Natural killer cell activity in the rat. V. The circulation patterns and tissue localization of peripheral blood large granular lymphocytes (LGL)

Large granular lymphocytes (LGL) and T cells were separated from blood by centrifugation on discontinuous gradients of Percoll, were labeled with [3H]uridine or [111In]oxine, and were injected i.v. into syngeneic euthymic or athymic nude rats. The tissue distribution of these labeled cells was monitored for up to 24 hr after transfer by scintillation counting of tissue homogenates and autoradiography of tissue sections. In normal euthymic rats, the main sites of LGL localization were the alveolar walls of the lungs and spleen red pulp; however, they were not detectable in the major traffic areas of T lymphocyte recirculation, the spleen white pulp, and lymph nodes. Furthermore, the density of labeled LGL was very low in the small intestine, thymus, kidney, and liver, although on a per-organ basis, about 10% of the injected radioactivity was found in the liver by 24 hr post-injection. When 111In-labeled LGL were injected i.v. into rats with an indwelling thoracic duct cannula, they completely failed to enter the thoracic duct lymphocyte (TDL) population over an observation period of 6 days. This finding was markedly different from the results obtained with T cells and was consistent with the lack of natural killer and antibody-dependent cellular cytotoxicity activity observed among TDL, even in rats pretreated with the biological response modifier, poly I:C. LGL in athymic nude rats also failed to recirculate between blood and lymph. However, in contrast to normal euthymic animals, a significant increase in the localization of radiolabeled LGL to lymph nodes was observed in nude rats between 30 min and 24 hr. Taken as a whole, these findings define the areas within the lungs and spleen in which blood LGL normally localize, and clearly demonstrate that LGL do not normally recirculate between blood and lymph.     

One-signal requirement for interferon-gamma production by human large granular lymphocytes

This laboratory has been investigating IFN-gamma gene expression by highly purified human large granular lymphocytes (LGL) and T cells. We report here that within 1 hr after interleukin 2 (IL 2) treatment of freshly isolated human LGL, IFN-gamma mRNA can be detected, with IFN-gamma protein in the culture medium within 4 to 6 hr of treatment. CD3- Leu-11+ LGL require only a single signal for IFN-gamma production because phytohemagglutinin (PHA), phorbol myristate acetate (PMA), IL 2, or ionomycin can each independently induce IFN-gamma production. In addition, PHA and ionomycin (but not IL 2) show significant synergy with PMA as a stimulus to LGL. In contrast, CD3+ T cells require two stimuli for high levels of IFN-gamma production, and not only are PMA plus ionomycin or PHA synergistic, but in addition, IL 2 and PHA demonstrate some synergy. Furthermore, we have found by fractionation of peripheral blood lymphocytes that IL 2-induced IFN-gamma production is associated with the LGL population and not T cells. These results indicate that with certain stimuli, LGL may be the predominant source of IFN-gamma from peripheral blood lymphocytes.     

Protective effects of differentiation inducers on natural killer sensitivity of K 562 cells: Analysis at a single-cell level

K 562 cells induced to differentiate by sodium butyrate (SB) or 12-O-tetradecanoyl-phorbol-13-acetate (TPA) were studied for their capacities to be bound and killed by large granular lymphocytes (LGL) in a single-cell cytotoxicity assay in agarose. After SB treatment, K 562 cells were less efficient in binding to LGL, whereas the frequency of killer cells among bound LGL was unaffected. When TPA was used to induce K 562 differentiation, the binding of LGL to their target and the lytic efficiency of the bound LGL were both diminished when compared to control K 562 cells. It has been demonstrated that the expression of structures involved in the binding of natural killer (NK) effectors to their targets could be correlated with the target-differentiation stage. It is shown that phorbol-ester treatment can also affect NK target structures involved in the killing step.     

