The relationship of host immune cells , cytokine and nitric oxide production to tumor cells in ovarian carcinoma

Solid tumors consist of malignant cells and stroma. Malignant cells elicit stroma formation and this is essential for neoplasia growth. 1 Many tumors of epithelial origin contain, in the tumor stroma, a significant number of infiltrating host leukocytes, especially macrophages and lymphocytes. 2 Also, these cells obtained from neoplastic effusions are extremely useful in evaluating the interactions between immune and cancer cells in the tumor microenvironment. 3

natural killer (NK) cells and mast cells are present in low numbers.Neutrophils are largely confined to blood vessels and eosinophils are seen occasionally.In general, the infiltrating cell density is higher in the stroma than in the tumor compartment. 2In peritoneal or pleural effusions secondary to cancers at different sites, including ovarian carcinoma, cells consist mainly of T lymphocytes, but NK cells, B cells and macrophages are also present. 3It has also been reported that neutrophils are frequently found in the ascites of ovarian carcinoma. 5

Immunological efficiency of TAMs
It is not known whether TAMs are a cell population that participates in the immune response against the tumor, or whether these cells might even enhance the development of the tumor. 7In this context, it is known that the recruited leukocytes can both recognize and kill malignant cells and establish an immune memory against them or produce factors that help tumor growth and vascularization through paracrine loops. 8The concept of a macrophage balance was introduced by Mantovani et al. 9   to encapsulate the notion that macrophages may aid or inhibit tumor growth according to their state of activation.
In a study of 25 patients with gynecological tumors, of whom 15 had ovarian carcinoma, the autologous peripheral blood mononuclear cells (PBMCs) differed from TAM subset populations in having a significantly higher potential to proliferate in response to mitogen stimulus and also in presenting higher cytotoxicity. 7Agreeing with this data, TAMs obtained from peritoneal or pleural effusions secondary to cancer at different sites had a lower proliferative response to mitogens than autologous PBMCs, which in turn was lower than that of control PBMCs, indicating a functional immunological impairment in patients with advanced carcinoma disease. 3 Another indicator of the immune state is the pattern of cytokine release by TAMs.Most of the available data was obtained from advanced ovarian carcinoma.In a recent s t u d y, i t w a s f o u n d t h a t t h e c u l t u r e d supernatants of mitogen-stimulated TAMs produced less IL-1α and β, TNF-α, IL-4 and IL-10, compared to those of autologous and control PBMCs, as assessed by the ELISA test. 3In a similar study, IL-4 was the major cytokine expressed by TAMs, while IFN-γ production was predominant in PBMCs. 7The analysis of cytokine expression by reverse transcriptase polymerase chain reaction (RT-P C R ) s h o w e d t h a t TA M s h a d r e d u c e d expression of genes for IL-2, IFN-γ and IL-4, and inversely, had increased IL-10 gene expression relative to normal PBMCs. 4Taken together, these results suggest that the incomplete activation of TAMs in vivo may be due to the accumulation of Th2 cells instead of Th1 cells, and it is plausible that the increased IL-10 contributes to downwards regulation of the Th1 cytokines.But the role of TAMs studied to date varies considerably, even in ovarian carcinoma.In a study of advanced ovarian carcinoma, TAMs produced appreciable amounts of IL-6 and spontaneously released significantly higher amounts of IL-8, compared to PBMCs. 5 In patients with ovarian or breast cancer, cytotoxic T cell cultures, isolated from tumors and then further stimulated with autologous tumor cells, lysed these cells and also secreted cytokines such as TNF-α, IFN-γ and GM-CSF. 10In a prospective study of 17 ovarian carcinomas, it was observed that patients in relapse had a significant reduction in TAMs, which were unable to respond to the tumor as evidenced by the correlation between tumor growth and a decreased number of infiltrating cells.6 These differences in cellular composition and the variable prognostic significance of leukocytes that infiltrate many human tumors suggest that different types of interactions are possible between tumor and host cells, possibly resulting in heterogeneous responses. 8Since TAMs are located at the tumor-host interface and have the potential to exert anti-tumor activity, these cells may constitute an attractive target for therapeutic intervention.

