255
Bi~him~ca et Biophysics Act4 1042 (1990) 255-258 Elsevier BBALIP 53306
The formation of thromboxane B,, leukotriene B4 and 12-hydroxy~icosatetra~noic acid by alveolar macro~hages after activation during tumor growth in the rat J.E. Vincent It M,A. Vermeer I, W.J. Kort 2 and F.J. Zijlstra ’ ~eFor~ment of Pharmaco~o~ and 2 ~bor~to~~or ~~~er~menta~Surgery, Facu&y of Medicine,
Ervsmw
’
~~jue~s~~~ ~~~~~~~a~
Rotterdam (‘IThe~etber~an~~ (Received 8 September 1989)
Key words: ~romboxane
3,: Leukotriene B4; 12-Hydrox~cosatetetraeno~c
acid; Tumor growth; (Rat alveolar macropha~e)
Pieces of tumor tissue were irnpl~t~ su~utaneously in the right flank of BN female rats. After 3, 7, 10, 12, 14 and 17 days the hmgs were lavaged and the alveolar macrophages colfeeted. The cells were activated with the calcium ionophore A23187 and the formation of thromboxane B, (TxB,), leukotriene B4 (LTB4) and 12-hydroxyeicosatetraenoic acid (12~HETE) determined. The formation of TxB, decreased considerably until day 7. Thereafter, no changes occurred. The formation of LTB4 increased after the ttmror implantation until day 10 and remained stable for the rest of the period. 12~HEm formation was approximately similar, with a decrease at day 12 but continued to increase after day 14. These results suggest that during tumor growth an inhibition of the cycle-oxygenase or t~~boxane synthase occurs and an activation of the C5- land ClZlipoxygenases of the alveolar macrophages,
In~~uction
The mutual effects of tumor tissue and macrophages have been the subject of a number of recent studies [1,2]. Alveolar macrophages participate in the defense of the lung in the inhalation of foreign substances. It has also been demonstrated that these cells, after activation, play a role in the elimination of neoplastic cells [3]. This is of particular interest in relation with the formation of metastases in lung tissue. After activation of alveolar macrophages, a number of arachidonic acid metabo~tes are formed, both by the cyclooxygenase and lipoxygenase pathways. In other experiments, we found that after labelling of the alveolar macrophages with [i4Cfarachidonic acid and activation with the calcium ionophore A23187, TX%, 12-hydroxy-5,8,10-heptadecat~enoic acid (HHT), LTB, and 12-HETE were the main metabolites formed. PGE,, PGF,, and 15-HETE were formed in lower amounts [4]. In the experiments described here, the formation of metabolites from endogenous arachidonic acid by alveolar macrophages during tumor growth in the rat was determined. The formation of TX%, a cyclooxygenase
Correspondence: J.E. Vincent, Department of Pharmacology, Faculty of Medicine, Erasmus University Rotterdam, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands. ~~-27~/~/$03.~0
derivative and of LTB, and 12-HETE, lipoxygenase derivatives were measured after activation by the calcium ionophore A23187. The results indicate that during the initial stages of tumor growth, 71x6, formation is decreased and that of LTB, and I2-HETE increased. Materials and Methods Experimental design. 42 inbred BN female rats, 100-150 g (body wt.), were randomly divided into groups of six. 36 of these rats received a piece of tumor tissue subcutaneously in the left flank, the six remaining were killed to serve as point 0. At day 3, 7, 10, 12, 14 and 17, at each day, another six animals were killed. Animal ~~b~~~~. BN rats were obtained from Harlan (The Netherlands). The animals were housed in Makrolon cages on presterilized woodchips, in groups of not more than four, receiving commercial rat chow, and acidified tap water ad lib. The animal room was air-conditioned and had a controlled 12 h light/l2 h dark rhythm. Tumor inoculation. BN472, which is a transplantable rna~~ adenoc~cinoma, was impl~ted subcutaneously in the right flank of rats under light anesthesia. Pieces of tumor tissue (2 X 2 X 2 mm3) were obtained from syngenic rats, in which the tumor was kept in passage. The tumor tissue was removed from the latter
