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Interaction of cancer chemotherapy agents with the mononuclear phagocyte system By Alberto Mantovani and Annunciata Vecchi Istituto di Ricerche Farmacologiche "Mario Negri", Via Eritrea 62, 20157 Milan, Italy

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Effect of chemotherapeutic agents on mononuclear phagocytes . Glucocorticoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . GC and mononuclear phagocytes . . . . . . . . . . . . . . . . . . GC and tumor growth and metastasis . . . . . . . . . . . . . . . . Antimetabolites . . . . . . . . . . . . . . . . . . . . . . . . . . . . Alkylating agents . . . . . . . . . . . . . . . . . . . . . . . . . . . Intercalating agents . . . . . . . . . . . . . . . . . . . . . . . . . . Plant alkaloids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Modulation of tumor cell susceptibility to macrophage cytotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Modulation of mononuclear phagocyte and antitumor efficacy . Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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C. R. Pfaltz et al., Progress in Drug Research / Fortschritte der Arzneimittelforschung / Progrès des recherches pharmaceutiques © Springer Basel AG 1990

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Introduction

Cytotoxic agents used in the medical treatment of neoplasia have profound effects on host immunity. Traditionally the interaction of cancer chemotherapy agents with immunity has been considered in connection with immunodepression. Immunodepression is in fact a prime determinant in the pathogenesis of some of the acute and longterm toxicities associated with cancer chemotherapy, including infections and second malignancies. Interference with immunity has also been considered an intrinsic limiting factor in the efficacy of anticancer agents. In this perspective, chemotherapeutic agents are, in a way, self-defeating. More recently, evidence has accumulated that the interaction of cancer chemotherapeutic agents with immunity is more complex than originally envisaged and that these drugs should be considered from the wider perspective of immunomodulation rather than from the narrower angle of simple immunodepression. In this light, it appears that the relationship between antitumor efficacy and modulation of host resistance is more complicated than expected on the somewhat simplistic assumption that chemotherapeutic agents act as pure depressants of immunity and that no substantial differences exist among antitumor drugs in their interaction with host defence mechanisms. Cells of the mononuclear phagocyte lineage playa central role in the immunobiology of neoplasia [1-3]. Appropriately activated macrophages can kill extracellularly tumor cells and have been suggested to act as a surveillance mechanism [1-3]. In established malignancy, mononuclear phagocytes represent a major component of the lymphoreticular infiltrate of neoplastic tissues. Thus, being strategically located at the very interface between tumor and host, tumor-associated macrophages are crucial cells in the light of therapeutic intervention. The purpose of this review is to analyze the interaction of cancer chemotherapy agents with cells of the monocyte-macrophage lineage. Previous reviews on the modulation of specific immunity by chemotherapeutic agents [4, 5] provide a framework for the present effort. We will extend and update previous analysis [6-8] on the effects of cancer chemotherapeutic agents on the mononuclear phagocyte system and discuss available information as to the in vivo relevance of modulation at this level.

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Effect of chemotherapeutic agents on mononuclear phagocytes G1ucocorticoids (G C) G C and mononuclear phagocytes

