Function of Liver Activation-Regulated Chemokine/CC Chemokine Ligand 20 Is Differently Affected by Cathepsin B and Cathepsin D Processing

Reagents, cell lines, and bacteria

Cath-B, Cath-D, cathepsin H (Cath-H), and antisera to human Cath-B or Cath-D were obtained from Athens Research and Technology. Biotinylated goat anti-human CCL20 was from R&D Systems. IL-1α, IL-1β, and TNF-α were obtained from PeproTech. Synthetic chemokines were prepared as described previously (34). CCL20_59–70 was prepared by C. Servis (Protein and Peptide Chemistry Facility, University of Lausanne, Switzerland). Pasteurized human plasma protein was provided by ZLB Bioplasma. Chondroitin sulfate (CS), other glycosaminoglycan (GAG) components, benzoylxycarbonyl (Z)-Arg-Arg-AMC hydrochloride (substrate for Cath-B), leupeptin and pepstatin A were purchased from Sigma-Aldrich. d-F-S(benzoyl)-F-F-A-A-pAB (substrate for Cath-D) was from Bachem. CA-074, a specific inhibitor for Cath-B, was obtained from Serva. Aminopeptidase M was from Roche Applied Science. Human melanoma cell lines A375 (solid tumor metastatic line; ATCC CRL-1619), A2058 (lymph node metastatic line; ATCC CRL-11147), and SK-MEL-2 (skin metastatic line; ATCC HTB-68) were from American Type Culture Collection. Escherichia coli BL21 was obtained from S. Didichenko (Institute of Immunology, Bern, Switzerland).

Chemokine cleavage by cathepsins and characterization of cleavage products

Five micromolar chemokines were incubated with 50 ng of Cath-D or Cath-B for 90 min at 37°C in 50 μl of buffer I (50 mM sodium citrate, 50 mM NaCl; pH 4.0) or buffer II (PBS containing 4 mM EDTA and 2 mM l-cysteine; pH 6.8), respectively. In control experiments, protease inhibitors were preincubated with the enzyme for 3 min at 37°C. Reactions were stopped by heating to 90°C, and the chemokine cleavage products were separated by SDS-PAGE in 10–20% Tris-Tricine gradient polyacrylamide gels and stained with Coomassie blue.

For cleavage site determination, the proteolytic fragments were acidified with trifluoroacetic acid and then loaded onto a reversed-phase C2/C18 column (Amersham Biosciences). Peptides were eluted with a linear gradient of 0–80% acetonitrile in 0.1% trifluoroacetic acid. The molecular masses were determined by MALDI-TOF mass spectrometry using a Voyager-DE STR system. The cleaved scissile bonds were then confirmed by Edman N-terminal sequencing.

Chemokine binding to GAG

Ninety-six-well enzyme immunoassay/radioimmunoassay (EIA/RIA) plates (Costar) were coated overnight at 4°C with different concentrations of GAG. The wells were washed with PBS containing 1 mM CaCl₂ and 1 mM MgCl₂ (PBS⁺), and then blocked with 5% FCS for 2 h at room temperature. CCL20 in PBS containing 1% FCS was added and incubated for 2 h. After washing, the wells were treated with biotinylated goat anti-human CCL20 (0.55 μg/ml) in PBS⁺ containing 1% FCS and 0.05% Tween 20 for 1 h, followed by incubation with streptavidin-conjugated alkaline phosphatase (Promega, Catalys AG). To quantify the reaction, Blue Phos (Kirkegaard Perry Laboratories Bioreba) was added, and the OD₅₉₅ was read in a microtiter plate reader. To determine the effect of Cath-D or Cath-B on chemokines bound to GAG, the wells were coated with GAG, washed as described above, and then blocked with 5% BSA. CCL20 in PBS⁺ containing 0.05% Tween 20 and 1% BSA was added to the GAG-coated wells and incubated for 2 h. After washing, the wells were incubated overnight at 37°C with 0.25 μg/ml Cath-D or Cath-B, and washed again, and CCL20 was probed with biotinylated goat anti-human CCL20. Bound CCL20 was then measured using streptavidin-conjugated alkaline phosphatase and Blue Phos as described above.

Intracellular calcium ([Ca²⁺]_i) mobilization

[Ca²⁺]_i changes were measured in CCR6-expressing mouse 300-19 pre-B cells (35) loaded with 0.1 nmol of fura 2-AM per 10⁶ cells as described previously (36).

Cell migration

Chemotaxis was assayed in 48-well chambers (Neuro Probe) using 3-μm pore size polycarbonate filters (Osmonics). Briefly, chemokines in chemotaxis buffer (RPMI 1640, 20 mM HEPES (pH 7.2), containing 1% pasteurized human plasma protein) were added to the lower wells, and 10⁵ cells were resuspended in the same buffer to the upper wells and incubated for 90 min at 37°C. Cells migrating to the lower side of the filter were stained and counted in five high-power fields. Cells migrating into the lower wells were also counted. Studies were performed at least three times with triplicate wells per experiment.

