Interleukin-1 production by immunologically hyporeactive tumour-bearing mice.

Mice bearing progressively growing syngeneic methylcholanthrene-induced sarcomas are immunologically hyporeactive. However, both basal (steady-state) and bacterial lipopolysaccharide (LPS)-induced synthesis of mRNA for interleukin-1 (IL-1) in peritoneal exudate cells (PEC) or spleen cells were comparable in control and tumour-bearing animals. Furthermore, the production of IL-1 by PEC stimulated with LPS in the presence of indomethacin was same in control and tumour-bearing mice. The results thus demonstrate that LPS-stimulated cells from animals bearing progressively growing syngeneic sarcomas synthesise the same quantities of mRNA for IL-1 and produce comparable amounts of IL-1 as do cells from normal animals, in spite of the profound immunological hyporeactivity of the former. ImagesFigure 2Figure 3Figure 4Figure 5

Animals or humans bearing progressively growing syngeneic tumours show a decrease in immunological responsiveness (North, 1985). The defects affect various components of the immune system including synthesis and production of lymphokines and cytokines. It has been demonstrated in various model systems that tumour bearers have impaired production of interleukin-1 (IL-1) (Pollack et al., 1983;Herman et al., 1984;Santos et al., 1985;Economon et al., 1988;Garraud et al., 1988).
IL-1 is thought to play a central role in host defence by virtue of its ability to augment the replication of activated T lymphocytes and to mediate several aspects of inflammation (Durum et al., 1985). Since the direct cytostatic (Gaffney & Tsai, 1986) or cytotoxic (Onozaki et al., 1985;Lachman et al., 1986) effects of IL-1 on tumour cells have been described, attempts with varying degrees of success to treat cancers with exogenous IL-1 have been reported (Nakamura et al., 1986;Braunschweiger et al., 1988;Bubenik et al., 1988;Castelli et al., 1988;Nakata et al., 1988;North et al., 1988).
Here we show, however, that both normal and immunologically hyporeactive mice bearing progressively growing syngeneic tumours synthesise comparable levels of mRNA for IL-1. They also produce similar amounts of IL-1, but the biological effects of IL-1 produced by tumour-bearing animals are masked by indomethacin-sensitive immunosuppressive molecules.

Animals
Male mice of the inbred strain C57BL/lOSn (here after called BlO) from the breeding colony of our Institute were used in all experiments.

Tumours
Sarcoma MC 11 originally induced by methylcholanthrene in a BlO male (Bubenik et al., 1978) was used. Tumour cells were grown in tissue cultures in Eagle's minimal essential medium supplemented with antibiotics, glutamine and 10% fetal calf serum. To induce tumours, 1 x 10' MC 11 cells in a volume of 0.1 ml of phosphate-buffered saline were injected subcutaneously into 7-8-week-old B1O mice. When the tumours reached a size of approximately 2 cm2 (roughly 3 weeks after cell inoculation), the animals were killed and used as donors of cells.
Thymocyte co-stimulatory assay Biological activity of IL-1 was tested in the thymocyte assay (Mizel et al., 1983). Thymocytes (5 x 10' per well) from 4-week-old B1O mice in 0.2 ml of complete RPMI 1640 medium were stimulated with Con A (2 ig ml'). Several dilutions of supernatants to be tested for IL-I activity were added to the wells. Cell proliferation was determined by adding 3H-thymidine (I piCi ml-') for the last 6 h of the 72 h incubation period.
IL-I production Unstimulated peritoneal exudate cells (PEC) from individual control and tumour-bearing mice were adjusted to a concentration of 2 x 106 ml' and cultured in 0.2 ml of complete RPMI 1640 medium in 96-well tissue culture plates (Sterilin). The cells were cultured alone, or with addition of 20 fig ml LPS (Difco) or 10-' M indomethacin (Sigma Chemical Co., St Louis, MO, USA) and/or in the presence of LPS and indomethacin. After 24h of culture the supernatants were collected and tested in the thymocyte assay. Northern blot analysis Total RNA was prepared from 2 x 107 spleen cells or PEC from control or tumour-bearing mice. The cells were freshly taken from animals or were incubated for 24 h unstimulated or were stimulated for the last 8 h of the 24 h incubation period with 20 pg ml-' LPS (Difco). Because the cells could transiently express some IL-1 mRNA upon isolation (Koide & Steinman, 1987), they were pre-incubated for 16 h before stimulation. RNA was extracted using the guanidine isothiocyanate method (Chirgwin et al., 1979) and subjected (30 jg per lane) to electrophoresis in 1% agarose, blotted on to Hybond N nitrocellulose membranes (Maniatis et al., (Lomedico et al., 1984) for 16 h at 42°C using the conditions of hybridization described elsewhere (Lipoldovia et al., 1989). The IL-la probe was a kind gift from Dr P.T. Lomedico (Hoffmann-La Roche Inc., Nutley, NJ, USA). Membranes were exposed at -70°C to Kodak XAR-5 film. The blots were then stripped and rehybridised with a cDNA probe for actin to demonstrate comparable quantities of RNAs in individual lanes.

