Differential Effects of Interleu kin 1-a (IL-1a) or Tumor Necrosis Factor-a (TNF-a) on Motility of Human Melanoma Cell Lines on Fibronectin
Sybren K. Dekker,* Jacqueline Vink,* Bert Jan Vermeer,t Jan A. Bruijn,:j: Martin C. MihmJr.,* and H. Randolph Byers* 'Dermatopathology Division, Department of Pathology, Harvard Medical School, and Massachusetts General Hospital, Boston, Massachusetts, U.S.A.; and Departments of tDermatology and :j:Pathology, University of Leiden, Leiden, The Netherlands
Interleukin-la (IL-la) and tumor necrosis factor-a melanoma cell lines tested; downward shift of the a 2 , a 3 , a4 , (TNF-a) induce a motogenic response in a number of benign and PI integrin subunits was detected among three of the and malignant cells. We examined the chemokinetic effects melanoma cell lines as were upward shifts of the a 4 , as, and of these cytokines on the cell migration of four melanoma a6 integrin subunits among three of the melanoma cell lines. cell lines on fibronectin using modified Boyden chambers IL-la and TNF-a induced enhanced migration on fibronec and video-time lapse analysis. Flow cytometry analysis of tin in one of the melanoma cell lines and were related to an
IL-l receptors, TNF receptors, and shifts in PI integrin ex upward shift in the a 4 and as integrin subunit expression. pression were correlated with the effects of these cytokines Taken together, the findings indicate that expression of a on cell migration on fibronectin. The four melanoma cell particular receptor for IL-l or TNF does not necessarily sig lines exhibited heterogeneous expression of types I and II nal a motogenic response in melanoma cells, but induces IL-l receptors as well as p60 TNF receptors. Scant p80 TNF heterogeneous shifts in PI integrin expression. However, up receptor expression was detected on only one cell line. Three regulation in a 4 and as integrin subunits appears to relate to of four melanoma cell lines demonstrated type I IL-l recep enhanced migration on fibronectin. Key words: migration/ tors by Western blotting. IL-la and TNF-a induced hetero tumor necrosis factor/interleukin 1/integrin. ] Invest Dermatol geneous modulation of PI integrin expression in the four 102:898-905, 1994
alignant cells, like many benign cell types during molecules in cell adhesion and motility in normal and malignant embryogenesis, exhibit a capacity for cell migra cells [6]. Tumor cell invasion requires attachment to and migration tion. Regulation of cell motility is complex, and through a number of extracellular matrix molecules [7], and inte involves an interplay of cytoskeletal proteins, ad grins belonging to the very late antigen (VLA) family, those with a hesion molecules, ion fluxes, and cell-surface re common P! subunit, are known to exhibit changes in levels of ex Mceptors that relay signals to set the cell in motion. Several cytokines pression on neoplastic transformation [B,9] . Cell transformation or that have potent motogenic properties have recently been identified exposure to certain cytokines have in common an alteration in cell [1] . Lymphocytes, fibroblasts, and endothelial or tumor cell produc behavior on extracellular matrix (ECM) proteins that is the result of tion of cytokines may have dramatic effects on the motility of ma shifts in integrin expression [2,4,9,10]. lignant cells, providing the malignant cells express the appropriate The expression of P! integrins in human melanoma is heteroge receptors. Exactly how cytokines signal motility is unclear; how neous [11 J and the pattern of expression dictates differential adhe ever, recent findings indicate that interleukin (IL)-1P, tumor necro sion [4,12] or migration behavior on specific extracellular matrix sis factor (TNF)-a, and transforming growth factor (TGF)-P are molecules [13]. Exposure of melanoma cells to TNF-a has recently able to modulate integrin expression on a variety of cells [2-5]' been reported to result in their increased adhesion to fibronectin [4]. Integrins are a family ofheterodimeric transmembrane receptors Specifically, upregulation of the asP! integrin was reported. This
