Defective NK cell activity following thermal injury.

Peripheral blood mononuclear lymphocytes (PBL) from thermal injury patients were examined for their ability to mediate natural killer (NK) cell activity against K562 tumour cells and against herpes simplex virus type 1 (HSV-1) infected Raji tumour cells. Using fluorescein isothiocyanate-conjugated monoclonal antibodies, the number of T3, T4, T8, Leu11, and Leu7 positive cells in PBL obtained from patients and normal controls was determined. Thermal injury patients had decreased levels of T3+ cells and a T4:T8 ratio which was significantly lower than that found in normal control individuals. Although patients had normal percentages of Leu7+ and Leu11+ cells, they had depressed NK cell activity against both K562 tumour cells and HSV-1 infected Raji cells. NK cell activity against K562 tumour cells was severely depressed during the first 20 days after injury. This defective NK cell activity did not appear to be due to a defect in PBL binding to the K562 tumour cells. In patients, the level of NK cell activity against HSV-1 infected cells did not correlate with the level of NK cell activity against K562 tumour cells. This finding further supports previous reports showing that NK cells which kill K562 tumour cells are different from the NK cell population which kills HSV-1 infected cells. Pretreatment of PBL obtained from patients with IL-2 or IFN-alpha, in some cases greatly enhanced NK cell killing of K562 tumour cells. However, IL-2 or IFN-alpha did not enhance NK cell activity in patients who had severely depressed levels of NK cell activity. Interestingly, in some patients, differential responsiveness to IL-2 and IFN-alpha was observed. In some patients, NK cell activity was enhanced by IL-2 but not by IFN-alpha. These results, while only suggestive, may indicate that different populations of NK cells respond preferentially to IL-2 and that IFN-alpha and/or IL-2 enhance NK cell activity in PBL obtained from some, but not all, thermal injury patients. Finally, this study clearly shows that thermal injury patients have defective NK cell activity not only against K562 tumour cells but also against virus-infected cells.     

Depressed natural killer cell activity in acute myocardial infarction.

Natural killer (NK) cell activity against K562 target cells was measured in patients within 24 h of acute myocardial infarction (AMI) and regularly thereafter for 6 weeks. NK cell activity was suppressed on days 1, 3, and 7 (P less than 0.01), day 14 (P less than 0.05) and at 6 weeks (P = 0.05) when compared to controls. Interferon, interleukin 2 and indomethacin enhanced NK cell activity on all days measured, but did not completely restore the defective NK cell activity. Serum from the patients did not suppress the NK cell activity of healthy mononuclear cells. The number of NK cells, identified as large granular lymphocytes (LGL), measured on days 1, 3, and 14 and at 6 weeks was not reduced in comparison to that of controls. Thus, the defective NK cell activity can be characterized as functional.     

Azathioprine suppression of natural killer activity and antibody-dependent cellular cytotoxicity in renal transplant recipients

The relative effects of azathioprine (AZA) and prednisone (PRED) on natural killer (NK) activity and the antibody-dependent cellular cytotoxicity (ADCC) of K (killer) cells and the number of FcR and other lymphoid cells were examined in renal transplant recipients. In addition to both long-term (>6 months) and short-term (<6 months) transplant recipients receiving conventional AZA-PRED therapy, an important group of long-term recipients receiving PRED but not AZA was studied for the first time. Both NK activity and ADCC are profoundly reduced in the long-term AZA-PRED group but are normal in the long-term PRED-alone (no-AZA) group. The short-term AZA-PRED group exhibits NK and ADCC levels significantly lower than normal but not as low as those of the long-term AZA-PRED group. Patient groups with low NK and ADCC also have low circulating Fc receptor-bearing (FcR) cells. A single patient in the long-term AZA-PRED group was removed from AZA therapy, and approximately 3 months was required for the patient's suppressed NK and ADCC to return to normal. These findings indicate that AZA rather than PRED is the major drug important in suppressing ADCC and NK activity in renal transplant recipients. Several months are required for combination AZA-PRED therapy to reduce these cytotoxic activities. Similarly, several months are required for suppressed ADCC and NK activity to return to normal upon discontinuation of AZA.     

