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Annals of Surgical Oncology 10:469-479 (2003)
© 2003 Society of Surgical Oncology

ORIGINAL ARTICLES

Prostaglandin E2 Suppresses NK Activity In Vivo and Promotes Postoperative Tumor Metastasis in Rats

Ilan Yakar, MA, Rivka Melamed, MSc, Guy Shakhar, MSc, Keren Shakhar, MA, Ella Rosenne, MSc, Naphtali Abudarham, MA, Gayle G. Page, DNSc and Shamgar Ben-Eliyahu, PhD

From the Neuroimmunology Research Unit (IY, RM, GS, KS, ER, NA, SB-E), Department of Psychology, Tel Aviv University, Tel Aviv, Israel; and School of Nursing (GGP), Johns Hopkins University, Baltimore, Maryland.

Correspondence: Address correspondence and reprint requests to: Shamgar Ben-Eliyahu, PhD, Department of Psychology, Tel Aviv University, Tel Aviv 69978, Israel; Fax: 972-3-640-9547; E-mail: shamgar{at}post.tau.ac.il


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: Prostaglandins (PGs) were shown in vitro to suppress several functions of cellular immunity. It is unclear, however, whether physiological levels of PGs can suppress cellular immunity in vivo and whether such suppression would compromise postoperative host resistance to metastasis.

Methods: Fischer 344 rats were administered PGE2 in doses (18 to 300 µg/kg subcutaneously) that increased the serum levels approximately 2- to 4-fold. We then assessed the number and activity of circulating natural killer (NK) cells, as well as rats’ resistance to experimental metastasis of a syngeneic NK-sensitive tumor (MADB106). To study whether endogenously released PGs after surgery compromise these indices, we tested whether laparotomy adversely affects them and whether a cyclooxygenase-synthesis inhibitor, indomethacin (4 mg/kg), attenuates these effects.

Results: PGE2 dose-dependently suppressed NK activity per NK cell and dose-dependently increased 4- and 24-hour MADB106 lung tumor retention (LTR); 240 µg/kg of PGE2 quadrupled the number of lung metastases counted 3 weeks later. Selective depletion of NK cells abrogated the promotion of LTR by PGE2. Surgery significantly suppressed NK activity and increased MADB106 LTR, and indomethacin halved these effects without affecting nonoperated rats.

Conclusions: PGE2 is a potent in vivo suppressor of NK activity, and its postoperative release may promote tumor recurrence.

Key Words: Animals • NK cells • Cytotoxicity • Tumor immunology


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Recent studies have confirmed a role for natural killer (NK) cells in resisting tumor development1 and have started to characterize the molecular mechanisms underlying NK/tumor cell interactions.2 Both human and animal studies have indicated that surgery (oncological and nononcological) can suppress NK cytotoxic activity (NKCA),3 and various soluble mediators have been suggested.4 It is important to note that animal studies also suggested that such suppression underlies promotion of metastasis by surgery.5–8 Thus, the suppression of NKCA in oncological surgeries may constitute a risk factor for the development of metastases.

A group of hormone-like substances that may mediate suppression of NKCA by surgery or stress is prostaglandins (PGs). PGs are quickly synthesized after tissue damage or sympathetic activation,9,10 and in vitro studies have indicated that PGE2 can suppress various elements of cellular immunity, including NKCA11,12 and cytotoxic T lymphocyte (CTL) activity.13,14 This suppression is carried via membrane receptors that trigger the synthesis of cyclic adenosine monophosphate (cAMP), which eventually interferes with the cytotoxic activity of NK cells and CTLs.15,16

However, despite ample research into the effects of PGs on cellular immunity, it is still unclear whether physiological levels of PGs suppress NKCA in vivo17,18 and whether such suppression promotes the risk of metastasis. First, the lowest effective in vitro concentrations of PGE2 (typically 10-9 to 10-8 M)19–21 are 10- to 100-fold higher than physiological plasma levels,17,22 although local PG levels may far exceed plasma levels. Second, some researchers,23–25 but not others,26,27 have found increased systemic levels of PGs after surgery, and it is unclear whether the local release of PGs can cause systemic suppression of cellular immunity. Third, the in vivo milieu contains various cytokines and hormones (e.g., interferon or interleukin [IL]-2) that are known to protect NK cells from the suppressive effects of PGE2 and other factors that increase intracellular cAMP.16,28,29 Last, the biological and clinical ramifications of changes in NK activity detected by in vitro studies are unclear.30 Consequently, clinical practice does not endorse prophylactic measures against such possible suppression, even when there is an increased risk of postoperative infection or metastasis.

