Spontaneous cytotoxicity of human intraepithelial lymphocytes against epithelial cell tumors.

Human intraepithelial lymphocytes are T cells primarily of the CD8+ phenotype located between intestinal epithelial cells. The cytotoxic and suppressor activities of these lymphocytes are largely unexplored. The spontaneous cytotoxic activity of these cells is evaluated in this study. Jejunal intraepithelial lymphocytes spontaneously lysed a variety of epithelial cell tumor lines (colonic and pancreatic adenocarcinomas and bladder epidermoid carcinoma) but not the highly natural killer-sensitive K-562 cells. Cold target inhibition studies showed that these lymphocytes preferentially bind the DLD-1 colonic adenocarcinoma cells rather than the K-562 cells. Pretreatment of the effector cells with interferon-gamma did not change their cytotoxic activity. The cytotoxic cells are T lymphocytes (expressing CD2, CD3, and CD8). In contrast, the spontaneous cytotoxic activity of peripheral blood lymphocytes is directed against both epithelial cell targets and K-562 cells, is enhanced by interferon-gamma, and is effected by natural killer cells (expressing CD2, CD16, and NKH1). Thus, the spontaneous cytotoxicity of intraepithelial lymphocytes differs from that of peripheral blood lymphocytes in their target cell restriction, lack of response to interferon gamma, and effector cell phenotype. Lymphocytes in the intestinal epithelium may have a novel and important role in recognizing and destroying transformed epithelial cells and colon cancers.     

Sialyl SSEA-1 antigen as a carbohydrate marker of human natural killer cells and immature lymphoid cells

The distribution of a carbohydrate antigen, the sialyl SSEA-1 (sialyl Lex-i), in human lymphoid cells was investigated by flow cytometry with a specific monoclonal antibody, MoAb FH-6. We concluded that the lymphocytes positive for the sialyl SSEA-1 antigen present in normal peripheral blood (PB) are natural killer (NK) cells since the positive cells had an NK activity toward K562 cells, and most of the sialyl SSEA- 1+ cells were simultaneously positive for Leu-11 (CD-16) and Leu-19. Essentially, no T and B cells, defined by Leu-4 (CD3) and Leu-16 (CD20), were positive for the sialyl SSEA-1 antigen in PB samples taken from healthy donors and patients with disorders unrelated to lymphoid malignancies. Among the malignant lymphoid cells, many sialylated SSEA- 1+ cells were observed in large granular lymphocyte (LGL) leukemia cells and some acute lymphoblastic leukemia (ALL) blasts, but not in CLL cells or malignant lymphoma cells. Sialyl SSEA-1 was also positive in some cultured human lymphoid cell lines. We conclude that expression of the sialyl SSEA-1 antigen is strictly limited to a distinct population of NK cells among the mature lymphocytes in normal PB, but the antigen is present in a wide range of immature lymphoblasts of T- and B-cell lineages as well as the NK-cell lineage. The sialyl SSEA-1 antigen disappears from the surface of immature lymphocytes of T- and B- cell lineages during the course of maturation.     

Human natural killer cells can inhibit clonogenic growth of fresh leukemic cells

The effect of allogenic human natural killer (NK) cells on fresh leukemic cells from three patients was investigated. The low levels of leukemic target cell lysis in the conventional 51Cr-release assay contrasted with a pronounced inhibitory effect on the colony growth of the clonogeneic leukemic target cells (L-CFC). The ability of allogeneic lymphocytes to inhibit L-CFC increased if they were pretreated with interferon (IFN), which also increased their NK activity, monitored in parallel cytotoxicity assay, against K562. Furthermore, cell separation procedures, based on differences in density among nonadherent lymphocytes, revealed that only NK cell containing fractions were inhibitory. We have also compared the susceptibility to NK-mediated L-CFC inhibition of IFN pretreated leukemic target cells with that of nontreated target cells. As in the case of NK lysis in general, this pretreatment of target cells abolished the presumably NK-mediated L-CFC inhibition. In conclusion, these data provide the first indication that NK cells can inhibit the in vitro growth of fresh clonogenic leukemia cells from patients with nonlymphocytic leukemia. The identity of NK cells as effector is strongly suggested by Percoll separation and responsiveness to interferon; the final proof awaits more sophisticated purification of these cells.     

