0192-0561/92 $5.00 + .00 Pergamon Press Ltd. International Society for Immunopharmacology.
Int. J. lmmunopharmac., Vol. 14, No. 6, pp. 1061-1068, 1992. Printed in Great Britain.
I N VITRO ACTIVATION OF MURINE PERITONEAL EXUDATE CELLS (PEC) AND PERITONEAL MACROPHAGES BY ST 789 PIERO FORESTA,* VITO RUGGIERO, CLAUDIO ALBERTONI, LUCREZIA PACELLO, BARBARALEONI and EDOARDO ARRIGONI MARTELLI Sigma-Tau S.p.A., Research and Development, Department of Microbiology and Immunology, via Pontina km 30.400, 00040 Pomezia, Italy (Received 15 January 1992)
ST 789 is a new synthetic compound characterized by an amino acidic group joined to the N9 position of the hypoxanthine ring, which has been shown recently to have immunomodulating properties and minimal toxicity. The drug has been reported to protect immunosuppressed mice from microbial infections and turnout growth, and to restore the mitogen-induced proliferation of splenocytes from immunosuppressed young mice. In this study, we show that in vitro addition of ST 789 is able to markedly augment the sheep red blood cells (SRBC) phagocytosis by PEC, and to potentiate the cytotoxic activity of peritoneal exudate (PE) macrophages (Mdp) vs the L-M tumour cell line. We also found that ST 789 enhanced the rlFN-gamma-induced NO2- release from cultured PE Md?. Similarly, in vitro addition of ST 789 to the latter cultures significantly increased the production of interleukin 1 (IL-1) and tumour necrosis factor (TNF) induced by lipopolysaccharide (LPS). These studies demonstrate that ST 789 is a potent phagocyte activator for the induction of cytokine release, phagocytosis and cytotoxic activity against tumour cells in vitro.
Abstract --
ST 789 (formerly denominated PCF 39) is an L-Arg terminal synthetic compound belonging to a novel family of hypoxanthine derivatives characterized by an amino acidic group joined to the N9 position of the hypoxanthine ring (Stradi, Rossi, Perezzani, Migliorati, Riccardi & Cornaglia-Ferraris, 1990). Observations in animal studies showed that ST 789 may have effects on the immune system (CornagliaFerraris et al., 1987) in that it was able to reduce mortality and prolong survival time in experimentally immunosuppressed mice infected with different bacterial and fungal pathogens. It was also reported that ST 789 parenteral administration in mice induced an increase in spleen and lymph node cellularity resulting in a significant resistance to the growth of two distinct syngeneic transplanted tumours (Giovarelli et al., 1987). Furthermore, other reports showed that ST 789 was able to restore the responsiveness of lymphocytes to mitogens in experimentally immunodepressed animals (Ruggiero, Albertoni, Manganello, Foresta & Arrigoni Martelli, 1991) and to stimulate natural
killer cell progenitor differentiation in lethally irradiated mice (Ponzoni et al., 1987). However, nothing has yet been reported about the direct effects of this hypoxanthine derivative on activities typically exhibited by Md? or other phagocytic cells. Therefore, this study was undertaken with the aim to assess in vitro whether ST 789 was able to affect phagocytosis by PEC and to modulate both cytokine and NO2 release by PE Mdp as well as their cytotoxic activity vs a tumour cell line.
EXPERIMENTAL
PROCEDURES
Mice
Male hybrid B6D2FI (7 weeks of age) and C 3 H / H e J mice were purchased from Charles River (Calco, Italy) and kept under specific pathogen-free conditions. The latter mouse strain was used within the age range of 6 - 8 weeks as a source of thymocytes for IL-1 assay.
*Author to whom correspondence should be addressed. 1061
1062
P . FORESTA el al.
Reagents Phytohaemagglutinin (PHA; Wellcome Research, Beckenham, U.K.); LPS (Escherichia coli 055:B5, Difco Laboratories, MI, U.S.A.); ST 789, i.e. N-a-5-(1,6-dihydro-6-oxo-9-purinyl) pentyloxycarbonyl-L-arginine was manufactured by Sigma-Tau Chemical Laboratories (Pomezia, Italy); recombinant murine TNF, recombinant human IL-1, and recombinant murine interferon-gamma (IFN-gamma) were all from Genzyme (Boston, MA, U.S.A.). Tritiated thymidine (3H-TdR, 5 Ci/mmol) was purchased from Amersham (Arlington Heights, IL, U.S.A.).
