Interleukin 1beta mediates the modulatory effects of monocytes on LNCaP human prostate cancer cells.

Proliferative and secretory responses in androgen-sensitive prostate cancer LNCaP cells are regulated by steroid and peptide hormones and by differentiation-promoting substances. In the present study, we evaluated whether peripheral blood monocytes that exhibit anti-tumour activity in haematopoietic and solid tumours influence growth and secretion in the LNCaP cell line. For this purpose, LNCaP cells were incubated with monocyte-conditioned medium (MCM), and proliferation as well as expression of androgen receptor (AR) and secretion of prostate-specific antigen (PSA) were assessed. Conditioned medium from monocytes reduced proliferation in a dose-dependent manner. Incubation with 40% MCM caused a 50% reduction in cell proliferation. AR protein decreased by 70% and PSA levels in supernatants from LNCaP cells were reduced by approximately 80% following treatment with MCM. We focused on the contribution of two major products of activated monocytes, prostaglandin E2 and interleukin 1beta (IL-1beta), to the MCM modulatory action. LNCaP cells treated with prostaglandin E2 showed neither a reduction in proliferation nor a down-regulation of AR and PSA levels. The effects of MCM on cellular proliferation, AR protein and PSA secretion were abolished by pretreatment of MCM with a neutralizing anti-IL-1beta antibody. In addition, recombinant IL-1beta was able to replace MCM for the inhibition of proliferation and down-regulation of AR and PSA proteins. LNCaP cells were shown to express the IL-1beta receptor type 1, which transduces IL-1beta signal. Our findings reveal that monocyte-derived IL-1beta inhibits the proliferation of androgen-responsive prostate tumour cells and reduces AR and PSA levels.

The LNCaP cell line. which xas derived from a lymph node metastasis of a patient who did not respond to endocrine therapy. is frequently used as a model for studying human prostate cancer (Horoszexxicz et al. 1983). It is know-n that steroid and peptide hormones as well as differentiation-promoting substances regulate proliferative and secretorv responses in LNCaP cells. The cells respond to androgen stimulation by acceleratinc division and producing prostate-specific antigen (PSA). The patterns of androaenic reaulation of growth and secretory function in the LNCaP cell line are different (Lee et al. 1995). The maximal growth rate is achiexed at 0.1 nm of dihydrotestosterone (DHT). whereas higher doses of this androgen induce grow-th arrest. It w as proposed that the inhibition of cellular proliferation by high androgen doses is mediated by transforming growth factor j (TGF-P) (Kim et al. 1996). In contrast to the proliferative response. the percentage of PSA-positix e cells and the levels of PSA protein in LNCaP supernatants increase at concentrations of 1 nmi dihydrotestosterone and bevond (Lee et al. 1995). In addition to androgens. polypeptide growth factors such as epidermnal growth factor (EGF). TGF-at. insulin-like growth factors and basic fibroblast growth factor (bFGF) are mitonenic for LNCaP cells (Wildinn et al. 1989: MacDonald and Habib. 1992: Nakamoto et al. 1992: Ritchie et al. 1997). Positix e effects of triiodothvronine and inhibitorx effects of xitamin D. retinoic acid. luteinizin5 hormone-releasing hormone. phenylacetate and actixin on grrow-th of LNCaP cells has-e also been described (Limonta et al. 1992: Skoxxronski et al. 1993: Young et al. 1994: Esquenet et al. 1995: Dalkin et al. 1996: Walls et al. 1996. Secretion of PSA ,A-as found to be enhanced after treatment with triiodothvronine. vitamin D and pheny lacetate (Skowronski et al. 1993: Esquenet et al. 1995: W'alls et al. 1996. wxhereas divergent results on PSA regulation by retinoic acid wA-ere reported (Fona et al. 1993: Youna et al. 1994. LNCaP cells express a mutant androgen receptor (AR). wxhich binds oestrogenic and progestagenic steroids and non-steroidal anti-androgens hN-droxvflutamide and nilutamide with hiaher affinitv than the wild-type AR. These substances induce reporter gene actixitx in the presence of LNCaP AR more efficientlI than in the presence of the %vild-trpe AR (Veldscholte et al. 1990).
Studies on interactions betw een leucocytes that produce X arious cvtokines and differentiation factors and prostate tumour cells max improve understandinc of prostate cancer biologx. In one of the initial studies in this field Hsieh et al (1995) have shoxxn that phytohaemagglutinin (PPHA )-stimulated lymphocvtes produce substance(s) that reduce cell prohiferation. increase expression of cytoskeleton proteins and down-regulate AR and PSA in LNCaP cells. These effects A-ere obserxed after treatment with xarious concentrations of T-lymphocyte-conditioned media (TCM1). Hoxx exer. the T-cell-derixved factor(s) that mediate these effects hax e so far not been identified. Interactions betxween other cells of the immune system and various tumours haxe been described (Wang et al. 1996: Iversen et al. 1997. For example. monocvtes were found to suppress cell viability and colony formation in human leukaemias and lung tumours. In this study. we address the issue of w hether peripheral blood monoc tes influence groxxth and secretion in androoen-responsive prostatic epithelial cells. We Interleukin-1f effects on prostate tumour cells 1005 showx that monocyte-derived interleukin 1 B (IL-I5> is a mediator of the modulatory effects on LNCaP cells.

