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10.1245/s10434-006-9078-9
Annals of Surgical Oncology 14:841-852 (2007)
© 2007 Society of Surgical Oncology
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Original Article

Amelioration of Operation-Induced Suppression of Marginating Pulmonary NK Activity using Poly IC: A Potential Approach to Reduce Postoperative Metastasis

Guy Shakhar, PhD1, Naphtali Abudarham, MA2, Rivka Melamed, MSc2, Yossi Schwartz, MA2, Ella Rosenne, MSc2 and Shamgar Ben-Eliyahu, PhD2

1 Department of Immunology, Weizmann Institute of Science, Rehovot 76100, Israel
2 Neuroimmunology Research Unit, Department of Psychology, Tel Aviv University, Tel Aviv 69978, Israel

Correspondence: Address correspondence and reprint requests to: Shamgar Ben-Eliyahu, PhD; E-mail: shamgar{at}tau.ac.il


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background and objectives: Pulmonary metastasis is a major cause of death in cases of operable cancer, and evidence suggests that postoperative immunosuppression contributes to this complication. In this study, we aimed to circumvent this risk and identify immunocytes critical in preventing pulmonary metastases.

Methods: F344 rats were treated with either vehicle or repeated low doses of poly I-C (0.2 mg/kg i.p., days 5, 3, and 1 preoperatively), a Th1-cytokine-inducing agent, then subjected or not to laparotomy. Using a non-immunogenic syngeneic mammary adenocarcinoma line (MADB106) we studied: (a) NK cytotoxicity (NKC) in marginating-pulmonary (MP) and in circulating leukocytes; (b) resistance to experimental lung metastasis; and (c) in vitro susceptibility of NKC to corticosterone and prostaglandin-E2, substances thought to mediate postoperative immunosuppression.

Results: MP but not circulating leukocytes showed significant NKC against MADB106 cells. Surgery suppressed this MP-NKC per NK cell and promoted MADB106 metastasis, and poly I-C treatment completely abolished both effects. Poly I-C quadrupled the numbers of MP-NK cells without causing apparent side effects, and protected MP-NKC from in vitro suppression by corticosterone and prostaglandin-E2.

Conclusions: MP-NK cells are unique in their ability to kill this apparently immunoresistant tumor. Low doses of synthetic ds-RNA (poly I-C), and potentially Th1 cytokines, can expand this MP-NK population and protect it from immunosuppression. The novelty of such a prophylactic approach is targeting the immediate postoperative period, which is characterized by high vulnerability to residual disease, and protecting critical anti-metastatic immunity against postoperative suppression. Testing such a potentially innocuous intervention in oncology patients preparing for surgery may reduce metastatic recurrence.

Key Words: Rodents • NK cells • Lung • Tumor Immunology • Neuroimmunology


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
It has been well established clinically that major operations introduce a period of immunosuppression that lasts for several days.1,2 Although the surgical wound itself is a site of intense immunological activity, in the periphery immune functions are inhibited.1,3 This inhibition probably represents an adaptive regulatory response intended to restrict inflammation to the site of injury3. It is now believed that crucial for initiating and maintaining this peripheral immunosuppression is the neuroendocrine response to tissue damage, inflammation, and nociception.1,4 Postoperative immunosuppression compromises cellular immunity, but generally spares humoral immunity. Thus, patients exhibit lower counts of Th1, Tc, and NK cells, and manifest deficiencies in NK activity, T cell proliferation, type-I cytokine secretion, and delayed type hypersensitivity response1.

Although in cancer patients immunity clearly fails to eradicate the primary tumor, it is becoming clearer that cellular immunity can reduce minimal residual disease after surgery.5 This raises concerns that postoperative immunosuppression might put these patients at greater risk of metastatic recurrence1,6. While this premise is extremely difficult to test clinically, it has received ample support from studies in animals, although many researchers justifiably criticize these studies.1,6 Because removal of the primary tumor is usually indispensable, we believe efforts should be made to prevent surgery from suppressing immunity.

In search of the mechanisms involved in postoperative immunosuppression, we have employed a rat model of experimental metastasis. In this model, we test the postoperative resistance of F344 rats to experimental metastases of a syngeneic mammary adenocarcinoma—MADB1067. This cell line homes to the lungs following intravenous inoculation and is highly malignant and poorly- or non-immunogenic8. The clearance of MADB106 cells from the lungs critically depends on NK cells.7–10 In the lung, NK cells bind to MADB106 cells11, and selective in vivo depletion of NK cells greatly increases MADB106 lung retention and colonization.8–11

Using the MADB106 model, we established that abdominal surgery increases the retention and colonization of tumor cells in the lungs, probably by suppressing NK activity.12 Various aspects of the surgical procedure and a range of neuroendocrine responses seem to produce this suppression. Apparently, tissue damage, pain,13,14 anesthetics15, and hypothermia16 trigger the release of catecholamines, prostaglandins17, and corticosteroids18, which compromise host immune functions either directly or by shifting cytokine balance toward a Th2 dominance6,19. Given the complexity of this response, administering a single antagonist or modulating the surgical procedure would probably not suffice to prevent the metastasis-promoting effects of surgery. An alternative approach would be to switch cellular immunity into a mode of resistance to suppression. This might be achieved by administering an immunomodulator to fool the immune system into believing there is an ongoing systemic viral infection—under such conditions, it would seem adaptive to override mechanisms of immunosuppression. Indeed, in vitro studies of immunostimulation with Th1 cytokines have provided some support for this notion20.