To measure the effect of Cath-D on the activity of immobilized CCL20, the polycarbonate filters were first coated for 75 min at 37°C with 50 nM CCL20 in 50 mM sodium citrate, 50 mM NaCl (pH 4.0), and then incubated overnight with 0.1–1 μg/ml Cath-D or Cath-H as control. The filters were washed with PBS, air-dried for 30 min, and rehydrated for 20 min in fresh chemotaxis buffer. Then, the filters were mounted into the chemotaxis chamber as indicated above and 10⁵ cells were added to the upper wells. The lower chamber contained chemotaxis buffer with no chemoattractant. Chemotaxis was then performed as indicated above.

Antibacterial activity

To measure bactericidal activities, ∼1 × 10⁴ exponentially growing E. coli BL21 cells were incubated with different concentrations of synthetic CCL20 or CCL20_59–70 in 100 μl of 10 mM potassium phosphate buffer (pH 7.4) supplemented with 1% (v/v) Luria broth medium. The mixture was incubated for 3 h at 37°C (250 rpm), diluted in previous buffer, and plated on Luria broth agar plates as described previously (5). Surviving bacteria were quantitated as CFU/milliliter on plates after incubation at 33°C for 14 h. Bactericidal activity was expressed as the ratio of colonies counted to the number of colonies on a control plate (with no peptide). The LD₉₀ is the concentration of protein that reduces the number of colonies by 90%. All results represent mean values of duplicates from at least three independent experiments.

Patients

The three patients included in this study had histologically confirmed malignant melanoma with skin metastases removed by surgery. The protocol was approved by the Medical Ethics Committee of the Canton of Bern, Switzerland. All three patients signed informed consent before study entry.

Melanoma cells

Melanoma cell lines were seeded into 100 × 20-mm tissue culture plates (Sarstedt) and fed with fresh culture medium (DMEM supplemented with 15% FCS; Invitrogen) every 2–3 days until 80% confluence. After aspiration of culture medium followed by washing with PBS, cells were supplied with 6.5 ml of DMEM containing 10% FCS and treated for 1–3 days with IL-1α and TNF-α. When secreted chemokines were further purified, cells were stimulated with DMEM containing the proinflammatory cytokines and 2% FCS.

Skin metastases were surgically removed from melanoma patients. After removing adipose and fibrous tissue, the metastasis were cut into small pieces and single-cell suspension was prepared by carefully disrupting the tissue pieces between the frosted ends of two microscope slides. Contaminating lymphocytes and macrophages/monocytes were removed by incubating the cell suspension with purified monoclonal mouse anti-CD3 and anti-CD14 Ab (BD Pharmingen) as first-stage reagent and sheep anti-mouse IgG-coated Dynabeads (Dynal Biotech) as second-stage reagent. Depletion was performed according to the manufacturer’s indication. Contamination of the final melanoma cell preparation by CD3- and CD14-positive cells varied from 0.5 to 12% as assessed by FACScan analysis. Melanoma cells adhered overnight in a 6-well plate (Costar 3506) at a concentration of 1–2 × 10⁶/well in 15% FCS. Adherent and suspended cells were stimulated for 48 h with 2 ml DMEM/10% FCS containing IL-1α and TNF-α.

Purification of CCL20 from culture medium

A375, A2058, and SK-MEL-2 cells were grown for 24, 48, and 72 h as described above. CCL20 was measured in the conditioned medium by a sandwich-type ELISA using commercially available kit (R&D Systems). The detection range was typically between 20 pg/ml and 1 ng/ml. Supernatants were centrifuged for 20 min at 1800 × g and then passed through a 0.22-μm filter before applying to a 1-ml HiTrap heparin column (Amersham Biosciences) equilibrated with 40 mM KHPO₄, 20 mM NaCl, 1 mM EDTA, 1 M urea, and 5% glycerol (pH 7.2). Proteins were eluted stepwise with 0.2, 0.5, and 1 M NaCl. CCL20-containing fractions were identified by ELISA and further purified on a reversed-phase C2/C18 column (Amersham Biosciences) with a flow rate of 0.1 ml/min and a continuous gradient of 0–80% acetonitrile in 0.1% trifluoroacetic acid. CCL20-containing fractions were used for cell migration and Ca²⁺-releasing experiments.