composed of an a and P subunit and are believed to be important integrin, along with a 4P! and a.jJ!, is known to bind the extracellu lar matrix protein fibronectin [14 -17]. We hypothesize that a cytokine-induced shift in asP! expression Manuscript received June 2B, 1993; accepted for publication January 14, may relate to motogenic properties of certain cytokines. Therefore, 1994. we tested the effect ofTNF-a as well as another known motogenic Reprint requests to: Dr. H. Randolph Byers, Immunopathology, Massa cytokine, IL-1a, on the migration of a number of melanoma cell chusetts General Hospital, 100 Blossom St., Boston, MA 02114. lines using micropore transmembrane and video time-lapse migra Abbreviations: (d)BSA, (denatured) bovine serum albumin; DOC, deoxy tion assays. Heterogeneity of response among the cell lines could cholic acid; FN, fibronectin; IL-1a, interleukin 1-alpha; IL-1R, interleukin- relate to differential expression of cytokine receptors. Therefore, we 1 receptor; MFI, mean fluorescence intensity; MM-AN, metastatic mela noma cell line-code; MM-RU, metastatic melanoma cell line-code; p60 used indirect immunofluorescence and flow cytometry utilizing TNF-R, p60 tumor necrosis factor receptor; pBO TNF-R, pBO tumor necro monoclonal antibodies against the two recently described types of sis factor receptor; PM-WK, primary melanoma cell line-code; RPM-EP, TNF receptors-p60 and pBO TNF-R [1B-21]-and the two recurrent primary melanoma cell line-code; TRIS, tris-hydroxymethyl types ofIL-l receptors-types I and II IL-1R [22,23]. In addition, amino methane; VLA, very late antigen. Western blotting with type I IL-l R was performed on the four cell
0022-202X/94/S07.00 Copyright © 1994 by The Society for Investigative Dermatology, Inc. 898 VOL. 102, NO.6 JUNE 1994 IL-la OR TNF-a AND MOTILITY IN MELANOMA 899
40~------~1 14.------. • Control (Fibronectin coated) • Control (Fibronectin coated) ..... 1'21 + TNF-a. 12 -..c II +TNF-a. 1IIIII+IL-1a. 30 (/) III + IL-1 a. E c 10 ~
Figure 1. Representative video time-lapse migration assay of the four mel Figure 2. Representative modified Boyden chamber transmembrane mi anOIna cell lines on fibronectin following exposure to TNF-a and IL-1a. gration assay of the four melanoma cell lines on double-sided fibronectin Whereas the PM-WK, RPM-EP, and MM-AN cell lines exhibit no signifi coated micropore filters after exposure to TNF-a and IL-1a. No significant cant increase in motility, a significant increase in mean migration rate of the effects are seen with either cytokines on the migration of the PM-WK. MM-RU cell line is observed with both TNF-a ("p < 0.005) and IL-1a RPM-EP. or MM-AN cell lines; however, both TNF-a and IL-la signifi incubation (' p < 0.001). cantly enhance the transmembrane migration of MM-RU melanoma cells ('p < 0.001). lines tested. Finally, TNF-a- and IL-la-treated melanoma cell lines were examined by quantitative flow cytometry to determine control MoAb anti-human leukocyte antigen (HLA) class I (W6/32) was whether cytokine-induced motogenic effects related to specific obtained from American Type Culture Collection. Negative control MoAb shifts in integrin expression. included an anti-Fe receptor antibody [26], fluorescein-conjugated anti mouse (or anti-rat) immunoglobulin (Ig)G alone [27], Fe fragment (Rock land Inc.), or unrelated mouse IgG isotype-matched fluorescein-conjugated MATERIALS AND METHODS antibodies (Becton-Dickinson, Mountain View, CAl. Melanoma Cell Lines and Culture Conditions The primary mela noma cell line PM-WK, isolated from the radial growth phase, the recurrent Transmembrane Cell Migration Through Double-Sided Fibronec melanoma cell line, RPM-EP, from the vertical growth phase, and the tin-Coated Micropore Membranes Both sides of the modified Boyden metastatic cell lines, MM-AN and MM-RU, from lymph nodes. were iso chamber po~ycarbonate membranes (Transwell 24, 8-,um pore, 3422; Co lated in permanent culture and maintained using standard culture media star, Cambndge, MA) were coated and washed three times with PBS as with 2% fetal bovine serum and 8% newborn bovine serum as previously described above. Cells of each melanoma cell line culture were added to each s described [24]. upper chamber unit of the transwell plate at 1 X 10 cells and cultured for 24-48 h with or without IL-1a (1000 U/ml) or TNF-a (5 ng/ml; 100 Extracellular-Matrix Coating Procedure Microcover glasses used in U/ml). Each membrane was then washed with PBS. fixed for 10 min inl0% the cell migration assay (see below) were coated with fibronectin (FN) or formalin. and incubated for 5 min with 0.5% Triton X-I00. After cotton larninin (LN) (Collaborative Research Inc .