Interleukin-8 stimulates the migration of human colonic epithelial cells in vitro

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1 Clinical Science (1999) 97, (Printed in Great Britain) 385 Interleukin-8 stimulates the migration of human colonic epithelial cells in vitro Andrew J. WILSON*, Keith BYRON and Peter R. GIBSON* *Department of Medicine, The Royal Melbourne Hospital, University of Melbourne, Parkville, Victoria 3050, Australia, and Department of Biochemistry, The Royal Melbourne Hospital, University of Melbourne, Parkville, Victoria 3050, Australia A B S T R A C T The migration of colonic epithelial cells (restitution) is an important event in the repair of mucosal injuries. Interleukin-8 (IL-8) is a physiological initiator of the chemotactic migration of leucocytes. This study aimed to determine whether IL-8 had a similar effect on migration in an in vitro model of wounded colonic epithelium. Cell migration over 24 h was assessed in circular wounds made in confluent monolayers of the human colon cancer cell line LIM1215. This migration was stimulated in a concentration-dependent manner by IL-8, with maximal effects of approx fold above basal migration. The motogenic effect of IL-8 was mediated independently of effects on cell proliferation. In contrast, it was partially dependent upon gene transcription and protein synthesis and involved the activation of pertussis-toxin-sensitive G- proteins. The short-chain fatty acids, acetate, propionate, butyrate and valerate, the activator of protein kinase C (phorbol-12-myristate-13-acetate) and tumour necrosis factor-α (TNF-α) all stimulated the secretion of IL-8. However, only the motogenic effect of TNF-α was dependent upon IL-8. In conclusion, IL-8 stimulated cell migration in an in vitro model of colonic epithelium, whereas the motogenic effect of at least one physiologically relevant factor was dependent upon an increase in its endogenous levels. If IL-8 stimulates colonic epithelial restitution in vivo, this would have ramifications for the control of repair processes following wounding of the colonic mucosa. INTRODUCTION The migration of gastrointestinal epithelial cells is important for the repair of superficial mucosal injuries, a process termed restitution [1], and for the repair of mucosal ulcers [2]. A process which models restitution can be elicited in vitro using mechanically wounded monolayers of gastrointestinal epithelial cells. Recent work in our laboratory has identified short-chain fatty acids (SCFAs), particularly butyrate [3], and phorbol-12- myristate-13-acetate (PMA), which activates protein kinase C (PKC) [4], as factors that stimulate the migration of human colon cancer cell lines (i.e. motogens). However, whether these effects require increased endogenous expression of soluble motogenic peptides is unknown. Interleukin-8 (IL-8) is a potent chemotactic factor for leucocytes, such as neutrophils and natural killer cells [5,6]. However, it also promotes the movement of cells of different lineages, such as keratinocytes [7] and fibroblasts [8]. Although IL-8 binds with equal affinity to two subtypes of the seven transmembrane domain G-proteincoupled receptor, designated IL-8RA and IL-8RB [9], Key words: interleukin-8, colon, cell migration, short-chain fatty acids, protein kinase C, epidermal growth factor, tumour necrosis factor-α. Abbreviations used: IL, interleukin; TNF-α, tumour necrosis factor-α; SCFAs, short-chain fatty acids; PMA, phorbol-12- myristate-13-acetate; PKC, protein kinase C. Corresponding author: Associate Professor Peter Gibson ( p.gibson medicine.unimelb.edu.au).

