School of Biotechnology, Banaras Hindu University, Varanasi. India. Summary Cisplatin (CP), a widely used anticancer drug activates cells of the immune ...
Immunology and Cell Biology (1997) 75, 492^96
Cisplatin-stimulated murine bone marrow-derived macrophages secrete oncostatin M AJIT SODHl, SHISHIR SHISHODIA and ANJU SHRIVASTAVA School of Biotechnology, Banaras Hindu University, Varanasi. India Summary Cisplatin (CP), a widely used anticancer drug activates cells of the immune system to a tumoricidal state, atid thus functions as a potent biological response modifier. Expression of oncostatin M (OSM), a novel cytokine having a growth regulatory effect, was studied in bone marrow-derived macrophages treated with cisplatin. Supematants from CP-stimulated macrophages were found to be cytostatic for OSM-sensitive A375 melanoma cells. Immunoblot analysis with anti-OSM antibody revealed that expression of OSM in macrophages upon CP stimulation is a rapid process and within 30 min of CP treatment, a significant amount of OSM is secreted into the culture supernatant. These results therefore indicate that CP can stimulate murine bone marrow-de rived macrophages to produce OSM which can be implicated as one of the cytostatic/ cytocidal factors in the antitumour action of cisplatin-stimulated macrophages. Key words: bone marrow-derived macrophages, cisplatin, cytostasis, oncostatin M.
Introduction
Materials and methods
Cisplatin (a>dichlorodiamineplatinum (11); CP), a widely used anticancer drug, activates murine macrophages and NK cells to a tumoricidal state and thus functions as a potent biological response modifier.'^ Macrophages. one of the major effector cells with tumoricidal activity, produce a variety of cytotoxic/cytostatic factors upon CP treatment.^"^ Much interest has been centred on the growth regulatory polypeptides isolated from activated leucocytes that selectively inhibit the growth of cancer ceils, while not inhibiting the growth of surrounding normal tissue. Oncostatin M (OSM). a glycoprotein of 28 000 kDa. is a novel growth regulator that was isolated from serum-free supernatants of phorbol ester-treated U937 histiocytic leukaemia cells.^ It was originally identified by its ability to inhibit the growth of A375 melanoma and other solid tumours, but not normal human fibroblasts.^"" Further, it has been reported that OSM, in combination with some other cytostatic polypeptide growth inhibitors like IFN-y or TGF-pl, plays a significant role in the immune regulation of tumour growth.'' In this study, we were interested to know whether cisplatin-treated macrophages are induced for OSM release. It is observed that supematants from CP-stimulated bone marrow-derived macrophages (BMDM) were indeed highly cytostatic to A375 melanoma cells. It is further observed that expression of OSM in CP-stimulated macrophages is a rapid process and within 30-60 min of CP treatment, a significant amount of OSM is secreted by the macrophages.
Cell culture and reagents Inbred strains of BALB/c mice. 8-10 weeks old were used for obtaining BMDM. L929 (murine fibroblast), A375 (human melanoma) and U937 (human histiocytic lymphoma) were obtained from the National Tissue Culture Facility, Pune, India. These cells were cultured in RPMI-1640 medium containing 10% FCS, penicillin (!OOU/mL), streptomycin (lOOpg/mL), and gentamicin (20 ^ig/mL). Foetal calf serum was purchased from Biological Industries, Haemek, Israel. Anti-OSM (N-1) antibody was purchased from Santa Cruz Biotechnology Inc.. (CA. USA). Medium RPMI-1640. and other chemicals were purchased from Sigma Chemical Company. St Louis. MO, USA. [^Hl-thymidine (specific activity: 59.2 x 10'^ Bq/mmol) was obtained from the Board of Radiation and Isotope Technology. Bombay. India. Chemiluminescence kit was purchased from BoehHnger Mannheiin Gmbh. Mannheim, Germany, All die reagents were endotoxin free as determined by the Limulus amoebocyte lysate assay (sensitivity limit, 0.1 ng/mL).
Isolation and activation of BMDM Non-adherent bone marrow cells (NABMC) were cultured for 7 days in 24-well plates (A/S Nunc, Roskilde, Denmark) in medium containing L929 conditioned medium (M-CSF) (20% v/v) to obtain BMDM monolayers.'^ Macrophage monolayers were then incubated with medium alone or with respective treatments in serum-free conditions for different time intervals as shown in the results section. For control, medium or medium containing CP (10 pg/mL) were incubated in similar conditions.
