Large granular lymphocytes or "pit cells" from rat liver: isolation, ultrastructural characterization and natural killer activity

Considerable numbers of large granular lymphocytes (LGL) were isolated from rat liver by a simple method consisting of sinusoidal lavage at elevated (50 cm water column) perfusion pressure. This method gave a yield comparable with the enzymatic dissociation method commonly used for the isolation of nonparenchymal liver cells, but was shorter in time and had the advantage of avoiding the potentially harmful effects of the dissociating enzymes. The isolated LGL were highly cytotoxic against YAC-1 lymphoma cells and this cytolytic activity was blocked by treatment of the effector cells with an antibody against natural killer cells (anti-asialo GM1). We characterized the hepatic LGL as nonphagocytic, nonadherent, peroxidase-negative and acid phosphatase-positive cells which could be enriched in the low-density fraction of a Percoll gradient. At the light microscopic level, they showed characteristic azurophilic granules which corresponded to strongly osmiophilic granules with a specific morphology in electron microscopy. It is concluded that these LGL are identical to the "pit cells" which were formerly described by electron microscopy in situ as normal components of the liver sinusoids and which are easily recognized by their fine structure. It is also proposed that the liver may represent one of the major natural killer organs.     

The role of human spleen interferon on natural killer activity of peripheral blood lymphocytes enriched in large granular lymphocytes

The elimination of monocytes as well as B- and T-lymphocytes by forming rosettes with high affinity for sheep red blood cells yielded an enriched population of both natural killer (NK) activity (cytotoxicity: 65.4 +/- 9.9% with an E/T ratio of 12:1, P less than 0.005) and large granular lymphocytes (LGL: 76 +/- 13%) compared to the untreated lymphocyte population where NK activity is 35.7 +/- 17.3% (E/T 12:1) and the percentage of LGL of 26 +/- 6%. We studied the action of type I interferon (IFN) obtained from human spleens, on NK activity of 9 peripheral blood lymphocyte populations and 9 enriched in LGL. NK activity of the total lymphocyte population is significantly increased (P less than or equal to 0.05) in 6 out of 9 cases after treatment by interferon. Cell populations enriched in LGL showed increased NK activity in only one case after treatment by interferon, but no increased activity was found in the other cases. These results are compatible with the notion of cellular cooperation in increased NK activity by interferon.     

A lymphoproliferative disorder of the large granular lymphocytes with natural killer activity

This paper reports the case of a patient with an abnormally expanded population of circulating lymphoid cells displaying the features of the so-called large granular lymphocytes (LGL). These cells were peroxidase negative and nonphagocytic, formed rosettes with sheep erythrocytes, had receptors for IgG, and contained azurophilic (electron-dense) granules. Like normal LGL, the patient cells were positive for two acid hydrolases (acid phosphatase and β-glucuronidase) but did not stain for α-naphthyl acetate esterase (ANAE), which is present in normal LGL. Ultrastructural studies revealed that the patient cells were rich in Golgi-derived vesicles, coated vesicles, multivesicular bodies, and immature granules, indicating that, unlike normal LGL, they were engaged in granulogenesis. These features, together with the absence of ANAE activity, are suggestive of some degree of cell immaturity. The patient cells displayed natural killer (NK) and antibody-dependent cellular cytotoxicity (ADCC) activities comparable to those of normal peripheral blood mononuclear cells, or even higher, and did not respond to T-cell mitogens or allogeneic cells. Furthermore, they were incapable of suppressing normal T-cell proliferation or pokeweed mitogen-induced B-cell differentiation. Analysis of the NK activity at the single-cell level revealed that a large proportion of the patient cells bound to the K562 target cells but could not accomplish the entire lytic process. This finding supports further the possibility that the patient cells were immature LGL. The surface phenotype of the patient cells (as defined by a battery of monoclonal antibodies) was somewhat different from that usually observed in the majority of the normal LGL because, in addition to the HNK-1 marker, the cells were OKT3⁺, aLeul⁺, aLeu4⁺, OKT8⁺, aLeu2a⁺, and 3A1⁺ but were OKM1^? and 4F2^?. This phenotype could correspond to that of maturing LGL.     

