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.     

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.     

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.     

Growth and metastatic behavior of human tumor cells implanted into nude and beige nude mice

The growth and metastatic behavior of several human tumor lines grown in adult nude mice, nude mice pretreated with antiserum against asialo GM1 glycoprotein, and beige nude mice were studied. The cell lines were all injected s.c. and i.v. A human colon carcinoma line was also injected into the spleen, and two human renal carcinoma lines were injected into renal subcapsule. All the tumor lines grew as fast or faster in adult nude mice compared with beige nude mice. There were no discernible differences in the production of experimental lung metastasis among the three groups of mice, but human colorectal carcinoma cells and human renal carcinoma cells produced more metastases in nude mice than in beige nude mice after intrasplenic or renal subcapsule injection, respectively. In vitro cytotoxicity assays confirmed that adult nude mice had high levels of natural killer (NK) cell activity whereas nude mice pretreated with anti-asialo GM1 serum and beige nude mice did not. The in vitro NK cell activity of nude mice was demonstrable against mouse lymphoma cells but not against human leukemia cells which were sensitive to lysis by human NK cells. These results suggest that the implantation of human tumor cells into beige nude mice, which are deficient in NK cell activity does not provide an advantageous model for the study of growth and metastasis of human neoplasms.Abbreviations NK natural killer - HCC human colon carcinoma - HRCC human renal cell carcinoma - anti-asGM1 anti-asialo GM1 - i.s. intrasplenic - RSC renal subcapsule - HBSS Ca²⁺- and Mg²⁺-free Hanks' balanced salt solution     

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.     

Recombinant interleukin 2 allows the differentiation of Thy 1.2+ LAK cells from nude mouse spleen cells

Culture of nude mouse spleen cells with recombinant human interleukin 2 (r-IL 2) resulted in the proliferation and generation of lymphokine-activated killer (LAK) cells which could lyse a variety of tumor cells. Flow cytometry study indicated that nude mouse spleen cells contained almost no Thy 1.2+ cells at the initial times of the culture, whereas LAK cells obtained from nude mouse spleen cells by culture with r-IL 2 (nude-LAK cells) expressed high intensity of Thy 1.2 antigen and lymphokine-activated cell-associated (LAA) antigen. The cytotoxic activity of nude-LAK cells was greatly reduced by treatment with anti-Thy 1.2 antibody plus complement but not with anti-Ly 1.2 or Ly 2.2 antibody plus complement treatment. Moreover, nude-LAK cells were resistant to the treatment with anti-asialo GM1 antibody plus complement, in contrast to resident nude natural killer (NK) cells. These data strongly suggested that r-IL 2 allowed the nude mouse spleen cells to differentiate into Thy 1.2+, Ly 1-,2-, asialo GM1- LAK cells which were distinct from Thy 1.2+, Ly 2+, asialo GM1- LAK cells induced from normal mouse spleen cells.     

Natural killer (NK) cells and graft-versus-host disease (GVHD): no correlation between the NK cell levels and GVHD in the murine P----F1 model

Graft-versus-host disease (GVHD) was induced in (CBA X C57BL/6) F1 mice by i.v. injection of 50 X 10(6) parental spleen cells. The GVHD induced an enhanced NK (anti-YAC-1) cytotoxicity during the first 2 weeks after the spleen cell transfusion. This cytotoxic activity was shown to be mediated by asialo GM1-positive, partially Thy-1-positive and nylon-wool (NW) non-adherent cells, thus being classical NK cells. Depletion of NK-cell activity from donor and/or recipient mice with anti-asialo GM1 antibody prior to the spleen cell transfer did not prevent the GVHD as judged by the splenomegaly assay. Also, when NK activity was potentiated with polyinosinic-polycytidylic acid (pIC), no effect on the GVHD was seen. These data suggest that NK cells are not crucial for the development of GVHD in this model.     

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.     

Enhanced natural killer cell activity in experimental murine encephalitozoonosis.