To test this approach in vivo, we developed a treatment based on chronic administration of low doses of poly I-C (polyriboinosinic acid-polyribocytidylic acid). This synthetic compound resembles viral double-stranded RNA and triggers an antiviral immune response. In a pilot study, we employed a pharmacological stressor to suppress NK activity and to reduce resistance to MADB106 metastases9. We have gradually reduced the dosage of poly I-C, minimizing side effects while still protecting the host from the deleterious effects of the pharmacological stressor. The final poly I-C regimen was so mild it hardly affected the appetite of the rats nor caused fever. The present study had two aims: (a) to test the efficacy of this regimen in preventing operation-induced immune suppression of NK activity and promotion of MADB106 metastasis, and (b) to begin to elucidate underlying immunological mechanisms of the deleterious effects of surgery and the potential protective effects of poly I-C. Because, in our experience, circulating and splenic NK cells cannot lyse MADB106 cells in vitro, although the tumor is sensitive to NK activity in vivo,7–10 in the current study we focused on a population of NK cells that has not yet been characterized—marginating pulmonary (MP)-NK cells—which seems uniquely located to destroy MADB106 cells in vivo.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals, timings of procedures, and counterbalancing
Adult F344 male rats (Harlan, Jerusalem, Israel) were housed four per cage with free access to food and water. Animals were kept under a 12 h:12 h light:dark cycle at 22 ± 1°C for at least 4 weeks before an experiment. At the time experiments were conducted, animals were 14–16 weeks old. To reduce procedural stress, all rats were handled for 4 consecutive days in a procedure room adjacent to the vivarium. To ensure that the specified treatments were the only factors that differed among the experimental groups, the order of drugs and tumor injections, and the order of blood withdrawal were counterbalanced among the different experimental groups (i.e., one rat from each group was first treated, then a second rat from each group, etc.). In all experiments, laparotomy was conducted during the first half of the dark period. The Institutional Animal Care and Use Committee of Tel Aviv University approved the experimental procedures and housing conditions.

Materials
Poly I-C, prostaglandin E2 (PGE2), and corticosterone were purchased from Sigma (Rehovot, Israel). Poly I-C was dissolved in saline. PGE2 and corticosterone were dissolved in ethanol and diluted in PBS and further in complete media (RPMI-1640 media supplemented with 10% heat-inactivated FCS, 50 µg/ml gentamicin, 2 mM L-glutamine, 0.1 mM non-essential amino acids, and 1 mM sodium pyruvate—Sigma).

Experimental laparotomy
Anesthesia was induced with halothane (Rhone-Poulenc, Bristol, UK) in room air, and maintained at 2–3% halothane in room air via a vaporizer. Rats breathed spontaneously throughout the anesthesia, and halothane concentration was adjusted according to the animal’s respiratory pattern and response to incision.

After hair trimming and scrubbing with alcohol, a 4-cm midline abdominal incision was performed, and the small and large intestines were externalized and rubbed with PBS-soaked gauze in four places. The intestines were then covered with PBS-soaked gauze, and the abdomen was left open. Finally, the abdomen was closed in one layer with 3–0 nylon sutures. Total procedure time from induction of anesthesia to the last stitch was set to 1 h. Rats awoke 5 min later.

MADB106 tumor cells and their preparation for in vivo administration
MADB106 is a selected variant cell line obtained from a pulmonary metastasis of a mammary adenocarcinoma (MADB100) chemically induced in the inbred F344 rat.21 To radiolabel tumor cells (for studying lung tumor retention), we cultured MADB106 cells in 0.5 µCi/ml of 125iododeoxyyuridine for 1 day. We separated the cells from the culture flask with a 0.25% trypsin solution and washed them in PBS (supplemented with 0.1% BSA) before injection9.

Induction and counting of tumor metastases
Rats were lightly anesthetized with halothane, and 105 MADB106 cells in 0.5 ml PBS (supplemented with 0.1% BSA) were injected into their tail vein. The injection procedure, from initiation of anesthesia to awakening took approximately 2 min. Lungs were removed 3 weeks later and placed in Bouin’s solution (72% saturated picric acid solution, 23% formaldehyde [37% solution], and 5% glacial acetic acid) for 24 h. The lungs were washed in ethanol, and two researchers ignorant of their origin counted visible surface metastases9.

