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Monocyte derived macrophages were isolated from the peripheral blood of 66 patients with either breast, colon, gynecological or hematological cancers. The macrophages from the breast and gynecologic cancer patients generally did not acquire enhanced cytotoxicity for human tumor cells after incubation with bacterial lipopolysaccharide (LPS). However, when the macrophages isolated from colon and hematologic cancer patients were studied, more than 50% of these patients possessed cytotoxic macrophages. LPS induced macrophage mediated cytotoxicity was also found to be inhibited by factors present in many cancer patient's plasma. Twenty-three of the 50 cancer patients studied possessed a plasma inhibitor capable of suppressing macrophage mediated cytotoxicity by more than 50%. Furthermore, of these 50 patients, 47 were incapable of killing the tumor cells in vitro either because they possessed nonresponsive macrophages and/or they possessed a plasma inhibitory factor. Thus, although macrophage cytotoxic function may be normal in some patients with cancer, cytotoxicity may be inhibited in some patients by factors in autologous plasma thereby rendering the macrophages ineffective in vivo.

The role of enkephalins, endorphins and other neuropeptides produced by the nervous system in the alteration of immune responsiveness is generally unknown. The present studies were undertaken to investigate the role of these neuropeptides in the modulation of cytotoxicity induced by LPS activated macrophages obtained from normal donors as well as breast cancer and Hodgkins disease patients. When the macrophages from normal donors were pretreated with these neuropeptides for 1 hr prior to co-culturing with target cells, macrophage mediated cytotoxicity was enhanced with 10(-6) M and 10(-8) M of [met]-enkephalin, 10(-6) M of [leu]-enkephalin and 10(-6) M and 10(-12) M of alpha-endorphin. However, when the macrophages were co-cultured with target cells in the presence of the neuropeptides, it was observed that 10(-6) M and 10(-12) M of alpha-endorphin enhanced cytotoxicity whereas no enhancement in cytotoxicity was observed when [met]-enkephalin or [leu]-enkephalin were added to the cultures. In fact, it appears that 10(-10) M of [met]-enkephalin and 10(-12) M of [leu]-enkephalin actually suppressed macrophage mediated cytotoxicity. When the opioid antagonist Naloxone was incubated with the neuropeptides in the presence of the macrophages the enhancement of macrophage killing produced by [met]-enkephalin, [leu]-enkephalin or alpha-endorphin was suppressed. When the breast cancer patients' macrophages were pretreated with these neuropeptides, enhancement in cytotoxicity was observed at 10(-10) M of [met]-enkephalin, and [leu]-enkephalin and at 10(-8) M and 10(-12) M of alpha-endorphin.(ABSTRACT TRUNCATED AT 250 WORDS)

It has been previously reported that human peripheral blood monocyte derived macrophages acquire enhanced cytotoxicity for tumor cells after incubation in either lymphocyte mediators or lipopolysaccharide. When the macrophages obtained from renal transplant recipients were studied we observed that the macrophages obtained from 1 to 4 year post-transplant survivors were cytotoxic for tumor cells whereas, macrophages obtained from renal transplant recipients at 10 days to 7 months post-transplantation have a depressed capacity to kill human tumor cells in vitro. Furthermore, in a long term study, eight renal patients were tested repeatedly up to 1 year post-transplant. All 5 renal transplant recipients showing no clinical evidence of rejection during this 1 year study were found to possess macrophages which were non-cytolytic for tumor cells whereas 2 of the 3 patients undergoing rejection crises did possess cytotoxic macrophages. Thus, it appears that at times of rejection crises, these patients' macrophages are converted into cytolytic cells.

Gel filtration and immunoadsorption techniques have been utilized to purify tumoricidal factors from sturgeon sera as well as human plasma. Two pure proteins with cytotoxic activity were isolated from the sturgeon serum (factors A2 and B2) and two additional cytotoxic factors were isolated from human plasma (Factors A and B). In another study, human factors A and B were quantitated in both normal donor and cancer patients' plasma. It was found that the cancer patients produced at least 50% less tumoricidal factors than did the normal donors. Furthermore, when the secretions from various lymphoid cell populations were purified utilizing immunoadsorbent techniques, it was found that the macrophages generally were producing the human tumoricidal factors. Both the sturgeon and the human factors were enzymatically cleaved into several small molecular weight fragments. Sephadex G-75 column chromatography was then utilized to isolate the enzymatic cleavage fragments. Three fragments with cytotoxic activity were isolated from both sturgeon factor A2 and B2. Similarly, human factor A was digested into two fragments with cytotoxic activity and factor B was digested into three fragments with cytotoxic activity. The molecular weights of the cytotoxic sturgeon and human fragments ranged from 14,000 to 23,000 whereas the intact sturgeon and human factors had a molecular weight slightly greater than 67,000. Ultimately, if these small molecular weight fragments can be sequenced, then it would be possible to prepare a synthetic analog which might have future potential as a mode of treatment for cancer patients.