• Bedford. MA) diluted in phos swab removal of upper chamber melanoma cells. the cells on the bottom side phate-buffered saline (PBS) to 10,ug/ml for 1 hat 37'C following estab were stained with Gill's hematoxylin. All membranes were analyzed with an lished techniques [25]. Overnight coating with 20 mg/ml heat-denatured Image analYSIS system (Microcomp. Southern Micro, Atlanta. GA), which bovine serum albumin (dB SA) at 4 'C was performed to block nonspecific was used to obtain densitometry readings of four different 7-mm2 areas binding sites. The upper and bottom side of the micro pore membranes in mcorporatmg nearly the entire membrane, using the 2 X objective. Stained modified Boyden chambers (see below) were similarly coated with FN and ~ells r ~du~e pixel intensity; therefore. the reciprocal of the integrated pixel dBSA. Throughont the paper and figures. FN/dBSA-coated snbstrates are mtenslty IS proporti.onal to the number of migrated cells. Controls versus terrned FN substrates. cytokme-treated urnts were analyzed using the Student t test.
cytokines and Monoclonal Antibodies Human recombinant IL-la Video Time-Lapse Cell Migration Assay Subconfluent cultures of the was a generous gift of Hoffmann-LaRoche Inc. (Nutley, NJ) and human melanoma ce!l lines were obtained in suspension after trypsinization or TNF-a (catalogue number 40066) was purchased from Collaborative Bio ethylenedlanune tetraacetic acid (EDTA) treatment. Approximately 4 X medical Products (Bedford, MA). The mouse anti-human type I IL-IR !O' cell.s were. plated in the center of each FN-coated coverslip and incubated (Catalogue number 1592-01). type II IL-IR (Catalogue number 1994-01), m medIUm Without cytokine or medium containing 1000 U/ml IL-la or 5 and anti-human p60 TNF-R (Catalogue number 1995-01) and rat anti ng/ml (100 U/ml) TNF-a. The concentrations used were derived from human p80 TNF-R (Catalogue number 1888-01) monoclonal antibodies titra~ion assays that elicited maximal motility response. Cell migration was were purchased from Genzyme (Cambridge. MA). The fluorescein-conju studied during similar 3-h intervals after 24. 48, and 72 h of control or gated goat anti-rat (Catalogue number 311-1202) was purchased from cytokine incubation using a Nikon Diaphot inverted microscope with a lO X Rockland Inc. (Gilbertsville, PAl and the fluorescein goat anti-mouse (Cata phase-contrast objective and attached mcubator as described previously logue number 55493-12110081) from Cappel Laboratories (Durham. NC). [24,.26]. Briefly, the cell s were maintained at 37°C in a 5% CO2/95% air The monoclonal antibody (MoAb) agai nst the extracellular domain of the e~IVuonment and the cell migration recorded using a Dage-MTI 65DX common PI subunit (AIIB2) of the VLA family and function blocking MoAb Video camera and a Hitachi TLC1550 time-lapse video cassette recorder. For against the as integrin subunit (BIIG2) were a kind gift of Dr. Caroline H. each experimental condition. migration of at least 20 cells was analyzed. Damsky (University of California, San Francisco. CAl. Dr. Martin Hemler Data were analyzed as described above. (Dana-Farber Cancer Institute, Boston. MA) kindly provided the MoAb to the a l subunit (Ts2/7), which binds to the extracellular domain but does not Immunofluorescent Staining for Types I and II IL-1Rs and p60 and functionally block cell adhesion, and the rat MoAb to the a6 subunit (GoH3) p80 TNF-Rs Melanoma cells were plated in eight-well tissue tek was a generous gift of Dr. Arnoud Sonnenberg (Central Laboratory of The chambers (Nunc. Naperville. IL). Following 24-h incubation. cells were Netherlands, Amsterdam, The Netherlands). The function-blocking fixed for 2 min with 4% paraformaldehyde in PBS. To block nonspecific MoAbs against the a4 (P4G9), a2 (PIE6). and a3 (PIB5) integrin subunits binding sites. cells were incubated for 30 min at 37'C with 50 tl12% BSA in were purchased from Telios Pharmaceuticals. Inc. (San Diego. CAl. Positive PBS, briefly rinsed with PBS, and incubated for 30 min at 37' C with 50,u1 of 900 DEKKER ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
mouse/anti-rat IgG alone, or incubation with the appropriate rat or mouse IgG isorype-FITC antibodies. Positive controls included incubation of nor mal peripheral blood lymphocytes and monocytes iso lated by Ficol gradient separation with all four anti-receptor MoAbs tested. Samples were analyzed with a Becton-Dickinson Facscan cytometer.
