Natural cytotoxicity of NC-2+ cells against the growth and metastasis of WEHI-164 fibrosarcoma.

We have previously reported a new receptor (NC-2) for natural cytotoxicity (NC) on murine leucocytes, identified by monoclonal antibody D9 (mAb D9). Pretreatment of mouse spleen cells with different concentrations of mAb D9 in vitro blocked NC against WEHI-164, whereas natural killing (NK) activity against YAC-1 was unaffected. This paper reports the immune surveillance against the growth of WEHI-164 tumour cells in mice by NC-2(+) Cells. The kinetics of in vivo reduction in NC activity were investigated by treating BALB/c and (CBA × C57BL/6) F1 mice with a single injection of 40 µg of mAb D9 and monitoring splenic NC activity by (51) Cr-release assay at intervals from 24 h to 3 weeks. Control mice were injected with OKT8 irrelevant antibody. Results showed a significant (P < 0.05) reduction in splenic NC activity within 24 h which persisted for up to 1 week. Similar results were also obtained when (CBA × C57BL/6) F1 mice were employed (P<0.001). In vivo tumour studies were undertaken to investigate the role of NC-2(+) cells in surveillance against tumour growth and metastasis of the WEHI-164 fibrosarcoma. When syngeneic BALB/c mice were injected with 40 µg of mAb D9 and then challenged with 5 × 10(5) WEHI-164 cells, results showed significantly increased growth rate of the transplanted WEHI-164 fibrosarcoma and tumour nodules in the lungs of animals, when compared to control mice with normal NC activity. Our data support an innate surveillance in metastasis and growth of WEHI-164 fibrosarcoma in mice.

Monoclonal antibody anti-NC-2 identifies a second receptor on cells mediating natural cytotoxicity in mice.

We have previously reported the identification by the murine monoclonal antibody (mAb) 1C4 of the first leucocyte receptor which is involved in natural cytotoxicity (NC) against WEHI-164; the NC-1.1 receptor. We report herein the identification and characterization of a second leucocyte receptor which is involved in NC, NC-2 (MW 50,000), identified by a rat anti-mouse mAb D9 (immunoglobulin G2a; IgG2a). Flow cytometric analysis showed that NC-2 was expressed on < 6% of splenic leucocytes of different inbred mouse strains and 96% of the cells of a mast-cell line which has high NC activity. In vitro treatment of splenic leucocytes with the D9 mAb blocked effector cell-WEHI-164 target cell conjugation and NC by approximately 50% without affecting natural killing (NK). Western blot analysis of affinity purified NC-2 and NC-1.1 using the D9 and 1C4 mAbs showed specific reactivity of the proteins with D9 and 1C4, respectively. Pretreatment of splenic leucocytes with both mAbs blocked NC 84%, a result which almost doubled that caused by either mAb alone. Flow cytometric screening of 16 different mouse cell lines showed that 19% of the cell lines expressed both receptors, 6% expressed only NC-2, 44% expressed mainly or only NC-1.1 and the remaining cells expressed neither receptor. These data indicate that D9 identifies a xeno-antigen, NC-2, which is expressed on cells mediating NC and not NK, and that it is not the previously described NC-1.1 allo-antigen. We conclude that NC-2 is likely to be one of a number of receptor molecules on cells mediating NC against tumour cells.

The antitumor effects of levamisole in mice are mediated by NC-1.1+ cells.