2 386 A. J. Wilson, K. Byron and P. R. Gibson the IL-8RB subtype has recently been demonstrated to play a larger role in its effects on neutrophil chemotaxis [10]. Several studies indicate that IL-8 may be of relevance to the colonic epithelium. It is secreted constitutively by isolated colonic crypts and colon cancer cell lines [11,12], and a range of factors, such as SCFAs, PMA, IL-1 and tumour necrosis factor-α (TNF-α), are known to upregulate this secretion [11 15]. Despite this, the influence of IL-8 on colonic epithelial migration has not been established. A possible role in intestinal repair for other cytokines involved in the inflammatory response is suggested by studies in rat small intestinal IEC-6 cells, where IL-1β, interferon-γ and IL-2 stimulate cell migration [16,17]. The present study aimed to determine the effect of IL-8 on migration in wounded in vitro models of colonic epithelium, to examine putative mechanisms by which it acts, to determine the effect of TNF-α on migration and to examine the role that IL-8 plays in the motogenic effects of SCFAs, PMA and, putatively, TNF-α. MATERIALS AND METHODS Cell culture The moderately differentiated human colon cancer cell line, LIM1215 (passage 20 25), was a gift from Dr. Robert Whitehead (Ludwig Institute for Cancer Research, Parkville, Victoria, Australia). Growth medium used for cell culture was RPMI 1640 (Life Technologies, Grand Island, NY, U.S.A.), supplemented with 2 g l NaHCO (BDH Chemicals, Kilsyth, Victoria, Australia), 4 mm glutamine (Trace Biosciences, Castle Hill, NSW, Australia), 50 units ml penicillin and streptomycin (Life Technologies) and 10% (v v) fetal calf serum (Commonwealth Serum Laboratories, Parkville, Victoria, Australia). All cell-culture ware was obtained from Becton Dickinson (Lane Cove, NSW, Australia). Wounding assay Cell migration was assessed in circular wounds created in confluent cell monolayers by a rotating Teflon tip [3], a method modified from that of Watanabe et al. [18]. The cell-free area of LIM1215 wounds was calculated as described previously [3]. Migration over 24 h was expressed as the migration differential, the mean change in cell-free area of ten replicate wounds. Factors used to modulate cell migration Human recombinant IL-8 (72 amino acid residue form) was purchased from R&D Systems (Minneapolis, MN, U.S.A.) and human recombinant TNF-α was obtained from Auspep (Parkville, Victoria, Australia). Known motogens used were the sodium salts of the SCFAs, acetate, propionate, butyrate and valerate prepared in PBS, ph 7.4, under sterile conditions and PMA (all from Sigma-Aldrich, St. Louis, MO, U.S.A.) [3,4]. The following inhibitors were used: a mouse monoclonal neutralizing antibody raised against human IL-8 (R&D Systems), the protein synthesis inhibitor cycloheximide (Calbiochem, San Diego, CA, U.S.A.), the RNA synthesis inhibitor actinomycin D (Sigma-Aldrich), the DNA synthesis inhibitor mitomycin C (Crown Scientific, Burwood, Victoria, Australia) and the G-protein inhibitor pertussis toxin (Sigma-Aldrich). With the exception of mitomycin C, which was applied as described previously [3], the inhibitors were added to wounded monolayers 2 h before, and also throughout, exposure to motogenic factors. All migration experiments were conducted using serum-free medium supplemented with 1 mg ml BSA (Sigma-Aldrich). [ 3 H]Thymidine-uptake studies The effects of IL-8 on [ H]thymidine uptake, adherent cell number and Trypan Blue exclusion were examined from 0 6, 6 12 and h after its addition to wounded LIM1215 monolayers, as described previously [3]. IL-8 assay The effect of a 24 h exposure to SCFAs, PMA, TNF-α and the inhibitor of histone deacetylase, trichostatin A (ICN Pharmaceuticals, Costa Mesa, CA, U.S.A.), on IL-8 secretion from wounded LIM1215 monolayers was examined. IL-8 levels in cell supernatants were assayed using an in-house ELISA (developed and performed by K. B.). The supernatant was carefully aspirated. The sample (100 µl well) was added to microtitre plates precoated with polyclonal IL-8 antibodies (R&D Systems) and incubated at 25 C for 2 h. The plate was washed and 100 µl of anti-mouse immunoglobulin biotinylated conjugate (DAKO, Botany, NSW, Australia), diluted 1:20000, was added to each well. After incubation for 90 min at 25 C, the plates were washed and 100 µl of streptavidin peroxidase, diluted 1: 10000, was added to each well. After a further 30 min incubation, the wells were washed and 100 µl tetramethylbenzidine was added. The colour reaction was stopped by the addition of 1 mol l H PO and the A was read using standards calibrated against World Health Organization reference material. Values were expressed relative to the protein content of the relevant cell homogenate (ng mg of protein), which was determined as described previously [3]. Statistical evaluation Experimental and control groups were compared using a two tailed paired Student s t test, and P 0.05 was considered statistically significant. All statistical analyses

