Interactions between rnacrophage cytokines and eicosanoids in expression of antitumour activity

Cytokines and eicosanoid products of macrophages play an essential role in expression of antitumour activity of macrophages either in a cell-to-cell contact system between the effector and the target cell or as cell-free soluble products. In this review the relationship between three main monokines, namely TNF-α, IL-1 and IL-6 and the interrelationship between these monokines and eicosanoids (PGE2, PGI2, LTB4, LTC4) in their production and in expression of antitumour activity is discussed. Emphasis is given to the effect of tumour burden on production of the monokines and of the eicosanoids and on the production of these compounds by the tumour cells. Finally, the therapeutic implications drawn from animal studies and clinical trials is discussed.


Introduction
Since the pioneering work of Metchnikoff, it became more and more evident that macrophage cells play an essential role in a wide array of biological activities. This includes among others the events of nonspecific resistance against invasion of foreign cells (including tumour cells), their function as a crucial mediator in the development of immune response and participation in the process of inflammation (for review see Ref. 2).
The various functions of macrophages are exerted either in a cell-to-cell contact set-up with the target cells or by various biologically and pharmacologically active factors released by these cells. Among the most salient factors released by macrophages are various monokines and products of arachidonic acid (prostaglandins and leukotrienes). An extensive amount of work has been done on the pharmacological and biological effects of macrophage cytokines and eicosanoids. The present review is by no means intended to provide a full coverage of all the activities of macrophage cytokines and eicosanoids. The aim is to discuss only the topic of interrelation between certain macrophage cytokines and eicosanoids in the context of their expression of antitumour activity. The main emphasis will be on the role of macrophage cytokines tumour necrosis factor-(TNF-) interleukin-1 (IL-1) and interleukin-6 (IL-6) and on the role of prostaglandin E 2 (PGE2), leukotriene B 4 (LTB4) and leukotriene C4 (LTC4) and their interrelation in the production and expression of antitumour activity. Another aspect (C) 1992 Rapid Communications of Oxford ktd to be discussed is the effect of tumour burden on their production by macrophages, production of these compounds by tumour cells and their therapeutic effectiveness in experimental turnout models and in cancer patients.
The first indications on induced release of certain antitumour factors by bacteria-free filtrates came for the early work of Coley (for review see Ref. 3). Later on, Carswell et a]. 4 reported the occurrence of an antiturnour cytotoxic factor in the serum of mice which had undergone treatment with bacterial lipopolysaccharide (LPS), coined the term tumour necrosis factor (TNF) and suggested that TNF is produced by activated macrophages in response to LPS. The occurrence of a lymphocyte activated factor produced by macrophages was first reported in 19725, 6 and was named lymphocyte activating factor (LAF). In 1979, the nomenclature of various cytokines was revised 7 and the former LAF was given the name of IL-1 which is still used today. IL-6 was initially described as a factor derived from fibroblasts with antiviral activity. 8 The term IL-6 was first suggested by Poupart and colleagues 9 and its production by human monocytes was reported. 1 Interactions in the production of macrophage cytokines and eicosanoids Interactions in production between TNF-, IL-1 and IL- 6: It has been reported that production from blood mononuclear cells and from peritoneal macrophages of TNF-, IL-1 and IL-6, can be induced by the same stimulatory agents. This applies to LPS, >3 phytohaemagglutinin (PHA) 3 Mediators of Inflammation. Vol and Staphylococcus epidermis. 13 It should be noted also monocytes was also reported by other authors. 26 By that the same monokine can be induced by different working with bone marrow derived mouse mechanisms. Thus, itwas reported that Mycoplasma macrophages it was found that LPS induces capricolum membranes induced TNF-o by a secretion of both TNF-oq IL-1 and IL-6. 27 IL-1 was mechanism different from induction by LPS. 14 able to stimulate IL-6 synthesis in human blood In spite of the similarities in stimulation of monocytes but not in monocyte derived macrothe production of the monokines, some differences phages whereas TNF-o had no effect on IL-6 were reported in the context of their production, synthesis in monocytes or macrophages. 28 Human These differences indicate that mechanisms of their alveolar macrophages and blood monocytes proproduction may be different. Thus, human duced large amounts of IL-6 in response to LPS monocytes stimulated with pneumococcal cell and monocytes produced lesser amounts of IL-6 in surface components produced IL-1 but not response to rIL-1. 