Murine natural cytotoxicity, which is a major component of the innate immune response in cancer, is mediated by leukocytes that express the NC-1.1 receptor. Mice depleted of natural cytotoxicity by treatment with an anti-NC-1.1 mAb show enhanced growth of certain transplantable tumors, so agents that enhance natural cytotoxicity by NC-1.1+ cells have the potential to be effective anticancer therapeutic agents. We have examined the immunomodulatory effect of levamisole on natural cytotoxicity mediated by NC-1.1+ cells against the BALB/c WEHI-164 murine fibrosarcoma. Administration of levamisole to BALB/c mice significantly enhanced in vitro splenic natural cytotoxicity against 51Cr-labeled WEHI-164 tumor cells. The effect was most marked 48 h after levamisole treatment, at a dose of 10 mg/kg body weight. This enhancement of natural cytotoxicity by levamisole could be completely abrogated by pretreatment of mice with an anti-NC-1.1 mAb. Treatment of BALB/c mice with 10 mg/kg levamisole significantly reduced the growth of WEHI-164 and this effect was abrogated by pretreatment of mice with anti-NC-1.1, indicating that the antitumor effect of levamisole was mediated, at least in part, via NC-1.1+ cells.

The antiabortive effect of progesterone-induced blocking factor in mice is manifested by modulating NK activity.

Immunologic effects of progesterone are mediated by a protein named the progesterone-induced blocking factor (PIBF), which inhibits NK activity and displays an antiabortive effect in mice. Our previous data provide indirect evidence for the importance of PIBF in the maintenance of normal gestation. This study was aimed at investigating whether neutralization of endogenous PIBF production influences pregnancy outcome and if so, what are the mechanisms that participate in this process. Syngeneically pregnant Balb/c mice on Day 8.5 of pregnancy were injected ip with 0.3 mg/kg of RU 486 or with 0.5 mg of rabbit anti-PIBF IgG alone, or together with anti-NK monoclonal antibodies. Mice treated with the same amount of normal rabbit serum or untreated mice of similar gestational age were used as controls. On Day 10.5 the ratio of living and resorbed embryos and NK activity of the spleen cells were determined. In mice treated with anti-PIBF the ratio of resorbed fetuses was significantly higher than that in untreated controls. In RU 486-treated mice we also observed significantly increased resorption rate, which was associated with the inability of spleen cells to produce PIBF. Both anti-PIBF treatment and that with progesterone receptor blocker resulted in increased splenic NK activity. There was a positive relationship between NK activity and the rate of resorptions. All the above effects were corrected by simultaneous treatment with anti-NK or anti-NC (natural cytotoxic) antibodies. These data allow the conclusion that PIBF contributes to normal gestation in mice and that the effect of PIBF is manifested via blocking NK and/or NC activity.

TNF-alpha is not the sole mediator of WEHI-164 tumour cell killing in natural cytotoxicity.

Using a mAb to NC-1.1, a receptor involved in recognition of tumour targets, the authors have examined the dogma that murine natural cytotoxicity (NC) is exclusively mediated by TNF-alpha. Three different NC-1.1+ spleen cells, WEHI-3BR1 myelomonocytic cells and an uncloned mast cell line-MCL) were reacted with NC-sensitive WEHI-164 targets in vitro, and the induction of TNF-alpha mRNA, surface expression of TNF-alpha, and the appearance of apoptotic bodies in the culture were simultaneously measured. NC-1.1+ spleen cells and WEHI-3BR1 cells showed marked induction of TNF-alpha mRNA within 30 min and this was maintained for up to 18 h. Only transient TNF-alpha mRNA induction was observed in MCL cells at 30 min. Surface TNF-alpha was detected on WEHI-3BR1 cells by 4 h, but was not detected on MCL cells. All three effector cell types mediated NC against WEHI-164 targets within 18 h, but they responded differently to the addition of anti-TNF-alpha mAb: anti-TNF-alpha completely blocked WEHI-3BR1 NC, blocked NC-1.1+ spleen cell NC by approximately 70%, and did not block NC by MCL cells. This indicates that TNF-alpha is induced during NC by WEHI-3BR1 effectors and NC-1.1+ spleen cells, is the sole mediator of NC by WEHI-3BR1, and appears to play no role in NC by MCL cells.

Phosphorylation of the NC-1.1 receptor and regulation of natural cytotoxicity by protein kinase C and cyclic GMP-dependent protein kinase.

