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INTERLEUKIN-8 AS AN AUTOCRINE GROWTH FACTOR FOR HUMAN COLON CARCINOMA CELLS IN VITRO
Article No. cyto.1999.0518, available online at http://www.idealibrary.com on
INTERLEUKIN-8 AS AN AUTOCRINE GROWTH FACTOR FOR HUMAN COLON CARCINOMA CELLS IN VITRO Robert Brew,1 John S. Erikson,1 David C. West,1 Anne R. Kinsella,2 John Slavin,2 Stephen E. Christmas1 Cell lines derived from human colon carcinomas secrete interleukin 8 (IL-8) in vitro and this chemokine has also been detected immunohistochemically in human colon carcinoma specimens, in which it is tumour cell associated. In these experiments, IL-8 was shown to comprise an important component of the angiogenic activity of colon carcinoma cell line supernatants. The effect of modulating IL-8 activity upon the growth of the colon carcinoma cell lines HCT116A, HT29 and CaCo2 was investigated. Supplementing endogenously produced IL-8 by recombinant chemokine led to stimulation of cell growth. Neutralization of the effect of endogenously produced IL-8, either with the specific antagonist peptide AcRRWWCR or with blocking anti-IL-8 antibody, resulted in around 50% inhibition of cell growth (P<0.05). All of the colon carcinoma cell lines tested expressed mRNA for both IL-8RA and RB when grown at confluence. At the protein level, all cell lines expressed IL-8RA. Expression of IL-8RB was weak, although increased expression was seen in HCT116A cells as they approached confluence. Antibodies to IL-8RA and RB did not affect proliferation at low cell density but were strongly inhibitory when cells were cultured at a higher density. These data suggest that IL-8 acts as an autocrine growth factor for colon carcinoma cell lines and would support the concept that a similar autocrine loop operates in vivo. 2000 Academic Press
The chemokines are a family of low molecular weight cytokines with leukocyte chemotactic activity.1 They are divided into several subfamilies, dependent upon the position of one or two pairs of cysteine residues involved in intramolecular disulfide bond formation. Members of the , or C-X-C, chemokine subfamily have a pair of cysteine residues separated by a single amino acid. Those members that have an ELR motif immediately preceding the first cysteine, including interleukin 8 (IL-8) and melanoma growth stimulatory activity (MGSA; Gro), are predominantly neutrophil chemoattractants.1 However, at higher concentrations IL-8 is also capable of attracting a subset of T lymphocytes.1 From the Departments of 1Immunology and 2Surgery, University of Liverpool, Medical School, Daulby Street, Liverpool L69 3GA, UK Correspondence to: S. E. Christmas, Department of Immunology, University of Liverpool Medical School, Daulby Street, Liverpool L69 3GA, UK; E-mail: [email protected] Received 11 August 1998; received in revised form 23 March 1999; accepted for publication 20 April 1999 2000 Academic Press 1043–4666/00/010078+08 $35.00/0 KEY WORDS: colon carcinoma/interleukin 8/interleukin 8 receptors/monoclonal antibodies/peptides 78
In addition to their chemotactic function, chemokines exert a variety of other effects. IL-8 is able to induce a respiratory burst, degranulation and proteolytic enzyme release in neutrophils2,3 and also has angiogenic activity in vitro4,5 and in vivo.6 Like MGSA/Gro, IL-8 can also act as an autocrine growth factor for certain melanoma cell lines.7,8 Two receptors for IL-8, IL-8RA (CXCR1) and RB (CXCR2), have been identified on human neutrophils, both of which are members of the seven transmembrane domain family of G-protein-associated receptors.9 Both bind IL-8 with high affinity but whereas IL-8RA is more specific for IL-8, IL-8RB also binds MGSA and other C-X-C chemokines with a similar affinity.10 IL-8RA is expressed on a range of cell types, including neutrophils, T cells, monocytes, fibroblasts and melanoma cells, whereas IL-8RB expression is rather more restricted.9 An additional, promiscuous chemokine receptor has recently been identified on human erythrocytes that is capable of binding IL-8. This is the Duffy blood group antigen, which has been found to serve as a receptor for the malarial parasite Plasmodium vivax.11 This receptor is only weakly homologous to IL-8RA and RB and has CYTOKINE, Vol. 12, No. 1 (January), 2000: pp 78–85
IL-8 in colon carcinoma cell growth in vitro / 79
RESULTS Constitutive secretion of IL-8 by colon carcinoma cell lines Several human colon carcinoma cell lines, including CaCo2 cells, produce IL-813,14 and an ELISA was used to quantify the constitutive production of immunoreactive IL-8 by a panel of cell lines at confluence. All three cell lines tested constitutively secreted between 0.2 and 2 ng/ml IL-8 into culture supernatants over a 48-h period. HCT116A, HCT116B and HT29 cells produced 880, 260 and 1770 pg/ml of immunoreactive IL-8, respectively. That culture supernatants from colon carcinoma cell lines were functional in stimulating neutrophil migration was demonstrated using an in vitro migration assay. Supernatants from HT29, HCT116A and CaCo2 lines all induced significant neutrophil chemotaxis and chemokinesis (data not shown).
Determination of angiogenic activity in vivo Supernatants from HCT116A and HT29 cell lines were both angiogenic in the chorioallantoic membrane assay (Table 1), giving the classical spoke-wheel of fine capillaries centered on the site of application (not shown). The angiogenic activity of HT29 supernatants was only weak to moderate, as compared with the moderate to strong reactions seen with the HCT116A supernatants (Table 1). IL-8 gave a strong angiogenic reaction as did VEGF (Table 1). Excess of neutralizing anti-IL-8 mAb strongly inhibited the angiogenic activity both of rIL-8 and the two colon carcinoma cell
TABLE 1. The angiogenic activity of colon carcinoma cell supernatants and control recombinant angiogenic molecules and the inhibitory effects of anti-IL-8 mAb on these activities Angiogenesis: no. positive/no. tested (grade of activity) Sample HCT116A HT29 IL-8 (2 ng) VEGF (4 ng)
Alone
+anti-IL-8
15/19 (strong) 3/6 (moderate) 8/9 (strong) 3/3 (strong)
6/13 (weak) 1/3 (weak) 2/5 (weak) 3/3 (strong)
100 * Stimulation of proliferation (%)
not as yet been demonstrated to be capable of transducing signals.9 Production of IL-8 has been reported by neutrophils, monocyte/macrophages and T cells but also by a range of non-haematopoietic cell types, including fibroblasts, endothelial cells, keratinocytes, hepatocytes, chondrocytes and mesothelial cells.1 Normal gastric epithelial cells12 and colon carcinoma cell lines13,14 also produce IL-8 constitutively and production is enhanced during inflammation12 and following cytokine stimulation.14 Gastric carcinoma cells12 can also express IL-8 and we have recently reported that tumour cells within human colorectal carcinomas contain IL-8 mRNA and protein.15 Extensive infiltration with neutrophils is not a common feature of colorectal tumours, indicating that the primary function of IL-8 synthesized by these tumours is not concerned with leukocyte chemoattraction. Here, we have utilized colorectal carcinoma cell lines to investigate the hypothesis that IL-8 acts as an autocrine growth factor for these tumour cells.