Culture of peripheral blood monocytes
Peripheral blood was collected from three healthy volunteers on several occasions. Monocvtes were obtained from peripheral blood mononuclear cells after Ficoll separation by standard adherence (90 min. 37-C( and cultured in RPMI-1640 medium that was supplemented wxith 10%e charcoal-stripped FCS (CS FCS). The purity of monocytes as determined by staining for naphthol acetate esterase and by measuring CD14 expression wxas > 90%. For preparation of monocyte-conditioned medium (NICM). 2.5 x 10monocvtes ml-' were cultured w-ith and x-ithout PHA. After 48 h. the supernatants were cleared by centrifugation and frozen at -20 C until use.

Proliferation assays in LNCaP cells
The LNCaP cell line w-as purchased at passage 21 from the American Type Culture Collection (Bethesda. MD. USA). LNCaP cells were seeded into 24-svell plates at 2 x 10cells per well in MCDB-131 medium supplemented with 10%7e FCS. Medium wxas changed 24 h later and the final concentration of CS FCS was 3%. Culture medium >-as supplemented with MCM (10-60%c. prostaglandin E, (I-i0l.tM) or IL-l1 (0.1-lOng ml Control experiments w-ere carried out in the absence of any supplement in medium or in the presence of PHA-M. In neutralization experiments. MCM w-as preincubated with the antibody against IL-1,B oxvernight. It has prexiously been determined that I mg of this antibody is capable of neutralizing approximately 1000 units of natural or recombinant IL-1 P. Cell proliferation A-as determined after 72 h incubation by means of the MITT assax. This assax is based on the abilitv of lixini cells to reduce slightly coloured tetrazolium salts to intensely coloured formazan derixatixves. The assay wxas performed as described previously (Cronauer et al. 1996). In selected experiments the MTT assav results Awere compared w-ith those obtained with a cell counter and were found to be identical.

Immunohistochemical analyses
Following treatment with MCNI or medium w ithout supplements. LNCaP cells were trxpsinized. cytospun. resuspended in PBS. fixed in 1% paraformaldehx de. permeabilized by adding 0.1%c Triton X-100 and stained for cvokeratin expression. Cy-tokeratin immunohistochemistry w as performed according to a streptaxidin-biotin-peroxidase protocol. The immunohistochemical procedure Ax as described prexviously (Hobisch et al. 1995).