Mannose 6-, fructose 1-, and fructose 6-phosphates inhibit human natural cell-mediated cytotoxicity.

In vitro human natural cell-mediated cytotoxicity (NCMC) to K-562, Molt-4, and F-265 cells is inhibited in a dose-dependent manner by mannose 6-phosphate, fructose 1-phosphate and fructose 6-phosphate. This inhibition is not observed with mannose, glucose, fucose, glucose 6-phosphate, mannose 1-phosphate, galactose 1-phosphate, or galactose 6-phosphate. Preincubation of the effector cells, obtained from fresh whole blood, with mannose-6-phosphate, fructose-1-phosphate, or fructose-6-phosphate did not inhibit cytotoxicity, which indicated that these hexose phosphates are not nonspecifically toxic towards the effector lymphocytes. Mannose-6-phosphate and the stereochemically similar fructose-1-phosphate are more potent inhibitors than fructose-6-phosphate in terms of concentration required and time of onset of effect. Inhibition of cytotoxicity by mannose-6-phosphate varied with target cell type: F-265 is protected at much lower concentrations of mannose-6-phosphate (less than 1 mM) than is either Molt-4 or K-562. The inhibition of NCMC is also observed with the inhibitors of lysosomal function, NH4Cl, and chloroquine. The presence of a functional mannose-6-phosphate receptor on target cells was demonstrated: (i) Gelonin, a seed protein that inactivates the eukaryotic ribosome but is nontoxic to intact cells, was covalently linked to monophosphopentamannose, and this conjugate ws toxic to both K-562 and F-265 target cells, the latter being by far the more sensitive; and (ii) chloroquine, NH4Cl, and mannose-6-phosphate all inhibited the toxicity of gelonin-monophosphopentamannose. These results suggest either that a cytolytic lymphokine contains a hexose phosphate residue and may be taken up by target cells through the lysosomal/mannose 6-phosphate pathway or that such a residue is involved in target cell-effector cell recognition.     

Analysis of natural killer (NK) cell activity and adhesion molecules on NK cells from umbilical cord blood

The activity of natural killer (NK) cells in human umbilical cord blood (CB) has been reported to be low, compared with that in adult peripheral blood (PB) in vitro. To examine the cause of this, after dividing the CD56+/CD3- cells in CB and PB into CD56bright and CD56dim NK cells, the NK cell activities and the expression of various surface antigens were assayed for each fraction. The NK cell activity of CD56dim NK cells in CB was significantly lower than that in PB (P = 0.0003), whereas, there was no significant difference between the NK cell activity of CD56bright NK cells in PB and CB. The expression levels of adhesion molecules (CD2, CD11a, CD18, DNAX accessory molecule-1), CD16, and CD57 for CD56dim NK cells in CB were significantly lower than those in PB, and approximately one-third of CB CD56dim NK cells were capable of forming conjugates with K562 cells, compared with PB CD56dim NK cells. Furthermore, the inhibition of both the NK cell activities and binding of CD56dim NK cells in PB and CB by monoclonal antibody against each of these adhesion molecules suggests that they play an important role in NK cell activity. These findings show that the low NK cell activity in CB is caused by the low NK cell activity of CD56dim NK cells and that the low expression level of adhesion molecules on CB CD56dim NK cells may contribute to this low NK cell activity.     

