Natural killer cells generated from bone marrow culture

By using anti-Nk-1 antiserum, we detected a significant proportion of Nk-1+ cells in bone marrow (BM) with low lytic activity that can be slightly enhanced with interferon (IF). These BM Nk-1+ cells also bind to YAC-1 cells. Because Nk-1 antigen has been found to mark NK cells, BM appears to harbor immature precursors to natural killer (NK) cells. We therefore used concanavalin A (Con A)-conditioned medium to culture BM cells to induce differentiation of the putative NK precursors. After 3 to 4 days in culture, cytotoxic activity to YAC, that peaked at 6 to 7 days, was consistently generated, and the activity still could be detected after 10 to 14 days in culture. In contrast, experiments using spleen cultures, performed in a similar manner, showed peak activity after 4 to 5 days and the activity declined thereafter. The cytotoxic activity of cultured BM cells was also higher than that of cultured spleen cells. Cultures of BM cells from old mice have good cytotoxic activity. The cytotoxic cells generated were Nk-1+ and Qa-5+. Furthermore, these culture conditions did not maintain the proliferation of CFU-C cells.     

Lymphokine-activated killer cells in rats: generation of natural killer cells and lymphokine-activated killer cells from bone marrow progenitor cells

The coculture of rat bone marrow cells with recombinant interleukin-2 induced the generation of cells mediating natural killer (NK) activity and subsequent lymphokine-activated killer (LAK) activity depending upon the dose of IL-2 and time of culture. NK activity was detected as early as 4 to 5 days after the addition of IL-2 and could be evoked with as little as 5 to 50 U/ml. The induced NK cells had large granular lymphocyte (LGL) morphology and expressed 0X8 and asialo GM1 surface markers but did not express 0X19 or W3/25 markers. LAK activity was detected only after 5 days of culture, and required above 100 U/ml IL-2. Cells mediating LAK activity also expressed 0X8 and asialo GM1 but not 0X19. The generation of detectable NK and subsequent LAK activity was due to induction of early progenitor cells and not contaminating mature LGL/NK cells within the bone marrow population since of removal of such mature NK cells with L-leucine methyl ester (L-LME) did not affect the subsequent generation of either activity. Moreover, the removal of actively dividing cells as well as mature NK cells from the bone marrow by treatment with 5-fluorouracil (5-FU) in vivo enriched the remaining bone marrow population for both NK and LAK progenitor cells. The phenotype of the L-LME- and 5-FU-resistant NK and LAK progenitor cells within populations of bone marrow was determined by antibody plus complement depletion analysis. Although treatment of normal bone marrow with anti-asialo GM1 + C reduced the induction of NK and LAK activity in 5-day cultures, treatment of 5-FU marrow with anti-asialo GM1 + C did not affect either activity. Treatment with a pan-T cell antibody + C did not affect the development of NK or LAK activity under any conditions. Thus, the 5-FU-resistant NK/LAK progenitors were asialo GM1 negative but became asialo GM1+ after induction by IL-2. Finally, evidence that bone marrow-derived LAK cells were generated directly from the IL-2-induced NK cells was obtained by treating the IL-2-induced LGL/NK cells with L-LME.(ABSTRACT TRUNCATED AT 400 WORDS)     

Suppression of natural killer activity in human blood and bone marrow cultures by bone marrow-adherent OKM1-positive cells

