Int. J. Cancer: 60,300-307 (1995) Publication of the International Union Against Cancer *’ Publication de I’Union Internationale Contre le Cancer 0 1995 Wiley-Liss, Inc.

EFFECTS OF GRANULOCYTE-MONOCYTE COLONY-STIMULATING FACTOR (GM-CSF) ON EXPRESSION OF ADHESION MOLECULES AND

CANCER-ASSOCIATED MACROPHAGES Sergio BERNASCONI’, Christian MATTEUCCI’, Marina SIRONI’, Mario CONNI’, Francesco COLOTTA’, Monica MOSCA’, Nicoletta COLOMBO*, Cristina BONAZZI’, Fabio LANDONI:, Giuseppe CORBETTA3, Alberto MANTOVANI’, and Paola ALLAVENA1,4

‘Istituto di Ricerche Farmacologiche, “Mario Negn”, ” via Eritrea 62, 20157, Milan, 2Dipartimetito Ostetricia e Ginecologia, Ospedale S. Gerardo, Monza Milan, ”ando2 Phamia Ltd., Milan, Italy.

PRODUCTION OF CYTOKINES IN BLOOD MONOCYTES AND OVARIAN

The present study was aimed at characterizing the effects of in vitro exposure to GM-CSF on blood monocytes and tumor- associated macrophages (TAM) in human ovarian cancer. Puri- fied populations of TAM from ovarian cancer patients were studied in terms of expression of surface molecules, cytokine production and tumor cytotoxicity after overnight incubation with GM-CSF or IFNy and LPS, used as reference activators. GM-CSF augmented the surface expression of ICAM-I and CD I8 in TAM and in blood monocytes. Stimulation was more prominent in monocytes than in TAM, which showed higher baseline expression of this adhesion molecule. ICAM-3 was not influenced by GM-CSF or by IFNy/LPS. GM-CSF-augmented ICAM-I expression was associated with higher levels of mRNA transcripts. The protein synthesis inhibitor cycloheximide super- induced basal and GM-CSF-induced ICAM- I transcripts, thus excluding a role for secondary polypeptide mediators. In the absence of stimuli, TAM produced higher levels, compared to monocytes, of IL-6 and IL-8 but not of IL-l and TNF. GM-CSF augmented the production of IL-6 and IL-8 (but not that of IL-l and TNF) in TAM, whereas it had little effect on blood monocyte. Tumoricidal activity was tested against two ovarian tumor cell lines (OVCAR3 and SW626). GM-CSF more promi- nently augmented monocyte cytotoxicity, while only 2 of 6 TAM preparations were stimulated by GM-CSF. These results sug- gest that GM-CSF selectively regulates the function of blood monocytes and TAM, the effect of this cytokine varying with the parameter and cell population examined. These data provide a rational and biological endpoint for further studies with GM- CSF as an activator of mononuclear phagocyte function in ovarian cancer. o 1995 Wiley-Liss. Iric.

Granulocyte-macrophage colony stimulating factor (GM-CSF) regulates the growth and differentiation of hematopoietic cells bclonging to the myelomonocytic pathway. As such, it is used to counteract the myelotoxic effects of cytoreductive therapies. In addition to influencing bone marrow precursors, GM-CSF has been shown to regulate several functions of mature mononuclear phagocytes, including expression of membrane structures, migration, antigen presentation, cytokine release and anti-tumor activity (see for review Hamilton, 1993).

Macrophage5 are a major component of the lymphoreticular infiltrate of tumor tissues (Mantovani et al., 1992). They play a complex, ambiguous role in the immunobiology of tumors and their stroma. Since they are located at the tumor-host inter- face and have the potential to exert anti-tumor activity, tumor-associated macrophages (TAM) are an attractive target for therapeutic intervention.

In terms of characterization of the leukocyte infiltrate, ovarian cancer is one of the most extensively studied human tumors (Mantovani et al., 1992). Based on immunobiology studies, macrophage activating agents, including bacteria, IL-2 and, more recently, IFNy, have been given i.p. in ovarian carcinoma patients, with some evidence of anti-tumor activity (Mantovani et al., 1981; Bast et al., 1983; Berek et al., 1985; Rambaldi et al., 1985; Bezwoda et al., 1989; Allavena et al.,

1990; Pujade-Lauraine et al., 1990; Steis et al., 1990; Stewart et al., 1990; Colombo et al., 1992).

