Epithelial cells respond to proteolytic and non-proteolytic detachment by enhancing interleukin-6 responses

Tabbi L Miller; Dennis W McGee

doi:10.1046/j.0019-2805.2001.01352.x

. 2002 Jan;105(1):101–110. doi: 10.1046/j.0019-2805.2001.01352.x

Epithelial cells respond to proteolytic and non-proteolytic detachment by enhancing interleukin-6 responses

Tabbi L Miller ¹, Dennis W McGee ¹

PMCID: PMC1782634 PMID: 11849320

Abstract

Intestinal inflammatory disease or infection often results in the loss of the epithelial layer as a result mainly of the action of proteases, including the leucocyte serine proteinases (neutrophil elastase), lysosomal cathepsins and the matrix metalloproteinases from recruited inflammatory cells. Previous studies have shown that bronchial or intestinal epithelial cells (IEC) can respond to proteolytic attack by producing cytokines. In this study, we have determined the effect of protease treatment on interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) production by IEC lines. Both neutrophil elastase and trypsin treatment induced elevated levels of mRNA for IL-6 in rat IEC-6 cells. Non-proteolytic detachment of the IEC-6 cells also induced elevated levels of IL-6 mRNA, suggesting that the effect was not caused by a specific protease or degradation product, but probably by an effect on cell shape or cell detachment. Similar results were seen with the IEC-18 cell line. Trypsin treatment of the IEC-6 cells also enhanced unstimulated and IL-1β costimulated IL-6 secretion, but not MCP-1 secretion or mRNA levels. Finally, nuclear levels of the CCAAT/enhancer binding protein-β (C/EBP-β) were rapidly enhanced after proteolytic detachment of the IEC-6 cells, suggesting a mechanism for the enhancement of IL-6 mRNA responses. These data indicate that epithelial cells can respond to proteolytic attack or cell detachment by producing IL-6, a cytokine with several anti-inflammatory and antiprotease effects, which may be important in moderating the loss of the epithelial layer by its effects on nearby epithelial or inflammatory cells.

Introduction

Epithelial cells normally exist as adherent cells attached to a basement membrane that consists of various extracellular matrix (ECM) proteins. However, a mucosal inflammatory response, such as in inflammatory bowel disease (IBD) or mucosal infections, often results in a degradation of the mucosal surface and shedding of the epithelial layer. In addition to the proinflammatory cytokines, such as interferon-γ (IFN-γ), interleukin (IL)-1 and tumour necrosis factor-α (TNF-α), produced during inflammatory diseases, activated macrophages, neutrophils, T cells and intestinal stromal cells also produce several powerful proteases that can degrade the ECM surrounding the cells. These proteases include, among others, the leucocyte serine proteinases [i.e. neutrophil elastase (NE)], lysosomal cathepsins and the matrix metalloproteinases (MMP).¹ Mucosal damage and shedding of the epithelial layer, which are characteristic of intestinal mucosal inflammatory diseases, have been linked to the presence of several of these MMPs.²^–⁵ Recently, serine proteases (such as NE) and MMPs were also found to contribute to disease progression and epithelial damage in a murine model of colitis.⁶ Damage to the mucosal epithelium may then permit entry of infectious agents, toxins or resident bacteria into the lamina propria and inflammatory responses to these agents may then exacerbate the disease process.⁷

Intestinal epithelial cells (IEC) have been shown to secrete a variety of inflammatory cytokines and chemokines, and the levels of these cytokines may be enhanced in response to proinflammatory cytokine stimulation, microbial infection, or the presence of bacterial toxins.⁸ A recent study has suggested that IEC may be capable of responding to proteolytic detachment from a surface by increasing expression of both IL-1 and the IL-1 receptor type II (IL-1RII), an anti-inflammatory factor that binds to and inactivates IL-1.⁹ However, most studies agree that IEC may not secrete IL-1 and may only release this cytokine after cell damage.¹⁰^,¹¹ Other studies have suggested that bronchial epithelial cells may be able to up-regulate production of the neutrophil chemoattractant, IL-8, in response to proteolytic attack by NE,¹²^,¹³ and they suggest that this response may be a result in large part to cell shape deformation.¹³ However, it is unknown at this time whether this type of effect can be induced in IEC with regard to other cytokines.

Isolated normal IEC¹⁴^,¹⁵ and IEC cell lines¹⁶^,¹⁷ have been shown to produce IL-6 in response to inflammatory cytokines or bacterial infection. IL-6 is known to possess several anti-inflammatory characteristics, such as its ability to down-regulate LPS-induced monocyte IL-1 and TNF-α mRNA expression in vivo¹⁸^,¹⁹ and other proinflammatory cytokine production in acute inflammatory responses.²⁰ IL-6 has also been shown to induce IEC cell lines to produce protease inhibitors such as the α-1 proteinase inhibitor,²¹ which inactivates NE, and to induce other cell types to produce tissue inhibitor of metalloproteinases-1 (TIMP-1).²²

We have previously examined various mechanisms that regulate IL-6 secretion by IEC.¹⁶^,¹⁷^,²³^,²⁴ The anti-inflammatory and antiprotease functions of IL-6 have inspired us to examine whether proteolytic treatment of IEC cell lines could affect IL-6 mRNA expression and subsequent IL-6 secretion. For these studies, we have used the non-transformed small intestinal rat IEC-6 and IEC-18 cell lines, as these cell lines have been previously shown to be excellent models for cytokine studies on IEC.⁹^,¹⁰^,¹⁶^,²³^,²⁴ Our studies show that IEC cell lines yielded increased levels of IL-6 mRNA and protein secretion in response to proteolytic treatment. Furthermore, similar enhanced levels of IL-6 mRNA were seen after detachment of the cells by a non-proteolytic treatment, suggesting that the effect may occur in response to cell detachment or changes in cell shape. A similar enhancement in secreted protein or mRNA levels was not seen, however, with the proinflammatory chemokine, monocyte chemoattractant protein-1 (MCP-1). Finally, proteolytic detachment of the IEC-6 cells was found to rapidly enhance nuclear levels of the CCAAT/enhancer binding protein [C/EBP-β; also known as nuclear factor (NF-IL6)], suggesting a possible mechanism for the enhancement of IL-6 mRNA responses. This increase in IEC IL-6 production in response to cell shape deformation or detachment may then play a role in the overall inflammatory response at the intestinal mucosal surface.

Materials and methods

Cell culture

IEC-6 (CRL 1592), IEC-18 (CRL 1589) and A549 (CCL 185) cells were obtained from the American Type Culture Collection (Rockville, MD). The IEC-6 and IEC-18 cells were cultured in Dulbecco's modified Eagle's medium (complete DMEM) containing 5% fetal calf serum (FCS; Hyclone Laboratories, Logan, UT), 0·1 µg/ml bovine insulin (Sigma, St Louis, MO), 25 IU/ml penicillin and 25 IU/ml streptomycin (Mediatech, Washington, DC). The A549 cells were maintained in F12K medium (complete F12K medium) containing 10% FCS (Hyclone), 1·5 g/l sodium bicarbonate, 25 IU/ml penicillin and 25 IU/ml streptomycin (Mediatech). Cultures were incubated in a humid, 10% CO₂–90% air atmosphere at 37°. IEC-6 and IEC-18 cells were utilized at or before the 19th or 21st passage, respectively.

Proteolytic treatment of cells to determine the effect on mRNA expression

IEC-6 or IEC-18 cells were added to wells of 12-well culture plates at 5 × 10⁵ cells/well in complete DMEM and were allowed to grow for 2 days. The culture supernatants were then removed and replaced with serum-free DMEM containing insulin, transferrin and selenium (ITS; Collaborative/Becton-Dickinson, Bedford, MA), l-glutamine, penicillin and streptomycin (designated as ITS-DMEM). A549 cells were cultured as described above in complete F12K medium, allowed to grow for 2 days, then culture supernatants were removed and replaced with serum-free F12K medium containing ITS (ITS-F12K). ITS-DMEM or ITS-F12K was added to wells with or without the appropriate protease – either bovine pancreatic trypsin (Sigma) at 500 µg/ml, or human neutrophil elastase (Calbiochem, La Jolla, CA) at 5·9 µg/ml (200 nm). In some cases, culture supernatants were replaced with MatriSperse (Collaborative/Becton Dickinson) instead of ITS-DMEM to determine the effect of non-proteolytic detachment. After the appropriate time interval, supernatants containing the non-adherent cells were removed and placed on ice. Adherent cells were removed by brief treatment with trypsin and EDTA (Sigma), then combined with the appropriate non-adherent cells from the same culture and washed in cold phosphate-buffered saline (PBS) prior to isolation of RNA, as described below.

