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Keywords:

  • local anaesthetics;
  • cytokines;
  • chemokines;
  • IL-1 receptor antagonist;
  • intestinal;
  • epithelial cells

SUMMARY

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES

Lidocaine and related local anaesthetics have been shown to be effective in the treatment of ulcerative colitis (UC). However, the mechanisms underlying their therapeutic effect are poorly defined. Intestinal epithelial cells play an important role in the mucosal inflammatory response that leads to tissue damage in UC via the secretion of pro-inflammatory cytokines and chemokines. The aim of this study was to evaluate the direct immunoregulatory effect of lidocaine on pro-inflammatory cytokine and chemokine secretion from intestinal epithelial cells. HT-29 and Caco-2 cell lines were used as a model system and treated with lidocaine and related drugs. The expression of IL-8, IL-1β and the IL-1 receptor antagonist (RA) were assessed by ELISA and quantification of mRNA. In further experiments, the effect of lidocaine on the secretion of IL-8 from freshly isolated epithelial cells stimulated with TNFα was tested. Lidocaine, in therapeutic concentrations, inhibited the spontaneous and TNFα-stimulated secretion of IL-8 and IL-1β from HT-29 and Caco-2 cell lines in a dose-dependent manner. Similarly, suppression of IL-8 secretion was noted in the freshly isolated epithelial cells. Other local anaesthetics, bupivacaine and amethocaine, had comparable effects. Lidocaine stimulated the secretion of the anti-inflammatory molecule IL-1 RA. Both the inhibitory and the stimulatory effects of lidocaine involved regulation of transcription. The results imply that the therapeutic effect of lidocaine may be mediated, at least in part, by its direct effects on epithelial cells to inhibit the secretion of proinflammatory molecules on one hand while triggering the secretion of anti-inflammatory mediators on the other.


INTRODUCTION

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES

Ulcerative colitis (UC) is characterized by chronic inflammation that involves the colon to variable extents. While the aetiology of UC is unknown, it is suggested that tissue damage in UC results from an uncontrolled inflammatory process within the intestinal mucosa. Dysregulation of the mucosal immune system leads to elevated levels of pro-inflammatory mediators that may perpetuate the immune response even further. In support of such a scenario, several studies have demonstrated elevated concentrations of chemokines such as IL-8 [1–4] and pro-inflammatory cytokines such as TNFα in the intestinal tissue of UC patients [5,6]. Furthermore, it has been shown that the ratio between the naturally occurring anti-inflammatory molecule IL-1 receptor antagonist (RA), which is constitutively expressed by intestinal epithelial cells, and the pro-inflammatory cytokine IL-1 is reduced in active inflammatory bowel diseases (IBD) [7,8]. These observations suggest that excess of pro-inflammatory mediators may play a role in the tissue damage associated with UC.

Intestinal epithelial cells play an important role in the mucosal immune system. These cells form the first line of defense between the microbial-rich lumen and the sterile internal milieu. One of the important mechanisms by which epithelial cells protect the mucosa is their ability to initiate a transient beneficial inflammatory response [9]. Intestinal epithelial cells secrete a wide array of pro-inflammatory cytokines and chemokines that can induce intestinal inflammation, which in turn recruits additional inflammatory cells into the inflamed tissue. In support of their physiological role, epithelial cells were shown to secrete cytokines following their stimulation by pro-inflammatory mediators such as TNFα and bacterial invasion [10,11], as well as after exposure to other pathogens like Entamoeba histolytica[12] and the parasite Trichinella spiralis[13]. Furthermore, following stimulation by TNFα, IL-1α or Salmonella, the secretion of different chemokines by epithelial cells was shown to vary between one to six hours, suggesting a dynamic role in the different phases of the ongoing mucosal inflammation [9]. Despite its protective effect and potency, the epithelial pro-inflammatory response should be tightly regulated in order to avoid the detrimental effects of sustained inflammation.

