TNF-alpha and IFN-gamma reverse IL-4 inhibition of lymphokine-activated killer cell function

Recombinant IL-4 inhibits IL-2-induced lymphokine-activated killer (LAK) cell development of PBMC. We evaluated the effect of various cytokines in reversing IL-4-mediated LAK inhibition. PBMC were cultured in IL-2 (10-1000 u/ml) with or without IL-4 (2-100 u/ml) and tested for cytotoxicity against the NK-sensitive K562 cells and NK-resistant UCLA-SO-M14 cells. Addition of IL-4 at the beginning of culture suppresses LAK activity in a dose-dependent fashion. Addition of IFN-gamma or TNF-alpha partially reverses IL-4-mediated inhibition (30-100%) in a dose-dependent fashion. IFN-gamma and TNF-alpha must be added within the first 24 hr of initiating culture in order to reverse IL-4 inhibition. Furthermore, IFN-gamma and TNF-alpha are most effective at reversing IL-4 inhibition at low concentrations of IL-2 (less than 100 u/ml). Addition of other IL-2-induced cytokines such as GM-CSF (50 u/ml), M-CSF (250 u/ml), and IFN-alpha (10-10,000 u/ml) fails to reverse IL-4 inhibition. In addition to suppression of LAK induction, IL-4 also inhibits IL-2-induced IFN-gamma and TNF-alpha protein production in PBMC. The reversal of IL-4-mediated LAK inhibition by TNF-alpha and IFN-gamma may therefore be due to resupply of these endogenously suppressed cytokines.     

Augmentation of murine lymphokine (rIL-2)-activated killer cell activity by indomethacin

The effect of indomethacin on murine lymphokine-activated killer (LAK) cell activity was investigated using a natural killer-resistant, spontaneously developed, weakly immunogenic, and highly tumorigenic syngeneic murine mammary adenocarcinoma, mimicking that of human disease, as the target. When used in combination with human recombinant interleukin-2 (rIL-2), indomethacin was found to augment LAK cell activity, which was generated from culture of the normal mouse splenocytes with rIL-2, as compared to that with rIL-2 alone. This increase in LAK cell activity was shown to be indomethacin dose-dependent, and was demonstrated only when indomethacin was added to the rIL-2-containing medium at the beginning of culture. The enhancement of LAK cell activity by indomethacin was abrogated when the nylon-wool nonadherent "macrophage-poor" splenocytes were incubated with rIL-2 plus indomethacin. These results indicated that the rIL-2-induced LAK cell activity generated from murine splenocytes could be augmented by indomethacin, and the macrophages may be involved as the mediator.     

Inhibition of murine lymphokine-activated killer (LAK) cell activity by adherent cells

The effects of adherent cell depletion, indomethacin, and prostaglandin E2 (PGE2) on murine LAK cell activity were investigated. Removal of plastic adherent cells from splenocyte suspensions either prior to 5-day culture with 1000 U/ml of recombinant human IL-2 (rIL-2) or prior to assay resulted in an enhanced LAK cell cytotoxicity compared to that of whole spleen cell suspensions. Indomethacin enhanced LAK cell cytotoxicity of whole splenocyte suspensions if present during the culture period, but had no effect on whole splenocyte or adherent cell-depleted cell suspensions if added just prior to assay. PGE2 suppressed LAK cell activity of nonadherent splenocyte but not whole splenocyte suspensions when present during the culture period. In vivo treatment of mice with indomethacin enhanced cytotoxicity directed toward both LAK sensitive, natural killer (NK) resistant (P-815) and LAK, NK sensitive (YAC-1) tumor cell targets. Splenocytes from indomethacin-treated mice cultured with additional indomethacin and rIL-2 exhibited highest LAK cell activity. The results from this study indicate that LAK cells are regulated by adherent cells which suppress LAK cell activity. This suppression can be reversed both in vitro and in vivo by indomethacin. This study has important implications for the possible clinical use of indomethacin in the potentiation of in vivo and in vitro LAK cell activity for immunotherapeutic protocols.     