Maintenance and regulation of natural killer (NK) cell activity in human bone marrow cultures were studied using K562 leukemia cells as targets. Culture of bone marrow cells in medium supporting long-term generation of myeloid cells resulted in a rapid loss of NK activity in 1-3 days. In contrast, antibody-dependent cytotoxicity to an NK-resistant tumor was maintained for more than 7 weeks. Horse serum, a component of the myelopoietic culture medium, was found to diminish NK cytotoxicity of blood and bone marrow cultures whereas hydrocortisone supplement did not. In addition, an adherent cell is present in bone marrow which greatly inhibits NK activity. Nonadherent bone marrow cells exhibited higher cytotoxicity than unfractionated cells at all days of culture; adherent cells were not cytotoxic to K562. Purified adherent marrow cells inhibited the cytotoxic capacity of nonadherent blood or marrow mononuclear cells during coculture. Indomethacin, an inhibitor of protaglandin synthesis, augmented levels of NK activity in cultures of bone marrow cells, indicating that macrophages may be suppressing this effector function via prostaglandins. Further identification of the adherent suppressor cells came from experiments in which suppression was prevented by treatment of the adherent cells with monoclonal OKM1 antibody plus complement. This study shows that bone marrow-adherent OKM1-positive cells, presumably macrophages, negatively regulate NK activity, and it defines conditions for analysis of the generation and/or positive regulation of NK cells in human bone marrow.     

Characterization of natural killer cells and their precursors in the murine bone marrow

We have fractionated murine bone marrow cells according to their density on bovine serum albumin (BSA) gradient and studied (a) the NK activity against YAC-1 targets, (b) the proportion of asialo GM1+ lymphocytes, (c) and the presence of large granular lymphocytes (LGL) in the different fractions (A, B, C, D). The NK activity was found mainly in the C fraction, but the proportion of asialo GM1+ cells was the same in every fraction. No LGLs were found in the bone marrow. Cells from the various fractions were also transplanted into irradiated recipients. Seven days later the highest NK activity was found in the spleens of mice injected with cells from the A + B fractions indicating that the immediate precursors for NK cells reside in the low density fractions of the BSA gradient. Mice transplanted with C or D fractions needed longer time to develop normal NK levels. The treatment of bone marrow cells before transplantation with anti-asialo GM1+ complement did not inhibit the development of NK activity, so it can be concluded that the precursor for NK is asialo GM1-.     

Natural killer cells in rhesus monkeys: properties of effector cells which lyse Raji targets

Spontaneously occurring natural killer cell activity of rhesus monkey peripheral blood mononuclear cells was assayed against five human cell lines, three of which were Epstein-Barr virus (EBV) positive, including the human B cell line Raji. The lytic activity to Raji cells was high, significantly higher than to any other cell line tested. Raji cells are normally insensitive to spontaneous lysis by human NK cells, and the contrasting vigor of the rhesus monkey cytolytic activity to Raji prompted us to investigate the properties of this effector cell. We found the effector cell-mediating lysis of Raji to be nonadherent and phagocytic with lytic activity slightly enhanced in the E-rosette-forming cell (ERFC+) fraction and decreased in the ERFC- fraction. Further isolation of FcIgG receptor-positive and FcIgG receptor-negative subsets by rosetting resulted in significant enrichment of NK activity to Raji in the positive fraction and a loss of activity in the negative fraction. Depletion studies with various monoclonal antibodies (mAb's) confirmed that nearly all lytic activity was contained in the CD16+ (Leu 11b+) population, while subsets of effector cells expressed CD2 (9.6) and CD8 (OKT8). Depletion of CD4 (OKT4)-, HLADR (OKIa)-, or LFA1 (MAC-1)-positive populations failed to reduce NK activity. We compared the phenotypic properties of alloimmune effector cells exhibiting specificity for allogeneic donor targets with those exhibiting lysis of Raji targets. Results indicated that allospecific cytotoxic T lymphocytes expressed a CD16-, CD2+ phenotype, a pattern distinct from that of the effector cell population recognizing Raji targets. The presence of CD2 mAb's in the culture had no effect on NK lytic activity. In contrast, mAbs CD8 and Leu 11b were inhibitory. This would suggest a functional role for CD8 and FcIgG molecules in the lysis of Raji cells by rhesus effectors. In summary, these studies describe a distinct population of effector cells in the blood of rhesus monkeys which exhibit spontaneous lytic activity to Raji cells and exhibit the properties of NK cells.     