The role of cytokine expression in the tumor
Although the role of infiltrating cells in malignant tumors is controversial, a likely stimulus for their presence is the local production of chemokines, so that the leukocyte content of a tumor may depend on the expressed cytokines.In this context, a variety of human and murine tumor cells produce monocyte chemoattractant factors and there is a correlation between the amount of activity in cultured supernatants and the number of TAMs, when these cells produce tumors in vivo. 11Several lines of evidence suggest that monocyte chemoattractant protein-1 (MCP-1) is an important determinant of macrophage infiltration into tumors (review 9 ).The presence of messenger RNA for MCP-1 in ovarian carcinoma was first demonstrated by an in situ hybridization technique, 11 and this study also demonstrated the expression of chemokines such as macrophage inflammatory protein-1 (MIP-1α), MIP-1β and RANTES activity (regulation upon activation: normal T cell expression and stimulation) by these tumors. 2Furthermore, a direct topographical association was observed between the number of chemokine-expressing cells and the leukocyte infiltrate at the epithelialstroma interface. 11 In general, the major components of TAMs have been described as cells resembling Th0 cells, i.e. producing both Th1-and Th2-type cytokines, or with a gradual shift from Th1 to Th2 cells occurring during progressive tumor growth. 3The secretion of Th1-like cytokines, as opposed to Th2, could potentially further enhance the endogenous immune response to ovarian cancer. 10Analysis of cytokine expression using RT-PCR techniques on total RNA isolated from ovarian carcinoma showed that the majority expressed TGF-β and IL-10, with absence of expression of IFN-γ.Half of these tumors expressed GM-CSF and IL-8, 6 which has also been described in vitro. 8In accordance with this, higher concentrations of IL-10 have been demonstrated in neoplastic effusions secondary to cancers at different sites, including ovarian carcinoma and, surprisingly, higher IFN-γ compared to autologous serum. 3It is known that IL-10 strongly inhibits the production of TNF-α and β , GM-CSF and IFNγ by peripheral blood monocytes. 6 The differences in the expression of cytokines described in the literature may be related to histological types of ovarian carcinoma analyzed in each study.For example, TNF-α and IL-2 are generally described as not being consistently detected in those tumors, in contrast to IL-10 and GM-CSF. 6The analysis of 13 cases of ovarian carcinomas showed that in only four cases were cells expressing messenger RNA for TNF-α and IL-2 observed, a number considerably lower than that observed in inflammatory conditions such as salpingitis or in normal peripheral lymphoid tissue. 12The gene for TNF was studied in biopsies of human epithelial ovarian cancer and a positive correlation was found between TNF expression and tumor grade, suggesting that TNF production may enhance tumor development. 13 Ovarian carcinoma cells produce cytokines that attract monocytes and promote their survival; TAMs in turn produce cytokines which can stimulate cancer cell growth.So there is an ambivalent relationship between tumor cells and TAMs.In the absence of effective therapeutic intervention, evidence suggests that the balance is shifted in favor of the tumor.

A role for NO in tumor biology
Nitric oxide (NO) is an essential physiological signaling molecule mediating various cell functions, including the cytotoxic/ cytostatic effects of the immune system against debilitating factors like infection and tumor (review 14 ).But, when produced for a long period and in high concentrations, an excess of NO could damage DNA leading to gene mutations and cancer. 15 Several human cancers are associated with chronic viral, bacterial and parasitic infections, with NO formation being elevated in these infections. 16Also, most of the cellular components of the tumor mass (tumor cells themselves and the immune cells infiltrate) have been shown to generate NO in vitro (review 17 ).
The expression of NO synthase (NOS) in tumors provokes the question about a physiological role for tumor-associated NO production.
Several reports suggest a tumoricidal role for NO in vivo.A chronic inhibition of NO synthesis with N-monomethyl-L-arginine (L-NMA) resulted in increased tumor growth and delayed immune recognition in mice, implicating endogenous NO in the impaired ability of tumor cells to proliferate. 18Moreover, the daily intraperitoneal administration of L-NMA prevented the tumoricidal activity in mice which were preinoculated with bacillus Calmette-Guérin (BCG) and subsequently transplanted with syngenic or xenogenic ovarian tumor cells.Since it has been demonstrated that NO mediates the BCGinduced host resistance to tumor grafts in mice, it is most likely that NO accounts for the tumoricidal activity. 19Also in mice, the administration of cytokine-stimulated tumor cells caused a two-fold increase in subcutaneous tumor growth and experimental pulmonary metastases, in relation to control cells.Nmonomethyl-L-arginine acetate reduced tumor size and the number of lung metastases to the control levels, suggesting that tumor cell NO production was responsible for this effect.