Since the aetiology of UC is unknown, current approaches to the treatment of this disease are based on nonspecific suppression of the immune system, assuming that uncontrolled activity may be the direct cause for tissue damage. UC patients are treated with the anti-inflammatory agents 5-aminosalicylic acid and glucocorticoids as well as other immunosuppressive agents. However, not all patients respond to these regimens and there is a continuous search for new, potent and less toxic approaches to treat UC. Recently, a number of studies have demonstrated a good clinical response of UC patients to a treatment based on enemas of local anaesthetic drugs such as lidocaine and ropivacaine [14–16] Although favourable clinical outcomes were observed, the mechanisms by which these agents exert their beneficial clinical effect and whether it involves the regulation of the immune system are poorly defined. Previous studies have suggested several mechanisms of action for these agents, including inhibition of the adhesion of granulocytes to the inflammatory sites [17–19], the reduction of lysosomal activity and production of superoxide [20,21] and the suppression of metabolic activation and secretion of LTB4 and IL-1 from granulocytes [22,23].

The intestinal epithelial cell population is extensively exposed to medications administered by enemas. Despite the potential interaction between lidocaine and intestinal epithelial cells, no information is available regarding the effect of local anaesthetic drugs on these cells. Therefore, the aim of this study was to determine whether lidocaine and related local anaesthetics exert a direct immunomodulatory effect on intestinal epithelial cells.

MATERIALS AND METHODS

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES

Reagents

TNFα was obtained from Boehringer Mannheim (Indianapolis, USA). Lidocaine, bupivacaine and amethocaine were obtained from Sigma (St Louis, MO, USA).

Cell lines and culture

HT-29 (ATCC HTB38) and Caco-2 (ATCC HTB27) cells were obtained from the American Type Tissue Culture Collection (Rockville, MD, USA). The cells were maintained in culture (at 37°C, 5% CO2 incubation) in DMEM media (Bet-Haemek, Israel) supplemented with 10% fetal calf serum (Bet-Haemek), 1% glutamine and 1% penicillin and streptomycin (Bet Haemek). Caco-2 cells were supplemented with Neut-mix F12 (Life Technologies, Wien, Austria). Cells were routinely tested for mycoplasma infection by the MYCOTRIM TC kit (Irvine Scientific, Santa Ana, CA, USA) according to the instructions supplied by the manufacturer.

Isolation of fresh intestinal epithelial cells

Epithelial cells were isolated from resected bowel specimens by dispase treatment. Briefly, after vigorous washing in RPMI-1640, the mucosal layer is dissected from the underlying submucosa. The dissected mucosal tissue is washed several times in RPMI supplemented with 1% penicillin-streptomycin and 2 mM glutamine, and minced into small pieces followed by treatment with the mucolytic agent DTT (1 mM) (Sigma) in RPMI 1640 for 5 min at room temperature. Tissue pieces are washed in RPMI and subjected to dispase (1 mg/ml; Boehringer Mannheim) treatment for 30 min at 37°C. During this 30 min incubation, tissue pieces are agitated every 5 min by vortexing, resulting in the liberation of epithelial cells and intraepithelial lymphocytes into the solution. This cell suspension is collected and immediately diluted 10-fold in RPMI to minimize any effect of dispase. Cells are spun down and washed twice in RPMI. Epithelial cells are further separated from the contaminating IELs and RBCs by percoll density gradient centrifugation. The cells on top of the 30% layer typically contain 95% pure epithelial cells (< 1% CD45+ cells or CD14+ cells) with a viability of 95–100%.

This part of the study was approved by the local Helsinki committee.

