Indirect stimulatory effects of murine interleukin-12 on in vitro production of nitric oxide by mouse peritoneal cells.

The effect of murine interleukin-12 (IL-12) on L-arginine-dependent biosynthesis of nitric oxide (NO) by mouse peritoneal cells was evaluated. Interleukin-12 was found to trigger considerably enhanced production of NO in a dose-dependent manner. Antibody neutralization studies indicated that the effect of IL-12 was mediated by IFN-gamma without apparent participation of TNF-alpha. Synergistic effects of IL-12 plus lipopolysaccharide (LPS) were also observed. Our data thus provide evidence that IL-12 is a powerful but indirect modulator of NO formation. These findings may contribute to the better understanding of various biologic effects of IL-12.

Ethics of research training for NIH T32 surgical investigators.

As part of the revised curriculum of the NIH T32 Training Grant mechanism, 6 hr of formal instruction in ethics of research are now required. We therefore implemented a four-session seminar (6 hr total time) structured around assigned readings, didactic presentations, and group discussions. The objective of this research project was to assess whether this new program provided our trainees with a framework for ethical conduct in research. Twelve trainees completed the ethics course; 8 trainees who had not yet taken the ethics course served as a control group. All trainees answered a 72-item questionnaire of our own design that examined a variety of issues in research ethics. We compared the responses of seminar participant and nonparticipant groups using the Fisher exact test and Student t test for nominal and ordinal data, respectively. Both groups of trainees perceived that too much emphasis was placed on quantity rather than quality of publications. Both groups felt that this pressure emanated from department chairmen rather than laboratory mentors (P < 0.0001). In contrast to these shared perceptions, the two groups demonstrated many differences in their comprehension of research ethics. For example, compared to the controls, trainees who participated in the ethics course believed that they could define potential NIH standards for data storage and research mentorship (P < 0.05), understood gratuitous manuscript authorship (P < 0.05), were comfortable in dealing with outlier or discordant data (P < 0.10), and, most pertinently, were fully prepared to seek third-party input into an ethical dilemma involving their own work (P < 0.006).(ABSTRACT TRUNCATED AT 250 WORDS)

Comparative studies of taxol and taxotere on tumor growth and lymphocyte functions.

Taxotere, a semisynthetic derivative similar to taxol, has shown promising results in vitro and in preliminary in vivo studies. We have previously reported that taxol at 10 micrograms/ml suppressed the cytotoxic function and activation of natural killer cells and major histocompatibility nonrestricted (MHC-NR) T cells against the ovarian cell line OV-2774 and the erythroleukemia cell line K-562. In this paper, we investigated the effect of taxotere on lymphocyte function and found that a suppression of MHC-NR cytotoxicity of naive lymphocytes is only seen at higher doses of taxotere (50 micrograms/ml). At the concentrations of 10 and 50 micrograms/ml, taxotere did not have any effect on the cytotoxic function of IL-2-preactivated lymphocytes. On the contrary, both taxol and taxotere caused a significant suppression of lymphocyte growth and activation with IL-2 at 10 and 50 micrograms/ml. Both drugs (at the concentration of 10 micrograms/ml) were potent inhibitors of the growth of the tumor cell lines OV-2774 and K-562. In conclusion, despite the fact that taxotere shares with taxol a potent antitumor effect, it seems to be less suppressive for lymphocyte cytotoxicity, an effect clearly desirable in cancer therapy.

Natural immunity in breast cancer patients during neoadjuvant chemotherapy and after surgery.