The suppression of NKCA by PGs may dissipate quickly after PGs are removed, as is the case with other cAMP-inducing agents.31 Thus, shortening the delay between harvesting NK cells (from the in vivo PG-rich environment) and assessing their cytotoxicity could prove instrumental. Additionally, it would be desirable to complement the in vitro NK assay with an in vivo assessment of NK activity. Such an assessment should ideally reflect the tumoricidal activity of NK cells against autogenic target cells in the natural milieu and in the presence of PGs.

Taking into account these considerations, in this study we investigated the in vivo effect of physiologically relevant levels of PGs on NKCA, as well as their biological significance. Fischer 344 (F344) rats were injected with doses that increased PGE2 plasma levels approximately 2- to 4-fold, as was observed by some researchers after surgery and other traumas.23–25,27,32 To assess the effect of endogenously released PGs in a clinically relevant setting, rats were operated on and were treated either with a PG-synthesis inhibitor (indomethacin) or with vehicle. Two different approaches were used to assess the consequences of these manipulations on NKCA: (1) an ex vivo approach, in which we assessed both the number and activity of circulating NK cells (to assess NKCA, we used a whole-blood assay that enables quick in vitro assessment of cytotoxicity without the removal of other leukocytes and without losing selectivity for NK activity33,34); and (2) an in vivo approach, in which we studied experimental metastasis of a syngeneic mammary adenocarcinoma, the MADB106. The metastatic process of this tumor line is restricted to the lungs, is highly sensitive to NK activity,35–37 and, as shown by several groups, reflects in vivo changes in levels of NKCA.5,37–39 After intravenous inoculation, we quantified the retention of MADB106 cells in the lungs at 4 and 24 hours and counted lung metastases 3 weeks later.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals
F344 male rats (Harlan Laboratories, Jerusalem, Israel) were used for all experiments, except for experiment 5, in which only females were used, and experiment 2 (rats purchased from Harlan Sprague-Dawley, Indianapolis, IN) and experiment 7, in which male and female F344 rats were used. Rats were housed four per cage with free access to food and water and were kept under a 12:12-hour light-dark cycle at 22°C ± 1 C°. At the time experiments were conducted, animals were 12 to 16 weeks old. In any given experiment, all animals were of the same age. The Institutional Animal Care and Use Committee of Tel Aviv University approved the experimental procedures and housing conditions.

Prostaglandin E2
PGE2 (Sigma, Rehovot, Israel) was first dissolved in ethanol and then diluted in saline or phosphate-buffered saline (PBS). For in vivo subcutaneous (SC) administration, PGE2 was injected in a volume of 1 mL/kg in a solution of 90% saline 10% ethanol. Control rats were injected with the same vehicle. For the in vitro study, ethanol-dissolved PGE2 was diluted in PBS and further in complete medium (see below) to reach a final concentration of <.01% ethanol in the assay medium. Control conditions contained equal quantities of ethanol, which is known not to affect NKCA at these concentrations.40

Indomethacin
Indomethacin (Sigma) was dissolved in propenyl glycol and injected intraperitoneally (IP; 1 mL/kg) in a dose of 4 mg/kg.

Measurement of Plasma PGE2 Levels
An enzyme-linked immunosorbent assay kit (Amersham Pharmacia Biotech, Piscataway, NJ; sensitivity 2.5 pg per well) was used to determine serum levels of PGE2.

Flow Cytometry
An aliquot of 100 µl of blood was combined with 50 µl of PBS (supplemented with 2% fetal calf serum [FCS] and .1% NaN3) and .1 µg of fluorescein isothiocyanate–conjugated anti–NKR-P1 (PharMingen, San Diego, CA). Samples were kept in the dark at room temperature thereafter. After a 15-minute incubation period, 2 mL of fluorescence-activated cell sorter (FACS) lysis solution was added (Becton Dickinson, Franklin Lakes, NJ), and 10 minutes later, samples were centrifuged for 5 minutes at 500 x g and the lysis solution was aspirated. Cells were washed again with 2 mL of PBS (5 minutes of centrifugation at 300 x g) and resuspended in 300 µL of PBS for flow cytometry analysis by using a FACScan (Becton Dickinson). The criterion for positive identification of spontaneously active large granular lymphocytes/NK cells was defined as being above a level of fluorescence intensity that distinguishes between brightly and dimly stained populations of NKR-P1–positive cells, as described previously by Chambers et al.41 These previous studies also demonstrated that NKR-P1 is expressed by 94% of blood large granular lymphocytes cells and that the NK cytolytic activity was totally contained in the NKR-P1 bright-cell population. Polymorphonuclear leukocytes were found to express low levels of NKR-P1 and were categorized as dim cells, and macrophages and mast cells were found to be negative.42 In our studies, bright cells were defined as showing >150 relative fluorescence intensity units, a level that distinguished between the two nonoverlapping populations of the dim and bright NKR-P1–positive cells. Nonspecific binding was assessed by using nonspecific immunoglobulin G1 that consistently yielded 0% of brightly stained cells.