Spleen cells from mice infected with the protozoan parasite Encephalitozoon cuniculi demonstrated enhanced in vitro cytolysis of YAC-1 lymphoma cells. Selective cell depletion experiments showed that the dominant cell population mediating cytolysis of YAC-1 tumor cells expressed the characteristic phenotype of murine natural killer (NK) cells because (i) pretreatment of spleen cells with anti-asialo GM 1 antiserum plus complement abolished the cytotoxic activity; (ii) augmented cytolysis was found in athymic nude mice; (iii) pretreatment of spleen cells with anti-Thy 1.2 plus complement did not affect the level of cytolysis; and (iv) nylon wool removal of adherent cells did not reduce the augmented cytolysis. The augmented cytolysis peaked 7 days after infection, gradually diminished, and finally returned to control levels by 21 days postinfection. The parasite-induced augmentation of NK cell activity was dose-dependent: inoculation of 10(7) parasites gave maximum enhancement, whereas 10(5) or 10(4) parasites had an insignificant effect on spontaneous NK cell cytolysis. The augmented NK cell cytotoxicity was dependent upon viable parasites; inoculation of killed parasites failed to stimulate a significant increase in spontaneous cytolysis. An active infectious process was an important component of this process. The peak of NK activity in euthymic mice was closely correlated with the active stage of infection, and reduction of NK cell activity coincided with recovery from infection. By contrast, athymic nude mice were unable to control E. cuniculi infections yet maintained persistently elevated NK responses. The present data, along with previous reports, indicate that infection with E. cuniculi evokes transient modulation of host immune functions.     

Expression of perforin in murine natural killer cells and cytotoxic T lymphocytes in vivo.

We have previously detected perforin expression in a subpopulation of asialo GM1+ natural killer (NK) cells and CD8+ T lymphocytes in murine spleen cells by immunocytochemical staining with an anti-perforin monoclonal antibody. In the present study, more detailed analyses of perforin expression in murine cytotoxic lymphocyte subpopulations were performed. The expression of perforin in asialo GM1+ spleen cells was predominantly confined to the NK1.1+ subset, where all NK activity also resided. Perforin expression was also studied on alloreactive cytotoxic T lymphocyte (CTL) induced in vivo. The cells expressing perforin in peritoneal exudate lymphocytes predominantly resided in the CD8+ T cell subpopulation co-expressing asialo GM1 where an allospecific CTL activity also resided. Furthermore, the percentage of perforin-positive cells in this population was greatly reduced after stimulation with anti-CD3 or anti-T cell receptors antibodies, which induce serine esterase release from the cytoplasmic granules. These findings highly suggest that perforin is involved in in vivo NK cell- and CTL-mediated cytolysis.     

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.     

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.     

Phenotypic characterization of two cell populations involved in the acquisition of suppressor activity by cultured spleen cells from Mycobacterium lepraemurium-infected mice.

The impairment of cellular immunity in mice infected with Mycobacterium lepraemurium was shown to correlate with the development of suppressor cells. We have previously reported that before suppressor activity is detectable in freshly harvested cell suspensions, suppressor cell precursors accumulate in the spleen of infected mice. Upon overnight culture in the presence of a regulatory cell subset, these precursor cells acquire the capacity to impair the concanavalin A (Con A)-induced proliferation of normal spleen cells. The purpose of this study was to determine the phenotype of the cells involved in this phenomenon. This was done by following the development of suppressor activity in spleen cell suspensions depleted of defined cell subsets of the adherent or the non-adherent cell fractions with selected MoAbs and immunomagnetic beads or by in vivo treatment. Our results indicate that the acquisition of suppressor activity requires the interaction of Ia+CD11b+Fc gamma R+IgG- asialo GM1- adherent cells with Thy1-CD4-CD8-IgG-Ia- asialo GM1-Fc gamma R+CD11b+ non-adherent cells. It is also shown that the development of suppressor activity is impaired by preventing cell-cell contact between these two cell subsets through coculture in 'Transwell chambers'. These observations support the conclusion that the in vitro acquisition of suppressor activity is a consequence of the maturation of suppressor cell precursors of the monocytic lineage induced by a receptor-ligand type interaction with a non-adherent cell subset that is clearly distinct from mature T, B and natural killer (NK) cells.     