@ 1990 Elsevier Science Publishers B.V. (E~omedical Division)
256 two weeks after the implantation, when it had a diameter of 20 mm. The tumor is lethal after 3 to 4 weeks. The tumor is a fast, invasively growing tumor that metastasizes spontaneously to distal lymph nodes and lungs. Tumor size was established at termination with the use of calipers. Alveolar macrophage lavage. After pentobarbital anesthesia, the animals were exsanguinated intra-abdominally. The trachea was exposed and cannulated with a 19-gauge needle which was tied. The lungs were lavaged 6-times with cold 5-ml aliquots of sterile PBS. The chest was massaged gently with each infusion. The tubes containing the lavage fluid were centrifuged for 10 min at 250 x g at 4°C. The cells were resuspended in cold PBS and washed twice. More than 98% of the cells were positive for nonspecific esterase activity. From the counted cells, 0.5 . lo6 cells were used for the determinations of the stable eicosanoid degradation products. Macrophage activation. Macrophages were diluted to 0.5 . lo6 ml and preincubated during 2 min at 37” C under shaking. Calcium-ionophore A23187 was added (final concentration 2 PM) and the suspension further incubated during 10 min. The tubes were centrifuged 2 min at 2800 X g and the supernatants collected and frozen at - 70 o C until RIA was performed. Radioimmunoassay. lo-p1 aliquots were used in RIA tests. Anti-sera to TxB,, LTB, and 12-HETE were obtained from Advanced Magnetics via BioLab (Brussels, Belgium). 3H-Labelled compounds were purchased from Amersham International (U.K.), standards of TxB, from Sigma (U.S.A.) and 12-HETE from Advanced Magnetics (U.S.A.). LTB, was obtained from Dr. J. Rokach (Merck-Frosst Labs, Canada). Statistics. A parametric one-way analysis of variance was used, followed by a Duncan’s new multiple-range test. A difference was considered statistically significant when the calculated P values were smaller than 0.05.
I I
25
0
5
10
Fig. 1. Formation of TxB, by alveolar macrophages activated by the calcium ionophore A23187 during tumor growth. Amounts in ng per million cells. Statistical significance * P < 0.05 compared to day 0.
1
01
0
5
10
‘5
20 tumOr
(days)
Fig. 2. Formation of LTB, (A) and 12-HETE (0) by alveolar macrophages after activation during tumor growth. LTB4: * * P c 0.01; *** P i 0.005compared to day 0. 12-HETE: * P c 0.05;** * P c 0.005compared to day 0; * * P i 0.025 compared to day 7; P -c0.01 compared to day 14.
Results In the animals inoculated with tumor tissue, tumor growth occurred. At day 3, a small elevation in the subcutaneous tissue can be felt. After 7 days the size of the tumor was significantly increased (Fig. 3). The number of alveolar macrophages was determined at different times. At day 0, the value was (5.37 + 0.63) 106/5 ml. There is little change until day 10 when a decrease occurs to 65% of the original value. This is followed by a return to the original value at day 12. In the formation of TxB, after activation with the calcium ionophore A23187, a decrease occurs in the initial stages of tumor formation, until day 7. Thereafter, a steady-state level was reached at approx. 68% of the control non-tumor-bearing rats (Fig. 1). The formation of LTB, increases up to day 10, at a value of 190%
30
0.75
25
0.65
20
055
15
045
035
5
0.25
0
015 0
5
10
‘5
20 runor
/days)
Fig. 3. Ratio of TxB,/LTB, formed by activated macrophages during tumor growth. * P r;0.01 compared to day 0. A, Tumor size; 0, TxB, /LTB,.