Glucocorticoid hormones are used not only in the treatment of neoplastic diseases, but also to suppress immunity and inflammation under a variety of clinical conditions. Thus the significance of modulation of mononuclear phagocyte function by GC extends beyond the field of cancer chemotherapy. We have recently reviewed the immunosuppressive activity of GC [7]. Here we will concisely summarize available information on the interaction of GC with the mononuclear phagocyte system and emphasize recent results of the mode of action of these agents. Mononuclear phagocytes are a major target of the immunoregulatory and antiinflammatory effects of glucocorticoid hormones as indicated, for instance, by peripheral blood counts of monocytes that are affected more than those of other leucocyte populations [8a-IO]. Cells of the monocyte-macrophage lineage have high-affinity binding sites for GC. Glucocorticoid-binding macromolecules have been demonstrated in monocytes, macrophages and macrophage cell lines [11, 12]. The dissociation constant of the receptors was within physiological ranges and the specificity and affinity correlated with biological function [12]. In vivo administration of GC causes dramatic monocytopenia both in man and mice [10, 13, 14]. Although GC have the capacity to affect monocyte production from bone marrow precursors, this is not the major determinant of the rapid fall of monocyte counts following GC administration. GC are not directly toxic for mature mononuclear phagocytes, interfere with the release of newly formed monocytes from the blood compartment and probably cause a redistribution of cells out of the circulation into unidentified compartments. The proliferation of myeloid precursors in colony stimulating factor (CSF)-stimulated cultures of bone marrow cells is suppressed by GC in vitro [15, 16]. It is of interest that in this in vitro system the myelosuppressive effect of GC is prevented by IL-l [16]. GC also inhibit the proliferative capacity of mature macrophages. GC alter the differentitation of mononuclear phagocytes. In an in vitro system consisting in culturing human monocytes, Rinehart et al.

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[17] reported that the transition from monocytes to macrophage-like

cells was inhibited by GC. Similarly, differentiation of an established monocytic cell line, was partially blocked by dexamethasone [11]. Along the same line in vivo or in vitro exposure to GC prevented the spontaneous fusion of rabbit alveolar macrophages to form multinucleated giant cells [18]. Recruitment of mononuclear phagocytes into tissues is a major target of the immunosuppressive and antiinflammatory action of GC [19, 20]. Rinehart et al. [21] reported that hydrocortisone inhibits the responsivencess of human monocytes to lymphocyte-derived chemoattractants. In vivo, GC have been shown to inhibit monocyte recruitment at sites of inflammation, infection and neoplastic growth [20-22].

In addition to causing monocytopenia and to inhibiting recruitment of mononuclear phagocytes in tissues, GC interfere with various functions of differentiated mature macrophages, including production of prostaglandins, plasminogen activator and collagenase [18,23,24]. Of particular relevance in the perspective of GC as antitumor agents is the effect of these compounds on the cytotoxic activity of mononuclear phagocytes. GC have been shown to reduce the cytostatic and cytotoxic activity of in vivo and in vitro activated macrophages of rodent or human origin [25-27]. Recent evidence indicates that GC interfere both with priming (IFN y) and triggering (LPS) signals of macrophage activation for tumor cytotoxicity [27]. Correspondingly in vivo treatment with cortisone acetate abolishes the non-specific protection induced by Corynebacterium parvum in mice bearing the P 815 mastocytoma [28]. Analysis of the interaction of GC with the production of cytokines by macrophages as well as with their capacity to respond to soluble mediators of immunity acting in autocrine or paracrine loops has considerably improved our understanding of the mode of action of these agents. Snyder and Unanue [29] reported that hydrocortisone inhibited the production of interleukin 1 (ILl) by mouse macrophages exposed to bacterial lipopolysaccharides (LPS). In the same study they also showed that GC blocked the lymphokine-induced expression of Ia antigens by macrophages. Similar results were reported by Smith [30], who studied mouse macrophages triggered by LPS under treatment with dexamethasone. Spontaneous or carrageenan-induced production of IL-I by rat macrophages was suppressed by hydrocorti-

