Natural killer cells in the blood and bone marrow of the rhesus monkey

Natural killer (NK) cells in peripheral blood lymphocytes (PBL) and bone marrow (BM) cells of the rhesus monkey were detected by their functional activity against K562 cells. Animals could be grouped into "high" or "low" NK responders, a trait found to be consistent over a period of 2 years. NK active cells in PBL were in the nonadherent population, with the majority bearing Fc receptors and a further subdivision of these into CR+ (complement receptor) and CR- NK cells. Of 10 monoclonal antibodies directed against different epitopes of human lymphocytes, OKT11, OKT10, and Leu 11 showed reactivity with rhesus NK cells. Only OKT10 was reactive with the effector site of the cell, as shown by its capability to block NK function. Of the Leu 11 monoclonal antibodies (a, b, c), Leu 11c was nonreactive while Leu 11a and Leu 11b were shown by immunofluorescence to bind to 7 to 21% of PBL; Leu 11b was also cytotoxic to the NK cells. Leu 11b did not prevent binding of Leu 11a to PBL, suggesting reactivity of these antibodies with different epitopes. Percoll fractionation of PBL and BM revealed a greater enrichment of NK activity with BM; also, with PBL peak NK activity occurred in fractions 4 and 5 while this occurred in fraction 5 with BM. Although Percoll PBL fractions contained a higher percentage of Leu 11b cells, the NK activity of the BM fractions was proportionately greater. The majority of PBL cells with NK activity were FcR+ while significant activity could be attributed to FcR- cells of BM, in both the unseparated and Percoll fractions of each tissue. The data suggest NK active cells of BM may be distinct from those found in PBL.     

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-.     

Purification and properties of cytoplasmic granules from cytotoxic rat LGL tumors

To evaluate the role of NK cell granules in the lytic activity of NK cells, cytoplasmic granules of rat NK tumors were purified by centrifugation of the cell homogenates in a Percoll gradient. Analysis of such gradients showed a band of light-scattering material near the bottom of the tube; assay of gradient fractions for lytic activity against SRBC showed a potent lytic activity giving a sharp peak in this region. Complete lysis of SRBC was achieved with less than 1 microgram/ml protein of the most active fractions. Examination in the electron microscope showed that a pool of fractions containing lytic activity consisted of pure cytoplasmic granules showing similar morphology to those found in the LGL tumors. The lytic band was associated with a peak in the activity of four different lysosomal enzymes. Analysis of Percoll gradient fractions showed that marker enzymes for mitochondria, plasma membrane, and cytosol were well separated from this activity peak. Analysis of the Percoll gradient fractions by SDS gel electrophoresis showed that this granule fraction was free of contamination of proteins from other parts of the gradient. The granules contained major protein bands of 62, 58, 30, 29, and 28 kilodaltons. In addition to protein, the purified granule fractions contain hexose and uronic acid, but no nucleic acids or phospholipids were detected in chemical assays. Major amounts of chymotryptic, tryptic, and elastase activities were not present, nor were peroxidase or lysozyme activities detectable in substantial amounts. These data show that NK tumor cell cytoplasmic granules contain a potent lytic activity and have biochemical properties that distinguish them from granules present in granulocytes and mast cells.     

Estrogen and antiestrogen modulation of the levels of mouse natural killer activity and large granular lymphocytes

We investigated the time course of the 17β-estradiol effect on mouse natural killer (NK) activity and the number of splenic large granular lymphocytes (LGL), a cell population recently associated with natural cytotoxicity and enriched in low density fractions of Percoll discontinuous density gradients. After 7 days of in vivo treatment with estrogen, an increased cytotoxicity against YAC-1 lymphoma cells was observed using only as effectors cells recovered from higher density fractions, usually devoid of NK activity. In contrast, after a 30-day treatment, augmented NK activity and an increase in LGL number were observed in the lower density Percoll fractions. Similar results were observed after a 30-day treatment with the antiestrogen tamoxifen. The cytotoxicity of both low density and high density splenocyte fractions was totally abrogated by treatment with antiserum to asialo GM₁ plus complement, whereas anti-Thy 1.2 antibody treatment only partially decreased the reactivity. Further estrogen administration up to 60 days decreased both NK activity and LGL number. It is concluded that estradiol can induce opposite effects on NK activity depending on the time of treatment, with stimulation of NK activity during the first 30 days after treatment followed by depressed NK activity 1 month later.     

Differential distribution of nitric oxide synthase between cell fractions isolated from the rat gastric mucosa.