257 of the controls, and remains at a steady level thereafter. The amounts of 12-HETE formed are higher than those of TxB, and LTB,. Here also, an increase occurs in the initial period of the formation of the tumor. The highest value is reached at day 7 at 228% of the controls. This is followed by a decrease, with a lowest value at day 12 and a rise again at day 14 and 17 (Fig. 2). The ratio TxB,/LTB,, which is a measure of the activities of the cyclooxygenase and CS-lipoxygenase, decreases rapidly in the first days (Fig. 3). TxB, is the ‘most important cyclooxygenase compound. The ratio TxB,/12-HETE, indicating the cyclooxygenase and ClZlipoxygenase activities, shows the same effect (data not shown). Discussion The different aspects of the interaction between macrophages and neoplastic tissue have been the subject of a considerable amount of recent studies [2]. The events that have been described indicate that this interaction is rather complex and comprises several reactions. In the initial period of tumor formation, macrophages are attracted by an infiltrate into the tissue [2]. The macrophages have the capacity to produce cytotoxic substances, such as tumor necrosis factor (TNF) and IL-2 after activation, which can eliminate the neoplastic tissue. It has also been reported, that tumors can produce substances with anti-inflammatory action which inhibit the activation of cells and in this way the release of these substances [2]. This also results in the inhibition of macrophage chemotaxis. In several experimental set-ups, it has been demonstrated that in the presence of a tumor, the mobilization of macrophages is diminished. A protein with anti-inflammatory properties has been isolated [2]. Other possible candidates for the inhibition of cell activation are PGE, and prostacyclin. A high production of PGE,, PGF,, and 6-keto PGF,,, the metabolite of prostacyclin has been demonstrated in a number of malignant tissues [5,6]. The growth of a fibrosarcoma in the rat was associated with high plasma levels of PGE, and TxB, [7]. PGE, has two different effects on the release of TNF-a! from rat resident peritoneal macrophages. At low doses, stimulation occurs and at high doses inhibition [8]. As TNF-(U induces the formation of PGE, a feed-back mechanism may exist. The decrease of TxB, formation by the alveolar macrophages in our experiments after the implantation of the tumor tissue could be induced by one of the anti-inflammatory factors. These substances would inhibited the cell activation and the release of arachidonic acid. This is not very likely as in our experiments the activity of the cyclooxygenase is reduced and that of the C5- and ClZlipoxygenase increased. LTB, induces increased vascular permeability and enhanced granulocyte adherence and migration. The effects of 12-HETE are the induction of chemotaxis for neutrophils, chemokine-
sis for smooth muscle cells and an increase in mononuclear procoagulant activity [9]. One explanation for the increased formation of the lipoxygenase derivatives, would be that when the same amounts of arachidonic acid are released after activation of the macrophages, a diminished formation of cyclooxygenase derivatives will lead to an increased formation of lipoxygenase products. Lipogenase activity has been related to natural killer-cell-mediated cytotoxicity. Effects of leukotrienes, LTB, and 1ZHETE on IFN-y production of T-cells have been described [lo]. IFN-y induces the production of IL-l, IL-2 and TNF [11,12]. Inhibitors of the lipoxygenase reaction also inhibited the cytotoxicity. This was enhanced by 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid (5-HPETE) and LTB, [13]. It could be that the same relationship exists in alveolar macrophages and that lipoxygenase activity is necessary for the cytotoxic effect of these cells. It has been demonstrated that the inhibition of 5-lipoxygenase of peritoneal macrophages activated by the calcium ionophore A23187 inhibited the cytotoxic activity towards tumor cells [14]. At day 12, a dip occurs in both LTB, and 12-HETE curves. The difference in the latter is statistically significant. We are still seeking an explanation for this effect. It could be, that at this time, an additional stress occurs in one of the processes of tumor formation, leading to a release of corticosteroids. These substances induce the formation of lipocortin, a phospholipase A, inhibitor. In this way, eicosanoid synthesis will be decreased. In future experiments, this will be investigated. The results of these experiments indicate that a relationship exists between lipoxygenase activity and cytotoxic action. It is possible, that in the presence of the tumor the lipoxygenase in the alveolar macrophages is activated in order to increase the cytotoxic action in an attempt to destroy the tumor cells infiltrating the lung tissue. This could be an explanation for the increased formation in our experiments of LTB, and 12-HETE. It would seem of interest to investigate, whether a humoral factor secreted by the tumor tissue induces the change in cyclooxygenase/lipoxygenase ratio in the alveolar macrophages. Acknowledgement We would like to thank Mrs. B.H.M. Busscher-Lauw for typing the manuscript. References 1 2 3 4
Lloyd, J.O. (1985) Science 230, 630-632. Cianciolo, G.J. (1986) Biochim. Biophys. Acta 865, 69-82. Sone, S. (1986) Biochim. Biophys. Acta 823, 227-245. Kort, W.J., Zijlstra, F.J. and Weyma, J.M. (1989) Prostaglandins, Leukotrienes and Essential Fatty Acids 37, 113-120.
