REVIEW ARTICLE

Tumour-associated macrophages (TAMS): disordered function, immune suppression and progressive tumour growth

B. AL-SARIREH and O. EREMIN
Section of Surgery, Queens Medical Centre, Nottingham, U.K.

Evidence currently available suggests that in established, progressively growing solid tumours, tumour associated macrophages (TAMs) are reprogrammed to induce immune suppression of host defenses in situ, through release of specific cytokines, prostanoids and other humoral mediators. This disordered response, results in the inhibition of effective anti-cancer cell-mediated immune mechanisms. Concurrently, TAMs produce tumour growth promoting factors. The summation of this complex interplay of biological factors results in progressive tumour growth and tumour cell dissemination. A better understanding of these complex inter-relationships should form the basis of novel strategies designed to eradicate tumour cells in man and animals. These various biological aspects and processes are discussed in detail and critically evaluated in this review article.

Key words: macrophage, chemotactic factors, promotion of tumour growth, growth factors, anti-tumour activity, cytokines inter-leukin-1, tumour necrosis factor-a, interleukin-10, transforming growth factor-b, ecosanoids, free radicles, apoptosis, immunosuppression

J.R.Coll.Surg.Edinb., 45, February 2000, 1-16

INTRODUCTION

Lineage

The monocyte develops from a pluripotent stem cell in the bone marrow under the influence of soluble haematopoietic growth factors and by physical interactions with stromal cells and extra cellular matrix proteins. The monoblast, which is the earliest cell committed to the monocytic lineage, differentiates via a promonocyte into a mature monocyte, which, after only a short period (< 24 hrs), leaves the bone marrow and enters the blood stream as a quiescent (G0/G1) cell.¹ Such monocytes may then differentiate further into resident tissue macrophages and, depending on the local microenvironment, acquire specialised phenotypic characteristics and diverse functions. Furthermore, blood monocytes can be recruited to sites of acute and chronic inflammation, where they become activated into cells expressing the macrophage phenotype. Activated macrophages have a wide range of biological functions, depending on their mode of activation.

Secretory Functions

Monocytes and macrophages have well developed secretory functions and are an important source for a wide variety of cytokines. During the process of extravasation in vivo monocytes initially adhere to capillary endothelial cells and subsequently to various extracellular matrix components, finally differentiating into tissue macrophages. When monocytes adhere to plastic or extracellular matrix components (e.g. fibronectin) in vitro, interleukin (IL)-1, IL-6, tumour necrosis factor (TNF)-a, colony stimulating factor (CSF)-1 and granulocyte macrophage-colony stimulating factor (GMCSF) expression is induced.^2-4 Moreover, monocytes respond to soluble inflammatory mediators, such as bacterial lipopolysaccharide (LPS), by the production of IL-1, IL-6, TNF-a and GM-CSF.^3,4 Thus, at sites of inflammation and infection, macrophages become involved in complex interactions with the various cells present at the site. As a result, macrophages of diverse functions are produced. The macrophages of man and experimental animals are known to produce and secrete more than 100 biologically active substances

Biological Functions

Macrophages have a pleiotropic biological role which includes antigen presentation, target cell cytotoxicity, removal of debris and tissue remodeling, regulation of inflammation, induction of immunity, thrombosis and various forms of endocytosis.⁵ In the setting of tumours, tumour-associated macrophages (TAMs) have a range of functions with the capacity to affect diverse aspects of neoplastic tissues including angiogenesis and vascularisation, stroma formation and dissolution, and modulation of tumor cell growth (enhancement and inhibition). When activated, they can induce neoplastic cell death (cytotoxicity, apoptosis) and/or elicit tumour destructive reactions through alterations of the tumour microvasculature.⁶

Leukocytes and Tumours

Virchow first identified leukocytes within and at the edge of tumour tissue in 1863.⁷ He proposed that the frequent occurrence of a lymphoreticular infiltrate in human melanoma reflected the origin of cancer at sites of previous chronic inflammation. However, when Handley reported subsequently that normal cell infiltration in malignant melanoma indicated a ‘regressive process’, opinions regarding the significance of the lymphoreticular infiltrate began to change.⁸ Numerous ensuing reports have discussed its relevance to the pathological process in tissues and/or prognosis. Conflicting views of the relationship between leukocyte infiltration and malignancy have appeared in the literature reflecting the pleiotropic, as yet inadequately defined function of infiltrating cells, aspects that are now being explored at the molecular level.

Solid tumours, both primary lesions and metastases, are infiltrated by large numbers of tumour-associated leukocytes. These are a heterogeneous population of cells consisting of various (and variable) subsets of T cells (helper, suppressor and cytotoxic), B cells, natural killer (NK) cells, and macrophages.⁹ However, the biological relevance and clinical significance of these different cellular infiltrates remains the subject of conflicting reports and continuing debate. In view of their normally defensive and beneficial role in vivo, leukocytes infiltrating tumours were originally believed to herald an immune response to the growing neoplasm. However, the spontaneous regression of an established tumour is a rare event, suggesting that in progressively growing tumours, the immunocompetence and anti-cancer effects of these cells within the tumour cell milieu may be compromised. Moreover, subpopulations of tumour-associated leukocytes have been shown to be detrimental to the host and beneficial to progressive tumour growth.^10,11

MACROPHAGES AND CANCER

Introduction

It is widely accepted that tumour-associated macrophages (TAMs) represent a major and important component of the leukocyte infiltrates of many tumours and metastatic deposits.^12,13 It has been established that TAMs have pleiotropic functions, which can influence tumour growth, both in terms of progression and regression. This differential effect of TAMs is believed to be regulated by modulation of the host immune system^14,15 (Figure 1). Tumour growth reduction by TAMs can be mediated by non-specific anti-tumour cytotoxic mechanisms or induction of specific cell lytic effects.^16,17 On the other hand, there is convincing data for TAMs demonstrating tumour cell growth-promoting effects through release of various cytokines and prostanoids.^18,19 Also, macrophages have been shown to suppress many T cell^20,21 and NK cell²² anti-tumour responses. What determines the outcome of these competing interactions and subsequent defined macrophage functions during tumour growth, as yet, is still poorly defined. An understanding of the complex regulatory mechanisms, therefore, that control macrophage functions during tumour growth is critical to planning therapeutic approaches to achieving improvement of patient well being and control of the malignant process.