3 Interleukin-8 and migration 387 were performed on raw data using Minitab release 8 (Minitab Inc., State College, PA, U.S.A.). RESULTS Effect of IL-8 on cell migration As shown in Figure 1, IL-8 stimulated the migration of LIM1215 cells in a concentration-dependent manner, with maximal effects of approx fold stimulation at 50 ng ml. No effects were observed at IL-8 concentrations below 10 ng ml. An IL-8 neutralizing antibody was used to confirm the specificity of this effect to the action of IL-8. An antibody concentration of 25 µg ml, which was determined to be optimal in preliminary experiments (results not shown), ablated the motogenic effect of IL-8 by over 80% (Figure 2). However, the antibody did not significantly affect basal, unstimulated migration [4% below control (n 4; S.E.M. 7; P 0.36)]. Mechanisms of the motogenic effect of IL-8 Role of cell proliferation Compared with control levels, 50 ng ml IL-8 exerted no significant effects on [ H]thymidine uptake or adherent cell number in wounded LIM1215 monolayers from 0 6, 6 12 and h after wounding (results not shown). Trypan Blue exclusion experiments indicated that the cells had 95% viability at each time point. Consistent with these results, 2 h pre-treatment with the DNA synthesis inhibitor, mitomycin C (20 µg ml), did not Figure 2 Effect of a neutralizing antibody against IL-8 (25 µg/ml), the protein synthesis inhibitor cycloheximide (5 µm), the RNA synthesis inhibitor actinomycin D (1.25 ng/ml) and the G-protein inhibitor pertussis toxin (10 ng/ml) on the motogenic effect of IL-8 (50 ng/ml) in LIM1215 cells The values are the means S.E.M. of 3 4 experiments and show the percentage inhibition of IL-8-stimulated migration (, P 0.05;, P 0.01 relative to stimulatory effect alone; paired Student s t test). inhibit the motogenic effects of a range of IL-8 concentrations (Figure 1). The concentration of mitomycin C used inhibits [ H]thymidine uptake under basal conditions in wounded LIM1215 monolayers by 90% [3]. Role of protein and RNA synthesis The motogenic effect of 50 ng ml IL-8 was examined when protein synthesis and transcription were blocked by optimal concentrations of cycloheximide (5 µmol l) and actinomycin D (1.25 ng ml) respectively [3]. As shown in Figure 2, the magnitude of IL-8-mediated motogenesis after 24 h was reduced by approx % by either inhibitor. Consistent with previous observations [3], both cycloheximide and actinomycin D inhibited basal wound closure by approx. 40%. Figure 1 Effect of increasing concentrations of IL-8 alone ( ) or after 2 h pre-treatment with the DNA synthesis inhibitor mitomycin C ( ) (20 µg/ml) on the migration of LIM1215 cells The values are the means S.E.M. of four experiments (*, P 0.05; **, P 0.01 relative to control; paired Student s t test). There were no significant differences between the motogenic effect of IL-8 alone or after mitomycin C pretreatment for any of the concentrations tested. Role of G-proteins The motogenic effect of 50 ng ml IL-8 was inhibited by approx. 50% by the G-protein inhibitor pertussis toxin (10 ng ml) (Figure 2), without evidence of cellular toxicity, as assessed by cell morphology and adhesion to the substratum. This concentration of pertussis toxin was determined to be optimal in preliminary experiments (results not shown). Basal wound closure was inhibited by 38% (n 3; S.E.M. 6; P 0.02) with 10 ng ml pertussis toxin. IL-8 secretion by LIM1215 cells The cellular secretion of IL-8 from wounded LIM1215 monolayers after 24 h, and the resultant IL-8 supernatant concentration, under basal conditions and in the presence

4 388 A. J. Wilson, K. Byron and P. R. Gibson Table 1 Secretion of IL-8 from LIM1215 cells under basal and stimulated conditions Cellular IL-8 secretion and resultant supernatant IL-8 concentrations were determined in wounded LIM1215 monolayers after incubation for 24 h. Values are expressed as means S.E.M. *, P 0.05; **, P 0.01 relative to basal levels (paired Student s t test). Cellular IL-8 secretion (ng/mg) Supernatant IL-8 concentration (ng/ml) % Stimulation n Basal Acetate (16 mmol/l) * 3 Propionate (8 mmol/l) * 3 Butyrate (2 mmol/l) ** 7 Valerate (4 mmol/l) * 3 PMA (10 nmol/l) * 3 TNF-α (10 nmol/l) * 5 butyrate (412% above controls; n 4; S.E.M. 52; P 0.03). PMA (10 nmol l) and TNF-α (10 nmol l) increased the secretion of IL-8 by approx. 