Spontaneous and TNFα-induced cytokine secretion

HT-29 or Caco-2 test cells were grown as confluent monolayers in 24-well tissue culture plates. HT-29 cells were used after the cells reached confluence. Caco-2 cells were maintained in culture for 4–6 days until the cells differentiated, as judged by staining sample cultures for the appearance of small intestinal morphology of the cells. Following culture, the medium was replaced by fresh medium, and supplemented with either lidocaine, bupivacaine or amethocaine. In experiments determining the spontaneous secretion of IL-8, IL-1β and IL-1 RA, the culture was continued for 24 h, after which the supernatants were collected and analysed. In other experiments, the effect of the drugs was tested in cells stimulated by TNFα. In these experiments, the various compounds were added and incubated with the cells for 30 min. Thereafter, TNFα was added at a concentration of 200 ng/ml and the cells incubated for another 24 h. The cells were then harvested, the supernatants collected and the concentrations of IL-1β, IL-8 or IL-1 RA were determined by ELISA.

To test the effect of lidocaine on freshly isolated epithelial cells, 106 cells were plated in 24-well plates in RPMI containing 10% FCS (Sigma), 1% penicillin-streptomycin and 2 mM glutamine. Thereafter, lidocaine was added for 24 h and the supernatants collected and assayed for the concentration of IL-8 by ELISA. To test the effect of lidocaine on TNFα-stimulated cells, epithelial cells were incubated with lidocaine for 30 min at 37°C, after which TNFα (100 ng/ml) was added for 24 h. Subsequently, the supernatants were harvested and assayed for IL-8 secretion.

For RNA extraction, cells were grown in 10-cm tissue culture dishes until they reached confluence. The medium was then replaced by fresh medium and supplemented with lidocaine and TNFα as previously described. After 2 h of incubation, the cells were harvested and their RNA was extracted.

Determination of IL-8, IL-1β and IL-1 RA secretion and cell viability

IL-8 was measured by ELISA as previously described [11]. Briefly, 96-well plates were coated with polyclonal goat antihuman IL-8 antibody (R & D Systems, Minneapolis, MN) serving as capture antibodies. After incubating with the tested supernatants and washing, polyclonal rabbit antihuman IL-8 detecting antibodies (Endogen, Boston, MA) were added. Alkaline phosphatase-labelled mouse antirabbit IgG (Sigma) was used as a secondary antibody. Quantification of bound antibodies was carried out using p-nitrophenylphosphate (Sigma). IL-1β was measured by ELISA using a commercially available kit (Genzyme, Cambridge, MA) in accordance to the manufacturer’s protocol. The concentrations of IL-1 RA were determined using a commercially available ELISA kit (Biosource Euorope S.A., Nivelles, Belgium). Three replicate samples were included in each experiment. Cell viability was determined using the MTT method [24].

RNA extraction, RNAse protection assay and screening with cDNA expression arrays

RNA extraction was performed using the Tri Reagent kit (MRC Cincinnati, OH) and mRNA levels were measured by the RiboQuant multiprobe RNA protection assay (RPA) (Pharminigen, San Diego, CA), as per the manufacturer’s protocol.

Briefly, antisense RNA probes were transcribed using the cDNA template set hCK-5. For transcription, 1 μl of the template was incubated for 1 h at 37°C in a mixture containing 1 μl RNasin, 1 μl GACU pool, 2 μl DTT, 4 μl 5X transcription buffer, 10 μl α-32P]UTP, and 1 μl T7 RNA polymerase. The reaction was terminated by adding DNAse. Labelled RNA probes were extracted using phenol/chloroform/isoamyl alcohol, and were precipitated using ethanol. The level of α-32P]UTP incorporated was determined using a scintillation counter.

For hybridization, 20 μg of RNA were precipitated by ethanol and the pellet dried using a vacuum evaporator centrifuge. The RNA samples were then resuspended in 8 μl of hybridization buffer (80% formamide, 1 mM EDTA, 400 mM NaCl, 40 mM Prpes preeazine-N,N′-Bis-Ethanesulphonic acid]) at 56°C, mixed with 2 μl of probe prepared as previously described, heated to 90°C, and then incubated at 56°C for 12 h.