Western Blot Analysis of Type I IL-lRs in Four Melanoma Cell Lines Melanoma cel ls were detached by scraping into a 15-ml tube and spun at 1500 X g for 5 min. Human T-lymphocytes, a kind gift of James Kurnick, were cultured in suspension and pelleted at 1500 X g. The cel ls were lysed in 0.5 ml RIPA buffer (150 mM NaCl, 1.0% Nonidet P-40, 0.5% deoxycholic acid, 0.1 % sodium dodecyl su lfate [SDS), 50 mM TRIS; pH B.O). Cell lysate were added to sample buffer and loaded onto SDS 4-12% polyacrylamide gradient gels. Electroclution onto nitrocellulose filters was performed in 50 mM TRIS, 380 rnM glycine, 20% methanol, and 0.1 % SDS. The filter was removed and dried, incubated for 30 min in 2.5% skim milk to block nonspecific binding sites, and then incubated for 2 h with the MoAb diluted in 0.5% skim milk at a concentration of20 pg/ml. Following two washes in TRIS-buffered saline (TBS) with 0.1 % Triton X-tOO and one in TBS, each for 15 min, the filter was incubated with the secondary anti body and developed using avidin-biotin peroxidase (ABC kit; Vector Labo ratories, Burlingame, CAl.
Flow CytoDlctric Evaluation of Expression of /1. Integrins After IL-la or TNF- the primary antibody against the specific cytokine receptor (see Monoclol/a! RESULTS Alltibody section), diluted 1 : 50 in 0.5% BSA in PBS. Preparations were then Effect ofIL-la or TNF-a on Motility of Melanoma Cells on incubated for 30 min at 37 °C with 50 III of a 1 : 40 dilution in 0.5% BSA in PBS of the appropriate secondary fluorescein-labeled antibody (see MOl/oe/o- FN-Coated Substrates After 48 h exposure to IL-la orTNF-a, 110/ Alltibody section) following three washings in PBS. Negative controls the mean migration rates on FN of the four cell lines were compared were incubated with the secondary fluorescein-labeled antibody alone or to cytokine nonexposed cells using the video time-lapse migration were previously incubated with an unrelated IgG isorype antibody. Finally system. Three of the melanoma celJ lines (PM-WK, RPM-EP, and the cells were embedded in mounting medium, placed under the 20 X MM-AN) did not show a significant increase or decrease in mean objective on a Nikon epifluorescent microscope (microphot), and photo migration rate, which ranged from 4 to 14 JLmjh (Fig 1). In con graphed with a fixed exposure time using 400 TMAX Kodak film. trast, the eel/line that had a high baseline migration rate (MM-RU; Flow Cytometry: Types I and U IL-lRs and p60 and pSO TNF-Rs 18 JLmjh) exhibited a significant increase in mean migration rate in Melanoma cells pl ated in T25 Corning tissue culture flasks (catalog number response to TNF-a (p < 0.005) and IL-Ia (p < 0.001). This re 25106, Falcon, Fischer Scientific) were cultured to subconfluence for 24, 4B, sponse of the MM-RU cell line was substrate specific; cytokine and 72 h in regular medium or medium containing 1000 U/ml IL-lfr or 5 treated cells that were plated on LN showed no significant increase ng/ml (100 U/ml) TNF-a at 37°C in a 95% air-50/o CO2 atmosphere. After in migration rate compared to controls. trypsinization or EDTA treatment, cells were washed in PBS, incubated for Similar findings were observed using the micropore transmem 30 min with fluorescein-labeled goat-anti-rat or anti-mouse antibody (t : 40 brane assay system (Fig 2). After 48 h, densitometry readings of dilution), and then incubated for 30 min at 4 ° C with MoAb against the rat anti-human TNF receptor pBO antibody or one of the mouse anti-human stained cells, proportional to the number of stained transmembrane antibodies against either the p60 TNF-R, the rype I IL-l R, or the rype II migrated cells, showed that TNF-a and IL-la had no effect on the IL-R. Negative controls included all cells pre incubated with the human IgG migration of the same three melanoma cell lines that did not re Fc fragment, incubation with the fluorescein isothiocyanate (FITC)-anti- spond in the video time-lapse assay. In contrast, the MM-RU cell VOL. 102, NO.6 JUNE 1994 IL-la OR TNF-a AND MOTILITY IN MELANOMA 901 type 11-1R type II 11-1R p60 TNF-R pao TNF-R PM-WK RPM-EP \'\~ " ~ / \ " ,./ .. <,., \, MM-AN MM-RU log Fluorescence intensity FigUre 4. Flow cytometric analysis ofIL-1Rs and TNF-Rs on four melanoma cell lines tested. Each melanoma cell line shows a distinct iii fry I . d II IL-l R, and p60 and p80 TNF-R expression. Ordinate is relative cell number. Thick lirze control secondary antibod onl II ' p~~ eo. 