Cells isolated from the rat gastric mucosa were resolved into two fractions on a percoll density gradient, and into five fractions using counterflow centrifugation (elutriation). Ca(2+)-dependent nitric oxide synthase (NOS) activity was found in the high density percoll fraction but not in the parietal cell enriched low density fraction. This activity was inhibited by NG-monomethyl-L-arginine with an IC50 of 3.7 microM. Cells in the elutriator fraction rich in mucous-epithelial cells exhibited the highest NOS activity, while the smaller cell fractions had no detectable NOS yet had the highest basal release of prostaglandin E2. The parietal cell enriched elutriator fraction again had low NOS activity. The activity of a constitutive NOS in the mucous-cell fraction may indicate a role of NO in the regulation of epithelial cell integrity or secretion.     

Distribution of terminal deoxynucleotidyl transferase in bovine serum albumin gradient-fractionated thymocytes and bone marrow cells of normal and leukemic mice.

The distribution of terminal deoxynucleotidyl transferase (TdT) peaks I and II, in single cell suspensions of thymuses, bone marrow, and peripheral lymphoid organs fractionated in discontinuous bovine serum albumin gradients, was examined in a variety of mouse strains and Fischer 344 rats to relate the normal patterns of thymocyte differentiation to the leukemic process. TdT peaks I and II were found in fractions A (10 to 23%), B (23 to 26%), and C (26 to 29%) of the thymus of both normal and leukemic C57BL/6 mice, whereas only peak I was found in the same fractions of AKR mice. TdT in bone marrow was found mainly in fraction A in both normal and leukemic mice. The specific activity of TdT in this fraction, which comprises only 1 to 5% of the total bone marrow cell population, was similar to that of the thymus. The cell population of fraction A of the bone marrow was found to increase (10 to 15-fold) in leukemic mice. Only low levels of TdT activity were found in either whole or fractionated bone marrow of athymic NIH Swiss (nu/nu) mice.     

Impaired natural killer cell function in hemophiliacs with or without continuous substitution

In the present study, 28 hemophiliacs substituted continuously and 5 hemophiliacs who had received almost no blood products were investigated. Cells of OKT 3+, OKT 4+, and OKT 8+ subsets were counted. Percoll separated fractions of peripheral blood mononuclear cells were examined by morphological criteria and were tested for NK cell activity. We found that the NK cell activity of both groups of hemophiliacs was decreased on testing Ficoll separated cells or low density Percoll separated cells. Normal NK cell activity was found in medium density cells of hemophiliacs. Two possible explanations are discussed: first, the NK cell activity may be suppressed in hemophiliacs and secondly, there may be a block in maturation of NK cell activity. It is unlikely that chronic substitution by blood products counts for these alterations. The possible role of chronic infections is discussed.     

Suppression of natural killer (NK) cell activity of spleen cells by thymocytes

The in vitro influence of thymus cells on natural killer cell activity of spleen cells against prelabeled target cells (YAC-I and RL♂I) has been studied in syngeneic as well as in allogeneic murine models. In mixing experiments to demonstrate suppression, total thymocytes have been found to have no effect on NK activity of syngeneic or allogeneic spleen cells. Among several thymocyte fractions separated by velocity sedimentation, a relatively faster sedimenting fraction showed remarkable suppression of NK activity by spleen cells against two target cells. The suppressive effect of this particular fraction on NK activity was demonstrated to be proportional to the cell dose. The suppressive function was resistant to irradiation at 1000 or 2000 rad administered in vitro and was not restricted by the major histocompatibility complex. Moreover, the thymocyte fraction which induced suppression was not sensitive to NK-mediated cytolysi? by syngeneic spleen cells. The suppression of NK cytolysis in vitro by certain subpopulations of thymocytes as observed in the present studies may be consistent with a role for the thymus in regulating NK activity in vivo.     

Suppressive effect of human natural killer cells on pokeweed mitogen-induced B cell differentiation