Androgen receptor-binding assay
LNCaP cells w-ere cultured in the absence (untreated control) or presence of respective supplements in 5%c CS FCS for 72 h. Then they were scraped off. washed once. resuspended in medium and incubated with ['H]methyltrienolone. at concentrations of 0.3-5 nst. for 90 min at room temperature. Non-specific bindingxwas measured in the presence of a 200-fold molar excess of unlabelled methx Itrienolone. The pellets w-ere recoxered after incubation by centrifugiation (3800g. 3 min) and w-ashed twice w-ith 500 gl of ice-cold medium. The cell pellets w-ere then lysed in 1 ml of scintillation liquid and the radioactix ity \-as determined in a P-counter. Cellular protein w-as determined according to the method described by Bradford (1978). B and K, wxere calculated by Scatchard analx sis.
Determination of prostate-specific antigen in supematants from LNCaP cells The cells were grown on 24-%vell plates in the presence of 5% CS FCS w-ith or x ithout supplements. The medium A-as remoxved after 72 h and the PSA lexvel x as measured by an enzy me immunoassax. PSA Xalues xxere corrected for cell number according to the results of the proliferation assay.
Determination of IL-1 ,B concentration in monocyte conditioned media IL-1 in MCM was determined by a solid-phase enzy me amplified sensitivity immunoassav. The assay is based on an olicoclonal sx stem in vvhich sexeral monoclonal antibodies directed a2ainst distinct epitopes of IL-iI5 are used. The use of sexeral distinct monoclonal antibodies avoids assay hyperspecificity. The assay xxas performed on a microtitre plate. Samples containing IL-i react with capture antibodies coated on a plastic xxell and with monoclonal antibodies labelled wxith horseradish peroxidase. After Fluorescence intensity Figure 1 Phenotypic charactenzatin of penpheral blod monocytes and LNCaP cells. Monocytes (A) and LNCaP cells (B) were labelled with monoclonkk antibodies specific for the antigen indicated follwed by FITCconjugated secondary antibody. and surface expression (bold lines) was determined with a flow cytometer as described in Maternals and methods.
Fluorescence intensity (x-axis) is plotted against the number of cells (y-axis) an incubation penrod of 2 h at room temperature the microtitre plate was washed three times to remove unbound enzyme-labelled antibodies. The revelation solution (tetramethylbenzvdinehydrogen peroxide) was added and incubated for 15 min at room temperature. The reaction was stopped with sulphuric acid and the microtitre plate was read at 450 nm. The concentrations of IL-1o in MCM ranged between 1.6 and 2.4 ng ml-'.

Measurement of surface antigen expression by flow cytometry
To determine surface antigren expression. penpheral blood monocvtes were labelled with primary mouse monoclonal anti-CD14 antibody MEM18 followed bv fluorescein isothiocyanate (FITC)conjugated goat anti-mouse Ig. LNCaP cells were labelled with biotinylated primary mouse monoclonal anti-IL-1 receptor antibody 6B5 followed by FHTC-conjugated streptavidin. Washes were in PBS containing 0.2'% boxine serum albumin (BSA). After the last wash. the cells were stored in PBS containing 0.2%7c BSA and 2%7c formaldehyde. The samples were analysed on a FACS Calibur. Data were analysed and presented using CellQuest software.

Monocyte-conditioned medium-treated LNCaP cells show changes in cell shape
To study the influence of monocv-tes on prostate cancer cells. LNCaP cells w-ere exposed to medium conditioned by peripheral blood monocytes homogenously positive for CD14 (Firure 1A). Distinct morphological changes in LNCaP cells were observed after treatment with monocyte-conditioned medium (MCM). The cells became elongated and show ed dendrite-like processes. hich were connected to each other (Figure 2). These morphological changes were notable even Awith 10%7 MCM in LNCaP culture medium. Expression of cytoskeletal proteins w-as evaluated by semiquantitatixe immunohistochemistr-. A monoclonal antibodv directed against luminal cy-tokeratins 8 and 18 was used for this purpose. The expression and staining intensit-of these cvtokeratins 'Aere previously reported to increase in the more differentiated prostatic luminal epithelium ). In the case of LNCaP cells treated with MCM. we obserxed neither an increase in the percentagre of cytokeratin-immunopositive cells nor a change in staining intensitv.

IL-1i is responsible for the modulatory effects of MCM
We focused on the contribution of individual factors to the MCM modulatory effects. Initially, we investigated the role of prostaglandin E2, which is a major product of activated monocytes (Venkataprasad et al, 1996). LNCaP cells treated with prostaglandin E2 showed neither a reduction in proliferation nor a down-regulation of AR and PSA levels. Thus, prostaglandin E2 can be excluded as a mediator of the inhibitory effects of monocytes on androgen-responsive prostate cancer cells. Next, we examined the role of the proinflammatory cytokine IL-1p, which exhibits anti-tumour activity in several models (Kilian et al, 1991;Braunschweiger et al, 1996), in the interaction between the immune system and prostate cancer cells. In order to determine whether IL-1 1 mediates MCM effects on LNCaP cells, MCM was preincubated with a neutralizing polyclonal anti-IL-l,B antibody. The effectiveness of this antibody was previously demonstrated in other assay systems (Salem et al, 1990;Lisak and Bealmear, 1991). MCM pretreated with the antibody did not reduce LNCaP cell proliferation ( Figure 6A). The MCM effect on AR protein was completely abolished after preincubation of MCM with the polyclonal anti-IL-15 antibody ( Figure 6B), and the PSA levels were almost completely restored ( Figure 6C) werealmst dentical to those of MCM. Cell proliferatio' xa reduced dose dependently ( Figure 6A). the AR protein lexvels decreased ( Figure 6D3) and PSA secretion xxas dirminished (Ficrure 6C(. Finally. LNCaP cells xxere shoxxn to express the IL-l13 receptor type I (Figure 1 B). xx hich transduces IIL-113 sigrnal (Curtis et al. 1989).