Impaired release of natural killer cytotoxic factor in patients with primary Sjögren''s syndrome.

The impaired natural killer (NK) cell activity against K562 target cells of patients with primary Sjögren''s syndrome (primary SS) was re-examined in a 2-year follow-up study of 10 patients and 10 normal controls. The ability of blood mononuclear cells (BMNC) to form effector/target cell conjugates and to release NK cytotoxic factor (NKCF) were studied. NK cell activity of the patients was unchanged low (P less than 0.01) compared with the controls. The number of effector/target cell conjugates did not differ between patients and controls, whereas NKCF-release from interferon-stimulated BMNC was significantly (P less than 0.01) reduced in the patients with primary SS and positively correlated to the reduced NK cell activity (r = 0.85, P = 0.0002). The permanently low NK cell activity of patients with primary SS appears therefore, at least in part, to be due to an impaired release of NKCF and not to a defective ability of effector cells to recognize and/or adhere to target cells.     

Concomitant Effect of 2''-Deoxycoformycin on Natural Killer Cell Activity and Tumour Cell Sensitivity to Lysis in Hairy Cell Leukaemia—Discordant Effects of Alpha Interferon

The effects of 2'-deoxycoformycin (dCF) and alpha interferon (IFN-alpha) on natural killer (NK) cell-enriched fractions and hairy cell (HC) targets from three patients with HC leukaemia (HCL) were investigated. There was no significant increase in NK activity when either the HC targets or NK-enriched cells were preincubated with dCF. However, preincubation of both HC and NK cells with dCF resulted in increased NK activity. Culture of enriched NK cells with IFN-alpha enhanced their activity. However, preincubation of HC targets with this drug in the presence or absence of dCF resulted in a protective effect. Maximal NK activity towards HCL was obtained when the target tumour cells were separately precultured with dCF and the NK-enriched effectors precultured with dCF + IFN-alpha. The effect of dCF and IFN-alpha was also measured using the standard K562 cells as targets for NK activity. dCF enhanced NK activity following preculture of both effector and target K562 cells, but IFN-alpha did not reduce K562 cell susceptibility to NK lysis as it did for HC cells. Our findings suggest that (a) dCF and IFN-alpha, which are used to treat HC, could function via activation of NK cells, (b) effects on both effector and tumour target cells should be taken into account, and (c) caution should be exercised in extrapolating the effects of NK-cell activity against K562 cells to those on HC targets.     

Lack of effect of LTB4 on human natural killer cell activity and T lymphocyte transformation

Human natural killer (NK) cell activity was not significantly affected by leukotriene (LT) B4 over a wide concentration range (10(-6)-10(-14) M), whether added directly to the NK cell assay or after preincubation of effector cells for 2 h with the drug before addition of Cr labelled K562 target cells. In addition, LTB4 did not affect the kinetics of NK cell mediated cytotoxicity. Addition of LTB4 (10(-6)-10(-10) M) to concanavalin A (Con A) or phytohaemagglutinin (PHA)-stimulated human peripheral blood mononuclear cells (PBMC) had no significant effect on proliferation measured by tritiated thymidine incorporation, using both optimal and sub-optimal mitogen concentrations. Whilst it is clear that LTB4 is an important mediator of inflammation involving polymorphonuclear (PMN) leukocytes in vivo, it does not affect NK cell or T cell function in vitro.     

Autoantibodies as Predictive and Diagnostic Markers of Idiopathic Inflammatory Myopathies

The purpose of the present study was to determine whether patients with euthyroid Graves’ exophthal-mopathy have an impaired NK cell function compared to patients with Graves’ hyperthyroidism and healthy controls.