Cell lines and tissue culture media L929 and L-M cell lines (two murine fibrosarcomas) were grown.in RPMI-1640 medium (Gibco, Grand Island, NY, U.S.A.) supplemented with 10°70 heat-inactivated foetal calf serum (Myoclone, Gibco), 25 mM HEPES (Sigma, St. Louis, MO, U.S.A.), 2 mM L-glutamine (Gibco), 50 ~g/ml gentamicin (Sigma), and 10/ag/ml tylocin (Gibco).
Isolation o f PEC and PE M+ Murine PEC were.harvested by peritoneal lavage using cold Hank's balanced salt solution Ca 2+ and Mg:+-free (HBSS, Gibco). PEC were then washed twice and resuspended in RPMI-1640 medium containing 10% FCS. M+ monolayers were prepared by plating PEC suspensions either in 24-well or 96-well plates (Falcon Primaria plates, B e c t o n Dickinson, Oxford, U.K.), and incubating at 37°C for 2 h in a humidified atmosphere of 5% CO2 to allow for M+ adherence. The non-adherent cells were then removed by washing three times with warm RPMI-1640 medium. This procedure resulted in an adherent cell population that was 85-95°70 macrophages, as determined by morphology and esterase staining.
Phagocytos& assay PEC were harvested as described above, and then resuspended at 4 × 10 6 cells/ml in supplemented RPMI-1640 medium. PEC suspensions were first incubated at 37°C for 2 h in polypropylene tubes (Falcon, Becton-Dickinson) in the absence (control) or in the presence of varying concentrations of ST 789. Then, volumes of 0.25 ml of PEC suspension were mixed with equal volumes of a 0.4°7o sheep red blood cells (SRBC) suspension, previously
opsonized with hyperimmune serum, and further incubated at 37°C for 1 h. After incubation, nonphagocityzed SRBC were lysed by brief hypotonic shock and after restoring the osmolarity the phagocytes were cytocentrifuged for 3 min at 800 rev/min (Cytospin-2, Shandon, Runcorn, Cheshire, U.K.) onto glass slides subsequently stained by a differential dye (Diff-Quick, Baxter Dade, Dudlingen, Switzerland). The slides were then examined under oil immersion (100×) and were subjected to two separate microscopic screenings of 100 phagocytes each. The percentage of phagocytes carrying ingested red cells, the mean number of ingested red cells per phagocyte, and the total number of phagocytized red cells were evaluated.
Cytotoxicity assay The tumoricidal activity of PE M~ was assessed by preparing M+ monolayers as described above. Then, 3H-TdR-labelled (18-h pulse with 1.0/aCi/ml) target L-M ceils were added to the wells yielding three different effector : target cell ratios (2.5 : 1, 5 : 1, and 1 0 : 1 ) . The volume of each well of the microplate was brought up to 0.3 ml by adding either culture medium (control) or serial dilutions of ST 789, and the microplate was incubated at 37°C in a humidified atmosphere of 5°70 CO2. After different incubation times (48 and 72 h) the uppermost 0.1 ml of supernatant was withdrawn from each well, and its radioactivity was counted in a 0-counter. Results are expressed as the mean (triplicate samples) of °7o specific 3H-TdR release = experimental c o u n t s / m i n -
spontaneous c o u n t s / m i n
total counts/min - spontaneous counts/rain
× 100.
Experimental counts/min and spontaneous counts/ min were evaluated from wells containing Mdp plus target cells and target cells alone, respectively. Total counts/min radioactivity was assessed by lysing target cells with 1°70 sodium dodecyl sulphate. Spontaneous lysis was always less than 15°70 of total radioactivity.