Assessment of MADB106 lung tumor retention
We anesthetized the rats in a similar fashion to that described above and intravenously injected each of them with 105 radiolabeled MADB106 cells. The lungs were removed 24 h later and placed in a {gamma}-counter for assessment of radioactive content. To estimate the percentage of intact tumor cells in the lungs, we examined the ratio between lung radioactivity and injected radioactive content10. Our previous studies have indicated that the levels of lung radioactivity reflect the numbers of viable tumor cells in the lungs. For more information, see the study by Ben-Eliyahu and Page (1992)10.

Harvesting of marginating pulmonary leukocytes
Rats were anaesthetized with an overdose of halothane, their thoracic cavities opened, and 6–8 ml of blood drawn from the right cardiac ventricle into a syringe containing heparin (200 U). To harvest leukocytes adhering to the pulmonary endothelium (MP leukocytes), we perfused the lungs by injecting heparinized PBS (30 U/ml) into the right ventricle (approximately 0.5 ml/s) and collecting 30 ml of perfusate from the left ventricle. To reduce contamination with circulating blood, we discarded the first 3 ml of perfusate. The perfusate was then concentrated to approximately 0.5 ml, washed in 4 ml complete media, and concentrated to a final volume of 1 ml.

Preparation of effector cells from the blood and spleen
Of the blood collected, a volume of 1 ml was washed once with PBS (adding 3 ml of PBS, centrifuging at 300 g for 10 min, and reducing supernatant levels to original volume) and twice with complete media. Spleens were pressed through a 1-mm stainless-steel screen, filtered through a 70-µm strainer (Becton Dickinson, Franklin Lakes, New Jersey), washed twice in complete media, and concentrated to 6.6 x 106 leukocytes/ml22.

In vitro assessment of cytotoxicity
A 4-h chromium release assay was used a to assess NK-mediated lysis of YAC-1 and of syngeneic MADB106 cells22. Effector cells were serially diluted and co-incubated, at different effector to target (E:T) ratios, with a fixed number of target cells (5000 per well). In the last experiment, we added PGE2 or corticosterone at increasing concentrations to wells containing effector cells and to wells assessing spontaneous chromium release. Our previous studies 23,24 indicate that cytotoxicity in this assay depends on NK cells, since their selective depletion nullified all specific killing.

Specifically, to assess NK cytotoxicity at different E:T ratios, an aliquot of 150 µl containing NK cells (washed blood, splenocytes suspension, or lung perfusate) was placed in the first row of a microtiter plate, and another aliquot of 150 µl was successively diluted twofold in complete media in the following rows. Five thousand 51Cr-radiolabeled MADB106 (or YAC-1) target cells in 100 µl complete media were added on top of the effector cell layer. Spontaneous and maximal releases of radioactivity from target cells were determined by substituting blood with the culture medium or with Triton-X 100 (Sigma Chemicals, St. Louis, MO, USA), respectively. In the last experiment, hormones were added to all E:T ratios, as well as to wells determining the spontaneous releases of radioactivity from target cells. Plates were centrifuged at 500 g for 10 min (in the case of blood to create a buffy coat layer of leukocytes and target cells on top of the red blood cells) prior to a 4-h incubation period. Following incubation, plates were again centrifuged, and 100-µl aliquots of the supernatant were recovered from each well for assessment of radioactivity in a {gamma}-counter. Specific killing was calculated as [(sample release x HCF-spontaneous release)/(maximal release-spontaneous release)] x 100. Hematocrit correction factor (HCF) compensates for changes in the hematocrit to supernatant volume over different E:T ratios. This correction factor is included when assessing whole blood NK activity in order to consider the changing volume of cell-free medium into which radioactivity is released.

Radiolabeling of MADB106 and YAC-1 target cells for cytotoxicity assays
10 x 106 cells were incubated for 1.5 h with 100 µCi 51Cr (in 100 µl saline), 100 µl FCS, and 50 µl complete media. Following incubation, cells were washed three times in complete media (300 g, for 10 min) and adjusted to the desired concentration in complete media.