Natural cytotoxicity (NC) against cancer involves receptor-ligand interactions between lymphohemopoietic cells that mediate NC against tumor cells. The only candidate for a receptor on cells mediating NC is NC-1.1, identified using mAb 1C4. In this study we showed that mAb 1C4 blocked NC-1.1+ cell conjugation to WEHI-164 tumor cells, indicating that NC-1.1 is a surface protein required for cell-cell interaction. Affinity-purified NC-1.1 was a 45-kDa monomeric protein. It was a good in vitro substrate for cyclic GMP (cGMP)-dependent protein kinase (PKG) and protein kinase C (PKC) and a relatively poor substrate for cAMP-dependent protein kinase (PKA). Phosphopeptide mapping revealed one phosphopeptide phosphorylated by PKG and PKA, and two additional peptides phosphorylated by PKC. Phosphorylation by PKG or PKA abolished phosphorylation at the PKC sites, while coincubation of NC-1.1 with both PKG and PKC reduced phosphorylation of all sites. NC-1.1 was also a phosphoprotein after immunoprecipitation from intact spleen cells and its phosphorylation was increased after cell stimulation with PKC or PKG activators (phorbol esters or 8-bromo-cGMP). The possible consequences of intracellular signaling were tested in functional assays for NC. Phorbol ester activation of spleen cells increased NC, while 8-bromo-cGMP and 8-bromo-cAMP had little effect. However, coincubation with both phorbol ester and either 8-bromo-cGMP or 8-bromo-cAMP virtually abolished NC without affecting cell conjugation. These results suggest that NC-1.1 is a receptor for a ligand on certain tumor cells and reveal that key intracellular signaling pathways involving PKC, PKG, and PKA interact to effect a coordinated control of NC.

Natural cell-mediated cytotoxicity (NCMC) against NK-sensitive tumours in vitro by murine spleen Ly-6C+ natural T cells.

Ly-6C+ cells constitute 13 +/- 3% of freshly isolated (CBA x C57BL/6)F1 mouse spleen leukocytes. Three distinct populations were identified: CD3 epsilon +NK-1.1- conventional T cells (6%), CD3 epsilon -NK-1.1- granulocytes (5%) and CD3 epsilon +NK-1.1+ T cells (approximately 2%). The CD3 epsilon +NK-1.1+ cells displayed a predominantly large granular leukocyte morphology and were the only Ly-6C+ cell subset identified by MAb 2B6-F2 to spontaneously lyse the NK-sensitive YAC-1 tumour in vitro. On further phenotypic analysis, these cells co-expressed high levels of TCRV beta 8.1/8.2 and CD11b, moderate levels of CD90 and low levels of CD4 or CD8. The removal of CD4+ and CD8+ cells prior to Ly-6C+ cell sorting showed that it was the CD4-CD8- double-negative (DN) CD3 epsilon +NK-1.1+ T-cell subset which was responsible for killing YAC-1. These results indicate that we have identified a DN Ly-6C+ subset of the recently designated NK-1.1+TCR alpha beta low natural T (NT) cells, which are capable of natural cell-mediated cytotoxicity (NCMC) against the NK-sensitive YAC-I tumour in vitro. Additionally, these cells mediated the in vitro killing of 2 further NK-sensitive tumours, murine B16 melanoma and human Jurkat T lymphoma. YAC-1 and Jurkat expressed Fas and were susceptible to anti-Fas MAb or rhuman Fas ligand (rhFasL)-induced lysis. Furthermore, anti-human Fas MAb M3 was shown to block sorted Ly-6C+ splenocyte in vitro killing of Jurkat. In contrast, B16 did not express cell-surface Fas and was resistant to anti-Fas MAb-induced lysis. Taken together, these results show that not only do Ly-6C+ NT cells kill NK-sensitive tumours in vitro but they mediate this activity via multiple cytotoxic mechanisms including Fas.

