine activation of MPhi . A direct effect of TNF-alpha on HT-29 cells has been demonstrated (38). However, a number of studies have not been able to illustrate a direct action of TNF-alpha on T84 function after an acute exposure (<= 72 h; 3-6 ng/ml) (20, 24, 48). In contrast, a recent study has shown a direct effect of TNF-alpha (100 ng/ml) on transepithelial resistance, but only in the presence of interferon-gamma (47). Another study demonstrated that chronic treatment (4 days) of T84 monolayers with 100 ng/ml recombinant TNF-alpha caused a significant increase in inulin movement across the T84 monolayers (21).

In exploring the role of TNF-alpha in our model of epithelial disfunction, we found that addition of recombinant TNF-alpha at the concentrations measured in the supernatant from activated MPhi did not affect epithelial physiology. Moreover, neutralization of TNF-alpha in activated MPhi CM did not prevent the disrupted secretory responses or the increased epithelial permeability, whereas inclusion of cA2 at the time of preparation of the CM resulted in a CM that evoked significantly less epithelial abnormalities. This suggested that the TNF-alpha was autocrinely affecting the MPhi , and not the epithelium directly. To furthur examine this possibility, recombinant TNF-alpha was added to MPhi -T84 cocultures, and this resulted in elevated baseline Isc and decreased transepithelial resistance of T84 monolayers. These results were very similar to those evoked by LPS/FMLP activation of MPhi . Taken together, these results support the hypothesis that TNF-alpha affected MPhi in an autocrine manner (7, 45), causing the release of other as yet unidentified monocyte-derived mediators, the net result of which was altered epithelial function. Integrating these findings with previous studies, we suggest that TNF-alpha can modulate epithelial function both directly (studies with HT-29 cells, Ref. 21) and indirectly (our study) via immune cell activation.

In summary, we have demonstrated that MPhi activated by common bacterial products can stimulate Cl- secretion, alter ion transport responses, and impair the barrier function of the epithelium. We have also shown that TNF-alpha is a key factor mediating the MPhi -induced pathophysiology, although not affecting T84 cells directly. It has been suggested that during active IBD, LPS passes through the mucosal barrier, gaining access to MPhi , and primes them such that subsequent contact with luminal bacteria results in excessive production of potent inflammatory mediators, particularly TNF-alpha , resulting in pathology/pathophysiology of intestinal tissue (2). Modeling this scenario in vitro, we have demonstrated that LPS/FMLP activation of MPhi has significant consequences for epithelial ion transport and permeability functions.

    ACKNOWLEDGEMENTS

We thank P. Singh, P. Stetsko and D. Steele-Norwood for expert technical assistance.

    FOOTNOTES

This research was funded in part by Centocor, Astra Draco Pharma, the Crohn's and Colitis Foundation of Canada, and the Medical Research Council, Canada.

Address for reprint requests: M. H. Perdue, Intestinal Disease Research Program, HSC-3N5, McMaster University, 1200 Main St. West, Hamilton, Ontario, Canada L8N 3Z5.

Received 23 November 1997; accepted in final form 10 June 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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