Results
Hyporeactivity of spleen cells from tumour-bearing mice Spleen cells from mice bearing progressively growing MC 11 sarcomas were significantly hyporeactive (P<0.001 by Student's t test) in proliferative responses to both T-cell (Con A) and B-cell (LPS) mitogens (Figure 1).
Synthesis of IL-la mRNA in spleen cells No difference between quantities of IL-1 mRNA in cells from control and tumour-bearing mice was found when the activation of spleen cells from tumour-bearing mice was tested at the level of expression of the gene for IL-la. The LPSstimulated spleen cells from tumour-bearing mice produced a level of IL-la mRNA comparable to that observed in cells from control mice (Figure 2). When the basal (steady-state) synthesis of mRNA for IL-la was tested in freshly isolated and in cultured unstimulated spleen cells, no difference between cells from control and tumour-bearing animals was observed (Figures 3 and 4). Synthesis of IL-lI mRNA in PEC PEC from control and tumour-bearing mice was cultured unstimulated or stimulated with LPS for 8 h. As shown in Figure 5, the LPS-stimulated PEC from both control and tumour-bearing mice produced high and comparable levels of mRNA for IL-la.  (lanes 4-6) B10 mice were isolated and total RNA was immediately prepared. RNA was hybridised with IL-la probe and after stripping with an actin probe.
Production of IL-I by PEC Unstimulated PEC from both control and tumour-bearing mice did not produce any amounts of IL-1 measurable in the thymocyte assay. Nor was any biological activity of IL-1 detected in the supernatants obtained from cultures of LPSstimulated PEC from tumour-bearing mice and only low titres of IL-1 were found in supernatants from LPSstimulated PEC from control animals (data not shown).
Since the biological activity of IL-1 could be masked by prostaglandins produced by macrophages, we next tested the production of IL-1 in the presence of 10-5 M indomethacin. As shown in Figure (0) or five tumour-bearing (0) BIO mice were incubated at a concentration of 2 x 106 cells ml-' in medium containing LPS (20 1tg ml ') and 10-5 M indomethacin for 24 h and the supernatants obtained were tested in the thymocyte co-stimulaory assay for the ability to support Con A-induced proliferation of mouse thymocytes. -18S The cells of MC 11 tumour have never been detected in spleen or peritoneum of mice bearing subcutaneously growing tumours. Therefore, the possibility of IL-1 and/or prostaglandin production by MC 11 cells in vitro can be excluded. Discussion -18S Figure 5 Expression of mRNA for IL-la in PEC from control and tumour-bearing mice. PEC were washed out from groups of 20 control or tumour-bearing B10 mice and were cultured for 8 h unstimulated or stimulated with LPS (201agml-'). Total RNA was isolated and hybridised with IL-la probe and after stripping with an actin probe. The RNAs were from unstimulated (lanes 1 and 2) or LPS-stimulated (lanes 3 and 4) cells from control (lanes 1 and 3) or tumour-bearing (lanes 2 and 4) mice. Position of molecular weight marker (18S) is shown.
inhibitor of prostaglandin synthesis (indomethacin), produced significant and comparable titres of IL-1. Indomethacin alone did not stimulate detectable IL-I activity in PEC from control or tumour-bearing mice (data not shown).
Progressive growth of syngeneic tumour is generally associated with gradual decrease of immunological responsiveness in the host. We show here that this tumour-induced hyporeactivity is not associated with reduced production of IL-1. Mice bearing methylcholanthrene-induced sarcomas synthesised the same amount of mRNA for IL-1 and produced comparable amounts of IL-I as normal animals.
There are, however, a number of papers demonstrating impaired production of IL-1 in tumour bearers (Pollack et al., 1983;Herman et al., 1984;Santos et al., 1985;Economon et al., 1988;Garraud et al., 1988). The IL-I production had been detected by bioassays. Using these tests we detected no IL-1 also in the supernatants from LPS-stimulated PEC from our tumour-bearing mice. Only in the presence of indomethacin, a potent inhibitor of prostaglandin synthesis, could the production of IL-1 by tumour bearers be detected. The finding thus shows that immmunologically hyporeactive tumour-bearing mice have a potential to produce a normal level of IL-1, but the production and/or activity of IL-1 are masked by higher production of prostaglandins. The immunosuppressive effects of prostaglandins have been demonstrated (Goodwin & Ceuppens, 1983) and the synthesis of prostaglandins has been considered as one of the mechanisms of action of non-specific suppressor cells present in tumour bearers (Young & Hoover, 1986;Parhar & Lala, 1988). Also in our model of methylcholanthrene-induced sarcomas in the mouse we observed that spleen cells from tumour-bearing animals inhibited the mitogen-induced proliferation of cells from normal donors (our unpublished results). These obser-

IL-1
Actin -fi|l--vations indicate that the suppressor mechanism in tumour bearers does not irreversibly inhibit IL-1 production since mitogen-stimulated cells from tumour bearers produce IL-1 in vitro as well as do cells from normal animals.
IL-1 is a cytokine that has a number of biological functions in the body and that is produced by different cells (Durum et al., 1985). Although impaired produciton of IL-1 has been described in various immunopathological situations, such as bacterial infections (Czuprynski & Brown, 1987), aplastic anaemia (Gascon & Scala, 1988) and tumour growth (Nakamura et al., 1986;North et al., 1988), the possibility cannot be excluded that the biological effects of IL-1, when tested in an attempt to detect IL-I, were masked by immunosuppressive molecules produced by the immunologically compromised subjects.
The results reported here show that LPS-stimulated cells from immunologically hyporeactive mice bearing syngeneic, progressively growing tumours synthesise IL-1 to the same extent as do cells from normal animals. The lower production of IL-1 by tumour bearers or by other immunologically compromised subjects, as had been generally demonstrated by the bioassays, could be due to the presence of indomethacin-sensitive immunosuppressive molecules. Indeed, the therapeutic anti-tumour effects of IL-1 have been augmented by indomethacin (Nakata et al., 1988).