TAM, including those in ovarian cancer, have peculiar functional properties. including responsiveness to activation signals (Mantovani et al., 1992). Most studies on the interac- tion of GM-CSF with mononuclear phagocytes have used human blood monocytes or mouse peritoneal macrophages (Gasson, 1991). Transfer of cytokine genes in a murine model has revealed that GM-CSF produced in situ has a unique ability to promote anti-tumor immunity (Dranoff et al., 1993). These considerations prompted us to analyze the effects of GM-CSF on TAM from ovarian carcinoma, since local admin- istration of cytokines is feasible in this disease, mostly confined to the peritoneal compartment. In vitro studies included the expression of adhesion molecules, cytokine production and tumor cytotoxicity.

MATERIAL AND METHODS

Cell culture and reagents The following reagents were used for culture of cell lines,

separation of effector cells and cytotoxicity assay: pyrogen-free saline for clinical use and pyrogen-free distilled water (Bieffe, Sondrio, Italy); medium RPMI 1640 (lox concentrated; Bio- chrom, Berlin, Germany); glutamine (Biochrom); gentamicine (Gentalyn) was a generous gift of Schering-Plough (Comazzo, Italy); aseptically collected FCS (Hyclone, Logan, UT). The routinely employed tissue culture medium was RPMI 1640 with 2 mM glutamine, SO ng/ml gentamicin, 10% FCS, hereafter referred to as complete medium. All reagents were checked for endotoxin contamination by the Limulus amebo- cyte lysate assay (Microbiological Associates, Walkersville, MD) with a sensitivity of 0.02-0.05 ngiml of Escherichia Cali Westphal lipopolysaccharides (LPS). Sera were tested after 1:3 dilution and heating at 100°C. All reagents were negative for endotoxin contamination. LPS (E. coli 0.SS:BS) was pur- chased from Difco (Detroit, MI). Human recombinant (rh) IFNy was kindly donated by Roussel Uclaf (Romainville, France). rhGM-CSF was from Sandoz (Basel, Switzerland).

Patients Twelve patients with histologically confirmed ovarian carci-

noma and ascites admitted to the S. Gerardo Hospital, Monza (Milan) formed the case list of this study. Six patients were untreated and six had received cisplatin-based chemotherapy. Samples were collected at least 1 month after the last cycle of chemotherapy. Normal healthy donors were used as control population. We could study monocytes from only 4 ovarian

4To whom correspondence and reprint requests should be ad- dressed, at Dept. Immunology, “Mario Negri” Institute, via Eritrea, 62.20157 Milan, Italy. Fax: (2)3546277.

Received: May 18,1994 and in revised form September 20, 1994.

ICAM-1 Mnl GM-CSF AND TUMOR-ASSOCIATED MACROPHAGES

MONOCYTES TAM

301

CDl la 82 K

FIGLIRE 1 - Expression of adhesion molecules by blood monocytes and TAM from ascitic ovarian carcinomas. Cells were exposed for 20 hr (100 ngiml GM-CSF; 100 Uiml IFNy and 100 ngimlLPS), and expression of adhesion molecules was assessed by FACS after staining with appropriate MAb. Numbers indicate net percent positive cells (after subtraction of percent positive cells treated with an irrelevant antibody, shown as dotted vertical line) and mean channel of fluorescence (MCF) on linear scale.

500

400

300

U 2 200

U

103

0

-100

MONOCYTES T A M

500

400

0

0

0

300

200

100

0

I I -100 GM-CSF LPS+IFNy

0 0

0

0 I 0

0

GM-CSF LPS+IFNy

FIGURE 2 - ICAM-I expression by blood monocytes (7 donors) and TAM (5 donors) from ascitic ovarian carcinomas after 20-hr stimulation with GM-CSF (100 ngiml) and LPS (100 ngiml) + lFNy (100 Uiml). Results are expressed as increase in MCF calculated as MCF of stimulated cells - MCF of cells incubated with medium alone.

cancer patients, with results comparable to normal blood cells, as expected on the basis of previous findings (Peri et al., 1981; Economou et al., 1988; Erroi et d., 1989).

Monocytes Monocytes were obtained from the peripheral blood of

normal healthy volunteers or patients with ovarian adenocarci- noma, as described (Colotta et al., 1984). Briefly, monocytes were obtained from Ficoll-Hypaque-separated mononuclear cells by centrifugation on a 46% gradient of isosmotic (285 mOsml) Percoll (Pharmacia, Uppsala, Sweden). Monocyte preparations were usually > 90% pure as assessed by positivity with monoclonal antibody (MAb) anti-CD14.

