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CYTOKINES INDUCED BY BCG OWAND IFN a-2B IN BLADDER CANCER

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FIG. 1. Effects of 3-day expos ure to BCG (100 pg.lml.1 and/or IFNu-2b (50,000 dm l. ) on message level of IL-6 in bladder cancer cell lines,MGH, SD, 582. Lane C, I, B , B&I showed control, with IFN a-2b, wit h BCG and with combinatio n of the se two agents respectively. Data arerepresented by one samp le in two independent experi ments, each with triplicate.

known, it is believed that BCG immunotherapy for bladdercancer involves a local non-specific T-cell dependent immu-nological reaction which is modulated by Th1- and Th2-likecytokines. Since the first report on urinary IL-2 in BCGhm uno the rap y, several other groups have carried out var-ious investigations of urina ry cytokines. These cytokines in-clude IL -Ip, IL-6, IL-8, IL-10 which c an be detected af ter thefirst instillation, an d IL-2, TNF-a, IFN-y which become de-tectable after several intravesical BCG instillation^.^ Re-cently, we observed that GM-CSF and TGF-pl were alsodetectable (data not shown). Sander et al suggested thateertain cytokines might locate in endothelium, malignant

urothelium a nd leukocytes but no phenotypic staining of thecytokine positive cells was performed.

In this study, all five bladder cancer cell lines could pro-duce various cytokines constitutively. No obvious tre nd wasapparent between production of these cytokines and parenttumor grade. Both BCG and IFNa-2b enhanced the produc-tion of IL-6 and IL-8 from th e high grade cell lines MGH, 582and SD. Furthermore, BCG but not IFNa-2b induced GM-CSF production in these th ree cell lines. In contra st, IFNa-2b, but not BCG, modulated production of these cytokinesfrom the low-grade cell lines RT4 an d RT112. Interestingly,in our studies, the sensitivity of bladder cancer cells to theeffect of BCG or IFNa-2b on cytokine production correlatedwith their susceptibility to the direct cytolytic and anti-proliferative effects. The differential sensitivity of ceil line sto BCG or IFNa-2b may be rela ted to tumor grade. Interest-ingly, BCG also stimulated the production of small amountsof IFN -a from all t hese five cell lines.

All the cytokines produced by tumor cells after treatment

with BCG o r IFNa-2b are inflammatory cytokines (IL-6,TNF-a a nd IL- lp), chemokines (IL-81 or growth factors (GM-CSF). No Th-l-like cytokines namely, IL-2, IFN-y o r IL-12were produced eithe r constitutively or after stimulation. It isnoteworthy that production of cytokines, such as IL-6, IL-8and GM-CSF, which can be detected in patients' urine afterthe first instillation, are also increased in bladder cancer celllines by BCG treatment. It is possible that certain urinarycytokines detected after BCG therapy may not only be fromimmunocompetent cells like macrophages, neutrophils andlymphocytes but also from tumor cells or urothelial cells.However, Th-1 like cytokines (IL-2 and IFN-y), which usuallycannot be detected in pa tients' u rine until the la ter instilla-tions, may not be produced by urothelial cells and are prob-

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980 CYTOKINES INDUCED BY BCG OWAND IFN a-2B IN BLADDER CANCER

FIG. 3. Effects of 3-day exposure to BCG (100 pg./ml.) andor IFNa-2b (50,000 d ml . ) o n message level of GMCSF in bladder cancer celllines, RT112, MGH, SD, 582. Lane C, I, B B&I showed control, with IFNa-2b, with BCG and with combination of these two agentsrespectively. Data are represen ted by one sample in two independent experiments, each with triplicate.

FIG. 4. Effects of 3-day exposure to BCG (100 pg./ml.) an d o rIFNn-2b (50,000 dm l. ) on IL-8 release from five cell lines, RT4,RT112, MGH, SD and 582. Results are shown as mean ? SEM ofthree Independent experiments.

ably produced primarily by immunocompetent cells. How-ever, it should be noted that the different exposure time ofcells to BCG and IFN-a (3 days in our study compared w ith2 hours in clinical practice) may re sul t in a different profile ofcytokine production in urothelial cells in patients.