Differential expression of lymphokine-activated killer cells and natural killer cells in adoptive transfer experiments utilizing fractionated bone marrow

Precursors of murine natural killer (NK) cells and lymphokine-activated killer (LAK) cells can be distinguished by utilizing an adoptive transfer system in which donor bone marrow is fractionated on Percoll discontinuous gradients. Although precursors of LAK cells are present in all fractions, one fraction (greater than 65% Percoll) contains LAK precursors and is depleted of NK precursors. Both in vitro NK activity and in vivo hybrid resistance is abrogated in recipients of bone marrow from the greater than 65% Percoll fraction, whereas LAK activity can be readily demonstrated.     

Rhythms in human bone marrow and blood cells

In 24h studies of bone marrow (BM), circadian stage-dependent variations were demonstrated in the proliferative activity of BM cells from subsets of 35 healthy diurnally active men. On an average, the percentage of total BM cells in deoxyribonucleic acid (DNA) synthesis phase was 188% greater at midday than at midnight (circadian rhythm: p = 0.018; acrophase or peak time of 13: 16h). Patients with malignant disease (n = 15) and a normal cortisol circadian rhythm showed higher fractions of BM cells in S-phase at midday. Colony-forming units--granulocyte/macrophage (CFU-GM), an indicator of myeloid progenitor cells, showed the same circadian variation as DNA S-phase (average range of change or ROC = 136%; circadian rhythm: p < 0.001; acrophase of 12:09h). Deoxyribonucleic acid S-phase and CFU-GM in BM both showed a circannual rhythm (p = 0.015 and 0.008) with an identical acrophase of August 12. The daily peak in BM glutathione content, a tripeptide involved in cellular defense against cytotoxic damage, preceded BM proliferative peaks by 4-5 h (ROC = 31-90%; circadian rhythm: p = 0.05; acrophase of 08:30h). Myeloid (ROC = 57%; circadian rhythm: p = 0.056; acrophase at 08:40h) and erythroid (ROC = 26%; circadian rhythm: p = 0.01; acrophase of 13:01h) precursor cells were positively correlated (r = 0.41; p < 0.001), indicating a circadian temporal relationship and equal influence on S-phase of total BM cells. Yield of positive selected CD34+ progenitor stem cells also showed significant circadian variation (ROC = 595%; circadian rhythm: p = 0.02; acrophase of 12:40h). Thus, the temporal synchrony in cell cycling renders BM cells more sensitive at specific times to hematopoietic growth factors and cell cycle-specific cytotoxic drugs. Moreover, proper timing of BM harvesting may improve progenitor cell yield. When using marker rhythms in the blood to allow for individualized timing of BM procedures, the times of low values in white blood corpuscles, neutrophils, and lymphocytes and high values in cortisol were predictive of the times of highest BM erythroid, myeloid, and total S-phase numbers occurring in the following 12 h.     

Natural killer cell-mediated antitumor reactivity of rhesus monkeys

We have analyzed natural killer (NK) cell-mediated antitumor activity or peripheral blood mononuclear cells of rhesus monkeys. All monkeys displayed significant NK cell cytolytic activity against the human tumor cell lines K-562, Daudi and CEM in a short-term (3 h) 51Cr-release assay. Similar to NK cells described in other species, the cytotoxic cells of monkeys were relatively nonadherent to nylon wool columns, exhibited low density after separation on discontinuous Percoll density gradients, and displayed large granular lymphocyte (LGL) morphology. Analysis of the mechanism of NK cell cytotoxicity of rhesus monkeys demonstrated that on the average, 7.1% (range: 3.1-13.2%) of lymphocytes bound to K-562 tumor, and that approximately 14.8% (range: 7.9-26.3%) of these tumor-binding cells (TBC) were cytolytically active. Examination of TBC on cytocentrifuge slides indicated that the majority of binders displayed LGL morphology. The cytotoxic reaction mediated by monkey NK cells exhibited Michaelis-Menten kinetics pattern; the maximum rate of lysis (Vmax) of K-562 was found to be 1-2 X 10(4) following 3 h of incubation. Using similar culture conditions, the recycling capacity of NK cells of this species was estimated at 2-6 times. Finally, it was observed that the NK cell activity of most monkeys could be potentiated following in vitro exposure to the biological response modifier, interleukin-2.     