'pes anh anti-cytokine receptor MoAb. ' , . y y; 1111 Ifle, stammg Wit line exhibited a significant increase in transmembrane migration t~e four cy.tokine receptors (Fig 4). Cells that were detached by over 48 h in response to TNF-a (p < 0.001) and IL-la (p < either trypsmjEDTA or EDTA alone gave similar results to the data 0.001), similar to the video time-lapse assay results. on the attached fixed cells outlined above. In the PM-WK cell line, types I and 1.1 IL-1 Rand p80 TNF-R were not detected; however, a Immunofluorescent Staining of Types I and II IL-1Rs and low expressIOn of p60 TNF-R was observed. In the RPM-EP line, p60 and p80 TNF-Rs in Attached Melanoma Cell Lines In the type I IL-1R and p60 TNF-R exhibited low expression whereas direct immunofluorescent staining of attached melanoma cell lines ~pe ~I IL-.1R ~as highly expressed. No detectable p80 TNF-R was revealed heterogeneous expression of IL-l and TNF receptors. m this This pattern of expression compared well Whereas no receptors could be detected on one cell line (PM-WK; Ident1fie~ ~e lliin e. with the mdlre~t Immlll1ofluorescent staining of attached cells. The see representative photomicrographs in Fig. 3), the other lines ex MM-.AN cellime exhibited a pattern different from the RPM-EP hibited differential expression of types I and II IL-1Rs and p60 and lme, and also exhibited expression of receptors similar to the p80 TNF-Rs. The cell line RPM-EP exhibited slight staining for ~e ll unmunofluorescent staining pattern of the attached cells. The types the type I IL-IR, strong staining for the type II IL-IR, slight stain I and II IL-1Rs and p60 TNF-R exhibited low expression, whereas ing for the p60 TNF-R, and was also negative for the p80 TNF-R no p80 TNF-R was detected. Finally, MM-RU exhibited low to (Fig 3D). The cell line MM-AN exhibited slight staining for the moderate expression of types I and II IL-lR and p60 TNF-R and type I IL-IR, mild staining for the type II 1L-1R and p60 TNF-R, low but detectable p80 TNF-R. and negative staining for the p80 TNF-R (Fig 3P). Finally, the . The expressions of these cytokine receptors in the melanoma cell MM-RU cell line, which was the only cell line that demonstrated a lmes were using peripheral blood lymphocytes and mon • motility response to IL-1a and TNF-a, exhibited slight type I ~onfirmed ocytes, which served as positive controls. All four cytokine recep IL-l R staining, moderate type II IL-l Rand p60 TNF-R staining, tors were detected on these positive controls, with mean fluores and slight p80 TNF-R staining (Fig 3H). To better quantify the cence values rangin~ from relatively low expression of p80 TNF-R expression of these receptors, we performed flow cytometry as out to moderate expressIOn of type I IL-l R on lymphocytes (similar to lined below. that detected on the MM-RU cell line) and high expression of the Flow Cytometric Analysis of Types I and II IL-lRs and p60 P?O TNF-R to very high expression of type II IL-IR on monocytes and p80 TNF-Rs To verify the immunofluorescent findings on (sumlar to that detected on the RPM-EP cell line). the fixed, attached cells, we performed quantitative flow cytometric To study the kinetics of receptor expression following addition of analysis of all four cell lines following indirect immunolabeling of IL-l or TNF-a, flow cytometry was performed for each of the four 902 DEKKER ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY Q,. demonstrated slight or marked shifts in the melanoma cell lines, and ~ W Z ::J ;: , « a: '" '" included a marked upward shift in the (l'1 subunit induced by IL-1(l' , ::E , , CI.I CI.I ::E Q,. ::E ::E 0, 0, in RPM-EP, a marked downward shift in the a 3 subunit in MM Q,. a: ::E ::E l- I- AN, and a marked upward shift of the (l'6 subunit in MM-RU in duced by IL-1(l'. The latter two shifts are confirmed by a concomi tant marked downward shift of the PI subunit in the RPM-EP cell line and marked upward shift in the PI subunit in the MM-RU cell line following treatment with only IL-l but not TNF-(l'. Interest ingly, the marked upward shift in the a l subunit and the downward shifts of (l'4 and a 6 subunits induced by IL-l(l' in RPM-EP have the 100 - effect of canceling each other out and resulting in no detectable shift in the PI subunit expression. ... Type I IL-1R To study the kinetics of integrin expression following the addi tion of IL-1 or TNF-(l', flow cytometry was repeated on each of the 68 - four cell lines after 24, 48, and 72 hand cytokine-treated cells were compared to untreated cells over the same time period. We con 45 - firmed in the MM-RU cell line that at 24 and 48 h the (l'4 and (l's subunits demonstrated an upward shift in expression similar to that observed at 72 h with IL-l or TNF-a. Furthermore, the RPM-EP cell line showed a similar downshift in expression of only the (l'4 subunit with IL-1 , but not TNF-(l', after 24 h. The other two cell 2 3 4 5 6 lines showed no detectable shifts in these integrin subunits follow ing exposure to IL-l or TNF-(l' over the time course studied. Figure 5. Western blot of melanoma cell lines (Ialles 1-4) and cultured T To address whether increased or (l'sPl relates to enhanced lymphocytes (lanes 5,6) with monoclonal antibody against the type IIL-IR a 4PI receptor. Bands are identified at 80-85 kDa in RPM-EP (Iallc 2), MM-AN motility, we performed a series of experiments using function (Ialle 3), and MM-RU (lane 4) and co-migrate with bands in the T-Iympho blocking MoAbs against the (l'4 or as subunit. We found that en cyte lysates (lan es 5,6) . No band is detected in the PM-WKccllline (lane 1). hanced motility on FN in the time-lapse video image analysis sys tem induced by TNF-(l' (19 ± 8.0 ,urn/h) could be inhibited significantly by 10 - 20 ,ug/ ml of the function-blocking anti-(l'4 sub receptors following exposure of the cells to these cytokines after 24, unit (9.5 ± 7.5 ,um/h; p < 0.001) or the function-blocking anti-(l's 48, and 72 h. Compared to cytokine-untreated control cells after 24, subunit (11 ± 6.5 ,um/h; p < 0.001). Likewise, the IL-1(l' 48, and 72 h, a slight downregulation of the type I IL-IR was enhanced motility (21 ± 4.0,um/h) could be inhibited significantly detected at 24 h and was sustained during exposure to IL-l. How by the function-blocking anti-(l'4 (6.0 ± 4.0 ,um/h; p < 0.001) or ever, no detectable shifts of the type II IL-IR, p60 TNF-R, or p80 anti-(l's (7.5 ± 4.5 ,um/h; p < 0.001) MoAbs. TNF-R were observed with this cytokine. TNF had no effect on the DISCUSSION expression of all four receptors over the 3-d time course studied. The production of cytokines by lymphocytes, macrophages, and Detection of Type I IL-1R in Melanoma Cells by Western other cells is integral to the cellular immune response for control Immunoblot To confirm that the low to moderate expression of ling the growth or dissemination of tumor cells [29,30]. Certain type I IL-IR detected by flow cytometry correlated with the actual cytokines may induce alterations of cell-surface molecules on tumor receptor as characterized by Western blotting techniques, we char cells. This may playa key role in metastasis, as in the expression of acterized the expression of type I IL-IR on the four melanoma cell growth receptors and cell adhesion molecules that regulate cell lines