Melanoma cell membranes

Cells at 80% confluence were washed with PBS and then detached from the culture dish with trypsin. For the isolation of membranes, cells were centrifuged for 5 min at 180 × g, washed with PBS, and resuspended in 2 ml of ice-cold hypotonic buffer containing 10 mM Tris-HCl and 0.5 mM MgCl₂ (pH 7.6). After sonication, 0.25 vol of 10 mM Tris-HCl, 0.5 mM MgCl₂, and 0.6 M NaCl (pH 7.6) was added to restore tonicity. Cell debris and nuclei were removed by centrifugation at 560 × g; the supernatant was adjusted to 5 mM EDTA and centrifuged at 150,000 × g. The membrane pellet was resuspended in 100 μl of PBS and stored at −20°C until used for CCL20 processing or Cath-B and Cath-D activity assays. Protein concentration was determined by the bicinchoninic acid assay (Pierce).

Cath-D and Cath-B activities

Cath-D activity was determined in cells and cellular fractions using a variation of the method described by Heylen et al. (37). Briefly, the Cath-D substrate d-F-S(benzoyl)-F-F-A-A-pAB (1.4 mM) was incubated for 4 h at 37°C with membranes or cytosol (25-μg protein equivalent) or conditioned medium (30 μl) in 0.1 M sodium citrate buffer (pH 4.0). This step was followed by incubation for 2 h at 37°C with aminopeptidase M (7 μg/ml). The reaction was stopped by adding TCA (14% v/v final). Released 4-aminobenzoic acid (pAB) was measured at 546 nm. The activity of Cath-D is expressed as picomoles of pAB produced per minute per 10⁵ cells. Cath-D specificity was confirmed using, in parallel assays, 2 μM pepstatin A, an inhibitor of aspartic proteases. Cath-B activity in cells and cellular fractions was determined as described previously (38). Shortly, cells were first washed with PBS, and 0.5 × 10⁶ cells were then transferred to 96-well plates and resuspended in PBS containing 4 mM EDTA and 2 mM l-cysteine (pH 6.8). Alternatively, 15 μg of membrane proteins or 200 μg of cytosolic protein or 200 μl of conditioned medium were used. The assay was initiated by the addition of 100 μM Z-Arg-Arg-AMC (Cath-B substrate) in the absence or presence of 1 μM CA-074 and followed for 50 min in a fluorescence plate reader with an excitation wavelength of 355 nm and an emission wavelength of 460 nm. To measure intracellular Cath-B activity, assay buffer was supplemented with 0.1% Triton X-100. Activity inhibited by CA-074 was considered to represent Cath-B activity. The activity of Cath-B is expressed as picomoles of 7-amino-4-methylcoumarin (AMC) produced per minute per 10⁵ cells. A standard curve of free AMC or pAB was used for quantification.

Immunoblotting

Proteins were resolved by SDS-PAGE (12 or 10–20% gradient polyacrylamide) and electroblotted onto polyvinylidene difluoride membranes. CCL20 was detected using polyclonal goat anti-human CCL20 (180 ng/ml), and Cath-B and Cath-D were detected with polyclonal rabbit anti-human Cath-B or Cath-D (1:5000) Ab.

Immunohistochemistry

Formalin-fixed, paraffin-embedded tissue samples were cut at 2–3 μm, dewaxed, rehydrated, and incubated with a polyclonal rabbit-anti-Cath-D Ab (DakoCytomation) in TBS containing 0.5% casein and 5% normal goat serum, for 60 min at room temperature. Sections were then washed with TBS. Next, a 1/500 dilution of biotinylated goat-anti-rabbit Ig antiserum (DakoCytomation) was applied for 45 min. The primary Ab was omitted in control sections. Thereafter, the sections were incubated with streptavidin-conjugated alkaline phosphatase (DakoCytomation). Staining was developed with fuchsin-naphthol AS-BI (Sigma-Aldrich) for 30 min, and counterstaining was performed with hematoxylin.

RNA preparation and RT-PCR

Total RNA from cultured melanoma cells was extracted with RNeasy Midi kit (Qiagen) according to the manufacturer’s instructions. After treatment with RNase-free DNase, total RNA (1 μg) was reverse transcribed using oligo(dT)₁₇ primer and AMV reverse transcriptase (Promega). Resulting first-strand DNA was amplified in a final volume of 20 μl containing 10 pmol of each primer and 1 U of Taq polymerase (Promega). The primers used were the following: +5′-accatgtgctgtaccaagagtttg-3′ and −5′-ctaaaccctccatgatgtgcaagtga-3′ for human CCL20; +5′-gtcaacggatttggtcgtatt-3′ and −5′-agtcttctgggtggcagtgat-3′ for human G3PDH. Amplification conditions were denaturation at 94°C for 30 s (5 min for the first cycle), annealing at 60°C for 30 s, and extension at 72°C for 30 s (5 min for the last cycle) for 35 cycles, and 27 cycles for human G3PDH. Amplification products (10 μl each) were subjected to electrophoresis on 2% agarose and stained with ethidium bromide.