29 Monocytes aged in vitro TNF-o. 15 Both protein kinase C (PKc) and produced little detectable IL-6 in response to LPS calmodulin (CAM) kinase dependent pathways or rIL-1, which might suggest that release of IL-6 were found to be involved in the induction of under stimulus is correlated to the degree of IL-1 mRNA by LPS, whereas TNFexpression maturity of macrophage cells. 29 TNF-o or rIL-6 seemed to be PKc dependent but not CaM kinase itself did not modulate IL-6 production in human dependent. 16 Other authors reported that TNFperipheral blood mononuclear cells. 3 No evidence and IL-1 production andsecretion by mononuclear was found that TNF-o acts to amplify the phagocytes can be modulated differentially. 17 production of IL-6 or IL-1 by murine macrophage Differences between the kinetics of production of cell lines. 31 It was also claimed that synthesis and IL-10, IL-lfl and TNF-o by murine peritoneal secretion of IL-1 either by human monocytes 32 or macrophages during the peritoneal exudative by mouse bone marrow derived macrophages 33 are response, were also described in relation to the two different biological events. optimal culture conditions and sequence of Similarly, LPS-induced production of TNF-o 34 appearance. 18 The production of TNF-o and IL-1 and IL-135 was also reported in human peritoneal in alveolar human macrophages was found to be macrophages collected from patients on continuous regulated differentially by LPS. 9 A different pattern ambulatory peritoneal dialysis (CAPD) during an of regulation was also observed in the case of episode of infectious peritonitis. human macrophages: during the initial phase of The conclusion from the above-mentioned data maturation of human blood monocytes (up to 7 is that TNF-oq IL-1 and IL-6 can mutually affect days in culture), IL-lfl and IL-6 were down-their production. The production and release of regulated whereas TNF-o levels markedly in-these cytokines are also affected by other agents creased. 2 A synergistic effect of interferon-z (including other cytokines) but these findings are (IFN-z) and LPS was observed in relation to the beyond the scope of the present review. A release of IL-lfl, IL-6 and TNFfrom human schematic representation of the interrelationship macrophages.eHowever, the LPS-inducedlevels of in production between the three cytokines is these cytokines differed during prolonged cultivagiven in Fig. 1. tion of macrophages (up to 28 days). 2 Differences in levels of production of IL-loq IL-lfl and TNF-o versus lower levels of IL-6 were also reported following stimulation by various agents of human blood mononuclear cells. 13 The relationship in production between TNF-o, IL-lfl and IL-6 is expressed by the findings that each one of these monokines can affect the production of the other monokines. Thus TNF-o was found to induce release of IL-1 in vilro 2'22 and in vivo. 23 TNF-o and IL-1 induced 1L-6 production in PiPo. 24 Stimulation of human monocytes by IL-1 caused a rapid down-regulation of IL-6 mRNA levels and concomitant enhancement of IL-6 mRNA expression. 24 IL-6 itself was found to suppress the IL-6-R at high concentrations. 25 IL-6 suppressed IL-lfl and TNFproduction induced by LPS or PHA in human blood mononuclear cells. 13 Inhibition of tPS-induced Interactions in production between cytokines and eicosanoids A series of findings indicated that production of TNFby macrophages can be regulated by both endogenous and exogenous PGE2. Thus, in response to LPS, murine peritoneal macrophages release concomitantly increased amounts of TNFand PGE2 .36'37 However, addition of exogenous PGE2 strongly suppressed the release of TNFby macrophages. 36'37 PGE2 down-regulated the expression of TNFgene in human blood monocytes. 38 In another work it was reported that low amounts of PGE2 enhance release of TNFfrom macrophages whereas high doses of PGE2 suppress its release. 39 Some authors reported recently that the gene expression of TNFwas enhanced by low doses of PGE. and by cGMP, and suggested that cGMP may represent one of the positive signals for TNFsynthesis. 4 Suppression of LPS-induced TNFproduction by PGE. was also reported by other authors. 41'42 The inhibitory effect of PGE2 on TNFproduction was correlated with induced augmentation of cAMP in macrophage cells. 36'39 In contrast to PGE., leukotrienes induce increases in TNFrelease from macrophages. Thus, human monocytes exposed to graded concentrations of LTB4 release high amounts of TNF-. 43 The enhancing effect of leukotrienes on TNF-a may be related to increases in cGMP levels by leukotrienes 44 and is also supported by findings that lipoxygenase inhibitors suppress formation of TNFin vitro and in vivo. 45 Treatment of macrophages with LPS enhanced the increase of a lipoxygenase product which counteracted the suppression of TNFsynthesis by a lipoxygenase inhibitor when added to macrophages exogenously. 46 Recent data suggest that endogenous prostaglandins and leukotrienes do not play a role in the regulation of TNFproduction. 