Figure 1: Paracrine interactions between TAM, tumour and lymphocyte cells. Tumour cells attract monocytes by producing chemotactic agents, including MCP-1, M-CSF, TGF-bb and as yet undefined mediators (tumour-derived chemotactic factors, TDCF). Tumour-derived signals can induce both pro- and anti-tumour effector functions in TAMs. Depending on the activation signals and the susceptibility of tumour target cells, TAMs can either enhance (pro-tumour) or inhibit (anti-tumour) tumour growth. As anti-tumour effectors, TAMs can mediate direct anti-tumour cytotoxicity, produce of cytotoxic molecules (H2O2, IL-1, TNF-aa, NO, ROI) and stimulate lymphocyte responsiveness through presentation of TAAs and production of immunostimulatory cytokines (e.g. IL-12). On the other hand, as pro-tumour effectors, TAMs produce growth factors that promote cancer cell proliferation and dissemination, enhance angiogenesis, and suppress lymphocyte responsiveness via production of immunosuppressive cytokines (e.g., IL-10) and prostanoids (e.g. PGE2)

Irrespective of their role in the pathophysiology of neoplasia, macrophage-derived cytokines and pro-inflammatory substances are critical regulators of macrophage activities. The discovery that tumours also produce pro- and anti-inflammatory cytokines and other regulatory molecules provides an insight as to how tumours may subvert macrophage anti-tumour functions and redirect activities to favour tumour cell growth. To further our understanding of this rapidly evolving field, current understanding of the tumour content and function of TAMs and interactions of TAMs with other effector immune cells, will be discussed.

Macrophage Content in Tumours

Immunohistological analysis of tissue sections and isolation of cells by mechanical disaggregation and enzymatic digestion of excised tumours are the established methods used to study the composition of tumours.^23,24 The general consensus from published studies is that the number of macrophages is substantial but varies greatly (10 to 65%) among the different tumours studied.9,11,23 However, the percentage of TAMs is usually maintained at a relatively stable level for a particular tumour type during transplantation into and growth in syngeneic hosts.⁶ Macrophages also infiltrate metastatic lesions, although TAMs have been less extensively studied in secondary tumour deposits.^25,26 Secondary tumour lesions can vary considerably in the degrees of macrophage infiltration. Tumour cell clones derived from the same primary tumour are heterogeneous in their capacity to attract monocytes²⁷ and this may account for the heterogeneity of TAM content in secondary foci. Even in macrophage-rich anatomical sites such as the liver, macrophage infiltration in metastases depends to a large extent upon the recruitment of monocyte precursors.²⁶ There is little published data regarding macrophage numbers during the various stages of tumour development. This is an unfortunate deficiency in knowledge since we know that the effect of macrophage biological products varies greatly between the different stages of tumour growth.^28,29

A major criticism against these various studies is that histological examination is a non-quantitative (at best semi-quantitative) assessment and not all cell types can be identified with certainty on the basis of their morphology alone. Also, the yield of cells prepared by mechanical disaggregation and enzymatic digestion, as regards content and type, is variable; cell selection during preparation is a distinct possibility. From analogous studies in other tissues it is known that similar techniques can produce inaccurate estimates of the relative numbers of cells isolated from such tissues.³⁰

Experiments in which tumours were transplanted into animals with defective T or NK cell immunity have shown the latter not to be a major determinant of macrophage infiltration. It appears that factors derived from tumour cells themselves play a pivotal role in the regulation of macrophage levels in poorly immunogenic tumours.⁶

Regulation of TAM Levels

The infiltration of mononuclear cells, as determined in most tumours, is generally restricted to the stromal areas with few cells infiltrating into tumour cell nests and between the tumour cells themselves.³¹ This migration is mediated by chemotactic factors, inducing inflammatory cells to leave the vascular compartment and egress into the surrounding areas. The migration of inflammatory cells within tumour cell nests in situ, however, is restricted. Several types of murine and human cells produce chemotactic factors for mononuclear phagocytes.^32,33 These factors are capable of inducing chemo-tactic migration of monocytes. Some tumours, however, produce inhibitors of chemotaxis.³⁴ The regulation of these and other factors, reflected in the varying numbers of macrophages in different types of tumours and at various stages of tumour growth, is largely unknown. The mechanisms modulating these processes are poorly defined.