Natural Killer Cells Contribute to Inflammation but Do Not Appear to be Essential for the Induction of Clinical Lymphocytic Choriomeningitis

The inflammatory exudate found in cerebrospinal fluid (CSF) of mice 6 days after intracerebral infection with lymphocytic choriomeningitis virus (LCMV) contains substantial populations of both cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. Removal of NK cell activity by in vivo treatment with antibody to the asialo GM1 ganglioside and studies with NK-deficient bg/bg mice did not clearly determine whether NK cells contribute in any way to the development of clinical LCM. However, the LCM disease process induced in cyclophosphamide-suppressed, LCMV-infected recipients by the adoptive transfer of LCMV-immune spleen cells occurs in the absence of NK cell effector function in spleen, lymph nodes, or CSF of the recipients, though potent CTL populations are present in all of these sites. In this situation, NK cells are apparently not required for the induction of neurological symptoms that are indistinguishable from those of classical LCM.     

Selective expression of asialo GM1 on maturational subsets of lymphocytes in normal and athymic mice

In an attempt to clarify the restriction of asialo GM1 expression, the presence of asialo GM1 positive cells in the lymphoid tissues of normal and athymic mice was examined using affinity-purified monospecific anti-asialo GM1 antibodies and a FACS-II. The results presented herein demonstrate that a significant frequency of asialo GM1-positive cells can be detected in the primary lymphoid tissues (bone marrow and thymus) as well as in the secondary lymphoid tissues (spleen and lymph nodes). The observed expression of asialo GM1 within the T-cell lineage is, in general, consistent with the previously proposed restriction of asialo GM1 expression to mature T cells. However, the demonstration that a significant frequency of asialo GM1-positive cells is detectable in bone marrow of normal mice, as well as in spleen and bone marrow of athymic mice, establishes that a substantial number of lymphocytes not committed to the T-cell lineage and/or pre-T cells also express the asialo Gm1 antigen. These results indicate that the asialo GM1 marker may be an important tool for following T differentiation from the multipotential stem cell to the functionally mature T cell.     

Two natural killer cell populations which belong to T cell lineage in nude mice

Enriched natural killer (NK) cell populations from BALB/c nude mice obtained by using nylon wool column (NWC) separation were stained with anti-Thy-1 and anti-asialo GM1 (GA1), and then were fractioned by flow cytometry into 4 cell populations: Thy-1+GA1+ cells, Thy-1+GA1- cells, Thy-1-GA1+ cells and Thy-1-GA1- cells. These cell populations were sorted out by flow cytometry and were examined for NK activity. Of the sorted cells, high NK activity was found in the two populations, Thy-1+GA1+ cells and Thy-1-GA1+ cells. In the other populations expressing Thy-1 antigen alone or expressing neither GM1 nor Thy-1, NK activity was almost undetectable. The two cell populations with NK activity were lineaged with respect to rearrangement of the T cell receptor genes. Southern gel analysis using a cDNA probe containing the D beta 1, J beta 1.3 and C beta 1 genes of the T cell receptor showed a reduction of the germ-line band of DNA in the 3 fractionated populations other than Thy-1-GA1- cells. Since these observations were demonstrated in nude spleen cells, it is indicated that the cells with NK activity belong to T cell lineage even if they lack Thy-1 expression and that they may develop out of the thymus, if they express Thy-1 antigen.     

Higher level expression of lymphocyte function-associated antigen-1 (LFA-1) on in vivo natural killer cells

Normal mouse spleen cells express low levels of lymphocyte function-associated antigen-1 (LFA-1) as well as other lymphoid cells. However, fractionation of spleen cells with Percoll discontinuous gradients resulted in the appearance of lymphocytes expressing high levels of LFA-1 molecule (LFA-1 high lymphocytes) in parallel with the enrichment of natural killer (NK) activity. Lower density spleen cells (fractions 1 and 2) expressed higher level of LFA-1 antigen than unfractionated spleen cells and showed a higher NK activity. In contrast, higher density spleen cells (fractions 3 and 4) expressed lower levels of LFA-1 antigen and revealed lower NK activity. LFA-1 high lymphocytes possessed a high level of asialo GM1, which was the cell surface marker for NK cells. Moreover, sorting of LFA-1 high lymphocytes from spleen cells caused a great enrichment of NK cells. These results demonstrated that in vivo NK cells expressed higher levels of LFA-1 molecule, which was an important adhesion molecule for NK cell-mediated cytotoxicity.     

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.