Cell growth inhibition assay Correspondence: Dr Ajit Sodhi, School of Biotechnology, Banaras Hindu University, Varanasi 22 1(X)5, India. Received 6 April 1997; accepted 14 July 1997.
The assay was perfonned in flat bottomed 96-wel! plates (A/S Nunc). Human A375 melanoma cells were used a.s a sensitive indicator cell Thymocytes, L929 or A375 cells (3 x 10-' cells) in 0.1 niL of
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RPMI-1640 medium supplemented with 10% (v/v) FCS and antibiotics were plated in each well. Three hours later, 0.1 mL of test samples were added to each well. Plates were incubated at 37°C for 3 days. I^HJ-thymidine (18.5 x lO^ Bq/well) was added to each well for the final 6 h of incubation and [-Hl-thymidine incorporation was determined by liquid scintillation counting. Anti-OSM antibody was added in some experiments as indicated in the results.
Preparation of cell lysates and anti-OSM immunohlotting After stimulation, macrophages were wa.shed in situ with ice-cold PBS, then lysed in 50 pL of lysis buffer (20 mmol/L Tris-Cl. pH8/ 137 mmol/L NaCl/ 10% (v/v) glycerol/ 1% (v/v) Triton X-100/ 1 mmol/L Na^VO^ 2 mmol/L EDTA/ 1 mmol/L PMSF/ 20 pmol/L leupeptin containing aprotinin at 0.15 U/mL) for 20 min at 4°C.'-' Proteins were separated on 10% SDS-PAGE at 20 mA. The separated proteins were transferred to nitrocellulose (S h at 125 mA: for dot blot analysis. 2.5 pg protein/dot of test supematants and cell lysates were blotted onto a nitrocellulose membrane) and immunoblotted as described'^ with the anti-OSM antibody, incubated with second antibody, and visualized by the BM Chemiluminescence western blotting kit (Boehringer Mannheim).
Statistical analysis The statistical significance of difference between the test groups was analyzed by Student's /-test (two-tailed).
Results CP induced murine BMDM to produce OSM Bone marrow-derived macrophages treated with CP elicited enhanced production of OSM compared to macrophages incubated in medium alone. Supematants collected from macrophages treated with CP (10 )ig/mL) for 24 h were found to inhibit the proliferation of A375 melanoma cells, very much comparable to PMA-induced U937 cell supernatant, a known source of OSM (Table 1). In several experiments. 50% inhibition of [^H]-thymidine incorporation into A375 cells was observed with concentrations of supematants ranging from 10-25% (v/v). In contrast, neither undiluted supernatant nor supernatant concentrated 50-fold from untreated Table 2
Table 1 Growth inhibitory effect of cisplatin macrophage supernatant on A375 cell lines
(CP)-treated
Treatments
%' Inhibition
Conlrol RPMI-1640 CP Macrophage RPMI-1640 CP U937 RPMI-1640 PMA
[•'Hl-TdR incorporation (c.p.m.)
24 780 ± 832 23 236 ±731
06.23
20 346 ± 1099 11 206 ± 1096*
17.89 54.77
24 269 ±2013 12 956 ± 1196*
02.06 47.71
Bone marrow-derived macrophages and U937 (1 x 10" cells/well) were incubated with CP (lOpg/mL) or PMA (lOng/mL) for 24 h and 72 h. respectively. Supematants were collected and tested for their ability to inhibit ['Hl-thymidine incorporation into A375 cells. Values are representative of three independent experiments done in triplicate ± SD. ^P < 0.05, significantly different from respective control.
Kinetics of cytostatic effect of cisplatin (CP)-treated BMDM supematants against A373 cell lines
Duration of treatment
0 min 1 min 10 min 15 min 30 min 60 min 12 h 24 h
macrophage possessed significant anti-proliferative activity against A375 cells (data not shown). These results indicate that growth inhibitory factors are induced or increased in expression in macrophages upon CP treatment. Kinetic studies revealed that the significant inhibitory effect is expressed in the supernatant within 15 min of CF treatment to macrophages which increased up to 1 h and attained a plateau thereafter (Table 2). As OSM is reported to be heat and acid stable,** CP-treaied macrophage supernatant was heated at 56°C for 1 h to inactivate the heat-labile cytokines like TNF. IL-1 and its effect was studied on growth of different cells. It was observed that the heat-inactivated supernatant had a similar effect on A375 cells; it resulted in significant inhibition of proliferation. L929 murine fibroblasts showed proliferation with heat-inactivated supernatant which otherwise was found to be inhibited by treatment with normal CP-treated macrophage supernatant. The thymocyte proliferation with heat-inactivated test supematants was inhibited (Fig. 1). The growth inhibitory and stimulatory effects on A375 and L929 cells, respectively, were neutralized in the presence of anti-OSM antibody.