Human macrophages, derived from peripheral blood monocytes, acquire enhanced cytotoxicity for human target cells after incubation in mediator-rich supernates from antigen-stimulated lymphocytes. Maximum cytotoxicity was observed after 24-h incubation in mediators. In comparison to normal macrophages, mediator-activated macrophages were cytotoxic to five of the six malignant cell lines tested but had no effect on five nonmalignant cell lines. In 20 experiments with one target (SK-BR-3), mean cytotoxicity was 23 +/- 2.7% and with another target (MA-160), was 29 +/- 3.4%. Macrophages became cytotoxic after 8-h incubation with mediators and the enhanced cytotoxicity persisted for at least 40 h after the lymphocyte mediators were removed. These findings are consistent with the hypothesis that macrophages, activated by antigen-induced lymphocyte mediators, can contribute to the host resistance to tumor growth in man.

Continuous DEAE-Sephadex ion-exchange gradients were utilized to purify 2 human plasma factors (A and B) which possessed cytotoxic activity in vitro against human tumor cells but not against normal cells. Antibodies were elicited in rabbits against human factors A and B. After insolubilization of the rabbit anti-human tumoricidal factor antibodies, the 2 factors, could then be purified utilizing immunoadsorbent techniques. Human plasma was adsorbed to the insolubilized antibodies so that factors A and B could be isolated in quantities large enough to conduct biologic studies. When the 2 factors purified by DEAE ion-exchange chromatography were examined by SDS-polyacrylamide gel electrophoresis, both factors A and B consisted of 1 protein band with an approximate molecular weight of 67,000. Both factors A and B were destroyed by heating at 100 degrees C. However, when heated at 56 degrees C for 30 min, factor A was destroyed whereas factor B retained its cytotoxic effects. Furthermore, the mechanism of tumor killing by the human factors appears to be by means of a soluble mediator. The addition of arginine to the incubation mixture had no effect on cytotoxicity. However, incubation with catalase or superoxide dismutase suppresses cytotoxicity. Thus it appears that O2- or H2O2 functions as an anti-tumor substance in our system. When the kinetics of tumor killing were studied, it was found that human factors were non-cytotoxic after 4 hr incubation, possessed slight cytotoxicity at 8 hr but reached maximal cytotoxicity levels after 24 hr incubation.

Human monocyte derived macrophages obtained from both normal donors and cancer patients can carry out antibody dependent cellular cytotoxicity (ADCC) to human tumor targets. The macrophages from breast and hematologic cancer patients with Stage I, II or III disease and colon cancer patients with Stage III disease killed tumor cells less effectively than the macrophages from normal donors. In contrast, the macrophages from colon cancer patients with Stage I and II disease killed the tumor targets as well as the macrophages from normal donors. Macrophages from head and neck cancer patients with Stage I and II disease killed tumor cells less effectively than the macrophages obtained from normal donors. In contrast, macrophages obtained from head and neck cancer patients with Stage III disease killed the tumor targets as well as the macrophages from normal donors. It must also be noted that individual patients did not always conform to the group pattern.

Monocyte-derived macrophages isolated from the peripheral blood of 17 patients with cancer were studied for their cytotoxic effects on a sensitive allogeneic tumor target as well as autologous tumor cells. Macrophages from four of the cancer patients were cytotoxic towards the allogeneic tumor target. Five patients had macrophages which were cytotoxic towards the autologous targets. When indomethacin, a prostaglandin inhibitor, was added to the noncytotoxic macrophages during the cytotoxicity assay, macrophages from 5 of the 11 cancer patients studied became cytotoxic for the allogeneic target, and macrophages from 4 of the 11 patients became cytotoxic for the autologous tumor cells. However, it was also noted that those cancer patients who possessed cytotoxic macrophaged in the absence of indomethacin, became noncytotoxic towards the tumor cells after addition of indomethacin. These results indicate that addition of indomethacin to cytotoxic macrophages suppresses macrophage mediated cytotoxicity whereas addition of indomethacin to noncytotoxic macrophages greatly enhances macrophage mediated cytotoxicity. This theory was further substantiated when it was observed that the cytotoxic macrophages obtained from normal donors also became noncytotoxic for a sensitive tumor cell line after the addition of indomethacin during the cytotoxicity assay.

Previously, we had demonstrated that plasma inhibitory factors obtained from colon cancer patients suppressed macrophage mediated cytotoxicity. When this inhibitor was further purified, four fractions (A, B, C and D) with inhibitory activity were isolated. In this study, it was found that these four purified inhibitory factors also dramatically decreased the extent of macrophage mediated cytotoxicity induced by LPS when added during the activation phase of the assay. When the secretions from a variety of fresh tumors as well as several established tumor cell lines were purified utilizing immunoadsorbent techniques, those same inhibitory factors were recovered. It was found that 8 of the 11 secretions obtained from a variety of fresh tumors and established tumor cell lines secreted inhibitor A; 6 secreted inhibitor B and 5 secreted inhibitor C. (The presence of inhibitor D was not studied.) Furthermore, one of the 11 tumors studied secreted none of these inhibitory factors. These findings lend support to the theory that these inhibitory factors which are initially produced by the tumor and subsequently shed into the sera ultimately lead to in vivo suppression of macrophage mediated cytotoxicity.