Characterization of IL-2-induced murine cells which exhibit ADCC activity

The incubation of murine splenocytes in recombinant interleukin 2 (IL-2) gives rise to both lymphokine-activated killer (LAK) cells capable of lysing fresh tumor cells and cells capable of mediating antibody-dependent cellular cytotoxicity (ADCC) in the presence of anti-H2 allosera. A similarity between these two IL-2-induced cell populations was found. The precursors of the cells mediating these activities were shown to be ASGM1 positive, Thy 1 negative, and radiosensitive. Cells taken from the spleen, thymus, and bone marrow were able to mediate ADCC after culture in IL-2. The effector cell was either Thy 1 positive or negative and was less affected by anti-Thy 1 plus C' treatment than cells which mediated LAK activity. In addition ADCC was exhibited in IL-2-cultured splenocytes from various murine strains and correlated with their LAK activity with one exception. While IL-2-cultured C57BL/6 splenocytes exhibited LAK activity similar to that of C3H LAK cells, no ADCC activity could be demonstrated in C57BL/6 cells. Study of the difference in the ability of these two strains to mediate ADCC revealed that IL-2-induced FcR+ cells in C3H thymocytes, but not in C57BL/6 thymocytes. Based on FACS analysis and on the radiosensitivity of the induction of both FcR+ cells and ADCC, it was suggested that IL-2 was expanding a small FcR+ cell population rather than inducing an increase in FcR density on the cell surface. The relationship between the IL-2-induced ADCC mediator and other IL-2-induced cells, as well as ADCC effector cells, and the possible implications of the results for the in vivo therapy of cancer based on ADCC are discussed.     

Synergistic antitumor activity of interleukin-2 and cimetidine against syngeneic murine tumor

Summary Cimetidine, an H2 histamine receptor antagonist, is a potent immunomodulating agent, which acts by inhibiting suppressor T lymphocyte function. The present work investigated the effect, if any, of cimetidine on interleukin-2 (IL-2)-induced natural killer (NK) and lymphokine-activated killer (LAK) cell activities, and on in vivo antitumor activity using syngeneic colon 26 adenocarcinoma as the model. Mimicking the clinical conditions, all in vitro experiments were evaluated with the splenocytes prepared from tumor-bearing BALB/c mice. Ten days after subcutaneous inoculation of tumor cells (5 × 10⁵), animals were treated intraperitoneally daily with phosphate-buffered saline (PBS), cimetidine (2 mg kg^–1 day^–1), IL-2 (300 000 IU/day), or cimetidine plus IL-2 for 7 consecutive days. The treatment of IL-2 plus cimetidine increased NK and LAK cell activities significantly and synergistically at the end of the treatment (i.e. on day 18) as well as 1 week after the treatment (i.e. on day 25), in comparison with those of the control groups (PBS, cimetidine alone, IL-2 alone). Also, in vivo antitumor activity, as analyzed by a Kaplan-Meier life table with the log-rank test, revealed a significantly prolonged survival in the group treated with IL-2 plus cimetidine compared to the control groups. Phenotyping performed on the murine splenocytes on day 18 indicated a significant reduction in Lyt2-positive cells in the cimetidine-treated group in comparison with the PBS group. A significant increase in asialo GM1-positive cells and IL-2-receptor-positive cells was detected in the group treated with IL-2 plus cimetidine in comparison with the PBS and IL-2 control groups. Therefore, this study indicates a synergistic enhancement of IL-2-induced NK and LAK cell activities in tumor-bearing hosts by cimetidine, a noncytotoxic inhibitor of suppressor T function, and a significantly prolonged survival of tumor-bearing animals treated by IL-2 plus cimetidine. It also suggests the clinical potential of combination therapy of IL-2 with cimetidine.     

Indomethacin up-regulates the generation of lymphokine-activated killer-cell activity and antibody-dependent cellular cytotoxicity mediated by interleukin-2

Prostaglandins can inhibit the generation of lymphokine-activated killer (LAK) cells by interleukin-2 (IL-2) whereas indomethacin augmented the induction of LAK cells by inhibiting prostaglandin synthesis. In the present study we demonstrate that prostaglandin E2 substantially inhibited the generation of both LAK and antibody-dependent cellular cytotoxicity (ADCC) activity by IL-2. In addition, indomethacin enhanced the induction of LAK activity and ADCC in splenocytes exposed to IL-2 in vitro. The effect of indomethacin was dose-dependent, reaching an optimal effect at 1 microM when 100-1000 units/ml IL-2 were employed. The effect of indomethacin on the generation of ADCC was seen in cells taken from both tumor-bearing mice and normal mice. ADCC induced by IL-2 was augmented by culturing cells from the spleen, liver and lungs, in the presence of indomethacin. ADCC induced in the presence of IL-2 and indomethacin was mediated by cells that were mainly plastic non-adherent cells and expressed the asialo-GM1 glycolipid. The potential of indomethacin in combined therapy with cytokines and specific anti-tumor monoclonal antibodies is discussed.     