The NK cell activity measured against K562 target cells was significantly suppressed (p < 0.01) in patients with euthyroid Graves’ ophthalmopathy, whereas the NK cell activity of patients with Graves’ hyperthyroidism was not. Although interferon-α, interleukin-2 and indomethacin significantly enhanced (p < 0.01) the NK cell activity in all three groups, none of these agents fully restored the defective NK cell activity in euthyroid Graves’ ophthalmopathy. The concentrations in the blood of large granular lymphocytes and CD 16 positive cells did not differ between the three groups, furthermore an immunosuppressive serum factor was not detected. The number of effector/target cell conjugates did not differ between patients and controls, whereas the interferon-α -induced production of a soluble natural killer cytotoxic factor (NKCF) with specificity for NK sensitive target cells was suppressed in patients with Graves’ euthyroid ophthalmopathy. We conclude that one of the mechanisms underlying the defective NK cell activity in patients with euthyroid ophthalmopathy may be an impairment of the release of NKCF from the NK cells.     

Suppressed natural killer cell activity in patients with euthyroid Graves' ophthalmopathy

The purpose of the present study was to determine whether patients with euthyroid Graves' exophthalmopathy have an impaired NK cell function compared to patients with Graves' hyperthyroidism and healthy controls. The NK cell activity measured against K562 target cells was significantly suppressed (p less than 0.01) in patients with euthyroid Graves' ophthalmopathy, whereas the NK cell activity of patients with Graves' hyperthyroidism was not. Although interferon-alpha, interleukin-2 and indomethacin significantly enhanced (p less than 0.01) the NK cell activity in all three groups, none of these agents fully restored the defective NK cell activity in euthyroid Graves' ophthalmopathy. The concentrations in the blood of large granular lymphocytes and CD16 positive cells did not differ between the three groups, furthermore an immunosuppressive serum factor was not detected. The number of effector/target cell conjugates did not differ between patients and controls, whereas the interferon-alpha induced production of a soluble natural killer cytotoxic factor (NKCF) with specificity for NK sensitive target cells was suppressed in patients with Graves' euthyroid ophthalmopathy. We conclude that one of the mechanisms underlying the defective NK cell activity in patients with euthyroid ophthalmopathy may be an impairment of the release of NKCF from the NK cells.     

Immunoregulatory effects of sizofiran (SPG) on lymphocytes and polymorphonuclear leukocytes.

The effect of SPG on leukocytes has been studied in 20 patients with oral carcinoma and the actions have been analysed in vitro. SPG 1 mg/kg was administered intramuscularly twice weekly. Peripheral venous blood was collected before, and 1 week and 2 weeks after the initiation of SPG treatment. Both CD16+CD57- and CD16-CD57+ cell populations were significantly increased after treatment, but no T cell subset varied. While enhancement of lymphokine-activated killer activity could not be found, an increase in natural killer (NK) activity was observed in 15 of the subjects, and the mean NK level was significantly increased from an initial 34.7 +/- 18.7% to 46.4 +/- 16.5% after two weeks of injections. O2-production by polymorphonuclear leukocytes (PMNL) was stimulated 6 h after SPG injection. When PMNL were treated in vitro with SPG 32 micrograms/ml, enhanced O2-generation was induced and protein kinase C (PKC) activity in a membrane fraction increased. SPG did not directly affect non-specific PMNL killing of K562 cells or antibody-dependent cell mediated cytotoxicity against Raji cells, but non-specific PMNL killing was enhanced by culture-conditioned medium from peripheral blood mononuclear cells (PBMC) containing 10 micrograms/ml SPG. Interleukin-1 beta, -3, -4, -6, tumour necrosis factor-alpha, granulocyte-macrophage colony stimulating factor and IFN-gamma levels in the conditioned medium were not increased compared with medium from PBMC not treated with SPG. No clear increase of these cytokines was found in serum from the SPG-treated patients. From the above results, enhancement of PMNL O2-generation by SPG seems to be a direct action of SPG, but the mechanism of elevation of the non-specific killing activity of PMNL and NK cells is not known. Perhaps other cytokines than those assayed have participated in increasing non-specific cytotoxicity.     

Suppressed natural killer cell activity during episodes of erythema nodosum leprosum in lepromatous leprosy.