47 In addition indomethacin (IND) (a cyclooxygenase inhibitor), exogenous arachidonate and MK-886 (a novel inhibitor of 5-1ipoxygenase product formation) do not affect TNFproduction. 47 The interrelationship between TNFand PGE2 production is also supported by the finding that TNFstimulates PGE2 production in murine resident peritoneal macrophages. 22 Other data indicate that enhancement of TNFactivity may be independent of PGE2 production. Thus IFN-y in combination with LPS enhanced TNFproduction but addition of IFN-3 to LPS had no effect on PGE2 levels produced in human monocytes. 48 With regard to IL-1, it was found that the same stimulator, namely PHA, induced production of both IL-1 and prostaglandin E in human monocyte monolayers. Products of the cyclooxygenase pathway of arachidonate metabolism seem not to be involved in the mechanism by which IL-1 stimulates thymocyte proliferation, whereas pro-ducts of the lipoxygenase pathway may mediate the thymocyte proliferative response induced by IL-1. Similarly, with TNF-, it seems that an arachidonate lipoxygenase product is important in the sequence of events leading to the production of IL-1. Additionally lipoxygenase inhibitors affected production of IL-1 in human peripheral blood monocytes. 49 An enhancing role of leukotrienes in production of IL-1 was also advocated by other authors. 5'5 Recently, it was reported that addition of exogenous LTB 4 to monocytes stimulates IL-1] transcription and mRNA accumulation. 52 A selfregulatory mechanism of IL-1 production was suggested by data showing that exogenous IL-1 induces increases in the levels of PGE2 in murine macrophage cultures, whereas exogenous PGE2 or prostacyclin (PGI2) (measured as its stable metabolite 6-keto prostaglandin F=) suppressed macrophage IL-1 production. 53 The inhibitory effect of PGE2 was correlated to induce increases in cAMP levels. 54 IL-1 was found, on the other hand, to stimulate 5-1ipoxygenase activity and in this way induces increases in PGE2 synthesis. 55 In contrast to these data, it was also reported 56 that PGE. had no effect on IL-1 synthesis in murine resident peritoneal macrophages but rather had a direct inhibitory effect on thymocyte proliferation. 5 In spite of the similarities in the stimulating conditions for production of TNFand IL-1 some differences were also reported. Thus, it was claimed that PGE2 suppresses expression of cell-associated TNFin murine peritoneal macrophages but had no effect on cell-associated IL-1 activity. 58 PGE2 suppressed accumulation of TNF mRNA but not of IL-I and 1L-lfl mRNA accumulation. 58 The conclusion from these experiments was that synthesis of TNF appears to be regulated at the level of transcription whereas synthesis of IL-I and IL-lfl is regulated post-transcriptionally. 58 Recently, it was shown that PGE2 inhibits release of TNFbut not of IL-1/8 from human peritoneal macrophages. 42 Interrelation between production of prostaglandins and of IL-1/g in human peritoneal macrophages was recently reexamined, s9 It was found that PGI2 (measured as its stable metabolite 6-keto-PGF=) declined sharply during episodes of peritonitis both in the presence or absence of LPS in the culture medium of human peritoneal macrophages. 59 On the other hand, PGE2 was released in the same amounts in cultures of macrophages collected during peritonitis and during an infection-free period. 59 These results suggest that PGI2 and PGE2 may play a different role in the regulation of IL-lfl production by human macrophages. 59 The production of IL-6 concomitantly to production of PGE2 in LPS-stimulated rat Kupffer cells was examined. 6  parallel with PGE2 before decreasing as PGE2 continued to rise. 6 Blocking of PGE2 production by IND increased IL-6 levels significantly thus showing that PGE2 produced by Kupffer cells down-regulates IL-6 secretion. 6 However, cyclooxygenase inhibitors inhibited production of IL-6 by human peripheral blood mononuclear cells, 61 but no direct relationship between inhibition of IL-6 and production of PGE. was found. 61 Leukotrienes stimulate production of IL-6 in cultures of human monocytes. 62'63 LTB 4 stimulates production of IL-6 and induces accumulation of IL-6 mRNA. 62'63 Finally, regarding the interrelation between the production of IL-1 and PGE., it was reported that IL-1 and PGE2 are produced by separate subsets of human monocytes. 64 The interrelationship between the production of cytokines and eicosanoids is given in Fig. 2.
Effect of tumour burden on production of macrophage cytokines and eicosanoids The functions of tumour associated macrophages (TAM) have been investigated extensively (for reviews see Refs. 65,66). It was suggested that in situ macrophages may affect the biology of neoplastic tissues in various ways besides tumour killing, by producing growth factors, by interaction with haemostatic mechanisms, by release of mutagenic reactive oxygen intermediates and neutral proteinases, and by their capacity to induce angiogenesis. 65 The effect of tumour burden on production of macrophage cytokines and eicosanoids was examined in a series of experimental systems and in cases of human cancer.