In situ hybridization

Formaldehyde-fixed and paraffin-embedded melanoma tissues from 10 patients were retrieved from the archives of the Institute of Pathology, University of Bern (Bern, Switzerland). The human CCL20 cDNA in pBluescriptII KS vector was a gift from B. Moser (University of Bern). Digoxigenin-labeled riboprobes (Roche Applied Science) for CCL20 were transcribed in vitro from linearized, gel-purified plasmids using T7 polymerase (antisense cRNA probe) and T3 polymerase (sense cRNA probe). In situ hybridization was performed as previously described (39), except that sections were prehybridized (30 min, 50°C) and hybridized (overnight, 50°C) in DIG Easy Rib (Roche Applied Science), and washed at 55°C in 2× SSC, 1× SSC, and 0.1× SSC.

Statistical analysis

All results represent mean values of triplicates. Values of p were calculated by Student’s t test using Microsoft Excel software. Correlation coefficients were calculated by linear regression analysis. A value of p < 0.05 was considered statistically significant.

Processing of selected chemokines by Cath-D and Cath-B

We tested a panel of 24 chemokines as potential substrates for Cath-D and Cath-B (Table I⇓). In addition to the previously reported processing of CCL3, CCL4, and CCL21 (7), we now describe CCL20 and CCL27 as additional substrates for Cath-D. None of the CXC chemokines was processed by Cath-D. In addition to Cath-D, overexpression of Cath-B has also been associated with poor prognosis in cancer. We tested therefore whether chemokines might also be substrates for this protease. We found that Cath-B, when measured at pH 6.8, cleaved a large spectrum of chemokines, including all non-ELR CXC chemokines (lacking Glu-Leu-Arg sequence preceding the first cysteine) and 6 of the 14 analyzed members of the CC family. CCL20, CCL21, and CCL27 were the only chemokines that were cleaved by both proteases.

Time courses of CCL20 processing by Cath-D and Cath-B are presented in Fig. 1⇓A. Cleavage by both proteases led to stable products that were not further degraded after an incubation time of 6 h and 90 min, respectively. Cleavage of CCL20 by Cath-D occurred at different sites, and five fragments were isolated by reversed-phase C2/C18 column chromatography (Fig. 1⇓B). Analysis of these peptides by mass spectroscopy and N-terminal sequencing led to the identification of three N-terminal peptides, CCL20_1–19, CCL20_1–52, and CCL20_1–55, an internal peptide, CCL20_23–55, a C-terminal peptide of 12 aa, CCL20_59–70 and full-length CCL20. CCL20_1–19 and CCL20_23–55 were held together by disulfides as demonstrated by MALDI analysis of this column fraction after reducing with DTT. Cleavage by Cath-B was very rapid and also complete and generated the distinct cleavage product CCL20_1–66 (Fig. 1⇓, B and C).

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FIGURE 1.

CCL20 processing by Cath-D and Cath-B. A, Time course of CCL20 processing by Cath-D and Cath-B. CCL20 (5 μM) was incubated with 50 ng of Cath-D or Cath-B at 37°C for the indicated times in buffer I (pH 4.0) or buffer II (pH 6.8), respectively. Pepstatin A (2 μM) or CA-074 (5 μM) were used as inhibitors for Cath-D and Cath-B, respectively (I). The cleavage products were separated by SDS-PAGE and stained with Coomassie blue. B, Reverse phase-HPLC profile of the cleavage products of CCL20 after Cath-D and Cath-B processing. C, CCL20 cleavage sites for Cath-D and Cath-B. The positions of the amino acids located at the cleavage sites are indicated.

Inactivation of CCL20 after Cath-D but not Cath-B cleavage

The degradation products of CCL20 were functionally tested by cell migration and intracellular calcium mobilization experiments. After incubation with Cath-D, CCL20 did not attract CCR6-transfected pre-B mouse cells. By contrast, exposure to Cath-B had no influence on the chemotactic activity of CCL20 (Fig. 2⇓A). Migration experiments were also performed in the presence of Cath-H, a protease that does not cleave CCL20. As expected, there were no significant differences between experiments with control buffer and buffer containing Cath-H (data not shown). We next tested the activity of the purified fragments resulting from Cath-D cleavage, CCL20_1–55 and CCL20_1–52. CCL20_1–55 was entirely inactive and could neither mobilize intracellular Ca²⁺ (Fig. 2⇓B) nor induce cell migration (C). Furthermore, we could demonstrate that CCL20_1–55 does not block the receptor or act as an antagonist, because subsequent addition of intact CCL20 yields a full Ca²⁺ response (Fig. 2⇓B, right trace). The shorter peptide CCL20_1–52 was inactive as well (data not shown). By contrast, the product of Cath-B cleavage, CCL20_1–66 retained full activity, mobilized intracellular Ca²⁺ (Fig. 2⇓B) and induced cell migration with similar potency as full-length CCL20 (Fig. 2⇓C). Overall, the above results show that Cath-D inactivates CCL20, whereas removal of four C-terminal amino acids by Cath-B has no functional consequences on CCL20 indicating that the extreme C-terminal part of this chemokine is not essential for proper CCR6 activation. We also assessed whether cleaved CCL20 might use an alternative receptor than CCR6. Transfected murine cells expressing CXCR2, CXCR3, CXCR4, CCR1, CCR5, and CCR7 were incubated with the cleavage products, CCL20_1–55 and CCL20_1–66, as well as intact CCL20 and monitored for calcium-releasing activity. None of these receptors responded to CCL20 or to its truncated products.