IEC-6 cell survival following protease treatment was assessed by Trypan Blue exclusion to determine cell viability. Also, the percentage of apoptotic/necrotic cells was estimated using the terminal dUTP nick end-labelling (TUNEL) apoptosis detection system (Promega, Madison, WI). IEC-6 cells were removed from culture flasks using trypsin and EDTA treatment and washed in serum-containing medium. The cells were then added at 2·5 × 10⁵ cells/well to uncoated plastic wells of 24-well culture plates, to allow readherence, or to wells precoated with 1% denatured bovine serum albumin (dBSA) in PBS. Precoating of the wells was accomplished by adding 250 µl of 1% dBSA (which had been denatured by boiling for 15 min) in PBS to each well and incubating for 1 hr at room temperature. The solution was then removed and the wells were allowed to air-dry. The cells were cultured at 37° for the appropriate time interval before collection, as described above. The cells were then cytocentrifuged onto microscope slides at 5 × 10⁴ cells/slide and stained for the presence of fragmented DNA. The percentage of stained cells was determined by viewing slides using a Zeiss Axiovert 100 inverted fluorescent microscope.

Determination of mRNA levels by reverse transcription–polymerase chain reaction (RT–PCR)

Cells collected from the above experiments were first washed in PBS and then total RNA was extracted by using either the acid guanidium thiocyanate method, as described previously,²⁵ or by a similar method using Trizol (Life Technologies, Grand Island, NY). Total RNA was quantified by measuring the absorbance of the samples at 260 nm, and samples containing 0·5, 0·25 and 0·125 µg of RNA were reverse transcribed (RT), using the Gene Amp RNA PCR Kit (Perkin-Elmer, Norwalk, VT) with oligo d(T)₁₆, at 42° for 60 min followed by 5 min at 99°. The resulting cDNA was then amplified by polymerase chain reaction (PCR) using primers specific for rat IL-6 (Clontech, Palo Alto, CA), rat MCP-1 (Biosource International, Camarillo, CA), or human IL-6 (Clontech). Primers for human or rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Clontech) were also used as controls for RNA content.

PCR was performed for the appropriate number of cycles using a Perkin-Elmer DNA Thermal Cycler 480. Each cycle consisted of 1 min at 94°, 2 min at 60–65°, and 3 min at 72°. PCR products were then separated by gel electrophoresis using 2% agarose gels (Fisher, Fair Lawn, NJ) and the resulting gels were stained with ethidium bromide and photographed. The PCR product band densities were quantified from photographic negatives using a UVP Gel Documentation system (UVP, Upland, CA) and the Labworks 3.02 Analysis sofware (Media Cybernetics, Del Mar, CA).

Proteolytic treatment to determine the effect on cytokine secretion

IEC-6 cells were cultured for 2 days in 24-well tissue culture plates at 2·5 × 10⁵ cells/well. Culture supernatants were then removed and the cells washed with serum-free medium. Each well then received 500 µl of complete-DMEM (untreated), 250 µl of complete DMEM plus 250 µl of trypsin–EDTA (trypsin added), or 250 µl of trypsin–EDTA (Sigma) in PBS (detached). Following a 10-min incubation, 250 µl of DMEM containing 5% FCS was added to the wells containing only trypsin–EDTA in PBS (detached). The contents of these detached wells were transferred to a 24-well culture plate that had been precoated with 1% dBSA to inhibit adherence of the cells. All cultures were then incubated in either the presence or absence of 1 ng/ml recombinant human IL-1β (R & D Systems, Minneapolis, MN) as an inflammatory stimulus. At 24 hr, the supernatants were harvested and stored at −80° for determining cytokine secretion levels.

Measurement of cytokine secretion

Levels of bioactive IL-6 secreted by the IEC-6 cells were measured by a bioassay utilizing the IL-6-dependent 7TD1 hybridoma,²⁶ which has been described previously.¹⁶ Two-fold serial dilutions of the culture supernatants were cultured for 4 days in 96-well tissue culture plates with 2 × 10³ 7TD1 cells/well. Proliferation of the 7TD1 cells was then quantified using the MTT colorimetric assay²⁷ and compared to a standard of recombinant murine IL-6. The addition of an anti-rat IL-6 antibody (Pierce-Endogen, Woburn, MA) to neutralize IL-6 in culture supernatants demonstrated the specificity of the assay.

Secreted levels of MCP-1 were measured by specific enzyme-linked immunosorbent assay (ELISA) for rat MCP-1 (Biosource). The resulting optical densities for both the 7TD1 bioassay and the ELISA were measured utilizing a BioTek EL 312 microplate reader (Winooski, VT).

Isolation of nuclear protein

Nuclear protein was extracted from isolated nuclei following a previously described protocol.²⁸ The culture supernatants were removed at the appropriate time-point, and 500 µl of cytoplasmic extraction buffer containing 10 mm Tris–HCl, 60 mm KCl, 1 mm EDTA, 1 mm dithiothreitol (DTT; Sigma), 0·4% IGEPAL (Sigma) and 1 µl/ml of a Protease Inhibitor Cocktail Set III (Calbiochem), containing 100 mm aminoethylbenzenesulphonic acid hydrochloride, 5 mm bestatin, 1·5 mm E-64, 2 mm leupeptin hemisulphate and 1 mm pepstatin A, was added and incubated for 5 min on ice. The culture supernatants were centrifuged at 4° for 5 min at 8000 g, then the contents of the wells were added to the cell pellets and incubated on ice for 5 min. The samples were then centrifuged at 4° for 3 min at 510 g and the supernatants removed before adding 500 µl of nuclear extraction buffer containing 50 mm Tris-HCl, 420 mm NaCl, 1·5 mm MgCl₂, 25% glycerol, 0·5 mm phenylmethylsulphonyl fluoride (PMSF; Sigma) and 1 µg/ml of the Protease Inhibitor Cocktail Set III (Calbiochem). The samples were then incubated for 10 min on ice before centrifuging at 4° for 10 min at 11750 g. The supernatants were then removed and Laemmli buffer was added to the nuclear protein. The protein samples were stored at −80° and Western blotting was performed as described below.

Western blotting analysis of nuclear proteins

The protein content of samples was determined using the Bio-Rad DC Protein Assay Kit (Bio-Rad, Melville, NY) or quantified by measuring the absorbance at 280 nm and 260 nm using a Perkin-Elmer Lambda Bio UV/Vis spectrophotometer (Perkin-Elmer). Equal concentrations of protein samples were then separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) at 200 V for 45 min at room temperature with a Bio-Rad Mini Protean III Apparatus (Bio-Rad) using a 12% resolving gel with a 4% stacking gel. The proteins were then transferred to nitrocellulose membranes using a Bio-Rad Trans-Blot apparatus at 100 V for 1 hr at room temperature. The blots were then blocked for 2 hr at room temperature or overnight at 4° in Tris-buffered saline containing 0·1% Tween-20 (TBST) and 5% BSA (Sigma). The blots were then treated overnight at 4° with 133 ng/ml of a cross-species reactive rabbit anti-human p65 (Rel-A) antibody (Santa Cruz Biotechnologies, Inc., Santa Cruz, CA), 133 ng/ml rabbit anti-human phospho-c-Jun antibody (Cell Signalling Technology, Beverly, MA), or 667 ng/ml of rabbit anti-rat C/EBP-β antibody (Santa Cruz Biotechnologies, Inc.). The blots were then washed three times for 5 min in TBST and treated for 1 hr with a 1 : 1500 dilution of a peroxidase-labelled anti-rabbit IgG secondary antibody (Cell Signalling Technology, Beverly, MA). The blots were then visualized using the chemiluminescent Phototope Western Detection System (Cell Signalling Technology) followed by exposure to X-ray film, and the band densities were quantified as described above.