Autoclaved BCG (Tokyo strain) had the same tumorgrowth-inhibiting activity in C,H/H, mice as live BCG. 3However, in vivo, the autoclaved BCG showed a markeddecrease in ability t o attac h to MBT-2 cells.23 In contr ast,

FXG. . Effects of 3-day exposure to BCG (100 pg./ml.) andorIFNa-2b (50,000 dml .) on IFN-a release from five cell lines, RT4,RT112, MGH, SD and 582. Results are shown as mean Z SEM ofthree independent experiments.

Morales reported th at heat-inactiv ated BCG (lyophilized andheated at 65C for 1 hour) failed to induce a nti-tu mor activityin C3WHeN mice challenged with MBT-2 cells.24 Also, Bohleet a1 showed th at heat-killed Pasteu r strain BCG (boiled atlOOC for 10 minutes) was significantly less effective in stim-ula ting PBMNC toward BCG-activated kille r (BAK) cell ac-tivity.' In the prese nt study, autoclaved BCG did not inhibit

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CYTOKINES INDUCED BY BCG OWAND IFN a-2B IN BLADDER CANCER 98 1

TARI.E Effects o f 3 - d a yexposure to BCG (100 p g /mi.) andlorIFNa-2 h (50,000 plm1.l on I L - l p and T G F -p l release from five cell

lines, RT4, RT112 , MGH, S D and 5 8 2

Treatment/Cell Line Control I F N BCG B I

I L - ~ o p g / l O ells)RT4 42 20 539 204* 46 t 23 929 ? 397*RT112 3 2 2 2 c 0.3 2 0.7 2 t 1MGH 0 8 0 5 0.3 0.3 2.5 1.2 5.4 t 1582 0 0.8 t 0 8 0.7 c 0.7 3 1SD 2.1 0.8 4.4 0.8 2.6 1.5 1.4 0.8

TGF-p1 (pg/106 cells)MGH 11 1.5 1 6 1 2 t 2 0

RT112 12 5 21 L 12 3 t 1.7 O

Results are shown as mean SEM of three independent experiments. * P<0.05

the cytokines produced by the tumor cells could directly orindirectly attract immunocompetent cells, such as mono-cytedmacorphages, lymphocytes and neutrophils, to tumorsites and stimulate them. This could be one of the reasonswhy BCG needs to come into contact with tu mor cells in vivo,to elicit an optimal anti-tumor re sp on ~e .' ~ ~' ~

Furthermore, several recombinant cytokines have directanti-proliferative and cytotoxic effects on bladder cancer cellsin vitro. For example, recombinant IL-6, IL-8 and GM-CSF

have direct anti-proliferative effect on 582 cells. As thesensitivity of bladder cancer cells to the effect of BCG orIFNa -2b on cytokine production correlated with t hei r suscep-tibility to th e direct cytolytic and anti-proliferative effects, itmay be possible that cytokines released from tumor cells,which are increased by BCG or IFNa -2b, may inhibit growthin an autocrine or parac rine manne r. However, the activity ofsecreted cytokines in either inhibiting or stimulating thegrowth of bladder cancer cells needs to be demonstrated.Furthermore, varying expression of soluble and membranebound cytokine receptor in different cell lines may result indifferent sensitivities to the modulatory effects of these se-creted cytokines.

ARer BCG treatment, all the bladder cancer cell linesproduce IFN-a. The effects of IFN-a on tumors in vivo canresult either from direct effects on the tumor cells, or indi-rectly, via th e activation of several incompletely defined ef-fector mechanisms. These include macrophage activationand stimulation of T cells and NK cell activity. Moreover,IFN-a may enhance the specific cytotoxicity of sensitizedlymphocytes aga inst tumo r cellsz6 and is known to stimula tethe stimulatory effect of IFN-a on the expression of delayedhypersensitivity.26