Breast cancer is the most common cancer in women. Surgery, and more recently neoadjuvant chemotherapy, are being utilized as the initial treatment for breast cancer; however little is known about their effects on the natural immune system. The natural immune system (natural killer [NK] cells) is thought to be important in immune surveillance, including protection from metastasis during the intravascular tumour seeding that occurs during surgery. To investigate the effects of surgery on the natural immune system, we studied the pre-operative and post-operative peripheral blood lymphocytes (PBL) of 10 patients with stage I or II breast cancer: there was a 71.6 +/- 25.3% post-operative reduction in NK cell function (P < 0.005, Student's paired t-test). To investigate the effects of neoadjuvant chemotherapy and surgery, we examined PBL from five patients with stage III breast cancer: NK cell function dropped 95.7 +/- 1.9% after neoadjuvant chemotherapy, and there was a further 51.0 +/- 23.4% decrease after surgery (P < 0.05, Student's paired t-test). Neither group of patients had decreased numbers of NK cells, changes in the percentage of T helper or suppressor cells, or alterations in the production of cytotoxic factor by NK cells. These findings suggest that the impairment in NK cell function reflects a defect in the ability of NK cells to recognize and/or bind to tumour target cells. We conclude that the initial treatment of breast cancer patients, whether it involves surgery alone or with neoadjuvant chemotherapy, profoundly impairs their natural immune system and could increase the risk of metastasis. Further studies are needed to delineate the mechanism of this derangement in natural immunity and possibly alter its course.

Oncolytic activity of NK cells against SW-756 squamous cervical carcinoma cell line: role of interferons alpha and gamma and CD54 adhesion molecule in oncolysis.

We investigated the sensitivity of a cervical tumor cell line SW-756 to lysis by peripheral blood mononuclear cells (MNC), natural killer (NK) cells, and major histocompatibility complex (MHC)-nonrestricted (MHC-NR) T cells from cervical cancer patients and normal donors. We found that SW-756 was resistant to lysis mediated by naive (unstimulated) MNC and MHC-NR T cells, but sensitive to lysis by naive NK cells. However, the cytotoxic function of MNC could be activated with interleukin-2 (IL-2) and interferon (IFN) alpha or gamma. Although IFNs were effective in enhancement of effector cell cytotoxicity and inhibited proliferation of cervical tumor cells, they also exerted an adverse effect on cytotoxicity; specifically, pretreatment of SW-756 cells with IFNs significantly decreased their susceptibility to lysis by effector cells. Analysis of surface phenotype of SW-756 cells after treatment with IFNs showed up-regulation of expression of HLA class I determinants, the phenomenon that may be responsible for decreased sensitivity of this tumor to MHC-NR NK cells. The studies on the involvement of CD54 adhesion molecule in cytotoxic functions indicated that expression of this molecule on effector cells (but not on target cells) was important for cytotoxicity against SW-756 tumor cells. The therapeutic implication of these studies for patients with cervical cancer is discussed.

Alteration of lymphocyte microtubule assembly, cytotoxicity, and activation by the anticancer drug taxol.

We studied the effect of the anticancer drug taxol on the cytotoxic mechanism of major histocompatibility complex nonrestricted lymphocytes and their activation with interleukin 2. Unseparated lymphocytes or highly enriched natural killer or T-cells were treated with 0.2-10 micrograms/ml of taxol for various times and tested for cytotoxicity against the K562 cell line and the ovarian cell line, OV-2774. Taxol caused a dose- and time-dependent suppression of lymphocyte cytotoxicity. The most pronounced suppression was noted after treatment with 10 micrograms/ml of taxol for 6 h; a lower but significant decrease in cytotoxicity was observed after treatment with 2 and 5 micrograms/ml of taxol. In addition, taxol inhibited activation of lymphocytes with interleukin 2; however, the cytotoxicity of interleukin 2-preactivated lymphocytes was less sensitive to taxol treatment. Mechanism studies showed that taxol was not directly toxic to lymphocytes and did not alter their ability to form conjugates with target cells. Taxol treatment decreased a rate of kinetics of lysis and lymphocyte recycling ability. The immunofluorescence and electron microscopic analysis showed polymerization of microtubules in taxol-treated lymphocytes. These data demonstrate that taxol impairs lymphocyte cytotoxic function and activation and indicate the role of microtubules in these functions. Clinically, these findings suggest that activation of lymphocytes prior to taxol treatment may increase the therapeutic benefit of this drug.

Analysis of interleukin-2-activated killer cells of rhesus monkeys: striking resemblance to the human system.