Induction of IL-1 and TNF release by PE M~b PE M+ prepared as described above were seeded in a 24-well plate at a density of 1.5 × 106 Mdp/well and incubated for 2 h at 37°C. Adherent M+ were then treated with serial dilutions of ST 789 either alone or together with 2 tag/ml of LPS, and incubated for 24 h at 37°C in a humidified atmosphere of 5°70 CO~.
In Vitro Activation At the end of this incubation, the cultured Md~ supernatants were harvested, sterilized by passage through 0.45/am Millipore filters (Millipore Corporation, Bedford, MA, U.S.A.), and used as such for TNF assays. Conversely, supernatants to be assayed for IL-1 activity were first dialysed in a membrane of pore size allowing passage of molecules up to approximately 10 kDa (Serva, Heidelberg, F.R.G.) and then filtered as described above. All samples were stored at - 8 0 ° C until assayed.
1L-1 assay IL-1 activity was determined by enhancement of thymocyte proliferation in the classical costimulation assay (Gery, Gershon & Waksman, 1972). Thymocytes from 6 to 8-week-old C3H/HeJ mice (hyporesponsive to endotoxin) were used. Briefly, thymocyte suspensions were prepared in RPMI-1640 containing 10°70 FCS and 50/aM 2-mercaptoethanol by teasing the thymus gently and finely followed by passage through a sieve to remove clumps. The thymocyte suspensions were then placed into the wells (1.0 x 10 6 cells/ml in 100/al medium) of a flat-bottomed 96-well microtitre plate. Serial twofold dilutions of dialysed M+ supernatants were then added to wells (in triplicate) in the presence of a suboptimal concentration of PHA (2/ag/ml). The final volume of each culture was 200 tal. The cultures were incubated for 72 h at 37°C in a 5070 CO2 atmosphere, and were labelled with 3H-TdR (0.5/aCi/well) during the last 18 h of incubation. The cells were then harvested onto glass fibre filtermats (Pharmacia, Turku, Finland) using an automated cell harvester (Pharmacia) and 3H-TdR incorporation was assessed by a 0-plate counter (Pharmacia). In each experiment, a corresponding standard curve was made by adding varying concentrations of recombinant human IL-1 to control wells.
TNFa assay TNFa levels in PE Md~ supernatants were determined by using the L929 cytotoxicity bioassay described by Flick & Gifford (1984) with minor modifications. Briefly, 100/al of L929 cells (3.2 x 105 cells/ml) in RPMI-1640 containing 1°70 FCS were distributed into each well of a flatbottomed 96-well microtitre plate and incubated overnight at 37°C in a humidified atmosphere of 5°7o CO2. After incubation, spent medium was discarded and serial dilutions (carried out in RPMI-1640 1°70
1063
FCS) of M~ supernatants were added to the cells in the presence of Actinomycin D at a final concentration of 2/ag/ml. The L929 cell cultures were then further incubated for 18 h at 37°C in a humidified atmosphere of 5°7o CO2. Following this incubation, the medium was discarded, and the plates were first washed twice with 0.9% NaCI and then stained for 15 min at room temperature with Crystal Violet (0.5°7o in 20°7o ethanol and 8°70 formaldehyde). After discarding the stain, the wells were gently washed in running tap water. The uptaken dye was then eluted with 33°7o acetic acid and the relative absorbance monitored at 590 nm. TNF activity was determined by using recombinant murine TNFa as a reference standard.
N O 2- production and assay PE M+ prepared as described above were seeded ( 1 . 0 X 10 6 M+/well) in a 24-well plate (Falcon,
Becton-Dickinson) and incubated 2 h at 37°C to allow them to adhere. After washing the M+ monolayers, ST 789 serial dilutions were added to the wells without or together with 10 U/ml of IFN-gamma, and the plate was further incubated for 48 h at 37°C in a humidified atmosphere of 5°7oCO2. After incubation, supernatants were collected and the nitrite concentration was measured by a microplate assay method (Ding, Nathan & Stuehr, 1988). Briefly, aliquots of supernatants were transferred to a microplate (0.1 ml/well), then mixed with an equal volume of Griess reagent (1% sulfanilamide/0.1% naphtylene diamine dihydrochloride/2.5% H3PO4) and incubated at room temperature for 10 min. The absorbance at 540 nm was measured with a Multiskan MCC/340 ELISA reader (Flow Laboratories, McLean, VA, U.S.A.) using a 620 nm reference filter. NO: was determined by using sodium nitrite as a standard. The nitrite concentration in the culture medium alone was also determined and subtracted from the values obtained in the experimental conditions.