Whole-Blood NK Cytotoxicity Assay
This 4-hour cytotoxicity procedure assesses antitumor NKA per milliliter of blood without prior purification of peripheral blood mononuclear cells (or the exclusion of any cell population). It reduces the time between blood withdrawal and assessment of cytotoxicity and lessens the potential interference with NK cell function. Our previous studies33,34 indicate that cytotoxicity in this assay depends on NK cells, because their selective depletion nullified all specific killing.

Blood was drawn into a syringe containing 50 U of preservative-free heparin per milliliter of blood. Blood was washed once with PBS (diluted 1/4 v/v and centrifuged at 300 x g for 10 minutes; supernatant was aspirated to the original blood volume) and washed twice with complete media (RPMI-1640 media supplemented with 10% heat-inactivated FCS, 50 µg/mL of gentamicin, 2 mM of L-glutamine, .1 mM of nonessential amino acids, and 1 mM of sodium pyruvate). To assess NK cytotoxicity at different effector-target (E:T) ratios, an aliquot of 150 µl of the washed blood was placed in the first row of a microtiter plate, and another aliquot of 150 µl was successively diluted 2-fold in complete medium in the following rows. A fixed number (5000) of chromium-51 (51Cr)–radiolabeled YAC-1 target cells was added on top of the blood in 100 µl of complete medium. Spontaneous and maximal releases of radioactivity from target cells were determined by substituting blood with the culture medium or Triton-X (Sigma Chemical Co., St. Louis, MO), respectively. In the in vitro study (experiment 1), 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 x g for 10 minutes to create a buffy-coat layer of leukocytes and target cells on top of the red blood cells before a 4-hour incubation period. After incubation, plates were again centrifuged, and aliquots of 100 µl of the supernatant were recovered from each well for assessment of radioactivity in a gamma counter. Specific killing was calculated as equation


(1)

Hematocrit correction factor (HCF) compensates for changes in the hematocrit-supernatant volume over different E:T ratios. This correction factor is included to consider the changing volume of cell-free medium in which the released radioactive molecules are dispersed.

Radiolabeling of YAC-1 Target Cells
A total of 20 x 106 YAC-1 cells were incubated for 1 hour with 100 µCi of 51Cr (in 100 µl of saline), 100 µl of FCS, and 50 µl of complete media. After incubation, cells were washed three times in complete medium (centrifuged at 300 x g for 10 minutes) and adjusted to the desired concentration in complete medium.

MADB106 Tumor Line (In Vivo Studies)
MADB106 is a selected variant cell line obtained from a pulmonary metastasis of a mammary adenocarcinoma (MADB100) chemically induced in the inbred F344 rat.35 After intravenous inoculation, MADB106 tumor cells seed and colonize only to the lungs, and the number of tumor cells retained in the lungs at different time points after inoculation (e.g., at 4 and 24 hours), as well as the consequent metastases enumerated weeks later, are highly dependent on NK activity.35,37 The MADB106 cell line was maintained in 5% CO2 at 37°C in monolayer cultures in complete medium and was separated from the flask by using .25% trypsin.

Radiolabeling of MADB106 Tumor Cells and Assessment of Lung Tumor Retention
A dose of .4 µCi/mL of 125Iododeoxyuridine (ICN Radiochemicals, Irvine, CA) was added to the growing cell culture 1 day before harvesting the cells for injection. For tumor cell injection, rats were lightly anesthetized with halothane, and 4 x 105/kg 125Iododeoxyuridine-labeled MADB106 tumor cells in 2 mL/kg of PBS were injected into the tail vein. Four or 24 hours later, rats were killed with halothane, and their lungs were removed and placed in a gamma counter for assessment of radioactive content. The percentage of tumor cells retained was calculated as the ratio of radioactivity measured in the lungs to total radioactivity in the injected tumor cell suspension. 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 Ben-Eliyahu and Page.43

Induction and Counting of Tumor Metastases
Rats were lightly anesthetized with halothane, and 105 MADB106 tumor cells (approximately 4 x 105/kg) were injected into the tail vein in .5 mL of PBS supplemented with .1% bovine serum albumin. Rats were killed with halothane 3 weeks after tumor inoculation, and their lungs were removed and placed for 24 hours in Bouin’s solution (72% saturated picric acid solution, 23% formaldehyde [37% solution], and 5% glacial acetic acid). After lungs were washed in ethanol, visible surface metastases were counted.