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FIGURE 2.

Influence of cathepsin treatment on chemotactic and calcium mobilization activity of CCL20. A, Migration of CCR6-transfected cells in response to CCL20 (control) and CCL20 after incubation for 90 min with buffer I (pH 4.0) and Cath-D or Cath-H, or with buffer II (pH 6.8) and Cath-B or Cath-H. Results are expressed as means ± SD of the numbers of cells observed per five high-power fields. No migrated cells were detected in the absence of chemoattractant. Statistically significant decreases in migrated cell number below the control values are indicated by asterisks (∗∗, p < 0.01). B, [Ca²⁺]_i-dependent fluorescence changes in CCR6-transfected cells by CCL20, CCL20_1–66, and CCL20_1–55. C, Migration in response to increasing concentrations of CCL20, CCL20_1–55, and CCL20_1–66. Results are expressed as means ± SD of the numbers of cells observed per five high-power fields.

It has been demonstrated that CCL20 has potent antimicrobial activity similar to β-defensins (40), which is ascribed to positive charges in the C-terminal part (5, 40). We therefore tested whether the C-terminal peptide CCL20_59–70 generated by Cath-D cleavage maintains the bactericidal activity of intact CCL20. Using a standard colony-forming assay with E. coli BL21 bacteria, we could show that CCL20_59–70 killed E. coli in a dose-dependent manner with a LD₅₀ of 3.2 μg/ml, whereas a concentration of 0.53 μg/ml full-length CCL20 was able to kill 50% of bacteria (data not shown). The activity of CCL20_1–66, the fragment obtained after cleavage with Cath-B, was not significantly different from that of intact chemokine (data not shown).

Binding of CCL20 to glycosaminoglycans does not prevent processing by Cath-D

Chemokine activity is further regulated by binding to GAGs located on endothelial cells and extracellular matrix. GAGs immobilize and enhance local concentrations of CCL20, thereby generating a solid-phase haptotactic concentration gradient. To determine whether binding to GAG may protect CCL20 from proteolytic processing by Cath-D, we first established that GAG molecules like CS, bind CCL20. Our microwell binding assay showed that CCL20 bound to CS-A-coated wells in a dose-dependent manner, whereas this specific increase in binding was not observed for the control GAG chondroitin (Fig. 3⇓A). Incubation of GAG with Cath-D did not affect subsequent CCL20 binding (data not shown). When GAG-immobilized CCL20 was treated with 0.25 μg/ml Cath-D, the chemokine no longer reacted with the polyclonal CCL20 Ab, suggesting proteolysis. It should be noted that in ELISA or dot-blot assays, polyclonal CCL20 Ab were able to recognize the CCL20 cleavage products after Cath-B but not Cath-D exposure (data not shown). Conversely, treatment with Cath-B, which is known to also have exopeptidase activity at acidic pH (41), did not significantly affect the recognition of CCL20 by anti-CCL20 at acidic nor neutral pH (Fig. 3⇓B). This result suggests that the last four residues of CCL20 are not included in the epitopes of the Ab. Incubation of polycarbonate filters with 50 nM CCL20 created a haptotactic chemokine gradient, which was able to attract CCR6-transfected cells (Fig. 3⇓C). This gradient was destroyed when the CCL20-coated filters were incubated with 0.1 μg/ml Cath-D for 16 h. Conversely, incubation with Cath-H did not inhibit migration of the CCR6 cells (data not shown). We did not test the effect of Cath-B on the haptotactic gradient of CCL20, because CCL20 activity was not affected after cleavage with this protease. As expected, a haptotactic chemokine gradient of CCL20_1–66, the product of Cath-B processing, was able to attract CCR6-transfected cells, whereas CCL20_1–55, the product of Cath-D processing, was inactive (data not shown). Taken together, these experiments indicate that Cath-D processes not only soluble CCL20 but also CCL20 attached to GAGs, which is the mostly likely condition in vivo.

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FIGURE 3.