For RNase digestion, 6 μl of a mixture containing RNase A and RNase T1 were added and reacted for 45 min at 30°C. After digestion, the samples were mixed with Proteinase K and an appropriate buffer (Pharmingen) for 15 min at 37°C, after which they were extracted by phenol/chloroform/isoamyl alcohol and precipitated with ethanol. The samples were then air-dried, resuspended in loading buffer, and size-separated using polyacrylamide gel electrophoresis. Appropriate bands representing IL-8, IL-1β and GAPDH RNA were measured using a phospho-imager.

The expression of IL-1 RA mRNA was tested using the Atlas cDNA expression array (Clontech, Palo Alto, USA), according to the manufacturer’s instructions. The results were analysed with the Atlas Image 1·01a software and normalized by global normalization (using the sum method).

Statistical analysis

All statistical analysis was performed using an unpaired, two tailed t-test. P-values greater than 0·05 were considered not significant. Error bars represent the variation between three different experiments.

RESULTS

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES

Lidocaine inhibits the secretion of IL-8 and IL-1β from HT-29 and Caco-2 cells

To test whether lidocaine exerts a direct suppressive immunomodulatory effect on intestinal epithelial cell lines, the colonic epithelial cell line HT-29 was incubated with increasing concentrations of lidocaine and the levels of IL-8 in the supernatant were assessed. As shown in Fig. 1a, lidocaine inhibited the spontaneous secretion of IL-8 from the epithelial cells in a dose-dependent manner. The suppressive effect was apparent at a concentration of 50 μM (from baseline 7·9 ± 0·351 ng/ml to 5·83 ± 0·071 ng/ml). Suppression of secretion was maximal at a lidocaine concentration of 1 mM (0·738 ± 0·038 ng/ml). The effective concentrations were within the reported therapeutic range of lidocaine [25].

image

Figure 1. The effect of lidocaine on spontaneous secretion of IL-8 and IL-1β from intestinal epithelial cells. Lidocaine was added for 24 h to fresh medium of cultured HT-29 (a, b) and Caco-2 cells (c, d) at the indicated concentrations. Thereafter, the supernatants were collected and assayed for the concentrations of IL-8 (a, c) and IL-1β (b, d) by ELISA.The results are presented as the mean and standard deviation of three different experiments. The effect was significant at a concentration of 50 μM (for a, c, d, P < 0·009; for b, P < 0·04).

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We next determined whether the inhibitory effect of lidocaine was specific to IL-8 or whether it held true for the pro-inflammatory cytokine IL-1β. Fig. 1b demonstrates the dose-dependent manner in which lidocaine inhibited the secretion of IL-1β. Baseline levels of IL-1β (0·78 ± 0·155 ng/ml) were decreased by a 50 μM concentration of lidocaine (0·33 ± 0·07 ng/ml) and a maximal effect was noted at a concentration of 1 mM lidocaine (0·143 ± 0·035 ng/ml).

To test whether the inhibitory effect of lidocaine on the secretion of IL-8 and IL-1β was limited to HT-29 cells, the drug was applied to Caco-2 cells. As illustrated in Fig. 1c and Fig. 1d, lidocaine inhibited the secretion of IL-8 (baseline 8·81 ± 0·44 ng/ml to 2·34 ± 0·1 ng/ml) and IL-1β (baseline 0·527 ± 0·026 ng/ml to 0·277 ± 0·013 ng/ml) from the Caco-2 cells in a dose-dependent manner, resembling its effect on the HT-29 cells. The viability of the cells was tested by MTT assays in order to rule out the possibility that lidocaine was toxic to the cells and therefore causing the reduced secretion of IL-8 and IL-1β. Lidocaine had no effect on cell viability (data not shown), strongly suggesting that it directly inhibited the secretion of IL-8 and IL-1β from viable intestinal epithelial cells.