An IL-3-induced splenic NC-1.1+ mast cell line mediates natural cytotoxicity independent of TNF-alpha.

Long-term culture of mouse spleen cells in IL-3-conditioned medium induced a stable mast cell line. This mast cell line (MCL) which could not be cloned expressed NC-1.1, a receptor on cells which mediate natural cytotoxicity (NC), and CD32/CD16 but no markers of T cells, B cells, macrophages, or NK cells. The MCL cells were large and granular, with abundant cytoplasm and >90% stained with Alcian blue, a mast cell-specific stain. Probing total RNA with cDNA encoding a mast cell-specific proteinase mMCP-5 identified the approximately 1-kb mMCP-5 transcript which was confirmed at the protein level. MCL cells mediated very high NC against WEHI-164 which increased with time in culture and which was blocked by anti-NC-1.1 but not by anti-TNF-alpha. When incubated with WEHI-164 tumor cells, MCL cells showed transient induction of TNF-alpha mRNA, but no detectable surface protein. Thus long-term culture of murine spleen cells in IL-3 induces mast cells which express the NC-1.1 receptor of cells which mediate NC, and utilize a cytotoxic effector mechanism which is not TNF-alpha.

Castanospermine, an oligosaccharide processing inhibitor, reduces membrane expression of adhesion molecules and prolongs heart allograft survival in rats.

The inhibition of intracellular oligosaccharide processing is a new approach to immunosuppression in allotransplantation. The net effect of such inhibition is reduction in the membrane expression of certain glycoproteins. Hence cell-cell interaction in allorejection may be impaired in the presence of glycoprotein processing inhibitors because the expression of key ligand-receptor pairs of N-linked glycoproteins including adhesion molecules is inhibited. The aims of this study were to measure the immunosuppressive ability of castanospermine (CAST) in a rat heart allograft model, to measure its effect on membrane expression of adhesion molecules (LFA-1 alpha, LFA-1 beta, ICAM-1), class I and class II MHC antigens and on other T cell associated molecules (CD4, CD8, CD39, CD45, W3/13), to test its tolerogenic potential and its toxicity. Membrane expression of these molecules was measured by flow cytometry for single cells and by immunoperoxidase staining for the allograft. In grafted rats CAST significantly reduced the expression of LFA-1 alpha on lymphoid cells in the thymus, lymph node, spleen and heart allografts. ICAM-1 expression on endothelial cells of the allograft vasculature, class I and class II MHC expression on lymphoid cells in the thymus, class II MHC expression on lymphoid cells in the allograft; and CD4, CD8, CD45 and W3/13 expression on lymphoid cells in some organs. By contrast, in non-grafted rats CAST significantly upregulated expression of class I MHC and CD45 in the thymus, lymph node and spleen, ICAM-1 and CD4 on lymphoid cells in the spleen, but reduced expression of LFA-1 alpha on lymphoid cells in the thymus. It also prolonged rat heart allograft survival in a dose-dependent manner and with limited testing was relatively non-toxic. In conclusion, CAST is an immunosuppressive molecule which may work by downregulation of the ligand-receptor adhesion molecule pair, LFA-1 alpha-ICAM-1 although subtle downregulation of class I and II MHC, CD4 and CD8 molecules could also contribute to its immunosuppressive activity. Hence, both lymphocyte-endothelial cell binding and lymphocyte activation may be inhibited by CAST. This work suggests that CAST may hold significant potential as a transplant immunosuppressant probably as an adjuvant agent to inhibitors of interleukin 2 secretion.

Ly-6C+ natural T (NT) cells mediate immune surveillance against NK-sensitive and NK-resistant transplantable tumours in certain strains of mice.