Flow cytometry
Standard FACS analysis was used to identify CD161bright lymphocytes.12 Specifically, an aliquot of 50 µl containing NK cells (washed blood, splenocyte suspension, or lung perfusate) was combined with 50 µl PBS++ (PBS supplemented with 2% FCS and 0.1% NaN3) and 0.1 µg FITC-conjugated anti-CD161 (anti-NKR-P1) (PharMingen, San-Diego). Samples were kept in the dark at room temperature thereafter. Following a 15-min incubation period, 1 ml FACS lysing solution (Becton Dickinson) was added and, 10 min later, samples were centrifuged for 5 min at 670 g and the lysate was aspirated. Cells were washed again with 2 ml PBS++ (5 min centrifugation, 670 g) and resuspended in 300 µl PBS++ for flow cytometry analysis using a FACScan (Becton Dickinson). The criterion for positive identification of NK cells was defined as being above a level of fluorescence intensity that distinguishes between bright and dim stained populations of CD161 positive cells, as described previously by Chambers et al.25 These previous studies also demonstrated that CD161 is expressed by 94% in blood LGL cells of the rat, and that the NK cytolytic activity was totally contained in the CD161 bright cell population. Polymorphonuclear (PMN) leukocytes were found to express low levels of CD161 and categorized as dim cells, and macrophages and mast cells were found to be negative26. In our studies, bright cells are defined as showing above 150 relative fluorescence intensity units, a level that distinguishes between the two non-overlapping populations of the dim and bright CD161 positive cells. Nonspecific binding was assessed using nonspecific IgG1 that consistently yielded 0% of brightly stained cells.

To assess the total number of NK cells per microliter of effiector cells, we added to each sample 30,000 polystyrene microbeads (20 µm in diameter, Duke Scientific, Palo Alto, CA) (i.e., 600 microbeads/µl of sample). Following cytometry, the formula "#CD161bright/#microbeads x 600" was used to calculate the number of NK cells per microliter. The coefficient of variation for this method was found in our laboratory to be 6% for identical samples12.

Statistical analysis
One- or two-way ANOVA was used, with an additional repeated-measures factor (E:T ratios) for analysis of cytotoxicity studies. Provided that ANO-VA indicated significant group differences, planned contrasts (PLSDF) were conducted to compare specific groups. For the final experiments, in which the in vitro suppression of NK activity by PGE2 and corticosterone was studied, a pool of leukocytes from different rats was used and aliquots were exposed to different concentration of hormones. Thus, a within-subject, repeated-measures ANOVA was used, as this analysis is more conservative than a mixed model. P values less than 0.05 were considered significant in all studies.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The chronic regimen of poly I-C and its limited side effects
The regimen consists of low doses of poly I-C (0.2 mg/kg dissolved in saline) given intraperitoneally on days 5, 3, and 1 before surgery. In humans, the side effects of poly I-C treatment consist of fever, chills, and anorexia. In high doses, poly I-C can cause hypotension and severe myalgia. To study the side effects of different regimens of poly I-C in rats, we assessed core body temperature and body weight. While a standard dose of poly I-C (4 mg/kg) increased body temperature, disrupted circadian rhythm, and decreased body weight by approximately 5%, the chronic low dose had no impact on body temperature, and reduced weight only in some of the experiments conducted, and only by up to 1.3%.

Preventing operation-induced lung colonization by MADB106 cells
To assess whether our poly I-C regimen can prevent laparotomy from promoting MADB106 metastases, we used a 2 x 2 design in which rats were treated with either poly I-C or vehicle, and then either underwent operation (laparotomy) or were left undisturbed. At 5 h after laparotomy, we injected all rats with an intravenous bolus of MADB106 cells, and 3 weeks later counted lung metastases. A total of 57 rats were used in the study.

Surgery increased metastasis threefold, and poly IC treatment completely abolished this effect, although it did not reduce baseline levels of metastasis significantly (Fig. 1aGo). ANOVA indicates a significant interaction between the effects of surgery and poly IC (F(1,53) = 7.7, P < 0.05), and planned contrasts indicate that the poly I-C treatment significantly reduced number of metastases in operated rats (P < 0.05). Thus, this regimen fares better in preventing surgery from promoting metastasis than in improving baseline host resistance.


Figure 1
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  		FIG. 1. a,b. The effects of laparotomy and repeated pre-treatment with poly I-C on MADB106 tumor colonization of the lungs. a The number of MADB106 lung metastases (± SEM) counted 3 weeks after i.v. inoculation. Surgery dramatically increased colony formation, and poly I-C pre-treatment completely prevented surgery from promoting metastasis (n = 57). b Tumor retention in the lungs 24 h after inoculation of radiolabeled MADB106 tumor cells (percentage of injected tumor, mean ± SEM). Here again, poly I-C reversed the detrimental effect of surgery, but also reduced retention in control rats (n = 70). An asterisk indicates a significant difference from the respective saline-treated group.

