Marginating pulmonary-NK activity and resistance to experimental tumor metastasis: suppression by surgery and the prophylactic use of a β-adrenergic antagonist and a prostaglandin synthesis inhibitor☆
Introduction
A large proportion of cancer-related deaths are caused by metastatic recurrence, despite successful excision of the primary tumor which is imperative for eliminating the major source of mutating and metastasizing tumor cells (Shakhar and Ben-Eliyahu, 2003). Unfortunately, the surgical procedure and the removal of the primary tumor have long been suspected to facilitate the growth of preexisting micrometastases and the dissemination of tumor cells during the perioperative period (Ben-Eliyahu, 2003). Potential mechanisms for such adverse effects include a drop in the levels of anti-angiogenic factors (Zetter, 1998), shedding of tumor cells due to the physical manipulation of the malignant tissue or its vasculature (Eschwege et al., 1995, Yamaguchi et al., 2000), local and systemic release of growth factors (Hofer et al., 1999), and suppression of cell mediated immunity (CMI) (Sietses, 1999). It has been suggested that synergy between these risk factors during the immediate postoperative period may temporarily render the patient susceptible to metastasis that would have been controlled otherwise (Shakhar and Ben-Eliyahu, 2003).
Suppression of CMI is a well-established postoperative clinical complication in major operations (Weighardt et al., 2000). Animal studies have provided direct evidence that such suppression, alone or in conjunction with other risk factors, can promote the survival and growth of minimal residual disease (MRD) after the primary tumor has been removed (Allendorf et al., 1999, Ben-Eliyahu et al., 1999, Colacchio et al., 1994, Da Costa et al., 1998, Shiromizu et al., 2000). MRD includes single tumor cells (which are found following surgery in the circulation, lymphatic system, or bone marrow of most cancer patients), as well as preexisting micrometastases. Nevertheless, many of the animal studies have been justifiably criticized for using immunogenic tumor lines (Hewitt, 1983, Killion et al., 1998), and clinical studies have so far provided only correlative evidence supporting the notion that postoperative immunosuppression can promote cancer recurrence (Shakhar and Ben-Eliyahu, 2003). Consequently, the clinical practice does not endorse prophylactic measures against postoperative immunosuppression in cancer patients.
Animal studies have indicated that immune control over circulating tumor cells and micrometastases is carried mainly through CMI, including cytotoxic T lymphocytes (CTL), NK, NK-T cells, dendritic cells, and macrophages (Smyth et al., 2001). Specifically, molecular mechanisms of NK recognition of tumor cells have been recently revealed, and the role of NK cells in controlling leukemia and metastasis in humans has received substantial support (Brittenden et al., 1996, Cerwenka and Lanier, 2001, Moretta et al., 2000). It is now believed that although the immune system has clearly failed to control the primary tumor in cancer patients, CMI can still eliminate MRD after the primary tumor has been removed, especially if surgery is conducted early, before elaborated tumor escape mechanisms have evolved (Shakhar and Ben-Eliyahu, 2003).
Several aspects of surgery have been proposed to underlie postoperative suppression of CMI, among them are anesthetic agents (Galley et al., 2000), hypothermia (Beilin et al., 1998; Ben-Eliyahu et al., 1999), tissue damage (Lennard et al., 1985), blood loss and transfusion (Klein, 1999), nociception and pain (Koltun et al., 1996), and perioperative distress (Cohen and Herbert, 1996, Larson et al., 2000). Several humoral factors could be suggested to mediate the immunosuppressive effects of these aspects of surgery (Ben-Eliyahu, 2003), central to the current study are prostaglandins (PGs) and catecholamines. Most of the above aspects of surgery cause activation of the sympathetic nervous system during the perioperative period (Koltun et al., 1996), and PGs are abundant following intrusive procedures due to tissue damage (Baxevanis et al., 1994). Both substances were reported to suppress various aspect of CMI in vitro and in vivo (Chambrier et al., 1996, Elenkov et al., 2000, Faist et al., 1990, Faist et al., 1996).