75
50 * 25
* *
* *
0
–25
0
–7
10
10–6 10–5 rIL-8 (mg/ml)
10–4
10–3
Figure 1. The effect of exogenous human rIL-8 on colon carcinoma cell proliferation. Results represent the mean of three experimentsSEM. *P<0.05. HCT116A ( ); HT29 ( ); CaCo2 ( ).
line supernatants but had no effect on the angiogenic activity of VEGF (Table 1).
Influence of exogenous IL-8 on colon carcinoma cell growth The colon carcinoma cell lines used constitutively produced between 0.8 and 2 ng/ml IL-8 over the 48-h culture period but exogenous human rIL-8 significantly increased the proliferation of HT29, CaCo2 and HCT116A cells at the highest rIL-8 concentration used (1 g/ml; Fig. 1). CaCo2 and HT29 cells also showed significantly enhanced proliferation at lower rIL-8 concentrations.
Inhibition of colon carcinoma cell proliferation by neutralizing anti-IL-8 mAb The effect of a neutralizing anti-IL-8 monoclonal antibody on proliferation of HT29, CaCo2 and HCT116A cell lines was tested by measuring [3H]thymidine incorporation in the presence of increasing amounts of antibody. In all three lines, this led to significant inhibition of cell growth (Fig. 2) which
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CYTOKINE, Vol. 12, No. 1 (January, 2000: 78–85)
A
*
25
50
100
*
*
*
*
* 30
75
100
0
–6
10
–5
–4
10 10 Anti-IL-8 mAb (mg/ml)
–3
10
Figure 2. The effect of anti-IL-8 mAb on colon carcinoma cell proliferation. Results represent the mean of five experimentsSEM. *P<0.05. HCT116A ( ); HT29 ( ); CaCo2 ( ).
peaked at around 40–50% inhibition at 1 g/ml antibody, a concentration exceeding that capable of neutralizing all of the IL-8 constitutively secreted by these cells. Some of the cell lines also showed significant inhibition of proliferation at lower antibody concentrations (Fig. 2).
Inhibition of colon carcinoma cell proliferation by an IL-8 antagonist peptide The 6-mer peptide AcRRWWCR has previously been reported to inhibit the chemotactic function of IL-8 upon human neutrophils.16 It was tested for its ability to inhibit colon carcinoma cell growth in comparison to an unrelated control peptide. An antagonist peptide concentration of 10 4 M gave >50% inhibition of growth of HT29 and HCT116A cells (Fig. 3), whereas the unrelated control peptide failed to show inhibition of proliferation at 10 4 M. At concentrations of 10 3 M, both IL-8 antagonist peptide and control peptide were apparently toxic and strongly inhibited cell growth (data not shown).
Expression of IL-8 receptors by colon carcinoma cells The expression of mRNA for the IL-8 receptors IL-8RA and RB by colon carcinoma cell lines at confluence was assessed using the reverse transcriptase PCR reaction. HT29, CaCo2 and HCT116A cells, as well as peripheral blood neutrophils, were all found to express transcripts for both of these receptors, whereas these were only weakly detectable in fresh peripheral blood mononuclear cells (Fig. 4). Dilutions of anti-IL8RA and RB mAbs were optimized on cytocentrifuge smears of normal human peripheral blood neutrophils, which express both forms of the IL-8R. Adherent
Control proliferation (%)
Inhibition of proliferation (%)
0
10
–7
0
10
–6
10
–5
10
–4
10
B
100
* 30
10
0
10–7
10–6
10–5
10–4
Concentration of peptide (M) Figure 3. The effect of the IL-8 antagonist peptide AcRRWWCR (solid line) and unrelated control peptide (broken line) on colon carcinoma cell proliferation. Results represent the mean of three experimentsSEM. (A) HCT116A cells; (B) HT29 cells. *P<0.05.
Figure 4. Reverse transcriptase PCR for IL-8 receptors in colon carcinoma cell lines. Lanes 1–6, IL-8RA; lanes 7–12, IL-8RB. Lanes 1 and 7, CaCo2; lanes 2 and 8, HT29; lanes 3 and 9, HCT116A; lanes 4 and 10, peripheral blood mononuclear cells; lanes 5 and 11, peripheral blood neutrophils; lanes 6 and 12, negative control (no template). M, molecular weight markers.
microcultures of HT29, CaCo2 and HCT116A cells that had not yet reached confluence were tested for expression of IL-8RA and RB protein in the same way. All three lines expressed IL-8RA, with >90% of cells in the population being strongly positive. However, these
IL-8 in colon carcinoma cell growth in vitro / 81
was little or no growth inhibition of cells initially seeded at 105 cells/ml but cells initially seeded at 3105/ml and that had reached confluence by the end of the experiment showed substantial dose-dependent inhibition of growth with both anti-IL-8RA and RB antibodies.
A 1 × 105/ml
0
50
Inhibition of proliferation
DISCUSSION 3 × 105/ml * *
100 0
1
5
10
B 1 × 105/ml
0
50 3 × 105/ml
* *
100 0
1 5 Anti-IL-8 receptor antibody (µg/ml)
10
Figure 5. The effect of anti-IL-8 receptor mAbs on colon carcinoma cell proliferation at initial seeding concentrations of 105 cells/ml (closed symbols) and 3105 cells/ml (open symbols). IL-8RA ( , ); IL-8RB ( , ) Results represent the mean of three experimentsSEM. *P<0.05. (A) HCT116A; (B) HT29.
cell lines were only weakly positive for IL-8RB at a mAb dilution that led to strong staining of neutrophils (data not shown). This was investigated further in HCT116A cells which were cultured for varying times until confluence was reached. Whereas IL-8RA was strongly expressed by almost all cells throughout the culture period, IL-8RB expression was very low early in the culture period and was only expressed substantially as cultures approached confluence (data not shown).