RESULTS
Effects o f S T 789 on SRBC phagocytosis by PEC In this experiment, murine PEC prepared as described in Experimental Procedures were pretreated for 2 h with ST 789 at concentrations ranging from 1 to 100/~g/ml before performing SRBC phagocytosis. As shown in Table 1, ST 789 was able to significantly affect SRBC phagocytosis
1064
P. FORESTA et al. Table 1. ST 789 effect on SRBC phagocytosis by murine PEC Phagocytizing cells*
Treatment Control ST 789 (1 ~g/ml) ST 789 (10 pg/ml) ST 789 (100 lag/ml)
36 34 46 48
No. SRBC phagocytized ~
+_ 0.6 + 2.0 + 2.0 + 2.0
138 139 176 205
SRBC per phagocyte*
+ 11 +_ 05 _+ 15 _+ 11
3.72 4.05 3.77 4.22
PEC (4 _+ 106 cells/ml) were treated in vitro either without or with the addition of ST 789 for 2 h at 37°C before testing their phagocytic activity. Each value represents the mean _+ S.D. of triplicate determinations. *Percentage of cells which have actively phagocytized with respect to the total number of phagocytizing cells. t M e a n value of SRBC phagocytized by 100 phagocytes. *Mean value of SRBC phagocytized by each phagocyte.
75
75-
~ 0
50-
r.
,.A
cj
o9 2 5 ¢)
j¢;:';;'
25
.......f S i "~'''"" ~
,
,
2.5:1
5:1 E:T
10:1
Fig. 1. Induction of PE M+-mediated cytotoxicity against L-M t u m o r cells by ST 789. PE M+ were cultured with L-M cells at three different effector : target cell ratios in the presence of 10/~g/ml (m) and 100/~g/ml ( A ) for 48 h at 37°C. Open circles represent the results o f L-M and M+ cultures without test samples.
2,5:1
5:1 E:T
Fig. 2. Induction of PE M+-mediated cytotoxicity against L-M tumor cells by ST 789. PE M+ were cultured with L-M cells at three different effector : target cell ratios in the presence of l0/~g/ml ( I ) and 100 ~g/ml ( A ) for 72 h at 37°C. Open circles represent the results of L-M and M+ cultures without test sample.
by resident phagocytes increasing the percentage of p h a g o c y t i z i n g cells f r o m 36 ( c o n t r o l ) u p to 46 a n d 48°7o at c o n c e n t r a t i o n s o f S T 789 o f 10 a n d 100/ag/ml, respectively. Furthermore, the number of S R B C p h a g o c y t i z e d b y P E C i n c r e a s e d u p to 127 a n d 148°70 f o l l o w i n g t r e a t m e n t w i t h 10 a n d 100 ~ g / m l o f S T 789, r e s p e c t i v e l y .
Treatment
--
--*
--
Effects o f S T 789 on P E M+ cytotoxicity
2 ----
PE M+ were cultured with ~H-TdR-labelled L-M t a r g e t cells e i t h e r a l o n e o r in t h e p r e s e n c e o f ST 789 f o r 48 a n d 72 h p r i o r to a s s e s s i n g t h e M + - i n d u c e d c y t o t o x i c i t y at t h r e e d i f f e r e n t e f f e c t o r : t a r g e t r a t i o s . F i g u r e 1 i l l u s t r a t e s t h e lysis o f L - M t a r g e t s b y u n t r e a t e d o r S T 7 8 9 - t r e a t e d P E M + at i n c r e a s i n g E : T r a t i o s , a n d a c o n s t a n t t a r g e t cell n u m b e r a f t e r
10:1
Table 2. Levels of IL-1 in culture supernatants of PE M+ treated with varying concentrations of ST 789 or LPS
LPS (/ag/ml)
ST 789 (/~g/ml)
1 10 100
IL-1 (U/ml)
Int. J. lmmunopharmac., Vol. 14, No. 6, pp. 1061-1068, 1992. Printed in Great Britain.