Experimental studies: It was reported that during tumour growth in rats (subcutaneous implantation), cyclooxygenase or thromboxane synthase is inhibited, whereas C5 and C12-1ipoxygenases of the alveolar macrophages are activated. 67'68 A transient increase of 12-HETE and LTC 4 production in murine peritoneal macrophages was also reported in mice implanted subcutaneously with B16 melanoma cells. 69 Tumour-host derived macrophages were found to suppress a series of events including activation of T cells, natural killing (NK) cells and lymphocyte activated killer (LAK) cells, and generation of tumoricidal activity in normal syngeneic murine splenic macrophages cultures in the presence of LPS. 7 The suppressive effects were correlated with an increase in PGE2 secretion by tumour-bearing host macrophages. 7 The role of prostaglandin secretion by macrophages from mice bearing syngeneic tumours was also supported by other authors. Thus, splenic and peritoneal macrophages collected from mice bearing different syngeneic tumours secreted large amounts of PGE2 as a result of their interaction with the tumours. 71 These macrophages were immunosuppressive and their suppressive activity was significantly reduced by IND thus proving that the suppressive effect was due to increased release of prostaglandin. 71 In another study, it was shown that inhibition of spleen cell cytotoxic capacity toward murine Lewis carcinoma was due to an increase in PGE2 levels. 72 The inhibitory effect was prevented by pretreatment of tumour-bearing mice with IND. 72 The effect of tumour burden on the ability of macrophages to secrete cytokines was also examined. Continuous alveolar macrophage (AM) and tumour infiltrated (TIM) cell lines were generated from C57BL/6J mice and tested for their potential to secrete IL-1, TNFor IL-1 following exposure to rMulFN-2 and LPS. 73 Neither cell line secreted substantial amounts of IL-1 or TNF<z but secreted large amounts of IL-6. 73 Peritoneal macrophages from sarcoma-bearing mice produced progressively less IL-1 as tumour burden increased. TM Administration of LPS to tumour-bearing mice early after tumour transplantation, resulted in reduced tumour growth and prevented the down-regulation of in vitro IL-1 production by peritoneal macrophages. TM Thus, it seems that a specific defect in IL-1 production was associated with increasing tumour burden. TM In another study it was concluded that murine tumour-infiltrating macrophages isolated from the lungs of mice bearing lung B16F10 metastases responded as normal alveolar macrophages to biological response modifiers in relation to induction of tumoricidal activity and to secretion of IL-1 and TNF-. 75 However, tumour associated macrophages (TAM) isolated from five cases of murine sarcomas showed a limited capacity to produce and release IL-1 as compared to peritoneal macrophages upon stimulation with LPS. 6 The role of TNF-z release by TAM in specific defence against the inoculated tumour was questioned by various authors. Thus TAM from mice bearing EMT6 tumours exhibited high anti-WEHI-164 activity 77'78 due to release of TNF-x 78 but was not effective against the EMT6 tumour. 77'78 Similarly, TAM in a murine fibrosarcoma model produced TNF-o but this production did not affect the tumour growth. 79 Human studies: Indirect indication of the role of prostaglandin production by macrophages from cancer patients was provided by data showing that monocyte-derived macrophages isolated from blood of cancer patients can be rendered cytotoxic by treatment with IND. 8'81 Apparently not all macrophages collected from cancer patients could be rendered cytotoxic against allogeneic or autologous tumour target cells, presumably because they were nonresponsive and/or because of the presence of a plasma inhibitory factor. 81 In another work, it was reported that peripheral and bone marrow enriched fraction of monocytes produce high levels of PGE2. 82 However, the increased release of PGE2 was not correlated to clinical or pathological findings. 82 The production of IL-1 and TNF-z by tumourassociated mononuclear leukocytes (TAML) and peripheral blood mononuclear leukocytes (PBML) in cancer patients was determined. Stimulation by LPS induced production of similar levels of TNF<z in TAML and PBML but production of IL-1 was markedly suppressed in LPS-stimulated tumourassociated mononuclear leukocytes (TAML). 83 In another study the release of IL-1 and IL-6 by TAM from ascites and solid tumours of human ovarian carcinoma was investigated. It was found that TAM release spontaneously or upon LPS stimulation high amounts of IL-6 whereas they were poor producers of IL-1. 84 LPS-induced production of TNFby peripheral blood macrophages was impaired in cases of breast cancer. 85 On the other hand, secretion of TNFby monocytes from patients with malignant brain tumours was significantly greater by comparison with monocytes from normal individuals. 6 Increase in secretion of TNFand of IL-1 was also reported by alveolar macrophages from patients with lung cancer as compared with secretion by peripheral blood monocytes from the same patients or by alveolar macrophages from patients with nonmalignant disorders. 87 However, alveolar macrophages from lung cancer patients were found to be impaired in their ability to develop antitumour cytotoxic activity compared with either the peripheral blood monocytes from the same patients or alveolar macrophages from patients with nonmalignant lung disorders. 8v An increase in the level of TNF-o was also found in tumour-infiltrating macrophages in human colorectal adenocarcinoma. 88 The increase in TNF-o levels correlated with an increase in the size of the primary turnout. 88 Some work was dedicated to determining the interrelationship between the production of TNF-o and PGE2 by monocytes from cancer patients. correlated with high metastatic potential for neoplastic cells. 93 Successful therapy with IND and with a combination of IND and IL-2 was explained by abrogation of prostaglandin-mediated suppression of NK activity and IL-2 production. [94][95][96][97] Production of eicosanoids and cytokines by tumour cells In view of the role played by eicosanoids and cytokines in expression of antitumour activity it seems likely that intrinsic production of these compounds by tumour cells may affect the resistance to tumour development in turnoutbearing animals and in human neoplasia.