Tumor-associated macrophages Purified preparations of TAM from peritoneal effusions

were obtained, after therapeutic paracentesis, by centrifuga-

tion on discontinuous Ficoll-Hypaque gradients (75%-loo%), as described previously (Allavena et al., 1986). Tumor cells were harvested at the 75% medium interface; lymphocytes and macrophages were collected at the 75%-100% interface. TAM were separated from tumor-associated lymphocytes (TAL) by centrifugation on a 42% Percoll gradient. The purity of TAM preparations was usually > 905% as assessed morphologically on May-Grunwald Giemsa-stained smears; when the separa- tion was not satisfactory, the discontinuous Ficoll or Percoll gradients were repeated.

In vitro cell activation Monocytes and TAM were activated by culturing 3 to 7 ml of

a cell suspension containing 3 x loh cellsiml complete medium in 5-cm diameter Petriperm hydrophobic dishes (Heraeus,

302 BERNASCONI ETAL.

Vienna, Austria) for 20 hr in the presence of 100 U/ml of IFNy and 100 ngiml of LPS or various concentrations of GM-CSF.

Sit$ace markers Cells were stained with saturating amounts of MAb and,

after washing with saline, with fluorescein-conjugated F(ab')z goat anti-mouse Ig (Techno Genetics, Turin, Italy). The following primary MAbs were used: MAb directed against the common p2 subunit (CD18) of leukocyte integrin was obtained from hybridoma cell line T S l i l 8 (IgGi) from the ATCC (Rockville, MD); MAb clone CLB-LFAI / 2 (IgCzd) directed against the a L subunit (CDl la) of LFA-1 (Dr. R. Van Lier, CLB Amsterdam, the Netherlands); MAb clone 44a (IgG2,) directed against the a M subunit (CDl lb) of Mac-IiCR3 (Dr. R. Todd, Ann Arbor, MI); iMAb clone L29 (IgGI), recognizing the ax subunit (CDl lc) of p150,95 (Dr. L. Lanier, DNAX, Palo Alto, CA); MAb anti-VLA4, clone HP2/1 (IgG1) (Dr. F. Sanchez-Madrid, University of Madrid, Spain); MAb anti- ICAM-1, clone LB2 (IgG2b) (Dr. M. Patarroyo, Stockholm, Sweden); MAb anti-ICAM-3, clone CBR-IC3/2 (IgG2,) (Dr. T. Springer, Boston, MA) and clones TP1/24, TP1/25 and HP2119 (Dr. F. Sanchez-Madrid); MAb anti-MHC class I1 molecule, clone L243 from the ATCC. Analysis of fluorescence was performed using a FACSTAR-Plus instrument (Becton Dickinson, San Jose, CA). calibrated with Calibrite Beads (Becton Dickinson). Ten thousand events within the forward/ orthogonal light scatter monocyte gate were analyzed for each sample. Cells stained with an irrelevant antibody, MAb anti- IFGCP (IgGi) obtained from the ATCC, accounted for non- specific staining. Results are expressed as net number of percent positive cells after subtraction of percent positive non-specifically stained cells and as mean channel fluores- cence (MCF).

Cytokzrie assays IL-lp and IL-8 were measured by competitive binding

radioimmunoassay (RIA), except that the precipitation with second antibody was carried out using Amerlex magnetic beads coated with donkey anti-rabbit antibody (Amersham, Aylesburg, UK). '251L-lp was from Amersham, rhIL-lp was a gift from Dompe (L'Aquila, Italy) and rabbit anti-human IL-1p was from Sclavo (Siena, Italy). The sensitivity of the assay was 20 pgiml. '251L-8 was from Amersham, rhIL-8 was a gift of Dai-Nippon (Osaka, Japan); rabbit anti-h-IL-8 poly- clonal antibody was raised in our laboratory. The sensitivity of the assay was 20 pgiml.

IL-6 assay IL-6 was measured as hybridoma growth factor activity using

7TDI cell line (a kind gift of Dr. Van Snick, Brussels, Belgium) as described previously (Sironi et al., 1989). The sensitivity of the assay was 50 Uiml. The cell line 7TD1 responded specifi- cally to IL-6 and did not respond to IL-1, IL-2, IL-3, IL-4, IL-5, GM-CSF, M-CSF, TNF and lymphotoxin. Moreover, hybri- doma growth factor activity was blocked by an anti-IL-6 anti-serum (Sironi et al., 1989).

