Systemic administration of IL-2 induces lymphokine-activated killer (LAK) cells capable of killing macrophages in various tissues.

Murine lymphokine-activated killer (LAK) cells induced by systemic high-dose recombinant human interleukin 2 (IL-2) administration lysed fresh syngeneic peritoneal macrophages (M phi). LAK cells lysed resident peritoneal M phi and M phi activated in vivo with thioglycollate (TG), Corynebacterium parvum (C. parvum), or Bacillus Calmette-Guérin (BCG). The induction of anti-M phi cytolytic activity was seen in the spleen, liver, lung, lymph nodes and peritoneal cavity, but was not observed in the thymus. Fluorescence analysis revealed that the majority of infiltrated cells in the peritoneal cavity of IL-2-administered mice were Thy-1+, asialo GM1+, L3T4-, Ly2-. Surface marker analysis on peritoneal exudate cells (PEC) from IL-2-administered mice with depletion techniques using antibody (Ab) and complement (C) indicated that Thy-1+, asialo GM1+, L3T4-, Ly 2- cells were responsible for anti-M phi lysis. These studies indicate that the in vivo administration of IL-2 induces LAK cells capable of killing M phi in various tissues.     

The precursor cells of mouse lymphokine-activated killer (LAK) cells

Culture of mouse spleen cells with recombinant human interleukin 2 (r-IL 2) resulted in the generation of lymphokine-activated killer (LAK) cells, which could lyse a variety of tumor cells. Negative selection study using various kinds of antibodies and complement indicated that LAK precursor cells existed in both mature spleen lymphocytes and immature spleen null cells. LAK cells were also induced from lymph node cells but not from unfractionated thymocytes. However, hydrocortison (HC)-resistant thymocytes or PNA- thymocyte subpopulations were highly responsive to r-IL 2 and maturated into LAK cells after 5 day-culture with r-IL 2. Moreover, it was demonstrated that r-IL 2 allowed the induction of LAK cells from nude mouse spleen cells, but not from bone marrow cells and fetal liver cells.     

Induction of nonspecific cell-mediated cytotoxic reactivity from non-immune spleen cells treated with aloctin A

Culturing of mouse spleen cells with aloctin A (Alo A), a lectin having anti-tumor activity, resulted in the induction of cells cytotoxic to syngeneic and allogenic tumor cells in vitro. Alo A-induced killer cells could be generated from spleen cells of natural killer cell-deficient beige mice but not from those of T cell-deficient nude mice. The results of cytotoxicity assay after treatment with antisera plus complement indicated that the killer cells mainly consisted of Thy 1+, Lyt 1-2+, asialo GM1(1)-T cells. Their cytotoxic activity was markedly augmented by the presence of Alo A during the assay for cytotoxicity. The assay of IL2 in the culture fluid from Alo A-treated spleen cells and the effect of dexamethasone (DEX) on IL2 production from Alo A-stimulated spleen cells and on generation of killer cells suggested that generation of Alo A-induced killer cells was closely associated with the amount of IL2 released from the spleen cells in culture.     

Inhibition of natural killer activity by calcitonin gene-related peptide

The effect of calcitonin gene-related peptide (CGRP) on natural killer (NK) cell activity in spleen cells from Balb/c mice and nude mice was studied. CGRP dose-dependently (10(-9) to 10(-7) M) inhibited NK activity of spleen cells from both strains of mice. This inhibitory effect was observed at the effector to target ratios of 12.5:1 to 100:1. Maximum inhibition by 10(-7) M CGRP was about 60%. The inhibition of NK activity by CGRP was also observed in anti-Thy 1.2 plus complement treated Balb/c spleen cells. Furthermore, when cells were treated with 10(-9) to 10(-7) M CGRP the concentration of intracellular cyclic AMP increased in spleen cells of nude mice. The characteristics of these cells were similar to those of NK cells, (1) being petri dish and nylon wool nonadherent, (2) expressing asialo GM1 antigen, and (3) lacking readily detectable Thy 1 antigen and immunoglobulin. In addition, the intravenous injection of asialo GM1 completely abolished NK activity in spleen cells from nude mice and the increase in intracellular cyclic AMP in spleen cells by CGRP was less in spleen cells from mice given an anti-asialo GM1 injection. Our present study suggests that CGRP inhibits NK cell activity by increasing the intracellular cyclic AMP concentration. CGRP may be implicated in the regulation of NK function.     