Mantovani and his group were the first to study monocyte chemoattractants released by tumour cells.³² They identified a cytokine of about 12 KDa, called tumour-derived chemotactic factor (TDCF), which is chemotactic for monocytes and is released by various murine and human tumour cells. Subsequently, the sequence and structure of this cytokine, now referred to as monocyte chemotactic protein-1 (MCP-1), was determined.^35,36 As anticipated from its in vitro chemo-tactic activity, injection of MCP-1 in vivo induces extravasation of monocytes.³⁶ There is also evidence that it regulates the level of TAMs in vivo. Using murine tumours and biopsies from human ovarian carcinomas (and other human tumours) transplanted into nude mice, significant correlation was found between the number of TAMs and MCP-1 expression, produced by the tumour cells.^27,37,38 Also, MCP-1 gene transfer resulted in higher levels of TAMs in a murine melanoma model.³⁷ In addition to its role in recruitment of monocytes, MCP-1 affects several aspects of mononuclear phagocyte function related to effector activity. For example, MCP-1 induces production of gelatinase, urokinase type plasminogen-activator (uPA) and expression of the cell surface receptors for uPA (uPA-R) by monocytes.^39.40 Natural MCP-1 was reported to induce IL-1 and IL-6 but not TNF production in monocytes.⁴¹ Also, MCP-1 was found to induce oxidative activity in human monocytes but inhibited nitric oxide (NO) synthase in the macrophage cell line J774.⁴² While MCP-1 is an important tumour-derived factor for the regulation of TAMs in certain neoplasms, there is also evidence for MCP-1 independent recruitment of TAMs. This pathway involves other cytokines and mediators such as MCP-2, MCP-3^39,43,44 monocyte colony stimulating factor (M-CSF)⁴⁵, granulocyte-macrophage colony stimulating factor (GM-CSF)^46,47, vascular permeability factor (VPF)⁴⁸ and IL-12p40.⁴⁹

The mechanisms involved in the maintenance of significant and constant levels of macrophages in growing tumours are complex, multifactorial and poorly understood. It has been reported that TAMs have increased proliferative capacity⁴⁵ and in situ proliferation may contribute to the macrophage content of tumour tissues. In an effort to define the molecular basis of TAM proliferation, the expression of M-CSF and of the c-fms proto-oncogene, which encodes the M-CSF receptor, was studied in murine sarcomas. TAMs expressed high levels of c-fms mRNA and sarcoma cells expressed M-CSF.⁴⁵ M-CSF is known to promote survival and/or proliferation of mononuclear phagocytes both in animals and humans.^45,50 These observations suggest the existence of a paracrine circuit in the regulation of TAM survival and proliferation, at least in this model, involving M-CSF secreted by tumour cells and acting on c-fms expressing TAMs. In man, it has been shown that breast carcinomas secrete macrophage-specific chemotactic cytokines based on the findings of a positive correlation between the degree of CSF-1 expression by tumours of a high grade and marked monocyte infiltration.⁵¹ Also, direct correlation was found between c-erbB-2 oncogene product overexpression and the presence of a large number of TAMs.²⁸ The mechanisms by which tumours that are c-erbB-2 positive and of a high grade, two biological features that are strongly associated⁵², recruit macrophages remains to be established. One possibility is MCP-1 deregulation after cerbB-2 gene amplification as both genes are present on the same arm of chromosome 17. Whatever the underlying mechanisms, it is likely that in situ survival and proliferation of monocytes recruited from blood contributes to the maintenance of the high and constant levels of TAMs in most growing tumours.

TAMS AND CANCER CELL INTERACTIONS

TAM Promotion of Tumour Growth

TAMs have been shown to play a key role in tumour growth and dissemination and in some of the systematic manifestations of neoplasia (Table 1). Mononuclear phagocytes can produce a range of growth factors, including platelet-derived growth factor (PDGF), epidermal growth factor (EGF) and transforming growth factor (TGF)-b, as well as cytokines such as TNF-a and IL-1, that have been shown to enhance metastatic spread in several animal tumour models.^39,53-55 It is not surprising, therefore, that TAMs can promote the growth of tumour cells, an activity best observed in vitro at low effector to target cell ratios or with tumour cells in suboptimal culture conditions.^5,12

Table 1: TAMs and tumour progression

The seemingly paradoxical activity of tumours in promoting macrophage infiltration actually benefits cancer growth through a number of mechanisms that have been elicited both in vitro and in vivo. For example, TAMs have been shown to have direct angiogenic activity^18,19 and, by enhanced fibrin deposition via pro-coagulant activity⁵⁶, may indirectly promote new vessel formation. Furthermore, there is evidence to suggest that lack of macrophages may inhibit angiogenesis and, as a consequence, induce tumour cell death.^18,53 TAMs also may enhance tumour growth through enhancement of tumour spread and the metastatic process.⁵⁷ Cells of the monocyte-macrophage lineage are potent producers of proteinases. For example, TAMs from various mouse sarcomas have increased amounts of the neutral serine protease, plasminogen activator⁵⁸; tumour cells can express receptors for plasminogen activator. Thus, macrophage-derived proteases may contribute to tissue invasion and spread by tumour cells.

Another possibly important growth promoting mechanism is the production of L-arginine-derived polyamines.¹⁴ L-Arginine is the substrate molecule for macrophage biosynthesis of the cytotoxic molecule nitric oxide (NO) through the activity of the inducible form of nitric oxide synthase (iNOS). However, some tumours markedly inhibit intra-tumoural macrophage production of NO59 by shunting L-arginine metabolism in favour of the biosynthesis of ornithine¹⁴, a precursor for polyamine synthesis and a key factor required for tumour cell replication.

The tumour promoting effects of macrophages are further augmented by their ability to synthesise and secrete inhibitory factors, which suppress host anti-tumour effectors, if and when present. TAMs produce prostanoids, including prostaglandin E2 (PGE2), which have strong immunosuppressive effects on cell-mediated immune mechanisims. Macrophages also produce the immuno-suppressive cytokines TGF-b and IL-10. The latter, in particular, inhibits T helper (TH1) activity with resultant suppression of cytotoxic T cell generation and activity, as well as inhibition of NK and lymphokine-activated killer (LAK) cell cytotoxicity.^60-62

The in vivo function of TAMs has been studied by the administration of compounds that specifically inhibit macrophage function or by adoptively transferring macrophages. The administration of relatively specific macrophage toxins, such as silica, carrageenan and glucocorticoid hormones enhance the establishment of metastases in various tumour models by affecting early stages of tumour cell implantation and growth.¹² In addition, the subsequent growth of metastatic deposits is also increased. Adoptive transfer studies, using mixtures of tumour cells and macrophages in normal or monocytopenic hosts, results in the earlier appearance and/or faster growth of various murine tumours.^53,64 Thus, in some tumours and at defined stages of tumour cell growth and progression, TAMs may provide the essential microenvironment in situ for optimal neoplastic proliferation. Conversely, fully activated cytotoxic macrophages may inhibit tumour growth.^5,65,66