Lidocaine inhibits the secretion of IL-8 and IL-1β from HT-29 cells stimulated by TNFα

The cytokine TNFα plays a major role in the intestinal inflammatory immune response [10,11] and elevated levels of TNFα have been found within the intestinal mucosa of UC patients [5,6] Previous studies have also shown that stimulation of epithelial cells by TNFα induces the secretion of an array of pro-inflammatory mediators, which may perpetuate the inflammation within the intestinal mucosa [9]. Based on these observations and the fact that lidocaine inhibited the spontaneous secretion of IL-8 and IL-1β from HT-29 and Caco-2 cells, we wished to determine whether lidocaine would also inhibit the secretion of pro-inflammatory mediators induced by TNFα. Stimulation of HT-29 cells by TNFα increased the baseline secretion of IL-8 from 7·9 ± 0·35 ng/ml to 28·87 ± 2·5 ng/ml and the IL-1β baseline secretion from 0·78 ± 0·15 ng/ml to 1·68 ± 0·3 ng/ml. As shown in Fig. 2a, lidocaine inhibited the TNFα-induced secretion of IL-8 from 28·87 ± 2·5 ng/ml to 3·4 ± 0·735 ng/ml in a dose-dependent manner. Similarly, lidocaine inhibited the TNFα-induced secretion of IL-1β from a baseline level of 1·68 ± 0·3 ng/ml to 0·163 ± 0·009 ng/ml (Fig. 2b). Thus, lidocaine inhibits both the spontaneous and the TNFα-stimulated secretion of pro-inflammatory mediators from intestinal epithelial cells.

image

Figure 2. The effect of lidocaine on the secretion of IL-8 and IL-1β from epithelial cells stimulated by TNFα. HT-29 cells were grown to confluence. Following culture, lidocaine was added to fresh medium at the indicated concentrations for 30 min, after which TNFα (200 ng/ml) was added and the culture was continued for 24 h. Thereafter, the concentrations of IL-8 (a), and IL-1β (b) were determined by ELISA. The results are presented as the mean and standard deviation of three different experiments. The effect was significant at a concentration of IL-8 at 100 μM (P < 0·002), IL1β at 50 mM (P < 0·03).

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Lidocaine inhibits the secretion of IL-8 from nonstimulated and TNFα-stimulated freshly isolated epithelial cells

To further support the applicability of our results to nontransformed cells, the effect of lidocaine on the secretion of IL-8 was assessed using freshly isolated colonic epithelial cells. Lidocaine inhibited the secretion of IL-8 from these cells from 5158 pg/ml at baseline to 3854 pg/ml using 0·5 mM lidocaine. Stimulation of the epithelial cells by TNFα increased the baseline secretion of IL-8 from 5158 pg/ml to 6975 pg/ml. As shown in Fig. 3, lidocaine suppressed the secretion of IL-8 from the TNFα-stimulated cells in a dose-dependent manner similar to its effect on the nonstimulated cells and the HT29 and Caco-2 cell lines. Since TNFα is thought to play an important role in the inflammatory response of the mucosa, the results suggest that lidocaine may exert a similar effect in vivo in diseases in which the intestinal mucosal is inflamed.

image

Figure 3. The effect of lidocaine on the secretion of IL-8 from TNFα- stimulated freshly isolated intestinal epithelial cells. Freshly isolated epithelial cells were incubated for 30 min with lidocaine, after which TNFα was added (100 ng/ml) and the culture was continued for 24 h. Following culture the supernatants were collected and the concentration of IL-8 was determined by ELISA.