Statistical analysis of data

Statistical significance between conditions was determined by the Student's t-test utilizing the StatView 4.01 (SAS Institute, Inc., Cary, NC) data analysis program. A probability (P-value) of <0·05 was considered as a significant difference between groups.

Results

IEC-6 cell survival following proteolytic treatment

Prior to determining the effect of proteolytic treatment of IEC on cytokine mRNA levels and secretion, our concern was whether these cells could survive for a significant period of time following such proteolytic treatment. To simulate proteolytic attack, IEC-6 cells were treated for 10 min with trypsin and EDTA. To achieve detachment, the cells were washed and replated in 24-well tissue culture plates that had been precoated with 1% dBSA in PBS to inhibit reattachment of the cells. For comparative purposes only, some cells were allowed to reattach by plating them in uncoated wells. The total number of viable cells harvested and counted at 6 and 12 hr after replating in dBSA-coated wells (detached cells) represented (at both time-points) 91% of the total viable cells harvested from uncoated wells (attached cells) at similar time-points (calculated from three separate experiments). This suggests that the detached IEC-6 cells could survive for a significant length of time after detachment. However, at 24 hr, the number of viable detached cells recovered from dBSA-coated wells represented only 57% of the total cells in the uncoated wells, suggesting that the cells had a limited survival time when not attached to a substrate and began to die rapidly between 12 and 24 hr.

In addition, the TUNEL staining technique was performed to obtain an estimate of the proportion of cells with fragmented DNA, which is characteristic of cells undergoing apoptosis or necrosis. Culture of the detached IEC-6 cells in dBSA-coated wells for 6 hr did result in an increase of the number of cells staining for fragmented DNA; however, this increase was only 8% of the total cells (Table 1). Even at 24 hr following culture on dBSA, only 23% of cells contained fragmented DNA (Table 1). Similar results were seen with cells that had been stimulated with IL-1β. Although these values were significantly higher than those of cells cultured in uncoated wells which reattached, this data supports the hypothesis that significant numbers of IEC may retain viability for long periods of time (at least 6 hr; possibly 12 hr) following detachment and therefore may continue to produce cytokines during these periods.

Table 1.

Percentage of adherent or detached cells demonstrating fragmented DNA

		TUNEL-stained cells (%)^*

		Exp. 1	Exp. 2

Well coating	IL-1β stimulation	6 hr	24 hr
Uncoated	–	2 ± 0	4 ± 1
	+	2 ± 1	8 ± 0
Denatured BSA	–	8 ± 0^†	23 ± 2^†
	+	11 ± 1^†	20 ± 4^†

Open in a new tab

IEC-6 cells were added at 2·5 × 10⁵ cells/well to ITS-DMEM, with or without 1 ng/ml recombinant human interleukin-1β (rhIL-1β) to wells of 24-well culture plates that were uncoated or precoated with 1% denatured bovine serum albumin (BSA). At the time-points indicated, the cells were collected, cytocentrifuged onto glass slides and stained. Data shown represent the mean ± SD values for triplicate cultures.

^†

Significant difference from similar cultures grown in uncoated wells (P < 0·005).

The effect of proteolytic and non-proteolytic detachment of IEC-6 cells on IL-6 mRNA levels

Once we had demonstrated that IEC-6 cells could in fact survive for significant periods of time following proteolytic treatment, our next goal was to determine if such treatment had an effect on the ability of IEC-6 cells to express mRNA for IL-6. In order to simulate a more natural situation, human NE was chosen for treatment of the IEC-6 cells. Treatment of the IEC-6 cells with NE for 5 hr resulted in enhanced levels of IL-6 mRNA as compared to cells cultured without NE (Fig. 1). Analysis of PCR band densities from the figure shown (determined as a ratio of IL-6 band density to GAPDH band density) demonstrated a 2·4-fold increase for the 0·5-µg RNA samples from NE-treated cells as compared to similar samples from untreated cells. Of note, treatment of the cells with NE for 5 hr resulted in complete detachment of the IEC-6 cells from the culture well and the cells displayed a characteristic rounded morphology.

The effect of proteolytic and non-proteolytic detachment of intestinal epithelial cells (IEC) on interleukin-6 (IL-6) mRNA expression. IEC-6 cells (5 × 10⁵ cells/well) were cultured for 2 days prior to removal of the culture supernatant and addition of ITS-DMEM, with or without 200 nm human neutrophil elastase (NE), 500 µg/ml bovine pancreatic trypsin, or MatriSperse. After 5 hr, the total RNA was extracted and aliquots of 0·5 µg (lane A), 0·25 µg (lane B), or 0·125 µg (lane C) of RNA were reverse transcribed and polymerase chain reaction (PCR) amplified with primers for rat IL-6 or glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The cDNA was then separated by agarose-gel electrophoresis and stained with ethidium bromide. Results shown are representative of three experiments each.

Interestingly, cells treated with bovine pancreatic trypsin, an enzyme commonly used to dissociate cells from tissues or culture flasks, also showed a similar 3·6-fold increase in IL-6 mRNA levels (Fig. 1). This suggests that the effect seen was not specific to the protease NE, but may be common to other proteases that result in detachment of the cells. In addition, time-course experiments with trypsin treatment were performed which showed that IL-6 mRNA levels were enhanced within 1 hr after treatment and remained elevated beyond 5 hr (data not shown).

Although the above experiments suggest that proteolytic treatment of the cells results in an enhancement of IL-6 mRNA levels, the effect was not a specific property of the protease used for treatment. Still, the question remained as to whether the enhancement occurred as a result of the protease itself or to the presence of a proteolytic cleavage product. To address this question, a non-proteolytic cell detachment solution called MatriSperse (Collaborative/Becton Dickinson) was used, which caused detachment of the cells by non-enzymatic dissociation from the ECM, as indicated by the manufacturer. Non-proteolytic detachment of the IEC-6 cells with MatriSperse also resulted in enhanced IL-6 mRNA levels at 5 hr (Fig. 1), as was seen with proteolytic detachment. Analysis of PCR band densities for the figure shown (determined as a ratio of IL-6 band density to GAPDH band density) demonstrated a fourfold increase for the 0·5-µg RNA samples from MatriSperse-treated cells as compared to similar samples from untreated cells. This result suggests that the enhancement of IL-6 mRNA levels may not necessarily be in response to a protease, but may have been a result of the detachment of the cells from the culture well or possibly the rounding-up or shape deformation that accompanied the detachment.

Effect of proteolytic treatment on IL-6 responses by other epithelial cell lines

Next, the IEC-18 non-transformed rat small IEC line was used to determine if the effects noted were common to other IEC cell lines. Proteolytic treatment with trypsin, as well as non-proteolytic detachment with MatriSperse, resulted in an enhancement of IL-6 mRNA levels after 5 hr (Fig. 2), as was seen with similar treatment of IEC-6 cells. An analysis of PCR band densities for the figure shown (determined as a ratio of IL-6 band density to GAPDH band density for the 0·25-µg RNA samples) revealed a 1·8-fold increase with the trypsin-treated cells and a 2·7-fold increase with the MatriSperse-treated cells as compared to similar samples from untreated cells. These results suggest that the enhancement of IL-6 mRNA levels seen with the IEC-6 cells following cell detachment/shape deformation may be a normal response common to IEC, although there may be some differences in the extent of the effect with the different cells.

The effect of proteolytic or non-proteolytic detachment on the interleukin-6 (IL-6) mRNA levels of the IEC-18 cell line. IEC-18 cells were cultured as described in the legend to Fig. 1 and treated with ITS-DMEM alone, ITS-DMEM containing 500 µg/ml bovine pancreatic trypsin, or MatriSperse. The cells were removed after 5 hr of incubation and total RNA was isolated for reverse transcription–polymerase chain reaction (RT–PCR) amplification. The results shown are representative of two separate experiments.