TAM Inhibition of Tumour Growth

Macrophages, particularly when activated, can be cytostatic or cytotoxic for tumour cell growth^67-69 (Table 2). In 1959, Biozzie observed that Bacille Calmette-Guérin (BCG)- a non-specific activator of the ‘reticulo-endothelial system-when inoculated into mice, was able to decrease the growth of certain tumours. Since these early observations, many reports have demonstrated that non-specific stimulants, known to activate macrophages, may inhibit tumour growth in animals when administered at an appropriate time interval in relation to inoculation of tumour cells. Also, it was observed that when grafts of tumour cells, transfected with the genes encoding for interferon (IFN)-g, IL-2 and TNF-a, were rejected, activated macrophages were present in the tissues at the sites of inoculation. Furthermore, specific antibodies that blocked the recruitment of macrophages inhibited the rejection response.⁷⁰

Table 2: TAMs and tumour regression

Various in vitro systems have been designed to induce macrophages to express anti-tumour activity and to investigate the mechanisms involved in this activity. The main aspects investigated have been the role of arachidonic acid derivatives (PGs and leukotrienes (LTs)), macrophage cytokines, and nitrite production from L-arginine. In view of the fact that the anti-tumour activity of macrophages is non-species specific, some studies were carried out using human macrophages and murine tumour cells.

It has been found that phorbol myristate acetate (PMA), in combination with the calcium ionophore A2318771 or calcium ionophore alone⁷², primed peritoneal macrophages for anti-tumour function. The ability of the calcium ionophore to induce anti-tumour activity was correlated with increased production of LTC4.⁷² Activated murine peritoneal macrophages treated with PMA bound significantly more tumour cells than did untreated macrophages.⁷¹ Normal murine peritoneal macrophages expressed anti-tumour cyto-static activity, when endogenous production of PGE2 was inhibited and production of LTs was enhanced.⁷³

In another model, culture conditions that induced increased PGE2 production by activated macrophages led to inhibition of their tumouricidal activity, whereas increased PGE2 production by resident non activated macrophages was associated with an increase in anti-tumour activity.⁷⁴ LPS induces production of both PGE2 and PGI2, but only PGE2 has a negative regulatory effect on macrophage activation of tumour cell killing.⁷⁵ Blockade of PG synthesis by indomethacin prevented LPS stimulation of macrophage anti-tumour activity, again showing the inhibitory effect of PGs.⁷⁶ The suppressive effect of PGE2 on induction of anti-tumour activity was also reported in models using human peritoneal macrophages and was postulated to be due to inhibition of IL-1 production.⁷⁷ It has been shown that PGE2 regulates macrophage-derived TNF gene expression⁷⁸, lipoxygenase inhibitors suppressing formation of TNF in vitro and in vivo.⁷⁹ In another study, PGE2 was found to suppress TNF but not IL-1 production.⁸⁰ These various aspects are discussed in more detail in the following sections.

MACROPHAGE ANTI-TUMOUR ACTIVITY

Introduction

The tumouricidal action of activated monocytes/macrophages has been the subject of many studies since the early investigations several decades ago.^81,82 A range of released products from macrophages can show cytostatic and cytotoxic activities for tumour cells.^83,84 Tumour cytostatic effects are mediated by various cytokines (INFs, TNF-a, IL-6, IL-1(a/b), reactive intermediates of oxygen or nitrogen, enzymes (e.g. arginase) metabolising essential amino acids, prostanoid metabolites (e.g. PGE2) and nucleotides (e.g. thymidine). Cytotoxic effects are mediated either by soluble factors (see above) or require close contact between the macrophage and the targeted tumour cells. The involvement of a particular effector pathway in macrophage anti-tumour activity depends upon the activation signals and susceptibility of tumour target cells selected for the experiments. In this review only major mechanisms of macrophage anti-tumour activity will be considered.

Direct Cellular Cytotoxicity

One of the first direct indications that killing of tumour cells by macrophages involves direct contact between them and tumour cells was provided by Bucana et al.⁸⁵ The latter group produced morphological evidence showing translocation of lysosomal organelles from cytotoxic macrophages into the cytoplasm of target cells. Electron microscopic observations have shown that destruction of tumour cells by activated macrophages is a non-phagocytic, lytic process.85 The inter-action between the activated macrophages and the neoplastic cells involved intimate cellular binding, was temperature dependent and required viable, metabolically active effector cells.⁸⁶

The binding of tumour cells to activated macrophages has been reported to be selective. For example, BCG-activated macrophages bound in vitro to tumour cells, whereas other types of macrophages did not bind tumour cells.⁸⁷ Cytolysis of the tumour cells by bound macrophages was reduced by decreasing the efficacy of the binding (e.g. with chelators or by trypsinisation of the macrophages) and was increased by agents, such as concanavalin A or neuraminidase with galactose oxidase, which increased binding affinity.⁸⁷ These studies indicate that intimate binding of macrophages to tumour cells is an essential step in the process of cytolysis. In further investigations of the mechanism(s) of binding, membrane preparations of murine, non-adherent neoplastic cells were found to contain structures that inhibited the binding of homologous and heterologous neoplastic target cells to activated macrophages.⁸⁸ These findings suggest that certain tumour cells may escape cell damage through mechanism(s) that inhibit/prevent effective binding to macrophages.