using cultured T lymphocytes as a positive control (Fig 5) . We migration [6,7,31]. Melanoma cells are extraordinarily pleiotro did not detect type I IL-l R in the cell lysate of one of the melanoma phic, and the expression of receptors for cytokines that induce cell cell lines (PM-WK; Fig 5, lane 1); however, three of the melanoma migration may select malignant cells that undergo dissemination. cell lines demonstrated a band at 80-85 kilodaltons (kDa) (Fig 5, Tumor cells may mimic lymphoid cells in that they express certain lanes 2-4). This band co-migrated with a prominent band detected adhesion molecules to permit trafficking into distant tissue sites. in the T-Iymphocyte lysate (5 X dilution lysate, Fig 5, lane 5; 1 X Cytokine-activated endothelial cells express cell adhesion molecule lysate, Fig 5, lane 6). These findings correlated with the flow cy (VCAM)-l (also named INCAM-Il0) [32], which binds to the (l'4 tometry detection of this receptor in three of the four cell lines. subunit (VLA-4). Some melanoma-endothelial interactions use the VLA-Integrin Expression Following TNF-a and IL-la identical adhesion receptors to bind to endothelium as do the lym Treatment Following TNF-a or IL-la incubation we examined phocytic cells [33,34]. IL-1 increases melanoma metastasis in nude VLA-integrin subunit expression in the four cell lines using flow mice [35], and this may be due to enhancement of certain adhesion cytometry. We hypothesized that shifts in integrin subunits known molecules or cytokine receptors. Indeed, certain cytokines have to bind FN could account for the difference in melanoma cell mi been able to induce modulation of integrin molecules on normal and gration. Figure 6 is a composite of histograms ill ustrating shifts in malignant cells [2- 5]. The shifts in integrin expression are largely a 3 , (l'4' and (l's integrin subunits in the four cell lines following dependent on TNF-(l' and IL-1, and these shifts have been found to TNF-(l' or IL-l(l' incubation. The thick lined peaks represent con coincide with altered phenotypic adhesive behavior to FN [2,4] . trol fluorescence with the secondary antibody alone; the fine lines We addressed a number of questions regarding the role ofIL-1(l' and represent the histograms following TNF-(l' or IL-l(l' incubation. TNF-a on migration of a number of established cell lines. These TNF-(l' or IL-l(l' induced a downward shift in the (l'3 subunit in the cell lines show marked differential-intrinsic migration rates [24] PM-WK cell line. In contrast, TNF-(l' had no effect on integrin and exhibit heterogeneous integrin expression [13,26], as reported expression in the RPM-EP and MM-AN cell lines, whereas IL-la in other melanoma cell lines [11,12]. First, we asked whether IL-1a induced a marked downward shift of the (l'4 subunit in RPM-EP and or TNF-(l' are motogenic for these melanoma cell lines. Second, we a3 in MM-AN. On the other hand, MM-RU was the only cell line examined whether IL-1 or TNF receptors could be related to be with marked upward shifts in integrin subunits involved in FN havioral responses to these cytokines. Third, does IL-1(l' or TNF-(l' binding. Both TNF-a and IL-1(l' induced marked upward shifts in induce shifts in integrin expression? And finally, does this relate to a 4 and (l's subunit expression. Table I summarizes the heteroge alterations in motility? neous effects ofTNF-a or IL-l(l' on all the VLA integrin subunits in We found that the melanoma cell line tested had differential the four melanoma cell lines. Only a few other integrin subunits migration responses afer IL-l(l' or TNF-(l' incubation, using the two VOL. 102, NO.6 JUNE 1994 IL--1O' OR TNF-O' AND MOTILITY IN MELANOMA 903 IL-1 a TNF-a a3 a4 a5 a3 a4 a5 PM- WK ~JA .J.A I A lA RPM-EP IA 1;\ MM-AN I 1\ IA ~ MM-RU log Fluorescence Intensity Figure 6. R:epresentative experiment showi~g a?, a., and as .integrin subunit expression