Selective In Vivo Depletion of NK Cells
Two days before tumor inoculation, approximately 1.5 mg/kg of anti–NKR-P1 were injected intravenously under light halothane anesthesia. In a previous study that used the above-mentioned dose of the anti–NKR-P1, we showed a complete abolition of blood and splenic NK cytotoxicity and a 100-fold increase in the lung retention and metastatic colonization of MADB106 tumor cells.43 In addition, we have used other monoclonal antibodies (R73, W3/25, and ED2), mouse serum, and saline as controls for the administration of anti–NKR-P1 and have found them to have no effect.43

Surgical Stress
The laparotomy procedure has been described elsewhere.34 Briefly, rats were anesthetized with 2.5% halothane, and a 4-cm midline abdominal incision was made. The intestine was externalized for 40 minutes, during which time it was kept moisturized and gently rubbed with a gauze pad. The intestine was then returned to the abdominal cavity, and the wound was sutured.

General Procedures
Before all experiments, rats were acclimatized to the vivarium for a minimum of 3 weeks. All experiments were conducted during the light phase, with minimal unintended disturbance to the animals. To reduce procedural stress, rats were habituated to the experimental routines. Specifically, all rats were handled for four consecutive days in a procedure room adjacent to the vivarium. The orders of drug and tumor injection and blood withdrawal in different groups of each experiment were counterbalanced (i.e., procedures were conducted in parallel in all groups).

Statistical Analysis
For statistical analysis, either factorial or within-subject repeated-measures analysis of variance (experiments 3 and 7, using E:T ratios as repeated measures) was used. Provided that significant group differences existed, Scheffé or Bonferroni post hoc tests were used to identify specific differences. The alpha level was set to .05 for all analyses.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Experiment 1: Dose-Dependent In vitro Suppression of Whole-Blood NKCA by PGE2
The in vitro effects of different concentrations of PGE2 on NKCA were studied by using a within-subject design and the whole-blood NKCA assay. Blood from each rat was assessed for NKCA in the presence of different concentrations of PGE2—0 M (control), 10-10 M, 3 x 10-10 M, 10-9 M, 3 x 10-9 M, 10-8 M, 3 x 10-8 M, and 10-7 M. Seven rats were studied, and control levels in each rat were assessed in triplicate. To ascertain that PGE2 had no direct effect on spontaneous release of 51Cr from target cells, this measure was also assessed in the presence of different concentrations of PGE2.

PGE2 suppressed NKCA in a dose-dependent manner, and this suppression became statistically significant in concentrations >=3 x 10-9 M (Fig. 1; P < .05). No direct effect of PGE2 on spontaneous release of 51Cr from YAC-1 target cells was evident in any of the concentrations used.



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  		FIG. 1. Natural killer (NK) cell cytotoxicity (mean ± SEM) in blood co-incubated (in vitro) with increasing concentrations of prostaglandin E2 (PGE2). Suppression of NK cytotoxic activity was dose dependent, becoming statistically significant at doses >=3 x 10-9 M.

 
Experiment 2: Serum Levels of PGE2 Induced by Laparotomy or by Exogenous Administration of PGE2
In the first study, we assessed serum levels after the administration of PGE2. F344 males and females were randomly assigned to four groups. Three groups were injected SC with 120 µg/kg of PGE2 at 1, 2, or 4 hours before blood collection. The fourth group was used to assess baseline levels of PGE2 and was injected with saline 1 hour before blood collection (total of 21 males and 26 females; n = 10–14 per group). The second study was conducted simultaneously and assessed the effects of laparotomy and of the PG synthesis inhibitor indomethacin on endogenous serum levels of PGE2. Rats underwent laparotomy or were only anesthetized and were treated with indomethacin (4 mg/kg IP) or vehicle. One or 4 hours later, blood was collected (total of 48 males and 46 females; n = 14–23 per group). For blood collection, rats were killed with halothane (60-second exposure to an overdose), and 1.5 mL of blood was withdrawn by cardiac puncture into syringes containing 50 µl of EDTA and indomethacin (20 and .07 mg/mL, respectively). The 1- and 4-hour time intervals were used in both studies, because experiments 3 through 7 used these time points for MADB106 inoculation or blood withdrawal. To accumulate enough animals, four replicates were conducted, each containing all groups of both studies.