Effect of anti-IL-8 receptor mAbs on colon carcinoma cell growth In initial experiments, mAbs against IL-8RA or IL-8RB led to variable inhibition of growth of HT29 and HCT116A cells (data not shown). However, when HCT116A cells were tested at different initial seeding densities, it became clear that both mAbs were only able to inhibit proliferation when the cells were approaching confluence. As shown in Figure 5, there
A feature of cells of epithelial origin, notably keratinocytes, is their constitutive production of IL-8.17 Normal human colonic crypt cells constitutively produce IL-818 and production by primary and malignant colonic epithelial cells is upregulated following stimulation with bacteria or bacterial toxins19,20 or in inflammatory bowel disease.18 Hence, epithelial cellderived IL-8 may mediate neutrophil chemoattraction in infection or inflammation and also, by virtue of its angiogenic properties,4,5 revascularization in the subsequent resolution of inflammation. Where epithelial cells also express IL-8 receptors, IL-8 may have an autocrine function in epithelial regeneration, as has been proposed for MGSA in wound healing.21 Continued expression of IL-8 by colonic epithelial cells following malignant transformation is suggestive of its involvement in carcinoma progression. It may be that IL-8 acts indirectly to support tumour growth. For example, IL-8 has been shown to be a major angiogenic mediator in vivo in a lung carcinoma model6 and to correlate with vascularity in human gastric carcinomas.22 Here, we have shown here that it contributes to the angiogenic activity present in HCT116A and HT29 colon carcinoma cell line supernatants. We have previously shown that IL-8 is expressed in a majority of human colorectal carcinomas.15 As well as having an indirect effect on tumour progression, several lines of evidence suggest that IL-8 can also play an autocrine role in the growth of colon carcinoma cells in vitro. First, the addition of exogenous IL-8 stimulated proliferation of all cell lines tested in a dose-dependent fashion. Second, antibodymediated neutralization of constitutively produced IL-8, or blockade of its action with a specific antagonist peptide, both inhibited cell proliferation. Maximal doses of IL-8 neutralizing antibody or antagonist peptide were only able to decrease cell proliferation by around 50%, indicating that IL-8 is not an absolute requirement for colon carcinoma cell growth. This peptide has been shown to suppress the MGSA/Gro dependent growth of melanoma cell lines at the same concentration found to be inhibitory in the present experiments.23 Experiments designed to block the effects of IL-8 at the level of its receptor gave complex results. When
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colon carcinoma cells were grown at low density, both anti-IL-8RA and RB antibodies had no effect on cell growth despite IL-8RA being strongly expressed. However, at higher cell density both anti-IL-8RA and RB antibodies were strongly inhibitory. Parallel studies on IL-8 receptor expression showed that although IL-8RB was only very weakly expressed by colon carcinoma cells seeded at low densities, as cells approached confluence around 50% of cells upregulated expression of IL-8RB. In contrast, IL-8RA was expressed strongly by most cells throughout the culture period. The inability of the neutralizing anti-IL-8 antibody to inhibit colon carcinoma cell growth by more than 50% might also be because the cells had not yet approached confluence, had not upregulated IL-8RB and hence were less dependent on an IL-8RB-mediated autocrine growth stimulatory pathway. Studies of neutrophils have shown that IL-8RB is more readily downregulated by IL-8 (or MGSA/ GRO) and is re-expressed more slowly following ligand-mediated modulation than is IL-8RA.24 As colon carcinoma cells become confluent and IL-8RB expression is upregulated then IL-8 levels may become more important in the control of cell growth. IL-8 expression has indeed been shown to be downregulated following the spontaneous differentiation of postconfluent CaCo2 cells25 and in differentiated human T84 intestinal epithelial cells.26 In melanoma cells, antibodies to IL-8RB were inhibitory to Grodependent proliferation in vitro27 and an antisense oligonucleotide to IL-8RB inhibited the growth of non-small cell lung carcinoma cells in vitro and in vivo.28 In preliminary experiments, we have found that a panel of 20 colon carcinomas all expressed high levels of IL-8RA (A. Reading et al. in preparation). Tumour cell expression of IL-8RA and RB has recently been reported in all breast carcinoma samples studied.29 Extensive infiltration with neutrophils is not a feature of human colorectal tumours. Although colon carcinoma cell lines secrete IL-8 in vitro,13,14 it has yet to be demonstrated that this occurs in vivo and it remains possible that the immunoreactive IL-8 found within colorectal tumours is stored intracellularly rather than secreted. Alternatively, if IL-8 is secreted by these tumour cells in vivo, it may be rapidly bound and consumed by endothelial cells during angiogenesis or by the tumour cells themselves during autocrine growth stimulation. A number of other growth factors produced by colon carcinoma cells have been shown to mediate autocrine growth stimulation in vitro. These include insulin-like growth factor-230,31 and transforming growth factors 32and 133,34 However, the latter can also mediate an autocrine growth inhibitory function in HCT116A cells,35 as can IL-8 in non-small cell lung carcinoma cells.36 Thus the same growth factors may
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have opposing effects in different tumour cell types. It will be of interest to extend studies into the role of IL-8 in other tumour systems in vitro and in vivo.
MATERIALS AND METHODS Cell lines The human colon adenocarcinoma cell lines HT29, CaCo237 and HCT116A and 116B38 were grown as adherent monolayers in RPMI1640+10% heat inactivated fetal calf serum containing 2 mM -glutamine and antibiotics (RPMI-CS). Cells were subcultured weekly by removing from culture flasks with non-enzymatic cell dissociation solution (Sigma, Poole, UK) and re-seeded at a concentration of 1105 cells/ml.
Reagents Recombinant human IL-8 (rhIL-8), vascular endothelial growth factor (VEGF) and mouse monoclonal antibodies (mAb) against human IL-8 were purchased from R & D Systems (Abingdon, UK). The anti-IL-8 mAb at a concentration of 10 g/ml was able to neutralize the chemotactic function of 1 g/ml rhIL-8. Mouse mAbs against IL-8 receptors A & B (CD128a & b) were purchased from Serotec (Bicester, UK). The IL-8 antagonist peptide AcRRWWCR16 and an unrelated control bcr–abl fusion peptide GFKQSSKAL, were synthesized using standard f-moc chemistry by Dr J. A. Smith (Department of Biochemistry, University of Liverpool).
Immunocytochemistry Cell surface antigen localization was performed using an immunoalkaline phosphatase kit (Zymed, S. San Francisco, CA, USA). Briefly, colon carcinoma cell lines were grown in sterile chambers mounted on glass microscope slides (GibcoBRL, Paisley, UK) until almost confluent. They were then air-dried overnight and fixed for 2 min in acetone. After blocking with non-immune goat serum for 10 min, primary mouse mAb was applied at a range of dilutions (1 in 50 to 1 in 250) for 30 min at room temperature. After washing, biotinylated goat anti-mouse immunoglobulin was added for a further 30 min followed by extravidin -alkaline phosphatase (Sigma, Poole, UK) for 30 min. Antibody localization was then visualized using naphthol-AS-MS-phosphate and fast red (Sigma). Negative control slides were processed in an identical manner but omitting the primary antibody. Cytocentrifuge preparations (Cytospin 2, Shandon, Runcorn, UK) of normal human neutrophils were used as a positive control for staining with anti-IL-8RA and RB mAbs.
IL-8 ELISA Tissue culture supernatants were harvested from colon carcinoma cell lines cultured at confluence for 48 h, centrifuged at 400g for 10 min and stored for up to 1 month at 20C prior to assay. Supernatants were then assayed for IL-8 content, along with RPMI-CS as a negative
IL-8 in colon carcinoma cell growth in vitro / 83
control, using a capture ELISA kit (CLB, Amsterdam, Netherlands), according to the manufacturer’s instructions, using several dilutions of duplicate samples. Concentrations of immunoreactive IL-8 in the supernatants were estimated by interpolation from the calibration curve obtained with the IL-8 standard. The detection limit for the assay was around 10 pg/ml.