Natural killer (NK) cell activity was investigated in peripheral blood mononuclear cells of patients with leprosy. The NK activity of patients with borderline or lepromatous leprosy did not differ significantly from that of normal subjects. However, in a group of patients with lepromatous leprosy undergoing an episode of erythema nodosum leprosum (ENL), NK activity was significantly depressed. In four patients with ENL, NK activity was virtually abolished. Depressed NK activity could not be attributed to the effects of corticosteroid therapy, nor did a serum factor appear to be responsible. Evidence was obtained that depressed NK activity in patients with ENL was not due to dysfunction of the NK cells themselves; they functioned normally when separated from the individual's total mononuclear cell population. Additional cell depletion studies suggested that the patients' monocytes were responsible for the observed depression of NK activity.     

Prevention and reversal of delta-9-tetrahydrocannabinol induced depression of natural killer cell activity by interleukin-2

The effects of delta-9-tetrahydrocannabinol (THC) on NK cell activity were studied. Previously, we reported that incubation of human peripheral blood mononuclear cells in THC resulted in an inhibition of natural killer (NK) cell activity. The present study examined the mechanism(s) of the decrease in NK cell activity. The inhibition of killing by NK cells was not due to a failure of NK cells to bind to K562 target cells. Furthermore, indomethacin did not abrogate the THC-mediated effect, suggesting that prostaglandins are not involved in the process leading to suppression of NK cell activity. However, NK activity was partially restored if cells, pretreated with THC, were washed to remove excess drug and then incubated overnight in fresh medium before assay. Addition of 1-100 U IL-2, either during pretreatment with THC or during overnight incubation, precluded or promoted the reversal of the inhibition of NK cell cytotoxicity. We conclude that the regulatory mechanism(s) involved in depression of NK cell cytotoxicity by THC is significantly influenced by IL-2.     

Natural killer activity and antibody-dependent cellular cytotoxicity in patients with adult T-cell leukemia

Natural killer (NK) activity of peripheral mononuclear cells (PMNC) from patients with adult T-cell leukemia (ATL), anti-HTLV-I antibody positive healthy carriers, and anti-HTLV-I antibody negative healthy persons (Ab-negative persons) was investigated using various target cells. PMNC from patients with ATL and healthy carriers had reduced NK activity against the NK-sensitive non-HTLV-I producing target cells, compared with the controls. In contrast, PMNC from patients with ATL, healthy carriers, and Ab-negative persons did not exhibit significant NK cell lysis against HTLV-I producing cells. However, one Ab-negative person exhibited increased NK cell lysis against an HTLV-I producing target cell. The effector cells involved in this enhanced cytolysis were found to be CD3+, HNK-1+, and CD8+. HTLV-I producing cells were lysed by PMNC from Ab-negative persons in the presence of anti-HTLV-I antibody (antibody-dependent cellular cytotoxicity; ADCC). The efficiency did not show significant difference between antibodies from patients with ATL and those from healthy carriers. The ADCC was specific to HTLV-I producing cells. PMNC from one patient with ATL in remission exhibited increased ADCC in the presence of autologous serum against HTLV-I, whereas PMNC from a patient with ATL or a healthy carrier did not exhibit ADCC. These results indicated that NK and K cells influence the immunological surveillance against HTLV-I infection or leukemic cells.     

Normal natural killer cell activity in Hodgkin''s disease patients in remission.

Patients with untreated active Hodgkin's disease (HD) have a defect in cell-mediated immunity. After therapy many HD patients still have long lasting abnormalities in T cell number and function. We examined whether NK activity is also permanently impaired in HD patients in remission. The mean NK activity of peripheral blood lymphocytes from 42 patients who were disease-free for 6-150 months was not different from that of healthy controls. Augmentation of NK activity after treatment of the cells by interferon in vitro was equal for patients and controls. Normal NK activity in HD in remission was independent of disease stage, age, remission duration and mode of therapy. Measuring PHA-induced lymphocyte proliferation and NK activity simultaneously demonstrates that patients with impaired cell-mediated immunity do not have concomitant reduction of NK activity. We conclude that NK activity in HD in remission is independent of decreased T cell mediated immunity. In addition, NK is resistant to long term suppression by the chemotherapy and radiation protocols that are used in HD.     

Immunological reconstitution after bone marrow transplant with Campath-1 treated bone marrow.