Natural killer activity in the rat. III. Characterization of transplantable large granular lymphocyte (LGL) leukemias in the F344 rat

Six transplantable large granular lymphocyte (LGL) tumor lines in F344 rats were examined for natural killer (NK) and antibody-dependent cell-mediated cytotoxicity. Tumor cells from all six lines were highly cytotoxic, even at low effector to target ratios, when tested against NK-susceptible targets, but were unreactive against an NK-resistant target (C58NT)D) and a macrophage-susceptible target (P815). Three lines showed significant levels of lysis against antibody-coated tumor cells. After in vivo transplantation, the levels of cytotoxicity steadily increased in three lines and decreased in one. The cytotoxic activity of one line (RNK-16) remained high through 12 transplant generations. Tumor cells injected i.p. spread via the lymphatics to regional lymph nodes, mediastinal nodes, blood, and eventually the bone marrow. Leukemia occurred concurrently with organ enlargement and increased levels of NK. Studies in (F344 X W/Fu)F1 rats clearly demonstrated that the cytotoxic cells from leukemic animals were the transplanted tumor cells themselves and not merely the activation of normal host LGL. These results demonstrate that naturally occurring, transplantable LGL leukemias are an easily obtainable and excellent source of materials for those studies requiring a large number of functionally active LGL.     

Fc gamma-receptor-bearing, non-B lymphocytes in human peripheral blood: cytophilic immunoglobulin binds almost exclusively to large granular lymphocytes

Cytophilic IgG (CYT-Ig) has previously been reported to bind to both the "TG" (E+, Fc gamma R+) and "L" (E-, Fc gamma R+) subsets of non-B lymphocytes in human peripheral blood. Present investigations show that IgG-binding cells, as detected by a sensitive antiglobulin rosetting reaction, are contained almost entirely within the large granular lymphocyte (LGL) subpopulation, and that fewer than 5% of other non-B lymphocytes acquire IgG from serum. Cell membrane-bound IgG sterically blocks the reaction of LGL with sheep red blood cells and therefore influences the proportions of these cells characterized as TG (E+) or L (E-) lymphocytes. Although the majority of TG lymphocytes are LGL, a further subpopulation of E+, Fc gamma R+ cells are detectable under particular test conditions. Unlike LGL, these lymphocytes do not react with rabbit IgG-coated ox RBC (EAG) in saline, but will form EAG rosettes when the reaction is enhanced in the presence of Ficoll. These Fc gamma R+ cells are mostly of typical small-lymphocyte morphology and do not bind detectable amounts of CYT-Ig, nor do they express the monoclonal antibody-defined VEP 13 determinant associated with Fc gamma R on LGL.     

Ultrastructure of large granule-containing lymphocytes possessing natural killer activity

By means of light and electron microscopy, ultrastructural cytochemistry and immune cytochemistry methods, contents and ultrastructure of large granule-containing lymphocytes (LGL) have been studied in human blood--this is cell population possessing natural killer and, partly, antibody-depending cytotoxicity. The LGL concentrates are isolated from blood applying successive physical-chemical methods, differential centrifugation in the density gradient of pack-phycoll and percoll included. Separate LGL populations are marked by means of rosette-forming reaction with sheep erythrocytes and monoclonal antibodies OKT4 and OKT8. Relative and absolute amount of the LGL in 1 1 of blood is 5.4 +/- 0.5% and 0.319 +/- 0.28 X 10(9), respectively. The LGL ultrastructure is characterized with a low nuclear-cytoplasmic ratio, with presence of osmiophilic (azurophilic) granules in cytoplasm and specific parallel-tubular structures, with a well developed Golgi complex, an essential number of mitochondria, vesicles with smooth wall and vacuoles, as well as multivesicular bodies and Gallo bodies. The LGL subpopulations, expressing various membrane antigens (E+, E-, OKT8+, OKT8-) differ in their ultrastructure, that is evidently stipulated by the degree of their differentiation and their function.     