A glycolipid on the surface of mouse natural killer cells

Cytotoxic treatment with rabbit antiserum raised against purified glycosphingolipid “asialo GM1” was capable of eliminating natural killer (NK) activity of spleen cells from different inbred mouse strains including CBA/J, C57BL/6, BALB/c, AKR, and athymic nude mice. The anti-asialo GM 1 antiserum showed little cross-reactivity with structurally related glycolipids, e.g. GM 1, GD 1 b and asialo GM 2 in the microflocculation test. The specific reactivity of this antiserum with NK cells was confirmed by the quantitative absorption of anti-NK activity with graded amounts of asialo GM 1 but not with other glycosphingolipids. The absorption of anti-brain-associated T cell antigen (anti-BAT) with asialo GM 1 also effectively diminished its anti-NK activity, leaving the ability to kill T cells intact. This suggests that the antibody to asialo GM 1 is responsible for the anti-NK activity contained in the anti-BAT antiserum. In contrast to the extreme sensitivity of NK cells to anti-asialo GM 1, alloreactive cytotoxic T killer cells generated in the mixed lymphocyte culture were not killed by anti-asialo GM 1 and complement. These results indicate that asialo GM 1 is expressed on mouse NK cells in a high concentration.     

Recombinant interleukin-2 inhibits growth of human tumor xenografts in congenitally athymic mice

Administration of human recombinant interleukin-2 (RIL-2) into congenitally athymic (nu/nu) mice carrying subcutaneous transplants of HeLa, HU 609T and T24B human carcinoma cells partially inhibited growth of the human tumor xenografts. In vitro activation of nu/nu spleen cells with human RIL-2 resulted in generation of killer cells showing in the 51Cr cytotoxicity assay similar levels of cytolysis as RIL-2-activated spleen cells from heterozygous (nu/+) mice. The RIL-2-activated (LAK) cells were cytotoxic for a variety of mouse and human tumors, reaching the peak of their cytotoxic activity after 3 days of cultivation in the RIL-2-containing medium. The cytotoxic activity of activated nu/nu spleen cells was significantly reduced by treatment with antibody against glycolipid asialo GM1, the differentiation antigen of natural killer (NK) cells. This finding suggests that in addition to the conventional, asialo GM1- LAK cells, asialo GM1+ activated NK cells participated in the cytotoxicity displayed by the IL-2-activated nu/nu killer spleen cells.     

Lymphokine-activated killer cells and aging in mice: significance for defining the precursor cell

The present study was undertaken to define the cell populations which mediate lymphokine-activated killer (LAK) cell activity in mice. Because old mice exhibit markedly decreased to nondetectable natural killer (NK) cell activity, this age-associated change provided an advantageous system to examine the contribution of NK and T cells to LAK activity. Spleen cells from either young (6-9 weeks) or old (20-26 months) mice were cultured with 1000 units/ml of recombinant interleukin 2 (rIL 2) for 3-5 days. The cells were then tested in a 51 Cr-release assay for their cytotoxicity against NK-resistant fresh tumor cells (MCA-102). The LAK activity exhibited by spleen cells from old mice following 5 days of culture was equivalent to that developed by spleen cells of young mice. This result was contrary to what would be anticipated if mature NK cells comprise the primary precursors of LAK activity, and required further elucidation. The Thy-1 and asialo GM1 (ASGM1) phenotypes of LAK precursor and effector cells were therefore examined by depletion techniques using the appropriate antibodies plus complement. The results using spleen cells harvested after 5 days of culture with rIL 2 showed that LAK effector cells which developed from spleen cells of both young and old mice were predominantly Thy-1+ (85.3% young; 91.8% old) and some coexpressed ASGM1. Spleen cells were treated prior to culture to study the precursor cells. Development of LAK activity by spleen cells from both young and old mice was greatly reduced by pretreatment with anti-ASGM1 plus complement. However, since spleen cells of old mice exhibit very low mature NK activity, these data suggest that the LAK precursors, at least in old mice, may be ASGM1+ NK precursor cells rather than mature ASGM1+ NK effector cells. In addition, treatment with anti-Thy-1 plus complement inhibited generation of a significant proportion of LAK activity only in the spleens of old mice, suggesting a qualitative difference in LAK precursor cells with age and supporting the heterogeneity of the cells which are capable of developing LAK activity.     