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The inhibitory effect of lidocaine is common to other local anaesthetic drugs

Lidocaine is a member of a class of local anaesthetic agents that are mostly amine compounds [27]. Although these drugs vary widely in their structure, they all penetrate the cells and exert similar therapeutic effects [28]. We next studied whether the other drugs in this class affect the epithelial cells in a manner similar to lidocaine. To test this possibility, intestinal epithelial cells were stimulated by TNFα and the effect of bupivacaine and amethocaine on the secretion of IL-8 and IL-1β was evaluated. As shown in Fig. 4, both bupivacaine and amethocaine exerted a marked inhibitory effect on the secretion of IL-8 and IL-1β from the cells (bupivacaine: IL-8 23·22 ± 2·05–3·77 ± 1·06 ng/ml, IL1β 1·44 ± 0·03–0·42 ± 0·11 ng/ml; after amethocaine treatment: IL-8 5·8 ± 0·9 ng/ml, IL-1β 0·425 ± 0·06 ng/ml). This effect was dose-dependent and resembled the effect of lidocaine. These results indicate that the other local anaesthetic drugs exhibit a comparable immunosuppressive effect to lidocaine on intestinal epithelial cells and therefore a similar mechanism of action may underlie their common beneficial therapeutic effects in UC patients.

image

Figure 4. The effect of bupivacaine and amethocaine on the secretion of IL-8 and IL-1β from intestinal epithelial cells. HT-29 cells were grown to confluence. Following culture, Lidocaine, bupivacaine or amethocaine were added to fresh medium for 30 min, after which TNFα (200 ng/ml) was added and the culture was continued for 24 h. Thereafter, the concentrations of IL-8 (a) and IL-1β (b) in the supernatant were determined by ELISA. The effect of the drugs at a concentration of 0·5 mM is presented. The results are the mean and standard deviation of three different experiments (all the results were significant at P < 0·01 relative to TNFα-stimulated secretion).

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Lidocaine inhibits the expression of IL-8 mRNA

The inhibition of IL-8 and IL-1β secretion from the intestinal epithelial cells could be envisioned to occur at a number of cellular levels. To test whether lidocaine inhibited the expression of IL-8 mRNA, HT-29 cells were preincubated with lidocaine and thereafter stimulated by TNFα. Two hours later, the expression of IL-8 mRNA in the cells was assessed by an RNAse protection assay. Of note, the determination of IL-8 mRNA levels was performed at this specific time point since previous studies have shown a maximal expression of IL-8-mRNA 2 h after stimulation of the cells with TNFα[11]. As shown in Fig. 5, treatment of the cells with lidocaine markedly reduced the level of expression of IL-8 mRNA. Thus, the effect of lidocaine appeared to be mediated, at least in part, by regulation of transcription.

imageimage

Figure 5. Expression of IL-8 mRNA following treatment of HT-29 cells with lidocaine. HT-29 cells were cultured until confluence was reached. Cells were incubated with lidocaine for 30 min, after which TNFα was added to the culture for two additional hours of incubation. The cells were then harvested and total RNA was extracted. IL-8 and GAPDH mRNA concentrations were measured using an RNA protection assay. (a) The autoradiography of the RNA protection assay. (b) A graphic representation of the ratios between IL-8/GAPDH mRNA. One experiment of two is shown.

Lidocaine induces the secretion of IL-1 RA

Previous studies have shown that the ratio of IL-1 to its naturally occurring antagonist, IL1 RA, is altered in inflammatory bowel diseases [7]. IL-1 RA is constitutively expressed in intestinal epithelial cells [7,29] Since lidocaine was shown to exert a beneficial therapeutic effect in UC and was found herein to suppress the secretion of IL-1β, we next tested whether it would also affect the expression of IL-1 RA in TNFα-stimulated cells. Initially, the expression of IL-1 RA mRNA was assessed by a cDNA expression array, in which the IL-1 RA gene was represented. Figure 6a shows that treatment with lidocaine induced a significant increase in the levels of the IL-1 RA message (2·3-fold). To test whether lidocaine also increased the secretion of the IL-1 RA protein, its concentration in the supernatants of the epithelial cell cultures was measured by ELISA. As can be seen in Fig. 6b,c, exposure of both cell lines to lidocaine induced a significant rise in the concentration of IL-1 RA. In the HT-29 cells the concentration rose from 170 ± 13·45 ng/ml to 370 ± 38·22 ng/ml and in the Caco-2 cells the concentrations of IL-1 RA were elevated from 194 ± 0·5 to 300 ± 7·73 ng/ml. Importantly, the fact that lidocaine induced protein synthesis in the cells, further indicates that its effect to inhibit the secretion of IL-1β and IL-8 was not mediated by cell death.