I N VITRO ACTIVATION OF MURINE PERITONEAL EXUDATE CELLS (PEC) AND PERITONEAL MACROPHAGES BY ST 789 PIERO FORESTA,* VITO RUGGIERO, CLAUDIO ALBERTONI, LUCREZIA PACELLO, BARBARALEONI and EDOARDO ARRIGONI MARTELLI Sigma-Tau S.p.A., Research and Development, Department of Microbiology and Immunology, via Pontina km 30.400, 00040 Pomezia, Italy (Received 15 January 1992)
ST 789 is a new synthetic compound characterized by an amino acidic group joined to the N9 position of the hypoxanthine ring, which has been shown recently to have immunomodulating properties and minimal toxicity. The drug has been reported to protect immunosuppressed mice from microbial infections and turnout growth, and to restore the mitogen-induced proliferation of splenocytes from immunosuppressed young mice. In this study, we show that in vitro addition of ST 789 is able to markedly augment the sheep red blood cells (SRBC) phagocytosis by PEC, and to potentiate the cytotoxic activity of peritoneal exudate (PE) macrophages (Mdp) vs the L-M tumour cell line. We also found that ST 789 enhanced the rlFN-gamma-induced NO2- release from cultured PE Md?. Similarly, in vitro addition of ST 789 to the latter cultures significantly increased the production of interleukin 1 (IL-1) and tumour necrosis factor (TNF) induced by lipopolysaccharide (LPS). These studies demonstrate that ST 789 is a potent phagocyte activator for the induction of cytokine release, phagocytosis and cytotoxic activity against tumour cells in vitro.
Abstract --
ST 789 (formerly denominated PCF 39) is an L-Arg terminal synthetic compound belonging to a novel family of hypoxanthine derivatives characterized by an amino acidic group joined to the N9 position of the hypoxanthine ring (Stradi, Rossi, Perezzani, Migliorati, Riccardi & Cornaglia-Ferraris, 1990). Observations in animal studies showed that ST 789 may have effects on the immune system (CornagliaFerraris et al., 1987) in that it was able to reduce mortality and prolong survival time in experimentally immunosuppressed mice infected with different bacterial and fungal pathogens. It was also reported that ST 789 parenteral administration in mice induced an increase in spleen and lymph node cellularity resulting in a significant resistance to the growth of two distinct syngeneic transplanted tumours (Giovarelli et al., 1987). Furthermore, other reports showed that ST 789 was able to restore the responsiveness of lymphocytes to mitogens in experimentally immunodepressed animals (Ruggiero, Albertoni, Manganello, Foresta & Arrigoni Martelli, 1991) and to stimulate natural
killer cell progenitor differentiation in lethally irradiated mice (Ponzoni et al., 1987). However, nothing has yet been reported about the direct effects of this hypoxanthine derivative on activities typically exhibited by Md? or other phagocytic cells. Therefore, this study was undertaken with the aim to assess in vitro whether ST 789 was able to affect phagocytosis by PEC and to modulate both cytokine and NO2 release by PE Mdp as well as their cytotoxic activity vs a tumour cell line.
EXPERIMENTAL
PROCEDURES
Mice
Male hybrid B6D2FI (7 weeks of age) and C 3 H / H e J mice were purchased from Charles River (Calco, Italy) and kept under specific pathogen-free conditions. The latter mouse strain was used within the age range of 6 - 8 weeks as a source of thymocytes for IL-1 assay.
*Author to whom correspondence should be addressed. 1061
1062
P . FORESTA el al.
Reagents Phytohaemagglutinin (PHA; Wellcome Research, Beckenham, U.K.); LPS (Escherichia coli 055:B5, Difco Laboratories, MI, U.S.A.); ST 789, i.e. N-a-5-(1,6-dihydro-6-oxo-9-purinyl) pentyloxycarbonyl-L-arginine was manufactured by Sigma-Tau Chemical Laboratories (Pomezia, Italy); recombinant murine TNF, recombinant human IL-1, and recombinant murine interferon-gamma (IFN-gamma) were all from Genzyme (Boston, MA, U.S.A.). Tritiated thymidine (3H-TdR, 5 Ci/mmol) was purchased from Amersham (Arlington Heights, IL, U.S.A.).