258 5 Bauknecht, T., Siegel, A., Meerpohl, H.G. and Zahradnik, H.P. (1985) Prostaglandins 29, 665-671. 6 McLemore, T.L., Hubbard, W.C., Litterst, CL., Liu, M.C., Miller, S., McMahon, N.A., Eggleston, J.C. and Boyd, M. (1988) Cancer Res. 48, 3140-3147. 7 Kort, W.J., Weyma, I.M., Bijma, A.M., Van Schalkwijk, W.P., Zijlstra, F.J. and Westbroek, D.L. (1987) Prostagl. Leukotr. Med. 28, 25-34. 8 Renz, H., Gong, J-H., Schmidt, A., Nain, M. and Gemsa, D. (1988) J. Immunol. 141, 2388-2393.
9 Lorenzet, R., Niemetz, J., Marcus, A.J. and Broekman, M.J. (1986) J. Clin. Invest. 78, 418-423. 10 Rola-Pleszcynski, M., Bouvrette, L., Gingras, D. and Girard, M. (1987) J. Immunol. 139, 513-517. 11 Farrar, W.L. and Humes, J.L. (1985) J. Immunol. 135, 1153-1159. 12 Philip, R. and Epstein, L.B. (1986) Nature 323, 86-89. 13 Bray, R.A. and Brahmmi, Z. (1986) J. Immunol. 136, 1783-1790. 14 Van Hilten, J.A., Elliot, G.R. and Bonta, I.L. (1988) Prostaglandins, Leukotrienes and Essential Fatty Acids 34, 187-192.
Bi~him~ca et Biophysics Act4 1042 (1990) 255-258 Elsevier BBALIP 53306
The formation of thromboxane B,, leukotriene B4 and 12-hydroxy~icosatetra~noic acid by alveolar macro~hages after activation during tumor growth in the rat J.E. Vincent It M,A. Vermeer I, W.J. Kort 2 and F.J. Zijlstra ’ ~eFor~ment of Pharmaco~o~ and 2 ~bor~to~~or ~~~er~menta~Surgery, Facu&y of Medicine,
Ervsmw
’
~~jue~s~~~ ~~~~~~~a~
Rotterdam (‘IThe~etber~an~~ (Received 8 September 1989)
Key words: ~romboxane
3,: Leukotriene B4; 12-Hydrox~cosatetetraeno~c
acid; Tumor growth; (Rat alveolar macropha~e)
Pieces of tumor tissue were irnpl~t~ su~utaneously in the right flank of BN female rats. After 3, 7, 10, 12, 14 and 17 days the hmgs were lavaged and the alveolar macrophages colfeeted. The cells were activated with the calcium ionophore A23187 and the formation of thromboxane B, (TxB,), leukotriene B4 (LTB4) and 12-hydroxyeicosatetraenoic acid (12~HETE) determined. The formation of TxB, decreased considerably until day 7. Thereafter, no changes occurred. The formation of LTB4 increased after the ttmror implantation until day 10 and remained stable for the rest of the period. 12~HEm formation was approximately similar, with a decrease at day 12 but continued to increase after day 14. These results suggest that during tumor growth an inhibition of the cycle-oxygenase or t~~boxane synthase occurs and an activation of the C5- land ClZlipoxygenases of the alveolar macrophages,
In~~uction
The mutual effects of tumor tissue and macrophages have been the subject of a number of recent studies [1,2]. Alveolar macrophages participate in the defense of the lung in the inhalation of foreign substances. It has also been demonstrated that these cells, after activation, play a role in the elimination of neoplastic cells [3]. This is of particular interest in relation with the formation of metastases in lung tissue. After activation of alveolar macrophages, a number of arachidonic acid metabo~tes are formed, both by the cyclooxygenase and lipoxygenase pathways. In other experiments, we found that after labelling of the alveolar macrophages with [i4Cfarachidonic acid and activation with the calcium ionophore A23187, TX%, 12-hydroxy-5,8,10-heptadecat~enoic acid (HHT), LTB, and 12-HETE were the main metabolites formed. PGE,, PGF,, and 15-HETE were formed in lower amounts [4]. In the experiments described here, the formation of metabolites from endogenous arachidonic acid by alveolar macrophages during tumor growth in the rat was determined. The formation of TX%, a cyclooxygenase