Baseline endogenous levels of serum PGE2 were higher in females than in males in both studies (P < .05). Administration of PGE2 approximately doubled baseline levels at 1 hour after injection (P = .002), reaching 106 and 150 pg/mL in males and females, respectively (i.e., 2.3 and 3.4 x 10-10 M, respectively), and dissipated within 2 hours (Fig. 2a). In the second study, neither laparotomy nor indomethacin had any significant effect on systemic (plasma) levels of PGE2 (Fig. 2b).



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  		FIG. 2. Serum levels of prostaglandin E2 (PGE2) (mean ± SEM) in male and female F344 rats (a) at 1, 2, and 4 hours after subcutaneous administration of 120 µg/kg of PGE2 and (b) after laparotomy. Females exhibited significantly higher baseline levels than males in both studies. The administration of PGE2 significantly increased serum levels of both sexes at 1 hour after injection, had a smaller and nonsignificant effect at 2 hours, and had no effect at 4 hours (a). Neither laparotomy (surgery) nor indomethacin (Indo) caused any significant changes in systemic (serum) levels of PGE2 (b).

 
Experiment 3: The Effects of PGE2 Administration on Numbers and Cytotoxicity of Blood NK Cells
Rats were randomly assigned into a control group injected with vehicle or one of two groups injected SC with either 18 or 180 µg/kg of PGE2. One hour later, rats were lightly anesthetized with halothane, and 1 mL of blood was drawn by cardiac puncture to assess both the number of NK cells per milliliter of blood (by FACS) and their activity (by using the whole-blood assay). The time interval between blood withdrawal and co-incubation with target cells was 2 to 3 hours (total of 44 rats; n = 10–12 per group).

PGE2 caused a dose-dependent reduction in NKCA per milliliter of blood (Fig. 3) without significantly affecting concentrations of NK cells. Only the highest dose of PGE2 (180 µg/kg) resulted in a significant suppression of NK activity (P < .05). The numbers of NK cells (mean ± SD) per microliter of blood in control, 18 µg/kg of PGE2, and 180 µg/kg of PGE2 were 268 ± 102, 294 ± 73, and 327 ± 137, respectively. Although there seems to be a trend for PGE2 to increase the numbers of circulating NK cells, it was not statistically significant. It is important to note that the significant decrease in NKCA per milliliter of blood caused by PGE2 administration cannot be attributed to changes in the numbers of NK cells, because these changes are in the opposite direction.



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  		FIG. 3. Levels of blood natural killer (NK) activity per NK cell 1 hour after in vivo administration of 0 (control), 18, or 180 µg/kg of prostaglandin E2 (PGE2). The higher dose caused a significant suppression of NK activity compared with control rats. PGE2 did not affect numbers of NK cells per milliliter of blood (not shown).

 
Experiment 4: The Effects of PGE2 on MADB106 Lung Tumor Retention
To investigate the in vivo effects of PGE2, rats were randomly assigned to one of two control groups (injected SC with vehicle) or one of three experimental groups injected with 75, 150, or 300 µg/kg of PGE2. PGE2 was administered simultaneously with intravenous inoculation of radiolabeled MADB106 tumor cells. Four hours after inoculation, one control group and two PGE2 groups (75 and 300 µg/kg) were killed to assess lung tumor retention (LTR). Twenty-four hours after tumor inoculation, the other control group and the third PGE2 group (150 µg/kg) were killed and assessed for LTR (total of 26 male rats; n = 5–6 per group).

Compared with the appropriate control group, PGE2 significantly increased levels of LTR at both the 4-hour (Fig. 4A) and the 24-hour intervals (Fig. 4B; P < .05). These effects were dose dependent, increasing from 2- to 3- to 10-fold effect with increasing PGE2 doses.



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  		FIG. 4. The effects of increasing doses of prostaglandin E2 (PGE2) on resistance to MADB106 metastasis (means ± SEM). (A) PGE2 dose-dependently increased MADB106 lung tumor retention (LTR) assessed 4 hours after tumor inoculation and (B) at 24 hours after tumor inoculation. (C) PGE2 also increased the number of lung metastases counted 3 weeks after inoculation. *Significant deviation from control levels.