image

Figure 6. The effect of lidocaine on the secretion and expression of IL-1 RA mRNA in intestinal epithelial cells. (a) Autoradiography of cDNA expression array. HT-29 cells were grown to confluence. Following culture, lidocaine was added to fresh medium for 30 min, after which TNFα was added for 2 h. Thereafter, the cells were harvested and RNA was extracted. The comparison in the expression of the mRNA of IL-1 RA between cells treated or untreated with lidocaine was assessed using a cDNA expression array. HT-29 (b) and Caco-2 (c) cells were grown to confluence. Following culture, lidocaine (10 mM) was added and the incubation was continued for 24 h. Subsequently, the supernatants were collected and the concentrations of IL-1 RA were determined by ELISA. The results are the mean and standard deviation of three different experiments. The differences were significant in both cell lines (HT-29, P < 0·03; Caco-2, P < 0·005).

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DISCUSSION

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES

Lidocaine and related drugs, which are used primarily as local anaesthetics and antiarrhythmic agents, were recently shown to have a beneficial therapeutic effect in UC [14–16]. Nevertheless, the mechanisms by which lidocaine improves the clinical symptoms in UC are unknown. Herein, we report that lidocaine directly inhibits the secretion of the cytokine IL-1β and the chemokine IL-8 from cultured intestinal epithelial cell lines and from freshly isolated colonic epithelial cells that were stimulated by TNFα. This suggests that the beneficial effect of lidocaine in UC may be mediated at least in part by its ability to down-regulate the secretion of epithelial-derived pro-inflammatory mediators. The direct effect of lidocaine on epithelial cells is of particular importance since it is administered in the form of enemas in the treatment of UC, thus exposing these cells to the highest concentrations of the medication. These findings are in agreement with other studies that have shown that lidocaine exerts immunoregulatory effects on a variety of cell types. For example, lidocaine-related local anaesthetics were shown to inhibit lymphocyte maturation and proliferation, the migration of macrophages into tissues [25], the expression of CD11b/CD18 by polymorphonuclear cells [19], the adhesion of leucocytes to injured venules [17–19] and the LPS-stimulated secretion of LTB4 and IL-1α from peripheral blood mononuclear cells [23]. Furthermore, the anti-inflammatory effects of lidocaine were shown in in vivo studies as well. Taniguchi et al. have shown that lidocaine treatment of rabbits injected with endotoxin reduced the serum concentrations of IL-8 and IL-6 [29]. Importantly, the anti-inflammatory effect of lidocaine was assessed in a number of animal-models of intestinal inflammation [14,15]. In these studies, lidocaine down-regulated mucosal inflammation and reduced the permeability of the intestinal mucosa. The authors suggested a correlation between these effects, and that they both result from the lidocaine-induced reduction in the release of substance P from nerve terminals [14,15]. No direct correlation between intestinal permeability and the levels of pro-inflammatory cytokines was suggested. However, previous studies have shown that inflammatory mediators may directly affect mucosal permeability. In one study, interferon γ was shown to increase the permeability of T84 monolayers [30]. In an additional study, exposure of Caco-2 cells to interferon γ, in combination with IL-1β and TNFα, increased the permeability of the monolayers [31]. Furthermore, using an in vivo model of intestinal ischemia and reperfusion, Sun et al. showed a correlation between the concentrations of IL-1β and dysfunction of the intestinal barrier [32]. Based on the above, we suggest a dual mechanism for the effect of lidocaine on the permeability of the intestinal mucosa; suppression of the release of substance P and suppression of cytokine and chemokine secretion from the epithelial cells.