Neutrophil migration assays Normal peripheral blood neutrophils were prepared using a standard laboratory technique.39 Filtered supernatants from colon carcinoma cell lines or Hanks balanced salt solution (HBSS) were pipetted either into the lower wells, the upper wells or both, of a 48-well chemotaxis chamber (Neuro Probe). An 8-m pore nitrocellulose filter was placed between lower and upper chambers. Immediately prior to loading into the wells of the upper chamber, the neutrophils were resuspended to a concentration of 106/ml in either HBSS or filtered supernatant and 50 l was then loaded into each well. Cells were placed at 37C in 5% CO2 and left to migrate for 90 min. Following incubation, filters were removed, washed in PBS, fixed in 2% paraformaldehyde and stained with haematoxylin. Migration into the filter was assessed by a single observer making 10 observations per well using the second cell leading front method.39 Results for each supernatant were expressed as the mean distance travelled through the membrane in comparison to cells responding to HBSS. Migration in response to a stimulus in the lower chamber only was considered as chemotaxis and that in the presence of a stimulus in the upper chamber or both upper and lower chambers was considered as chemokinesis.
Chick chorioallantoic membrane angiogenesis assay Culture supernatants from colon carcinoma cell lines were concentrated tenfold using centrifugal membrane filtration (‘‘microsep’’, 3-kDa cut-off; Filtron Technology Corp., Northborough, MA). They were then tested for angiogenic activity on chick chorioallantoic membranes (CAM), essentially as previously described.40 Briefly, test samples were mixed with an equal volume of sterile 1% methylcellulose (4000 centipoises; Sigma) in PBS and 8 l of this was applied onto a 2 mm diameter Teflon column and air-dried under sterile conditions to give a clear disc. These were then applied to chick CAM on day 10 post-fertilization and the degree of angiogenesis was assessed after 3 and 4 days of incubation at 37C.
RB mAb, or of IL-8 antagonist or control peptide were used. In all cases, control wells initially contained the same number of cells in RPMI-CS alone and samples were prepared in sextuplicate for each data point. After a further 72-h incubation at 37C and 5% CO2, wells were pulsed with 1 Ci [3H]-thymidine for 4 h, harvested and incorporated radioactivity counted using a Topcount (Packard, Meridien, CT). Means of sextuplicates were calculated and results expressed as a percentage of growth stimulation or inhibition in comparison to cells grown in RPMI-CS alone.
Reverse transcriptase PCR Total RNA was extracted from 5106 colon carcinoma cells at confluence, and peripheral blood mononuclear cells and neutrophils using Trizol (Gibco-BRL). This was then converted into cDNA using reverse transcriptase (GibcoBRL) in the presence of oligo-dT primer. Pairs of oligonucleotide primers specific for IL-8 receptors A and B were designed from the published sequences41 and synthesised as follows: IL-8RA sense: IL-8RA antisense: IL-8RB sense: IL-8RB antisense:
5 TGA GGT CCT GGG AAA ATG ACA CA 3 5 GGC CAT GCA TAG CCA GGA TC 3 5 CAT TCA GAG ACA GAA GGT GGA TAG 3 5 AAG GTT GGG TGG TAG TCA GAG 3
These primers span several exons and were predicted to give rise to products of 405 bp (IL-8RA) and 296 bp (IL-8RB) in length. They were used to amplify specific cDNA sequences by the PCR using an initial denaturation step of 1 min at 94C followed by 20 cycles of denaturation for 40 s at 94C, annealing for 45 s at 56C and extension for 60 s at 72C. This was followed by a further 14 cycles of denaturation for 40 s at 94C, annealing for 60 s at 56C and extension for 90 s at 72C. For the final cycle, denaturation was for 60 s, annealing was for 3 min and extension was for 10 min. Amplified products were electrophoresed in a 2% agarose gel containing ethidium bromide and observed under ultraviolet light. cDNA from human neutrophils was used as a positive control and reaction tubes containing all reagents except cDNA served as negative controls. The size of amplified products was estimated by comparison with a 100-bp DNA ladder.
Statistical analysis The statistical significance of differences between groups was tested using Student’s t-test.
Proliferation assays Colorectal tumour cell lines were removed from plastic culture flasks with non-enzymatic cell dissociation solution and seeded at a concentration of 1105 cells/ml in RPMI-CS in 96-well flat-bottomed plates. In some experiments, cells were also seeded at a concentration of 3105 cells/ml. After 24 h, the medium was carefully removed and replaced with fresh medium containing a range of concentrations of rhIL-8 in RPMI-CS. In other experiments, a range of concentrations of anti-IL-8 mAb, anti-IL-8RA or
Acknowledgements Synthetic peptides were made by Dr JA Smith, Dept. of Biochemistry, University of Liverpool. Neutrophil migration assays were performed by Mr AT Treweeke, Dept. of Haematology, University of Liverpool. This work was supported by the North West Cancer Research Fund.
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CYTOKINE, Vol. 12, No. 1 (January, 2000: 78–85) 20. Mahida YR, Makh S, Hyde S, Gray T, Borriello SP (1996) Effect of Clostridium difficile toxin A on human intestinal epithelial cells: induction of interleukin-8 production and apoptosis after cell detachment. Gut 38:337–347. 21. Nanney LB, Mueller SG, Bueno R, Peiper SC, Richmond A (1995) Distributions of melanoma growth stimulatory activity or growth-regulated gene and the interleukin-8 receptor B in human wound repair. Am J Pathol 147:1248–1260. 22. Kitadai Y, Haruma K, Sumii K, Yamamoto S, Ue T, Yokozaki H, Yasui W, Ohmoto Y, Kajiyama G, Fidler IJ, Tahara E (1998) Expression of interleukin-8 correlates with vascularity in human gastric carcinomas. Am J Pathol 152:93–100. 23. Hayashi S, Kurdowska A, Cohen AB, Stevens MD, Fujisawa N, Miller EJ (1997) A synthetic peptide inhibitor for alpha-chemokines inhibits the growth of melanoma cell lines. J Clin Invest 99:2581–2587. 24. Norgauer J, Metzner B, Schraffsta¨ tter I (1996) Expression and growth-promoting function of the IL-8 receptor in human melanoma cells. J Immunol 156:1132–1137. 25. Huang N, Katz JP, Martin DR, Wu GD (1997) Inhibition of IL-8 gene expression in CaCo-2 cells by compounds which induce histone hyperacetylation. Cytokine 9:27–36. 26. Schulte R, Autenrieth IB (1998) Yersinia enterocoliticainduced interleukin-8 secretion by human intestinal epithelial cells depends on cell differentiation. Infect Immunity 66:1216–1224. 27. Chuntharapai A, Kim KJ (1995) Regulation of the expression of IL-8 receptor A/B by IL-8: possible functions of each receptor. J Immunol 155:2587–2594. 28. Olbina G, Cieslak D, Ruzdijic S, Esler C, An Z, Wang X, Hoffman R, Seifert W, Pietrzkowski Z (1996) Reversible inhibition of IL-8 receptor B mRNA expression and proliferation in non-small cell lung cancer by antisense oligonucleotides. Anticancer Res 16:3525–3530. 29. Miller LJ, Kurtzmann SH, Wang Y, Anderson KH, Lindquist RR, Kreutzer DL (1998) Expression of interleukin-8 receptors on tumor cells and vascular endothelial cells in human breast cancer tissue. Anticancer Res 18:77–81. 30. Singh P, Dai B, Yallampalli U, Lu X, Schroy PC (1996) Proliferation and differentiation of a human colon cancer cell line (CaCo2) is associated with significant changes in the expression and secretion of insulin-like growth factor (IGF) IGF-II and IGF binding protein-4: role of IGF-II. Endocrinology 137:1764–1774. 31. Lamonerie T, Lavialle C, Haddada H, Brison O (1995) IGF-2 autocrine stimulation in tumorigenic clones of a human colon-carcinoma cell line. Int J Cancer 61:587–592. 32. Hirsch T, Eggstein S, Frank S, Farthmann E, von Specht BU (1996) Autocrine growth stimulation of SW403 colon carcinoma cell line is caused by transforming-growth-factor--mediated epidermal growth factor receptor activation. J Cancer Res Clin Oncol 122:328–334. 33. Huang F, Newman E, Theodorescu D, Kerbel RS, Friedman E (1995) Transforming growth factor 1 is an autocrine positive regulator of colon carcinoma U9 cells in vivo as shown by transfection of a TGF1 antisense expression plasmid. Cell Growth Differ 6:1635–1642. 34. Ciardello F, Damiano V, Bianco R, Bianco C, Fontanini G, De Laurentiis M, De Placido S, Mendelsohn J, Bianco AR, Tortora G (1996) Antitumor activity of combined blockade of epidermal growth factor receptor and protein kinase A. J Natl Cancer Inst 88:1770–1776. 35. Wang J, Sun L, Myeroff L, Wang X, Gentry LE, Yang J, Liang J, Zborowska E, Markowitz S, Willson JK (1995) Demonstration that mutation of the type II transforming growth factor receptor inactivates its tumor suppressor activity in replication error-positive colon carcinoma cells. J Biol Chem 270:22044–22049. 36. Wang J, Huang M, Lee P, Komanduri K, Sharma S, Chen G, Dubinett SM (1996) Interleukin-8 inhibits non-small cell lung cancer proliferation: a possible role for regulation of tumor growth by autocrine and paracrine pathways. J Interferon Cytokine Res 16:53–60. 37. Fogh J, Wright WC, Loveless JC (1977) Absence of HeLa cell contamination in 169 cell lines derived from human tumors. J Natl Cancer Inst 58:209–214.