Natural cytotoxicity of lymphocytes from lymph nodes draining breast carcinoma and its augmentation by interferon and OK432

Summary Lymphocytes isolated from axillary lymph nodes draining breast carcinoma were tested for natural killer (NK) activity against K562 in a 4-h ⁵¹Cr-release assay, and the in vitro effects of interferon (IFN) and OK432 (a streptococcal preparation) on their cytotoxicity were examined in comparison with NK activity of autologous peripheral blood lymphocytes (PBL). The levels of NK activity were lower in lymph node lymphocytes (LNL) than in PBL of the same patients. Significant levels of LNL-mediated lysis were recorded in 14 of 42 (33%) lymph node samples and in nine of 14 (64%) patients. Purification of large granular lymphocytes (LGL) from lymph node cells by discontinuous Percoll density gradient centrifugation resulted in an induction or enhancement of cytotoxic activity, with no reactivity in LGL-depleted, small T-lymphocyte populations. Positive reactions were observed with 10 of 13 (77%) LGL samples. The low reactivity of LNL was not attributable to coexistent suppressor cells for NK function, since lymph node cells failed to suppress NK activity of normal PBL. Partially purified human IFN and OK432 augmented NK activity of patients' PBL in approximately 70% and 90% of the cases, respectively, while LNL-mediated lysis was augmented in only 7% and 36% of the lymph node samples by IFN and OK432, respectively. These results indicate that K562-reactive NK cells and/or their precursors may frequently be present at subthreshold levels in the lymph nodes draining breast carcinoma, and that the augmentation of LNL-mediated cytotoxicity by OK432 might provide a local potentiation of natural immune function at the host-tumor interface rather than IFN.     

Identification and purification of NK cells with lysosomotropic vital stains: correlation of lysosome content with NK activity

The lysosome content of lymphocytes has been analyzed with lysosomotropic vital stains and the fluorescence-activated cell sorter (FACS). Large granular lymphocytes (LGL), which account for virtually all natural killing activity in peripheral blood, are quantitatively different from small lymphocytes (SL) in this respect. LGL obtained by Percoll gradient density centrifugation accumulate more of the lysosomotropic vital dyes than SL do, staining with either neutral red or mepacrine (quinacrine). Furthermore among the LGL-rich, low density lymphocyte population highly, granulated cells can be separated from less granulated ones by mepacrine staining and FACS. Thus, separated highly granulated LGL express very high natural killing, whereas the less granulated low density large lymphocytes do not kill.     

Comparison of the effect of IL-2 and IL-6 on the lytic activity of purified human peripheral blood large granular lymphocytes

The effects of IL-6 and IL-2 on highly purified, human peripheral blood large granular lymphocytes (LGL) were investigated and compared. IL-6 enhanced LGL NK activity in a dose-dependent manner against K562, however IL-2 was a more potent stimulus of LGL NK function. Neither IL-2 nor IL-6 increased LGL cytotoxic potential in a parallel estimation of heteroconjugated antibody (anti-CD16 x anti-nitrophenyl mAb)-dependent cytotoxicity against nitrophenyl-modified YAC. Unlike IL-2, IL-6 did not significantly induce LGL lymphokine-activated killer activity, LGL proliferation, or LGL lymphokine production. In particular, IL-6 did not stimulate detectable LGL IL-2 production or IL-2R modulation, and mAb to the p75 IL-2R had no effect on IL-6 induction of LGL NK activity. Therefore, in the absence of T cells, IL-6 provided an IL-2-independent signal to LGL that resulted in augmentation of their NK activity without stimulating their proliferation or other LGL functions.     