Functional properties of lymphocytes isolated from murine small intestinal epithelium

Intraepithelial lymphocytes (IEL) were isolated from murine small intestine using a modification of previously published procedures. Analysis of IEL by immunofluorescence using monoclonal antibodies showed they were predominantly Lyt-2+ cells, with relatively few B cells or macrophages present. IEL cultured at sufficiently high cell densities proliferated in response to concanavalin A (Con A), phytohaemagglutin (PHA) and lipopolysaccharide (LPS). IEL were also capable of recognizing alloantigens in an in vivo graft-versus-host assay and in an in vitro mixed lymphocyte reaction. These studies therefore confirm that murine IEL contain cells with immunologic properties characteristic of typical T lymphocytes.     

Large natural killer cell lymphoma arising from an indolent natural killer cell large granular lymphocyte proliferation

Natural killer cell large granular lymphocyte proliferation is a relatively rare disorder that typically runs a chronic, indolent course. We present a patient with a 3 1/2-year history of natural killer cell large granular lymphocyte proliferation terminating in large cell lymphoma with natural killer cell features. The diagnosis of natural killer cell large granular lymphocyte proliferation was based on flow cytometric demonstration of an expanded population of CD3- CD16+/CD56+ lymphocytes in the peripheral blood. The patient experienced various rheumatologic symptoms, but was hematologically stable for 3 1/2 years. He then developed fevers, night sweats, weight loss, and a left lower lobe lung mass. Resection of the mass showed a large cell lymphoma with immunohistochemical positivity for CD2, CD7, CD56, and T-cell intracellular antigen-1, compatible with natural killer cell origin. In situ hybridization for Epstein-Barr virus and polymerase chain reaction analysis for T-cell receptor gene rearrangement were negative. To our knowledge, this is the second documented report of chronic natural killer cell large granular lymphocyte proliferation terminating in an aggressive large natural killer cell lymphoma.     

Natural killer activity of human blood lymphocytes

Natural killer (NK) activity is an operational designation. It implies the in vitro cytotoxicities registered in short-term tests exerted by lymphocytes derived from donors with no known immunization history against the particular target. The strength of the effect exerted by unmanipulated blood lymphocytes shows an individual variation. Short-term in vitro treatment with interferon elevates the lytic potential of lymphocytes. Owing to the heterogeneity of the cytotoxic blood lymphocytes with regard of cell surface properties it is not possible to separate all active cells and inactive cells in clean populations. A considerable enrichment of active cells can be achieved if nylon wool non-adherent, large, granular Fc gamma receptor positive SRBC receptor negative--or low-avidity SRBC receptor positive--OKM1-reactive cells are separated. Negative cells are concentrated in the Fc receptor and OKM1-negative high-avidity SRBC receptor positive high cell density subset. The activity of lymphocytes in the former category is potentiated by interferon and the latter acquire the lytic function if PHA is added to the assay system. Freshly separated, non-cultured tumor cells are not or weakly sensitive to the effect of unmanipulated lymphocytes. However, when the lymphocytes are treated with interferon prior to the assay a lytic potential can be induced even against these in allogeneic effector target combinations. Cytotoxic cells which acquired the function after in vivo and/or in vitro immunization are designated as 'cytotoxic T-lymphocytes' (CTL), and were shown to act on the basis of antigen recognition. The expression of known T-markers on at least a fraction of the active cells and the recognition of alloantigens in NK systems suggest that the distinction between CTL and NK cells is not as sharp as initially suggested.     

Augmentation of murine natural killer activity after culture

The augmentation of murine natural killer (NK) cell activity after culture is described. Increased NK cell activity occurred when spleen cells were cultured for 18 or 42 h at high cell density in macro culture plates at 37°C. Similar results were also achieved using the same cell density when micro culture plates were employed. The simple modifications of culture conditions described in this paper should provide an excellent tool to study murine NK activity after culture. Furthermore, the micro culture system has the added advantage of enabling one to test large numbers of samples.     

Immuno-electron microscopic characterization of large granular lymphocytes (natural killer cells) from rat liver

The immunocytochemical characteristics of large granular lymphocytes (LGL), isolated from the liver sinusoids of euthymic and athymic (nude) rats, were investigated in electron microscopy by the immunoperoxidase technique. The LGL were found positive for MRC OX-8 (natural killer cells and cytotoxic/suppressor T cells) and negative for MRC OX-19 (pan-T marker) in both rat strains. The LGL were heterogeneous in the expression of the natural killer cell marker asialo-GM1 which was found on 56% of the LGL from euthymic and on 71% of the LGL from athymic rats. LGL were easily distinguished from the other cells in the preparations, "conventional" lymphocytes and monocytes, thanks to their highly characteristic ultrastructural features, in particular by the presence of specific electron-dense cytoplasmic granules and rod-cored vesicles. These features have been described formerly for the so-called "pit cells" and are more reliable than the classical LGL characteristics at the light microscopic level, i.e. the presence of azurophilic granules. Our results give further support for the existence of an important population of natural killer cells in the liver sinusoids.     