We have found numerous and exquisite homologies between the interleukin-2 (IL-2)-activated killing systems of rhesus monkeys and humans. Lymphocytes with high oncolytic and proliferative activity were generated from peripheral blood, spleen, and bone marrow of monkeys after culture with IL-2. The distribution of lymphocyte subsets in IL-2 cultures closely paralleled that seen in humans, including a decrease in CD4+ and increase in CD8+, CD38+, and CD25+ lymphocytes and an increase in density of CD2 molecules. We also describe three distinct subsets of monkey lymphocytes, CD16+,56-, CD16+,56+"dim", and CD16-,56+"bright", and show that the CD56+"bright" subset is substantially increased (to as high as 79%) after IL-2 activation. Furthermore, as in humans, the cells with oncolytic activity were characterized as CD56+, CD16+/-, and CD8+. This strong homology with humans indicates that the rhesus monkey may be a valuable preclinical model for evaluation of therapeutically relevant biological response modifiers.

Recent approaches to induction of cytotoxic lymphocytes against leukemia.

We have shown that therapy of AML patients in second remission with continuous infusion of IL-2 resulted in generation of cytotoxic lymphocytes in peripheral blood and bone marrow. However, these lymphocytes mediated cytotoxicity only against tumor cell lines, and not against AML blasts. Additional in vitro activation with higher dose of IL-2 was necessary for induction of AML blast oncolysis. This indicates that the concomitant treatment with continuous and periodic bolus IL-2 infusion may be necessary, to achieve higher levels of IL-2 and subsequently, more potent lymphocyte activation. Alternatively, it may not be possible to induce optimal antileukemia response in vivo with tolerable doses of IL-2, and the adoptive therapy with in vitro supremely activated lymphocytes may represent an option. In this article, we discuss several approaches to the supreme activation of cytotoxic lymphocytes against highly resistant AML blasts and their possibilities in leukemia therapy.

Role of natural killer cells in cancer.

This paper reviews recent publications and presents data dealing with natural killer (NK) cell activity in the tumor-infiltrating lymphocytes (TIL) and peripheral blood (PBL) of patients with solid tumors and leukemia. Cells with NK markers or function are not a prominent feature of lymphoid infiltrates in solid tumors and, when present, do not appear to correlate with other prognostic variables. Nevertheless, NK cells among IL-2-activated TIL mediate antitumor cytotoxicity. Many studies indicate that NK activity is reduced in patients with advanced cancer. In some of these studies, low NK activity has been shown to be an unfavorable prognostic variable. PBL, splenic and bone marrow NK activity in patients with acute myelogenous leukemia (AML) is also decreased. However, adherent IL-2-activated lymphocytes composed primarily of NK cells were found to be cytotoxic against AML blasts and could be generated from patients in remission and relapse. The question of whether low NK activity in solid tumors and leukemia is the result of the disease state or contributes to it, remains unanswered. Data are also presented here showing that treated, apparently disease-free patients with high PBL NK activity have a significantly longer metastasis-free survival time than those with low NK activity (n = 91, p < 0.026, Cox proportional hazards test).

Human natural killer cell development from bone marrow progenitors: analysis of phenotype, cytotoxicity and growth.

We have developed a culture system allowing for generation of NK cells from human CD34+ bone marrow progenitors. The appearance of NK cells expressing CD56+, CD3- phenotype and large granular lymphocyte morphology was observed after 2-3 weeks of culture with IL-2. The NK cell appearance coincided with development of lytic activity. NK cells generated in bone marrow cultures proliferated actively (expansion index ranged from 2- to 200-fold). The phenotype of NK cells generated from CD34+ bone marrow deviated from peripheral blood NK cells in that a lower percentage of the former cells expressed CD16, CD2, CD7, and CD8 antigens. NK cells were also generated from CD34+ populations depleted of the CD34+, CD33+ subset indicating that myeloid-committed progenitors are not required for NK cell development. The dose of IL-2 was not important for generation of NK cells; however, only high doses of IL-2 supported development of optimal NK cell cytotoxic potential. Addition of TNF-alpha facilitated IL-2-dependent NK cell generation. These data showed that NK cells can develop from early bone marrow progenitors and that this system may be instrumental in studies on NK cell lineage and differentiation.