The suppressive effect of human natural killer (NK) cells on B cell differentiation induced by pokeweed mitogen (PWM) was investigated. By using Percoll discontinuous density gradient centrifugation, peripheral blood nonphagocytic and nonadherent mononuclear cells were divided into low and high density fractions for which NK cells (Large granular lymphocytes, LGL) and T cells were enriched, respectively. These fractionated mononuclear cells were co-cultured with purified autologous B cells in the presence of PWM, and were examined for their helper and suppressor activities on differentiation of B cells to immunoglobulin-(IgM and IgG) producing cells by a highly sensitive reversed hemolytic plaque assay. The T cell-enriched high density fractions provided help for B cell differentiation to levels higher than that of unfractionated mononuclear cells. On the other hand, the NK-enriched low density fractions did not show helper activity, and when added to the culture of B cells plus helper T cells, they markedly suppressed B cell differentiation. This suppressive activity, as well as the NK cytotoxicity of the NK-enriched fractions, was abrogated by treatment of the cells with monoclonal antibody against human NK cells (HNK-1), but not against T cells (OKT3) in the presence of complement. NK cells also suppressed PWM-driven B cell differentiation in the presence of T4+ (helper/inducer T) but not T8+ (cytotoxic/suppressor T) cells; however, they showed no inhibition of soluble factor-induced B cell differentiation assayed in the absence of helper T cells. It is thus concluded that human peripheral blood NK cells exhibit an ability to suppress PWM-driven B cell differentiation, possibly by acting through the effect on helper T cells but not directly on B cells.     

Low molecular weight serine proteinase inhibitors of the human intervertebral disc

Human lumbar disc tissue when extracted with 4M GuHCl and subjected to dissociative CsCl density gradient ultracentrifugation yielded trypsin inhibitor activity in the low bouyant density fractions (rho less than or equal to 1.38 g/ml). Disc proteoglycans sedimented in the high bouyant density fractions (rho greater than or equal to 1.5 g/ml). Sephadex G75F gel filtration of the low bouyant density protein fractions afforded a major low molecular weight (Kav = 0.5) trypsin inhibitor pool which was further purified by trypsin affinity chromatography. This latter step facilitated separation of the trypsin inhibitors from neutral proteinase activity also present. The trypsin inhibitor fraction so isolated was shown to possess potent inhibitory activity against a range of human serine proteinases including leukocyte elastase and cathepsin G, urokinase, kallikrein, plasmin and thrombin. Significantly this serine proteinase inhibitor preparation effectively prevented degradation of proteoglycans by a neutral proteinase also isolated from the human intervertebral disc.     

Regulation of cell proliferation inhibitory and stimulatory factors diffused by 3T3 cultured cells

The growth rate of normal cells multiplied in vitro decreases as the cell density of the culture increases. Previous results suggested that this density-dependent inhibition of growth in nontransformed cells was due to the diffusion of growth inhibitory substances in the medium of dense cultures. In this paper, we demonstrate that dense cultures of 3T3 cells secrete inhibitory and stimulatory factors. Macromolecules of conditioned medium were fractionated on Biogel P150 and the different fractions were tested on quiescent cultures of 3T3 cells stimulated or not to proliferate by addition of alpha globulin. When target cells were not stimulated to proliferate by addition of exocrine growth factors, we observed the inhibitory activity of a large molecular weight inhibitor (IDF45) and the stimulatory activity of autocrine growth factors (fraction about 35 and 10 K molecular weight), on the incorporation of 14C inosine into nucleotide pool and RNA. However, DNA synthesis was significantly stimulated with fraction 10 K only. This discrepancy between the stimulation of RNA and DNA synthesis may be explained by the presence, simultaneously, of inhibitory and stimulatory factors in fraction 35 and 10 K molecular weight. The presence of inhibitory factor was demonstrated when the fractions were tested on target cells stimulated to proliferate by alpha globulin addition and labeled with 14C thymidine. In these conditions, the stimulatory activity of autocrine growth factors was not observable, and only the inhibitory activity on DNA synthesis of fractions 35 and 10 K appeared. It is tempting to assume that the regulation of in vitro cell proliferation is determined by the balance between these antagonist stimulatory and inhibitory autocrine growth factors.     

Origin and differentiation of natural killer cells. II. Functional and morphologic studies of purified NK-1.1+ cells

