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CancerImmunolImmunother(1992) 35:395-400

ancer

mmunology mmunothPapy

© Springer-Verlag 1992

Effects of lipopolysaccharide on interleukin-2-induced cytotoxic activity of murine splenocyte cultures: role of prostaglandin E 2 and interferons Dominique Vaillier 1, Richard Daculsil, and Norbert Gualdel, 2 1URA 1456 CNRS,Universit6de BordeauxII, and 2FondationBergoni6,Bordeaux,France Received 31 March 1992/Accepted2 June 1992

Summary. Splenocytes cultured for 24 h in the presence of interleukin-2 (IL-2), lipopolysaccharide (LPS) or both together expressed a cytotoxic acitvity against the YAC-1 lymphoma cell line and to a lesser extent against P815 mastocytoma cells. The association of IL-2 and LPS had an additive effect on induction of cytotoxicity. The IL-2-induced cytotoxic activity lasted for the whole of the culture; however, the addition of LPS at the initiation of the culture increased the cytotoxic activity during its the early phase, the increment being followed by a fall of lyric activity after 72 h of culture. Assessment of interferon (IFN) in the culture supematants showed (a) a production of IFN 7 by IL-2supplemented cultures, (b) a more potent IFN production by cultures treated with IL-2 plus LPS (including 20% IFN~/13), (c) and that indomethacin (lgM) potentiated the effect of either IL-2 or LPS used alone but did not significantly increase the cytotoxic activity of cultures treated with IL-2 plus LPS (the one that produced a high level of IFN). When cultures were treated by an anti-IFN7 antibody we observed no change in the cytotoxic activity; however, in the presence of anti-IFN~13 serum the cytotoxic activity of cultures treated with IL-2 plus LPS was inhibited after 24 h but stimulated after 72 h. When cultures treated with IL-2 plus LPS were supplemented with both indomethacin and anti-IFNc~/13 the cytotoxic activity assessed after 72 h of culture was maintained at the same level as that of IL-2-treated cultures, hence the fall after 72 h of the cytotoxicity of cultures initiated in the presence of LPS alone was affected by both the immune serum and the cyclooxygenase inhibitor. Altogether these data show that when splenocytes are cultured for more than 72 h in the presence of IL-2 and LPS their cytotoxic activity decreases, and it is likely that this diminution is linked to the endogenous prodution of prostaglandin E2 and IFNo~/~.

Correspondence to: D. Vaillier, URA 1456 CNRS, Universit6 de Bordeaux, 146, rue L6o-Saignat,33076 Bordeauxcedex,France

Key words: Cytotoxicity - IL-2 - LPS - Interferons Prostaglandins

Introduction Non-MHC(major histocompatibility complex)-restricted cytotoxic cells can be induced by culturing unsensitized lymphocytes in the presence of interleukin-2 (IL-2) [12]. Most lymphokine-activated killer (LAK) cells are in fact activated natural killer (NK) cells rather than a distinct subset of lymphocytes [13, 14]. It has been found that NK cells growing in IL-2 produced interferon y (IFNT) in culture [9, 18]. However, little is known about the mechanisms regulating LAK cell induction by IL-2 and the role played by the interferons on LAK activity. For instance, confusing data have been reported concerning IFNy, which has been found to induce a synergistic effect with IL-2 [17], suppressive effects [5] or no effect [33]. On the other hand bacteria and their products have been shown to modulate the immune response in vivo and in vitro. Lipopolysaccharide (LPS) is an inducer of cytotoxic activity either after i.v. injection into mice [6] or in short-term culture [2]; it is known as an initiator of IFN [27, 2, 28, 35] and prostaglandins [7, 21], which regulate NK activity [30, 36]. The mechanisms whereby LPS activates NK cells have been studied; they concern the increase of the binding of NK cells to tumor target cells [32] and the activation of cytotoxicity by the IFN produced [8] or by an interferonindependent mechanism [34]. It is likely that in vivo, under normal physiological conditions, even if very small amounts of bacterial products are resorbed from the intestinal tract, they act on the lymphoid system. On the other hand, LAK cells have been used in adoptive immunotherapy for the treatment of malignancies, therefore it is interesting to study the effects of association of LPS and IL-2 in cultures of murine spleen cells on the induction of

396 c y t o t o x i c activity a n d the role p l a y e d b y the i n t e r f e r o n s a n d prostaglandins.