Cell lines and tissue culture media L929 and L-M cell lines (two murine fibrosarcomas) were grown.in RPMI-1640 medium (Gibco, Grand Island, NY, U.S.A.) supplemented with 10°70 heat-inactivated foetal calf serum (Myoclone, Gibco), 25 mM HEPES (Sigma, St. Louis, MO, U.S.A.), 2 mM L-glutamine (Gibco), 50 ~g/ml gentamicin (Sigma), and 10/ag/ml tylocin (Gibco).
Isolation o f PEC and PE M+ Murine PEC were.harvested by peritoneal lavage using cold Hank's balanced salt solution Ca 2+ and Mg:+-free (HBSS, Gibco). PEC were then washed twice and resuspended in RPMI-1640 medium containing 10% FCS. M+ monolayers were prepared by plating PEC suspensions either in 24-well or 96-well plates (Falcon Primaria plates, B e c t o n Dickinson, Oxford, U.K.), and incubating at 37°C for 2 h in a humidified atmosphere of 5% CO2 to allow for M+ adherence. The non-adherent cells were then removed by washing three times with warm RPMI-1640 medium. This procedure resulted in an adherent cell population that was 85-95°70 macrophages, as determined by morphology and esterase staining.
Phagocytos& assay PEC were harvested as described above, and then resuspended at 4 × 10 6 cells/ml in supplemented RPMI-1640 medium. PEC suspensions were first incubated at 37°C for 2 h in polypropylene tubes (Falcon, Becton-Dickinson) in the absence (control) or in the presence of varying concentrations of ST 789. Then, volumes of 0.25 ml of PEC suspension were mixed with equal volumes of a 0.4°7o sheep red blood cells (SRBC) suspension, previously
opsonized with hyperimmune serum, and further incubated at 37°C for 1 h. After incubation, nonphagocityzed SRBC were lysed by brief hypotonic shock and after restoring the osmolarity the phagocytes were cytocentrifuged for 3 min at 800 rev/min (Cytospin-2, Shandon, Runcorn, Cheshire, U.K.) onto glass slides subsequently stained by a differential dye (Diff-Quick, Baxter Dade, Dudlingen, Switzerland). The slides were then examined under oil immersion (100×) and were subjected to two separate microscopic screenings of 100 phagocytes each. The percentage of phagocytes carrying ingested red cells, the mean number of ingested red cells per phagocyte, and the total number of phagocytized red cells were evaluated.
Cytotoxicity assay The tumoricidal activity of PE M~ was assessed by preparing M+ monolayers as described above. Then, 3H-TdR-labelled (18-h pulse with 1.0/aCi/ml) target L-M ceils were added to the wells yielding three different effector : target cell ratios (2.5 : 1, 5 : 1, and 1 0 : 1 ) . The volume of each well of the microplate was brought up to 0.3 ml by adding either culture medium (control) or serial dilutions of ST 789, and the microplate was incubated at 37°C in a humidified atmosphere of 5°70 CO2. After different incubation times (48 and 72 h) the uppermost 0.1 ml of supernatant was withdrawn from each well, and its radioactivity was counted in a 0-counter. Results are expressed as the mean (triplicate samples) of °7o specific 3H-TdR release = experimental c o u n t s / m i n -
spontaneous c o u n t s / m i n
total counts/min - spontaneous counts/rain
× 100.
Experimental counts/min and spontaneous counts/ min were evaluated from wells containing Mdp plus target cells and target cells alone, respectively. Total counts/min radioactivity was assessed by lysing target cells with 1°70 sodium dodecyl sulphate. Spontaneous lysis was always less than 15°70 of total radioactivity.