Natural killer cells vs cytotoxic T cells in the peripheral blood of virus-infected mice

The cell-mediated immune response of mice against various enveloped RNA and DNA viruses expressed by immune lymphocytes from the spleen and the peripheral blood (PBL) were compared. PBL from mice of various strains infected with vaccinia virus, vesicular stomatitis virus (VSV), or lymphocytic choriomeningitis virus (LCMV) were tested on histocompatible or incompatible target cells infected with the homologous virus. PBL from immune mice showed clear H-2 restriction, but additionally, they expressed high natural killing (NK) activity on YAC-1 cells. The high NK-cytolytic activity of PBL on YAC-1 differed significantly from that expressed by splenic lymphocytes. In both lymphocyte populations lysis was detected as early as 1 day after infection; NK activity decreased in the spleen after day 4 post infection, whereas that of PBL persisted at high levels for up to 10 days after infection. Treatment of mice with anti-asialo GM1 in vivo abrogated NK activity in PBL effector cells tested in vitro. These results may explain some of the difficulties to observe MHC-restricted cytotoxic T cells in PBL from humans or primates during primary infections with virus.     

Cholinergic neuronal differentiation of bone marrow mesenchymal stem cells in rhesus monkeys

The purpose of the present study was to determine the best cholinergic neuronal differentiation method of rhesus monkey bone marrow mesenchymal stem cells(BMSCs).Four methods were used to induce differentiation,and the groups were assigned accordingly:basal inducing group(culture media,bFGF,and forskolin);SHH inducing group(SHH,inducing group);RA inducing group(RA,basal inducing group);and SHH+RA inducing group(SHH,RA,and basal inducing group).All groups displayed neuronal morphology and increased expressio...     

重组杆状病毒转导恒河猴骨髓间充质干细胞

[目的]研究重组杆状病毒(Bac-CMV-EGFP)能否能有效转导恒河猴骨髓间充质干细胞(rhesus Bone marrow-derived Mesenchymal Stem Cells,rBMSCs),及杆状病毒转导后对细胞活力,增殖及分化能力的影响.[方法]体外原代培养rBMSCs,不同剂量的杆状病毒转导3代以后的细胞,并用流式细胞仪分别检测其转导效率.在较高的杆状病毒转导效率下,检测rBMSCs细胞活力,增殖及分化能力,并与正常对照组细胞进行比较.[结果]杆状病毒在感染指数(Multiplicity Of Infection,MOI)为300v.g/cell,孵育温度为25度,孵育时间为4h的转导条件下,对rBMSCs转导效率可达80%左右.进一步检测后发现,高效转导杆状病毒后的rBMSCs的细胞活力,增殖及分化能力与未转导病毒细胞组无明显变化.[结论]重组杆状病毒可安全有效地基因修饰rBMSCs,且不影响其生物特性,为今后的体内基因治疗灵长类动物模型试验奠定了基础.     

Natural killer cells, bone, and the bone marrow: studies in estrogen-treated mice and in congenitally osteopetrotic (mi/mi) mice.

Mice lose natural killer cells after 6 weeks of treatment with 17 beta-estradiol. We here demonstrate that the same protocol leads to loss of genetic resistance to bone marrow transplantation and to significant osteoproliferation with loss of bone marrow. We also show that mice with reduced marrow because of congenital osteopetrosis are deficient in natural killing. These findings are consistent with previous evidence that natural killing and genetic resistance to bone marrow transplantation are dependent upon the marrow. Temporal studies of bone histology and radiology during and after treatment with estrogen reveal that alterations in natural killing proceed more rapidly than changes in bone marrow volume. These studies also demonstrate that estrogens induce osteoproliferation only at endosteal surfaces that are adjacent to hematopoietic marrow. From these observations, we conclude that estrogens do not reduce natural killer cells simply by reducing the volume of bone marrow. Estrogens may instead have an effect on bone marrow. Estrogens may instead have an effect on bone marrow cells that leads both to osteoproliferation and to a deficiency of marrow-dependent cells.