 
MADB106 colonization of the lungs is sensitive to NK activity only during the first 24 h after inoculation10, whereupon other immune mechanisms assume their role11. To clarify the relative contribution of NK cells in the protective effects of poly I-C, we needed a short-term index of metastatic colonization. We therefore assessed pulmonary retention of radiolabeled MADB106 cells 24 h after inoculation (n = 70). Much as with metastases, surgery increased tumor retention sevenfold, and poly I-C completely abolished this increase (Fig. 1bGo). ANOVA indicates significant interaction between the effects of surgery and poly I-C (F(1, 66) = 8.1, P < 0.05), and planned contrasts indicate that the poly I-C treatment significantly reduced tumor retention in operated rats (P < 0.05). Poly I-C also reduced retention in non-operated rats by 64% (P < 0.05). The similar findings with both indices suggest that NK cells are likely mediators of the protective effects of poly I-C.

Protecting NK activity from suppression following surgery
To specifically elucidate the role of NK cells, we studied whether poly I-C treatment protects NK activity from suppression by surgery. We naturally wanted to use syngeneic MADB106 cells as targets in the in vitro NK cytotoxicity assay. However, in our experience, splenocytes and peripheral blood leukocytes fail to lyse this tumor in vitro in a 4-h 51Cr release assay. Since it has been well established that NK cells are critical for eliminating MADB106 cells from the lungs,7–10 we studied MP leukocytes, which include NK cells strategically located to protect the host from pulmonary MADB106 metastases.

We used the same design as above and, 12 h after laparotomy, harvested MP and circulating leukocytes. We assessed their cytotoxicity, using the 4 h 51Cr release assay, against MADB106 cells as well as against standard YAC-1 target cells. Additionally, we quantified the proportion and the total number of NK cells (CD161bright) within these leukocyte populations to evaluate cytotoxicity per NK cell (n = 29).

Comparing NK activity between MP leukocytes and circulating leukocytes
MP leukocytes, but not circulating leukocytes, showed significant lysis of MADB106 cells in a 4-h 51Cr release assay (NK assay) (Fig. 2b & 2aGo). This difference is accredited to higher NK cytotoxicity per MP-NK cell, rather than to an increased number of NK cells in the MP population: looking at the control-saline group, the number of NK cells per microliter of MP leukocytes and per microliter of circulating (blood) leukocytes (PBL) tested for cytotoxicity was similar (approximately 300/µl in both cases) (Fig. 2d versus 2cGo). Additionally, comparing the NK activity against the standard YAC-1 target cells between the MP and the circulating leukocyte populations of the control-saline group revealed that many fewer MP-NK cells are sufficient to achieve the same cytotoxicity levels induced by circulating NK cells (i.e., 30% cytotoxicity is achieved by less than one-quarter of the number of MP-NK cells than that achieved by the circulating NK cells) (Fig. 2f versus 2eGo). Notably, because other leukocytes are present in the NK cytotoxicity assay, this marked difference in NK cytotoxicity per NK cell could be related to interactions between NK cells and other leukocytes populations, or to differences in cytotoxicity per single NK cells (see Discussion).


Figure 2
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  		FIG. 2. a–f. The effects of surgery and poly I-C treatment on the numbers and activity of marginating pulmonary (MP) and circulating NK cells. a Peripheral blood leukocytes (PBL) failed to show a significant lysis of the syngeneic MADB106 target cell, even when rats were administered with poly I-C. b Marginating pulmonary (MP) leukocytes effectively lysed MADB106 cell in vitro. Surgery dramatically suppressed this NK cytotoxicity without changing numbers of MP-NK cells (d), suggesting suppression of NK activity per NK cell. Poly I-C treatment markedly increased the numbers of MP-NK cells (d), more so in control rats (d), and similarly increased cytotoxicity in control and operated rats (b), suggesting that surgery did not suppress NK activity per NK cell in the poly I-C-treated rats (see also Fig. 3Go). Similar effects are evident with respect to lysis of YAC-1 target cells by MP-NK cells (f). In PBL, poly I-C and surgery did not affect numbers of NK cells (c), but surgery significantly suppressed cytotoxicity against the YAC-1 target cell in both control and operated rats (e). Thus, surgery suppressed NK activity per NK cell, and poly I-C was not effective in preventing this effect. An asterisk indicates a significant difference from the respective saline-treated group (d). In b, e, and f, there were significant effects of surgery and of poly I-C, but no interaction. Values represent mean ± SEM. n = 29 rats.

 
The impact of surgery and poly I-C on numbers and cytotoxicity of MP-NK cells (Fig. 2b, 2d, 2fGo)
With respect to NK cytotoxicity against MADB106 target cells by MP population (Fig. 2bGo), in saline-treated rats, surgery ablated the lysis of MADB106 cells (Fig. 2bGo) without reducing their numbers significantly (Fig. 2dGo). Thus, surgery suppressed individual MP-NK cell activity against MADB106 cells. In the poly I-C treated rats, both the number of MP-NK cells and NK cytotoxicity were markedly elevated (Fig. 2d, 2bGo). In these rats, surgery reduced both the numbers and the cytotoxicity of MP-NK cells, although these levels were still higher than in non-operated saline-treated rats (Fig. 2b, 2dGo). Importantly, the reduction in NK cytotoxicity in these poly I-C treated rats seems mostly due to lower numbers of MP-NK cells, as seen when cytotoxicity curves are adjusted based on the actual MP-NK to MADB106 ratio (Fig. 3Go). It therefore appears that poly I-C had a dual effect on MP-NK cells. It recruited more cells to the lungs and rendered them resistant to suppression by surgery. With respect to killing of YAC-1 target cells by MP-NK cells (Fig. 2fGo), similar effects were found, although suppression by surgery was not as dramatic as with respect to cytotoxicity against the syngeneic MADB106 line.