DISCUSSION
T'he major findingr of the present study is that peripheral blood monocytes are capable of modulating, cellular exvents in androgenresponsixve LNCaP cells by secretingr IL-113. This ability of monocv-tes is similar to that prexviously reported for T lymphocytes in the same cell line (Hsieh et al. 1995). Both cell types secrete substances that cause a reduction in cellular proliferation. diminish lexvels of AR protein and doxxn-regulate PSA in LNCaP cells. In our experiments. MCM was effective regardless of the presence of PHA in monocvrte cultures. In contrast. unstimulated lymphocytes provoked only, a minimal growth inhfibition of LNCaP cells (Hsieh et al. 1995).
LNCaP cells became elongated and exhibited dendrite-like processes after contact xxith MCMI and TC.M. Similar changres in cell shape xxere described after treatment of LNCaP cells xxith analogues of cAMP (Bang, et al. 1994). There was, however, one difference betxxeen MCMand TCM-treated LNCaP cells. The expression of cy-tokeratins 8 and 18 and their staining intensity increased twofold after incubation xxith TCM as determined by% a tota intensitV score procedure (Hsieh et al. 1995). In the case of MCM xxe did not see any differences in the expression of these cytoskeleton proteins by semiquantitatixve immunohistochemisti-x. Contrasting, results on expression of cytokeratins may be due to use of different antibodies and immunohistocheniical techniques.
It wxas suggaested bv others that 1CMand cAMP analogyue-treated LNCaP cells undergo neuroendocrine differentiation (Bang et al. 1994: Hsieh et al. 1995. TCM treatment proxoked an increase in total intensity score of twxo neuroendocnine mark-ers. neuronspcfi'c enolase and serotonin. Hoxx ever, the concept of neuroendocrine differentiation of this cell line wvas questioned recently (Noordzij et al. 1996). In that publication and in our prexvious expenimental studies neuroendocrine cells within the LNCaP cell line were not identified (unpublished data). Thus. a differentiation process induced by leucocytes in androgen-responsixve prostate cancer cells is not well characterized to date.
T'he MCM-induced growth-inhibitory effect wxas associated with decreased AR expression. Association betwxeen AR expression and ggrowth regulation of prostate cancer cells can be studied in AR-positive LNCaP cells and in AR cDNA-transfected PC-3 cells. Itwxas demonstrated that the expression of AR in PC-3 cells leads to a decrease in proliferation (Yuan et al. 1993). We infer that more reliable information on AR involvement in garowxth regulation may be obtained in its natural cellular environment. i.e. in LNCaP cells. Growth inhibition and AR reduction A-ere prexviouslx observed in LNCaP cells incubated wxith retinoic acid and TCM (Youngy et al. 1994: Hsieh et al. 1995. Regarding AR expression.
conditioned media from T ly mphocytes and monocytes. and reioi cd differ from anti-androgens cyproterone acetate. hNdrox,% flutamide and bicalutamide.wxxhich are commonlv used in endocrine therapy for prostate cancer. These compounds bind to the AR wxith low-aff-inity and do not permit acquisition of a transcriptionally actixve form of the receptor (Kemppainen et al. 1992: Kallio et al. 1994. Howxever, they did not down-regulate AR protein itself. AR is express-ed in relapsed prostate tumours and in their metastases. wxhich wxere obtained before onset of therapy and durnenc tumour progression (van der Kxx ast et al. 1991: Hobisch et al. 1995. Several recent publications support the viewx that reduction in androgren concentration is not sufficient to prexvent transmission of extracellular signals xia the AR (Veldscholte et al. 1990: Culig, et al. 1993. 1994: Kokontis et al. 1994: Nazareth and Weigel1. 1996. Prostate cancer cells may adapt to an enxvironment A-ith verx lowx androgzen concentration by increasing their AR expression and transcriptional actixvity (Kokontis et al. 1994). Furthermore. mutant ARs discoxvered in prostate cancer frequently exhibit a gain in function: they are efficiently actixvated bx other steroi C ds and non-steroidal AR antagonists (Veldscholte et al. 1990: Culig, et al. 1993. Finally. AR activitv isu-eulated by sexveral non-steroi'dal substances such as polvpeptide groxwth factors and second messengers (Culig et al. 1994: Nazareth andWeigel. 