GAG-bound CCL20 is cleaved by Cath-D. A, Binding of CCL20 to CS-A. EIA/RIA plate wells were coated with 20, 50, or 100 μg/ml CS-A, or chondroitin as control. After blocking, increasing concentrations of CCL20 (25, 50, 100, or 150 ng/ml) were added to the wells and incubated for 2 h. The binding of CCL20 to GAG was assessed by ELISA. Results are expressed as the mean of OD ± SD. Statistically significant increases above 25 ng/ml CCL20 concentration values are indicated by asterisks (∗, p < 0.05; ∗∗, p < 0.01; and ∗∗∗, p < 0.001). B, Cath-D treatment inhibits recognition of CCL20 by anti-CCL20 Ab. EIA/RIA plate wells were coated with 100 μg/ml CS-A. After blocking, 150 ng/ml CCL20 was added to the wells, incubated for 2 h, and washed before treating with Cath-D or Cath-B (0.25 μg/ml) for 16 h at 37°C. CCL20 was then assessed by ELISA. Controls with buffer only, are indicated. The values are expressed as the relative OD of binding to CS-A compared with nonspecific binding to BSA. Statistically significant decreases are indicated by an asterisk (∗, p < 0.05). C, Haptotactic assays with CCR6-transfected cells. Polycarbonate filters were coated with 50 nM CCL20 and then incubated overnight with 0.1 μg/ml Cath-D, buffer I, or migration buffer (control). Results are expressed as means ± SD of the numbers of cells observed per five high-power fields. No migrated cells were detected in the absence of chemoattractant. Statistically significant decreases in migrated cells compared with control are indicated by an asterisk (∗, p < 0.05).

Expression of CCL20 in melanoma cell lines and human melanoma tissue

CCL20 is up-regulated in neoplastic human skin diseases, such as mycosis fungoides or under inflamed conditions, such as psoriasis and contact dermatitis (41, 42). So far, no expression of CCL20 in melanoma has been described; therefore, we investigated whether expression of this chemokine may also occur in human malignant melanoma cell lines and primary malignant melanoma of the skin. Total RNA was extracted from three melanoma cell lines, A375, A2058, and SK-MEL-2, from primary keratinocytes, the keratinocyte cell line NCTC 2544, and the epithelial cell line Caco-2, and subjected to RT-PCR analysis. Expression of CCL20 was evident in all cells (Fig. 4⇓A). Furthermore, CCL20 transcripts were also detected in paraffin-embedded sections of human malignant melanoma (Fig. 4⇓B). Keratinocytes of the epidermal layer served as internal positive control for CCL20 expression. CCL20 mRNA was detected in a small proportion of the melanoma cells of 6 of the 10 patients studied. Not all melanoma cells expressed CCL20, possibly due to the existence of functionally different subsets. Based on morphology, the CCL20 transcripts did not appear to be related to the presence of accumulated leukocytes.

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FIGURE 4.

Expression of CCL20 in melanoma cell lines and human malignant melanoma. A, RT-PCR analysis of CCL20 and G3PDH expression in melanoma cell lines A375, A2058, and SK-MEL-2. Positive control RT-PCR products were obtained from primary keratinocytes (Prim. Keratin.), human keratinocyte cell line (NCTC 2544), and Caco-2 cells. A 100-base DNA ladder (ladder) and a negative control (no cDNA; control −) are shown. B, Localization of CCL20 transcripts in human malignant melanoma by in situ hybridization. Hybridization signals of antisense CCL20 riboprobes are identified in keratinocytes and melanoma cells. No signals were detected in serial sections hybridized with the sense probe.

To test whether CCL20 transcripts are translated into a functional protein, we analyzed CCL20 secretion of human melanoma cell lines and assessed its activity. Resting melanoma cells produced relatively low amounts of CCL20. However, treating the cells for 48 h with the proinflammatory cytokines IL-1α (10 ng/ml) and TNF-α (50 ng/ml) increased CCL20 levels 3- to 50-fold in all cell lines (Fig. 5⇓A). CCL20 from conditioned medium of stimulated A375 and SK-MEL-2 cells was partially purified using heparin affinity chromatography and reversed-phase HPLC and tested for biological activity. CCL20 secreted by melanoma cells mobilized intracellular Ca²⁺ (Fig. 5⇓B) and was also able to induce cell migration with similar potencies as synthetic CCL20 (Fig. 5⇓C). Interestingly, melanoma cell lines did not secrete the skin-specific chemokine, CCL27, even upon stimulation with IL-1α, TNF-α, and IL-1β either alone or in combination (data not shown).

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FIGURE 5.