 
Experiment 5: The Effects of PGE2 on MADB106 LTR in NK-Depleted Rats
Normal and NK-depleted (anti–NKR-P1–treated) rats were injected with either saline or PGE2 (240 µg/kg SC), simultaneously with intravenous inoculation of radiolabeled MADB106 tumor cells. Twenty-four hours later, rats were killed and assessed for LTR (total of 55 female rats; n = 10–16 per group).

NK depletion in itself increased LTR by approximately 120 times, from .15% to 19.5%. PGE2 caused a marked and significant increase in LTR in normal rats while having almost no effect in NK-depleted rats (Fig. 5). When tumor retention is expressed as percentage of the mean of the saline-treated groups, there is a significant interaction between the effects of PGE2 and the effects of NK depletion (P < .0001), indicating the blockade of the effects of PGE2 by selective depletion of NK cells. Our previous studies suggest that such a lack of effect in NK-depleted rats cannot be attributed to a ceiling effect induced by the high levels of tumor retention in NK-depleted rats, because some treatments (e.g., plastic microspheres) enhance tumor retention in NK-depleted animals as effectively as in normal rats.43,44



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  		FIG. 5. The effect of prostaglandin E2 (PGE2) on lung tumor retention (LTR) of radiolabeled MADB106 tumor cells in natural killer (NK)-depleted or normal rats. Values are expressed as percentages of the relevant saline (control) group (±SEM). PGE2 significantly increased LTR in normal rats but not in NK-depleted rats. The absolute levels of LTR in the saline condition were approximately 120 times higher in NK-depleted rats than in normal rats (not shown). *Significant deviation (alpha < .0001) from control (100%).

 
Experiment 6: The Effects of PGE2 on the Number of MADB106 Metastases
Rats were randomly assigned to a control condition and injected with vehicle or to an experimental condition and injected SC with 240 µg/kg PGE2. MADB106 tumor cells were intravenously administered at the same time with PGE2/vehicle administration, and lung metastases were counted 3 weeks later (total of 32 male rats; n = 16 per group). PGE2 caused a significant 3.5-fold increase in the number of metastases compared with the control group (P < .0001; Fig. 4C).

Experiment 7: The Effects of Indomethacin (a PG Synthesis Inhibitor) on the MADB106 Metastasis-Promoting Effects of Surgery
To assess whether endogenously released PGs, in the context of surgery, also promote tumor metastasis, we used laparotomy, because surgical procedures are known to increase PG levels.23–25,27 F344 rats served as controls or underwent laparotomy and immediately afterwards were injected with vehicle or with indomethacin (4 mg/kg IP), a PG synthesis inhibitor. Rats were intravenously inoculated with radiolabeled MADB106 cells 4 hours after surgery, and 24 hours later they were killed to assess MADB106 LTR (total of 75 rats; approximately half from each sex in each group).

In untreated rats, surgery caused a 5-fold increase in LTR (P < .003). Indomethacin reduced this effect by approximately 60% (P < .05). No significant effect of indomethacin was evident in nonoperated rats (Fig. 6). The effects of surgery and indomethacin were similar in males and females.



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  		FIG. 6. Levels of MADB106 lung tumor retention (LTR) (mean ± SEM) in control and operated rats injected with saline or with the prostaglandin synthesis inhibitor indomethacin. Surgery significantly increased LTR, and indomethacin significantly attenuated this effect (*) without affecting nonoperated rats.

 
Experiment 8: The Effects of Indomethacin on the Suppression of NKCA by Surgery
To assess whether endogenously released PGs, in the context of surgery, suppress circulating levels of NKCA, F344 rats served as controls or underwent laparotomy and immediately afterward were injected with vehicle or with indomethacin (4 mg/kg IP). Twelve hours after surgery, rats were lightly anesthetized with halothane, and 1 mL of blood was drawn by cardiac puncture to assess both the number of NK cells per milliliter of blood (by FACS) and their activity (by using the whole-blood assay; total of 22 male rats; n = 5–6 per group).

Surgery significantly suppressed NKCA per milliliter of blood (Fig. 7) without significantly altering the numbers of circulating NK cells, and indomethacin markedly reduced this effect of surgery. The only group significantly different from the control/saline group was the surgery/saline group (P < .05). The numbers of NK cells per microliter of blood (mean ± SD) were 234 ± 126, 378 ± 182, 266 ± 194, and 365 ± 156 in the control/indomethacin, control/saline, surgery/indomethacin, and surgery/saline groups, respectively. Although there seems to be a trend for indomethacin to reduce the numbers of circulating NK cells, it was not statistically significant. It is important to note that the protective effects of indomethacin against suppression of NKCA by surgery cannot be attributed to changes in the numbers of NK cells, because these changes are in the opposite direction.