Lymphokine-Activated Killer Cells in Mouse Bone Marrow Chimaeras The Relationship to Natural Killer Cells and to Alloreactive Cytotoxic T Cells

Spleen cells from mouse bone marrow chimaeras were cultured in vitro in mixed lymphocyte cultures (MLC) or in the presence of interleukin 2 (IL-2) without the added alloantigen. Precursors for the nonspecific cytotoxic cells (in this study: lymphokine-activated killer (LAK) cells) lysing natural killer (NK) cell-sensitive YAC-1 lymphoma could be found 10-12 days after the bone marrow reconstitution, simultaneously with the appearance of the NK activity. The ability of LAK cells to lyse NK-resistant tumour targets as well was demonstrated using the P 815 mastocytoma cell line; reactivity against this target was demonstrable 1 week later than the appearance on YAC-1 lysing cells. Phenotypically LAK cells derived from spleen cell cultures of bone marrow chimaeras did not differ from LAK cells derived from normal spleen cell cultures: precursors resided within the Thy 1-, asialo-GM1+ cell population, and effectors expressed both of these antigens. Splenic NK cells of early bone marrow chimaeras (up to 14-18 days after the bone marrow reconstitution) were Thy 1+ cells, and thus LAK cells of bone marrow chimaeras were not derived from these Thy 1+ NK cells. The treatment of effector cells with anti-Thy 1 antibody plus complement (C) abolished the lytic activity totally. However, these cells were not cytotoxic T cells, since alloreactivity, as an indication of the T-cell cytotoxicity, could not be demonstrated until 4-5 weeks after the bone marrow reconstitution.     

Augmentation of lymphokine-activated killer cell activity in vitro by aloctin A

Culturing of mouse spleen cells with IL2-containing MLA144 conditioned medium (MLA144 CM) resulted in the induction of cells cytotoxic to syngeneic and allogeneic tumor cells in vitro. The cytotoxicity was markedly augmented by the presence of aloctin A (Alo A), a lectin having anti-tumor activity, during the assay for the cytotoxicity. The results of cytotoxicity assay after treatment with antisera and complement indicated that the killer cells mainly consisted of Thy 1+, Lyt 1-2-, asialo GM1-T cells.     

Suppression of pulmonary tumour metastasis in mice by recombinant human interleukin-2: role of asialo GM1-positive cells.

Recombinant human interleukin-2 (rIL-2) suppressed metastatic tumour colony formation in the lungs of C57BL/6 mice bearing Lewis lung carcinoma (3LL). In tumour-bearing mice given rIL-2, non-specific killer cells that were cytotoxic not only against natural killer-sensitive YAC-1 cells but also against 3LL cells in an in vitro 51Cr-release assay were concomitantly induced as tumour metastasis was suppressed. These non-specific killer cells were mostly removed by treatment with anti-Thy 1.2 or anti-asialo GM1 antibody plus complement (C) in vitro but not with anti-Lyt 1.2 or anti-Lyt 2.2 plus C, indicating that they were positive for Thy 1 and asialo GM1 but not for Lyt 1 and Lyt 2. In order to explore the mechanism by which rIL-2 suppressed tumour metastasis, we examined the clearance of intravenously injected 51Cr-labelled 3LL cells in the lungs of mice given rIL-2. The rate of tumour cell clearance was increased. This enhanced clearance was almost completely removed by injecting anti-asialo GM1 antibody. In addition, the injection of anti-asialo GM1 antibody also depleted most of the non-specific killer cells induced by administering rIL-2. These results indicate that asialo GM1-positive cells are not only cytotoxic in vitro but also play a critical role in the clearance of 3LL cells in the lungs in vivo. Our results indicate that asialo GM1-positive cells play an important role as anti-metastatic effector cells in suppressing the metastasis of 3LL cells in mice given rIL-2.     