before. and af~er treatment with IL-la (lift fa.llels) orTNF-a (righ! pa nels). Ordmate IS relative cell number. Tlllck litle, control, mcubatlon m regular medIUm; tlllll litle, mcubatlon m medium co ntammg cytokme. different assays. These findings are in accordance with data showing receptors on a number of melanoma cell lines tested. The type I that IL-la or TNF-a are motogenic in a variety of malignant cells IL-1R was detected in the majority of the melanoma cell lines [1,36 - 38]. This response was most likely via the IL-1Rs or TNF tested, using flow cytometry, immunofluorescence, and Western Rs, therefore we characterized the IL-1Rs and TNF-Rs in all the blotting. The low fluorescence intensity on the melanoma cells for melanoma cell lines. There are at least two types of IL-l receptors, the type I IL-l Rs suggests that relatively few receptors are expressed the type I (80-90 kDa) [22] and type II (60- 68 kDa) [23] receptors. compared to the type II IL-lRs on the RPM-EP cell line. The These two types of IL-l Rs have been identified in a variety of cell presence of detectable type I IL-IR alone did not correlate to the types. We report the presence of a heterogeneous pattern of these IL-la-induced enhanced migration. For example, the RPM-EP Table I. Summary of Cytokine Effects on PI lntegrin Expression (Flow Cytometry Analysis Showing Mean Fluorescence Intensity Values of the Four Melanoma Cell Lines Tested)- a, a2 a) a. as a6 p, PM-WK Control 9.04 5.74 18.01 5.50 17.81 14.92 48.90 +TNF-a 7.87 (4.93) (15.25) 4.86 16.64 13.50 42.29 + IL-la 7.40 (4.19) (16.91) 5.52 17.20 13.51 45.88 RPM-EP Control 30.64 45.83 55.19 11.57 9.97 17.13 97.82 +TNF-a 22.22 39.63 56.20 13.19 9.60 14.49 96.08 + IL-la 43.32 40.38 62.85 (8.45) 11.08 (12.63) 96.40 MM-AN Control 25.24 14.54 31.15 8.06 7.45 11.52 74.20 +TNF-O' 24.13 17.24 29.40 5.06 6.6 1 12.35 69.27 +IL-la 25.30 15.53 (24.18) 6.59 6.88 10.92 (53.78) MM-RU Control 2.39 10.53 3.07 14.97 6.42 4.88 49.93 +TNF-a 2.39 10.65 2.85 29.72 9.96 7. 18 53.1 2 + IL-10' 2.43 11.65 1.23 29.75 13.59 8.02 67.71 • Kolmogorov-Smirnov two-sample test as outlined in Ma terials a"d Meth ods. Plait! ,,,,,,,bers, absent or minimal shift; italics, slight upward shift, and ita lics ill parentheses, slight downward shift; boMface, marked upward shift and boldface in parentheses, marked downward shift. All italics and bold """,bers > 99.9% confidence level (a = 0.001). 904 DEKKER ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY cell line also had detectable type I IL-IR, similar to the MM-RU cell into these cytokine-induced motogenic effects on melanoma cells line; however, only the MM-RU cell line responded dramatically to may help in understanding the molecular mechanisms of melanoma the IL-1a. Likewise, the presence or quantity of type II IL-1R did cell dissemination. not correlate with a motogenic response to the IL-la. Indeed, the type II IL-1R was expressed abundantly in the RPM-EP cells (two orders of magnitude more than nearly all other receptors on all the Part oftlris work was supported by gra nt CA-45587 frolll the NatiollalltlstiWtes of other cell lines), yet this cell line did not respond to IL-1a, com Health (HRB) alld a Fellowship gra llt of the DUlch Callcer FOIltJdatioll "SticiJtillg pared to the MM-RU cell line, which expressed relatively small KOtlillgitl Wilhe[milla FOllds" (SKD) . amounts of type II IL-1 R. These findings underscore the difficulty We are deeply illdepted to Rick Gra/lSteill alld Stefall Grabbe for their JlsefJlI in correlating expression of these cytokine receptors with a particu suggestions and comments, alld to Dallid Bachillsky, Gallg Zhellg, alld Katillka lar response. Both IL-1 receptor types belong to the Ig superfamily Kerstell for their i/lllQ[Jlable colltribut.iollS. and have a large cytoplasmic domain, presumably involved in signal transduction. 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