In our study, we used two intestinal adenocarcinoma cell lines as model systems. These cell lines were used under similar conditions in previous studies and were shown to resemble freshly isolated epithelial cells in the array of cytokines they secrete in response to stimulation by pro-inflammatory mediators and bacterial invasion [9,10]. In addition to their efficacy in the previous human studies, our experiments showed that both undifferentiated HT-29 cells and differentiated Caco-2 cells responded to lidocaine in a similar manner, supporting the physiologic relevance of the findings. Moreover, lidocaine inhibited the secretion of IL-8 from nonstimulated and TNFα-stimulated freshly isolated epithelial cells. Taken together, although we did not use fresh epithelial cells from UC patients, our observations suggest that the direct effect of lidocaine on intestinal epithelial cells may account for some of its clinical effects in vivo.

Our results have shown that lidocaine not only inhibited the secretion of pro-inflammatory mediators, but also induced the secretion of the naturally occurring anti-inflammatory molecule IL-1 RA from the epithelial cell lines (for review see reference [33]). This observation is of particular importance since previous studies have shown that the mucosal ratio of IL-1/IL-1 RA is altered in inflammatory bowel diseases [7,8]. Furthermore, IL-1 RA was recently shown to be an important mediator in inflammatory conditions. Two different groups reported that mice deficient in IL-1 RA developed spontaneous arthropathy [34] as well as arterial inflammation [35]. Moreover, in two recent studies, IL-1 RA induced a significant clinical response when administered to patients suffering from rheumatoid arthritis [36]. Thus, the beneficial effect of lidocaine may be the result of its dual activity to reduce IL-1β secretion on the one hand and to induce the secretion of IL-1 RA on the other.

Previous studies suggested that IL-1 RA is expressed constitutively by epithelial cells [7] but mainly in its unglycosylated, intracellular form. This form is synthesized without a leader sequence that enables its secretion from the cell [37]. In contrast, others have shown that IL-1 RA is secreted by freshly isolated intestinal epithelial cells [38]. Such differences could result from the use of different experimental systems, i.e. different cell lines may behave in different manners due to varying culture conditions and experimental conditions. Our results are in agreement with the study by Daig et al.[38] and suggest that IL-1 RA is indeed secreted from the intestinal cells and importantly, that its secretion can be pharmacologically induced.

The molecular mechanism by which lidocaine regulates the secretion of IL-1 RA is not clear. Previous studies have shown that the transcription of the secreted or unsecreted form of IL-1 RA is regulated by the alternative use of one of two different first exons [39]. Herein, we have demonstrated that the secretion of IL-1 RA is regulated at the transcriptional level. Therefore, our results suggest that the exposure of epithelial cells to lidocaine triggers the transcription of IL-1 RA RNA which contained the appropriate exon allowing for the secretion of the IL-1 RA molecule. Our study does not address the mechanism by which the effect of lidocaine on the secretion of IL-1 RA was mediated. Others have shown that the synthesis of IL-1 RA can be up-regulated by other cytokines such as interferon α[40], interferon β[41], IL-4 and IL-13 [26] or TGFβ[42]. Since we have shown that lidocaine also regulates the expression of other pro-inflammatory mediators, its effect on the secretion of IL-1 RA may be mediated either directly, or indirectly via its effect on the secretion of other mediators from the cells.

Taken together, our study shows for the first time that local anaesthetics like lidocaine can directly suppress cytokine secretion from epithelial cells and augment the secretion of anti-inflammatory mediators. Understanding the molecular basis of their activity may allow to design novel agents for the treatment of IBD.

ACKNOWLEDGEMENTS

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES

This study was supported in part by the Center for the Study of Emerging Diseases, L. Mayer and L. Bassani were supported by NIH grants AI 24671, AI 23504 and AI 44236.

REFERENCES

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES
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