Administration of recombinant interleukin 2 to mice enhances production of hemopoietic and natural killer cells

Seven days of continuous perfusion of mice with human recombinant interleukin 2 (rIL 2) (approximately 3 X 10(4) U/day) increased the percentage of large mononuclear leukocytes (LML) among bone marrow, spleen, lymph node cells and liver interstitial cells (LIC). An increase in the lymphokine-activated killer (LAK) activity was evident in these organs. The greatest increase in the number of LML and in the LAK activity was observed among the liver interstitial cells (about 500-fold increase). The LML were nonphagocytic, Thy-1+, sIg-, Ly 2+, L3T4- and asialo Gm1+. Perfusion of athymic nude mice, or of thymectomized, irradiated radiation chimera, showed that the Thy-1+, LAK+ LML were the thymus and T lymphocyte-independent progeny of Thy-1- marrow precursors. The LML had no T cell function in a graft-vs.-host reactivity assay, neither did they have an inhibitory effect on T lymphocyte function in vivo. rIL 2 perfusion did not significantly affect the medullary hemopoiesis but did strongly enhance the extramedullary hemopoiesis, particularly within the interstice of the liver: the number of erythroid and myeloid cell was increased as well as the number of colony-forming units per spleen and colony-forming units per culture for various lineages (20-50-fold increment). These results show that in vivo, rIL 2 has a global enhancing effect on hemopoiesis together with a more selective influence on the production of LML.     

Analysis of splenic lymphocytes with high electrophoretic mobility in adult and aged nude mice

Electrophoretic mobility (EPM) and surface markers of splenic lymphocytes in adult (8 weeks old) and aged (over 1 year old) nude mice were investigated. Splenic lymphocytes in nude mice showed a bimodal pattern consisting of low mobility lymphocytes (LML) corresponding to B cells and high mobility lymphocytes (HML). The HML of nude mice showed the following immunological characteristics: (1) surface Ig- cells; (2) asialo GM1+ cells; (3) an increase in natural killer (NK) activity after depletion of B cells; (4) abrogation of the HML peak and NK activity after treatment with anti-asialo GM1 and complement. These findings suggested that HML in nude mice were NK cells. The mobility of NK cells was slightly lower than that of T cells in normal mice, although their histograms greatly overlapped each other. In the spleen cells of nude mice, there was a significant increase in the numbers of Thy-1+ cells and a decrease in the intensity of asialo GM1 antigen as a function of age. The surface markers of HML were Thy-1+- asialo GM1++ in adult nude mice, but were Thy-1+ asialo GM1+ in aged nude mice. However, although HML in aged nude mice became Thy-1+, these had almost the same EPM as those in adult nude mice.     

Inhibition of Natural Killer Activity by Calcitonin Gene-Related Peptide

The effect of calcitonin gene-related peptide (CGRP) on natural killer (NK) cell activity in spleen cells from Ba1b/c mice and nude mice was studied. CGRP dose-dependently (10 to 10 M) inhibited NK activity of spleen cells from both strains of mice. This inhibitory effect was observed at the effector to target ratios of 12.5:1 to 100:1. Maximum inhibition by 10^-7 M CGRP was about 60 %. The inhibition of NK activity by CGRP was also observed in anti-Thy 1.2 plus complement treated Ba1b/c spleen cells. Furthermore, when cells were treated with 10 to 10^-7 M CGRP the concentration of intracellular cyclic AMP increased in spleen cells of nude mice. The characteristics of these cells were similiar to those of NK cells, (1) being petri dish and nylon wool nonadherent, (2) expressing asialo GM₁ antigen, and (3) lacking readily detectable Thy 1 antigen and immunoglobulin. In addition, the intravenous injection of asialo GM₁ completely abolished NK activity in spleen cells from nude mice and the increase in intracellular cyclic AMP in spleen cells by CGRP was less in spleen cells from mice given an anti-asialo GM₁ injection. Our present study suggests that CGRP inhibits NK cell activity by increasing the intracellular cyclic AMP concentration. CGRP may be implicated in the regulation of NK function.     