1996) Therefore. strategies aimed at reducing, AR lexvels. such as administration of secretor-v prodlucts from the immune cells. may be beneficial in metastatic prostate cancer. AR dowxn-regulation was followed by reduction of PSA protein in LNCaP supernatants. Thus. the effect of MCM is different to that of pheny lacetate and xvitamnin D. substances that haxve been reported to inhibit proliferation of LNCaP ceHls and increase PSA secretion (Walls et al. 1996: Zhao et al. 1997.  w-as proposed that a protein in the molecular wveight range of 13-24 kDa is responsible for TCM action. It is not unlikelv that orowth-modulatorv effects of TCNI and MICM could be. at least in part. attributed to the same cytokine. Monocvtes are know-n as the major source of secreted IL-1 (Dinarello. 1988). Human IL-1 is a 1 7-kDa protein that is secreted durinnc an inflammatory process. Tw-o functionally almost equivalent forms of IL-1. IL-lct and IL-1,B. xxhich display about 27%homologx at the protein lex el. exist. Both forms of IL-I  same cell-surface receptor and therefore showsimilar biological activities. IL-I was found to exhibit anti-tumour activityin various neoplasms either by augmentation of cellular immune response or by inhibition of proliferation (Braunschweiger et al. 1988: Kilian et al. 1991. In aareement with our results. IL-1 was previously found to provoke a dose-dependent growth inhibition in LNCaP cells Chiao. 1995: Ritchie et al. 1997). IL-I diminished DHT effect on the proportion of the replicating LNCaP cells (Hsieh and Chiao. 1995). However. our results with recard to AR bindine differ from those of Hsieh and Chiao. These authors failed to show a negative effect on AR protein levels after IL-1 a treatment. Ritchie et al (1997) also treated LNCaP cells and androgenindependent PC-3 and DU-145 cells with increasing concentrations of IL-1[. Supplementation of prostate cell cultures with IL-15 caused a decreased proliferation of all three cell lines. However. the negative effect of IL-1[ on proliferation was far more pronounced in LNCaP cells than in the two androgen-independent cell lines.
Although determination of AR protein was not performed in that studv. a mark-ed effect in LNCaP cells suggyests an interaction between IL-1 [ and AR pathways. IL-1[ also decreased cell chemotaxis in the three prostate cancer cell lines (Ritchie et al. 1997).
Interestingly. inhibition of grow%th of MCF-7 breast cancer cells by IL-1 is associated with down-regulation of oestrogen receptor (Danforth and Sgagias. 1991) A different type of interaction between monocvtes and LNCaP cells was recently reported (Klein et al. 1997). Expression of matrilvsin. which is a member of the matrix metalloproteinase superfamily. in LNCaP cells is induced by conditioned medium from the monocvtic cell line THP-1. In addition. recombinant IL-1 up-regulated matrilysin expression. Biolo2ical neutralization experiments revealed that the effect of THP-1 conditioned medium was abolished by an anti-IL-[3 antibody. and it was concluded that IL-I [3 is a mediator of matrilysin induction. This up-regulation of matrilysin by IL-i may be harmful in prostate cancer. It was shown that matrily-sin-transfected prostate cancer cells have a hich invasive potential in immunodeficient mice (Powell et al. 1993).
IL-[3 activates several second-messen-er systems in target tissues (Munoz et al. 1990: Roberts et al. 1992: Carman-Krzan and Wise. 1993: Cole et al. 1995: Sjoholm 1995. For example. IL-I[ induces cAMP in human decidual cells. It stimulates secretion of nerve Lrowth factor in astrorlial cultures by activation of the phospholipase A2-lipoxygenase pathway. In pancreatic [cells ceramide may be invol-ed in transducing the cvtotoxic and cvtostatic actions of IL-1 [3. Involvement of protein kinase C and nitric oxide pathways in IL-1[ signalling was also described. It remains to be determined in future studies which of these sianalling pathways may be operative in prostatic epithelial cells.
In summary. this studv demonstrates that the monocvte secretorn product IL-i [3 is a potentially important negative regulator in LNCaP cells. Therapeutic application of this pleiotropic cvtokine in prostate cancer will probably be further explored.