Figure 3
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  		FIG. 3. NK cytotoxicity curves based on actual ratios of marginating pulmonary (MP)-NK cells to MADB106 target cells. The cytotoxicity curves of the four experimental groups (seen in Fig 2bGo) were shifted horizontally to align along the same NK to MADB106 ratios (based on the actual number of MP-NK cells in each sample tested for cytotoxicity), so that NK activity per NK cell can be easily compared between the groups. Surgery markedly suppressed NK activity per NK cell in the saline-treated groups, but not in the poly I-C treated groups, suggesting that poly I-C protects individual MP-NK cells from suppression by surgery. Additionally, the poly I-C-induced increase in NK activity evident in Fig 2bGo seems mostly attributable to the increase in the number of MP-NK cells, as it is not evident in this figure when adjusting for the numbers of MP-NK cells.

 
Statistical analysis (two-way ANOVA, repeated measures for cytotoxicity studies) yielded the following results: with respect to the number of MP-NK cells (Fig 2dGo), there was a significant interaction between the effects of poly I-C and surgery (F(1, 25) = 5.5, P < 0.05), and planned contrasts indicated that poly I-C increased the number of MP-NK cells in both the control and surgery condition; however, the surgery-poly I-C group was shown to have a lower number of cells than the control-poly IC group (P < 0.05). In both cytotoxicity studies (Fig 2b, & 2fGo), there were main effects of surgery (F(1, 25) = 9.8, 8.5, P < 0.05) and main effects of poly I-C (F(1, 25) = 26.4, 37.6, P < 0.05), but no interaction.

The impact of surgery and poly I-C on numbers and cytotoxicity of circulating NK cells against YAC-1 target cells (Fig. 2c & 2fGo)
There was no significant treatment effect of surgery on the numbers of circulating NK cells and no interactive effect between surgery and the administration of poly IC. In the cytotoxicity study (Fig 2eGo), there was a main effect of surgery (F(1, 25) = 23.8, P < 0.05) and a main effects of poly I-C (F(1, 25) = 9.6, P < 0.05), but no interaction, and planned contrasts indicated a significant NK-suppressing effect of surgery within the saline-treated groups and within the poly I-C-treated groups. Therefore, given that there were no differences in the numbers of circulating NK cells (Fig. 2cGo), the reduction of cytotoxicity caused by surgery, in both the saline- and poly I-C-treated animals (Fig. 2eGo), is mostly due to reduced cytotoxicity per NK cell. Thus, surgery reduced individual NK cytotoxicity, and poly I-C did not seem to protect against this reduction.

NK cells in other immune compartments
Because poly I-C may increase the numbers of MP-NK cell partly through migration of NK cells to the lungs, we investigated whether this was not at the expense of other immune compartments. We treated 16 rats with poly I-C or vehicle and studied numbers and activity of NK cells in blood, whole body perfusate, lung perfusate, and spleens. Except for the lungs, where our findings were replicated, no significant changes in numbers or activity were observed. The general trend was an increase in the total content of NK cells, relieving concerns of a trade-off between the lungs and other immune compartments.

Small versus large NK cells
Because MP leukocytes, but not circulating-leukocyte cells, could kill MADB106 target cells in an NK cytotoxicity assay, we studied the morphology of NK cells in these two populations, in an attempt to elucidate characteristics unique to MP-NK cells. Based on forward scatter of FACS analysis, we identified two populations of MP-NK cells that seem discrete in their size distribution, and classified them as small NK cells (8–15 µm) and large NK cells (16–25µm): While these populations differ in size and were seen as two different clusters, they showed almost no difference in CD161 florescence intensity. Importantly, while within MP cells large NK cells were observed to constitute 30–35% of the total NK cell population (SD = 5–8%, in the different groups), in the blood and spleen the large NK cell populations constituted only 9–11% (SD = 1–2%). There were no significant differences in these percentages among the four experimental groups in any of the immune compartments studied, and individual variance within each immune compartment was very small. Thus, it seems that one unique characteristic of the MP-NK population is that it contains a greater proportion of large NK cells.