Inhibition of Natural Killer Activity by Calcitonin Gene-Related Peptide

Abstract

The effect of calcitonin gene-related peptide (CGRP) on natural killer (NK) cell activity in spleen cells from Ba1b/c mice and nude mice was studied. CGRP dose-dependently (10 to 10 M) inhibited NK activity of spleen cells from both strains of mice. This inhibitory effect was observed at the effector to target ratios of 12.5:1 to 100:1. Maximum inhibition by 10^?7 M CGRP was about 60 %. The inhibition of NK activity by CGRP was also observed in anti-Thy 1.2 plus complement treated Ba1b/c spleen cells. Furthermore, when cells were treated with 10 to 10^?7 M CGRP the concentration of intracellular cyclic AMP increased in spleen cells of nude mice. The characteristics of these cells were similiar to those of NK cells, (1) being petri dish and nylon wool nonadherent, (2) expressing asialo GM₁ antigen, and (3) lacking readily detectable Thy 1 antigen and immunoglobulin. In addition, the intravenous injection of asialo GM₁ completely abolished NK activity in spleen cells from nude mice and the increase in intracellular cyclic AMP in spleen cells by CGRP was less in spleen cells from mice given an anti-asialo GM₁ injection. Our present study suggests that CGRP inhibits NK cell activity by increasing the intracellular cyclic AMP concentration. CGRP may be implicated in the regulation of NK function.     

Role of T and adherent cells in in vitro response of nude mouse spleen cells to bacterial lipopolysaccharide.

Thy 1.2-positive cells and adherent cells were depleted from nude mouse spleen cell suspensions by treatment with anti-Thy 1.2 antisera plus complement and by passage through Sephadex G-10 column, respectively. The results of 3H-TdR uptake and appearance of IgM containing cells induced in vitro by lipopolysaccharide (LPS) were similar in both untreated and depleted spleen cells. Our findings confirm T and adherent cell independence of LPS response in nude mice.     

Overnight incubation of mouse spleen cells in recombinant IL-2 generates cytotoxic cells with NK characteristics from precursors enriched with or devoid of LGL.

Interleukin-2 (IL-2) augments natural killer (NK) activity as well as generating effector cells named lymphokine activated killer cells (LAK) which are capable of lysing a wide spectrum of target cells. A large body of evidence has been accumulated to evaluate the relationship between NK and LAK cells and conflicting results have been reported. Our study was addressed to further analyse this relationship and in particular to investigate whether in a short incubation IL-2 is merely capable of augmenting the activity of pre-existing killer cells, or whether it can also promote the differentiation of precursor cells. Eighteen-hour culture of mouse spleen cells in human recombinant IL-2 induced a DNA-synthesis-independent generation of cytotoxic cells bearing an NK phenotype (aGM-1+, Thy1.2+/-, CD8-, CD4-). These were generated from precursor cells also bearing an NK phenotype, recovered either from low density Percoll fractions enriched in lytic cells with LGL morphology as well as from high density fractions devoid of LGL and cytotoxic activity.     

Expression of asialo GM1 by both Thy-1-positive and Thy-1-negative lymphocytes: evidence for modification of asialo GM1 by sialic acid

In order to determine the extent to which Thy-1-positive and Thy-1-negative lymphocytes express the asialo GM1 antigen, lymphocytes in normal spleen and lymph node were examined for simultaneous expression of the asialo GM1 and Thy-1.2 determinants. The results presented herein demonstrate that 55-57% of Thy-1.2-positive cells in spleen and 61-70% of Thy-1.2-positive cells in lymph node express asialo GM1. Furthermore, a significant frequency of Thy-1-negative cells in spleen (12-19%) and in lymph node (28-32%) also express asialo GM1. Since asialo GM1 has previously been shown to be absent on Ig-positive lymphocytes [9], these results establish that asialo GM1 is a marker shared by lymphocytes belonging to the T-cell lineage and lymphocytes apparently not committed to the T-cell to the T-cell lineage, most probably including natural killer (NK) cells. The implication of this finding as to the controversy regarding the possible relation of NK cells to T cells is discussed. Pretreatment of lymphocytes from spleen, thymus and lymph node with neuraminidase resulted in subsequent reactivity of 80-90% of these cells with anti-asialo GM1 anti-bodies. A smaller increase in asialo GM1 detection after neuraminidase treatment was seen with bone-marrow cells (65%). Protease treatment did not affect the subsequent reactivity of lymphocytes with anti-asialo GM1 antibodies. It is concluded that in situ enzymatic modification of asialo GM1 by the addition of sialic acid may be an important regulatory event in lymphocyte differentiation.     