Genesis of human oncolytic natural killer cells from primitive CD34+CD33- bone marrow progenitors.

We have found that fully functional CD56+CD3- NK cells can be generated from highly enriched populations of CD34+ human bone marrow progenitors in long term bone marrow culture (LTBMC) with IL-2. Strong lytic activity against K-562 cells was observed after 2 to 3 weeks of culture and coincided with the appearance of CD56+CD3- large granular lymphocytes, which comprised 55 to 84% of cells. In contrast, lymphocytes generated from CD34- bone marrow cells were predominantly CD56-CD3+ T cells. The phenotypic profile of NK cells generated in LTBMC differed from that of IL-2-cultured peripheral blood NK cells. Specifically, a lower percentage of LTBMC-derived NK cells coexpressed CD16, CD2, CD7, and CD8 Ag. NK cells generated in LTMBC proliferated actively, with an expansion index ranging from two- to 200-fold. Furthermore, NK cells were generated from the CD34+CD33- hematopoietic subset, which had been depleted of the myeloid-committed progenitors (CD34+CD33+). Generation of cytotoxic NK cells in LTBMC was dependent on the dose of IL-2; although CD56+CD3- NK cells were generated in LTBMC supplemented with a broad range of doses (10 to 10(3) U/ml) of IL-2, the acquisition of full cytotoxic function required a high concentration of IL-2 (10(3) U/ml). These observations demonstrate the IL-2-driven differentiation of NK cells from early bone marrow progenitors and indicate that LTBMC may provide an excellent model for studies of NK cell lineage and differentiation in normal and pathologic conditions.

Effect of new investigational drug taxol on oncolytic activity and stimulation of human lymphocytes.

Taxol is a new antineoplastic agent active in the treatment of drug-refractory ovarian and metastatic breast neoplasms. Extensive investigations have been concerned with the effect of taxol on a variety of tumor cells, but there is virtually no information about its effect on human lymphocytes. Since lymphocytes, especially natural killer (NK) cells, have been recognized to play an important role in the body's defense against tumors, we studied the effect of taxol on the cytotoxicity of naive (unstimulated) peripheral blood mononuclear cells (MNCs) and NK cells as well as on these cells' activation and growth in interleukin-2 (IL-2) cultures. We found that taxol impaired the cytotoxicity of naive MNC and NK cells against the NK-sensitive cell line K-562 and against an ovarian cancer cell line, OV-2774, in a concentration-dependent fashion. The highest impairment was observed after incubation of the effector cells with 10 micrograms/ml taxol. In addition, taxol also interfered with the induction of lymphokine-activated cytotoxicity and with lymphocyte growth in IL-2 cultures. However, IL-2 preactivated NK cells displayed substantial levels of cytotoxicity even after taxol treatment. These findings, which indicate that treatment with taxol should follow rather than precede immunotherapeutic intervention, may be important in planning combined chemo- and immunotherapy strategies for cancer patients.

Induction of cytotoxic lymphocyte subsets against leukemia by stimulation with AML blasts.

Although the application of interleukin-2 (IL-2) activated lymphocytes in immunotherapy of acute myelogenous leukemia (AML) is of therapeutic interest, the high resistance of AML blasts to lymphocyte lysis may represent an obstacle to this type of therapy. However, our data shows that the leukemia resistance can be conquered by concomitant culture of lymphocytes with IL-2 and AML blasts. This approach induces not only leukemia-directed cytotoxic cells, but also promotes their growth. Additionally, multiple cytotoxic lymphocyte populations with leukemia lytic activity are induced in AML/IL-2 cultures. These include natural killer (NK) cells and subsets of T cells with both the major histocompatibility complex (MHC)-restricted and MHC-nonrestricted cytotoxic function. Thus, this protocol, which is conducive to general stimulation of cellular immune responses against leukemia, may enhance the benefits of lymphocyte therapy.