Recognition of unusual presentation of natural killer cell leukemia

Expansion of the natural killer (NK) subset of lymphocytes represents a rare leukemia phenotype with variations in clinical presentation, morphology, surface phenotype, and effector function. This paper reports on a 5-year-old male patient who had an unusual presentation of an NK cell leukemia that was initially diagnosed as neuroblastoma. A bone marrow (BM) aspirate showed clumps of undifferentiated cells with the following phenotype: CD56^bright+, CD33^dim+, CD45-, CD2-, CD19-, CD16-, and CD57-. Cytochemistry was noncontributory. The patient, having failed to respond to conventional neuroblastoma chemotherapy, was subsequently diagnosed as having NK cell leukemia based on functional in vitro assays. The patient responded to acute lymphoblastic leukemia (ALL) chemotherapy but relapsed 4 weeks into treatment and eventually died 25 weeks after initial presentation. The cell surface phenotype observed is consistent with a rare NK cell subset, the biology of which has not been well defined. Freshly isolated BM cells killed K562 cells in a conventional ⁵¹Cr-release assay. Both interleukin-2 (IL-2) and interferon-α (IFN-α) induced LAK activity against the Daudi cell line. IL-2 induced proliferation of the leukemic cells. TNF-α, IFN-γ, IL-6, IL-1ra, and TGF-β levels were assessed and found to be concentrated in BM, in contrast to plasma samples. TNF-α was present at a high concentration in BM (150.9 pg/ml), probably a reflection of the associated disease pathology of severe bone pain and pyrexia. In summary, this paper details clinical and laboratory investigations of a leukemia of a rare NK cell subset.     

Agranulocytosis induced by macrolide antibiotics

Expansion of the natural killer (NK) subset of lymphocytes represents a rare leukemia phenotype with variations in clinical presentation, morphology, surface phenotype, and effector function. This paper reports on a 5-year-old male patient who had an unusual presentation of an NK cell leukemia that was initially diagnosed as neuroblastoma. A bone marrow (BM) aspirate showed clumps of undifferentiated cells with the following phenotype: CD56^bright+, CD33^dim+, CD45-, CD2-, CD19-, CD16-, and CD57-. Cytochemistry was noncontributory. The patient, having failed to respond to conventional neuroblastoma chemotherapy, was subsequently diagnosed as having NK cell leukemia based on functional in vitro assays. The patient responded to acute lymphoblastic leukemia (ALL) chemotherapy but relapsed 4 weeks into treatment and eventually died 25 weeks after initial presentation. The cell surface phenotype observed is consistent with a rare NK cell subset, the biology of which has not been well defined. Freshly isolated BM cells killed K562 cells in a conventional ⁵¹Cr-release assay. Both interleukin-2 (IL-2) and interferon-α (IFN-α) induced LAK activity against the Daudi cell line. IL-2 induced proliferation of the leukemic cells. TNF-α, IFN-γ, IL-6, IL-1ra, and TGF-β levels were assessed and found to be concentrated in BM, in contrast to plasma samples. TNF-α was present at a high concentration in BM (150.9 pg/ml), probably a reflection of the associated disease pathology of severe bone pain and pyrexia. In summary, this paper details clinical and laboratory investigations of a leukemia of a rare NK cell subset.     

Severe deficiency of natural killer activity in the peripheral blood of patients with hairy cell leukemia

Natural killer (NK) activity against K-562 tumor cells was evaluated in peripheral blood leukocytes (PBL) obtained from untreated patients affected by hairy cell leukemia (HCL). NK activity present in PBL from 10 HCL patients was at least six-fold lower (p less than 0.01) than that present in PBL from 15 healthy donors. Decreased NK activity in HCL PBL was not due to dilution of the NK effector cells by the neoplastic cells; in fact, NK activity of PBL from 4 HCL patients with less than 5% circulating neoplastic cells was still five-fold lower (p less than 0.01) than that present in normal PBL. Partial characterization of the NK defect in HCL patients indicated that: (A) defective cytotoxicity was not dependent on the duration of the assay; (B) HCL PBL added to normal PBL during the assay did not exert suppressor activity; (C) the NK activity of HCL PBL could be potentiated in vitro by interferon; and (D) low levels of NK activity were associated with reduced numbers of circulating monocytes (p less than 0.01) and of large granular lymphocytes (LGL) (p less than 0.01). In conclusion, our results indicate that the low levels of NK activity present in the peripheral blood of HCL patients may be related to reduced numbers of circulating effector cells.     