We have previously shown that anti-Ly-6C monoclonal antibody (MAb) 2B6-F2 identifies a subset of (CBA +/- C57BL/6)F1 splenic NK-1.1+ natural T (NT) cells which kill the NK-sensitive YAC-1 target in vitro. Furthermore, these Ly-6C+ cells are responsible for 40-50% of in vitro YAC-1 killing in all mouse strains tested. In BALB/c and DBA/2 mice, these cells killed not only YAC-1 but also the NK-resistant WEHI-164 M1/16 target via a receptor that recognises a shared determinant on these targets in vitro. In the present study, the anti-tumour role of Ly-6C+ cells against the NK-sensitive B16 melanoma and NK-resistant tumours WEHI-7 T lymphoma and WEHI-164/1C fibrosarcoma was studied in vitro and in vivo. In vitro, B16, WEHI-7 and WEHI-164/1C tumour cell lines were highly sensitive to Ly-6C+ cell killing. In vivo, these same tumours showed significantly increased growth when transplanted s.c. into syngeneic mice treated with 2B6-F2 (0.05 < or = p < 0.0005), and this was most marked in the first 15 days following tumour appearance, when tumours were <15 mm in diameter. Our results show that Ly-6C+ cells play a role in controlling the growth of transplantable NK-sensitive B16 melanoma, and in BALB/c mice, at least, the repertoire of susceptible tumours is extended to include NK-resistant WEHI-7 and WEHI-164/1C. We conclude that Ly-6C+ NT cells play a role in immunosurveillance against NK-sensitive as well as NK-resistant tumours in certain strains of mice.

Monoclonal antibody 2B6-F2 identifies a subset of murine natural killer cells with receptors for YAC-1 and WEHI-164 targets.

This study reports the generation of rat MoAb 2B6-F2 (IgG2a) that identifies a subpopulation of murine NK cells in the spleen, bone marrow, peripheral blood, lung, and peritoneal leukocytes. By flow cytometry, 2B6-F2 reacted with 10% of naive CBA (immunizing strain) spleen leukocytes of which 3% exhibited small lymphocyte morphology while 7% were large granular leukocytes. This pattern of binding was similarly obtained for BALB/c, C57BL/6, CE, and DBA/2 mice. 2B6-F2 and complement reduced splenic NK activity 40-57% in CBA, C57BL/6, BALB/c, CE, and DBA/2 strains. In CBA, C57BL/6, and CE mice < or = 15% reduction in splenic NC activity was observed while DBA/2 and BALB/c mice displayed 55-73% reduction. Cellular competitive inhibition studies showed that 2B6-F2+ NK cells in BALB/c and DBA/2 mice bind and kill YAC-1 and WEHI-164 target cells via a receptor that recognizes a shared determinant on these targets. Western blot and radioimmunoprecipitation studies indicated that 2B6-F2 identifies a 14-kDa monomeric cell surface molecule.

Natural killer and natural cytotoxic cells are present at the maternal-fetal interface during murine pregnancy.

NK cell activity has been detected in the early murine decidua, and hypothesized to be mediated by granulated metrial gland (GMG) cells. The possibility that natural cytotoxic (NC) cells are also present in the decidua has not been investigated. In this study mAb to NK cells (anti-NK-1.1) and NC cells (anti-NC-1.1) were used to characterize the decidual cells of days 8-14 pregnant (CBA x C57BL/6) F1 mice. Flow cytometric and immunohistological analyses showed predominantly NK-1.1+ and NC-1.1+ large and granular single nucleated decidual cells with abundant cytoplasm. A 'bright' and a 'dim' subset were identified for both NK-1.1+ and NC-1.1+ cells. The NC-1.1dim and NK-1.1dim cells increased in number and size as pregnancy progressed. When tested in 51Cr-release assays, the decidual cells showed significant levels of both NK and NC activities which increased with progression of pregnancy. The NK and NC activities were partially inhibited (47 and 34%) by preincubation of the decidual cells with anti-NC-1.1 and complement (C'), or anti-NC-1.1 alone. Results indicate that natural cell-mediated cytotoxicity in the decidua is in part, at least, mediated by NK-1.1+ and NC-1.1+ cells, and that the NK-1.1dim and NC-1.1dim cells are very likely to be GMG cells. This is the first report of NC cells in the mouse uterus.