Differential oncolytic effect of NK-enriched subsets in long-term interleukin-2 cultures.

The aim of this study was to characterize the oncolytic efficacy of human natural killer (NK) cell subsets generated from highly NK-enriched population in long-term IL-2 cultures. NK cells cultured for 3 weeks with interleukin-2 (IL-2) were separated into several subsets using two color fluorescence-activated cell sorting and CD2, CD8, CD16, and CD56 monoclonal antibodies. These individual NK cell subsets were then tested for cytotoxicity against various tumor target cell lines, including K-562, Daudi, and Ovcar-3. The CD16+/CD56+ NK cell subset was superior in its cytotoxic activity against all targets in comparison to the CD16-/CD56+ subset. Within CD16 population, CD16+CD2+ NK cells were most potent; however CD16+/CD2- subset was also cytotoxic, indicating that CD2 molecule is important, but not necessary for NK cell cytotoxic function. CD16+/CD8+ and CD16+/CD8- subsets showed variance in cytolytic efficacy, depending on the tumor target tested. Highest cytotoxicity against K-562 was observed in the CD16+/CD8+ population, while the CD16+/CD8- subset manifested highest cytotoxic activity against Daudi. No significant differences within these NK cell subsets were observed against Ovcar-3 targets. These data indicate that NK cell subsets are not equally oncolytic, and that the oncolytic effect may be tumor dependent.

Surgical stress impairs natural killer cell programming of tumor for lysis in patients with sarcomas and other solid tumors.

BACKGROUND: Natural killer (NK) cells may provide a first line of defense against the metastatic implantation of circulating tumor emboli. Because tumor emboli are discharged systemically in patients undergoing solid tumor resection, it is important to determine the nature of surgical-stress impairment of perioperative NK cell cytotoxic function. METHODS: The authors studied 85 patients undergoing surgical resection of solid tumors, most of whom had an abrupt and marked decrease in NK cell cytotoxicity that was detectable within 18 hours of surgical resection. RESULTS: This impairment was not caused by rapidly emerging suppressor cells (measured in autologous effector cell mixing studies) or decreased NK cell frequency in the peripheral blood (assessed phenotypically and morphologically). Instead, surgical stress exerted a direct "toxic" effect on NK cells that could be localized to a specific phase of the NK cell tumor lysis cycle. Tumor binding and the first round of tumor lysis were intact postoperatively (measured in single-cell assays). However, postbinding programming for lysis was decreased sharply after surgery (assessed by calcium pulse assays). In addition, the kinetics of lysis and the rate of lytic programming were slower after surgery (assayed in target saturation kinetic chromium-51 release tests). CONCLUSIONS: These latter defects probably were related to the programming for lysis deficiency because postprogramming NK cell maximal recycling capacity was not affected by surgical stress.

Murine Kupffer cells and hepatic natural killer cells regulate tumor growth in a quantitative model of colorectal liver metastases.

This investigation aimed to develop a biologically relevant murine model of colorectal liver metastases and determine if Kupffer cells (KC) and hepatic natural killer cells (hNKC) regulate tumor growth. The model involves the injection of murine colon adenocarcinoma 26 (MCA 26) tumor cells into the portal vein of female-specific pathogen-free BALB/c mice. Metastases developed in all animals, and the growth was limited entirely to the liver. To determine if KC and hNKC control the development of liver metastases, the in vivo function of these hepatic effector cells was modulated. Tumor growth was quantitated by the uptake of 125I into tumor DNA. Stimulation of the KC and hNKC produced a significant (P less than 0.01) dose-dependent decrease in 125I uptake in the liver in both treatment groups, which was associated with a significant improvement in survival (P less than 0.05). The in vivo cytotoxic function of the liver was inhibited with an intravenous injection of gadolinium chloride (for KC) or asialo GM1 antiserum (for hNKC). Inhibition of KC and hNKC cytotoxic function led to a significant (P less than 0.01) increase in 125I uptake in the liver and a significant decrease in survival (P less than 0.05).