The Assessment of NK Cytotoxicity and CD56+/CD16+ Phenotype by Flowcytometry in PBL Isolated from Women with Recurrent Spontaneous Abortion

Background: Human peripheral blood NK cells constitutively express CD56 and CD16 antigens. Peripheral blood NK cells seem to be strongly related with decidual NK cells, and may reflect the decidual NK cell functional status. There are varied reports concerning the relationship between NK cell cytotoxicity in women with recurrent spontaneous abortion. Objective: To study NK activity in women with history of RSA by using a non-radioactive cytotoxicity assay. Methods: Peripheral blood lymphocytes from RSA and healthy multiparous women were obtained. Lymphocytes were isolated and mixed with K562 NK-sensitive cell line. A non-radioactive method for NK cell cytotoxicity assessment was utilized. Dead K562 cell populations were detected by FACS Calibur flow cytometry, and the data obtained was analysed on cell-Quest software. The proportion of CD16+ /CD56+ cells was then calculated. Results: The proportion of NK cells were 9.21% ± 3.06 and 13.48% ± 4.09 in healthy women and RSA, respectively. The percentage of cytotoxicity was determined to be 19.3% ± 3.9 in healthy group and 27.1% ± 6.5 in RSA group with an effector:target ratio of 50:1. The data shows an increase in PBLs potential for in vitro cytotoxicity assay in RSA individuals. The analyses indicate that there is a weak positive correlation between NK cytotoxicity potential and the percentage of NK cells in PBL population. Conclusion: The present study demonstrates that the percentage of CD56+ /CD16+ cells increases in individuals with recurrent spontaneous abortion. We conclude that NK cytotoxicity as well as NK number is partially involved in RSA.     

Differential effects of ifosfamide on the capacity of cytotoxic T lymphocytes and natural killer cells to lyse their target cells correlate with intracellular glutathione levels

We established an in vitro model to study the influence of ifosfamide treatment on intracellular glutathione (GSH) levels in activated human effector cells with specific phenotypes and immunologic functions. Besides its role as the major intracellular reductant, GSH has been shown to affect the initiation and progression of lymphocyte activation after stimulation with lectins. An incubation of activated human peripheral blood lymphocytes (PBL) with 4-hydroxyifosfamide, the activated form of ifosfamide (4-OH-IF), resulted in a depletion of the intracellular GSH levels and a significant inhibition of the proliferative capacity in a dose-dependent manner. The cytotoxic activity of separated CD3- natural killer (NK) cells and CD3+ allospecific, cytotoxic T lymphocytes (CTL), either untreated or treated with 4-OH-IF at different concentrations, was compared in a standard 51chromium release assay (CML). There were three major findings. (1) The capacity of CD3+ major histocompatibility complex (MHC)-restricted CTL to lyse their specific allogeneic target cells was substantially reduced by preincubation of the effector cells with 4-OH- IF. This inhibition of the lytic activity in CD3+ CTL correlated with a substantial depletion of the intracellular GSH levels in this population. Rapid reconstitution of depleted GSH levels and restoration of cytotoxic activity of CTL was achieved by incubation of the effector cells with thiols, eg, glutathione ester (GSH-ester) or 2- mercaptoethanesulfonate (mesna). (2) In contrast, the lytic activity in CD3- NK cells was not substantially affected (up to 100 mumol/L 4-OH- IF). This result correlates with the capacity of NK cells to maintain their intracellular GSH levels after an ifosfamide treatment. (3) In comparison with CD3+ CTL, CD3- NK cells are more resistant to an ifosfamide treatment because they have higher initial GSH levels and a more than fourfold higher relative rate of GSH synthesis.     

The effect of anti-malarial drugs on human natural killer cells in vitro

Human natural killer (NK) cell cytotoxicity against K-562 target was found to be markedly depressed by quinacrine, mefloquine, pyrimethamine and chloroquine. The most potent were quinacrine and mefloquine. Two other drugs tested, quinine and primaquine, displayed no significant effects even after long incubation periods with effector cells. The results showed that the inhibitory effects were not related to an inhibition of effector cell binding to target or to a toxic effect on NK cells.     

