Prostaglandin E2 modulation of human monocyte antibody-dependent cell-mediated cytotoxicity against human red blood cells

CELLULAR

IMMUNOLOGY

71, 196-201 (1982)

SHORT COMMUNICATIONS Prostaglandin E2 Modulation of Human Monocyte AntibodyDependent Cell-Mediated Cytotoxicity against Human Red Blood Cells J. LEE MURRAY’ Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma 73102 Received March 2s. 1982; accepted June 16. 1982 The effect of inhibitors of prostaglandin E2 (PGE*) synthesis on monocyte as well as lymphocyte antibody-dependent cellular cytotoxicity (ADCC) against human red blood cells was studied. Effector cells were obtained from normal healthy controls and from patients with previously resected malignant melanoma who were receiving adjuvant immunotherapy with subcutaneous Corynebacterium parvum. Patient monocyte ADCC was significantly increased compared to controls (25% vs 8%) at effector-to-target ratios of 1:2; lymphocyte ADCC was similar in both groups. Indomethacin at 10S6M significantly augmented monocyte ADCC in patients, but not in controls; lymphocyte ADCC was not affected. Rabbit antibody to human PGEz had a similar effect on patient monocyte ADCC, suggesting that the mechanism of augmentation was via blocking of PGEr secreted during the course of the reaction. The data suggest that PGE2 acts as a feedback mechanism to limit monocyte and not lymphocyte ADCC. Furthermore, monocytes must be preactivated for augmentation by PGE2 inhibitors to occur.

INTRODUCTION Prostaglandins (PG) appear to have an important role in immune regulation (l-3). Those of the E series (PGE2, PGE,) have been demonstrated in vitro to inhibit mitogen-stimulated lymphocytes ( 1), antibody formation (2), and direct cytolysis by activated lymphocytes (3). Moreover, the role of endogenously produced PG as a regulator of antibody synthesis has been confirmed in vivo (4). The generation of human monocyte and lymphocyte antibody-dependent cellular cytotoxicity (ADCC) toward human 0+ antibody-coated erythrocytes has been well demonstrated in vitro (5). Several in vivo experiments suggest that ADCC may play a significant role in immune surveillance (6,7). In addition, recent studies have suggested that E-series prostaglandins interfere with macrophage tumoricidal activity (8, 9). In light of these findings we examined monocyte and lymphocyte ADCC in melanoma patients receiving an immunostimulant, along with healthy controls in the presence of inhibitors of PGE2. Our results suggest that endogenous PGEz acts to limit monocyte and not lymphocyte ADCC. Moreover, PGE2 production is an early event which follows monocyte activation and is dependent on it. ’ To whom correspondence should be addressed, Department of Clinical Immunology and Biological Therapy, M. D. Anderson Hospital and Tumor Institute, 6723 Bertner Ave., Houston, Tex. 77030. 196 OOOS-8749/82/110196-06$02SlO/O CopyrightQ 1982 by Academic Press, Inc. AU rigbu

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AND METHODS

Subjects. Fifteen patients with primary malignant melanoma, who were currently disease free and who were receiving adjuvant immunotherapy with subcutaneous Corynebacterium parvum (Burroughs Wellcome Co., Research Triangle Park, N.C.) were studied. Twelve healthy volunteers served as controls. C. parvum was administered at a dosage of 4 mg, either weekly, every 2 weeks, monthly, or every 6 months (maintenance) for 5 years; the majority of patients were studied either at 2 weeks, 1 month, or 3 months following injections. No patient had received previous chemotherapy. Monocytes and lymphocytes. Venous blood from patients and controls was drawn in plastic syringes containing 1 cc 5% EDTA in phosphate-buffered saline (PBS) Monocyte-enriched (M4) and lymphocyte-enriched (L) fractions were obtained using the method of Koller et al. (10). Peripheral blood mononuclear cells were allowed to adhere to plastic petri dishes (Falcon 3008, Falcon Products, Oxnard, Calif.) for 90 min at 37°C. Monolayers were washed six times with RPM1 containing 20% fetal calf serum (FCS; Grand Island Biological Co., Grand Island, N.Y.) to remove nonadherent cells, and overlayed with a solution of 0.1% EDTA, 0.2% BSA in Seligman’s balanced salt solution (SBSS, Grand Island Biological Co.) Adherent cells were gently scraped off with a rubber policeman. This procedure provided a monocyte-enriched population which was judged to be 85% pure by esterase stain (11) and 95% viable as judged using trypan blue. Monocyte purity was not significantly different between controls and patient populations. Both monocytes and nonadherent cells were pelleted, washed twice in RPMI, and resuspended to a concentration of 5 X lo6 cells/ml in Hanks’ balanced salt solution (HBSS) (Gibco). Nonadherent lymphocytes, in both patients and controls, averaged 90% purity, with 10% monocyte contamination. Viability was 95%, as assessed by trypan blue. Target cells. Human 0+ red cells collected weekly from single donors were stored in acid citrate dextrose at 4°C. From 50 to 150 X lo6 targets were incubated with 75 &i of “Cr (sodium chromate, New England Nuclear Co., Boston, Mass.) for 60 min at 37°C washed three times in HBSS, and resuspended to their original volume. Targets were antibody coated by incubating them for 30 min at 37°C with a 1:50 dilution of high-titer anti-D antiserum (Rhogam, Ortho Diagnostics, Raritan, N.J.) Non-antibody-coated targets were similarly incubated in HBSS alone. Targets were then washed three times and resuspended in HBSS at a concentration of 20 X lo6 cells/ml. Cytotoxicity assay. With a few modifications, the standard microcytotoxicity assay has been described previously ( 12). Briefly, 100 ~1 of effector cell suspension (0.25 X lo6 cells) was added with 25 ~1 of target cell suspension (0.50 X lo6 cells) to individual wells of flat-bottomed microtiter plates (Microtest II, Falcon Plastics) at an effector:target ratio (E:T) of 1:2. Additional HBSS and/or reagents (see below) were added to achieve a final volume of 200 pi/well. Cells were incubated without fetal calf serum or AB serum. To determine spontaneous “Cr release (SR), target cells were incubated in the absence of effecters. Total 5’Cr released was measured in wells where 100 ~1 detergent (Zap Isoton, Hilleleah, Fla.) was added in place of HBSS. Spontaneous release did not exceed 5% of total releasable chromium. Plates were centrifuged at 50g for 5 min to initiate cell contact, and then