Protection of NK activity from in vitro suppression by prostaglandin E2 (PGE2) and corticosterone
To more directly assess whether our poly I-C regimen protects NK cells from suppression, we studied in vitro suppression of NK activity by PGE2 and corticosterone following in vivo treatment of rats with poly I-C. These two substances are known to suppress NK activity in vitro,27,28 and their in vivo blockade ameliorates NK cell suppression following surgery.17,18 We treated half of the total number of rats with poly I-C as before, and used MP leukocytes, as well as circulating leukocytes and splenocytes, to assess the in vitro suppression of NK cytotoxicity against MADB106 and YAC-1 target cells in the presence of increasing in vitro doses of the drugs. The doses of PGE2 and corticosterone used in vitro ranged from 3 x 10–9 M to 10–6 M, which we expected to induce increasing levels of NK suppression. Although it is difficult to compare in vitro to in vivo levels, with respect to corticosterone these are physiological serum levels. With respect to PGE2, these are above physiological serum levels, but clearly tissue levels of locally released PGE2 are much higher than serum levels.

Overall, we used 64 rats in this study, which was conducted in 6 replications. Based on data from FACs analysis, we diluted the effector cells to achieve roughly equal concentrations of NK cells in the poly I-C and saline conditions. In each replication, we pooled the populations of MP leukocytes, circulating leukocytes, and splenocytes from all rats treated with poly I-C and from all treated with saline, and exposed aliquots of each pool to different doses of PGE2 and corticosterone. Most commonly, we conducted duplicates of each condition during each replication (quadruplets in controls). Thus, each point in Fig. 4Go represents at least six measurements. Cytotoxicity levels were converted to lytic unit and are presented as percentage of control levels (zero concentration of the drug) in the poly I-C and saline conditions.


Figure 4
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  		FIG. 4 a–d. Pharmacological suppression of the cytotoxicity of leukocytes collected from rats that were either treated or not with poly I-C. a Corticosterone dose-dependently suppressed NK cytotoxicity of marginating pulmonary (MP) leukocytes against MADB106 cells (data points represent lytic units as percentage of 0 concentration of corticosterone). In vivo poly I-C treatment increased the ID50 of corticosterone approximately tenfold. b Similar findings were obtained with PGE2. c and d In circulating leukocytes, in which NK activity was assessed against YAC-1 target cells, poly I-C treatment failed to reduce the suppression of cytotoxicity by either corticosterone or PGE2. Each data point represents the average of several assessments from at least 12 different rats.

 
Here again, MP leukocytes, but not circulating- or splenic leukocytes, could lyse MADB106 cells. Baseline levels of activity (in each of the three immune compartments) were similar between rats that received and those that did not receive poly I-C (as is also suggested in the previous study and indicated in Fig. 3Go) (data not shown). PGE2 and corticosterone dose-dependently suppressed NK cytotoxicity in all three immune compartments as seen in Fig. 4a,bGo (against MADB106 target cells), c, and d (against YAC-1 target cells) with respect to MP-NK and circulating NK cells exposed to different doses of corticosterone and PGE2 (e.g., 4a, F(6, 48) = 32.0, P < 0.05). Most importantly, poly I-C pre-treatment markedly and reproducibly reduced this suppression in MP leukocytes, increasing the ID50 of both corticosterone and PGE2 from ~ 10–8 M to ~10–7 M (Fig. 4a,bGo). Repeated-measures (drug dose) ANOVA indicates a main effect of poly I-C in each of the drugs (F(1, 48) = 9.1 for corticosterone; F(1, 44) = 9.8 for PGE2, P < 0.05). Poly I-C had a similar protective effect on lysis of YAC-1 targets by MP leukocytes and by splenocytes (data not shown). However, poly I-C did not protect circulating leukocytes from suppression by corticosterone and PGE2 (Fig. 4cGo, Fig. 4dGo).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Based on our current and previous results17,18, we propose a scenario in which surgery triggers the release of prostaglandins, glucocorticoids, and other factors, and suppresses the cytolytic activity of NK cells. Specifically, it renders MP-NK cells less efficient in lysing MADB106 cells that lodge in the pulmonary capillaries. Poly I-C treatment recruits more NK cells to these capillaries and protects them from suppression by the stress response to surgery.

We are currently investigating which cytokines and cellular interactions produce the response of MP-NK cells to poly I-C. We suspect that the following sequence of events takes place. Recognition of poly I-C by TLR-3 expressed on dendritic cells, fibroblasts, or macrophages initiates the response29. The resulting secretion of several innate cytokines, the most important of which are type-I interferons30,31, stimulates NK cells. This recruits more NK cells to the pulmonary vasculature by changing the expression pattern of adhesion molecules and renders these cells resistant to suppression by prostaglandins20, corticosteroids32, and potentially other immunomodulators (e.g., catecholamines). Our ongoing studies indeed indicate a marked increase in the number of MP-NK cells following chronic treatment with IL-12 (33). Intriguingly, in the current study, poly I-C administration did not increase NK activity per cell in non-stressful conditions, perhaps because of the relatively low dose we used.