Other workers have shown that binding between BCG-activated macrophages and target tumour cells can be weak or strong.⁸⁹ Initial weak binding, however, may develop into strong contact when the activated macrophages bound the neoplastic cells. Thus, the process of cell-cell contact and interaction inducing cytolysis is a complex and dynamic process.⁸⁹ Although it is generally believed that macrophages need to be activated in order to bind and destroy tumour cells, data from other studies have shown that unstimulated peripheral blood monocytes may also express cytolytic activity against melanoma cells.⁹⁰ Also, human monocytes and alveolar macrophages can be cytotoxic for tumour cells without prior activation.⁹¹ However, it is still possible that the monocytes and tissue macrophages had been primed in vivo prior to their isolation and assay in vitro.

A special type of macrophage anti-tumour activity is that mediated through antibody-dependent cellular cytotoxicity. In this cytotoxic process antibody-coated tumour cells bind to macrophages via the Fc receptor on the macrophage membrane. TAMs are potent effectors of this reactivity, as expected on the basis of their CD16 phenotypic expression.^7,92

Cytotoxicity of Secretory Products

Macrophages secrete a great many products: enzymes, complement components, reactive oxygen intermediates, arachidonic acid metabolites, coagulation factors, cytokines, nitrite and various other substances. Among these secretory products, arachidonic acid intermediates (PGs and LTs), cytokines (in particular IL-1 and TNF-a) and nitrites are believed to be intimately involved in anti-tumour activity.

Eicosanoids

Although it is generally recognised that increased production of PGs by macrophages is inversely correlated with their anti-tumour activity, it is well documented that in certain instances PGs may actually inhibit tumour cell metabolism and growth. Thus, it has been reported that PGE2 can inhibit tumour growth in vivo and in vitro.⁹³ Also, PGE2 enhanced IL-1 inhibition of WEHI-3B murine myelomonocytic leukaemia cell growth⁷³, and macrophage secreted IL-1 and TNF rendered WEHI-3B cells susceptible to cytostasis by PGs.⁹⁴

Eicosanoids also regulate the release of cytokines from macrophages. For example, PGE2 usually inhibits the production of TNF-a and IL-1, but enhancement of TNF-a production by low doses of PGE2 has also been reported. ⁹⁵ PGE2 was found to act differentially on the regulation of TNF-a and IL-1 gene expression.^96,97 With human peritoneal macrophages, PGE2 inhibited the release of TNF-a but not IL-1b.⁸⁰ In contrast to PGE2, LTs induce increased release of TNF-a from macrophages.⁹⁸ Thus, human monocytes exposed to graded concentrations of LTB4 released large amounts of TNF-a. Hence, in general PGE2 inhibits and LTs enhance macrophage cytotoxicity, which is at least partly mediated by the modulation of TNF-a release.

Cytokines

Cytokines are soluble glycoproteins secreted by different types of normal cells such as monocytes/macrophages, lymphocytes, endothelial cells and fibroblasts. Cytokines are also produced and released by different types of malignant cells.⁹⁹ Cytokines are important regulators of inflammation, tissue repair and host defenses. Normally, there is a low, steady level of cytokine production but enhanced amounts of cytokines are produced after cell activation as a result of tissue damage, sepsis, malignancy, etc. Monocytes/macrophages can be activated by LPS or IFN-g. Cytokines produced by activated cells can stimulate or inhibit the production of other cytokines by the same cells in an autocrine manner or those present in the vicinity by a paracrine mechanism. Thus, a complex network of cell interactions is established, with positive and negative feedback controls, regulating cytokine production. For example, TNF-a induces the synthesis of both IL-1 and IL-6 in macrophages.¹⁰⁰ In contrast, IL-10, which is also synthesised by macrophages, inhibits the synthesis of TNF-a, IL-1 and IL-6.¹⁰¹ TNF-a can induce its own synthesis in macrophages.¹⁰² Both IFN-g and TNF-a can activate macrophages¹⁰³, which subsequently synthesise TNF-a and IL-1.¹⁰⁴

Macrophage-derived cytokines have been shown to be cyto-toxic against a range of tumour cells.^105,106 IL-1 has been demonstrated to be cytotoxic and cytostatic for tumour cells.¹⁰⁷ Also, it has been demonstrated that IL-1 can act synergistically or additively with TNF-a in killing tumour cells. TNF-a is capable of causing haemorrhagic necrosis of certain transplantable tumors in vivo¹⁰⁸, and capable of direct tumour cytotoxicity in vitro. TNF-a is produced primarily by monocytes and macrophages. This has led to the postulate that TNF-a is involved in the delivery of the lethal hit of some tumours.^109,110 A crucial role for TNF-a in macrophage-mediated anti-tumour cell cytotoxicity has been demonstrated in two experimental studies. The first series of investigations demonstrated that UV-induced tumour clones, selected for resistance to macrophages, were resistant to TNF-a.¹¹¹ The second series of experiments showed that anti-TNF-a antibodies inhibited macrophage cytotoxicity. More definitive experiments should now be possible with the advent of knockout mice in which the genes for TNF-a and its receptors are deleted.¹¹²

Reactive Intermediates of Oxygen

Upon membrane perturbation or phagocytosis, mononuclear phagocytes produce reactive intermediates of oxygen that can play an important role in the destruction and removal of microbial organisms and tumour cells.^113,114 Reactive inter-mediates of oxygen are produced by the respiratory burst of monocytes and macrophages in response to ligand-receptor interactions and are involved in both antibody-dependent¹¹⁵ and independent^114,116 macrophage cytotoxicity. Reactive intermediates of oxygen produced by phagocytes can also be mutagenic and TAMs from murine mammary carcinomas have been reported to show mutagenic activity inducing DNA strand breaks in tumour cells.¹²

Reactive Intermediates of Nitrogen

Nitric oxide (NO) production occurs as the result of oxidation of one of the guanidino nitrogens of L-arginine through the action of nitric oxide synthase (NOS).¹¹⁷ Two basic isoforms of NOS have been described, to date, based on their constitutive (cNOS) or inducible (iNOS) expression. The inducible form of NOS can be found in several cell types including macrophages, hepatocytes, fibroblasts, endothelial and vascular smooth muscle cells, following stimulation with a variety of agents including microbes, microbial products and inflammatory cytokines. Expression of iNOS is often increased synergistically by the combined action of several of these agents.