Immunopotentiating activity of nigerooligosaccharides for the T helper 1-like immune response in mice

The immunopotentiating activity of nigerooligosaccharides (NOS), a mixture of nigerose, nigerosyl glucose and nigerosyl maltose, was studied in vitro and in vivo in mice. Mitogen-induced proliferation of splenocytes from normal mice was augmented in a dose-dependent manner by nigerose of NOS. NOS enhanced interleukin 12 (IL-12) and interferon-gamma (IFN-gamma) production by normal splenocytes in the presence of the potent IL-12 inducer, heat-killed Lactobacillus plantarum L-137, in vitro. Consistent with the in vitro finding, L. plantarum L-137-induced IL-12 production and IL-2-induced IFN-gamma production were augmented in mice fed with a 14.6% NOS diet for 2 weeks compared with mice fed with a control diet. Notably, mice fed with the NOS diet showed significantly longer survival time than the control mice after the induction of an endogenous infection by administering 5-fluorouracil in a lethal dose. Taken together, these results suggest that NOS may exert immunopotentiating activity through the activation of an IL-12-dependent T helper 1-like immune response.     

Postoperative administration of interleukin-12 significantly enhances the anti-tumor immune response of MBT-2 bladder cancer bearing mice

This study, using the MBT-2 murine bladder tumor model, mainly investigated the role of interleukin-12 (IL-12) in the specific antitumor immune response of a tumor-bearing host when systemically administrated after surgery. Day 17 tumor-bearing mice (D17TBM) along with non-tumor bearing naive mice were treated with daily intraperitoneal (i.p.) injection of IL-12 (0.25 microg/mouse) from day 18 to day 24 for a total of 7 doses. Their splenocytes were obtained on Day 31 for natural killer cells (NK), lymphokine activated killer cells (LAK) and cytotoxic T lymphocyte (CTL) activity assay and lymphocyte subsets phenotypic analysis. The tumor suppression effect of systemic IL-12 administration was evaluated based on the tumor outgrowth of the higher number of tumor cells rechallenged 24 hours after resectioning of the primary tumor. After evaluation on Day 31, the result of in vitro cytotoxicity assay revealed that systemic administration of IL-12 mainly enhanced the splenic LAK and CTL activities in non-tumor-primed naive mice, and the NK activity in tumor-primed D17TBM, respectively. However, in vivo administration of IL-12 with or without IL-2 failed to upgrade the proportions of either CD4+ CD44+ or CD8+ CD44+ T cells subsets in the spleens and regional inguinal lymph nodes (LNs) of both the D17TBM and naive mice. However, the splenic CD8+ CD44+ T-cell subset in the IL-12-treated D17TBM increased prominently after further culturing in the presence of IL-2 400 units/ml plus IL-12 25 ng/ml for 4 days. The fact that systemic administration of IL-12 significantly suppressed the outgrowth of Day-18 challenged tumor cells, especially in D17TBM, clearly indicates that the established specific antitumor immunity in tumor-primed D17TBM was efficiently augmented. From the results of this study, we conclude that, after surgical resection of a primary tumor, systemic administration of IL-12 can be an effective adjuvant therapy because it demonstrates a significant augmentation effect on the tumor-specific immune response in the tumor-primed host.     

Inhibition of human NK cell and LAK cell cytotoxicity and differentiation by PGE2

Activation of natural killer (NK) activity K562 target cells from nonadherent (NA) lymphocytes by interleukin 2 (IL-2) was inhibited marginally PGE2 (30-3000 nM). PGE2 did not effectively suppress the NK activity of IL-2-activated cells. The NK activation and acquisition of resistance to PGE2-mediated suppression of NK activity were dependent on protein synthesis. When NA cells were incubated with IL-2 for 3 or more days to generate lymphokine-activated killer (LAK) activity against Raji target cells, PGE2 only partially inhibited the activation of NK/LAK activity by an optimal dose of IL-2 (10 U/ml). The activation of NK/LAK activity by a suboptimal dose of IL-2 (0.1 U/ml) was inhibited by PGE2. When the NK/LAK activity of IL-2-activated cells was assessed in the presence or absence of PGE2, the LAK activity was more sensitive than the NK activity to PGE2-mediated suppression.     