TNF was measured by cytotoxicity on L929 cells in the presence of 1 pgiml of actinomycin D, as previously described (Agganval et al., 198s) using recombinant TNF (gift of BASF/ Knoll, Ludwighafen, Germany; specific activity 6.6 x lo6 Uimg) as a standard. Thc sensitivity of the assay was 6 Uiml.

Norfhem blot analysis Total RNA was extracted by the guanidine isothiocyanate

method (Chirgwin et al., 1979). Northern blot analysis was as described in detail elsewhere (Sironi et al., 1989). A human ICAM-1 cDNA was labeled by nick translation and '*[a]dCT- P(Amersham, sp. act. 5000 Ciimmol). The protein synthesis inhibitor cycloheximide (Sigma, St Louis, MO) was used at a concentration of 10 ngiml. Loading and transfer of total RNA

onto nylon membranes were chcckcd by ethidium bromide staining and examination under UV light. Cytotoxicity assay

The tumoricidal activity of monocytes and TAM was mea- sured in a 24-hr and a 48-hr 3H-TdR release assay, as described (Peri et al., 1981). Briefly, tumor cells were prelabeled by incubation for 20 hr with 0.5 pCiiml 3H-TdR ( 5 Ci/mM, Amersham), cells were detached by exposure to 0.05% trypsin- 0.02% EDTA, washed and seeded (104/well) in 96-well flat- bottomed trays. Unstimulated and IFN-y/LPS or GM-CSF- treated monocytes or TAM (2 x lo5 resulting in an effector to target ratio of 20:l) were added in a final volume of 0.3 ml complete medium. After 24 or 48 hr of incubation, 0.1 ml of the supernatant was collected and isotope release was determined. Results are presented as mean ( 2 S D ) percentage of specific lysis (3 replicates) after subtraction of spontaneous isotope release. Differences of 4% to 6% specific lysis were usually statistically significant. Spontaneous isotope release at 48 hr was < 20% of total incorporated radioactivity, and unstimu- lated monocytes pre-incubated for 20 hr without stimuli had little or no cytotoxicity.

Statistical analysis Baseline functions of mononuclear phagocyte populations

were compared using the Mann Whitney U test, whereas the stimulatory effect of GM-CSF was analyzed using Student's t test for paired data.

FIGURE 3 - Expression in GM-CSF-treated monocytes of ICAM-1 transcripts. Human blood monocytes (upper panel) or mononuclear cells (lower panel) were treated with 100 ngiml GM-CSF for 1-18 hr (upper panel) or with GM-CSF (100 ngiml) and CH (10 pgiml) for 1 hr (lower panel), then examined for ICAM-1 expression. The ethidium bromide stained total RNAs blotted onto the membranes are shown on the right.

GM-CSF AND TUMOR-ASSOCIATED MACROPHAGES 303

RESULTS Iridiiction of cidhesion molecules

We examined the effect of GM-CSF on expression of adhesion molecules in peripheral blood monocytes and ovar- ian cancer TAM. In agreement with previous studies on blood cells (Peri et al., 1981; Economou et al., 1988; Erroi et al., 1989) no substantial difference in terms of adhesion molecule expres- sion and cytokine production was observed between normal donors and patients in this small ‘case list. As illustrated in the representative experiment in Figure 1, TAM showed higher expression of ICAM-1 than blood monocytes: e.g., MCF 394 vs. 244 (11 < 0.03 when fluorescence intensities of all patients were analyzed) but not of other adhesion molecules. GM-CSF markedly increased the expression of ICAM-1 in blood mono- cytes, with MCF of 237 % 25 (mean ? SE) and 431 ? 44 for control and treated cells ( p < 0.01,9 experiments). To a lesser degree, GM-CSF augmented the surface expression of p2 integrins, with an increase better evident for the common p chain CD18 and for C D l l a . C D l l b and C D l l c did not vary consistently (not shown). GM-CSF increased the expression of adhesion molecules in TAM, though the effect was less marked than that observed in monocytes, with MCF of 320 ? 30 and

MONOCYTES CAM-1 “‘r?