Secretion of active CCL20 by melanoma cell lines. A, CCL20 in culture supernatant of melanoma cell lines after stimulation with IL-1α and TNF-α. Confluent A375, A2058, and SK-MEL-2 melanoma cells were stimulated for 24, 48, or 72 h with 10 ng/ml IL-1α and 50 ng/ml TNF-α in culture supernatant supplemented with 10% FCS. Culture supernatant from unstimulated cells was used as control. Production of CCL20 was determined by ELISA. The results are the means ± SD of three experiments. Absence of error bar means that the error bar is within the symbol. Statistically significant increases in CCL20 production are indicated by asterisks (∗, p < 0.05; and ∗∗, p < 0.01). B, CCL20 from culture supernatant of stimulated A375 cells was partially purified by heparin-Sepharose column and reversed-phase chromatography. [Ca²⁺]_i-releasing activity was analyzed with CCR6-transfected cells. C, CCL20 from culture supernatant of stimulated A375 cells was partially purified by heparin-Sepharose column. CCL20-containing fractions were 350-fold concentrated relative to culture supernatant (Sn) and analyzed for cell migration using CCR6-transfected cells (CCR6⁺; ▴). Nontransfected cells (CCR6⁻; ○) were used as negative control and synthetic CCL20 (80 nM) as positive control (CCR6⁺; ▪). Results of three experiments are expressed as means ± SD of the numbers of cells observed per five high-power fields.

In a more physiological setting, we stimulated human metastatic melanoma cells for 48 h with IL-1α and TNF-α, and we observed CCL20 secretion up to 0.5 nM per one million cells. This findings suggest that melanoma cells may produce sufficient CCL20 to attract immune cells.

Cathepsins in melanoma cell lines and human melanoma tissue

To localize Cath-D and Cath-B activities with respect to the cellular compartment and the extracellular environment, we determined proteolytic activities of these enzymes in cellular membranes, cytosol, and conditioned medium of the melanoma cell lines A375, A2058, and SK-MEL-2 using d-F-S(benzoyl)-F-F-A-A-pAB as substrate for Cath-D and Z-Arg-Arg-AMC as substrate for Cath-B. Cath-D activity was detected in all compartments of SK-MEL-2 cells, and low activities were found in membranes of A2058 cells (p < 0.05) (Fig. 6⇓A). However no Cath-D activity was found in A375 cells. Immunoblots of membranes, cytosol, and conditioned medium with anti-Cath-D Ab were in line with these findings (Fig. 6⇓B). Coomassie blue staining demonstrated that equal amounts of proteins were loaded (Fig. 6⇓B).

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FIGURE 6.

Cathepsins in melanoma cell lines and human malignant melanoma. A375, A2058, and SK-MEL-2 melanoma cells were stimulated with IL-1α and TNF-α for 48 h before isolation of subcellular fractions. A, Upper panel, Membranes, cytosol, and conditioned medium were incubated for 4 h with 30 mM d-F-S(benzoyl)-F-F-A-A-pAB to assess Cath-D activity. Results are expressed as picomoles of pAB released per minute per 10⁵ cells. Cath-D activity was also measured in the presence of pepstatin A (data not shown). A, Lower panel, Intact cells, cells lysed with Triton X-100, membranes, and cytosol were incubated for 50 min with 100 μM Z-Arg-Arg-AMC to assess Cath-B activity. Results are expressed as nanomoles of AMC released per minute per 10⁵ cells. No Cath-B activity was detected in conditioned medium or in the presence of CA-074 (data not shown). Statistically significant increases in activities above controls in the presence of inhibitor are indicated by asterisks (∗, p < 0.05; and ∗∗, p < 0.01). B, Membranes, cytosol, and conditioned medium of A375 (1), A2058 (2), and SK-MEL-2 (3) cells were immunoblotted with anti-Cath-D or anti-Cath-B Ab. Purified Cath-D and Cath-B from human liver were used as controls (Cathepsin). A Coomassie blue staining of similar samples is given as a control for equal loading of the samples. C, Cath-D was immunochemically detected in paraffin sections of human melanoma (left panel); the negative control was obtained by omitting the primary Ab (right panel).

As expected, Cath-B activity of melanoma cells was mainly associated with membranes. However, activity was not detected at the cell surface but was associated with intracellular membranes, as demonstrated by experiments with intact and Triton X-100-permeabilized cells (Fig. 6⇑A). Immunoblots with anti-Cath-B Ab revealed that A375 membranes contained a Cath-B doublet at 25/27 kDa (nonglycosylated and glycosylated forms of the H chain protein, respectively). Only the upper band of this doublet was detected in A2058 membranes, whereas SK-MEL-2 cells displayed only the lower band. In addition, the SK-MEL-2 cells contained the 31-kDa isoform both in membranes and cytosol (Fig. 6⇑B).