EBV-induced molecule 1 ligand chemokine (ELC/CCL19) promotes IFN-gamma-dependent antitumor responses in a lung cancer model

The antitumor efficacy of EBV-induced molecule 1 ligand CC chemokine (ELC/CCL19) was evaluated in a murine lung cancer model. The ability of ELC/CCL19 to chemoattract both dendritic cells and T lymphocytes formed the rationale for this study. Compared with diluent-treated tumor-bearing mice, intratumoral injection of recombinant ELC/CCL19 led to significant systemic reduction in tumor volumes (p < 0.01). ELC/CCL19-treated mice exhibited an increased influx of CD4 and CD8 T cell subsets as well as dendritic cells at the tumor sites. These cell infiltrates were accompanied by increases in IFN-gamma, MIG/CXCL9, IP-10/CXCL10, GM-CSF, and IL-12 but a concomitant decrease in the immunosuppressive molecules PGE(2) and TGFbeta. Transfer of T lymphocytes from ELC/CCL19 treated tumor-bearing mice conferred the antitumor therapeutic efficacy of ELC/CCL19 to naive mice. ELC/CCL19 treated tumor-bearing mice showed enhanced frequency of tumor specific T lymphocytes secreting IFN-gamma. In vivo depletion of IFN-gamma, MIG/CXCL9, or IP-10/CXCL10 significantly reduced the antitumor efficacy of ELC/CCL19. These findings provide a strong rationale for further evaluation of ELC/CCL19 in tumor immunity and its use in cancer immunotherapy.     

IL-4 inhibits the costimulatory activity of IL-2 or picolinic acid but not of lipopolysaccharide on IFN-gamma-treated macrophages

We reported previously that IL-2 induces tumoricidal activity in IFN-gamma-treated murine macrophages. The present study was performed to investigate the regulation of IL-2-dependent tumoricidal activity in murine macrophage cell lines. The v-raf/v-myc-immortalized murine macrophage cell lines ANA-1, GG2EE, and HEN-CV did not express constitutive levels of cytotoxic activity against P815 mastocytoma cells. Moreover, these macrophage cell lines did not become tumoricidal after exposure to IL-4, IFN-gamma, IL-2 or LPS. However, these macrophages developed cytotoxic capabilities after incubation with either IFN-gamma plus IL-2 or IFN-gamma plus LPS. IL-4 inhibited IFN-gamma plus IL-2- but not IFN-gamma plus LPS-induced tumoricidal activity. This effect of IL-4 was not restricted to v-raf/v-myc-immortalized macrophage cell lines because similar results were obtained by using a macrophage cell line that was established from a spontaneous histiocytic sarcoma. The suppressive activity of IL-4 on the ANA-1 macrophage cell line was dose-dependent (approximately 12-200 U/ml) and was neutralized by the addition of anti-IL-4 mAb. IL-4 decreased the IFN-gamma-induced expression of mRNA for the p55 (alpha) subunit of the IL-2R in ANA-1 macrophages. Therefore, at least one mechanism by which IL-4 may have inhibited IFN-gamma plus IL-2-induced tumoricidal activity was by reducing macrophage IL-2R alpha mRNA expression. We have previously reported that picolinic acid, a tryptophan metabolite, is a costimulator of macrophage tumoricidal activity. We now report that IL-4 also inhibited IFN-gamma plus picolinic acid-induced cytotoxicity in ANA-1 macrophages. We propose that IL-2 and picolinic acid may have a common mechanism of action that is susceptible to IL-4 suppression.     

Correlation of immunomodulatory and therapeutic activities of interferon and interferon inducers in metastatic disease