9-fold (P 0.03) and 13- fold (P 0.02) respectively. This concentration of PMA induces maximal motogenic effects in LIM1215 cells [4]. Figure 3 Effect of the neutralizing antibody against IL-8 (25 µg/ml) on the motogenic effects of acetate (Ace; 16 mmol/l), propionate (Pro; 8 mmol/l), butyrate (But; 2 mmol/l), valerate (Val; 4 mmol/l), TNF-α (10 nmol/l) and PMA (10 nmol/l) in LIM1215 cells The values show the stimulatory effect on migration of each factor alone ( ) or in the presence of the antibody ( ) and are the means S.E.M. of 3 4 experiments (, P 0.05 relative to stimulatory effect alone, paired Student s t test). of SCFAs, PMA and TNF-α are shown in Table 1. LIM1215 cells were observed to constitutively secrete IL-8, resulting in a basal supernatant concentration of approx. 3 ng ml. In descending order of potency, butyrate (2 mmol l), valerate (4 mmol l), propionate (8 mmol l) and acetate (16 mmol l) stimulated IL-8 secretion. These concentrations have been shown previously to induce maximal motogenic effects in these cells [3]. The stimulatory effect of butyrate on IL-8 secretion (approx. 5-fold) was concentration dependent (with maximal effects at 2 4 mmol l) and was first observed 12 h after its addition (results not shown). Trichostatin A increased the secretion of IL-8 at concentrations of 1 µmol l (499 % above controls; n 4; S.E.M. 36; P 0.01) and 2 µmol l (589% above controls; n 4; S.E.M. 64; P 0.01) to a greater extent (P 0.02 for either concentration) than did a parallel exposure to 2 mmol l Role of IL-8 in the motogenic effects of SCFAs, PMA and TNF-α As shown in Figure 3, the IL-8 neutralizing antibody (25 µg ml) failed to ablate the motogenic effects of acetate, propionate, butyrate, valerate and PMA. Two experiments using a higher concentration of the antibody (50 µg ml) were performed and the motogenic effects of butyrate and PMA were similarly unaffected (means of 2% and 3% respectively below stimulatory effect alone). The motogenic effect of 50 ng ml IL-8 (77%; n 4; S.E.M. 6; P 0.01) was significantly less potent (P 0.01) than that of optimal concentrations of butyrate (96%; n 4; S.E.M. 10; P 0.01) and PMA (421%; n 4; S.E.M. 30; P 0.01) when tested in parallel experiments. TNF-α stimulated the migration of LIM1215 cells when added at concentrations of 1 nmol l (25%; n 4; S.E.M. 3; P 0.04) and 10 nmol l (68%; n 7; S.E.M. 6; P 0.01). Higher concentrations did not induce greater motogenic effects (results not shown). As shown in Figure 3, the motogenic effect of 10 nmol l TNF-α was reduced by approx. 75% by an IL-8 antibody concentration of 25 µg ml (P 0.03 relative to the stimulatory effect alone). When used at 50 µg ml, the antibody induced a similar inhibitory effect on TNF-αmediated motogenesis in two experiments (mean of 81% below stimulatory effect alone). DISCUSSION The chemokine, IL-8, stimulated the migration of LIM1215 cells in a concentration-dependent manner. Studies with a neutralizing antibody against IL-8

5 Interleukin-8 and migration 389 indicated that its motogenic effect occurred as a result of a specific action on the cells. However, despite the constitutive secretion of IL-8 by LIM1215 cells, basal migration was not controlled by its endogenous activity. This is most likely a reflection of the fact that the supernatant levels of IL-8 present under basal conditions were lower than the lowest concentration at which exogenous IL-8 stimulated migration. The effects of other chemokines on the migration of colonic epithelial cells warrant further investigation, especially as isolated colonic crypts and colon cancer cell lines express chemokines such as growth related oncogene (GRO), epithelial cell-derived neutrophil activator 78 (ENA 78), monocyte chemotactic peptide-1 (MCP-1) and regulated on activation, normal T expressed and secreted (RANTES) [19,20]. Furthermore, chemokines structurally related to IL-8, such as growth-related oncogene and epithelial cell-derived neutrophil activator 78, bind to the IL-8RB subtype [9]. Wound closure stimulated by IL-8 was mediated independently of effects on cell proliferation, and a partial requirement for gene transcription and protein synthesis was demonstrated. In addition, IL-8-mediated motogenesis was suppressed by pertussis toxin. This is consistent with the fact that IL-8 binds with equal affinity to two subtypes of receptor associated with pertussistoxin-sensitive G-protein subunits [21]. A similar mechanism has been identified in its promotion of the chemotaxis of neutrophils [5] and natural killer cells [6]. Several key mediators of cell migration are known to be activated through G-proteins, such as focal adhesion kinase and its substrates paxillin and p130cas [22,23], Rho-family GTPases [23] and phospholipase A2 [24]. Since the IL-8RB subtype is known to play a larger role in the effect of IL-8 on neutrophil chemotaxis [10], identification of the subtype responsible for its motogenic effect in LIM1215 cells warrants further investigation. The relative potency of the SCFAs in stimulating IL-8 secretion was consistent with their effects