Because Cath-D was found to be secreted into the extracellular milieu of SK-MEL-2 cells and processing of CCL20 by this protease modified its activity, we investigated Cath-D expression in human melanoma tissue by immunohistochemical staining. As shown in Fig. 6⇑C, Cath-D immunostaining (red signals) is detected in cytoplasma of melanoma cells and probably also in the extracellular microenvironment of the tumor. The cells of the epidermis are negative.

Cleavage of CCL20 by membranes of IL-1α- and TNF-α-stimulated melanoma cells

We then studied cleavage of exogenously added CCL20 by membranes and conditioned medium of three melanoma cell lines and compared it to the cleavage pattern where purified cathepsins were used. CCL20 was incubated with membranes or conditioned medium of IL-1α- and TNF-α-stimulated cells, and the cleavage products were separated by SDS-PAGE and stained with Coomassie blue. In the presence of Cath-D-specific buffer, the membranes from all melanoma cell lines processed CCL20, but cleavage was only partially inhibited by pepstatin A (Fig. 7⇓A), indicating the presence of non-pepstatin A-sensitive protease activity. Noticeably, pepstatin A had no influence of CCL20 processing by membranes of A375 cells, further confirming the results presented in Fig. 6⇑A, which demonstrate that these cells do not express Cath-D. We then tested whether the non-pepstatin A-sensitive protease activity would depend on Cath-B, which is known to have exopeptidase activity at acidic pH (Fig. 7⇓B, left panel). SK-MEL-2 membranes were incubated with CCL20 in the presence of CA-074 and/or pepstatin A. Results of Coomassie blue staining show that cleavage of CCL20 was totally blocked when both inhibitors were present (Fig. 7⇓B) and establish that Cath-D (and/or Cath-D-like proteases) and Cath-B are the only proteases involved in the processing of CCL20 at pH 4.0. Pepstatin A-sensitive proteases degraded CCL20 totally, whereas the cleavage by Cath-B was limited. Such limited proteolysis was also observed when membranes of A375, A2058, and SK-MEL-2 cells were incubated at neutral pH (Fig. 7⇓C). Because processing was inhibited by the specific Cath-B inhibitor CA-074, these results suggest Cath-B to be the only protease involved. Conditioned medium of stimulated SK-MEL-2 cells processed CCL20 in a pepstatin A-dependent manner to the same truncation products as purified Cath-D (Fig. 7⇓D), which was verified by mass spectrometry. The migration experiment presented in Fig. 7⇓D, right panel, further supports the view that conditioned medium of melanoma cells inactivates CCL20. CCL20 was incubated for 8 h with conditioned medium of SK-MEL-2 cells in the presence and absence of pepstatin A, and aliquots were then used for migration experiments. In the presence of pepstatin A, no CCL20 degradation took place and only 5 nM was necessary to induce a maximal cell migration response; 50 nM, however, was necessary for a maximal response when incubation was performed in conditioned medium with active Cath-D. All of these results strongly point to Cath-D as the CCL20-inactivating protease in conditioned medium.

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FIGURE 7.

Cleavage of CCL20 by melanoma cell lines. A, Processing of CCL20 by membranes of A375 (1), A2058 (2), and SK-MEL-2 (3) cells. CCL20 (5 μM) was incubated for 4 h at 37°C with membrane equivalents of 3 × 10⁵ cells in 50 mM sodium citrate, 50 mM NaCl (pH 4.0), and pepstatin A as indicated. The cleavage products were separated by SDS-PAGE and stained with Coomassie blue. The molecular masses of intact CCL20_1–70 and CCL20_1–55 after Cath-D cleavage are 8025 and 6224 Da, respectively (arrows). B, Processing of CCL20 by purified Cath-B (left panel) or Cath-D (middle panel) for 1.5 h or membranes of SK-MEL-2 cells (right panel) for 4 h at pH 4.0 in presence of CA-074 and pepstatin A as indicated. The molecular masses of intact CCL20_1–70, CCL20_1–66 after Cath-B cleavage and CCL20_1–55 after Cath-D cleavage are 8025, 7552, and 6224 Da, respectively (arrows). C, As in A but incubation was with PBS containing 4 mM EDTA, 2 mM l-cysteine, 0.1% Triton X-100 (pH 6.8). D, Processing of CCL20 by conditioned medium of SK-MEL-2 cells. Conditioned medium was concentrated (2.5×) and incubated with CCL20 (5 μM) at pH 4.0 for the indicated times and in the presence of pepstatin A (left panel). Coomassie blue staining of CCL20 incubated for 8 h with purified Cath-D or conditioned medium is shown in the middle panel. The right panel shows migration in response to increasing concentrations of CCL20 after incubation for 8 h in conditioned medium with or without pepstatin A. Results are expressed as means ± SD of the numbers of cells observed per five high-power fields. A representative experiment of three is shown. IB, Immunoblotting; Cond. medium, conditioned medium.