Finally, we wanted to determine if the effect of proteolytic detachment/shape deformation on IL-6 mRNA responses could be seen with other epithelial cell types. For these studies, we used the human lung carcinoma cell line, A549, which has been used extensively in studies on lung epithelial cell IL-6 responses.²⁹^,³⁰ Proteolytic detachment of the A549 cells also resulted in enhanced IL-6 mRNA levels at 5 hr (Fig. 3). These results extend our findings to suggest that the enhancement of IL-6 mRNA levels in response to proteolytic detachment/shape deformation may be common to several epithelial cell types in addition to IEC.

Proteolytic detachment of human lung A549 cells also enhances interleukin-6 (IL-6) mRNA levels. The A549 cells were cultured as described in the legend to Fig. 1 and treated for 5 hr with or without 500 µg/ml bovine pancreatic trypsin in ITS-F12K prior to collecting the cells for reverse transcription–polymerase chain reaction (RT–PCR) amplification. The results shown are representative of two separate experiments.

The effect of proteolytic treatment of IEC-6 cells on IL-6 secretion

As the previous experiments had shown that detachment of IEC induced by proteolytic or non-proteolytic means resulted in an alteration in IL-6 mRNA levels, we then determined if such detachment also resulted in altered cytokine secretion levels. Unfortunately, the proteases contained in culture supernatants adversely affected the 7TD1 bioassay and the MatriSperse was found to inhibit proliferation of the 7TD1 cells. Therefore, we modified our procedure by proteolytically detaching the IEC-6 cells and then adding serum-containing medium to inhibit the trypsin. The detached cells were then transferred to dBSA-coated wells to prevent reattachment of the cells. Culture supernatants from these cultures could then be used in the 7TD1 bioassay. The specific procedure is outlined below.

The IEC-6 cells (2·5 × 10⁵ cells/well) were cultured in tissue culture plates for 2 days in DMEM containing 5% FCS. The cells were then treated with 500 µl of fresh DMEM containing 5% FCS (untreated) or were detached from the wells by brief treatment with 250 µl trypsin and EDTA followed by dilution with 250 µl of DMEM containing 5% FCS before transfer of the cells and media into wells that had been precoated with 1% dBSA to prevent readherence (detached). Additional cultures of cells were treated with 250 µl of DMEM containing 5% FCS and with 250 µl of trypsin and EDTA (trypsin added) as an additional control to determine the possible effect of trypsin remaining in the cell suspensions. The cells in these trypsin-added wells remained attached throughout the experiment. The detached cells showed a sixfold increase in IL-6 secretion levels as compared to the untreated cells (P < 0·05; Fig. 4a), whereas the trypsin-added cultures yielded no differences from the untreated cultures. Additionally, some cultures were stimulated with rhIL-1β to simulate an inflammatory situation. In the IL-1β-stimulated cultures, the detached cells showed a 15-fold increase in IL-6 secretion levels as compared to the untreated cells (P < 0·05; Fig. 4b). The addition of a neutralizing rabbit anti-rat IL-6 antibody (Pierce-Endogen) to some supernatants in the 7TD1 bioassay confirmed the specificity of the assay, as treatment with this antibody almost completely inhibited proliferation of the 7TD1 cells with the supernatants (data not shown).

Proteolytic detachment of IEC-6 cells enhances unstimulated and interleukin (IL)-1β-stimulated IL-6 secretion. IEC-6 cells (2·5 × 10⁵ cells/well) were cultured for 2 days prior to washing and treatment with DMEM containing 5% fetal calf serum (FCS)±recombinant human (rh)IL-1β (untreated), 5% FCS DMEM with trypsin and EDTA±rhIL-1β (trypsin added), or trypsin and EDTA for 10 min, and then 5% FCS DMEM±rhIL-1β was added (to inhibit the trypsin and EDTA) and the detached cells were transferred to dBSA-coated wells (detached). After 24 hr, the supernatants were collected for IL-6 determination. (a) The effect on unstimulated cells and (b) the effect on rhIL-1β (1 ng/ml)-stimulated cells from the same experiment. Data represent the mean±SD values for triplicate wells from one experiment, representative of three separate experiments. *Significant difference from the untreated cells (P ≤ 0·05).

Effect of protease treatment on MCP-1 responses by IEC-6 cells

As IEC have been shown to produce the monocyte chemoattractant, MCP-1, the level of which can be elevated in IBD,³¹ we also determined the effect of proteolytic detachment on MCP-1 secretion using a culture supernatant generated as described in Fig. 4. However, no significant difference in MCP-1 secretion levels was seen between the untreated cells and those that were detached and recultured on dBSA-coated wells with unstimulated or IL-1β-stimulated cultures (Fig. 5). This suggests that proteolytic detachment/shape deformation had no effect on IEC MCP-1 responses. As a confirmation, analysis of mRNA levels from IEC-6 cells treated with trypsin (as described in Fig. 1) also showed no effect of trypsin treatment on MCP-1 mRNA levels (data not shown).

Proteolytic detachment of IEC-6 cells has no effect on unstimulated and interleukin-1β (IL-1β)-stimulated monocyte chemoattractant protein-1 (MCP-1) secretion. IEC-6 cells were cultured under the same conditions as described in the legend to Fig. 4 and the culture supernatants were collected for determination of MCP-1 levels by specific enzyme-linked immunosorbent assay (ELISA). The data shown represent the mean±SD values for triplicate wells for each condition.

The effect of proteolytic detachment of the IEC-6 cells on nuclear NF-κB, c-Jun and C/EBP-β protein levels

Our next step was to investigate the intracellular mechanisms which may be responsible for the observed increases of IL-6 mRNA levels following proteolytic detachment of the IEC. The enhancement of IL-6 mRNA levels suggested a possible activation of transcription factors that are involved in regulating transcription of the IL-6 gene. Regulation of IL-6 gene transcription has been shown to be mediated mainly by the transcription factors NF-κβ, AP-1 and C-EBP-β (formerly known as NF-IL6).³² However, we found no effect on MCP-1 mRNA responses, and transcription of the MCP-1 gene is known to be regulated mainly by NF-κB and AP-1,³³ but not by C/EBP-β. Therefore, a probable candidate for the effect of proteolytic detachment on IL-6 responses was C/EBP-β.

Nuclei from untreated or trypsin-treated cells were prepared at various time-points during treatment and the nuclear protein was isolated for SDS–PAGE and Western blot analysis. An analysis of nuclear proteins was chosen as only activated NF-κB, free of the inhibitor IκBα, can be transported to the nucleus of cells³⁴ and a recent report has shown that C/EBP-β is found only in the nucleus of IEC-6 cells.³⁵ Shown in Fig. 6(a), nuclear protein extracts from untreated or trypsin-treated cells showed little differences in the nuclear RelA (p65) NF-κB subunit (the most common NF-κB subunit protein; see ref. 34) levels at each time period studied. In addition, nuclear levels of phosphorylated c-Jun, a component of the AP-1 transcription factor, were barely detectable and showed no change between untreated or trypsin-treated cells over the time-period studied (data not shown). However, the 44-000 molecular weight (MW) liver-activating protein (LAP) form of C/EBP-β was found to be absent in nuclear extracts from untreated cells and present in the trypsin-treated cells at the 10-min time-point, and the levels decreased somewhat over the time period studied (Fig. 6b). The 22-000 MW liver inhibitory protein (LIP) form of C/EBP-β was present in all samples and did not change over the same time period (data not shown). These experiments suggest that detachment of the IEC-6 cells may induce a rapid increase in the LAP form of C/EBP-β.