0 L 0 2 0 0 4 0 0 8 0 0 o o l l

CAM3

4 ; 94 % 243

0 2 0 0 4 0 0 8 0 0 o o 1 1 (

369 % 36 for ICAM-1 in control and GM-CSF-treated cells (5 experiments; Fig. 1).

The absolute increase of ICAM-I after GM-CSF treatment was 194 ? 43 and 49 ? 14 for monocytes and TAM, respectively (Fig. 2). The combination of IFNy and LPS, used as positive control in each experiment, was a better inducer of adhesion molecule expression with an absolute increase for ICAM-1 expression of 377 ? 37 and 137 2 51 for monocytes and TAM.

The effect of GM-CSF on ICAM-1 expression was dose- dependent, with an optimal effect at concentrations > 3 ng/ml and an incubation time of at least 12 hr (not shown).

It was interesting to explore whether induction of ICAM-1 was associated with increased mRNA and whether secondary polypeptide mediators (see below) could account for this induction. As shown in Figure 3 (representative of 2 experi- ments), GM-CSF induced augmented steady-state levels of ICAM-1 transcripts. The protein synthesis inhibitor cyclohexi- mide augmented ICAM-1 transcripts in unstimulated cells and reinforced GM-CSF-induced expression. GM-CSF has been reported to increase class I1 MHC in monocytes (Gosselin et

I 95% I 6 4

LPS + IFNy M r F l I 89 % I 264 I

I I

0 2 0 0 4 0 0 8 0 0 8 0 0 l m o

TAM CAM-1 CAM-3

0 u .

79 % 334

FIGURE 4 - Expression of ICAM-3 and ICAM-1 by blood monocytes and TAM from ovarian carcinomas. Cells from 1 representative experiment were exposed to medium or GM-CSF (100 ngiml) or IFNy (100 Uiml) and LPS (100 ngiml) for 20 hr and then examined for either ICAM-3 or ICAM-1 by FACS analysis. Numbers indicate the net percent positive cells (after subtraction of percent positive cells treated with an irrelevant antibody, shown as dotted vertical line) and the mean channel of fluorescence (MCF) on linear scale.

TABLE 1 -CONSTITUTIVE PRODUCTION OF CYTOKINES

Monncvtes T A M

IL-6 Uiml n = 8’ 30 10-737)‘ n = 9 271 I (195-57779) < 0.05’

IL-1 ngiml n = 7 0.04 (0.014.06) n = 9 0.07 (0.02-0.33) ns TNF Uiml n = 6 6 (4-9) n = 8 6 (4-8) ns

Blood monocytes and TAM from ascitic ovarian carcinoma were isolated as described in “Material and Methods.” Cells (3 x 1O6iml) were cultured in Petriperm dishes in complete medium for 20 hr. Supernatants were collected and tested for cytokine content.-lNumber of experiments.-?Median value, range in parenthesis.-?p value (Mann-Whitney test). ns = non-significant.

IL-8 ngiml n = 14 4 tO.33-80) n = 8 36(14-264) < 0.05

IL-6

1.0-

0.8 -

E 0.6- - . 0 4 -

0 2 -

IL-8

J

10000 : - E . 2

1000

100000

10000

10000

- E 1000 . 3

1 GO

10

100000

10000

1000

100

10 MEDIUM GM-CSF MEDIUM LPS + lFNy

TN F

MEDIUM GM-CSF

400 i

100

~ I d L i L 0 MEDIUM LPS + IFNy

IL-6

100000 100000 4 -

100- 100 MEDIUM GM-CSF

TNF

- MEDIUM LPS+IFNY

600 600

500 500

400

300 m = 200 200

F :::L 100 0 100 0

MEDIUM GM-CSF MEDIUM LPS + IFNy

IL-8

IL-1

50

30 40/ P

loooo 1 loooo 1

1' MEDIUM GM-CSF

3000

2000

1500 .

1- MEDIUM LPS+IFNy

I L - 1

Y MEDIUM GM-CSF MEDIUM LPS+IFN MEDIUM GM-CSF MEDIUM LPS + IFN Y FIGURE 5 - Cytokine production by rnonocytes ( a ) and TAM (h) from ascitic ovarian carcinomas after treatment with stimuli.

GM-CSF-induced IL-6 and IL-8 production in TAM were statistically significant compared to unstimulated cells:p < 0.02 a n d p < 0.04, respectively. Student's t test for paired data.