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sone in vitro [31]. In apparent contrast with these data in rodents, methylprednisolone did not affect the production of IL-I by human monocytes exposed to a phorbol ester [32]. However, subsequent studies [33, 34] revealed that methylprednisolone inhibited the production of lymphocyte-activating factor by monocytes cultured with PPD. It was not excluded that under those circumstances the glucocorticoid in fact acted upon contaminating T cells responsible for triggering the release of the mono kine by mononuclear phagocytes. Reconstitution experiments provided further evidence that block at the IL I level can be important in glucocorticoid-mediated immunosuppression at least in vitro. Exogenous IL 1 has been shown to prevent glucocorticoid inhibition of the murine primary humoral response in vitro and to protect helper but not suppressor T cells from these agents [35-38]. However, using human cells Bendtzen et al. [33] failed to reconstitute the production of leukocyte migration inhibitory factor (LlF), inhibited by methylprednisolone, with exogenous IL 1. IL 1 has also been reported to counteract the myelosuppressive effect of dexamethasone in vitro in a system of granulocyte/macrophage colony formation in response to CSF [16]. Renelletti et al. [39] studied the role of accessory cells in suppression of mitogen responsiveness in blood lymphocytes and thymus cells. Monocytes or IL 1 reconstituted the dexamethasone-inhibited response to phytohemagglutinin of blood lymphocytes, but mononuclear phagocytes or IL 1 failed to reconstitute the response of thymocytes. The isolation of cDNA clones encoding cytokines has provided the conditions for more in depth analysis of the mode of action of GC on mononuclear phagocytes. Dexamethasone has been shown to reduce expression of IL 1 mRNA transcripts. Reduced expression of IL 1 mRNA was related to inhibited transcription of the gene. A block in translation into protein of IL 1 mRNA in the presence of GC has also been reported [40, 41]. It is of interest that IL 1 production has been reported to be differentially regulated in macrophages versus vascular endothelium, a cell type that shares many properties with mononuclear phagocytes [42-44]. Dexamethasone was found to inhibit IL I production in macrophages but not in endothelial cells. Similarly, dexamethasone did not affect GM-CSF induction in endothelial cells while it strongly suppressed it in macrophages. In considering the modulatory effect of GC on monokine circuits, pharmacological concentrations of these agents, i. e. those used in

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chemotherapy protocols, should be considered. It must however be emphasized that under physiological conditions GC play a crucial role as modulators of the action of monokines. For instance, in mice, the acute toxicity of IL 1 is prevented by endogenous production of GC [45]. Moreover GC have a cooperative, permissive influence on the induction of certain acute phase proteins (e. g. fibrinogen) by monokines [7]. Tumor necrosis factor (TNF), a pleiotropic monokine functionally related to IL 1, is also affected by GC. GC inhibit the expression of TNF mRNA in macrophages by interfering with gene transcription [46]. Inhibition of TNF transcript expression by GC results in lower levels of mono kine being produced. Since TNF is involved in the cytotoxic action of mononuclear phagocytes, at least against certain tumor targets, (for review see ref. 2), inhibition of production of this monokine could at least partially account for the block of macrophage cytotoxicity by GC discussed above. In addition to interfering with TNF production, dexamethasone has recently been shown to inhibit the tumor necrotizing activity of this cytokine [47]. Dexamethasone blocked TNF-induced tumor necrosis only when the monokine was injected intratumorally. Necrosis of neoplastic tissues induced by local administration of TNF involves recruitment of leukocytes from the blood compartment and participation of vascular endothelium. It is conceivable that GC inhibit TNF-induced necrosis by blocking the endothelial cell responses to this cytokine and leukocyte extravasation. 2.1.2

GC and tumor growth and metastasis

Although it is clear that GC can inhibit growth of primary tumor, metastatic dissemination can be augmented by these agents [22, 48-59]. For instance, in the first (to the best of the author's knowledge) of these observations in a syngeneic model. Agosin et al. [48] found that cortisone inhibited the growth of C3H spontaneous mammary adenocarcinoma but induced the appearance of metastasis. A more widespread distribution of metastases after treatment with glucocorticoid hormones has also been reported in humans with recurrent malignancy [60, 61]. Enhanced metastasis is not caused by increased numbers of tumor cells leaving the primary site [49], although this point has not been extensively investigated. In fact, enhanced metastasis is also observed when tumor cells are injected intravenously, thus ex-