Correspondence: J.E. Vincent, Department of Pharmacology, Faculty of Medicine, Erasmus University Rotterdam, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands. ~~-27~/~/$03.~0
derivative and of LTB, and 12-HETE, lipoxygenase derivatives were measured after activation by the calcium ionophore A23187. The results indicate that during the initial stages of tumor growth, 71x6, formation is decreased and that of LTB, and I2-HETE increased. Materials and Methods Experimental design. 42 inbred BN female rats, 100-150 g (body wt.), were randomly divided into groups of six. 36 of these rats received a piece of tumor tissue subcutaneously in the left flank, the six remaining were killed to serve as point 0. At day 3, 7, 10, 12, 14 and 17, at each day, another six animals were killed. Animal ~~b~~~~. BN rats were obtained from Harlan (The Netherlands). The animals were housed in Makrolon cages on presterilized woodchips, in groups of not more than four, receiving commercial rat chow, and acidified tap water ad lib. The animal room was air-conditioned and had a controlled 12 h light/l2 h dark rhythm. Tumor inoculation. BN472, which is a transplantable rna~~ adenoc~cinoma, was impl~ted subcutaneously in the right flank of rats under light anesthesia. Pieces of tumor tissue (2 X 2 X 2 mm3) were obtained from syngenic rats, in which the tumor was kept in passage. The tumor tissue was removed from the latter
@ 1990 Elsevier Science Publishers B.V. (E~omedical Division)
256 two weeks after the implantation, when it had a diameter of 20 mm. The tumor is lethal after 3 to 4 weeks. The tumor is a fast, invasively growing tumor that metastasizes spontaneously to distal lymph nodes and lungs. Tumor size was established at termination with the use of calipers. Alveolar macrophage lavage. After pentobarbital anesthesia, the animals were exsanguinated intra-abdominally. The trachea was exposed and cannulated with a 19-gauge needle which was tied. The lungs were lavaged 6-times with cold 5-ml aliquots of sterile PBS. The chest was massaged gently with each infusion. The tubes containing the lavage fluid were centrifuged for 10 min at 250 x g at 4°C. The cells were resuspended in cold PBS and washed twice. More than 98% of the cells were positive for nonspecific esterase activity. From the counted cells, 0.5 . lo6 cells were used for the determinations of the stable eicosanoid degradation products. Macrophage activation. Macrophages were diluted to 0.5 . lo6 ml and preincubated during 2 min at 37” C under shaking. Calcium-ionophore A23187 was added (final concentration 2 PM) and the suspension further incubated during 10 min. The tubes were centrifuged 2 min at 2800 X g and the supernatants collected and frozen at - 70 o C until RIA was performed. Radioimmunoassay. lo-p1 aliquots were used in RIA tests. Anti-sera to TxB,, LTB, and 12-HETE were obtained from Advanced Magnetics via BioLab (Brussels, Belgium). 3H-Labelled compounds were purchased from Amersham International (U.K.), standards of TxB, from Sigma (U.S.A.) and 12-HETE from Advanced Magnetics (U.S.A.). LTB, was obtained from Dr. J. Rokach (Merck-Frosst Labs, Canada). Statistics. A parametric one-way analysis of variance was used, followed by a Duncan’s new multiple-range test. A difference was considered statistically significant when the calculated P values were smaller than 0.05.