It has been shown that activated murine macrophages induced cytostasis in several tumour cell lines only when the culture media contained L-arginine.¹¹⁸ This cytostatic response was found to be induced with the metabolism of L-arginine to NO₂ -and L-citrulline since the inhibition of this pathway by the L-arginine analogue, Ng-monomethyl-L-arginine (N-MMA), suppressed the anti-tumour effect. Further work has demonstrated that NO was the precursor for NO₂- and NO₃- produced by macrophages from L-arginine; NO could duplicate the cytostatic injury.¹¹⁹ Exposure of tumour cells to NO mimicked the L-arginine dependent anti-tumour effects of activated macrophages, including loss of a significant fraction of intra-cellular iron as well as inhibition of mitochondrial respiration and DNA synthesis^119,120 The provision of excess iron to these cells restored enzyme activity and prevented tumour cell death. Since none of the enzymes in the glycolytic pathway contain iron-sulphur complexes, glycolysis remains functional as an ATP-generating system in the presence of NO.¹²¹ The observed suppression of DNA synthesis could be explained by the NO-dependent inhibition of ribonucleotide reductase, a rate-limiting enzyme in DNA synthesis.¹²² These and other results have led to the conclusion that NO is a mediator of the cytotoxic activity of activated macrophages and to the proposal that NO kills or limits growth in tumour cells by inducing metabolic failure and inhibiting DNA replication through the perturbation of key enzymes via iron sequestration.

NO can also behave as a weak free radical. It is its reactivity with molecular oxygen, transition metals and superoxides that results in the formation of compounds with potentially profound cytotoxic effects.¹²³ These derivatives of NO include highly reactive intermediates such as its oxidation product nitrosonium ion (NO⁺), its reduction product nitroxynitrite (ONOO^-), and the secondary products generated from these precursors.^124,125 In reacting with dioxygen, NO forms nitrosamines and the nitrosation of thiols results in the generation of S-nitrosothiols. The nitrosation of amines and the deamination of DNA bases could contribute to the toxicity of NO.^126,127 The inhibition of iron/sulphur cluster containing enzymes by NO has already been mentioned. Interestingly, NO can also lead to metal ion loss from zinc containing proteins and, through the formation of disulfide bonds, NO can destroy Cys-type zinc fingers, potentially altering transcription factor binding to DNA.¹²⁷

Although an important role for macrophage-derived NO as an anti-neoplastic effector molecule has been established, it is also true that the generation of NO exerts significant effects on macrophages producing the NO molecules. Phagocytosis and the production of reactive oxygen intermediates are profoundly suppressed in rodent peritoneal macrophages cultured in conditions inducing NO production.¹²⁸ Moreover, NO producing macrophages exhibit reduced electron transport chain activity, enhanced reliance upon glycolysis for energy generation, a reduced ATP content and decreased protein synthesis. These metabolic alterations are similar to the known effects of NO in tumour cells. Also, macrophages activated with IFN-g and LPS rapidly lose their L-arginine dependent cytotoxic capacity, when cultured in media containing L-arginine, and this loss of tumouricidal activity correlates with macrophage death through enhanced apoptosis.¹²⁹

Macrophages and Apoptosis

More than twenty-five years ago, two transmission electron microscopy studies described the ultrastructural features of cells during macrophage-induced cell death.^130,131 The target cells showed chromatin condensation, swelling of the endoplasmic reticulum and the formation of extensive cell surface blebs, all features characteristic of an apoptotic cell death.¹³²

A further electron microscopy study, using SL2 lymphoma cells¹³³ incubated with peritoneal macrophages, also showed tumour cells with features typical of apoptosis. Chromatin condensation at the nuclear envelope and formation of surface protrusions, likely to be apoptotic bodies, were evident. Scanning electron micrographs revealed the dramatic membrane surface convolutions characteristic of apoptosis.

When U937 histiocytic lymphoma cells are subjected to the cytocidal activity of IFN-g-stimulated monocytes, they die with characteristic morphological features of apoptosis.¹³⁴ Furthermore, it has been reported that P815 mastocytoma cells and L929 fibroblasts are both induced to undergo apoptosis by activated macrophages. In this instance, apoptosis was demonstrated through the presence of a characteristic nucleosomal ladder in the genomic DNA.¹³⁵ Combined with earlier morphological studies, these experiments suggest that an important component of macrophage-mediated cytotoxicity involves the induction of target cell apoptosis.

The variety of mediators produced by macrophages suggest that there are multiple mechanisms which may be responsible for macrophage-mediated cell death in vivo. IL-1 has been shown to be cytotoxic to a human melanoma cell line.¹³⁶

The IL-1b isoform was demonstrated to induce apoptosis in murine thymoma cells.¹³⁷ Interestingly, the protease that processes IL-1b to its mature form, IL-1b converting enzyme (ICE), is an important intracellular regulator of apoptosis. The precise physiological role, however, of this and other proteases in apoptosis and IL-1b processing is still unclear.