Improvement in the proliferative capacity and natural killer cell activity of murine spleen lymphocytes by thyrotropin.

In the present study we investigated the effect of thyrotropin (TSH) on both the proliferative capacity and the natural killer (NK) cell activity of murine spleen lymphocytes. It was found that TSH at various concentrations significantly increased the proliferative response of mouse lymphocytes to both concanavalin A (Con A) and phytohemagglutinin (PHA). This increase was particularly evident when suboptimal concentrations of mitogens were used (40-50% increase). The administration to cell cultures of TSH alone could not induce a significant stimulation of proliferative capacity. In order to provide a better knowledge about the mechanism by which TSH improved the mitogen-induced lymphocyte proliferation, the effect of the pituitary hormone on lymphocytes directly stimulated with recombinant interleukin-2 (RIL-2), was examined. It was observed that there was a great increase in IL-2-induced lymphocyte proliferation by TSH. The improvement in proliferative capacity of lymphocytes was particularly evident by using suboptimal rIL-2 concentrations (25-30% increase). The studies carried out on the cytotoxic activity of NK cells showed that TSH was able to significantly increase the IL-2-induced NK cell activity without modifying the basal levels of cytotoxicity. The results support the immunoregulatory role of TSH and contribute towards understanding the mechanisms of interaction between neuroendocrine and immune systems.     

Augmentation of natural killer activity by neuraminidase treatment of lymphocytes from tumor-bearing mice

Spleen cells from tumor-bearing mice showed decreased natural killer (NK) activity and decreased binding to target cells with progression of the tumor. Treatment of spleen cells from tumor-bearing mice with vibrio cholerae neuraminidase (VCN) increased the cytotoxicity to a level twice or more as high as that of untreated cells, but the same treatment of spleen cells from normal mice had no or little effect. On the other hand, neither in spleen cells from tumor-bearing mice nor in those from normal mice, the VCN treatment had no effect on their binding to M-HeLa cells. The suppression of NK activity by preincubation with serum from tumor-bearing mice or prostaglandin E2 was completely abolished by VCN treatment. The above results indicate that VCN treatment of lymphocytes might augment NK activity by an antagonistic effect against an immune suppressive factor.     

Effect of eicosapentaenoic and docosahexaenoic acid on natural killer cell activity in human peripheral blood lymphocytes

The effects of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on natural killer (NK) cell activity in human peripheral blood lymphocytes were studied. The direct addition of trieicosapentaenoyl-glycerol (EPA-TG) or tridocosahexaenoylglycerol (DHA-TG) emulsion to a cytotoxicity assay system significantly suppressed NK cell activity. The addition of lipoxygenase inhibitor AA861 also inhibited NK cell activity. The inhibition was proportional to the concentration of EPA-TG emulsion. DHA-TG emulsion, or AA861. The presence of both EPA-TG emulsion or DHA-TG emulsion and AA861 at the same time led to a greater inhibitory effect on NK cell activity than when these emulsions were used separately. The inhibitory effect caused by these lipids or lipoxygenase blockade could not be reversed by adding back exogenous leukotrienes to the assay system. Preincubation of effector cells with EPA-TG or DHA-TG emulsion resulted in a significant inhibition of their NK cell activity. NK cell activity of human lymphocytes was markedly decreased after the infusion of EPA-TG emulsion into healthy volunteers. Thus, in vivo use of EPA-TG or DHA-TG emulsion may influence immune reactivity of the host, although the mechanism has not yet been elucidated.     

Suppression of murine natural killer cell activity by adherent cells from aging mice

Natural killer (NK) cell activity declines with age in mice. The purpose of this study was to investigate the effect of peritoneal and splenic adherent cells from young and old mice on NK activity to determine whether adherent cell suppressor function might contribute to this decline. Peritoneal adherent cells from old mice suppressed NK activity of young splenic non-adherent indicator cells more than peritoneal cells from young mice. Splenic adherent cells from old but not from young mice also suppressed this activity. That (1) the suppressive activity of the adherent cell populations was not affected by treatment with anti-Thy-1 plus complement, and that (2) the adherent cell population contained 77-92% cells positive for alpha-naphthyl acetate esterase activity, suggests that the active adherent suppressor cell may be a macrophage. Therefore, the age-related decline in NK activity in mice can be explained, in part, by an increase in adherent cell suppressor function.     