I I
25
0
5
10
Fig. 1. Formation of TxB, by alveolar macrophages activated by the calcium ionophore A23187 during tumor growth. Amounts in ng per million cells. Statistical significance * P < 0.05 compared to day 0.
1
01
0
5
10
‘5
20 tumOr
(days)
Fig. 2. Formation of LTB, (A) and 12-HETE (0) by alveolar macrophages after activation during tumor growth. LTB4: * * P c 0.01; *** P i 0.005compared to day 0. 12-HETE: * P c 0.05;** * P c 0.005compared to day 0; * * P i 0.025 compared to day 7; P -c0.01 compared to day 14.
Results In the animals inoculated with tumor tissue, tumor growth occurred. At day 3, a small elevation in the subcutaneous tissue can be felt. After 7 days the size of the tumor was significantly increased (Fig. 3). The number of alveolar macrophages was determined at different times. At day 0, the value was (5.37 + 0.63) 106/5 ml. There is little change until day 10 when a decrease occurs to 65% of the original value. This is followed by a return to the original value at day 12. In the formation of TxB, after activation with the calcium ionophore A23187, a decrease occurs in the initial stages of tumor formation, until day 7. Thereafter, a steady-state level was reached at approx. 68% of the control non-tumor-bearing rats (Fig. 1). The formation of LTB, increases up to day 10, at a value of 190%
30
0.75
25
0.65
20
055
15
045
035
5
0.25
0
015 0
5
10
‘5
20 runor
/days)
Fig. 3. Ratio of TxB,/LTB, formed by activated macrophages during tumor growth. * P r;0.01 compared to day 0. A, Tumor size; 0, TxB, /LTB,.
257 of the controls, and remains at a steady level thereafter. The amounts of 12-HETE formed are higher than those of TxB, and LTB,. Here also, an increase occurs in the initial period of the formation of the tumor. The highest value is reached at day 7 at 228% of the controls. This is followed by a decrease, with a lowest value at day 12 and a rise again at day 14 and 17 (Fig. 2). The ratio TxB,/LTB,, which is a measure of the activities of the cyclooxygenase and CS-lipoxygenase, decreases rapidly in the first days (Fig. 3). TxB, is the ‘most important cyclooxygenase compound. The ratio TxB,/12-HETE, indicating the cyclooxygenase and ClZlipoxygenase activities, shows the same effect (data not shown). Discussion The different aspects of the interaction between macrophages and neoplastic tissue have been the subject of a considerable amount of recent studies [2]. The events that have been described indicate that this interaction is rather complex and comprises several reactions. In the initial period of tumor formation, macrophages are attracted by an infiltrate into the tissue [2]. The macrophages have the capacity to produce cytotoxic substances, such as tumor necrosis factor (TNF) and IL-2 after activation, which can eliminate the neoplastic tissue. It has also been reported, that tumors can produce substances with anti-inflammatory action which inhibit the activation of cells and in this way the release of these substances [2]. This also results in the inhibition of macrophage chemotaxis. In several experimental set-ups, it has been demonstrated that in the presence of a tumor, the mobilization of macrophages is diminished. A protein with anti-inflammatory properties has been isolated [2]. Other possible candidates for the inhibition of cell activation are PGE, and prostacyclin. A high production of PGE,, PGF,, and 6-keto PGF,,, the metabolite of prostacyclin has been demonstrated in a number of malignant tissues [5,6]. The growth of a fibrosarcoma in the rat was associated with high plasma levels of PGE, and TxB, [7]. PGE, has two different effects on the release of TNF-a! from rat resident peritoneal macrophages. At low doses, stimulation occurs and at high doses inhibition [8]. As TNF-(U induces the formation of PGE, a feed-back mechanism may exist. The decrease of TxB, formation by the alveolar macrophages in our experiments after the implantation of the tumor tissue could be induced by one of the anti-inflammatory factors. These substances would inhibited the cell activation and the release of arachidonic acid. This is not very likely as in our experiments the activity of the cyclooxygenase is reduced and that of the C5- and ClZlipoxygenase increased. LTB, induces increased vascular permeability and enhanced granulocyte adherence and migration. The effects of 12-HETE are the induction of chemotaxis for neutrophils, chemokine-