GM-CSF AND TUMOR-ASSOCIATED MACROPHAGES 305

al.. 1993). This finding was confirmed in 2 experiments in the present study, but GM-CSF did not affect class I1 in TAM (not shown). ICAM-3, a leukocyte adhesion molecule structurally and functionally related to ICAM-1 (de Fougerolles and Springer, 1992: Fawcett et al., 1992; Vazeux et al., 1992), was not affected by GM-CSF (Fig. 4).

Cytokine prodiiction The production of certain cytokines (IL-1, TNF and IL-6)

had been previously studied in TAM from ovarian carcinoma (Economou et al., 1988; Erroi et al., 1989; Naylor et al., 1990). Although the present study bas performed on a limited number of patients whose cells were exposed iri vitro to GM-CSF, it offered an opportunity to re-examine and extend prcvious observations. As shown in Table I, ovarian cancer TAM, in the absence of deliberate stimulation, produced appreciable levels of IL-6 (median value 2711 Uiml, range 195-57.779,~ < 0.05 vs. monocytes) but not of IL-1 and TNF, in agreement with previous observations (Economou et al., 1988: Erroi et al., 1989). In the absence of stimuli, TAM also relcased IL-8 in amounts substantially greater than monocytes (mean value 36 ngiml, range 114-264, p < 0.05); monocytes from normal donors and cancer patients usually produced undetectable levels of IL-8, only occasionally exceeding 10 ngiml.

GM-CSF had selective effects on cytokine production (Fig. 5). It induced no TNF or IL-1 production in monocytes (Fig. 5a) and TAM (Fig. Sb), though a small TNF response was

found in 4 of 6 monocyte preparations (Fig. 5a) . In contrast, GM-CSF increased the production of IL-6 and IL-8. Interest- ingly, the stimulating effect of GM-CSF on IL-6 and IL-8 was more evident on TAM (statistical significance p < 0.02 and p < 0.04, respectively), while monocytes did not reach signifi- cance. For instance, induction of IL-8 by GM-CSF was only observed in 3 monocyte preparations (Fig. 5a) . whereas augmentation of production by TAM was observed in 7 of 8 patients tested in this respect (Fig. 56).

In a limited series of experiments, in which GM-CSF was tested in combination with IFNy or LPS, we failed to observe a clear and reproducible synergism in terms of cytokine produc- tion or ICAM-1 expression (not shown).

Tirrnor cytotoxicity In a relatively limited series of experiments, we examined

the effect of GM-CSF on the tumoricidal activity of monocytes and TAM using tumor lines of ovarian origin. As shown in Figure 6, the combination of IFNy and LPS, used as a classical inducer of tumor cytotoxicity, stimulated monocyte-mediated killing against both OVCAR3 and SW626 targets. GM-CSF caused stimulation of killing by monocytes against the OVCAR3 and SW626 targets but, by and large, was a less active stimulus than IFNy + LPS. IFNy + LPS also stimulated cytotoxicity of TAM but at lower levels than blood monocytes. With GM- CSF, only 2 of 6 TAM preparations showed a modest increase in killing of OVCAR3 and SW626.

OVCAR-3

70 Bo 1

O l MEDIUM GM-CSF

J 70 -

0 1 MEDIUM LPS+IFNY

SW626

,

1 10 -

60 - 60 - E " v, 2 50 - u- P

8

\ 0-

MEDIUM GM-CSF MEDIUM LPS+IFNy

FIGURE 6 - Cytotoxic activity of untreated or GM-CSF-treated (100 ngiml for 20 hr) blood monocytes (0-0) or TAM (0-0) against ovarian carcinoma cell lines OVCAR3 and SW626. Tumor targets were labeled with 'H-TdR and co-cultured with monocytes for 48 hr. Results are expressed as percent specific lysis at the effector: target of 20:1. Each dot represents an individual experiment.

306 BERNASCONI ETAL.

DISCUSSION

The purpose of the present study was to conduct an extensive analysis of the effects of GM-CSF on blood mono- cytes and TAM in ovarian carcinoma. GM-CSF has profound effects on mature myelomonocytic cells, but several aspects of its action have been the object of conflicting reports (see for review Hamilton, 1993). The present study also provided an opportunity to extend current knowledge on TAM in ovarian cancer by characterizing expression of adhesion molecules and production of IL-8, albeit in a limited number of subjects.