A monoclonal antibody with reactivity to asialo GM1 and murine natural killer cells

A monoclonal antibody (MAb) was prepared by the fusion of murine SP2-O myeloma cells with BALB/cByJ spleen cells that were immunized with the glycolipid asialo GM1 adsorbed to naked Salmonella. The specificity of the IgM antibody obtained was defined using various glycolipids, cell extracts and saccharides in ELISA assays and thin-layer chromatography (TLC) immunoblots. The non-reducing terminal galactose is the immunodominant residue for this antibody; however, there is undetectable reactivity to free galactose, galactosylceramide or compounds with an alpha-linked galactose. The SH-34 antibody specifically lyses asialo GM1-expressing macrophages in the presence of complement and removes NK cells in vitro from spleen cell populations. When the specificity of the MAb was compared to that of a commercially available rabbit antiserum to asialo GM1, it was found that both cross-reacted with GM1 and asialo GM3 at high antibody concns; however, the MAb did not bind asialo GM2 while the rabbit antiserum showed substantial reactivity to this glycolipid. It is anticipated that this MAb will be useful for the study of murine and rat natural killer cells.     

Toxoplasma-induced activities of peritoneal and spleen natural killer cells from beige mice against thymocytes and YAC-1 lymphoma targets.

Homozygous (bg/bg) and heterozygous (bg/+) beige mice were infected with Toxoplasma gondii, and splenic and peritoneal natural killer (NK) cell activities were assayed against YAC-1 lymphoma (NK-YAC) and thymocyte (NK-THY) targets. Although uninfected bg/bg mice were devoid of NK-YAC activity when compared with bg/+ mice, NK-THY activity was at a completely normal level. Both effector cells showed NK-1.2+ Thy-1.2 +/- asialo GM1+ asialo GM2+ phenotype. T. gondii infection induced a marked augmentation in splenic NK-YAC activity of bg/+ mice, whereas a slight increase was shown in the bg/bg mouse spleen cells. On the other hand, the infection did not change the splenic NK-THY activity of either strain of mice. An increased expression of Thy-1.2 antigen was shown on both NK-THY and NK-YAC effector cells from the infected mouse spleen. The T. gondii-induced augmentation was dramatic in the peritoneal cavity of the both mice. The activated peritoneal NK cells were of the NK-1.2- Thy-1.2+ asialo GM1 +/- asialo GM2+ phenotype and were considered to be generated from functionally inactive peritoneal cells. Splenic effector cells obtained from the infected mice were selectively depleted with target cell monolayer, whereas peritoneal cells from the infected mice were strongly absorbed by the target monolayers without selectivity. A weak but significant interferon (IFN) titer was detected in the peritoneum, but not in the spleen, of the infected mice. Most of the IFN titer was acid labile. Treatment with anti-IFN-alpha/beta resulted in partial decline of both NK and IFN activities of bg/bg mice, but not bg/+ mice. Thus, involvement of both IFN-alpha/beta and IFN-gamma in the generation of peritoneal NK cells and IFN-independent augmentation of splenic NK cells in toxoplasmosis were suggested.     

Characterization studies of suppressor cells in murine bone marrow chimaeras.

When BALB/c bone marrow cells were transferred to lethally X-irradiated C3H/He mice either intrasplenically (i.s.) or intravenously (i.v.), suppressor cells were detected by means of MLR coculture assays in the spleen of i.s. and i.v. chimaeras. Some but not all of the suppressor cells expressed a Thy 1.2 antigen, indicating that suppressor cells either sensitive or insensitive to anti-Thy 1.2 antibody plus complement treatment were generated in the spleen of i.s. and i.v. chimaeras. According to the examination of Lyt alloantigen expression on suppressor T cells, Lyt 1+2-, Lyt 1-2+, and Lyt 1+2+ suppressor T cells appeared to be present. The culture supernatants from several T-cell clones showed the suppressor activity against alloreactive MLR. Cell surface markers of these clones were composed of Lyt 1+2-, Lyt 1+2+ and Lyt 1-2+. In addition, since Carrageenan treatment abrogated the suppressor activity of plastic dish adherent cells, we conclude that some of them were composed mainly of macrophages.