Tumor necrosis factor induction by Candida albicans from human natural killer cells and monocytes

Other investigators have previously reported that TNF has been induced from macrophages by bacteria and, more recently, from NK cells by certain tumor cells. Sendai virus has also been reported to induce TNF from macrophages. We report here that an opportunistic fungi, Candida albicans, can also induce TNF, not only from human monocytes, but also from Percoll-fractionated large granular lymphocytes (LGL) which mediate NK function. Incubation of monocytes of LGL with C. albicans for 8 h was sufficient for detection of TNF release and peak induction was observed at 24 h. Induction of TNF from LGL did not require the participation of monocytes or T cells because treatment of the LGL with CD14 or CD15 to eliminate contaminating monocytes and CD3, CD4, or CD8 to eliminate contaminating T cells did not decrease the level of TNF produced from the treated LGL. Small T cells recovered from the denser fractions of the Percoll gradient had no ability to produce TNF, even when 10% monocytes were added to the T cells to provide accessory function. The phenotype of the TNF-producing LGL was CD2+, CD11+, CD16+, NKH1+, LEU7-. The TNF produced by both monocytes and LGL was neutralized by specific monoclonal and polyclonal anti-TNF but not by monoclonal antilymphotoxin. These results indicate that TNF production is a normal response of monocytes and LGL to stimulation by fungi such as C. albicans and that the release of TNF may be related to its ability to activate effector function to control Candida growth, which we have shown earlier for neutrophils with TNF.     

Effect of rutin on the cholesterol content of the lymph, blood and tissues of the dog

It has been discovered in experiments on mongrel dogs anesthetized with morphine and hexenal that intravenous injection of rutin (3 mg/kg bw) reduced the concentration of total, esterified and free cholesterol in blood serum, with the exception of free cholesterol level in arterial blood, which increased. Under the same conditions, the concentration of free and esterified cholesterol in thoracic duct lymph ascended. In the popliteal lymph node, gastrocnemius muscle and liver, the content of total cholesterol appreciably dropped as compared to control. Rutin has been found to accelerate the lymph flow and to raise cholesterol resorption from the interstitial space. It is inferred that lymph plays an important part in cholesterol transport in the body.     

Quantitative analysis of rod-cored vesicles and dense granules of large granular lymphocytes in the liver,spleen, and peripheral blood of rats

Large granular lymphocytes (LGL) comprise a natural defense system in the liver and exert an inhibitory effect on tumor cell metastasis. In order to demonstrate the maturation of LGL in the liver from the morphological aspect, we evaluated electron-microscopically the frequency of 0.2 m vesicles (rod-cored and empty vesicles) and dense granules in LGL from the liver, spleen, and peripheral blood of the rat. Both of these cell organelles are characteristic to LGL and may relate to natural killer-mediated cytolysis. On the average, there were 12.7 of the 0.2 m vesicles and 4.3 rod-cored vesicles (RCV) per cell section in the liver, 6.6 0.2 m vesicles and 1.6 RCV in the spleen, and 8.6 0.2 m vesicles and 0.9 RCV in the peripheral blood. The number of 0.2 m vesicles per cell section ranged from 0 to 19 with the exception of a few higher instances. Therefore, LGL were divided into vesicle-rich(>9 0.2 m vesicles per cell section) and vesicle-poor (<8 per cell section) populations. Hepatic LGL consisted mainly of a vesicle-rich population while splenic LGL consisted mainly of a vesicle-poor population, and peripheral blood contained equal proportions of both populations. In addition to diversity with regard to the number of 0.2 m vesicles, LGL obtained from various organs also displayed heterogeneity in the number and size of dense granules. Since the number of dense granules per cell section usually ranged from 1 to 13, LGL were diveded into 2 populations, i.e., LGL with many (>7 per cell section) granules and those with a few(<6 per cell section) granules. Specifically, splenic LGL had a few small (average diameter, less than 400 nm) dense granules, while sections of LGL from the liver and peripheral blood displayed many small dense granules and a few large (>400 nm) ones, respectively, in addition to the populations seen in the spleen. Thus, the present study has demonstrateda difference in the distribution of 0.2 m vesicles in LGL based on the tissue of origin. The present study has revealed the difference in the distribution of 0.2 m vesicles of LGL by tissue and indicated that immature LGL are predominant in the spleen, while hepatic LGL are generally more mature as defined by the number of vesicles. These data suggest that the microenvironment of the liver may contribute to the increased expression of these vesicles in LGL.     