GM-CSF augmented surface expression of the adhesion molecules ICAh4-1 and CD18. The effect was more evident for blood monocytes and ICAM-1. GM-CSF-induced surface expression of ICAM-1 was associated with induction of specific transcripts. Since GM-CSF induces cytokines which affect adhesion molecules (see below), it was important to assess whether inhibition of protein synthesis blocked induction by GM-CSF. Cycloheximide super-induced basal and GM-CSF- stimulated expression of ICAM-1, thus demonstrating that de nova protein synthesis is not required for ICAM-1 transcripts induction.

When compared with blood monocytes, TAM from ovarian carcinomas were reported to express substantial amounts of IL-6 but not of IL-1 and TNF (Erroi et al., 1989). These previous data were confirmed in this limited series of patients. In addition, TAM spontaneously released higher amounts (mean value 90 ng/ml) of IL-8 than monocytes, which only occasionally had detectable spontaneous release. As expected on the basis of this finding, IL-8 was also found in ascites, though other cell types besides TAM (e.g., neoplastic cells) could contribute to accumulation of this cytokine, IL-8 is a chemokine active on neutrophils and T lymphocytes (Oppen- heim et al., 1991). Neutrophils are frequently found in the ascites of ovarian carcinoma, and T lymphocytes are present in ascitic and solid tumors. It is tempting to speculate that TAM-derived IL-8 plays a role in regulating leukocyte infiltra- tion in ovarian cancer.

GM-CSF had different effects on cytokine production, depending on the cell population and mediator considered. It had little or no effect on IL-1 and TNF production in both monocytes and TAM. The effect of GM-CSF on IL-1 and TNF production by mononuclear phagocytes had been the object of conflicting reports (Hamilton, 1993). Monocyte and TAM production of IL-6 was increased by GM-CSF, though less than by IFNy + LPS. used as reference stimuli. When IL-8 was studied, somewhat different effects were observed in mono- cytes and TAM. GM-CSF was reported to induce IL-8 in blood monocytes (Takahashi et al., 1993). In the present study, low

levels of induction of IL-8 in monocytes were observed in only 3 of 6 donors. In contrast, GM-CSF augmented IL-8 produc- tion in 7 of 8 TAM preparations examined. Augmentation of chemokine production by in situ macrophages may act in concert with the attractant activity of GM-CSF itself (Wang et al., 1987) to amplify leukocyte recruitment.

GM-CSF has been reported to induce tumoricidal activity in human monocytes (Grabstein et al., 1986; Cannistra et al., 1988). This finding was confirmed and extended in the present study. GM-CSF augmented monocyte killing of the susceptible cell lines SW626 and OVCAR3. With TAM, a modest increase in tumor cytotoxicity against OVCAR3 and SW626 was ob- served only in 2 preparations. It has previously been shown that TAM are relatively refractory to augmentation of killing (Peri et al., 1981). This was confirmed in the present study with both GM-CSF and IFNy + LPS.

Transfer of the GM-CSF gene in the B16 melanoma was uniquely effective in eliciting protective anti-tumor immunity, possibly because of cell recruitment and interactions in the tumor micro-environment (Dranoff et al., 1993). Production of GM-CSF in certain tumors is associated with infiltration of dendritic/Langerhans cells (Tazi et al., 1993). ICAM-1 can serve as an accessory molecule in antigen presentation (Dustin and Springer, 1991), and IL-6 is a growth and differentiation factor for T and B cells (Kishimoto, 1989). Thus the modula- tion of TAM described here, in concert with recruitment of circulating precursors, may contribute to stimulation of immu- nogenicity associated with delivery of GM-CSF in the tumor micro-environment.

The effects of GM-CSF on mature mononuclear phagocytes and on related cell types, eg., dendritic cells (Tazi ef al., 1993), raise the interesting possibility that the function of this molecule in oncology may extend beyond counteracting rnyelo- suppression. Ovarian carcinoma has been extensively studied from the point of view of its leukocyte infiltrate, is uniquely suited for local administration of immune activators and shows clinical response to cytokines such as IFNy (Colombo et al., 1992; Pujade-Lauraine et al., 1990). The results reported here provide a rational as well as a biological endpoint for further study of GM-CSF in ovarian cancer.

ACKNOWLEDGEMENTS

This work was supported in part by the Program Italy- U.S.A. on Cancer Therapy, Consiglio Nazionale delle Ricer- che (finalized project ACRO to A.M.) and by the Italian Association for Cancer Research (AIRC).

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