IL-8 in colon carcinoma cell growth in vitro / 85 38. Brattain MG, Fine WD, Khaled FM, Thompson J, Brattain DE (1981) Heterogeneity of malignant cells from a human colonic carcinoma. Cancer Res 41:1751–1756. 39. Treweeke AT, Aziz KA, Zuzel M (1994) The role of G-CSF in mature neutrophil function is not related to GM-CSF-type cell priming. J Leukoc Biol 55:612–616.
40. West DC, Hampson IN, Arnold F, Kumar S (1985) Angiogenesis induced by degradation products of hyaluronic acid. Science (Washington DC) 228:1324–1326. 41. Ahuja SK, Shetty A, Tiffany HL, Murphy PM (1994) Comparison of the genomic organisation and promoter function for human interleukin-8 receptors A and B. J Biol Chem 269:26381–26389.
INTERLEUKIN-8 AS AN AUTOCRINE GROWTH FACTOR FOR HUMAN COLON CARCINOMA CELLS IN VITRO Robert Brew,1 John S. Erikson,1 David C. West,1 Anne R. Kinsella,2 John Slavin,2 Stephen E. Christmas1 Cell lines derived from human colon carcinomas secrete interleukin 8 (IL-8) in vitro and this chemokine has also been detected immunohistochemically in human colon carcinoma specimens, in which it is tumour cell associated. In these experiments, IL-8 was shown to comprise an important component of the angiogenic activity of colon carcinoma cell line supernatants. The effect of modulating IL-8 activity upon the growth of the colon carcinoma cell lines HCT116A, HT29 and CaCo2 was investigated. Supplementing endogenously produced IL-8 by recombinant chemokine led to stimulation of cell growth. Neutralization of the effect of endogenously produced IL-8, either with the specific antagonist peptide AcRRWWCR or with blocking anti-IL-8 antibody, resulted in around 50% inhibition of cell growth (P<0.05). All of the colon carcinoma cell lines tested expressed mRNA for both IL-8RA and RB when grown at confluence. At the protein level, all cell lines expressed IL-8RA. Expression of IL-8RB was weak, although increased expression was seen in HCT116A cells as they approached confluence. Antibodies to IL-8RA and RB did not affect proliferation at low cell density but were strongly inhibitory when cells were cultured at a higher density. These data suggest that IL-8 acts as an autocrine growth factor for colon carcinoma cell lines and would support the concept that a similar autocrine loop operates in vivo. 2000 Academic Press
The chemokines are a family of low molecular weight cytokines with leukocyte chemotactic activity.1 They are divided into several subfamilies, dependent upon the position of one or two pairs of cysteine residues involved in intramolecular disulfide bond formation. Members of the , or C-X-C, chemokine subfamily have a pair of cysteine residues separated by a single amino acid. Those members that have an ELR motif immediately preceding the first cysteine, including interleukin 8 (IL-8) and melanoma growth stimulatory activity (MGSA; Gro), are predominantly neutrophil chemoattractants.1 However, at higher concentrations IL-8 is also capable of attracting a subset of T lymphocytes.1 From the Departments of 1Immunology and 2Surgery, University of Liverpool, Medical School, Daulby Street, Liverpool L69 3GA, UK Correspondence to: S. E. Christmas, Department of Immunology, University of Liverpool Medical School, Daulby Street, Liverpool L69 3GA, UK; E-mail: [email protected] Received 11 August 1998; received in revised form 23 March 1999; accepted for publication 20 April 1999 2000 Academic Press 1043–4666/00/010078+08 $35.00/0 KEY WORDS: colon carcinoma/interleukin 8/interleukin 8 receptors/monoclonal antibodies/peptides 78
In addition to their chemotactic function, chemokines exert a variety of other effects. IL-8 is able to induce a respiratory burst, degranulation and proteolytic enzyme release in neutrophils2,3 and also has angiogenic activity in vitro4,5 and in vivo.6 Like MGSA/Gro, IL-8 can also act as an autocrine growth factor for certain melanoma cell lines.7,8 Two receptors for IL-8, IL-8RA (CXCR1) and RB (CXCR2), have been identified on human neutrophils, both of which are members of the seven transmembrane domain family of G-protein-associated receptors.9 Both bind IL-8 with high affinity but whereas IL-8RA is more specific for IL-8, IL-8RB also binds MGSA and other C-X-C chemokines with a similar affinity.10 IL-8RA is expressed on a range of cell types, including neutrophils, T cells, monocytes, fibroblasts and melanoma cells, whereas IL-8RB expression is rather more restricted.9 An additional, promiscuous chemokine receptor has recently been identified on human erythrocytes that is capable of binding IL-8. This is the Duffy blood group antigen, which has been found to serve as a receptor for the malarial parasite Plasmodium vivax.11 This receptor is only weakly homologous to IL-8RA and RB and has CYTOKINE, Vol. 12, No. 1 (January), 2000: pp 78–85
IL-8 in colon carcinoma cell growth in vitro / 79
RESULTS Constitutive secretion of IL-8 by colon carcinoma cell lines Several human colon carcinoma cell lines, including CaCo2 cells, produce IL-813,14 and an ELISA was used to quantify the constitutive production of immunoreactive IL-8 by a panel of cell lines at confluence. All three cell lines tested constitutively secreted between 0.2 and 2 ng/ml IL-8 into culture supernatants over a 48-h period. HCT116A, HCT116B and HT29 cells produced 880, 260 and 1770 pg/ml of immunoreactive IL-8, respectively. That culture supernatants from colon carcinoma cell lines were functional in stimulating neutrophil migration was demonstrated using an in vitro migration assay. Supernatants from HT29, HCT116A and CaCo2 lines all induced significant neutrophil chemotaxis and chemokinesis (data not shown).