Cultivation and characterisation of avian lymphocytes with natural killer cell activity

Conditioned medium (CM), as a presumed source of lymphokines including interleukin-2, was prepared from chicken spleen cell cultures stimulated with concanavalin A (con A). When CM was used to cultivate spleen cells from 6- to 8-week-old P-2 chickens, eight of nine spleens yielded cell lines which grew continuously for at least 50 days. Six of the cultures were tested for natural killer (NK) cell activity against LSCC-RP9, a lymphoblastoid cell known to be susceptible to NK cells, and against several Marek's disease lymphoblastoid cell lines (MDCC-CU2, -CU36 and -MSB-1). All six cultures lysed the RP9 cells in a chromium release assay with high levels of specific release (30 to 50%) at effector cell to target cell ratios of 5:1 or 10:1. CU2 and CU36, which are NK-cell resistant, were not lysed, while there was a low level of specific activity against MSB-1. The cells were characterised for surface and internal antigens with monoclonal antibodies and were negative for thymocyte antigen, IgM, a T cell antigen also present on granulocytes and red blood cells, and two antigens found in macrophages. Two of the six lines examined had a low number of cells expressing la antigen, while the other four were negative. An antigen present on circulating T cells and a macrophage (sub)population was present on all lines. The majority of the cells had the morphological appearance of mammalian large granular lymphocytes (LGL) with a rather small kidney-shaped nucleus. Granules and vacuoles were present in the cytoplasm. However, there was a variable percentage of lymphoblastoid (LB) cells also present. Cell lines established with CM from con A-stimulated spleen lymphocytes in other studies were also shown to have high levels of NK activity regardless of the relative proportions of cells with the morphological appearance of LGLs or LBs, or the relative frequency of expression of the two T cell markers or la antigen, all of which varied markedly.     

Comparative natural killer cell activities of thymic, bursal, splenic and intestinal intraepithelial lymphocytes of chickens

The natural killer (NK) cell activities of spleen, thymus, bursa, peripheral blood and gut intraepithelial lymphocytes (IEL) from FP and SC chickens were investigated in 4-hr and 16-hr 51Cr release assays. Target cells were 4 different tumor cell lines derived from either an avian leukosis tumor transplant (LSCC-RP9, LSCC-RP12) or from Marek's disease lymphomas (MDCC-MSB-1, MCDD-CU36). Great variability in cytotoxic potential was observed among NK cells of different lymphoid organs. NK cell cytotoxicity varied depending upon the type of effector cells, type of target cells, the ratio of effector to target cells, and the age and genetic background of chickens. Substantial levels of NK cell activity were detected in spleen and gut IEL of SC chickens in a 4-hr assay. In contrast, the NK cytotoxicity in gut IEL of FP chickens was not detectable until 16 hr after incubation. The ranges of target cell specificity demonstrated by IEL, spleen, thymus and bursa NK cells were similar to one another and, in general, the level of cytotoxicity increased with incubation time. Thymus and bursa NK cell activity of both SC and FP chickens was not detectable in a 4-hr assay but substantial NK cell activity was demonstrated in a 16-hr assay. The results of the present study demonstrate that various lymphoid organs of chickens, such as spleen, thymus, bursa, and gut intraepithelium, contain subpopulations of cells that can mediate spontaneous cytotoxicity.     

Characterization of macrophage subsets regulating murine natural killer cell activity

We examined the relationship of I-A expression by normal murine macrophages to their immunoregulatory role on natural killer cell activity. Macrophages were isolated on the basis of plastic adherence; characterized on the basis of conventional markers such as phagocytic ability, cytoplasmic non-specific esterase activity, surface MAC-1 and F4/80 antigen expression; and then used for functional studies relative to their expression of surface I-A. Two functional macrophage subsets were identified: NK-stimulatory and NK-suppressive subsets. The former function was associated with splenic macrophages, which were predominantly I-A+ as identified with a radioautographic immunolabeling technique; the latter function with peritoneal macrophages which were predominantly I-A-. Loss of macrophage I-A expression in vitro was delayed in the presence of indomethacin and enhanced in the presence of PGE2, indicating that PGE2 down-regulates I-A expression on macrophages. The NK stimulatory function of I-A+ macrophages was attributable to a soluble mediator, identified as IFN-gamma, since the stimulatory ability was abrogated with an anti-IFN-gamma antibody. I-A expression appears to be important for the stimulatory function, since some interference with this function was noted in the presence of anti-I-A antibody. The NK-suppressor function of I-A- macrophages was attributable to the soluble mediator PGE2, since this function was abrogated with indomethacin or anti-PGE2 antibody. These results are relevant to the understanding of normal in vivo immunoregulation by macrophages.     