['^H]-TdR incorporation (c.p.m.) RPMI-1640 CP 12 875 ±673 12 038 ±823 11 936 ± 896
12 078 ±936 12 036 ±832 11 886 ± 736 11 983 ± 789 11 583 ± 963
11 036 ± 932 10 326 ±911 9576 ± 672* 7376 ±531* 7031 ±468* 7213 ±632* 7123 ± 656*
Inhibition RPMI-1640
CP
06.50 07.29 06.19 06.51 07.68 06.92 10.03
14.20 19.79 25.62 42.71 45.39 43.97 44.67
Bone marrow-derived macrophages (BMDM; 1 x 10^ cells/well) were incubated with 10 |ig/mL of CP for various durations. After incubation, the supematants were collected and were assayed for cytostatic effect on A375 cell lines sensitive to oncostatin M. Values are representative of three Independent experiments done in triplicate ± SD. *P < 0.05, significantly different from respective control.
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Figure 1 Growth regulator)' effect of cisplatin (CP)-treated bone marrow-derived macrophage supematant was tested on A375 cells, L929 cells and normal murine thymocyte proliferation. Culture supematanth collected from CP-treated macrophages were divided in two parts. One was tested as such [CP (N)I and the other part was heated to 5 6 ^ for 1 h (CP (H)]. Target cells (3 x 10-'cells/well) were incubated in ( • ) medium alone or with ( • ) CP (N). ( 0 ) CP (H) or ( O ) CP (H) + anti-oncostatin M antibody for 72 h. Proliferation was measured by [*H|-thymidine incorporation as descnbed in Materials and methods. Values ± SD are representative of three independent experiments done in triplicate.
Expression of OSM in BMDM upon CP treatment Expression of OSM in macrophages upon CP treatment was further confirmed by immunoblotting with anti-OSM antibody. Macrophages were treated with different doses of CP for different durations. Cisplatin treatment rapidly increa.sed the expre.ssion of OSM. Oncostatin M expression was maximum at 1-5 min of CP addition. The response was detectable with 5 (jg/mL of CP and was maximum at lOpg/mL CP treatment (Fig. 2). These are the CP doses needed to induce other biological responses in macrophages.'-''-^ In contrast to the biological effects (Tables 1,2). the expression of cytoplasmic OSM upon CP treatment appeared transient (i.e. it decreased to basal level after 15 min even in the presence of CP; Fig. 2).
Kinetics of OSM expression in CP-treated macrophage lysate and culture supernatants Macrophages were treated with 10 pg/mL of CP for different durations of time. Culture supernatants were collected and macrophages were lysed as described in Materials and methods and both lysates and the respective supematants were analysed for the presence of OSM using dot blot analysis with anti-OSM antibody. The maximum expression of OSM
Figure 2 Kinetics of cisplatin- and LPS-induced oncostatin M expression in bone marrow-derived macrophages. Macrophages were stimulated with medium alone (lane 13), cisplatin 2 pg/mL (lanes 1,4,7,10), 5 pg/mL (lanes 2,5.8,11) and 10 pg/mL (lanes 3,6,9.12) or lOpg/mL LPS for 15 min (lane 14) for 1 min (lanes 1-3), 5 min (lanes 4-6), 15 min (lanes 10-12) and 30 min (lanes 7-9). Triton X-IOO soluble proteins (lO^ig/lane) were separated on a 10% SDS-PAGE. transferred to nitrocellulose, and probed with antioncostatin M antibody. This is one representative experiment of three.
was observed within 1-5 min of CP treatment in the cell lysate which decreased thereafter (Fig. 3a), similar to the western blot result (Fig. 2). Whereas, in the case of supernatants. OSM content was found to increase gradually and reached a maximum within 15-30 min of CP treatment (Fig. 3b).