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  		FIG. 7. Levels of blood natural killer (NK) activity (mean ± SEM) in control and operated rats injected with saline or with the prostaglandin synthesis inhibitor indomethacin. Surgery significantly suppressed NK activity, and indomethacin reversed this effect. No significant changes in the numbers of NK cells were caused by surgery or indomethacin (not shown).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study sought to elucidate whether physiological levels of PGs can suppress NKCA in vivo and whether such effects may underlie reduced resistance to blood-borne metastasis after surgery. To assess the in vivo effects of PGE2, we administered this prostanoid to the rat in doses that produce a physiologically relevant increase in plasma levels of PGE2 for a few hours. For example, after the administration of 120 µg/kg of PGE2 (an intermediate dose in our studies), plasma levels of PGE2 increased to 200% of baseline levels at 1 hour and 150% at 2 hours, and they returned to baseline within 4 hours. PGE2 caused a dose-dependent suppression of blood NKCA, a dose-dependent increase in MADB106 LTR, and a 4-fold increase in the number of lung metastases. Similar or greater increases in plasma levels of PGE2 were reported to occur physiologically in humans after various types of trauma, including surgery.23–25,27,32 However, although there is no doubt that PGE2 is excessively released in the vicinity of damaged tissue, several studies,26,27 including this one, have failed to detect increased systemic levels of PGE2 after surgery. This study clearly indicates the lack of such a systemic increase at the time points tested (1 and 4 hours after surgery) and the surgical procedure used (laparotomy)—other changes in levels of PGE2 were evident in our study, including a reliable sex difference and an increase in PGE2 levels after its systemic administration. Thus, to assess whether the endogenous local release of PGs in response to surgery has similar effects on immune competence, rats underwent laparotomy and were treated with the cyclooxygenase (COX)-inhibitor indomethacin or with vehicle. Surgery significantly suppressed NKCA and increased experimental metastasis of the MADB106, and indomethacin halved these effects without affecting nonoperated rats. These findings raise the possibility that local release of PGs initiates a cascade of events that eventually lead to the suppression of NKCA.

It is important to note that the two approaches we used to assess the in vivo effect of PGs are complementary: whereas administration of PGE2 reveals that this prostanoid is sufficient to compromise immunity and promote metastasis, blocking the synthesis of endogenously released PGs reveals that they are necessary to induce these effects in the more complex context of surgery. Taken together, these findings indicate that PGs, specifically PGE2, are potent in vivo suppressors of both NKCA and resistance to blood-borne NK-sensitive metastasis.

Many mechanisms, both immunological and nonimmunological, might mediate the effects of PGs on resistance to tumor development, including a direct effect of PGs on MADB106 tumor cells. However, it is our suggestion that, in this study, the reduction in NKCA was the major factor underlying the promotion of metastasis by PGs. Previous research points to NK cells as a pivotal factor controlling both LTR and the consequent lung metastases of the MADB106: pulmonary NK cells were observed interacting with MADB106 cells in situ,45 and marginating pulmonary NK cells were implicated in lysing MADB106 cells46 and in recruiting other leukocytes to the lungs.47 Substances that enhance NK cell activity (e.g., lipopolysaccharide and polyriboinosinic acid-polyribocytidylic acid [poly I-C]) improve resistance to MADB106 LTR and metastasis,43 and manipulations that compromise NK activity (e.g., intracerebral IL-1, components of cigarette smoke, prolonged hypothermia, alcohol consumption, laparotomy, and swim stress) interfere with both indices.5,39,44,48–50 Most directly, selective depletion of NK cells typically increases MADB106 LTR and the number of metastases more than 100-fold,37,38,44 as was also evident in this study, and adoptive transfer of NK cells, but not other types of leukocytes, restores resistance to MADB106 metastasis.35,36 Thus, suppression of NK activity by PGs, if it occurs in vivo, would be expected to render the rat more susceptible to MADB106 metastasis, as indeed occurred in this study.