The mechanism of therapeutic activity of recombinant murine interferon-gamma (rMu IFN-gamma) and the IFN inducer polyinosinic-polycytidylic acid solubilized with poly-L-lysine in carboxy methyl cellulose (pICLC) in treating metastatic disease was investigated by comparing effector cell augmentation with therapeutic activity in mice bearing experimental lung metastases (B16-BL6 melanoma). Effector cell functions in spleen, peripheral blood, and lung (the organ with tumor) were tested after 1 and 3 weeks of rMu IFN-gamma or pICLC administration (intravenous, three times a week). In these studies, natural killer (NK), lymphokine-activated killer (LAK), cytolytic T lymphocytes (CTL) (against specific and nonspecific targets), and macrophage tumoricidal and tumoristatic activities were measured. rM IFN-gamma and pICLC had therapeutic activity and immunomodulatory activity in most assays of immune function examined. Specific CTL activity of pulmonary parenchymal mononuclear cells (PPMC), but not in splenocytes or peripheral blood lymphocytes (PBL), during week 3 and not during week 1, correlated with the therapeutic activity of rMu IFN-gamma and of pICLC. Macrophage tumoricidal activity in PPMC, but not in alveolar macrophages, also correlated with the therapeutic activity of rMu IFN-gamma, but the opposite was true for the therapeutic activity of pICLC. NK activity of PPMC, but not of splenocytes or PBL, during week 1 correlated with the therapeutic activity of pICLC; in contrast, NK activity at any site did not correlate with the therapeutic activity of rMu IFN-gamma. LAK activity at any site did not correlate with the therapeutic activity of either agent.     

Immunomodulating effects in vitro of interleukin-2 and interferon-gamma on human blood and bone marrow mononuclear cells

Mononuclear cells (MNC) derived from peripheral blood (PBMNC) of 23 normal donors and 4 AIDS patients, and from bone marrow (BMMNC) of 15 normal donors were incubated at 37 degrees C in culture medium alone or in the presence of either natural or recombinant human interleukin-2 (IL-2) or recombinant human interferon-gamma (IFN-gamma; 1-1,000 U/ml). The cultured cells were washed on days 1, 4 or 7 and tested for various immune functions in vitro and for cell surface phenotype. IL-2, but not IFN-gamma, was found mitogenic for both PBMNC and BMMNC. The natural killer (NK) activity of both PBMNC and BMMNC was the only function tested that was markedly augmented (over 100-fold compared to medium control) by both lymphokines. Pretreatment of PBMNC with IL-2 at greater than or equal to 10 U/ml profoundly suppressed (up to 90%) various functions, such as mitogenic responses (phytohemmagglutinin, concanavalin A, pokeweed mitogen), allogeneic mixed leukocyte reaction, antibody production and T cell colony formation in agar. In contrast, some BMMNC functions were elevated at low doses of IL-2 and IFN-gamma, and significant suppression of BMMNC was seen only with high doses of IL-2 (greater than or equal to 100 U/ml) and IFN-gamma (1,000 U/ml). IL-2 was by far more effective than IFN-gamma in both the amplification of NK activity and the suppression of most of the other functions. IL-2, but not IFN-gamma, was found to activate/induce suppressor cells and increased the proportion of Leu-2+ (CD8) cells in PBMNC; the suppressive effect was time- and dose-dependent. The IL-2-induced suppression could be diminished by inclusion of anti-IL-2 antibody during the pretreatment phase. Similar suppressive effects were noted in PBMNC from AIDS patients. These findings suggest that: (a) high-dose IL-2 may elicit immunosuppression which can be mediated by nondiscriminative highly cytotoxic cells (i.e. lymphokine-activated killer cells) and/or by noncytotoxic, nonspecific suppressor cells, and (b) that PBMNC respond differently to the lymphokines than do BMMNC.     

Anti-tumor cytotoxic potential and effect on human bone marrow GM-CFU of human LAK cells generated in response to various cytokines.

The effects of various recombinant cytokines i.e. IL-1 alpha, IL-3, IL-4, IL-6, IFN-gamma, TNF-alpha and GM-CSF used either alone or in combination with IL-2, were investigated in this study. First, their capacity to induce killer cells from human PBL was examined by evaluating the degree of killing of human NK-sensitive K562 or NK-resistant Daudi cells. Second the effects of these cytokines, LAK cells (at 1/1, 2/1, 4/1 ratio LAK effectors/bone marrow cell targets) and of the supernatants from washed killer cell cultures, were examined on the colony forming ability of human bone marrow for GM-CFU in vitro. Various degrees of NK activity against K562 was observed in PBL stimulated with the cytokines, whereas LAK activity was found only with IL-2 alone. Culture of PBL with IL-2 + IL-1 alpha or IL-2 + IL-6 or IL-2 + GM-CSF resulted in the highest LAK killing. However, addition of TNF-alpha, or IFN-gamma to IL-2 in cultures resulted in a significant suppression of LAK cell activity. Addition of IL-1 alpha, IL-2, IL-3, and IL-4 to BM cultures had little or no effect on day 14 GM-CFU, whereas addition of IL-6 and GM-CSF resulted in a stimulatory effect. LAK cells induced with IL-2 alone had no significant suppressive effects on GM-CFU.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential expression and tissue compartmentalization of the inflammatory response following thermal injury