In our previous studies in F344 rats we have shown that following various stressful conditions, the release of adrenal catecholamines and activation of β1- and β2-adrenoceptors can suppress NK activity and increase susceptibility to experimental metastasis of the MADB106 tumor line (Ben-Eliyahu et al., 1999, Ben-Eliyahu et al., 2000, Stefanski, 1994). This mammary adenocarcinoma is a non-immunogenic syngeneic tumor line that establishes metastases only in the lungs following its intravenous inoculation (Barlozzari et al., 1983, Barlozzari et al., 1985, Ben-Eliyahu and Page, 1992). In addition to using stressful conditions, we were able to induce suppression of NK activity by administering a non-selective β-adrenergic agonist, or by administering PGE2 (Barlozzari et al., 1983, Barlozzari et al., 1985, Ben-Eliyahu and Page, 1992, Ben-Eliyahu et al., 1996b, Shakhar and Ben-Eliyahu, 1998, Yakar et al., 2003). However, NK activity was always studied in vitro against standard xenogeneic YAC-1 target cells, because the syngeneic MADB106 line was found to be resistant to the activity of NK cells from various cell populations. These populations included splenocytes, circulating leukocytes, PBMCs (Barlozzari et al., 1983, Barlozzari et al., 1985, Ben-Eliyahu and Page, 1992, Ben-Eliyahu et al., 1991), and leukocytes harvested from the bone marrow or from the lung interstitial and alveoli compartments (following dissection and collagenase digestion—unpublished data from our laboratory). This in vitro resistance of the MADB106 to NK activity, however, is in marked contrast to its in vivo susceptibility to NK cells that we and others have shown using various in vivo approaches, including selective depletion and selective replacement of NK cells (Barlozzari et al., 1983, Barlozzari et al., 1985, Ben-Eliyahu and Page, 1992, Ben-Eliyahu et al., 1996b, Shakhar and Ben-Eliyahu, 1998).
Thus, the current study in rats had two major aims: (a) to assess the use of a β-blocker and of a PG-synthesis inhibitor in the postoperative context as prophylactic measures against the immunosuppressive and metastasis-promoting effects of surgery, and to study whether catecholamines and PG act independently of each other; and (b) to study the potential role of a yet unrecognized population of NK cells, those adhering to the lungs vasculature—marginating-pulmonary (MP)-NK cells—in destroying MADB106 cells, and in mediating the effects of surgery and of the studied prophylactic measures.
Section snippets
Animals
Fisher 344 male and female rats 13–16 weeks old (age matched within each experiment) were purchased from Harlan laboratories, Jerusalem, Israel. Animals were housed 4 in a cage with free access to food and water on a 12:12 light:dark cycle. All studies were approved by The Institutional Animal Care and Use Committee of Tel Aviv University. Female were used only in Experiment 6, in which females and male were equally included in each experimental group.
Timing and counterbalancing of experimental procedures
Rats were acclimatized to the vivarium for
Experiment 1: lung tumor retention (LTR) of MADB106 tumor cells—the effect of surgery and its blockade by indomethacin and nadolol
An early index of host resistance to MADB106 lung colonization is the retention of these tumor cells in the lungs 24 h following their inoculation. These 24 h also delineate the period in which the experimental metastatic process of the MADB106 is sensitive to NK activity (Barlozzari et al., 1983, Barlozzari et al., 1985, Ben-Eliyahu and Page, 1992). Thus, relative to longer indices (e.g., actual metastases), the 24 h LTR index more exclusively reflects the in vivo levels of NK activity.
Discussion
Our findings demonstrate that a β-adrenergic blocker (nadolol), and a prostaglandins synthesis inhibitor (indomethacin), each attenuated the metastasis-promoting effects of surgery when used alone, and together almost completely abolished them. The combination of drugs was also effective in preventing the NK-suppressive effects of surgery on lung NK activity against the standard YAC-1 target line and against the syngeneic MADB106 line. Administration of physiologically relevant doses of either
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This research was supported by NIH/NCI grant CA73056 and by a grant from the Israel Science Foundation to Dr. S. Ben-Eliyahu.