Cells bearing the NK-specific marker NK-1.1 were purified from mouse spleens by utilizing a monoclonal anti-NK-1.1 antibody and cell sorting. In normal adult mice, all of the splenic NK activity against YAC-1 cells was found in the NK-1.1+ fraction, whereas NK-1.1- cells were depleted of NK activity. The NK activity of sorted NK-1.1+ cells was enriched 15- to 30-fold over unfractionated spleen cells. Light and electron microscopic studies of purified NK-1.1+ cells showed a homogeneous population of cells, each containing one to four cytoplasmic granules. Mice whose bone marrow has been destroyed by chronic exposure to 17-beta-estradiol have very low NK activity. However, spleen cells of estradiol-treated mice contained a normal frequency of NK-1.1+ cells which bound to YAC-1 cells, but failed to lyse them even after purification and subsequent exposure to interferon-alpha/beta in vitro. It appears, therefore, that in the absence of intact bone marrow, NK-1.1+ cells may be arrested in a nonlytic and interferon-unresponsive state. Spleens of neonatal mice which have low NK activity were analyzed to ascertain whether immature NK-1.1+ cells, similar to those found in estradiol-treated mice, could be demonstrated. Spleens of 8- to 9-day-old mice also contained NK-1.1+ cells which had very low NK activity even after purification. Sorted NK-1.1+ cells were examined for cytotoxicity in mice whose NK activity was suppressed by pretreatment with Corynebacterium parvum (-15 days). In contrast to cells from estradiol-treated and neonatal mice, NK-1.1+ from mice treated with C. parvum had normal functional activity. Similarly, although NK activity of unfractionated bone marrow cells is low, sorted NK-1.1+ cells were greatly enriched for lytic activity. Thus, we conclude that cell sorting with monoclonal anti-NK-1.1 antibody provides a powerful tool for examining the mechanisms underlying various states of low NK activity, and there exist NK-1.1+, nonlytic, interferon-unresponsive cells which apparently require an intact marrow microenvironment for differentiation into mature, lytic NK cells.     

Phenotypic characterization of cynomolgus monkey natural killer cells

Natural killer (NK) activity of cynomolgus monkey peripheral blood lymphocytes (PBL) was determined using B95-8 cells as target cells. Examination for the reactivity of human NK-related monoclonal antibodies (mAbs), anti-Leu-7, anti-Leu-11b, anti-NKH1A, and NC-1, with cynomolgus PBL revealed that Leu-11b (CD16) was the only antigen expressed on cynomolgus PBL. The percentage of Leu-11b-positive (Leu-11b+) cells correlated well with the level of NK activity when PBL taken from 21 monkeys were tested. After depletion of Fc receptor-positive (FcR+) cells, NK activity was lost concomitantly with the disappearance of Leu-11b+ cells. These results show that cynomolgus NK cells are mainly FcR+ which can be detected by mAb directed to Leu-11b. Cynomolgus PBL were separated by Ficoll-Hypaque centrifugation after E rosette formation with 2-aminoethylisothiouronium bromide-treated sheep red blood cells, and NK activities of both E rosette-forming (E+) and nonforming (E-) fractions were determined. The high level of killing was observed in the E- fraction, suggesting that the majority of cynomolgus NK cells was contained in the E- fraction. The separation of PBL by Percoll discontinuous density gradient showed cynomolgus NK cells were enriched in the low density fractions.     

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.     

Regulation of the cytotoxic T lymphocyte response against Qa-1 alloantigens

Spleen cells from B6.Tlaa (Qa-1a) mice primed against C57BL/6 (Qa-1b) splenocytes in vivo generate Qa-1-specific CTL when rechallenged with Qa-1b Ag in vitro. The addition of unirradiated Qa-1b splenocytes to these cultures inhibits the generation of Qa-1-specific CTL. By using highly purified cell populations, we demonstrate that the only cell population in resting spleen capable of causing this inhibition is NK1.1+. Although resting CD8 cells lack inhibitory activity, purified CD8 cells precultured with Con A and IL-2 inhibit anti-Qa-1 CTL. This inhibition is specific for the Qa-1b Ag expressed on the inhibitor cells, is not due to cold target competition, and is thus similar to that ascribed to veto cells. Although NK cells from resting spleen inhibit the generation of Qa-1-specific CTL, NK cells precultured in the presence of Con A and IL-2 show an approximate 30-fold increase in veto activity. Thus, NK cells represent the most likely cell population for down-regulating anti-self class I-reactive CTL.     

Subcellular localization of squalene synthase in human hepatoma cell line Hep G2.

Using the Hep G2 cell line as a model for the human hepatocyte the question was studied whether Hep G2-peroxisomes could be able to synthesize cholesterol. Hep G2 cell homogenates were applied to density gradient centrifugation on Nycodenz, resulting in good separation between the organelles. The different organelle fractions were characterized by assaying the following marker enzymes: catalase for peroxisomes, glutamate dehydrogenase for mitochondria and esterase for endoplasmic reticulum. Squalene synthase activity was not detectable in the peroxisomal fraction. Incubation of Hep G2 cells with U18666A, an inhibitor of the cholesterol synthesis at the site of oxidosqualene cyclase, together with heavy high density lipoprotein, which stimulates the efflux of cholesterol, led to a marked increase in the activity of squalene synthase as well as HMG-CoA reductase, whereas no significant effect on the marker enzymes was observed. Neither enzyme activity was detectable in the peroxisomal density gradient fraction, suggesting that in Hep G2-peroxisomes cholesterol synthesis from the water-soluble early intermediates of the pathway cannot take place. Both stimulated and non-stimulated cells gave rise to preparations where squalene synthase activity was comigrating with the reductase activity at the lower density side of the microsomal fraction; however, it was also present at the high density side of the microsomal peak, where reductase activity was not detected.     