First reports indicated that BP8/P1 murine ascitic tumour and, to a less extent a subcutaneously implanted S180 rat tumour, produced PGE2 .98 However, the role of PGE2 production in the development of the tumour remains uncertain because induction of a decrease in PGE2 levels by IND did not affect appreciably the tumour growth. 98 In more recent work it was shown that certain murine tumours produce prostaglandins and that their response to IND therapy was directly related to their ability to produce prostaglandin. 99 Production of PGE2 by EL4 leukaemia cells from C57BL/6 mice was also correlated to the extent of 100 migration and dissemination of the tumour. Prostaglandin biosynthesis was also found to occur in established cell lines derived from human lung, colorectal adenocarcinoma, and ovarian adenocarcinoma, lm A difference in the amount of PGE2 released was found between cancer cells metastasizing into rat liver or rat kidney. 1 It was assumed that this difference may be related to the mechanism of cancer metastases or to selection of 102 the organ in which metastases occur. Tumour cells were reported to also produce cytokines. Thus, tumours from cachectic mice produced both TNFand IL-1 in vivo as documented by the presence of TNFand IL-I 103 mRNA and immune-reactive protein for IL-I. The tumour cells also produced TNFand IL-I in long-term cultures but not IL-6.13 Secretion of TNF-0 by human leukaemic cells was also reported. 14 A myeloma cell line established from the pleural effusion of a myeloma patient secreted both TNFand IL-6 and these cytokines induced proliferation of the cell line. 15 In IL-6 production a dual effect was described: the murine MH134 tumour cells produced high amounts of IL-6 whereas the murine CSAIM tumour produced only marginal levels of IL-6. 6 However, both tumours induced production of IL-6 by T cells in the tumour-bearing host. 6 Leukaemic cells from patients with acute myeloid leukaemia produced both IL-6 and IL-1. 7 Prostatic carcinoma cell lines expressed the IL-6 receptor and secreted IL-6.18 Squamous cell carcinoma cell lines produced both IL-1 and IL-6. 9 An interesting situation was described with the murine P815 mastocytoma: this line produces TNF 1 but, at the same time, this was one of the first tumour cell lines which was found to be sensitive to exogenous TNF. 3'4 It seems likely that production of prostaglandins and macrophage cytokines as well as induction of their production by macrophages in tumourbearing hosts plays a role in the development of the tumour in vivo. However, the direct relationship between neoplasia and the ability of tumour cells to produce and/or induce production of macrophage eicosanoids and cytokines is not clear yet.

Interactions between macrophage cytokines and eicosanoids in expression of antitumour activity
Interactions between TNF-, IL-1 and IL-6: TNFalone is cytostatic or cytotoxic for a wide range of murine and human tumour-cell lines. 11'12 Oi1 the other hand, TNF had no effect on a wide variety of murine and human tumour-cell lines and enhanced the growth of various normal cell lines. 111 '112 Moreover, a heterogeneous cytotoxic response of TNF-0 was described for various cell lines isolated from the same single neoplasm of human colorectal carcinoma113 or renal cell carcinoma. 114 Membrane-associated TNF was shown to be the lytic principle of activated macrophages cytotoxic for TNF susceptible tumour cells. 115 The complexity of TNF antitumour activity is also shown by findings that TNFmediates the enhanced cytotoxicity induced in monocytes by IFN, IL-1 and by TNF itself. 16 Treatment of human monocytes with TNFincreased their cytostatic ability in a dose-dependent manner against P815 murine mastocytomas. 1 IL-1 was also reported to be cytocidal for several tumour-cell lines. 118 However, IL-1 can act also as an autocrine growth factor for acute myeloid leukaemia cells. 19 The fact that TNFand IL-1 are both produced by activated macrophages, and that TNFitself is an inducer of IL-1 production, prompted investigations devised to determine possible synergistic and additive antitumour effects of combinations of the two cytokines. Combination of TNFand IL-1 synergistically 12 or in additive manner 1 inhibited the growth of human A-375 melanoma cells. In another study, it was claimed that enhancement of antitumour human monocyte activity by combined TNFand IL-1/ was less than additive. 12 We found that a combination of TNFand IL-1 had an additive effect on antitumour cytostasis against WEHI-3B murine tumour cells. 123 It should be noted that TNF-o and IL-1 represent only a fraction of a wider spectrum of macrophage cytokines involved in expression of antitumour activity of macrophages. 124'125 Antitumour activity of IL-6 against human breast carcinoma and leukaemia/lymphoma cell lines 126 and in vivo against four murine metastatic tumours 12v has been reported. On the other hand, autocrine generation and requirement as a growth factor for human multiple myelomas 128 and for human renal cell carcinomas 129 has also been described.