Studies have shown that both animal tissue-fixed immune cells and human peripheral blood mononuclear cell (PBMC) functions are altered after burn injury. Additional studies suggest that the burn injury-induced alterations in these divergent cell populations from different species are similar. It remains unknown, however, whether the observed changes in animal tissue-fixed immune cell function following thermal injury also occurs to a similar extent in the PBMC population. The aim of our study was to compare PBMC and tissue-fixed immune cell functions from the same animal using a murine burn model. At 7 days post-burn, mice were more susceptible to sepsis and delayed type hypersensitivity responses were suppressed. Splenocytes isolated from injured mice displayed suppressed proliferation and increased IL-10 production. In contrast, PBMC from injured mice displayed suppressed proliferation, IL-2 and IFN-gamma production. Splenic macrophage nitric oxide, PGE(2), TNF-alpha, IL-6 and IL-10 production was enhanced post-burn and IL-12 production was suppressed. PBMC from such animals displayed enhanced PGE(2) production and suppressed IL-6 and IL-12 production. These results indicate that while an immunosuppressive Th(2) phenotype (increased IL-10 and/or suppressed IL-2, IFN-gamma) was induced in both the splenic and PBMC compartments post-injury, differential expression and dimorphism in the response also exists. Thus, the assessment of only PBMC function in burn patients may not accurately reflect the patient's actual immune status at the tissue level.     

Interferon-gamma reduces macrophage-suppressive activity by inhibiting prostaglandin E2 release and inducing interleukin 1 production

Normal peritoneal M phi of C3H/HeN mice were able to suppress lymphocyte proliferation in a dose-dependent fashion when added to Con A-pulsed spleen cell cultures. However, M phi-suppressive activity could be partially or completely reduced by in vitro pre-exposure to nonimmune IFN-alpha or immune recombinant IFN-gamma. For both IFN-alpha and IFN-gamma, reduction of M phi suppression was marginal at 10(1) U/ml and became highly significant at 10(2) to 10(3)/ml. The ability of IFN-alpha and IFN-gamma to modulate M phi suppression appears to be related to distinct mechanisms. In fact, impairment of M phi suppression by IFN-alpha occurred in parallel to the decrease of M phi capacity to produce PGE2 and the oxygen intermediate O2-, two molecules responsible for M phi-suppressive activity. In contrast, M phi exposed to IFN-gamma showed only impairment of PGE2 production, whereas O2- release was not significantly affected. Furthermore, at variance with IFN-alpha, IFN-gamma directly stimulated M phi to synthesize and release IL 1, a monokine known to promote lymphocyte proliferation.     

Murine lymphokine-activated killer (LAK) cells: phenotypic characterization of the precursor and effector cells

Murine and human lymphocytes incubated in recombinant interleukin 2 (RIL 2) generate a population of cytotoxic cells (lymphokine-activated killer cells [LAK]), which are able to lyse a wide array of fresh tumor cells but do not lyse fresh normal cells. Intravenous administration of these cells with the concomitant administration of RIL 2 can eliminate established pulmonary and hepatic metastases in mice. To characterize the cell that has in vitro LAK activity, we subdivided murine lymphocytes by lysing select subpopulations with the use of complement and antibodies against lymphocyte surface markers or by fluorescence-activated cell sorting. Thy-1.2-negative splenocytes were found to generate near normal amounts of LAK activity after RIL 2 incubation. Small and inconsistent LAK cell activity was generated from Thy-1.2-positive splenocytes. Ia-positive and surface immunoglobulin-positive splenocytes had little or no LAK precursor capability and did not appear to be necessary for LAK activation. Treatment of splenocytes with anti-asialo GM1 (anti-ASGM1) heterosera and complement markedly decreased their ability to generate LAK activity. At the effector stage, cytotoxic cells were of the Thy-1.2-positive, Ia-negative phenotype. Ia-depleted cells were separated into subpopulations bearing or not bearing the gamma Fc receptor (gamma FcR). The majority of cytotoxicity resided in gamma FcR-positive cells. Thus the precursors of murine LAK cells are "null" lymphocytes bearing neither T nor B cell surface markers but develop the Thy-1.2 cell surface marker in vitro, in association with the development of lytic activity for fresh tumor cells after stimulation by RIL 2.     