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A Comparison of Monocyte and Lymphocyte Antibody-Dependent Cellular Cytotoxicity (ADCC) between Patients and Controls L-ADCC MB-ADCC E:T 1:2 Treated patients ( 15) Untreated patients (6) Controls ( 12)

25 It 2 5fl se1 3

ET 1:2 II

E:T 1O:l

+4+c~

’ Results are expressed as mean percentage ADCC f SEM. MB-ADCC was significantly increased in patients on C. parvum compared to controls and untreated patients (P < 0.0001). ‘No significant difference in L-ADCC between patients and controls at both 1:2 and 10~1 effector:target ratios.

incubated for 4 hr at 37°C in a humidified atmosphere of 95% air, 5% CO*. Following incubation 100 ~1 of supernatant from the total 200 ~1 was removed and the radioactivity measured. Target cell lysis expressed as the percentage “Cr released was determined by dividing the total counts per minute released in the experimental wells by the total amount of “Cr released in wells with ZAP added. To ensure that cytotoxicity was antibody dependent Wr radioactivity from wells in which uncoated targets were added was subtracted from wells in which identically treated antibody-coated targets were present. All assays were performed in triplicate. Reagents. In selected experiments PGEz and indomethacin (Sigma Chemical Co., St. Louis, MO.) were added to the assay. PGEz (35 mg/ml) was dissolved in ethanol in lo-ml glass scintillation vials (Kimble Products, Toledo, Ohio), evaporated under liquid nitrogen, and stored at -20°C. On the day of the experiment 1 ml HBSS was added to an individual vial for a stock PGE2 concentration of 10m4 M. Serial dilutions were made in HBSS for final concentrations in the assay of lo-‘, 10p7, and 1Oe6M. Similarly, 10 mg indomethacin was dissolved in 1 ml ethanol as a stock solution and final dilutions ( 10m8through 10e6M) were made in HBSS. Final alcohol content was less than 0.01% and did not significantly affect spontaneous release of erythrocyte targets. Anti-PGE, antibody. Rabbit antibody to human PGEz (PGE,-AB) was obtained from Boehringer-Mannheim, Indianapolis, Indiana. Antibody was resuspended in 11 ml of PBS (concentration: 1 mg/ml protein, antibody fraction), from which 150 and 1:lOO dilutions were made in HBSS. Cross-reactivity with PGE, was 0.020%, PGA2 0.020%, PGB, O.Ol%, PGB2 O.Ol%, PGE, 3.2%, PGF1, O.Ol%, and PGF2, 0.06%. Statistical analysis. Statistical analyses were performed using Student’s t test for the evaluation of two means, as well as the paired t test. RESULTS AND DISCUSSION Monocyte antibody-dependent cellular cytotoxicity (M4 ADCC) was significantly elevated in melanoma patients treated with C. parvum compared to controls and untreated patients. In contrast, lymphocyte ADCC (L-ADCC) was similar between patients and controls at both effector-to-target ratios shown (Table 1).

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0 - 4 0

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0

I:lii% (6)

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t@)

FIG, 1. The effect of adding 10e6M indomethacin to the cytotoxicity assay at time 0. Control wells (0) had an equivalent amount of HBSS added. Open circles (0) represent the mean + SE of the sum of individual experiments. Closed circles (0) denote M#-ADCC, open triangles (A) L-ADCC at a constant E:T of 1:2. Significant augmentation of Md-ADCC occurred only in patients; control MgADCC as well as both patient and control L-ADCC were unaffected.