IL-6 production is induced by LPS which also stimulates production of TNFand IL-1, IL-1 induced synthesis of IL-6 in human blood monocytes 28'29 and TNFinduced IL-6 in sera of cancer patients and turnout-bearing mice. 13 Moreover, it was found that IL-6 is involved in IL-1 induced activities as pyrogenicity and stimulation of thymocyte proliferation. TM However, there are apparently few data on synergistic, additive or antagonistic effects of combination of IL-6 with either TNFor IL-1 on tumour cells. It was claimed that systemic administration of low doses of IL-6 in combination with sub-therapeutic doses of TNF to mice bearing a weakly immunogenic syngeneic tumour resulted in marked 127 regression and some cure.
Interactions between cytokines and eicosanoids: Modulation of production of various arachidonic acid derivatives is by itself related to induction of antitumour activity in macrophages. Thus, in vitro treatment of murine peritoneal macrophages with IND, a cyclooxygenase inhibitor, induced antitumour cytostatic activity against a murine plasmacytoma and this effect was increased when LTB4 was added to the cultures. 132'13 The IND stimulation of macrophage cytostasis against the murine plasmacytoma was enhanced by endogenous metabolites of lipoxygenase and counteracted by PGE2 .14 Induction of macrophage cytostasis towards P815 mastocytoma by calcium ionophore was reversed by specific inhibition of lipoxygenase. 15 In other work it was shown that LTC4 is an essential 5-1ipoxygenase intermediate in A23187-induced antitumour cytostatic activity 16 and that addition of L-serine to cultures stimulated by calcium ionophore increased both the accumulation of LTC4 in murine macrophages as well as their antitumour activity. 13v LPS-induction of macrophage tumour killing was counteracted by PGE2 but not by PGI1.138 It should be noted that there are contradictory reports concerning the effect of prostaglandin production by macrophages turnout cells, and the direct effect of prostaglandins on turnout growth. Thus, it was reported that PGE could inhibit DNA synthesis and tumour cell replication in vitro and tumour growth in vivo. 19'14 PGA also inhibited turnout growth in vitro and in vivo. 141'142 However, in spite of the fact that induction of antitumour activity in macrophages by LPS was associated with an increase in PGE2 and thromboxane production, these compounds did not seem essential for the expression of antitumour activity, as induction of antitumour activity took place and was even enhanced in the presence of indomethacin. 14 It has been reported also that addition of exogenous PGE2 to murine peritoneal macrophages did not alter the ability of these cells to produce high levels of tumour-cell lysis when stimulated with LPS. 144 Finally, contrary to suggestions in Refs. 143 and 144 blockade of prostaglandin synthesis by indomethacin prevented the effect of LPS and led to a substantial resumption of target growth in the presence of activated macrophages. 14s A differential effect of PGE2 on expression of macrophage antitumour activity was described in relation to the state of the macrophages: culture conditions that caused increased PGE2 production by activated macrophages resulted in inhibition of their tumoricidal activity but production of high levels of PGE2 by resident and peptone elicited macrophages was associated with an increase in antitumour activity. 146 The contradictory results described may be due to differences in sources of macrophages, in types of target tumour cells and in the physiological state of the effector cells.
We and others have investigated the interrelation between eicosanoids and TNFor IL-1/ in expression of antitumour activity. Human peritoneal macrophages collected from CAPD patients during an intercurrent infectious inflammation showed a sharp drop in cAMP and a decrease in production of cyclooxygenase metabolites. 147 On the other hand, they were primed in an in vivo inflammatory environment so that they were much more cytostatic against murine tumour ceils than macrophages collected during inflammation free periods. 148 When macrophages collected during inflammation were cultured with LPS, their antitumour cytostasis against two murine tumourcell lines was markedly increased and this increase was associated with increase in TNF-o and IL-lfl release. 34,35,148 Interrelation between eicosanoids and TNFor IL-lfl in expression of antitumour activity was also examined by addition of the cell-free compounds to cultures of turnout cells. Interestingly, concomitant addition of PGE2 enhanced the antitumour effect of IL-1]3 on a IL-lfl susceptible WEHI-3B murine turnout, whereas addition of LTC4 inhibited the antitumour effect of IL-1.149 The synergistic effect between prostaglandins and cytokines in expression Mediators of Inflammation. Vol of antitumour activity was also observed when the WEHI-3B murine tumour cells were first treated with the cytokine and afterwards with the prostaglandin: pretreatment with IL-lfl rendered the tumour cells susceptible to PGE 2 or PGI2 whereas only susceptibility to PGE2 was increased by pretreatment with TNF-. 123 Other authors found that Kupffer resident rat cells and Kupffer inflammatory murine liver cells produced both TNFand PGE2. Upon activation with IFN-y + LPS (for mouse resident Kupffer cells) or with LPS alone (for rat Kupffer cells and mouse inflammatory Kupffer cells) the cells produced more PGE2 and more TNF-o. 15 However, PGE2 did not play a role in tumour because treatment with indomethacin increased the TNF induced killing, is Therapeutic implications Work on therapeutic effectiveness was mostly concentrated on ways to affect in vivo prostaglandin production and on the possibility of using TNFeither alone or in combination with other agents for therapy. The therapeutic effectiveness was examined in three systems: against animal tumours, against xenogeneic transplants of human tumours in nude mice and in clinical trials in cancer patients.