Discussion We report that OSM induction is up-regulated in BMDM when treated with CP. Previous studies have shown that, in addition to anti-neoplastic activity, CP can activate macrophages as well as other cells of the immune system.'"^ In the present investigations we were interested to know whether macrophages, which are known to express oncostatin M," are induced for enhanced OSM secretion upon CP treatment. Westem blot analysis with anti-OSM antibody showed an increased expression and secretion of OSM by macrophages in response to CP treatment, which appeared to be a rapid process. Expression of OSM increased within 1-5 min of CP treatment to macrophages and within 30-60 min. a significant amount of OSM was secreted into the culture medium (Table 2, Figs 2, 3a,b). It has also been reported that OSM mRNA is present in macrophage-like cells and activated T lymphocytes." Whether CP treatment regulates OSM expression at the transcription or translation level remains to be determined. As OSM expression in BMDM on CP treatment was immediate, the possibility of the presence of precursor OSM molecules cannot be ruled out. Mononuclear cells are known to produce numerous cytokines and lymphokines, many of which share properties regulating cellular growth in vZ/ro.'-''"'^ Oncostatin M was originally identified as a growth inhibitor for a few human tumour cell lines.**"" However, studies with recombinant OSM have shown that it may inhibit or stimulate cell proliferation or affect the cell morphology of a wide variety of cultured nomial and tumour cells." Thus, OSM is one of the many factors released by lymphocyte.*; and macrophages that can function as a growth regulator.-'' As shown in Table 1, proliferation of A375 melanoma cell was inhibited following
Bone marrow-derived macrophages secrete oncostatin M
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of OSM against A375 cells was not attributable lo other known lymphokines. Oncostatin M has received an increasing amount of attention recently as one of the muttifaceled cytokines produced by a number of different cell types involved in a divergent array of cellular interactions as an effector and regulator molecule. Bone marrow-derived macrophages serve as a source of macrophages which do not exhibit as much of the macrophage heterogeneity and therefore are used as a tool for investigating various functions of macrophages. The novelty of the present investigation is that OSM produced by BMDM could be implicated as one of the additional effector molecules in the tumoricidal activity of CP-stimulated macrophages and may have interesting implications in the chemo-immunotherapeutic clinical trials with CP.
Acknowledgements This project was funded by grants from DST, New Delhi. Shishir Shishodia and Anju Shrivastava are recipients of SRF from CSIR, New Delhi.
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Figure 3 Doi blots showing oncostatin M (OSM) expression in cells and culture supematants of cisplatin-induced bone marrowderived macrophages. Macrophages were incubated with medium alone or with 10 pg/mL cisplatin for 1.5, 10, 15 or 30 min. Supernatants were collected and checked for the presence of secreted OSM as described in Materials and methods. Macrophage monolayers were washed with PBS and lysed. (a) Triton X-100 soluble proteins and (b) supematants were blotted on to nitrocellulose membranes and probed with anti-OSM antibody, (a) A is 10 x dilution of B (2.5 ^lg protein/dot).
treatment with CP-treated macrophage supematant. As CP-treated macrophages also produce TNF, IL-1 and NO,"^' contribution of these cytokines to the up-regulation of OSM induction is unlikely because in the presence of neutralizing antibodies against IL-I and TNF-a, antiproliferative activity against A375 cells was found to be stable (data not shown). Also, expression of OSM appears to be an early event whereas expression of TNF and lL-1 takes at least 8-24 h after CP stimulation.5'^ Previous reports suggest that OSM is a highly stable protein and retains its biological activity even at higher temperatures.^ We heated the test supematant to 56'^C for 1 h to inactivate the presence of other heat-labile cytokines such as IL-1 and TNF-a. Consistent with earlier reports, our observations show that heat-inactivated CP-induced macrophage supematant induced proliferation in L929 fibroblast cells which otherwise were inhibited with normal CP-treated macrophage supematant. The cytostatic effect on A375 cells as well as the growth stimulatory effect on L929 fibroblasts were abrogated in the presence of anti-OSM antibody (Fig. 1). Reports suggest that at concentrations of TNF that inhibit replication of L929 cells by 99%, no inhibition of A375 cell growth tiikes place.**-' Heat-inactivated CP-treated supematant lacked IL-1 activity as measured by a thymocyte proliferation assay (Fig. I). Therefore, the cytostatic activity
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