These data are consistent with other studies demonstrating enhancement of monocyte ADCC by C. parvum (13, 14). Our study differs, however, from that of Thatcher et al. (15), who noted an increase in both K cell and NK cell activity in patients immunized at 3-week intervals. Discrepancies could be due to schedule differences, route of C. parvum administration (iv versus SC) and the fact that patients in their study all had disseminated melanoma. As seen in Fig. 1, the addition of 10e6 M indomethacin significantly increased monocyte ADCC in 6 of the original 15 patients studied by 29% (i.e., from 17 to 22%; P < 0.03, paired t test). In contrast, lymphocyte ADCC and control monocyte ADCC were not affected. Indomethacin did not cause a significant increase in spontaneous “Cr release from red cell targets. Moreover, 3 patients who were not receiving C. parvum had M&ADCC similar to that of controls (3.7 f 0.9; mean f SEM) which was not significantly increased by indomethacin addition (4.7 f 0.9). Similar results were obtained in an additional six patients following the addition of 10e8 M indomethacin, although the effect was not as pronounced (Fig. 2). A significant increase in patient monocyte ADCC also occurred (23 - 29%; P < 0.03) in the presence of a 1:100 dilution of antibody to PGE2 (PGE,-AB). These data suggested that monocytes which had been “preactivated” by an in vivo stimulus secreted PGE2, which acted to limit the course of the ADCC reaction. Since PGE*-AB also caused a significant augmentation in patient M+ADCC, we speculated that the antibody attached to PGE2 binding sites as it was produced, decreasing its effective concentration in solution. Additional evidence for this hypothesis stems from the observation that augmentation of M&ADCC by PGE2AB was overcome by adding exogenous PGEz at 10e6 M (i.e., PGE1-AB alone

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= 29% ADCC; PGE2-AB + PGEz = 20% ADCC; P = 0.05) (Fig. 2). Additional studies which measure the actual concentrations of endogenous PGEz are needed to confirm these results. Findings similar to the above have been noted by other investigators (8, 16). Taffet and Russell (8) demonstrated that the loss of tumoricidal activity of mouse peritoneal macrophages was secondary to the effects of increased PGE, synthesis. In this case macrophages had to be initially primed with lipopolysaccharide (LPS) for inhibition to occur. Treatment of stimulated macrophages with indomethacin augmented cytotoxicity. Likewise, exogenously added prostaglandins of the E series were capable of inhibiting the tumoricidal activity of interferon-treated macrophages (16). In both of these studies exogenous PGEz inhibited cytotoxicity only if it was added simultaneously with or subsequent to the addition of activating agent. In neither case did PGE2 block the generation of ADCC. The addition of exogenous PGEz (lo-* and low6 M) had little effect on MbADCC (Fig. 2). Previous studies have suggested that endogenously produced PGE2 may eventually densensitize the macrophage to further suppression, depending on the nature of the activating stimulus (17). Moreover, macrophages treated with LPS respond to self-synthesized PGEl by elevating cyclic AMP levels, which, in turn, “down regulate” PGEl receptors (18). From these studies, and from ours, it appears that PGEl inhibition of monocyte function is dose dependent and selflimited. Although preliminary, these data may have relevance with respect to biologic responsemodifiers in tumor immunology. Tumors are also capable of prostaglandin synthesis (19); such a mechanism may play a role in inhibiting effector cell function. Enhancement of both antibody-dependent and non-antibody-dependent cytotoxicity

CONTROLS %

35 25

PATIENTS

FIG. 2. A comparison of control and patient M&ADCC in the presence of various dilutions of indomethacin, antibody to PGEl (PGE,-AB), and prostaglandin E2 (PGE,) alone. Each bar represents the mean + SE of six individuals studied. Indomethacin, as well as PGE,-AB, at the 1:lOO dilution augmented patient M#-ADCC. The 1:400 dilution of PGE,-AB was not effective. Modest inhibition of patient M&-ADCC occurred with the addition of 10e6M PGEr. Slight augmentation was observed at the lo-* M dose. Prostaglandin Ez abrogated the augmentation effect of PGE,-AB. Control M+ADCC was not affected by the addition of these agents.

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against tumor cells has been observed following addition of prostaglandin synthetase inhibitor (20). In this case the effect was due to inhibition of prostaglandin production by the target cell. In light of these findings, perhaps a more fruitful approach to immunotherapy trials might include both macrophage-stimulating agents along with prostaglandin synthetase inhibitors. ACKNOWLEDGMENTS I wish to thank Georgia Thomas, M.D., for manuscript preparation, and Nemat Morsy for expert technical assistance.

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