Experimental animal tumours Prostaglandins have been implicated as enhancers of tumour growth and spread. 93'151 The sources of prostaglandin were tumour cells 99 and/or cells of monocyte-macrophage lineage. Accordingly, it was assumed that inhibition of prostaglandin biosynthesis by cyclooxygenase inhibitors might have a beneficial therapeutic effect.
Most of the work on the basis of this assumption was done by looking on the therapeutic effect of the cyclooxygenase inhibitor IND as downgrading PGE2 production. Thus, it was shown that IND therapy prevents tumour metastasis of a mouse mammary carcinoma 94 cures B16F10 murine melanoma lung metastasis when given in combination with IL-2, 95 and cures murine Ehrlich ascites tumours when administered in combination with LAK cells and IL-2. 96 The effectiveness of IND therapy was correlated to the ability of murine tumours to produce prostaglandin. 99 However, it was doubted if the therapeutic effectiveness of indomethacin is due to inhibition of PGE2 production. 152  After its first characterization as tumour necrosis factor produced by macrophages, 4 its activity against a long series of human and murine tumours was demonstrated in vitro. '2 The next obvious step was to determine the therapeutic effectiveness of TNFin tumour-bearing hosts, rHuTNFwas shown to be effective against subcutaneously implanted murine MethA sarcoma but not against the same tumour injected i.p. 54 The curative effect of TNF-0 was attributed to its ability to induce local haemorrhages because of its effect on the endothelial cells. 54 It should be noted also that TNF-0 therapy involved generation of specific cell-mediated antitumour immunity by a still undefined mechanism. 154 In another experimental system the antitumour effect of recombinant murine TNFgiven either by continuous i.v. infusion or by repeated i.v.
injections was determined in a rat liver metastases model. 155 Only early continuous infusion had an effect on the number of liver metastases presumably because higher doses of TNF-0 were tolerated by this schedule. 155 The conclusion of the authors was that TNFby itself is not a very efficient antitumour agent and it might be necessary to use TNF-0 in combination with other antitumour agents. 155 A similar conclusion of more therapeutic effectiveness of TNF in combination with other treatments was in the case of TNF radiotherapy by comparison with TNF-0 alone in rat renal-cell carcinoma, 56 or a combination of TNFwith the interferon-inducer bropirimine in rat colon cancer. 57 Sequential use of anti-CD3, IL-2 and TNF for LAK induction and maintenance potentiated antitumour activity against a pulmonary metastatic model in mice. 158 Another interesting additive effect was described after combined treatment with activated macrophages and a low dose of TNF-0 in mice bearing Lewis lung carcinoma or EMT6 sarcoma. 159 A synergistic therapeutic effect was also described in tumour-bearing mice treated with low doses of IL-6 in combination with subtherapeutic doses of TNF-0. 127 The therapeutic use of TNF-0 in tumour-bearing mice was found to be affected by an increase in the toxicity of TNFin tumourbearing hosts 6 and by induction of tolerance to TNF-0. 16 A side effect of antibodies to TNFwas reported" passive immunization with anti-TNF antibodies abrogated part.ially IL-2 toxicity in tumour-bearing mice. 162 Finally, the effect of TNF therapy might differ in context to the strain of mice" the curative effect of TNF was stronger against MethA sarcomas implanted in BALB/c nu/+ mice than into BALB/c nu/nu mice, when TNF was injected i.v. and similar when injected i.t. 63 A new therapeutic approach was described recently: a novel chimera tumour necrosis factor (TNF-STt) constructed by connecting a modified recombinant human TNF-o (rTNF-S) with thymosin-/g 4 was suggested to be a promising approach for obtaining molecules that more favourably attack tumours than conventional rTNF. 164 Xenografts of human tumours in nude mice: TNF was also found to be effective against human malignant melanoma, human gastric cancer and nasopharyngeal carcinoma cell lines implanted in nude mice. 16 Combined therapy with IFN-, and TNF-o was found to be more effective than TNF-ot alone against human ovarian cancer cells inoculated in nude mice. 165 A similar synergistic effect between IFN (interferon-alpha) and TNF-o was described against a human tumour line causing lung metastasis and intra-abdominal carcinomatosis in nude mice. 166 In another study it was found that combined treatment with TNF-o and etoposide was efficient against a human renal cell carcinoma implanted in athymic mice. 16v Clinical trials: The findings on cytotoxic effects of cytokines (especially TNF-) on a wide array of murine and human turnout cell lines in vitro and the results on therapeutic effectiveness against murine turnouts and against human turnout cells implanted in nude mice prompted the start of clinical trials in various cancer patients. Unfortunately the results in clinical trials were not very spectacular. In one of the first studies a certain improvement due to TNF-o therapy was observed in three out of 18 patients: two cases of lymphoma and one case of Hodgkin'$.168 Febrile reactions and other side effects occurred in most of the patients and they could be prevented by steroids and IND. 168 However, according to the authors, "such prophylaxis may not be desirable because its influence on possible therapeutic benefits is unknown". Some beneficial response in cases of gastric cancer and non-Hodgkin's lymphoma were also reported by other authors. 