In this present study, poly I-C treatment protected MP-NK cells from both in vivo suppression by surgery and in vitro suppression by PGE2 and corticosterone. Although we used the same syngeneic target cell line in both experiments (the MADB106), we did not unequivocally prove that the protection from pharmacological suppression underlies the in vivo protective effects of poly I-C against the effects of surgery. Nonetheless, we believe this is indeed the case: prostaglandins and corticosteroids are released after surgery4,34 and are known to suppress NK activity in vitro and in vivo9,27,28. More directly, we have recently shown that blocking these substances with specific antagonists prevented surgery from suppressing NK cytotoxicity and host resistance to MADB106 lung metastasis17,18. The intracellular mechanisms employed by poly I-C to desensitize NK cells to immunosuppressive hormones are not yet clear. Leung and Koren have demonstrated that interferons protect NK cells from suppression by prostaglandins partly by blunting the cAMP response to these factors35. However, since in our study poly I-C also attenuated suppression by corticosterone, which is cAMP independent, protection may occur more downstream, at a stage where both pathways converge.

Our findings suggest that MP-NK cells are more active than splenic and circulating NK cells: they comprise the only NK cell population we found that can lyse MADB106 tumor cells in vitro; per NK cell they exhibit higher cytotoxicity against standard YAC-1 target cells; and the greater proportion of large NK cells in this population (35% within MP-NK versus 10% within blood or splenic NK cells) is consistent with activation of cytotoxic cells. Our future studies aim at characterizing purified MP-NK cells in terms of specific activation markers and cytotoxic activity against various tumor lines. Without purification of NK cells it will be difficult to determine whether MP-NK cells are individually more active than circulating NK cells, or whether other cells unique to the MP or to other immune compartments determine the different levels of cytotoxicity observed. These questions were beyond the scope of the current study. Importantly, peripheral lymphocytes collected from cancer patients often fail to lyse autologous cells from excised tumors36. We suspect that activated populations of effector cells in the lungs of such patients may more efficiently recognize and destroy circulating tumor cells. The positive effect of poly I-C on MP-NK cells was twofold: it increased their numbers markedly and rendered them resistant to suppression. Similar changes probably occur naturally in the lungs under conditions of systemic viral infection and may constitute a mechanism for trapping and destroying virally infected cells. Under such conditions, it seems adaptive for immunity to become impervious to the effects of stress or injury, even though in the steady-state peripheral immunosuppression may serve some biological purpose3.

Aside from its transitory immunosuppressive effects, surgery gives immunity a second chance to eradicate cancer1. Evidence is accumulating that after the primary tumor is removed, most patients still harbor minimal residual disease5. The fact that many patients do not develop metastases is, to our mind, at least partly ascribed to their immune system. Specifically, once the primary tumor is removed, systemic levels of tumor-derived immunosuppressive factors subside, a protective microenvironment no longer exists to shelter tumor cells, and isolated tumor cells remain vulnerable to attack by immunity until they proliferate and establish metastases1,5,37. During this critical period, NK cells carry out part of the tumoricidal activity36. These lymphocytes can lyse tumor cells that have lost MHC-I expression (probably to evade recognition by CTLs) or express markers of cellular stress38. While only indices related to NK cells were assessed in the present study, it is clear that other immunocytes, notably CTLs, play a role in resisting metastasis38,39. Whereas surgery is known to suppress cell-mediated immunity in general1,2, it remains to be seen whether poly I-C treatment can also protect cellular immune functions other then NK activity.

Our findings suggest that treatment with low doses of poly I-C might benefit oncology patients preparing for surgery. At low doses, this treatment is known to be a relatively safe therapeutic option40,41, which non-selectively activates cellular immunity, employing a wide cytokine network, and recruiting several effector cells. Although poly I-C treatment has yielded limited clinical success in the past, it was commonly used in advanced stages of the disease, against inoperable primary tumors or established metastases41. The potential clinical significance of the intervention we suggest lies with its context, i.e., the removal of a primary tumor, with specific reference to the immediate aftermath of surgery and the elimination of minimal residual disease. Preserving and enhancing immunocompetence throughout this critical period may give the patients’ immune system a final opportunity to eradicate residual tumor cells before they establish metastases. Since the lungs’ vasculature filters all circulating tumor cells, and the lungs form a major target site for prevalent metastatic tumors, the specific effects of our treatment on pulmonary immunity might prove especially beneficial.


   ACKNOWLEDGMENTS
 
This research was supported by an NIH/NCI grant CA73056 and by a grant from the Israel Science Foundation (both granted to S. Ben-Eliyahu).

Received for publication April 17, 2006. Accepted for publication May 18, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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