Induction of IL-1 and TNF release by PE M~b PE M+ prepared as described above were seeded in a 24-well plate at a density of 1.5 × 106 Mdp/well and incubated for 2 h at 37°C. Adherent M+ were then treated with serial dilutions of ST 789 either alone or together with 2 tag/ml of LPS, and incubated for 24 h at 37°C in a humidified atmosphere of 5°70 CO~.
In Vitro Activation At the end of this incubation, the cultured Md~ supernatants were harvested, sterilized by passage through 0.45/am Millipore filters (Millipore Corporation, Bedford, MA, U.S.A.), and used as such for TNF assays. Conversely, supernatants to be assayed for IL-1 activity were first dialysed in a membrane of pore size allowing passage of molecules up to approximately 10 kDa (Serva, Heidelberg, F.R.G.) and then filtered as described above. All samples were stored at - 8 0 ° C until assayed.
1L-1 assay IL-1 activity was determined by enhancement of thymocyte proliferation in the classical costimulation assay (Gery, Gershon & Waksman, 1972). Thymocytes from 6 to 8-week-old C3H/HeJ mice (hyporesponsive to endotoxin) were used. Briefly, thymocyte suspensions were prepared in RPMI-1640 containing 10°70 FCS and 50/aM 2-mercaptoethanol by teasing the thymus gently and finely followed by passage through a sieve to remove clumps. The thymocyte suspensions were then placed into the wells (1.0 x 10 6 cells/ml in 100/al medium) of a flat-bottomed 96-well microtitre plate. Serial twofold dilutions of dialysed M+ supernatants were then added to wells (in triplicate) in the presence of a suboptimal concentration of PHA (2/ag/ml). The final volume of each culture was 200 tal. The cultures were incubated for 72 h at 37°C in a 5070 CO2 atmosphere, and were labelled with 3H-TdR (0.5/aCi/well) during the last 18 h of incubation. The cells were then harvested onto glass fibre filtermats (Pharmacia, Turku, Finland) using an automated cell harvester (Pharmacia) and 3H-TdR incorporation was assessed by a 0-plate counter (Pharmacia). In each experiment, a corresponding standard curve was made by adding varying concentrations of recombinant human IL-1 to control wells.
TNFa assay TNFa levels in PE Md~ supernatants were determined by using the L929 cytotoxicity bioassay described by Flick & Gifford (1984) with minor modifications. Briefly, 100/al of L929 cells (3.2 x 105 cells/ml) in RPMI-1640 containing 1°70 FCS were distributed into each well of a flatbottomed 96-well microtitre plate and incubated overnight at 37°C in a humidified atmosphere of 5°7o CO2. After incubation, spent medium was discarded and serial dilutions (carried out in RPMI-1640 1°70
1063
FCS) of M~ supernatants were added to the cells in the presence of Actinomycin D at a final concentration of 2/ag/ml. The L929 cell cultures were then further incubated for 18 h at 37°C in a humidified atmosphere of 5°7o CO2. Following this incubation, the medium was discarded, and the plates were first washed twice with 0.9% NaCI and then stained for 15 min at room temperature with Crystal Violet (0.5°7o in 20°7o ethanol and 8°70 formaldehyde). After discarding the stain, the wells were gently washed in running tap water. The uptaken dye was then eluted with 33°7o acetic acid and the relative absorbance monitored at 590 nm. TNF activity was determined by using recombinant murine TNFa as a reference standard.
N O 2- production and assay PE M+ prepared as described above were seeded ( 1 . 0 X 10 6 M+/well) in a 24-well plate (Falcon,
Becton-Dickinson) and incubated 2 h at 37°C to allow them to adhere. After washing the M+ monolayers, ST 789 serial dilutions were added to the wells without or together with 10 U/ml of IFN-gamma, and the plate was further incubated for 48 h at 37°C in a humidified atmosphere of 5°7oCO2. After incubation, supernatants were collected and the nitrite concentration was measured by a microplate assay method (Ding, Nathan & Stuehr, 1988). Briefly, aliquots of supernatants were transferred to a microplate (0.1 ml/well), then mixed with an equal volume of Griess reagent (1% sulfanilamide/0.1% naphtylene diamine dihydrochloride/2.5% H3PO4) and incubated at room temperature for 10 min. The absorbance at 540 nm was measured with a Multiskan MCC/340 ELISA reader (Flow Laboratories, McLean, VA, U.S.A.) using a 620 nm reference filter. NO: was determined by using sodium nitrite as a standard. The nitrite concentration in the culture medium alone was also determined and subtracted from the values obtained in the experimental conditions.