More direct evidence for NK mediation of the tumor-promoting effects of PGE2 is derived from the ineffectiveness of PGE2 in suppressing tumor resistance in rats selectively depleted of NK cells. This finding indicates that in the absence of NK cells, the various physiological effects of PGE2 do not enhance MADB106 tumor metastasis. Ruling out a potential ceiling effect, we have shown on several occasions that other manipulations can enhance MADB106 tumor metastasis in NK-depleted rats43,44 (also see experiment 5). Additionally, our recent work indicates that in vivo immunostimulation with poly I-C protects NK cells from in vitro suppression by PGE2 and prevents surgery from suppressing NK activity and from enhancing MADB106 metastasis.46 Finally, it is unlikely that direct effects of PGs on MADB106 underlie our findings. Surgery did not increase systemic levels of PGE2 but did suppress resistance to MADB106 metastasis. Additionally, in a different study, we did not observe direct in vitro effects of various doses of PGE2 on MADB106.46 Therefore, in this study, preventing the release of PGs by indomethacin probably reduced postoperative metastasis by reducing the suppression of NKCA.

The suppression of blood NKCA by PGE2 and by surgery occurred at the level of individual NK cells—the numbers of blood NK cells were not significantly affected. This kind of NKCA suppression is most likely receptor mediated. However, it cannot be determined from this study, or from previous literature, whether PGE2 suppressed NKCA by activating prostanoid receptors on NK cells or by regulating the levels of other cytokines or hormones controlling NKCA. The feasibility of a direct effect is supported by in vitro studies that used enriched or purified populations of NK cells.11,12,20 However, these and other studies,19,21 as well as our study with whole blood, managed to demonstrate in vitro suppression of NKCA only if concentrations of PGE2 were one to two orders of magnitude higher than the physiological plasma levels (ranging from 10-10 M to 10-9 M).17,22 Nevertheless, the comparison between in vitro and in vivo concentrations of PGE2 may be misleading: PGs are quickly degraded in vivo, usually in their first pass through the lungs (approximately 90%),51 and therefore local levels in the wounded area (or in the injection site) are much higher, as are PGE2 levels in the vasculature that drains this area. Sampling systemic blood may thus provide an underestimation of the highest levels of PGE2 encountered by NK cells. Indirect effects of PGs on NKCA are clearly feasible. PGs are pivotal factors signaling tissue damage to the immune and central nervous systems. For example, PGE2 secretion triggers the release of the major anti-inflammatory cytokine IL-10,52 and COX inhibitors attenuate the hypothalamic-pituitary-adrenal axis response to surgery (including the release of cortisol).53 Thus, although it is clear that endogenous release of PGs after surgery can eventually lead to suppression of NKCA and promotion of NK-sensitive pulmonary metastasis, it is unclear whether these effects are attributable to a direct effect of PGs on NK cells or are mediated by various neuroendocrine and immunological mechanisms triggered by PGs.

Our findings, especially if corroborated in humans, may have significant clinical implications for cancer patients. Several tumors have been shown to release PGs or to recruit immunocytes to do so,11,54,55 apparently as a mechanism to escape destruction by cellular immunity. Indeed, cancer patients often exhibit low levels of NKCA and other cell-mediated immune functions.54 This unfortunate state is further aggravated by surgery: major surgical procedures in animals and in cancer patients have been shown to greatly compromise cellular immunity for up to a week.3,56 Animal studies demonstrated a key role for cellular immunity (e.g., NK, macrophage, and CTL activity) in controlling metastasis57–59 and indicated a strong association between surgery-induced suppression of cellular immunity and increased susceptibility to metastasis.34,46,49 In vitro studies using animal and human leukocytes indicated that PGs are potent suppressors of almost all aspects of cellular immunity.60 Human studies have shown that low perioperative levels of NK or CTL activity are associated with greater cancer-related morbidity and mortality in patients with colorectal,61 breast,62 lung,63 and head and neck64 cancers. Because surgery is indispensable for cancer treatment, prophylactic measures against suppression of cell-mediated immunity should be considered, and their effect on recurrence rates should be assessed. The results of this study suggest that the use of COX inhibitors can reduce perioperative immunosuppression. Because such treatment might increase bleeding, selective COX-2 inhibitors may be advantageous during the immediate perioperative period.


   Acknowledgments
 
Supported by National Institutes of Health grant CA73056 (SB-E), by a grant from the Israel Science Foundation (SB-E), and by National Institutes of Health grant NR03915 (GGP).

The acknowledgments are available online at www.annalssurgicaloncology.org.


   Footnotes
 
Tissue damage and some tumors induce the release of prostaglandins (PGs). This study demonstrates that endogenously released PGs after surgery, or systemic administration of physiologically relevant doses of PGE2, can reduce host resistance to metastasis, seemingly through their in vivo suppression of NK activity.

Received for publication August 23, 2002. Accepted for publication December 26, 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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