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eluding a contribution at the level of the primary tumor [22, 58]. Tumors comprise subpopulations heterogenous in various biological properties and metastases can differ both among themselves and from primary tumors in several biological properties [62]. Hence it could be argued that the different effects of GC on primary tumors and metastases may be related to differences in the intrinsic properties of tumor cells that populate these two sites. To test this hypothesis, cells from metastases were transplanted intramuscularly and the effects of hydrocortisone under these conditions were studied [22]. Hydrocortisone had similar effects on tumors originated by transplanting cells from the primary lesion and from metastases, with enhancement of secondaries and inhibition of intramuscular malignancy. Thus the divergent effects of GC on growth of primary tumors and metastases are not related to differences in the intrinsic properties of tumor cell populations at these sites [22]. Metastasis is a multistep event [62], and the augmentation by GC of secondary spread is not indiscriminate for all steps of the process. Release of neoplastic cells from the primary lesions does not seem to be affected [22, 49]. Moreover, once established, secondary deposits are not enhanced by hydrocortisone, but actually tumor growth at these sites is inhibited by GC as found at the primary lesion [22]. Thus GC augment metastasis by affecting some early step of implantation and growth at secondary sites [22]. In addition to augmenting metastasis at sites already involved by the secondary spread under "normal" conditions, GC alter the pattern of metastasis. In fact, glucocorticoid hormones caused the appearance of extrapulmonary metastases in tumors that otherwise colonize only in lungs [22, 50]. It is noteworthy that similar observations have been reported in humans [60, 61]. Host defense mechanisms have been suggested to promote tumor growth at least in some neoplasms and at certain phases of tumor progression [63]. In particular, evidence suggests that the macrophage infiltrate of neoplastic tissues can provide the conditions for optimal tumor growth, possibly by favoring angiogenesis and by providing growth factors for neoplastic cells [64, 65]. In a series of murine tumors, hydrocortisone diminished the macrophage infiltrate of neoplastic tissues and concomitantly the growth of primary tumors [22]. Since there was no correlation between in vivo macrophages suppression of tumor growth on the one hand and glucocorticoid receptor

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content or in vitro susceptibility of glucocorticoid cytotoxicity of neoplastic cells on the other, the possibility that interference with some host components was responsible for inhibition of neoplastic growth by hydrocortisone was explored. A role for NK cells or T lymphocytes was excluded on the basis of data obtained in mice with congenital or acquired defects of these reactivities. Hydrocortisone depleted the macrophage content of the neoplastic tissues, and admixture of macrophages to tumor cells reconstituted malignant growth in hydrocortisone-treated mice at least for the mFS6 sarcoma, but not for another sarcoma used in the same study [22]. Thus these and other [66] data strongly suggest that at least in some tumors glucocorticoid may inhibit neoplastic growth by reducing macrophage accumulation in neoplastic tissues, tumor-associated macrophages providing the optimal conditions for neoplastic proliferation by favoring angiogenesis and providing growth factor [67,68]. This mode of action of GC, formally demonstrated so far only in one tumor model [22], may be of interest in relation to the recently observed antitumor activity of combinations of these agents with heparin, which have been suggested to act by interference with angiogenesis [69]. As discussed in the previous section, glucocorticoid hormones enhance metastasis in experimental animals, and, possibly, in humans. The mechanisms of this effect of GC have not been elucidated. NK cells and macrophages have been suggested to act as a mechanism of restraint of the vascular phase of secondary spread of cancer cells and of the early steps of cell implantation and growth [for review see 65, 70]. It is at this (these) level(s) that GC augment metastasis [22, 49]. Therefore one can speculate that inhibition of NK cells and mononuclear phagocytes contribute to enhancement of metastasis by GC [22]. Effects on the vessel wall could also playa role in augmentation of secondaries by GC [58]. 2.2