Determination of angiogenic activity in vivo Supernatants from HCT116A and HT29 cell lines were both angiogenic in the chorioallantoic membrane assay (Table 1), giving the classical spoke-wheel of fine capillaries centered on the site of application (not shown). The angiogenic activity of HT29 supernatants was only weak to moderate, as compared with the moderate to strong reactions seen with the HCT116A supernatants (Table 1). IL-8 gave a strong angiogenic reaction as did VEGF (Table 1). Excess of neutralizing anti-IL-8 mAb strongly inhibited the angiogenic activity both of rIL-8 and the two colon carcinoma cell
TABLE 1. The angiogenic activity of colon carcinoma cell supernatants and control recombinant angiogenic molecules and the inhibitory effects of anti-IL-8 mAb on these activities Angiogenesis: no. positive/no. tested (grade of activity) Sample HCT116A HT29 IL-8 (2 ng) VEGF (4 ng)
Alone
+anti-IL-8
15/19 (strong) 3/6 (moderate) 8/9 (strong) 3/3 (strong)
6/13 (weak) 1/3 (weak) 2/5 (weak) 3/3 (strong)
100 * Stimulation of proliferation (%)
not as yet been demonstrated to be capable of transducing signals.9 Production of IL-8 has been reported by neutrophils, monocyte/macrophages and T cells but also by a range of non-haematopoietic cell types, including fibroblasts, endothelial cells, keratinocytes, hepatocytes, chondrocytes and mesothelial cells.1 Normal gastric epithelial cells12 and colon carcinoma cell lines13,14 also produce IL-8 constitutively and production is enhanced during inflammation12 and following cytokine stimulation.14 Gastric carcinoma cells12 can also express IL-8 and we have recently reported that tumour cells within human colorectal carcinomas contain IL-8 mRNA and protein.15 Extensive infiltration with neutrophils is not a common feature of colorectal tumours, indicating that the primary function of IL-8 synthesized by these tumours is not concerned with leukocyte chemoattraction. Here, we have utilized colorectal carcinoma cell lines to investigate the hypothesis that IL-8 acts as an autocrine growth factor for these tumour cells.
75
50 * 25
* *
* *
0
–25
0
–7
10
10–6 10–5 rIL-8 (mg/ml)
10–4
10–3
Figure 1. The effect of exogenous human rIL-8 on colon carcinoma cell proliferation. Results represent the mean of three experimentsSEM. *P<0.05. HCT116A ( ); HT29 ( ); CaCo2 ( ).
line supernatants but had no effect on the angiogenic activity of VEGF (Table 1).
Influence of exogenous IL-8 on colon carcinoma cell growth The colon carcinoma cell lines used constitutively produced between 0.8 and 2 ng/ml IL-8 over the 48-h culture period but exogenous human rIL-8 significantly increased the proliferation of HT29, CaCo2 and HCT116A cells at the highest rIL-8 concentration used (1 g/ml; Fig. 1). CaCo2 and HT29 cells also showed significantly enhanced proliferation at lower rIL-8 concentrations.
Inhibition of colon carcinoma cell proliferation by neutralizing anti-IL-8 mAb The effect of a neutralizing anti-IL-8 monoclonal antibody on proliferation of HT29, CaCo2 and HCT116A cell lines was tested by measuring [3H]thymidine incorporation in the presence of increasing amounts of antibody. In all three lines, this led to significant inhibition of cell growth (Fig. 2) which
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CYTOKINE, Vol. 12, No. 1 (January, 2000: 78–85)
A
*
25
50
100
*
*
*
*
* 30
75
100
0
–6
10
–5
–4
10 10 Anti-IL-8 mAb (mg/ml)
–3
10
Figure 2. The effect of anti-IL-8 mAb on colon carcinoma cell proliferation. Results represent the mean of five experimentsSEM. *P<0.05. HCT116A ( ); HT29 ( ); CaCo2 ( ).
peaked at around 40–50% inhibition at 1 g/ml antibody, a concentration exceeding that capable of neutralizing all of the IL-8 constitutively secreted by these cells. Some of the cell lines also showed significant inhibition of proliferation at lower antibody concentrations (Fig. 2).
Inhibition of colon carcinoma cell proliferation by an IL-8 antagonist peptide The 6-mer peptide AcRRWWCR has previously been reported to inhibit the chemotactic function of IL-8 upon human neutrophils.16 It was tested for its ability to inhibit colon carcinoma cell growth in comparison to an unrelated control peptide. An antagonist peptide concentration of 10 4 M gave >50% inhibition of growth of HT29 and HCT116A cells (Fig. 3), whereas the unrelated control peptide failed to show inhibition of proliferation at 10 4 M. At concentrations of 10 3 M, both IL-8 antagonist peptide and control peptide were apparently toxic and strongly inhibited cell growth (data not shown).
Expression of IL-8 receptors by colon carcinoma cells The expression of mRNA for the IL-8 receptors IL-8RA and RB by colon carcinoma cell lines at confluence was assessed using the reverse transcriptase PCR reaction. HT29, CaCo2 and HCT116A cells, as well as peripheral blood neutrophils, were all found to express transcripts for both of these receptors, whereas these were only weakly detectable in fresh peripheral blood mononuclear cells (Fig. 4). Dilutions of anti-IL8RA and RB mAbs were optimized on cytocentrifuge smears of normal human peripheral blood neutrophils, which express both forms of the IL-8R. Adherent
Control proliferation (%)
Inhibition of proliferation (%)
0
10
–7
0
10
–6
10
–5
10
–4
10
B
100
* 30
10
0
10–7
10–6
10–5
10–4
Concentration of peptide (M) Figure 3. The effect of the IL-8 antagonist peptide AcRRWWCR (solid line) and unrelated control peptide (broken line) on colon carcinoma cell proliferation. Results represent the mean of three experimentsSEM. (A) HCT116A cells; (B) HT29 cells. *P<0.05.
Figure 4. Reverse transcriptase PCR for IL-8 receptors in colon carcinoma cell lines. Lanes 1–6, IL-8RA; lanes 7–12, IL-8RB. Lanes 1 and 7, CaCo2; lanes 2 and 8, HT29; lanes 3 and 9, HCT116A; lanes 4 and 10, peripheral blood mononuclear cells; lanes 5 and 11, peripheral blood neutrophils; lanes 6 and 12, negative control (no template). M, molecular weight markers.
microcultures of HT29, CaCo2 and HCT116A cells that had not yet reached confluence were tested for expression of IL-8RA and RB protein in the same way. All three lines expressed IL-8RA, with >90% of cells in the population being strongly positive. However, these
IL-8 in colon carcinoma cell growth in vitro / 81
was little or no growth inhibition of cells initially seeded at 105 cells/ml but cells initially seeded at 3105/ml and that had reached confluence by the end of the experiment showed substantial dose-dependent inhibition of growth with both anti-IL-8RA and RB antibodies.