The effect of proteolytic detachment on IEC-6 cell nuclear factor (NF)-κB p65 and CCAAT/enhancer binding protein-β (C/EBP-β) liver-activating protein (LAP) protein levels. IEC-6 cells (5 × 10⁵ cells/well) were cultured for 2 days before the culture supernatants were removed and ITS-DMEM, with or without 500 µg/ml bovine pancreatic trypsin, was added. At the appropriate time-points, the cells were lysed and nuclei collected for isolation of nuclear proteins. Equal concentrations of nuclear proteins were separated by SDS–PAGE and Western blotted using antibodies specific for (a) the p65 (RelA) NF-κB protein or (b) the LAP C/EBP-β protein. Blots from the same experiment are shown, which are representative of two separate experiments. T, trypsin treated; U, untreated.

Discussion

Degradation of the ECM during an inflammatory response by infiltrating leucocytes allows these cells to migrate into the tissues, but it can have a devastating effect on surface epithelial cells in tissues such as the intestinal mucosa. Several studies have linked leucocyte-derived (and activated stromal cell-derived) proteases with the shedding of the epithelial layer during intestinal mucosal inflammatory disease.²^–⁶ This shedding of the mucosal epithelial layer can then exacerbate the disease process by allowing the entry of intestinal micro-organisms and food antigens.

In response to an inflammatory situation or proinflammatory cytokines, IEC have been shown to produce several cytokines.⁸ The fact that some of the cytokines secreted by IEC can function in an anti-inflammatory capacity suggests that these cells may have the potential to exert some measure of control by suppressing inappropriate or detrimental immune responses at the intestinal mucosal surface. An imbalance between proinflammatory and anti-inflammatory factors has been suggested to play a significant role in the disease pathology of IBD.³⁶ An indication that proteolytic treatment of IEC could result in the production of pro- and anti-inflammatory factors was shown by Waterhouse and co-workers who found that trypsin-treated IEC-18 cells produced enhanced levels of IL-1 as well as the anti-inflammatory IL-1RII.⁹ Another cytokine secreted by IEC (and which possesses some significant anti-inflammatory functions) is IL-6, and our studies have addressed the capacity of IEC cell lines to respond to proteolytic attack by producing IL-6.

Treatment of the IEC cell lines with NE or trypsin resulted in an enhancement of IL-6 mRNA levels, and trypsin treatment resulted in an increase in the secretion of bioactive IL-6. This suggests that IEC may well respond to proteolytic attack by producing IL-6, which has been shown to down-regulate proinflammatory cytokine responses in inflammation¹⁸^–²⁰ and induce other cells to produce the α-1 proteinase inhibitor and TIMP-1.²¹^,²² However, proteolytic treatment of the cells had no effect on MCP-1 responses, suggesting that the effect is not universal for all cytokines.

As mentioned above, IEC-18 cells have been shown to produce enhanced levels of IL-1 in response to trypsin treatment; however, the IL-1 was apparently associated with the inhibitor IL-1RII.⁹ This suggests a possible effect on both pro- and anti-inflammatory factors. Indeed, Shibata and co-workers have found that bronchial epithelial cells treated with NE also respond by producing elevated levels of IL-8 mRNA and protein.¹³ Our finding that lung A549 cells respond to trypsin treatment by producing enhanced levels of IL-6 mRNA is in line with this work on respiratory tissue and suggests that these effects may be common to many epithelial cell types.

Interestingly, the increase in IL-6 mRNA levels seen in our experiments was not dependent upon the protease used. Indeed, similar effects were seen even with non-proteolytic detachment of the cells, suggesting that the effect may occur in response to cell detachment or changes in cell shape. A previous study with bronchial epithelial cells presented evidence showing that an increase in IL-8 responses after NE treatment was caused by cell shape deformation.¹³ It was also found that a similar increase in IL-8 responses could be obtained by treating cells with the microtubule-dissociating drugs colchicine or vinblastine. Furthermore, pretreatment of the cells with taxol, which stablizes microtubules, before treatment with NE, prevented the increase in IL-8 responses, confirming that the effect was probably mediated through changes in microtubules.

We have found that treating the IEC-6 cells with colchicine could result in an enhancement of IL-6 mRNA levels, indicating that the effect may also be caused by changes in microtubule structures (data not shown). However, we have also found that the IEC-6 cells respond to taxol treatment by producing elevated levels of IL-6 mRNA so, unfortunately, this drug cannot be used together with trypsin or NE treatment to determine a direct link between microtubules and the effect. This stimulatory effect of taxol has been seen with other sensitive cell lines, which show taxol-mediated increases in IL-8 gene expression together with NF-κB and AP-1 activation.³⁷ Still, these experiments, along with the experiments using the non-proteolytic detachment solution MatriSperse, do tend to suggest that cell shape deformation may play a role in the effect. In addition, these studies indicate that cell shape deformation or detachment of IEC by non-proteolytic means, such as drug therapy, may also result in enhanced IL-6 responses that may have importance to the overall drug effect. However, we must use caution in extending the results presented here to IEC in vivo, as normal IEC, unlike the cell lines used in our study, are highly polarized and may show variations in their response.

Finally, proteolytic detachment of the IEC-6 cells was determined to have little, if any, effect on nuclear NF-κB or phosphorylated c-Jun levels, suggesting that these normally potent mechanisms for activating inflammatory cytokine genes did not participate in the response. However, detachment of the cells did appear to result in a rapid accumulation of the LAP form of C/EBP-β in the nucleus. As indicated before, C/EBP-β is known to be a regulator of IL-6 and IL-8, but not of MCP-1, gene activation. An increase in C/EBP-β levels could have resulted in an enhancement of IL-6 mRNA levels without affecting MCP-1 responses, as seen in our results. In addition, an increase in C/EBP-β levels could have played an important role in the synergistic enhancement of IL-6 secretion seen with the detached cells stimulated with IL-1β, as the C/EBP-β could have acted in synergy with NF-κB from the IL-1 stimulation to greatly enhance IL-6 gene transcription. A synergistic effect of C/EBP-β with NF-κB has previously been shown to be a powerful activator of both IL-6 and IL-8 gene transcription.³⁸ Furthermore, C/EBP-β is known to be involved in the regulation of several acute-phase proteins,³⁹ and cell detachment/shape deformation may even affect the production of these proteins through C/EBP-β. Although IL-6 is known to induce the expression of C/EBP-β,³⁹ the rapid increase in C/EBP-β levels was probably too early to be an autocrine effect induced by an increase in IL-6 secretion from the cell detachment/shape deformation, especially as increases in IL-6 mRNA levels were not detected until after 1 hr of treatment. Experiments are now in progress to better characterize the relationship between cell detachment/shape deformation and C/EBP-β for activation mechanisms and C/EBP-β gene expression.

Regardless of the underlying mechanism, either by attack from inflammatory or even possibly bacterial proteases or non-proteolytic means, our studies suggest that epithelial cells may respond to cell shape deformation or detachment by producing elevated levels of IL-6. This response is rapid, showing elevated levels of IL-6 mRNA as early as 1 hr after treatment. In addition, this effect appears to act in synergy with IL-1β to greatly enhance IL-6 secretion. Although the cells on the intestinal villus tips or colonic surface may have little chance of producing effective levels of IL-6, cells within the crypts may remain in the crypt lumen for longer periods of time and might produce effective levels of IL-6, especially in the presence of IL-1. This IL-6 could then affect nearby IEC, stromal cells, or even inflammatory cells in the crypt abscesses by inducing the production of protease inhibitors and down-regulating proinflammatory cytokine production. Indeed, this could even be an important mechanism to help protect intestinal crypt IEC, which would then be necessary for renewing the epithelial layer during wound healing.

Acknowledgments

The authors would like to thank Farah Lubin, Shari Hanifin and Wil Von Zagorski for their assistance with this study. This work was supported by US PHS Grant DK 54049.