Generation of cytotoxic NK cells in peripheral blood and bone marrow of patients with acute myelogenous leukemia after continuous infusion with recombinant interleukin-2

We have studied the cytotoxic profile and distribution of lymphocyte subsets of patients with acute myelogenous leukemia in second remission, after continuous infusion with recombinant interleukin-2 (IL-2). The patients received repetitive cycles of 1-1.25 x 10(6) U/m2/day of IL-2, given as 4 days continuous intravenous infusion followed by a 3-day treatment-free interval for the first 4 weeks. Patients receiving greater than 4 cycles were treated with the same dose of IL-2 continuously for 4 days, followed by a 10-day treatment-free interval. These studies showed that IL-2 treatment resulted in the generation of peripheral blood cytotoxic activity against both NK-susceptible, K-562, and NK-resistant Daudi cell lines. In most patients, enhancement of lytic activity increased with the number of IL-2 infusions. The cytotoxicity in some patients increased as much as 700-fold and 830-fold against K-562 and Daudi cells, respectively. It is of importance that oncolytic activity was also induced in bone marrow compartment (up to 182-fold against K-562). Some decline in cytotoxicity was observed within 14 days after initiation of IL-2 infusion in peripheral blood, but high levels of lytic activity persisted at this time in bone marrow. It is of interest to note that the cytotoxicity of in vivo IL-2 primed lymphocytes was further potentiated by IL-2 in vitro. Importantly, the cytotoxic cells induced in vitro displayed lytic activity against fresh leukemic blasts. Phenotypic analysis demonstrated that CD3-, CD56+ NK cells were significantly increased by in vivo IL-2 treatment (34 to 47-fold in absolute numbers), while CD3-, CD56+ T-cell subset remained low. Characterization of cytotoxic cells using the complement-dependent assay and monoclonal antibodies indicated that both the in vivo-induced and ex vivo-potentiated lytic function was mediated by CD3-, CD56+, CD16- -NK cells.     

Absence of circulating natural killer (NK) cells in a child with erythrophagocytic lymphohistiocytosis lacking NK cell activity

A 5-year-old girl who was diagnosed as having erythrophagocytic lymphohistiocytosis died at age 9 years. Peripheral lymphocytes from the patient persistently lacked natural killer (NK) cell activity during the 4-year observation period: the percent lysis values as measured by a 4-hr 51Cr release assay at a 40:1 effector:target ratio were below 1.0% against K562 and Molt-4 cells as compared with the normal lymphocyte value (mean +/- SD) of 46.2% +/- 5.8% and 43.9% +/- 6.7%, respectively. The patient's lymphocytes never developed NK cell activity by their incubation with target cells for longer time periods or by their stimulation with interferon-alpha, interleukin-2, or polyinosinic-polycytidilic acid. Single cell-in-agarose assay showed the absence of target-binding cells (TBCs): TBC numbers were below 0.3% as compared with the normal lymphocyte value of 8.1% +/- 1.3% (mean +/- SD). Flow cytometry showed a marked decrease in Leu-7+ cells (1.7%) and the absence of Leu-11+ cells (0.4%) in the peripheral blood. These results first demonstrate a case of erythrophagocytic lymphohistiocytosis in which there is the lack of NK cell activity due to the absence of circulating NK cells.     

Hematopoiesis in vitro coexists with natural killer lymphocytes

The role of natural killer (NK) lymphocytes in the regulation of human hematopoiesis is controversial. NK-mediated inhibition of colony formation of hematopoietic progenitor cells has been irregularly reported for various cell lineages. In an effort to clarify such disparate findings, we studied the interaction of clearly defined NK and partially purified progenitor cell populations. Cell sorter purified CD16 positive blood NK cells and enriched autologous marrow progenitors were co-incubated at various lymphocyte to marrow cell ratios and then cultured in methylcellulose. There was no inhibition of myeloid, erythroid, or mixed colony formation. Similarly, activation of CD16 positive lymphocytes by interleukin-2 (IL-2) before co-incubation and co-culture did not result in inhibition of colony formation. Furthermore, in a newly designed assay system, we demonstrated that NK cells, which did not modulate colony-formation, remained capable of recognizing and killing rare K562 target cells seeded within the marrow cell population. Our results indicate that unstimulated and IL-2 activated isolated blood NK cells coexist with functioning autologous marrow progenitors in vitro.     