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In human tumors the role of NO has not been established.NO has been reported as being diminished or absent in premalignant lesions and tumors of the large intestine, 14 while, conversely, an increased level of NOS expression and/or activity was observed in human gynecological tumors, and this fact was inversely associated with the differentiation grade of the tumor. 21In ovarian cancer, high levels of NOS activity were detected, while the enzyme activity was below detectable levels in gynecological tissue from non-cancer patients.
In addition, the immunoreactive proteins in which NOS activity was detected were localized to the tumor cells.Since cytokines and hypoxia can synergistically induce NOS expression, the premalignant and malignant tumor tissue may establish sustained NO production in a variety of tumor cells.However, the effect of NO production in tumor biology may change during tumor progression. 21This hypothesis is supported by data investigating the role of NO in cancer metastasis.After in vitro incubation with cytokines or LPS, non-metastatic cells exhibited a high level of inducible NOS activity and NO production, whereas metastatic cells did not.The transfection of tumor cells produced fast-growing and highly metastatic tumors, whereas functional iNOS-transfected cells produced slow-growing and nonmetastatic tumors in syngenic or nude mice.These data indicate that NO decreased survival of tumor cells in the circulation and inhibited tissue invasion.

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A hypothesis has been put for ward suggesting that loss of NO from a biological cell could enable it to evade cell-cycle arrest and terminal differentiation, resulting in a premalignant cell or predisposed cell.A loss of NO from a malignant cell could result in uncontrolled cellular division.Since persistent vasodilatation is a specific feature in tumor vasculature and in the surrounding tissue, NO generated by the vascular endothelium in the proximity of or within the tumor, under the control of a local growth factor, could regulate the tumor blood flow via vasorelaxation. 14,17In addition, the role of NO in angiogenesis is well documented.17 Cancer growth can be stimulated as well as inhibited by the immune system.The intratumor macrophage arginine metabolism is a molecular explanation for the dual ability of the immune system to inhibit or stimulate tumor growth.It has been proposed that arginine metabolism in the tumor bed yielding citrulline and NO favors tumor rejection, whereas production of ornithine and urea could promote tumor growth.

Interactions between cytokines and nitric oxide
One of the first recognized natural mechanisms for regulating NO synthesis was IL-4.When macrophages were activated with IFNγ and a low dose of LPS, they produced significant amounts of NO and expressed high levels of NOS.This production and expression were inhibited in a dose-dependent manner by preincubating the cells with IL-4.IL-10 and TGFb can also inhibit NO synthesis.In contrast, IFNγ and TNF-α, occupying their respective receptors, transmit a series of signals leading to the expression of NOS and the synthesis of NO. 25   In vitro, human MCP-1 was able to inhibit the production of NO by a macrophage cell line, suggesting that tumor-derived MCP-1 is likely to represent a mechanism for controlling NOmediated macrophage cytotoxicity, and for the recruitment and concomitant partial functional deactivation of TAMs.
26 Advanced neoplasia has long been associated with defective capacity to mount responses to inflammatory stimuli.Thus, a balance between chemotactic and inhibitory cytokines may regulate infiltrate in tissues, including neoplasms.In this context, it is known that in mice IL-8 and TNF-α cause defective neutrophil recruitment when administered in the sistemic circulation, [27][28][29] and the production of NO is involved in this inhibitory activity. 30This observation raises the possibility that cytokines, leaking from advanced tumors, play a role in systemic defects of inflammation and immunity associated with neoplasia. 1