References

1.Goetzl EJ, Banda MJ, Leppert D. Matrix metalloproteinases in immunity. J Immunol. 1996;156:1–4. [PubMed] [Google Scholar]
2.Bailey CJ, Hembry RM, Alexander A, Irving MH, Grant ME, Shuttleworth CA. Distribution of the matrix metalloproteinases stomelysin, gelatinase A and B, and collagenase in Crohn's disease and normal intestine. J Clin Pathol. 1994;47:113–6. doi: 10.1136/jcp.47.2.113. [DOI] [PMC free article] [PubMed] [Google Scholar]
3.Saarialho-Kere UK, Vaalamo M, Puolakkainen P, Airola K, Parks WC, Karjalainen-Lindsberg M. Enhanced expression of matrilysin, collagenase, and stromelysin-1 in gastrointestinal ulcers. Am J Pathol. 1996;148:519–26. [PMC free article] [PubMed] [Google Scholar]
4.Vaalamo M, Karjalainen-Lindsberg M, Puolakkainen P, Kere J, Saarialho-Kere U. Distinct expression profiles of stromelysin-2 (MMP-10), collagenase-3 (MMP-13), macrophage metalloelastase (MMP-12), and tissue inhibitor of metalloproteinase-3 (TIMP-3) in intestinal ulcerations. Am J Pathol. 1998;152:1005–14. [PMC free article] [PubMed] [Google Scholar]
5.Pender SL, Tickle SP, Docherty AJP, Howie D, Wathen NC, MacDonald TT. A major role for matrix metalloproteinases in T cell injury in the gut. J Immunol. 1997;158:1582–90. [PubMed] [Google Scholar]
6.Tarlton JF, Whiting CV, Tunmore D, Bregenholt S, Reimann J, Claesson MH, Bland PW. The role of up-regulated serine proteases and matrix metalloproteinases in the pathogenesis of a murine model of colitis. Am J Pathol. 2000;157:1927–35. doi: 10.1016/S0002-9440(10)64831-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
7.Sartor RB. Pathogenesis and immune mechanisms of chronic inflammatory bowel diseases. Am J Gastroenterol. 1997;92:5S–11S. [PubMed] [Google Scholar]
8.McGee DW. Inflammation and mucosal cytokine production. In: Ogra P, Mestecky J, Lamm ME, Strober W, Bienenstock J, McGhee JR, editors. Mucosal Immunology. San Diego: Academic Press; 1999. pp. 559–73. [Google Scholar]
9.Waterhouse CCM, Stadnyk AW. Rapid expression of IL-1β by intestinal epithelial cells in vitro. Cell Immunol. 1999;193:1–8. doi: 10.1006/cimm.1999.1468. [DOI] [PubMed] [Google Scholar]
10.Stadnyk AW, Sisson GR, Waterhouse CCM. IL-1α is constitutively expressed in the rat intestinal epithelial cell line IEC-6. Exp Cell Res. 1995;220:298–303. doi: 10.1006/excr.1995.1319. [DOI] [PubMed] [Google Scholar]
11.Eckmann L, Reed SL, Smith JR, Kagnoff MF. Entamoeba histolytica trophozoites induce an inflammatory cytokine response by cultured human cells through the paracrine action of cytolytically released interleukin-1α. J Clin Invest. 1995;96:1269–79. doi: 10.1172/JCI118161. [DOI] [PMC free article] [PubMed] [Google Scholar]
12.Nakamura HK, Yoshimura K, McElvaney NG, Crystal RG. Neutrophil elastase in respiratory epithelial lining fluid of individuals with cystic fibrosis induces interleukin-8 gene expression in a human bronchial epithelial cell line. J Clin Invest. 1992;89:1478–86. doi: 10.1172/JCI115738. [DOI] [PMC free article] [PubMed] [Google Scholar]
13.Shibata Y, Nakamura H, Kato S, Tomoike H. Cellular detachment and deformation induce IL-8 gene expression in human bronchial epithelial cells. J Immunol. 1996;156:772–7. [PubMed] [Google Scholar]
14.Jung H, Eckman L, Yang S-K, Panja A, Fierer J, Morzyka-Wroblewska E, Kagnoff MF. A distinct array of proinflammatory cytokines is expressed in colonic epithelial cells in response to bacterial infection. J Clin Invest. 1995;95:55–65. doi: 10.1172/JCI117676. [DOI] [PMC free article] [PubMed] [Google Scholar]
15.Panja A, Siden E, Mayer L. Synthesis and regulation of accessory/proinflammatory cytokines by intestinal epithelial cells. Clin Exp Immunol. 1995;100:298–305. doi: 10.1111/j.1365-2249.1995.tb03668.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
16.McGee DW, Beagley KW, Aicher WK, McGee JR. Transforming growth factor-β and IL-1β act in synergy to enhance interleukin-6 secretion by the intestinal epithelial cell line, IEC-6. J Immunol. 1993;151:970–8. [PubMed] [Google Scholar]
17.Vitkus SJD, Hanifin SA, McGee DW. Factors affecting Caco-2 intestinal epithelial cell interleukin-6 secretion in vitro. Cell Dev Biol. 1998;34:660–4. doi: 10.1007/s11626-996-0017-7. [DOI] [PubMed] [Google Scholar]
18.Aderka D, Le JM, Vilcek J. IL-6 inhibits lipopolysaccharide-induced tumor necrosis factor production in cultured human monocytes, U937 cells, and in mice. J Immunol. 1989;143:3517–23. [PubMed] [Google Scholar]
19.Ulich TR, Guo K, Remick D, Del Castillo J, Yin S. Endotoxin-induced cytokine gene expression in vivo. III. IL-6 mRNA and serum protein expression and the in vivo hematologic effects of IL-6. J Immunol. 1991;146:2316–23. [PubMed] [Google Scholar]
20.Xing Z, Gauldie J, Cox G, Baumann H, Jordana M, Lei X-F, Achong MK. IL-6 is an antiinflammatory cytokine required for controlling local or systemic acute inflammatory responses. J Clin Invest. 1998;101:311–20. doi: 10.1172/JCI1368. [DOI] [PMC free article] [PubMed] [Google Scholar]
21.Hafeez W, Ciliberto G, Perlmutter DH. Constitutive and modulated expression of the human α1 antitrypsin gene. J Clin Invest. 1992;89:1214–22. doi: 10.1172/JCI115705. [DOI] [PMC free article] [PubMed] [Google Scholar]
22.Richards CD, Shoyab M, Brown TJ, Gauldie J. Selective regulation of metalloproteinase inhibitor (TIMP-1) by oncostatin M in fibroblasts in culture. J Immunol. 1993;150:5596–603. [PubMed] [Google Scholar]
23.McGee DW, Elson CO, McGhee JR. Enhancing effect of cholera toxin on interleukin-6 secretion by IEC-6 intestinal epithelial cells: mode of action and augmenting effect inflammatory cytokines. Infect Immun. 1993;61:4637–44. doi: 10.1128/iai.61.11.4637-4644.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
24.McGee DW, Beagley KW, Aicher WK, McGee JR. A synergistic relationship between TNF-α, IL-1β, and TGF-β1 on IL-6 secretion by the IEC-6 intestinal epithelial cell line. Immunology. 1995;86:6–11. [PMC free article] [PubMed] [Google Scholar]
25.Chomczynski P, Sacchi N. Single step method of RNA isolation by acid guanidium thiocyanate–phenol–chloroform extraction. Anal Biochem. 1987;162:156–9. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
26.Van Snick J, Cayphas S, Vink A, Uyttenhove C, Coulie PG, Rubria MR, Simpson RJ. Purification and NH2-terminal amino acid sequence of a T-cell derived lymphokine with growth factor activity for B-cell hybridomas. Proc Natl Acad Sci USA. 1986;83:9679–83. doi: 10.1073/pnas.83.24.9679. [DOI] [PMC free article] [PubMed] [Google Scholar]
27.Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxic assays. J Immunol Methods. 1983;65:55–63. doi: 10.1016/0022-1759(83)90303-4. [DOI] [PubMed] [Google Scholar]
28.Jobin C, Haskill S, Mayer L, Panja A, Sartor RB. Evidence for altered regulation of IκBα degradation in human colonic epithelial cells. J Immunol. 1997;158:226–34. [PubMed] [Google Scholar]
29.Arnold R, Humbert B, Werchau H, Gallati H, King W. Interleukin-8, interleukin-6, and soluble tumor necrosis factor receptor type I release from a human pulmonary epithelial cell line (A549) exposed to respiratory syncytial virus. Immunology. 1994;82:126–33. [PMC free article] [PubMed] [Google Scholar]
30.Larsson BM, Larsson K, Malmberg P, Palmberg L. Gram positive bacteria induce IL-6 and IL-8 production in human alveolar macrophages and epithelial cells. Inflammation. 1999;23:217–30. doi: 10.1023/a:1020269802315. [DOI] [PubMed] [Google Scholar]
31.Reinecker HC, Loy EY, Ringler DJ, Mehta A, Rombeau JL, MacDermott RP. Monocyte-chemoattractant protein 1 gene expression in intestinal epithelial cells and inflammatory bowel disease mucosa. Gastroenterology. 1995;108:40–50. doi: 10.1016/0016-5085(95)90006-3. [DOI] [PubMed] [Google Scholar]
32.Akira S, Taga T, Kishimoto T. Interleukin-6 in biology and medicine. Adv Immunol. 1993;54:1–78. doi: 10.1016/s0065-2776(08)60532-5. [DOI] [PubMed] [Google Scholar]
33.Martin T, Cardarelli PM, Parry GCN, Felts KA, Cobb RR. Cytokine induction of monocyte chemoattractant protein-1 gene expression in human endothelial cells depends on the cooperative action of NF-κB and AP-1. Eur J Immunol. 1997;27:1091–7. doi: 10.1002/eji.1830270508. [DOI] [PubMed] [Google Scholar]
34.Baldwin AS. The NF-kappa B proteins. New discoveries and insights. Annu Rev Immunol. 1996;14:649–83. doi: 10.1146/annurev.immunol.14.1.649. [DOI] [PubMed] [Google Scholar]
35.Boudreau F, Blais S, Asselin C. Regulation of CCAAT/enhancer binding protein isoforms by serum and glucocorticoids in the rat intestinal epithelial crypt cell line IEC-6. Exp Cell Res. 1996;222:1–9. doi: 10.1006/excr.1996.0001. [DOI] [PubMed] [Google Scholar]
36.Casini-Raggi V, Kam L, Chong YJT, Fiocchi C, Pizarro TT, Cominelli F. Mucosal imbalance of IL-1 and IL-1 receptor antagonist in inflammatory bowel disease: a novel mechanism of chronic intestinal inflammation. J Immunol. 1995;154:2434–40. [PubMed] [Google Scholar]
37.Lee L-F, Haskill JS, Mukaida N, Matsushima K, Ting JP-Y. Identification of tumor-specific paclitaxel (taxol)-responsive regulatory elements in the interleukin-8 promoter. Mol Cell Biol. 1997;17:5097–105. doi: 10.1128/mcb.17.9.5097. [DOI] [PMC free article] [PubMed] [Google Scholar]
38.Matsusaka T, Fujikawa K, Nishio Y, Mukaida N, Matsushima K, Kishimoto T, Akira S. Transcription factors NF-IL6 and NF-κB synergistically activate transcription of the inflammatory cytokines, interleukin 6 and interleukin 8. Proc Natl Acad Sci USA. 1993;90:10193–7. doi: 10.1073/pnas.90.21.10193. [DOI] [PMC free article] [PubMed] [Google Scholar]
39.Poli V. The role of C/EBP isoforms in the control of inflammatory and native immunity functions. J Biol Chem. 1998;45:29279–82. doi: 10.1074/jbc.273.45.29279. [DOI] [PubMed] [Google Scholar]