Antimetabolites

This discussion will focus on thiopurine, 6-mercaptopurine (6-MP) and its nitroimidazole derivative azathioprine (AZA), which, possibly because of the widespread use of the latter in clinical practice as an immunosuppressant, have been extensively studied for their interaction with mononuclear phagocytes. AZA is rapidly transformed in vivo into its parent compound, 6-MP [71], and has similar immunosup-

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pressive properties [72, 73]. The available information concerning the effects of other antimetabolites on cells of the monocyte-macrophage series is scanty and fragmentary and will be mentioned only briefly. Treatment with relatively low doses of thiopurines resulted in a profound monocytopenia in various animal species [74-82]. The mechanisms responsible for the reduction in monocyte counts following treatment with AZA were extensively investigated by Van Furth and co-workers [81, 82]. These investigators found a decreased mitotic activity of promonocytes during AZA treatment in mice; however, the labeling index of promonocytes exposed to AZA increased and a higher percentage of these cells were tetraploid. Therefore, it was concluded that AZA arrests the cell cycle of the promonocytes late in the DNA synthesis phase or in the postsynthesis (G2) phase, thus preventing mitosis. During acute inflammation, the cell cycle time of bone marrow promonocytes decreased and monocyte production increased: relatively low doses of AZA (3 mg/kg) abolished this response elicited by inflammatory stimuli. Moreover, while in untreated mice 40% of the monocytic cells leaving the circulation entered the site of inflammation, this fraction was reduced to 10% in AZA-treated mice. It is of interest in this connection that Phillips and Zweiman [83] showed that high concentrations of 6-MP directly affected macrophage migration in guinea pigs. Similarly, in vitro exposure to AZA or to amethopterin inhibited the response of rabbit macrophages to MIF [84]. Interestingly enough, production of MIF by lymphocytes was not markedly affected by AZA in rabbits and baboons [84, 85]. In this line, we have observed that an other antimetabolite, 5-fluorouracil (5FU), only weakly affected IL-l production by peritoneal macrophages when cells were tested 24 hours after in vivo treatment and the effect was completely recovered 72 hours after treatment (Table 1). Treatment with AZA profoundly inhibited the accumulation of macrophages within a murine sarcoma [86]. The mechanism of this effect was not fully elucidated but the above-described interference of AZA with monocyte production and impairment of macrophage responsiveness to lymphokine could playa role in the reduction of tumor-associated macrophages. A reduced accumulation of macrophages in AZA-pretreated, tumor-transplanted animals was associated with a reduced growth of this sarcoma. This observation, together with data in X-irradiated mice [87], suggested that tumor-associated macrophages were providing a stimulus for tumor cell proliferation in vivo.

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Table 1 Effect of chemotherapeutic agents on IL-I production by murine macrophages Drug

Dose (mg/kg)

5FU

200i.p.

5FU

200i.p.

Cy

200i.p.

Days after treatment

c.p.m. at supernatant dilution 1/8 1/32

1 I 3 3 2 2

14453 ± 1043 9712± 635* 17281 ± 3160 19664 ±3357 24358 ±2078 6733 ± 1700**

6845 4643 5092 4278 6117 2708

± ± ± ± ± ±

1093 1018 983 1695 997 357**

c.p.m.: counts per minutes. Macrophages (1.5 x 106 /ml) from C3H/HeN control and treated mice were exposed to 20 ,ug/ml LPS for 24 hr. Supernatants were collected, centrifuged, dialyzed and frozen. IL-I was evaluated as LAF activity (lymphocyte activating factor) on thymocytes (7.5 x 105 / well) cultured with PHA (I ,ug/ml) and various dilutions of supernatants for 72 hr in a final volume of 0.2 ml/well. Proliferation was evaluated as 3H-thymidine incorporation during the last 6 hr of incubation. * p

Interaction of cancer chemotherapy agents with the mononuclear phagocyte system.

487 Interaction of cancer chemotherapy agents with the mononuclear phagocyte system By Alberto Mantovani and Annunciata Vecchi Istituto di Ricerche F...
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