Phenotype and morphology of murine NC-1.1+ natural cytotoxic cells.

The morphology and phenotype of cells identified by the anti-NC-1.1 mAb, 1C4, were studied in CBA and (CBA x C57BL/6) F1 mice. This mAb blocked splenic natural cytotoxic (NC) cell activity in vitro and in vivo. Single colour flow cytometric analysis showed that 6 +/- 1% of all CBA splenocytes were NC-1.1+, and that granular cells of varying sizes, as defined by their forward and side light scatter, contained the highest proportion of NC-1.1+ cells (29 +/- 7%). When analysed by two colour flow cytometry, the large and the granular NC-1.1+ spleen cells in CBA mice were shown to co-express Thy-1.2 and L3T4 (< or = 33%), Mac-1 (< or = 26%), IgM (< or = 60%) and FcR gamma II and J11d (< or = 100%). In contrast, T cell markers were not co-expressed on the small agranular NC-1.1+ spleen cells. This pattern of marker expression was evident in CBA spleens from 2 days of age. When (CBA x C57BL/6) F1 spleen cells were similarly analysed, 82% of granular NC-1.1+ cells also co-expressed NK-1.1. Single colour analysis of CBA bone marrow, thymus, lymph node and peripheral blood leucocytes revealed that < or = 10% of all cells examined were NC-1.1+ while the most granular cells in these organs were 16-43% NC-1.1+. These results support the 'horizontal lineage' theory that NC cells are cells of different lymphohaemopoietic cell lineages at particular stages of differentiation.

Anaerobic exercise causes transient changes in leukocyte subsets and IL-2R expression.

There is evidence that the stress of intense athletic competition and training depresses cellular immunity and predisposes athletes to increased infection. This paper reports changes in circulating leukocyte subsets of trained (group I: VO2max = 67.2 +/- 5.4 ml.kg-1min-1; age = 22.0 +/- 6.2 yr) and untrained (group II: VO2max = 55.0 +/- 4.9 ml.kg-1min-1; age = 21.4 +/- 2.0 yr) males following 1 min of bicycle ergometry at maximum effort. Significant post-exercise increases in concentrations of total leukocytes, monocytes, lymphocytes, CD3+, CD4+, CD8+ lymphocytes were observed in both groups (all P < 0.01). The CD4/CD8 ratio decreased significantly (P < 0.01) but the granulocyte concentration was not altered (P > 0.05). Despite groups I and II not differing in either peak power or total work performed during the exercise test (P > 0.05), group II had a significantly greater concentration and percentage of CD8+ lymphocytes immediately after exercise (P < 0.01). All of the early changes were transient, with normalization occurring within 1 h. Only trained subjects showed a significant decrease in the percentage of CD25+ lymphocytes following PHA stimulation of whole blood obtained 6 h post-exercise. Alterations in leukocyte subpopulations found in response to predominantly anaerobic exercise appear to be associated with a significant, but possibly transient, alteration in the mitogenic responsiveness of lymphocytes that is restricted to aerobically trained subjects.

Prefertilization treatment of mice with platelet activating factor affects pregnancy.

Embryo-derived platelet activating factor (EPAF) is thought to be either biologically similar to platelet activating factor (PAF) or responsible for PAF liberation in vivo. We have previously shown that premating PAF treatment in the mouse renders the platelets nonresponsive to EPAF, leading to a reduced implantation rate in these animals. In this study, we have shown that females, injected with PAF before mating, show altered embryo development invivo on day 4 postfertilization. This is manifested as an interruption of compaction, a reduced cell number per embryo, and reduced embryo number per mouse. Results suggest that EPAF represents an early pregnancy signal that supports embryo development. The most likely mechanism is via platelet activation, since only those mice that showed thrombocytopenia after fertilization were found to have normal embryos on day 4 postmating.