Table 1. Cytotoxic activity of spleen cells cultured for 24 h or 72 h in the presence of interleukin-2 (IL-2), lipopolysaccharide (LPS) or IL-2 plus LPS against YAC or P815 target cells Conditions of culture

Materials and m e t h o d s

24 h

Mice. C3H mice, 7 - 9 weeks old, were used throughout the experiments. Reagents. Lipopolysaccharide from Escherichia coli 055:B5 was purchased from Difco. Anti-IFN~[3 serum (1 : 320000) was a generous gift from Dr. Ion Gresser (IRCS, Villejuif, France). Anti-murine IFN7 antibodies were purchased from Genzyme (Boston, Mass.). rIL-2 was purchased from Cetus corporation (Emeryville, Calif.) and indomethacin from Sigma (St Louis, Mo.).

Celllines. YAC- 1, P815 and L929 cells were maintained in vitro; YAC- 1 is a Moloney-virus-induced lymphoma of A/Sn origin and P815 is a mastocytoma of DBA origin. L929 is a fibroblast cell line derived from an adult C3H mouse. Cells and culture medium. Spleens were harvested, filtered through gauze and the cell suspension obtained was suspended in culture medium. The culture medium was RPMI-1640 supplemented with 10% heat-inactivated fetal calf serum (FCS; Boehringer, Manheim, FRG), 2 mM L-glutamine, 10 mM HEPES, 100 U/ml penicillin, 100 gg/ml streptomycin sulfate and 0.05 mM 2-mercaptoethanol. Samples of 1 × 107 spleen cells were suspended in 2 ml culture medium and cultured for 24 h or 72 h in tissue-culture plates with 16-ram-diameter wells (Nunc, Denmark) in the presence of IL-2 (100 U/ml) or LPS (1 gg/ml) or both. Then the splenocytes were harvested, washed twice and used as effector cells in a 51Cr-release assay. Supernatants were collected, sterilized by filtration (0.22 gl) and kept frozen at-30 ° C until use. Cytotoxicassay. Effector spleen cells were assayed using 51Cr(Na52Cr04 Amersham, England)-labelledtarget cells. Cells from the YAC and P815 lines were used as targets. NK activity was measured for spleen/target cell ratios of 100 : I, 50 : 1 and 20 : 1 in a final volume of 200 gl. All assays were performed in triplicate. After 4 h at 37 ° C, 100 btl supernatant was removed from each well for counting. Specific lysis was calculated as: experimental release - spontaneous release lysis (%) =

total release - spontaneous release

Cytotoxic activitya (LU) after culture for

x 100

Spontaneous release was obtained by incubating targets alone and total release was determined by collecting 100 gl from wells in which the pellet had been resuspended Results of cytotoxicity assays were expressed as LU/107 cells. Lytic activity was calculated from linear regression curves plotted from the various E/T ratios. One lytic unit (LU) was defined as the number of effector cells required to lyse 20% of the targets.

1FNassay. The presence of IFN was assayed by anti-(vesicular stomatitis virus) activity. L929 cells were seeded in 96-well (3 x 104/well) flatmicrotiter plates (Nunclon). Serial dilutions of supernatants were added. After 18 h at 37 ° C, the cells were infected with vesicular stomatitis virus at a high multiplicity of infection. The plates were scored after 24 h. The antiviral effect was estimated by measuring the red neutral dye incorporation into living cells according to the method described by Hudson and Hay [ 15] and using a Titertek multiscan spectrophotometer. The IFN titer of a supernatant was estimated as the reciprocal of the dilution inhibiting 50% of the cytopathic effect.