Our previous studie s showed th at the combination of BCGand IFN a-2b had additive cytolytic and anti-proliferativeeffects on various bladder cancer cells in vitro.'6.20 In thisand our previous studies, we found the combination ofBCG and IFNa-2b additively increased the production ofIL-6, IL-8 and TNF-a. Interestingly, constitutive TGF-plproduction could be totally inhibited by the combination ofthes e age nts. Among the TGF-P isoforms, TGF-pl is a major

effector molecule in many inflammatory conditions. TGF-pldeficiency res ult s in a severe dysfunction of the immune andinflammatory systems and is accompanied by increased pro-duction of several cytokine mediators of inflammation, suchas IFN--y and TNF-a. TGF-p may also suppress Th-1-like andinflammatory 'flokine production. proliferation Of

some tumor cells may be enhanced by TGF-P, and TGF-Pmight also play a role in malignant transformation and tu-

FIG. 6. Effects of autoclaved BCG 100 glml.) and live BCG 100pg./ml.) on cell growth of two cell lines, MGH and 582 Results areshown as percentage of cell number relative to control cells. Resultsare shown as mean SD of two experiments.

mor developmentz6 Thus, it is-noteworthy that TGF-p1 pro-duction could be totally inhibited by the combination of BCGand IFNa-2b.rowth of the MGH cell line but was ab le to inhibit 5 82 cells

equally as effectively as live BCG. Interestingly, cytokineproduction in MGH cells in response t o autoclaved BCG was

cells, autoclaved BCG stimulated IL-6 and TNF-a productionas effectively as live BCG. Both rIL-6 and rTNFa havegrowth in hibiting effects on bladder cancer cell lines; rTNF-ais also cytotoxic. ,

recognized that ,-ytokines regulate im-mune and inflammatory responses, For example, IL-6, aninflammatoly cytokine, is involved in induction of IL-2 pro-duct ion, T-cell proliferation, cytotoxic T-cell diffe renti ationan d th e acute -phase reaction. The effects of IL-6 aregistic with IL-1 and TNF-a, while IL-8 is chemotactic foractivated T ymphocytes and neutrophils.2 6 GM-CSF func-tionally activates mature immunocompetent cells at an in-flammatory site and also inhibits their migration, ensuringtheir retention in the region of inflammation. GM-CSF alsoenhances proliferation and differentiation of progenitor cellsand induces expression of MHC class I1 molecules.26 Thus,

reduced, compared with that elicited by live BCG. In 582 C O N C L U S I O N

BCG and IFNa-Zb appear t o have independent grade-

rela ted Stimu latoV effects on cytokine production from blad-der tumor cells. This cytokine production appears to corre-spond to additive cytolytic and anti-proliferative effects ofBCG and IFNa-2b on thes e cell lines. Although recombinantpreparations of these cytokines have anti-proliferative and/orcytolytic effects on these bladder cancer cell lines, furt herstudies are required to determine whether these endog-enously secreted cytokines play a role in the direct cytolyticeffects of BCG and IFNa -2b on thes e cell lines.

Acknowledgments. We are grateful to Schering-Plow Pt.Ltd., Malaysia and Connaught Laboratories Limited, Can-ad a for the gift of INTRON A and Immucyst. We also thankDr. Hon Wei Min, Ms. Carmel Lawrencia and Dr. Ge rard Ngfor their help.

It is now

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FIG. 7. Effects of autoclaved BCG (10 0 pg./ml.) and live BCG 100 g./ml.) on IL-6, IL-8, GM-CSF and TN F-a rele ase from two cell lin es,MGH and 582. Results ar e shown as mean SEM of two independent experiments.

K E F E R E N C E S

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interferon i n superficial bladder cancer: a northern Californiaoncology group study. J. Clin. Oncol., 6: 476, 1988.

15. Sargent, E. R. and Williams, R. D.: Immunotherapeut ic alterna-tives in superficial bladder cancer: interferon, interleukin-2,and keyhole-limpet hemocyanin. Urol. Clin. North A m . 1 9581, 1992.

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attachment and viability of bacilius Calmette-Guerin to blad-der cancer cells. Cancer, 72: 558, 1993.

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