on this index in Caco-2 human colon cancer cells [15], which is known to be related to the magnitude of their inhibitory effect on a key enzyme involved in the negative regulation of transcription, histone deacetylase. This is likely also to apply in LIM1215 cells, since trichostatin A, a more potent inhibitor of histone deacetylase than butyrate, exerted greater effects on IL-8 secretion than an optimal butyrate concentration. As reported previously in other colon cancer cell lines [11,13], both PMA and TNF-α increased IL-8 secretion. The ability of TNF-α to stimulate migration contrasted with previous observations in IEC-6 cells, where it had no effect [16]. SCFAs (especially butyrate), PMA and TNF-α stimulated the supernatant concentration of IL-8 to levels where exogenous IL-8 was found to be motogenic. However, studies with the IL-8 neutralizing antibody indicated that only the stimulatory effect of TNF-α on migration was IL-8 dependent. Further evidence against a role for IL-8 in mediating SCFA- and PKC-induced motogenesis was that the maximal motogenic effects of butyrate or PMA were greater than that of IL-8. There was also a discrepancy in the time course of the effects of butyrate on IL-8 secretion and migration, since its initial motogenic effects occur within 6 h [3]. Work in our laboratory has indicated that migration can be stimulated by the activation of multiple signalling pathways. Examples include the epidermal-growthfactor receptor tyrosine kinase, which appears to control the motogenic effects of SCFAs and also regulates basal migration, PKC and the transforming-growth-factor-β receptor serine threonine kinase [3,4]. The present study has identified another, namely the activation of G-proteins following the binding of IL-8 to its receptor. It is likely, therefore, that SCFAs and PMA are stronger stimuli for motogenic pathways than IL-8. In contrast, TNF-α may only weakly activate these alternative pathways, resulting in motogenesis primarily through its effects on IL-8. As a result of differential N-terminal splicing, at least two forms of IL-8 (consisting of either 72 or 77 amino acids) are secreted by cells, depending upon their lineage. It has been demonstrated that these forms differ in their biological activity in vitro [25]. The type of IL-8 secreted by LIM1215 cells was not examined in the present study and there has been no published evidence of the form secreted by these cells, or others originating from the colonic epithelium. However, the fact that increased IL- 8 levels mediated the motogenic effect of TNF-α indicates that LIM1215 cells secrete an active form of IL-8. Although it is unknown whether the colonic epithelium expresses receptors for IL-8, these results nevertheless suggest that IL-8 and or other chemokines may play a role in the reparative events that follow mucosal injury. The secretion of IL-8 by colonic epithelial cells may be stimulated by wounding itself [12] or by physiologically relevant factors, such as TNF-α. A subsequent motogenic response to IL-8 would hasten restitution and help maintain epithelial integrity. Such actions have potential relevance for diseases characterized by mucosal ulceration, such as the inflammatory bowel disease, ulcerative colitis, where delivery of IL-8 to epithelial cells may be of therapeutic benefit. In conclusion, IL-8 stimulates the migration of LIM1215 colon cancer cells but does not regulate basal migration, despite its constitutive secretion. The motogenic effect of IL-8 is independent of effects on cell proliferation but is at least partially dependent upon gene transcription, protein synthesis and the activity of pertussis-toxin-sensitive G-proteins. The motogenic effect of at least one physiologically relevant factor, TNF-α, is dependent upon increased endogenous levels of IL-8. This contrasts with the mechanism of SCFA- and

6 390 A. J. Wilson, K. Byron and P. R. Gibson PKC-mediated motogenesis. These results suggest that local increases in the production of IL-8 may stimulate the migration of colonic epithelial cells, which has potential relevance for both the biology and pathobiology of the colonic mucosal barrier. ACKNOWLEDGMENTS A. J. W. was in receipt of an Australian Postgraduate Award scholarship REFERENCES 1 Silen, W. and Ito, S. (1985) Mechanisms for rapid reepithelialization of the gastric mucosal surface. Annu. Rev. Physiol. 47, Modlin, I. M., Basson, M. D., Soroka, C. J. et al. (1993) Ulcer-induced alterations in cell phenotype and matrix and growth factor expression. Eur. J. Gastroenterol. Hepatol. 5 (Suppl 3), S59 S67 3 Wilson, A. J. and Gibson, P. R. (1997) Short-chain fatty acids promote the migration of colonic epithelial cells in vitro. Gastroenterology 113, Wilson, A. J. and Gibson, P. R. 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