Separation of active and inactive fractions from starved culture of Vibrio parahaemolyticus by density dependent cell sorting

The co-existence of physiologically different cells in bacterial cultures is a general phenomenon. We have examined the applicability of the density dependent cell sorting (DDCS) method to separate subpopulations from a long-term starvation culture of Vibrio parahaemolyticus. The cells were subjected to Percoll density gradient and separated into 12 fractions of different buoyant densities, followed by measuring the cell numbers, culturability, respiratory activity and leucine incorporation activity. While more than 78% of cells were in lighter fractions, about 95% of culturable cells were present in heavier fractions. The high-density subpopulations also had high proportion of cells capable of forming formazan granules. Although this was accompanied by the cell specific INT-reduction rate, both leucine incorporation rates and INT-reduction rates per cell had a peak at mid-density fraction. The present results indicated that DDCS could be used to separate subpopulations of different physiological conditions.     

Organic synthesis in algal cells separated into age groups by fractional centrifugation

Summary Synthesis of organic matter was studied in cells of the green, high-temperature alga Chlorella 7-11-05 separated from a nonsynchronized population into fractions of predominantly small or large cells by centrifugation. Rates of synthetic activity were determined as changes in optical density, dry weight, and packed volume of cells in several suspending fluids and under various light intensity conditions.It was found that synthetic activity of the smaller (younger) cell fraction was invariably higher than that of the larger (older) cell fraction, provided a reasonably good separation of cells into size fractions was achieved during centrifugation and the difference between size composition of these fractions was maintained throughout observation.An occasional lack of difference in the performance of the small- and large-cell fractions, or even a higher synthetic activity of the originally large-cell fraction, was traced to cell division taking place during observation. Under conditions favorable to cell division, the large-cell fraction was progressively enriched with smaller (younger) cells, the average age composition of the originally large-cell fraction was shifted toward younger age, and the metabolic activity of the large-cell fraction increased to the extent that, in some experiments, it surpassed that of the originally small-cell fraction.The decline in synthetic activity of cells in the course of cell development previously observed on synchronized cells was thus substantiated on nonsynchronized populations in the absence of the light: dark synchronizing agent and is, therefore, characteristic of normal cell development.     

Synthesis of plasmalemmal glycoproteins in intestinal epithelial cells. Separation of Golgi membranes from villus and crypt cell surface membranes; glycosyltransferase activity of surface membrane

The relationship between Golgi and cell surface membranes of intestinal cells was studied. These membranes were isolated from intestinal crypt cells and villus cells. The villus cell membranes consisted of microvillus membrane, a Golgi-rich fraction, and two membrane fractions interpreted as representing lateral-basal membranes. The villus cell microvillus membrane was purified by previously published techniques while the other membranes were obtained from isolated cells by differential centrifugation and density gradient velocity sedimentation. The two membrane fractions obtained from villus cells and considered to be lateral-basal membranes were enriched for Na+,K+-ATPase activity, but one also showed enrichment in glycosyltransferase activity. The Golgi membrane fraction was enriched for glycosyltransferase activity and had low to absent Na+,K+-ATPase activity. Adenylate cyclase activity was present in all membrane fractions except the microvillus membrane but co-purified with Golgi rather than lateral-basal membranes. Electron microscopy showed that the Golgi fraction consisted of variably sized vesicles and cisternalike structures. The two lateral-basal membrane fractions showed only vesicles of smaller, more uniform size. After 125I labeling of isolated intact cells, radioactivity was found associated with the lateral-basal and microvillus membrane fractions and not with the Golgi fraction. Antibody prepared against lateral-basal membrane fractions reacted with the surface membrane of isolated villus cells. The membrane fractions from isolated crypt cells demonstrated that all had high glycosyltransferase activity. The data show that glycosyltransferase activity, in addition to its Golgi location, may be a significant property of the lateral-basal portion of the intestinal villus cell plasma membrane. Data obtained with crypt cells support earlier data and show that the crypt cell surface membrane possesses glycosyltransferase activity.