Typing of IFN activity. IFN-containing supernatants were incubated at 37 ° C for 1 h with medium alone or with appropriate amounts of anti-IFN antibodies. Following incubation, the remaining IFN was assessed as above. The amounts of anti-IFN~l~ serum and anti-IFN7 antibodies

Medium LPSb IL-2c IL-2plusLPS

72 h

YAC

P815

YAC

P815

2.1+ 0.7a 23 _+ 3.4 47.2+ 6 89 -+12

0.2+0.1 7.2_+4.4 15.7-+4.2 24 -+2

2 +1.3 0.2_+0.2 56 _+6.6 18.1-+4

0.3_+0.2 0.3_+0.2 12.2+__3.6 1.3_+0.4

a Results are expressed in lytic units (LU) calculated at 20% cytotoxicity/107 cells. Mean of five experiments _+ SEM b LPS: 1 btg/ml c IL-2:100 U/ml

employed were sufficient to neutralize 1000 units of the corresponding 1FN, completely.

Statistics. Results were analyzed by using paired Student's t-tests.

Results

Effects of LPS on cytotoxic activity induced by IL-2 during 24 h or 72 h of culture of spleen cells S p l e n o c y t e s were c u l t u r e d d u r i n g 24 h or 72 h a l o n e or with I L - 2 (100 U / m l ) or L P S (1 btg/ml) or with I L - 2 plus LPS. C u l t u r e d cells w e r e h a r v e s t e d after 24 h or 72 h a n d their c y t o t o x i c activity assessed a g a i n s t target cells. It is w e l l k n o w n that N K cells are c y t o t o x i c for Y A C - 1 cells a n d that L A K cells are able to lyse b o t h Y A C - 1 a n d P 8 1 5 cells. W e v e r i f i e d that the P 8 1 5 cells w e u s e d were resistant to N K lysis b y fresh s p l e n i c cells (data n o t s h o w n ) , w e h a v e m a d e a series o f c o m p a r a t i v e e x p e r i m e n t s u s i n g either Y A C - 1 or P 8 1 5 as target cells. C y t o t o x i c activity o f s p l e e n cells c u l t u r e d for 24 h or 72 h alone or with I L - 2 or L P S or b o t h was m e a s u r e d . R e s u l t s o f five e x p e r i m e n t s , p o o l e d in T a b l e 1, s h o w e d that w h e n s p l e e n cells were c u l t u r e d alone, the c y t o t o x i c activity was v e r y low. T h e c y t o t o x i c activity i n d u c e d b y I L - 2 (100 U / m l ) was s u p e r i o r to that i n d u c e d b y L P S (1 btg/ml) after 24 h of culture. T h e p r e s e n c e o f b o t h activators i n d u c e d an additive effect. A f t e r 72 h o f culture, c y t o t o x i c activity i n d u c e d b y I L - 2 was m a i n t a i n e d , w h i l e the c y t o t o x i c activity fell in the cultures g r o w n i n the p r e s e n c e o f L P S a n d I L - 2 plus LPS. P 8 1 5 cells w e r e less l y s e d t h a n Y A C - 1 cells b u t since the m o d i f i c a t i o n s o f the c u l t u r e c o n d i t i o n s h a d a s i m i l a r i n f l u e n c e o n b o t h cell lines w e u s e d o n l y Y A C - 1 cells as targets for the n e x t e x p e r i m e n t s .

Effects of indomethacin on cytotoxic activity and 1FN production induced by either IL-2 or IL-2 plus LPS L P S is k n o w n to i n d u c e p r o s t a g l a n d i n Ea (PGE2) p r o d u c tion, w h i c h c a n b e i m p l i c a t e d in the r e d u c t i o n o f c y t o t o x i c activity o b s e r v e d i n the cultures m a i n t a i n e d in the p r e s e n c e

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Fig. 1. Effect of the presence of indomethacin (1BM) on cytotoxic activity induced by lipopolysaccharide (LPS), interleukin-2 (IL-2), and IL-2 plus LPS in 24-h or 72-h cultures of spleen cells. Means of five experiments _ SEM. *P

Effects of lipopolysaccharide on interleukin-2-induced cytotoxic activity of murine splenocyte cultures: role of prostaglandin E2 and interferons.

Splenocytes cultured for 24 h in the presence of interleukin-2 (IL-2), lipopolysaccharide (LPS) or both together expressed a cytotoxic activity agains...
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