In vitro migration of rat large granular lymphocytes

Rat peripheral blood large granular lymphocytes (LGL) were isolated by fractionation on discontinuous Percoll gradients. LGL migration was studied using nitrocellulose filters. Rat LGLs migrated into nitrocellulose filters in response to N-formyl-methionyl-leucyl-phenylalanine (f-MLP), casein, and serum components. Percoll-enriched high-density lymphocytes had small, but significant, migratory capacity in response to stimuli under these conditions. Removal of OX-19+ contaminating cells by panning confirmed the migratory capability of rat LGL/NK cells under these conditions. Checkerboard analysis of the LGL response to chemoattractants revealed that induction of migration involved chemokinesis although a chemotactic component was also discernible. The prompt migration of rat LGL in response to different stimuli is consistent with the hypothesis that these cells may represent one of the first easily mobilizable lines of resistance against noxious agents. In the rat combined in vitro/in vivo studies may provide a better understanding of the regulation of LGL recruitment and extravasation.     

Co-sedimentation of chondroitin sulfate A glycosaminoglycans and proteoglycans with the cytolytic secretory granules of rat large granular lymphocyte (LGL) tumor cells, and identification of a mRNA in normal and transformed LGL that encodes proteoglycans

Rat large granular lymphocyte (LGL) tumor cell lines were analyzed for the presence of proteoglycans and glycosaminoglycans in their cytolytic secretory granules. When isolated rat LGL tumor cells were incubated in vitro for 1 to 3 hr with [35S]sulfate, and the 35S-labeled macromolecules were purified by density-gradient centrifugation, they filtered on Sepharose CL-4B columns predominantly as approximately 500,000 m.w. macromolecules. After 19 hr of incubation with [35S]sulfate, however, an 85,000 m.w. species predominated. Pulse-chase experiments revealed that the larger macromolecules were proteoglycans that with time were processed to glycosaminoglycan-sized macromolecules. As assessed by their susceptibility to chemical and enzymatic degradation and by high pressure liquid chromatography of the chondroitinase ABC-generated unsaturated disaccharides, the cell-associated rat LGL tumor cell proteoglycans bore almost exclusively chondroitin sulfate A glycosaminoglycans. Northern blot analysis using a gene-specific probe revealed that both normal peripheral blood and transformed rat LGL expressed the same approximately 1.3-kb mRNA that encodes the peptide core of the proteoglycans in the secretory granules of rat and mouse mast cells. In vivo radiolabeling of rat LGL tumor cells and isolation of their intact granules after nitrogen cavitation and density sedimentation established that glycosaminoglycans compartmentalized with cytolytic activity. Thus these negatively charged macromolecules may play a role in the regulation of the packaging and delivery of the cytolysins and basically charged serine proteases that have been identified in the cytolytic secretory granules of LGL.     

Cytotoxic activity against trophoblast and choriocarcinoma cells of large granular lymphocytes from human early pregnancy decidua

Large granular lymphocytes (LGL) are the most abundant cell type in first trimester human pregnancy decidua. We have shown previously that CD56-positive decidual LGL have cytotoxic activity against the natural killer (NK) target K562, and that this cytotoxicity is augmented by pretreatment with interleukin-2 (IL-2). We now report that flow cytometrically purified populations of CD56-positive decidual LGL have no cytotoxic activity against either the BeWo choriocarcinoma cell line or freshly isolated term trophoblast. Incubation of unfractionated decidual cells with IL-2 induced cytotoxicity against BeWo, but term trophoblast remained resistant to lysis. Both BeWo and trophoblast showed much lower binding frequencies to decidual or peripheral blood cells than K56 targets, and excess trophoblast did not inhibit cytotoxic activity against K562. This suggests that the resistance of trophoblast to lysis by either decidual or peripheral blood LGL is due to the lack of accessible NK target structures on the surface of trophoblast.     