RESULTS
Effects o f S T 789 on SRBC phagocytosis by PEC In this experiment, murine PEC prepared as described in Experimental Procedures were pretreated for 2 h with ST 789 at concentrations ranging from 1 to 100/~g/ml before performing SRBC phagocytosis. As shown in Table 1, ST 789 was able to significantly affect SRBC phagocytosis
1064
P. FORESTA et al. Table 1. ST 789 effect on SRBC phagocytosis by murine PEC Phagocytizing cells*
Treatment Control ST 789 (1 ~g/ml) ST 789 (10 pg/ml) ST 789 (100 lag/ml)
36 34 46 48
No. SRBC phagocytized ~
+_ 0.6 + 2.0 + 2.0 + 2.0
138 139 176 205
SRBC per phagocyte*
+ 11 +_ 05 _+ 15 _+ 11
3.72 4.05 3.77 4.22
PEC (4 _+ 106 cells/ml) were treated in vitro either without or with the addition of ST 789 for 2 h at 37°C before testing their phagocytic activity. Each value represents the mean _+ S.D. of triplicate determinations. *Percentage of cells which have actively phagocytized with respect to the total number of phagocytizing cells. t M e a n value of SRBC phagocytized by 100 phagocytes. *Mean value of SRBC phagocytized by each phagocyte.
75
75-
~ 0
50-
r.
,.A
cj
o9 2 5 ¢)
j¢;:';;'
25
.......f S i "~'''"" ~
,
,
2.5:1
5:1 E:T
10:1
Fig. 1. Induction of PE M+-mediated cytotoxicity against L-M t u m o r cells by ST 789. PE M+ were cultured with L-M cells at three different effector : target cell ratios in the presence of 10/~g/ml (m) and 100/~g/ml ( A ) for 48 h at 37°C. Open circles represent the results o f L-M and M+ cultures without test samples.
2,5:1
5:1 E:T
Fig. 2. Induction of PE M+-mediated cytotoxicity against L-M tumor cells by ST 789. PE M+ were cultured with L-M cells at three different effector : target cell ratios in the presence of l0/~g/ml ( I ) and 100 ~g/ml ( A ) for 72 h at 37°C. Open circles represent the results of L-M and M+ cultures without test sample.
by resident phagocytes increasing the percentage of p h a g o c y t i z i n g cells f r o m 36 ( c o n t r o l ) u p to 46 a n d 48°7o at c o n c e n t r a t i o n s o f S T 789 o f 10 a n d 100/ag/ml, respectively. Furthermore, the number of S R B C p h a g o c y t i z e d b y P E C i n c r e a s e d u p to 127 a n d 148°70 f o l l o w i n g t r e a t m e n t w i t h 10 a n d 100 ~ g / m l o f S T 789, r e s p e c t i v e l y .
Treatment
--
--*
--
Effects o f S T 789 on P E M+ cytotoxicity
2 ----
PE M+ were cultured with ~H-TdR-labelled L-M t a r g e t cells e i t h e r a l o n e o r in t h e p r e s e n c e o f ST 789 f o r 48 a n d 72 h p r i o r to a s s e s s i n g t h e M + - i n d u c e d c y t o t o x i c i t y at t h r e e d i f f e r e n t e f f e c t o r : t a r g e t r a t i o s . F i g u r e 1 i l l u s t r a t e s t h e lysis o f L - M t a r g e t s b y u n t r e a t e d o r S T 7 8 9 - t r e a t e d P E M + at i n c r e a s i n g E : T r a t i o s , a n d a c o n s t a n t t a r g e t cell n u m b e r a f t e r
10:1
Table 2. Levels of IL-1 in culture supernatants of PE M+ treated with varying concentrations of ST 789 or LPS
LPS (/ag/ml)
ST 789 (/~g/ml)
1 10 100
IL-1 (U/ml)