[b1] 1.Goetzl EJ, Banda MJ, Leppert D. Matrix metalloproteinases in immunity. J Immunol. 1996;156:1–4. [PubMed] [Google Scholar]

[b2] 2.Bailey CJ, Hembry RM, Alexander A, Irving MH, Grant ME, Shuttleworth CA. Distribution of the matrix metalloproteinases stomelysin, gelatinase A and B, and collagenase in Crohn's disease and normal intestine. J Clin Pathol. 1994;47:113–6. doi: 10.1136/jcp.47.2.113. [DOI] [PMC free article] [PubMed] [Google Scholar]

[b3] 3.Saarialho-Kere UK, Vaalamo M, Puolakkainen P, Airola K, Parks WC, Karjalainen-Lindsberg M. Enhanced expression of matrilysin, collagenase, and stromelysin-1 in gastrointestinal ulcers. Am J Pathol. 1996;148:519–26. [PMC free article] [PubMed] [Google Scholar]

[b4] 4.Vaalamo M, Karjalainen-Lindsberg M, Puolakkainen P, Kere J, Saarialho-Kere U. Distinct expression profiles of stromelysin-2 (MMP-10), collagenase-3 (MMP-13), macrophage metalloelastase (MMP-12), and tissue inhibitor of metalloproteinase-3 (TIMP-3) in intestinal ulcerations. Am J Pathol. 1998;152:1005–14. [PMC free article] [PubMed] [Google Scholar]

[b5] 5.Pender SL, Tickle SP, Docherty AJP, Howie D, Wathen NC, MacDonald TT. A major role for matrix metalloproteinases in T cell injury in the gut. J Immunol. 1997;158:1582–90. [PubMed] [Google Scholar]

[b6] 6.Tarlton JF, Whiting CV, Tunmore D, Bregenholt S, Reimann J, Claesson MH, Bland PW. The role of up-regulated serine proteases and matrix metalloproteinases in the pathogenesis of a murine model of colitis. Am J Pathol. 2000;157:1927–35. doi: 10.1016/S0002-9440(10)64831-6. [DOI] [PMC free article] [PubMed] [Google Scholar]

[b7] 7.Sartor RB. Pathogenesis and immune mechanisms of chronic inflammatory bowel diseases. Am J Gastroenterol. 1997;92:5S–11S. [PubMed] [Google Scholar]

[b8] 8.McGee DW. Inflammation and mucosal cytokine production. In: Ogra P, Mestecky J, Lamm ME, Strober W, Bienenstock J, McGhee JR, editors. Mucosal Immunology. San Diego: Academic Press; 1999. pp. 559–73. [Google Scholar]

[b9] 9.Waterhouse CCM, Stadnyk AW. Rapid expression of IL-1β by intestinal epithelial cells in vitro. Cell Immunol. 1999;193:1–8. doi: 10.1006/cimm.1999.1468. [DOI] [PubMed] [Google Scholar]

[b10] 10.Stadnyk AW, Sisson GR, Waterhouse CCM. IL-1α is constitutively expressed in the rat intestinal epithelial cell line IEC-6. Exp Cell Res. 1995;220:298–303. doi: 10.1006/excr.1995.1319. [DOI] [PubMed] [Google Scholar]

[b11] 11.Eckmann L, Reed SL, Smith JR, Kagnoff MF. Entamoeba histolytica trophozoites induce an inflammatory cytokine response by cultured human cells through the paracrine action of cytolytically released interleukin-1α. J Clin Invest. 1995;96:1269–79. doi: 10.1172/JCI118161. [DOI] [PMC free article] [PubMed] [Google Scholar]

[b12] 12.Nakamura HK, Yoshimura K, McElvaney NG, Crystal RG. Neutrophil elastase in respiratory epithelial lining fluid of individuals with cystic fibrosis induces interleukin-8 gene expression in a human bronchial epithelial cell line. J Clin Invest. 1992;89:1478–86. doi: 10.1172/JCI115738. [DOI] [PMC free article] [PubMed] [Google Scholar]

[b13] 13.Shibata Y, Nakamura H, Kato S, Tomoike H. Cellular detachment and deformation induce IL-8 gene expression in human bronchial epithelial cells. J Immunol. 1996;156:772–7. [PubMed] [Google Scholar]

[b14] 14.Jung H, Eckman L, Yang S-K, Panja A, Fierer J, Morzyka-Wroblewska E, Kagnoff MF. A distinct array of proinflammatory cytokines is expressed in colonic epithelial cells in response to bacterial infection. J Clin Invest. 1995;95:55–65. doi: 10.1172/JCI117676. [DOI] [PMC free article] [PubMed] [Google Scholar]