sis for smooth muscle cells and an increase in mononuclear procoagulant activity [9]. One explanation for the increased formation of the lipoxygenase derivatives, would be that when the same amounts of arachidonic acid are released after activation of the macrophages, a diminished formation of cyclooxygenase derivatives will lead to an increased formation of lipoxygenase products. Lipogenase activity has been related to natural killer-cell-mediated cytotoxicity. Effects of leukotrienes, LTB, and 1ZHETE on IFN-y production of T-cells have been described [lo]. IFN-y induces the production of IL-l, IL-2 and TNF [11,12]. Inhibitors of the lipoxygenase reaction also inhibited the cytotoxicity. This was enhanced by 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid (5-HPETE) and LTB, [13]. It could be that the same relationship exists in alveolar macrophages and that lipoxygenase activity is necessary for the cytotoxic effect of these cells. It has been demonstrated that the inhibition of 5-lipoxygenase of peritoneal macrophages activated by the calcium ionophore A23187 inhibited the cytotoxic activity towards tumor cells [14]. At day 12, a dip occurs in both LTB, and 12-HETE curves. The difference in the latter is statistically significant. We are still seeking an explanation for this effect. It could be, that at this time, an additional stress occurs in one of the processes of tumor formation, leading to a release of corticosteroids. These substances induce the formation of lipocortin, a phospholipase A, inhibitor. In this way, eicosanoid synthesis will be decreased. In future experiments, this will be investigated. The results of these experiments indicate that a relationship exists between lipoxygenase activity and cytotoxic action. It is possible, that in the presence of the tumor the lipoxygenase in the alveolar macrophages is activated in order to increase the cytotoxic action in an attempt to destroy the tumor cells infiltrating the lung tissue. This could be an explanation for the increased formation in our experiments of LTB, and 12-HETE. It would seem of interest to investigate, whether a humoral factor secreted by the tumor tissue induces the change in cyclooxygenase/lipoxygenase ratio in the alveolar macrophages. Acknowledgement We would like to thank Mrs. B.H.M. Busscher-Lauw for typing the manuscript. References 1 2 3 4
Lloyd, J.O. (1985) Science 230, 630-632. Cianciolo, G.J. (1986) Biochim. Biophys. Acta 865, 69-82. Sone, S. (1986) Biochim. Biophys. Acta 823, 227-245. Kort, W.J., Zijlstra, F.J. and Weyma, J.M. (1989) Prostaglandins, Leukotrienes and Essential Fatty Acids 37, 113-120.
258 5 Bauknecht, T., Siegel, A., Meerpohl, H.G. and Zahradnik, H.P. (1985) Prostaglandins 29, 665-671. 6 McLemore, T.L., Hubbard, W.C., Litterst, CL., Liu, M.C., Miller, S., McMahon, N.A., Eggleston, J.C. and Boyd, M. (1988) Cancer Res. 48, 3140-3147. 7 Kort, W.J., Weyma, I.M., Bijma, A.M., Van Schalkwijk, W.P., Zijlstra, F.J. and Westbroek, D.L. (1987) Prostagl. Leukotr. Med. 28, 25-34. 8 Renz, H., Gong, J-H., Schmidt, A., Nain, M. and Gemsa, D. (1988) J. Immunol. 141, 2388-2393.
9 Lorenzet, R., Niemetz, J., Marcus, A.J. and Broekman, M.J. (1986) J. Clin. Invest. 78, 418-423. 10 Rola-Pleszcynski, M., Bouvrette, L., Gingras, D. and Girard, M. (1987) J. Immunol. 139, 513-517. 11 Farrar, W.L. and Humes, J.L. (1985) J. Immunol. 135, 1153-1159. 12 Philip, R. and Epstein, L.B. (1986) Nature 323, 86-89. 13 Bray, R.A. and Brahmmi, Z. (1986) J. Immunol. 136, 1783-1790. 14 Van Hilten, J.A., Elliot, G.R. and Bonta, I.L. (1988) Prostaglandins, Leukotrienes and Essential Fatty Acids 34, 187-192.