TNF-a plays a crucial role in macrophage-mediated cytotoxicity.¹⁷ TNF-a mediated killing is believed to be via induction of apoptosis and has been described in many cell types in vitro.^138,139 Many of the biological effects of soluble TNF-a, including programmed cell death, can be mediated through the p55 TNF receptor I (TNFRI).^140,141 However, the membrane bound form of TNF-a was also found to activate TNFR II¹⁴² and lead to apoptosis, even in cells resistant to the cyto-toxic effects of soluble TNF-a. Thus, membrane-bound TNF-a may be crucial for induction of apoptosis. This is consistent with the observation that cell to cell contact appears essential in macrophage-mediated apoptosis.^143,144

As mentioned above, reactive intermediates of oxygen can play a role in the tumouricidal effects of macrophages. It has been observed that freshly isolated monocytes were able to induce reactive oxygen intermediate-dependent apoptosis in autologous natural killer cells.¹⁴⁵ The in vivo relevance of this is still unclear. NO is also an important molecule in macrophage-mediated host defences.¹⁴⁶ Cytokine-induced NO, produced by activated macrophages, can inhibit and kill different pathogens¹⁴⁷ as well as tumour cells.¹⁴⁸ NO-dependent cell lysis is known to result in the induction of apoptosis.¹³⁵ Evidence supporting the capacity of NO to induce apoptosis has been reported in many cell types.^{123,135,149,150} In view of the complex assay of molecular targets for NO in living cells, it is not surprising that the precise mechanism(s) for NO-dependent apoptosis, as yet, is unknown. NO-dependent apoptosis may be a consequence of NO-induced DNA damage^123,151 or NO-dependent oxidative injury.¹²¹ However, recent experimental data do not support the role for the latter as a mechanism for tumour cell apoptosis, at least in certain cell types.¹²³ It is important to emphasise that all the above observations about NO have been documented in rodent macrophages acting as effector cells. The ability of human macrophages to elicit NO-dependent tumour cell apoptosis requires elucidation.

Tumour Cell Inhibition of Macrophages

In spite of a substantial number of TAMs in a tumour mass and the propensity of tumours to activate macrophages, tumour cells escape macrophage anti-tumour activities by subverting macrophage functions to minimise anti-tumour effects and favour tumour progression. Tumour cells release in situ inhibitory cytokines that disrupt putative host anti-cancer defences, resulting in altered macrophage function and immune suppression.^15,152,153 The location, phenotype, developmental stage and activation state of the macrophage determines the variable effects of these tumour-derived molecules.

Tumours produce substances that both down-regulate^20,154 and up-regulate¹⁵⁵ macrophage cytotoxic and effector mechanisms. These macrophage-derived cytotoxic factors include IL-1⁸³, TNF-a^83,110, reactive oxygen intermediates^114,156 and NO.^157,158 Tumours can stimulate macrophages to produce cytotoxic molecules through tumour soluble or membrane-bound tumour-associated antigens (TAAs)^155,159 extracellular matrix proteins¹⁶⁰, or receptor-mediated binding of Fc portions of antibodies^161,162 attached to tumour cells.¹⁶³ Although macrophage production of TNF-a, reactive oxygen intermediates and NO imparts cytotoxic and suppressor activities, tumour growth also increases macrophage production of noncytotoxic inhibitory molecules such as PGE2, TGF-b and IL-10. The in vitro cytotoxic and suppressor functions of TNF-a, reactive oxygen intermediates and NO may be irrelevant or circumvented in vivo in man and animals with a prominent tumour burden. In many established tumours the production of TNF-a, reactive oxygen intermediates and NO does not necessarily lead to tumouricidal activity, but may instead lead to suppression of anti-tumour lymphocytes.¹⁵

Although in vitro investigations have established that macrophages can kill tumour cells, whilst leaving normal cells unharmed^164,165, macrophage-derived cytotoxic molecules may not be effective during in vivo tumour growth.^165,166 These findings suggest that tumours counteract the activity of and/or inhibit the production of cytotoxic molecules, leading to ineffectual macrophage function.^14,154 Many tumour-derived molecules, such as IL-4, IL-6, IL-10, macrophage-deactivating factor, TGF-b, PGE2, M-CSF and p15E, deactivate or suppress macrophage cytotoxic activity. Although TAMs normally are primed for enhanced cytotoxicity, various tumour factors produced suppress activated macrophage cytotoxicity.^20,167 Also, tumour-derived cytokines and chemotactic molecules may fail to stimulate macrophage TNF-a and NO production.^168,169 Furthermore, many tumours appear to posse mechanisms to resist damage from various macrophage-derived cytotoxic molecules.^161,163,165 The concurrent action of several macrophage-derived cytotoxic molecules, therefore, is required for lysis of tumour cells, suggesting that the inhibition of one type of cytotoxic molecule may be sufficient for the tumour to escape damage and survive.¹⁶³

In addition to direct inhibition of macrophage anti-tumour activity, tumours can modulate macrophage production of immunostimulatory molecules; for example, IL-18, a macrophage-derived cytokine that induces IFN-g production and promotes lymphocyte-mediated immune responses.¹⁷⁰ Another important humoral mediator is IL-12, which promotes T cell and LAK cell proliferation and cytotoxicity¹⁷¹ and induces the generation of TH1 cells.¹⁷² Although IL-12 does not directly affect tumour growth, IL-12 reduces the metastatic potential of many tumour types by promoting tumour entry of anti-tumour effector cells.^173,174 Direct in situ administration of IL-12 increases the number of infiltrating tumouricidal macrophages and T cells.¹⁷⁴ Exogenous IL-12 restores TAM immunocompetence; therefore, tumourinduced macrophage dysfunction may be manifested in reduced production of IL-12.