Analysis of natural killer activity and antibody-dependent cellular cytotoxicity in healthy volunteers and in patients with primary lung cancer and metastatic pulmonary tumors

Summary To clarify the contribution of ADCC and NK activities to host immune response against cancer, the characteristics of cells mediating these activities were examined in the peripheral blood lymphocytes of normal volunteers, and the changes of these activities were also evaluated in patients with lung cancer and metastatic pulmonary tumors before and after chemotherapy. OAT cells derived from small cell carcinoma of the lung and K-562 cells derived from erythroleukemia were used as target cells of ADCC and/or NK assay. ADCC and NK activities were not changed according to age, sex, and blood type. Mild and marked personal difference were observed in ADCC and NK activity, respectively. These activities were also influenced by environment. ADCC and NK activities of normal adult volunteers were diversely correlated at the coefficient of -0.426.NK activities were high against K-562 and CCRF-CEM cells, and low against BALL and OAT cells. NK activity against K-562 cells was strongly inhibited by K-562 or CCRF-CEM cells with high NK sensitivity, on the other hand, it was slightly inhibited by OAT and BALL cells with low NK sensitivity. NK activity against OAT cells was strongly inhibited by OAT, K-562 and CCRF-CEM cells, but not inhibited by BALL cells. The effector cells mediating NK activity were identified as non-adherent, E-receptor-positive, Fc-receptor-positive small lymphocytes.NK activity was not decreased before chemotherapy in patients with stage III primary lung cancer and metastatic pulmonary tumors. It was decreased only in patients of bad performance status, and it was significantly decreased in all patients after chemotherapy. ADCC also exhibited the tendency to decrease after chemotherapy in tumor-bearing patients. The recovery of NK-activity after chemotherapy well correlated with the effect of chemotherapy.     

Generation of lymphocytes potentiated against leukemic lymphoblasts by stimulation using leukemic cell lysate-pulsed dendritic cells in patients with acute lymphoblastic leukemia and measurement of in vitro anti-leukemic cytotoxicity

Using granulocyte-macrophage colony stimulating factor, interleukin4 and tumor necrosis factor α, we generated dendritic cells (DCs) from mononuclear cells isolated from the peripheral blood (PB) of eight patients with acute lymphoblastic leukemia (ALL), who were in complete remission (CR), and pulsed these DCs with leukemic cell lysates. Specific cytotoxicity assays were performed by incubation of effector cells (lymphocytes generated from cryopreserved mononuclear cells isolated in CR state of ALL) and targets (cryopreserved leukemic cells at diagnosis). Patients showing decreased cytotoxicity had poorer clinical courses. When we measured lymphocyte subsets, we found positive correlations between cytotoxicity levels and the proportions of T lymphocytes and CD8+ T lymphocytes, but negative correlations between cytotoxicity levels and the proportions of NK cells and regulatory T lymphocytes. In conclusion, we show here that leukemia-specific autologous DCs can be generated from the PB of ALL patients in CR, that the incubation of these DCs with leukemic cell lysates can generate lymphocytes potentiated against leukemic cells, and that relationships are evident among all of cytotoxicity, lymphocyte subsets, and patient prognosis.     

Melatonin-Induced Suppression of Human Lymphocyte Natural Killer Activity in Vitro

One of the important immune functions influenced by neuroendocrine factors is natural killer (NK) activity, which is directed against neoplastic and virus-infected cells. The effects of melatonin (Mel) and N-acetylserotonin (NAc-5HT) on NK activity of human peripheral blood lymphocytes were investigated. Leukocytes of healthy human subjects were used in the experiment. NK activity was estimated by measurement of radioactive chromium (51Cr) release from human leukemia cells K 562 (target cells). The previous exposure of human lymphocytes (effector cells) to Mel in concentrations of 10(-6) M and 10(-10) M resulted in an inhibition of NK activity (P less than 0.01) for all the examined effector-target cell ratios (10:1; 20:1, 40:1). NK activity was also suppressed by Mel (10(-8) M), but only if effector-target ratio equal to 20:1 was used (P less than 0.02), and by Mel (10(-12) M) for effector-target ratio equal to 40:1 (P less than 0.05). In none of the examined concentrations did NAc-5HT inhibit NK activity of human lymphocytes. On the basis of the data reported above, a direct suppressive effect of Mel (but not of NAc-5HT) on NK activity of human peripheral blood lymphocytes can be assumed.     