Isolation and functional characterization of chicken intestinal intra-epithelial lymphocytes showing natural killer cell activity against tumour target cells.

Intestinal intra-epithelial lymphocytes (IEL) of SC or FP chickens were isolated and examined for their natural killer (NK)-cell activity against chicken tumour cell lines, LSCC-RP9 (RP9), LSCC-RP12 (RP12), MDCC-MSB-1 (MSB-1) and MDCC-CU36 (CU36). In general, IEL of satisfactory yield and of good viability were obtained with EDTA treatment of the gut tissues, followed by rapid passages of the resultant cells through nylon-wool columns and centrifugation on two-step Percoll density gradients (45% and 80%). In 4-hr and 16-hr 51Cr-release assays, the NK-cell activity of chicken IEL depended not only upon the type of target cells but also upon the incubation time and the host genetic background. RP9, MSB-1 and CU36 were susceptible to NK lysis by IEL and by spleen cells, while RP12 was resistant to lysis even after a prolonged incubation. In kinetic studies the cytotoxicity was detactable from 2 hr after incubation and progressively increased up to 16 or 18 hr. The IEL of SC chickens revealed significantly higher levels of NK-cell activity against RP9 than FP-strain chickens, whereas their splenic NK-cell activity was not significantly different. Against MSB-1 targets, however, IEL of SC and FP chickens showed similar levels of NK-cell activity while their spleens did not (being higher in FP). When tested in FP chickens, IEL NK-cell activity was inhibited by the addition of unlabelled homologous target cells. In general, NK-cell activity was higher in the jejunum and ileum than in the duodenum and caecum. Efforts to enrich IEL NK-effector cells by discontinuous Percoll gradients were not successful. The results of the present study show that IEL of chicken intestine contain effector cells that can mediate NK-cell activity against chicken tumour cells.     

Dissociation of natural killer and lymphocyte-activated killer cell lytic activities in human CD3− large granular lymphocytes

CD3^? large granular lymphocytes (LGL) are known to display natural killer cell (NK) activity without prior sensitization or restriction by major histocompatibility antigens. Upon short-term exposure to interleukin-2, NK cells were shown to acquire lymphocyte-activated killer cell (LAK) activity. The aim of this study was to analyze the characteristics of these lytic activities. Our data indicated that both NK and LAK activities were Ca²⁺ dependent; however, they could be dissociated by a Ca²⁺ channel blocker or a Ca²⁺ channel competitor agent. Moreover, NK activity was associated with granule exocytosis of lytic proteins spontaneously present in CD3^? LGL, the most likely candidate being the pore-forming protein perforin. By contrast, LAK activity was found to be dependent on de novo protein synthesis and distinct from granule exocytosis. Our results strongly suggest that NK and LAK activities could be defined as two distinct pathways involving different lytic mediators.     

Selective modulation of the natural killer activity of murine intestinal intraepithelial leucocytes by the neuropeptide substance P.

Neuropeptides can influence immune effector cell function at both systemic and mucosal immune sites. We examined the ability of substance P (SP) to modulate the natural killer (NK) activity of intestinal intraepithelial leucocytes (IEL). Yac-1 killing by IEL but not splenic cells was increased after either 18 hr preincubation or 6 hr of co-incubation with SP. We also examined the NK activity of IEL and spleen isolated from mice treated with SP in vivo. The selective increase in NK activity of IEL occurred without any demonstrable change in the number or phenotype of the IEL. The IEL responsive to SP in vivo and induced in vivo by SP were both Thy-1- and did not kill the NK insensitive mastocytoma cell line P815. Lastly, we examined the ability of SP to induce the release of interleukin-2 (IL-2) and IL-4 from IEL after 6 and 18 hr of in vitro culture. No increase in the release of these cytokines was observed, suggesting that IL-2 and IL4 are not involved in the local augmentation of IEL NK activity by SP. These observations suggest that SP has a selective stimulatory effect on intestinal activity and may play a role in the regulation of intestinal cell-mediated immunity.