Dysregulation of IL-12 production has been shown to occur in tumour-associated immune cells.¹⁷⁵ For example, macrophages produce significant amounts of IL-10 and TGF-b^176,177 which may directly or indirectly block IL-12 synthesis. Also, NO induces macrophage IL-12 gene expression.¹⁷⁸ However, tumour-derived factors, such as TGF-b, reduce macrophage NO production by inhibiting iNOS activity¹⁷⁹ and NO production by TAMs is compromised.^59,180 In the absence of autocrine stimulatory signals, which are blocked by tumour-derived factors, macrophages may be incapable of producing IL-12. As a result, TH1 cells may fail to undergo the necessary activation and production of the key immunoregulatory cytokine IL-2. Furthermore, neutralisation studies suggest that the inhibition of macrophage IL-10, TGF-b, and NO production significantly reverse macrophage suppressor activity against T cells.20 This partial inhibition of macrophage suppressor activity may permit increased expression of IL-12, which would select for the growth of the key immunoregulatory TH1 cells.

Tumour growth may also partly decrease immunocompetence through macrophage hyporesponsiveness to proliferation signals, including GM-CSF. During malignancy and progressive tumour growth the role of GM-CSF is not well defined.¹⁸¹ GM-CSF normally enhances macrophage activation⁸³ and accessory function¹⁸² and increases MHC class II molecule expression.¹⁸³ However, these activities are inhibited by tumour growth.^163,184 In tumour bearing hosts, macrophages produce lower levels of, and are hyporesponsive to, GM-CSF.¹⁷⁷ Tumour-induced decreases in GM-CSF production may partly account for decreased macrophage accessory functions and reduced class II molecule expression. Tumour cells are believed to induce IL-10 release from TAMs, resulting in inhibition of GM-CSF synthesis.¹⁷⁷ IL-10, even in low concentrations, significantly decreases the production of other cytokines such as IL-1a, IL-6, IFN-g, and TNF-a.^185,186 Although IL-10 significantly inhibits the synthesis of macrophage-derived cytokines and reactive intermediates of oxygen^185,186 its importance as an inhibitor of macrophage proliferation and anti-tumour function is unclear and in need for further delineation.

TAMs and Immune Cells

There is substantial evidence that TAMs can suppress T cell^{20,60,180,187} and NK cell-mediated reactions^22,188, as well as their own functions. This may be mediated, in part, by the production of PGs and immunosuppressive cytokines such as IL-10.^187,189 During tumour growth, macrophages can adversely affect host anti-tumour responses and mediate lymphocyte suppression.^188,190 Macrophages can up- or down-regulate lymphocyte and other immune cell responses.¹⁹¹ For example, anti-tumour T lymphocytes can be induced by TAA presentation and interaction.¹⁶ However, in situ TAMs suppress many T cell and NK cell responses. Altered antigen presentation by TAMs may further subvert putative anti-tumour host responses.¹⁹²

Macrophages suppress T lymphocyte and NK cell functions by secreting various inhibitory molecules such as lipocortin¹⁹³, PGE2 ^20,194,NO^20,195,196,H₂O₂ ¹⁹⁷, TGF-b ^60,198, and IL-10.^62,199 However, only the inhibition of lipocortin, PGE2, reactive intermediates of oxygen, and NO production blocks macrophage-mediated suppression in the tumour bearing host. The increased macrophage output of autoinhibitory TGF-b1 and IL-10 during tumour growth controls the production of other macrophage suppressor molecules such as PGE2, NO, TNF-a^152,195; enhancing molecules such as GM-CSF¹⁷⁷ are triggered through autocrine induction. For example, normal host macrophages inhibit T cell proliferation, and antibody-mediated neutralisation of TGF-b1 and IL-10 unblocks normal host macrophage-mediated suppression of T cell proliferation.^152,195 In contrast, tumour-bearing host macrophages maintain active inhibitory function, even after TGF-b1 and IL-10 neutralisation, because their output of other macrophage-derived suppressor molecules is increased.^152,195

Tumours may inhibit macrophage cytotoxicity by either directly suppressing it or by inhibiting TH1 cells that induce macrophage anti-tumour effector mechanisms.^200,201 TH1 cells promote macrophage activation by producing IL-2 and IFN-g, whereas TH2 cells suppress macrophage accessory and cytotoxic activities by producing IL-4 and IL-10.²⁰² IL-10 inhibits macrophage activity by decreasing macrophage production of NO, TNF-a¹⁵² and PGE2 production²⁰³ and may also reduce secretion of IFN-a and IL-12, which is unfavourable for the generation of TH1 cells.^172,204,205 This suggests that macrophage-derived IL-10 may play a significant role in controlling T cell and macrophage activities during tumour-induced immunosuppression.

CONCLUSION

This review has emphasised the complexity of the interactions between macrophages, neoplastic cells and other immune cells in the tumour bearing host. Macrophages can affect tumour growth in several ways. They can enhance tumour growth, progression and invasiveness through release of growth factors (EGF, TGF (and cytokines), various mediators (cytokines, prostanoids and proteolytic enzymes) and/or through suppression of immune cells (T and NK cells) involved in anti-tumour responses. They can also damage the tumour cells directly or indirectly through release of eicosanoids, cytokines and free radicals, alone or in collaboration with other cellular and humoral components of the immune system. On the other hand, tumours also produce regulatory molecules that can modulate and subvert macrophage activities to minimise anti-tumour effects and favour tumour growth and progression.

The outcome of these competing interactions and subsequent specific macrophage functions during tumour growth is still poorly understood. Further studies, therefore, are required to define more precisely these complex interactions between the host and their growing solid tumours. A better understanding of the regulation and function of TAMs may help to establish more therapeutically efficacious novel therapies for cancer management.

ACKNOWLEDGEMENT

We wish to acknowledge the financial support of the Jordanian Royal Private Purse.

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Copyright date: 29th December 1999

Correspondence: Bilal Al-Sarireh. Section of Surgery, Floor E, West Block, Queens Medical Centre, University of Nottingham, Nottingham NG7 2UH, UK.
Email.Bilal.al-sarireh@nottingham.ac.uk

©2000 The Royal College of Surgeons of Edinburgh, J.R.Coll.Surg.Edinb.,45; 1: 1-16