IL-4 inhibits IL-2-mediated induction of human lymphokine-activated killer cells, but not the generation of antigen-specific cytotoxic T lymphocytes in mixed leukocyte cultures

Human rIL-4 was studied for its capacity to induce lymphokine-activated killer (LAK) cell activity. In contrast to IL-2, IL-4 was not able to induce LAK cell activity in cell cultures derived from peripheral blood. IL-4 added simultaneously with IL-2 to such cultures suppressed IL-2-induced LAK cell activity measured against Daudi and the melanoma cell line MEWO in a dose-dependent way. IL-4 also inhibited the induction of LAK cell activity in CD2+, CD3-, CD4-, CD8- cells, suggesting that IL-4 acts directly on LAK precursor cells. IL-4 added 24 h after the addition of IL-2 failed to inhibit the generation of LAK cell activity. Cytotoxic activity of various types of NK cell clones was not affected after incubation in IL-4 for 3 days, indicating that IL-4 does not affect the activity of already committed killer cells. No significant differences were observed in the percentages of Tac+, NKH-1+ and CD16+ cells after culturing PBL in IL-2, IL-4 or combinations of IL-2 and IL-4 for 3 days. IL-4 also inhibited the activation of non-specific cytotoxic activity in MLC, as measured against K-562 and MEWO cells. In contrast, the Ag-specific CTL activity against the stimulator cells was augmented by IL-4. Collectively, these data indicate that IL-4 prevents the activation of LAK cell precursors by IL-2, but does not inhibit the generation of Ag-specific CTL.     

Estrogenic compounds suppressed interferon-gamma production in mouse splenocytes through direct cell-cell interaction

Here, we reported the effects of 17beta-estradiol (E2), isoflavone genistein (Gen), and daidzein (Dai) on the production of interferon (IFN)-gamma by splenocytes isolated from C57BL/6N mice. When mouse splenocytes were stimulated with lipopolysaccharide, E2, Gen, and Dai suppressed the production of IFN-gamma. However, when only nonadherent cell populations of splenocytes were tested, none of these estrogenic compounds suppressed IFN-gamma production. This result indicates that IFN-gamma production by nonadherent cell populations of splenocytes treated with estrogens is regulated by adherent cell populations. Moreover, direct cell-cell interaction between both populations was necessary for suppression of IFN-gamma production by nonadherent populations. In addition, E2 conjugated with bovine serum albumin inhibited IFN-gamma production as well as E2. This result suggests that the plasma membrane-associated estrogen receptor plays a prominent role in this suppression mechanism.     

Mechanisms of strain-dependent development of mast cells from mouse splenocytes

Mast cell development from spleen cells was not triggered by prostaglandin E1 (PGE1) or dibutyryl cAMP (db-cAMP) during a 12 day culture when the spleen cells were obtained from C57BL/6N and DBA/1 mice, but mast cells did develop when the spleen cells were obtained from C3H/HeN, BALB/c and ICR mice. A lack of endogenous IFN-gamma in the initial 2 days of the culture period was responsible for the failure. This was confirmed by adding neutralizing anti-IFN-gamma antibody and rIFN-gamma to the cultures and by determining IFN-gamma levels in the spleen cell cultures. Th1 cells in the spleens of C57Bl and DBA/1 mice were much more sensitive to PGE1 and db-cAMP than Th1 cells from other inbred mice strains, and consequently, IFN-gamma production was inhibited in spleen cell cultures of C57BL and DBA/1 mice on addition of PGE1 or db-cAMP. Furthermore, the different sensitivities of Th1 cells to PGE and db-cAMP were dependent on the different levels of IL-12 p40 monomers or homodimers in the spleen cell cultures. As the endogenous specific inhibitors of IL-12 p70 (heterodimers of p40 and p35), large amounts of IL-12 p40 monomers or homodimers in the spleen cell cultures of C57BL and DBA/1 mice enhanced the ability of PGE1 and db-cAMP to inhibit IFN-gamma production by antagonizing the activity of IL-12 heterodimers. These results indicate that the strain-dependent development of mast cells from mouse splenocytes is related to endogenous IFN-gamma levels, which are regulated by PGE, db-cAMP, IL-12 p70 and IL-12 p40.