Impaired release of a soluble natural killer cytotoxic factor in systemic lupus erythematosus

Disease states characterized by abnormalities in immune regulation often demonstrate concomitant abnormalities in cytotoxicity mediated by natural killer (NK) cells. For example, some patients with systemic lupus erythematosus (SLE) have depressed NK activity despite the presence of normal numbers of effector cell:target cell conjugates. This study was designed to determine if defects in NK cell function were directly related to impaired release of a soluble cytotoxic factor. NK activity of peripheral blood mononuclear cells and large granular lymphocytes was measured using 51Cr-labeled K562 target cells in 4-hour release assays. The SLE patients had significantly decreased NK activity relative to normal controls. However, the number of effector cell:target cell conjugates was not different in SLE patients versus control subjects. The release of a soluble natural killer cytotoxic factor (NKCF) by peripheral blood mononuclear cells was measured by cytotoxicity induced in K562 cells. NKCF was released preferentially by suspensions enriched in NK cells (large granular lymphocytes). At a 1:1 dilution, NKCF release was significantly lower in SLE patients than in controls. The release of NKCF correlated well with NK activity. Thus, this study shows that the defect in NK cell activity in SLE patients may be related to an impairment in release of a soluble cytotoxic factor with specificity for NK cell-sensitive targets.     

Natural killer cell activity of rhesus macaques against retrovirus-pulsed CD4+ target cells

Rhesus peripheral blood mononuclear cells (PBMC) fail to demonstrate natural killer (NK) activity against the human T-cell lines CEM, CEM x 174, or SUP-T1. However, these cell lines could act as NK-sensitive target cells if they were pulsed with heat-inactivated, whole simian immunodeficiency virus (SIV). The ability of these SIV-pulsed T-cell lines to act as NK-sensitive target cells was directly related to the relative density of CD4 on their surface. Target cell generation was inhibited by preincubation of cell lines with CD4 monoclonal antibody (MAb) with specificity for the SIV binding site. In addition, NK activity was seen against target cells that had been prepared with human immunodeficiency virus type 1 (HIV-1) gp120, nonglycosylated gp120, env A of feline leukemia virus (FeLV), and simian type D retrovirus (SRV). Addition of leupeptin to target cells prior to SIV pulsing did not result in a significant decrease in cytotoxic activity, suggesting that processing is not required for the generation of target cells. The cells that mediate NK activity are nonadherent, do not form rosettes with AET-treated sheep red blood cells (SRBC), and are phenotypically CD16+ and CD8+. NK activity of SIV-infected macaques was significantly decreased against both K562 cells and SIV-pulsed target cells as compared with uninfected animals. However, treatment of PBMC with interleukin-2 (IL-2) resulted in a partial restoration of NK activity.     

Chronic Alcohol Consumption Is Associated With an Increased Cytotoxic Profile of Circulating Lymphocytes That May Be Related With the Development of Liver Injury

Background: Apoptosis has recently emerged as a key component of acute and chronic liver diseases and it could be related to alcoholic liver disease. In the present study, we attempted to analyze the cytotoxic profile of circulating lymphocytes in chronic alcoholic patients grouped according to ethanol intake status and presence of liver disease. Methods: We investigate the phenotypic and functional behavior of different compartments of peripheral blood (PB) cytotoxic T and natural killer (NK) cells in chronic alcoholic patients without liver disease and active ethanol intake (AWLD group; n = 22), and in subjects with alcohol liver cirrhosis (ALC group; n = 22). Results: AWLD patients showed an expansion of both CD4+/CD8+ cytotoxic T cells and NK/T cells, in association with an enhanced cytolytic activity against K562 cells and a higher ability to induce in vitro expression of the pro‐apoptotic protein APO2.7 in HepG2 cells. Conversely, ethanol intake in ALC patients was associated with decreased NK cell numbers, a reduced cytotoxic activity against K562 cells without significant changes in the expression of APO2.7, and a pro‐fibrotic profile of cytokine secretion. Conclusions: Overall, our results suggest that alcoholic patients display different phenotypical and functional changes in circulating PB cytotoxic lymphocytes according to the presence of alcoholic liver disease, which could be related to the development and progress of liver injury.