[b15] 15.Panja A, Siden E, Mayer L. Synthesis and regulation of accessory/proinflammatory cytokines by intestinal epithelial cells. Clin Exp Immunol. 1995;100:298–305. doi: 10.1111/j.1365-2249.1995.tb03668.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

[b16] 16.McGee DW, Beagley KW, Aicher WK, McGee JR. Transforming growth factor-β and IL-1β act in synergy to enhance interleukin-6 secretion by the intestinal epithelial cell line, IEC-6. J Immunol. 1993;151:970–8. [PubMed] [Google Scholar]

[b17] 17.Vitkus SJD, Hanifin SA, McGee DW. Factors affecting Caco-2 intestinal epithelial cell interleukin-6 secretion in vitro. Cell Dev Biol. 1998;34:660–4. doi: 10.1007/s11626-996-0017-7. [DOI] [PubMed] [Google Scholar]

[b18] 18.Aderka D, Le JM, Vilcek J. IL-6 inhibits lipopolysaccharide-induced tumor necrosis factor production in cultured human monocytes, U937 cells, and in mice. J Immunol. 1989;143:3517–23. [PubMed] [Google Scholar]

[b19] 19.Ulich TR, Guo K, Remick D, Del Castillo J, Yin S. Endotoxin-induced cytokine gene expression in vivo. III. IL-6 mRNA and serum protein expression and the in vivo hematologic effects of IL-6. J Immunol. 1991;146:2316–23. [PubMed] [Google Scholar]

[b20] 20.Xing Z, Gauldie J, Cox G, Baumann H, Jordana M, Lei X-F, Achong MK. IL-6 is an antiinflammatory cytokine required for controlling local or systemic acute inflammatory responses. J Clin Invest. 1998;101:311–20. doi: 10.1172/JCI1368. [DOI] [PMC free article] [PubMed] [Google Scholar]

[b21] 21.Hafeez W, Ciliberto G, Perlmutter DH. Constitutive and modulated expression of the human α1 antitrypsin gene. J Clin Invest. 1992;89:1214–22. doi: 10.1172/JCI115705. [DOI] [PMC free article] [PubMed] [Google Scholar]

[b22] 22.Richards CD, Shoyab M, Brown TJ, Gauldie J. Selective regulation of metalloproteinase inhibitor (TIMP-1) by oncostatin M in fibroblasts in culture. J Immunol. 1993;150:5596–603. [PubMed] [Google Scholar]

[b23] 23.McGee DW, Elson CO, McGhee JR. Enhancing effect of cholera toxin on interleukin-6 secretion by IEC-6 intestinal epithelial cells: mode of action and augmenting effect inflammatory cytokines. Infect Immun. 1993;61:4637–44. doi: 10.1128/iai.61.11.4637-4644.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]

[b24] 24.McGee DW, Beagley KW, Aicher WK, McGee JR. A synergistic relationship between TNF-α, IL-1β, and TGF-β1 on IL-6 secretion by the IEC-6 intestinal epithelial cell line. Immunology. 1995;86:6–11. [PMC free article] [PubMed] [Google Scholar]

[b25] 25.Chomczynski P, Sacchi N. Single step method of RNA isolation by acid guanidium thiocyanate–phenol–chloroform extraction. Anal Biochem. 1987;162:156–9. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]

[b26] 26.Van Snick J, Cayphas S, Vink A, Uyttenhove C, Coulie PG, Rubria MR, Simpson RJ. Purification and NH2-terminal amino acid sequence of a T-cell derived lymphokine with growth factor activity for B-cell hybridomas. Proc Natl Acad Sci USA. 1986;83:9679–83. doi: 10.1073/pnas.83.24.9679. [DOI] [PMC free article] [PubMed] [Google Scholar]

[b27] 27.Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxic assays. J Immunol Methods. 1983;65:55–63. doi: 10.1016/0022-1759(83)90303-4. [DOI] [PubMed] [Google Scholar]

[b28] 28.Jobin C, Haskill S, Mayer L, Panja A, Sartor RB. Evidence for altered regulation of IκBα degradation in human colonic epithelial cells. J Immunol. 1997;158:226–34. [PubMed] [Google Scholar]

[b29] 29.Arnold R, Humbert B, Werchau H, Gallati H, King W. Interleukin-8, interleukin-6, and soluble tumor necrosis factor receptor type I release from a human pulmonary epithelial cell line (A549) exposed to respiratory syncytial virus. Immunology. 1994;82:126–33. [PMC free article] [PubMed] [Google Scholar]

[b30] 30.Larsson BM, Larsson K, Malmberg P, Palmberg L. Gram positive bacteria induce IL-6 and IL-8 production in human alveolar macrophages and epithelial cells. Inflammation. 1999;23:217–30. doi: 10.1023/a:1020269802315. [DOI] [PubMed] [Google Scholar]

[b31] 31.Reinecker HC, Loy EY, Ringler DJ, Mehta A, Rombeau JL, MacDermott RP. Monocyte-chemoattractant protein 1 gene expression in intestinal epithelial cells and inflammatory bowel disease mucosa. Gastroenterology. 1995;108:40–50. doi: 10.1016/0016-5085(95)90006-3. [DOI] [PubMed] [Google Scholar]

[b32] 32.Akira S, Taga T, Kishimoto T. Interleukin-6 in biology and medicine. Adv Immunol. 1993;54:1–78. doi: 10.1016/s0065-2776(08)60532-5. [DOI] [PubMed] [Google Scholar]

[b33] 33.Martin T, Cardarelli PM, Parry GCN, Felts KA, Cobb RR. Cytokine induction of monocyte chemoattractant protein-1 gene expression in human endothelial cells depends on the cooperative action of NF-κB and AP-1. Eur J Immunol. 1997;27:1091–7. doi: 10.1002/eji.1830270508. [DOI] [PubMed] [Google Scholar]

[b34] 34.Baldwin AS. The NF-kappa B proteins. New discoveries and insights. Annu Rev Immunol. 1996;14:649–83. doi: 10.1146/annurev.immunol.14.1.649. [DOI] [PubMed] [Google Scholar]

[b35] 35.Boudreau F, Blais S, Asselin C. Regulation of CCAAT/enhancer binding protein isoforms by serum and glucocorticoids in the rat intestinal epithelial crypt cell line IEC-6. Exp Cell Res. 1996;222:1–9. doi: 10.1006/excr.1996.0001. [DOI] [PubMed] [Google Scholar]

[b36] 36.Casini-Raggi V, Kam L, Chong YJT, Fiocchi C, Pizarro TT, Cominelli F. Mucosal imbalance of IL-1 and IL-1 receptor antagonist in inflammatory bowel disease: a novel mechanism of chronic intestinal inflammation. J Immunol. 1995;154:2434–40. [PubMed] [Google Scholar]

[b37] 37.Lee L-F, Haskill JS, Mukaida N, Matsushima K, Ting JP-Y. Identification of tumor-specific paclitaxel (taxol)-responsive regulatory elements in the interleukin-8 promoter. Mol Cell Biol. 1997;17:5097–105. doi: 10.1128/mcb.17.9.5097. [DOI] [PMC free article] [PubMed] [Google Scholar]

[b38] 38.Matsusaka T, Fujikawa K, Nishio Y, Mukaida N, Matsushima K, Kishimoto T, Akira S. Transcription factors NF-IL6 and NF-κB synergistically activate transcription of the inflammatory cytokines, interleukin 6 and interleukin 8. Proc Natl Acad Sci USA. 1993;90:10193–7. doi: 10.1073/pnas.90.21.10193. [DOI] [PMC free article] [PubMed] [Google Scholar]

[b39] 39.Poli V. The role of C/EBP isoforms in the control of inflammatory and native immunity functions. J Biol Chem. 1998;45:29279–82. doi: 10.1074/jbc.273.45.29279. [DOI] [PubMed] [Google Scholar]

PERMALINK

Epithelial cells respond to proteolytic and non-proteolytic detachment by enhancing interleukin-6 responses

Tabbi L Miller

Dennis W McGee

Abstract

Introduction