169 Side effects to TNF-o were recorded and could be ameliorated by IND or ketoprofen. 169 In cases of B-lymphoma some improvement was detected in one out of 26 patients and side effects to TNFwere again observed, iv In another study no antitumour response was detected in 29 cancer patients, lvl In two cases out of 16 evaluable patients with disseminated cancer, TNF-0 therapy had some antitumour effect: regression of a neck lesion in one case and resolution of malignant ascites in another, iv2 The results on various clinical trials with TNF-0 until 1988 were summarized at a seminar of the 19th German Cancer Congress: "Although TNF as a single agent is unlikely to be of major benefit for patients with cancer, it has to be concluded that the great expectations which were particularly due to its dramatic effect in the murine sarcoma model leading to the designation 'TNF' have been disappointed", lv More clinical trials have been performed with TNF-0 in recent years. In a phase II trial with 22 eligible patients with metastatic colorectal adenocarcinoma, treatment with TNFwas not effective. 1v4 A more promising result was obtained in a clinical trial with TNF-0 including 29 patients with refractory malignant ascites: out of 29 patients, 22 responded with a complete (16) or partial (6) resolution of their ,ascites. lvs Recently, in one out of 53 patients with advanced malignancies a partial response to TNF was observed in one patient with colorectal carcinoma, lv6 In another recent study no clinical efficacy of TNF-0 was found in a phase I trial with patients with advanced cancer, lvv Rather disappointing results concerning the use of human recombinant TNF-0 for cancer therapy were reported recently by two groups: no objective responses were observed in 22 cases of advanced carcinoma of the pancreas iv8 and no significant antitumour activity of rHTNF-0 was detected in 127 eligible patients with diverse metastatic malignancies, iv9 Another group concluded that rHTNF-0 has only modest antitumour activity in 26 patients with renal cell carcinoma. 18 A more promising approach for therapy was suggested by using TNF-0 in combination with other interleukins. 81'182 This was suggested by results obtained in vitro and in experimental systems in vivo with such combinations. However, in only one patient with melanoma and one patient with mesothelioma (out of 36 patients), was some response observed to combined treatment of recombinant TNFwith recombinant IFN-2,18 and two partial responses were seen in a study with combined recombinant IL-2 followed by recombinant TNF therapy in 31 patients with metastatic malignancies. 84 The use of TNF-0 therapy is handicapped by the toxicity of the agent and by its extremely pleiotropic biological effects. It should be also noted that in most clinical trails with TNF-0 most of the patients were found refractory to other kinds of treatments and were in an advanced stage of disease. Moreover, it was reported that sera of cancer patients may contain factor(s) inhibiting TNF. 18 '86 It has been also reported that treatment with TNF-0 might induce decreases of NK cell activity and of monocyte production in cancer patients. 8v The general conclusion from clinical trials until now is that therapy with TNFis still in its infancy. Apparently, combinations of various interleukins for cancer therapy might be more promising. A new approach was suggested in the recent years consisting of therapy by monocytes from cancer patients induced to maturate in vitro to macrophages

Concluding Remarks
A vaste amount of material has accumulated on the interrelationship in production between macrophages cytokines, macrophage cytokines and eicosanoids, production of these products by turnout cells and the effect of tumour burden on their production. The expression of interrelationship between macrophage cytokines themselves and between macrophage cytokines and eicosanoids was also extremely investigated. In certain instances the results obtained in in vitro systems were also expressed in vivo in various therapeutic schedules in mice bearing syngeneic tumours on xenografts of human turnout-cell lines. Unfortunately, the promising results obtained in vitro and in experimental systems in vivo have not yet been well duplicated in clinical trials. Much more work has still to be done in defining optimal conditions for the use of single interleukins and (more likely) combinations of interleukins for effective anticancer therapy. Some of the negative and positive aspects of use of one of the most commonly tested cytokine (TNF-0 is schematically represented in Fig. 3). It is also possible that other approaches such as therapy with cells able to produce in vivo a wide array of cytokines (macrophages) or devising ways for effective induction of cytotoxic interleukins in vivo by a cancer patient's own cells might lead to more promising results.