Differential regulation of interleukin-1 gene expression in human CD3- large granular lymphocytes

Our laboratory analyzed the expression of lymphokine and cytokine mRNA in CD3- peripheral blood large granular lymphocytes (LGL). Herein we present evidence that this subset of lymphocytes can synthesize IL-1 beta mRNA constitutively and that the cytoplasmic mRNA levels of IL-1 beta can be increased rapidly by interleukin (IL)-2. IL-1 alpha mRNA is expressed constitutively very infrequently and increases in IL-1 alpha mRNA are seen only after prolonged incubation with IL-2. Furthermore, IL-1 activity could not be detected in LGL culture supernatants, indicating that other processes may be involved in releasing biologically active IL-1 from LGL. In addition, MAb to the p75 IL-2 receptor on LGL abrogated IL-2 induction of IL-1 beta mRNA, suggesting that IL-2 signaling via the p75 IL-2 receptor induced IL-1 beta gene expression in LGL. Since, in contrast to T cells, LGL are capable of mediating effector functions without prior stimulation, they are said to be already "primed" for response. Overall, these data suggest that constitutive lymphokine gene expression may be involved in the in vivo priming of LGL.     

Effects of transforming growth factor beta on the functions of natural killer cells: depressed cytolytic activity and blunting of interferon responsiveness

Type beta transforming growth factor (TGF-beta) is a unique polypeptide that has been isolated from a number of different tissues and can induce the phenotypic transformation of non-neoplastic fibroblasts as measured by the stimulation of their growth in soft agar. Recently, TGF-beta has been demonstrated to exert profound inhibitory effects on T and B lymphocyte proliferation. In this study, the effects of TGF-beta on natural killer (NK) cell function were investigated. After 20 hr of culture in the presence of TGF-beta, the NK activity of peripheral blood lymphocytes (PBL) was significantly reduced compared with PBL cultured in medium alone. Similarly, TGF-beta produced a significant depression in the cytolytic activity of highly enriched large granular lymphocytes (LGL). This effect of TGF-beta appeared to be mediated directly on the effector cells, because cultivation of the K562 target cells in TGF-beta did not affect target cell susceptibility to lysis. Binding studies with 125I-TGF-beta indicated that LGL possess approximately 1400 high-affinity (Kd = 1PM) receptors/cell, which represents a considerably higher affinity receptor for TGF-beta than that found on fibroblasts. Culturing of PBL and LGL in TGF-beta resulted in a marked blunting of the boosting of NK cytolysis by interferon-alpha but not by interleukin 2, which suggested that TGF-beta may down-regulate interferon-alpha receptors on NK cells. These results, indicate that in addition to inhibitory effects on T and B cells, TGF-beta also inhibits NK cell function. Although the in vivo role of TGF-beta is presently undefined, it may be an important immunoregulatory protein that has a negative influence on lymphocyte activation.     

Production of hemopoietic growth factors and gamma-interferon by large granular lymphocytes from patients with T gamma lymphocytosis

Two patients with T gamma lymphocytosis in whom expanded large granular lymphocyte (LGL) populations were detected in peripheral blood and bone marrow are described. The surface antigen phenotypes of the LGL from these patients were similar with a major portion of cells carrying T3, T8, T11 and Leu7 markers. However, whereas fresh LGL from both patients demonstrated antibody-dependent cell cytotoxicity (ADCC), natural killer (NK) cell function was present in one case but absent in the other. Supernatants from enriched suspensions of the LGL unstimulated by exogenous antigen or mitogens were shown to contain significant amounts of hemopoietic growth factors colony-stimulating activity (CSA) and burst-promoting activity (BPA). In one case gamma-interferon was also detected. This study contributes to the accumulating evidence that LGL are able to generate factors which have the capacity to influence the proliferation of hemopoietic progenitor cells in vitro.