A 1 × 105/ml
0
50
Inhibition of proliferation
DISCUSSION 3 × 105/ml * *
100 0
1
5
10
B 1 × 105/ml
0
50 3 × 105/ml
* *
100 0
1 5 Anti-IL-8 receptor antibody (µg/ml)
10
Figure 5. The effect of anti-IL-8 receptor mAbs on colon carcinoma cell proliferation at initial seeding concentrations of 105 cells/ml (closed symbols) and 3105 cells/ml (open symbols). IL-8RA ( , ); IL-8RB ( , ) Results represent the mean of three experimentsSEM. *P<0.05. (A) HCT116A; (B) HT29.
cell lines were only weakly positive for IL-8RB at a mAb dilution that led to strong staining of neutrophils (data not shown). This was investigated further in HCT116A cells which were cultured for varying times until confluence was reached. Whereas IL-8RA was strongly expressed by almost all cells throughout the culture period, IL-8RB expression was very low early in the culture period and was only expressed substantially as cultures approached confluence (data not shown).
Effect of anti-IL-8 receptor mAbs on colon carcinoma cell growth In initial experiments, mAbs against IL-8RA or IL-8RB led to variable inhibition of growth of HT29 and HCT116A cells (data not shown). However, when HCT116A cells were tested at different initial seeding densities, it became clear that both mAbs were only able to inhibit proliferation when the cells were approaching confluence. As shown in Figure 5, there
A feature of cells of epithelial origin, notably keratinocytes, is their constitutive production of IL-8.17 Normal human colonic crypt cells constitutively produce IL-818 and production by primary and malignant colonic epithelial cells is upregulated following stimulation with bacteria or bacterial toxins19,20 or in inflammatory bowel disease.18 Hence, epithelial cellderived IL-8 may mediate neutrophil chemoattraction in infection or inflammation and also, by virtue of its angiogenic properties,4,5 revascularization in the subsequent resolution of inflammation. Where epithelial cells also express IL-8 receptors, IL-8 may have an autocrine function in epithelial regeneration, as has been proposed for MGSA in wound healing.21 Continued expression of IL-8 by colonic epithelial cells following malignant transformation is suggestive of its involvement in carcinoma progression. It may be that IL-8 acts indirectly to support tumour growth. For example, IL-8 has been shown to be a major angiogenic mediator in vivo in a lung carcinoma model6 and to correlate with vascularity in human gastric carcinomas.22 Here, we have shown here that it contributes to the angiogenic activity present in HCT116A and HT29 colon carcinoma cell line supernatants. We have previously shown that IL-8 is expressed in a majority of human colorectal carcinomas.15 As well as having an indirect effect on tumour progression, several lines of evidence suggest that IL-8 can also play an autocrine role in the growth of colon carcinoma cells in vitro. First, the addition of exogenous IL-8 stimulated proliferation of all cell lines tested in a dose-dependent fashion. Second, antibodymediated neutralization of constitutively produced IL-8, or blockade of its action with a specific antagonist peptide, both inhibited cell proliferation. Maximal doses of IL-8 neutralizing antibody or antagonist peptide were only able to decrease cell proliferation by around 50%, indicating that IL-8 is not an absolute requirement for colon carcinoma cell growth. This peptide has been shown to suppress the MGSA/Gro dependent growth of melanoma cell lines at the same concentration found to be inhibitory in the present experiments.23 Experiments designed to block the effects of IL-8 at the level of its receptor gave complex results. When
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colon carcinoma cells were grown at low density, both anti-IL-8RA and RB antibodies had no effect on cell growth despite IL-8RA being strongly expressed. However, at higher cell density both anti-IL-8RA and RB antibodies were strongly inhibitory. Parallel studies on IL-8 receptor expression showed that although IL-8RB was only very weakly expressed by colon carcinoma cells seeded at low densities, as cells approached confluence around 50% of cells upregulated expression of IL-8RB. In contrast, IL-8RA was expressed strongly by most cells throughout the culture period. The inability of the neutralizing anti-IL-8 antibody to inhibit colon carcinoma cell growth by more than 50% might also be because the cells had not yet approached confluence, had not upregulated IL-8RB and hence were less dependent on an IL-8RB-mediated autocrine growth stimulatory pathway. Studies of neutrophils have shown that IL-8RB is more readily downregulated by IL-8 (or MGSA/ GRO) and is re-expressed more slowly following ligand-mediated modulation than is IL-8RA.24 As colon carcinoma cells become confluent and IL-8RB expression is upregulated then IL-8 levels may become more important in the control of cell growth. IL-8 expression has indeed been shown to be downregulated following the spontaneous differentiation of postconfluent CaCo2 cells25 and in differentiated human T84 intestinal epithelial cells.26 In melanoma cells, antibodies to IL-8RB were inhibitory to Grodependent proliferation in vitro27 and an antisense oligonucleotide to IL-8RB inhibited the growth of non-small cell lung carcinoma cells in vitro and in vivo.28 In preliminary experiments, we have found that a panel of 20 colon carcinomas all expressed high levels of IL-8RA (A. Reading et al. in preparation). Tumour cell expression of IL-8RA and RB has recently been reported in all breast carcinoma samples studied.29 Extensive infiltration with neutrophils is not a feature of human colorectal tumours. Although colon carcinoma cell lines secrete IL-8 in vitro,13,14 it has yet to be demonstrated that this occurs in vivo and it remains possible that the immunoreactive IL-8 found within colorectal tumours is stored intracellularly rather than secreted. Alternatively, if IL-8 is secreted by these tumour cells in vivo, it may be rapidly bound and consumed by endothelial cells during angiogenesis or by the tumour cells themselves during autocrine growth stimulation. A number of other growth factors produced by colon carcinoma cells have been shown to mediate autocrine growth stimulation in vitro. These include insulin-like growth factor-230,31 and transforming growth factors 32and 133,34 However, the latter can also mediate an autocrine growth inhibitory function in HCT116A cells,35 as can IL-8 in non-small cell lung carcinoma cells.36 Thus the same growth factors may
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have opposing effects in different tumour cell types. It will be of interest to extend studies into the role of IL-8 in other tumour systems in vitro and in vivo.
MATERIALS AND METHODS Cell lines The human colon adenocarcinoma cell lines HT29, CaCo237 and HCT116A and 116B38 were grown as adherent monolayers in RPMI1640+10% heat inactivated fetal calf serum containing 2 mM -glutamine and antibiotics (RPMI-CS). Cells were subcultured weekly by removing from culture flasks with non-enzymatic cell dissociation solution (Sigma, Poole, UK) and re-seeded at a concentration of 1105 cells/ml.
Reagents Recombinant human IL-8 (rhIL-8), vascular endothelial growth factor (VEGF) and mouse monoclonal antibodies (mAb) against human IL-8 were purchased from R & D Systems (Abingdon, UK). The anti-IL-8 mAb at a concentration of 10 g/ml was able to neutralize the chemotactic function of 1 g/ml rhIL-8. Mouse mAbs against IL-8 receptors A & B (CD128a & b) were purchased from Serotec (Bicester, UK). The IL-8 antagonist peptide AcRRWWCR16 and an unrelated control bcr–abl fusion peptide GFKQSSKAL, were synthesized using standard f-moc chemistry by Dr J. A. Smith (Department of Biochemistry, University of Liverpool).
Immunocytochemistry Cell surface antigen localization was performed using an immunoalkaline phosphatase kit (Zymed, S. San Francisco, CA, USA). Briefly, colon carcinoma cell lines were grown in sterile chambers mounted on glass microscope slides (GibcoBRL, Paisley, UK) until almost confluent. They were then air-dried overnight and fixed for 2 min in acetone. After blocking with non-immune goat serum for 10 min, primary mouse mAb was applied at a range of dilutions (1 in 50 to 1 in 250) for 30 min at room temperature. After washing, biotinylated goat anti-mouse immunoglobulin was added for a further 30 min followed by extravidin -alkaline phosphatase (Sigma, Poole, UK) for 30 min. Antibody localization was then visualized using naphthol-AS-MS-phosphate and fast red (Sigma). Negative control slides were processed in an identical manner but omitting the primary antibody. Cytocentrifuge preparations (Cytospin 2, Shandon, Runcorn, UK) of normal human neutrophils were used as a positive control for staining with anti-IL-8RA and RB mAbs.
IL-8 ELISA Tissue culture supernatants were harvested from colon carcinoma cell lines cultured at confluence for 48 h, centrifuged at 400g for 10 min and stored for up to 1 month at 20C prior to assay. Supernatants were then assayed for IL-8 content, along with RPMI-CS as a negative
IL-8 in colon carcinoma cell growth in vitro / 83
control, using a capture ELISA kit (CLB, Amsterdam, Netherlands), according to the manufacturer’s instructions, using several dilutions of duplicate samples. Concentrations of immunoreactive IL-8 in the supernatants were estimated by interpolation from the calibration curve obtained with the IL-8 standard. The detection limit for the assay was around 10 pg/ml.
Neutrophil migration assays Normal peripheral blood neutrophils were prepared using a standard laboratory technique.39 Filtered supernatants from colon carcinoma cell lines or Hanks balanced salt solution (HBSS) were pipetted either into the lower wells, the upper wells or both, of a 48-well chemotaxis chamber (Neuro Probe). An 8-m pore nitrocellulose filter was placed between lower and upper chambers. Immediately prior to loading into the wells of the upper chamber, the neutrophils were resuspended to a concentration of 106/ml in either HBSS or filtered supernatant and 50 l was then loaded into each well. Cells were placed at 37C in 5% CO2 and left to migrate for 90 min. Following incubation, filters were removed, washed in PBS, fixed in 2% paraformaldehyde and stained with haematoxylin. Migration into the filter was assessed by a single observer making 10 observations per well using the second cell leading front method.39 Results for each supernatant were expressed as the mean distance travelled through the membrane in comparison to cells responding to HBSS. Migration in response to a stimulus in the lower chamber only was considered as chemotaxis and that in the presence of a stimulus in the upper chamber or both upper and lower chambers was considered as chemokinesis.
Chick chorioallantoic membrane angiogenesis assay Culture supernatants from colon carcinoma cell lines were concentrated tenfold using centrifugal membrane filtration (‘‘microsep’’, 3-kDa cut-off; Filtron Technology Corp., Northborough, MA). They were then tested for angiogenic activity on chick chorioallantoic membranes (CAM), essentially as previously described.40 Briefly, test samples were mixed with an equal volume of sterile 1% methylcellulose (4000 centipoises; Sigma) in PBS and 8 l of this was applied onto a 2 mm diameter Teflon column and air-dried under sterile conditions to give a clear disc. These were then applied to chick CAM on day 10 post-fertilization and the degree of angiogenesis was assessed after 3 and 4 days of incubation at 37C.
RB mAb, or of IL-8 antagonist or control peptide were used. In all cases, control wells initially contained the same number of cells in RPMI-CS alone and samples were prepared in sextuplicate for each data point. After a further 72-h incubation at 37C and 5% CO2, wells were pulsed with 1 Ci [3H]-thymidine for 4 h, harvested and incorporated radioactivity counted using a Topcount (Packard, Meridien, CT). Means of sextuplicates were calculated and results expressed as a percentage of growth stimulation or inhibition in comparison to cells grown in RPMI-CS alone.
Reverse transcriptase PCR Total RNA was extracted from 5106 colon carcinoma cells at confluence, and peripheral blood mononuclear cells and neutrophils using Trizol (Gibco-BRL). This was then converted into cDNA using reverse transcriptase (GibcoBRL) in the presence of oligo-dT primer. Pairs of oligonucleotide primers specific for IL-8 receptors A and B were designed from the published sequences41 and synthesised as follows: IL-8RA sense: IL-8RA antisense: IL-8RB sense: IL-8RB antisense:
5 TGA GGT CCT GGG AAA ATG ACA CA 3 5 GGC CAT GCA TAG CCA GGA TC 3 5 CAT TCA GAG ACA GAA GGT GGA TAG 3 5 AAG GTT GGG TGG TAG TCA GAG 3
These primers span several exons and were predicted to give rise to products of 405 bp (IL-8RA) and 296 bp (IL-8RB) in length. They were used to amplify specific cDNA sequences by the PCR using an initial denaturation step of 1 min at 94C followed by 20 cycles of denaturation for 40 s at 94C, annealing for 45 s at 56C and extension for 60 s at 72C. This was followed by a further 14 cycles of denaturation for 40 s at 94C, annealing for 60 s at 56C and extension for 90 s at 72C. For the final cycle, denaturation was for 60 s, annealing was for 3 min and extension was for 10 min. Amplified products were electrophoresed in a 2% agarose gel containing ethidium bromide and observed under ultraviolet light. cDNA from human neutrophils was used as a positive control and reaction tubes containing all reagents except cDNA served as negative controls. The size of amplified products was estimated by comparison with a 100-bp DNA ladder.
Statistical analysis The statistical significance of differences between groups was tested using Student’s t-test.
Proliferation assays Colorectal tumour cell lines were removed from plastic culture flasks with non-enzymatic cell dissociation solution and seeded at a concentration of 1105 cells/ml in RPMI-CS in 96-well flat-bottomed plates. In some experiments, cells were also seeded at a concentration of 3105 cells/ml. After 24 h, the medium was carefully removed and replaced with fresh medium containing a range of concentrations of rhIL-8 in RPMI-CS. In other experiments, a range of concentrations of anti-IL-8 mAb, anti-IL-8RA or
Acknowledgements Synthetic peptides were made by Dr JA Smith, Dept. of Biochemistry, University of Liverpool. Neutrophil migration assays were performed by Mr AT Treweeke, Dept. of Haematology, University of Liverpool. This work was supported by the North West Cancer Research Fund.
84 / Brew et al.
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