Immunological reconstitution after allogeneic bone marrow transplant (BMT) was studied in 20 patients who received Campath-1 treated bone marrow. The peripheral blood lymphocyte phenotype was analysed with a panel of monoclonal antibodies at 3, 6 and 12 months. T cell proliferative capacity was evaluated by stimulation with PHA and Con A and in the mixed lymphocyte reaction (MLR). Natural killer (NK) cell activity was analysed against the K562 cell line at specified times after BMT in nine patients. Absolute numbers of T lymphocytes were reduced in all patients at 3 and 6 months. A marked decrease in the number of CD4+ cells persisted beyond 12 months. CD8+ cells regenerated more rapidly and reached normal at 6 months. No correlation was found between changes in lymphocyte subpopulations and the presence of graft-versus-host disease or cytomegalovirus infection. B cells recovered rapidly and maintained normal numbers throughout the study. A moderate increase in HNK1+ (Leu7) cells was observed at 3 and 6 months simultaneously with a low expression of NK15 (Leu11) and OKM1 antigens at 3 and 6 months, suggesting the presence of immature NK cells early after the transplant. A profound decrease of T cell proliferative capacity was observed both after mitogen stimulation and in the mixed lymphocyte reaction. NK cell activity was raised during the first month after transplant in all but one patient but no correlation was found with the presence of GVHD or cell marker analysis.     

Difference in effect of single immunosuppressive agents (cyclophosphamide, CCNU, 5-FU) on peripheral blood immune cell parameters and central nervous system immunoglobulin synthesis rate in patients with multiple sclerosis.

Cyclophosphamide (CY), 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) and 5-fluorouracil (5-FU) were given in single course schedules to chronic progressive multiple sclerosis (MS) patients clinically stable for 6 months. The following peripheral immune cellular parameters were measured before, during and after each drug administration: white blood count (WBC), polymorphonuclear count (PMN), lymphocyte count, percentage of T cells, T cell response to phytohaemagglutinin (PHA), percentage of B cells, percentage of cells bearing receptors for the Fc portion of immunoglobulin (% FcR cells), killer (K) cell activity defined by antibody-dependent cellular cytotoxicity (ADCC), and natural killer (NK) cell activity. Central nervous system (CNS) immunoglobulin G (IgG) synthesis was also measured. The patients were followed carefully by both quantitative and qualitative methods for any change in their neurologic condition. Selective reduction in NK activity was observed with CY and 5-FU while no significant alteration was seen in %FcR cells and K activity. CY differed from 5-FU in reducing lymphocyte count and B cell percentage while 5-FU decreased the percentage of T cells. CCNU, but not the other drugs, reduced T cell proliferative response to PHA. In addition, CCNU, which is known to penetrate well into the nervous system, caused a modest reduction in CNS IgG synthesis, while 5-FU had an uncertain effect. Clinically the patients were unchanged or continued to progress in their disability. The results suggest an independence of the CNS immune from the systemic immune system in MS in response to many immunosuppressive drugs.     

Characterization of the in vivo and in vitro Effects of Indomethacin on Human Natural Killer Cell Activity

The in vivo and in vitro effects of indomethacin on the natural killer (NK) cell activity against K 562 target cells were studied. In vivo administration of indomethacin, 3 X 50 mg for 7 days to normal donors did not influence baseline NK cell activity, which means that treatment with prostaglandin (PG) inhibitors can be allowed in studies on NK cell activity of persons with normal PG production. The NK cell activity of fresh mononuclear cells was boosted with pharmacological concentrations of indomethacin in vitro, while frozen cells were not. Our results indicate that indomethacin enhances the NK cell activity in vitro by blocking the prostaglandin production of monocytes, since monocyte depleted effector cells were not boosted by indomethacin.     

Dissociation of responses measured by natural cytotoxicity and chemiluminescence

Monocyte/macrophage-mediated cytotoxicity requires the generation of activated oxygen radicals, which can be measured by chemiluminescence (CL). To investigate whether natural killer (NK) cell activity required activated oxygen species, both cytotoxicity against K562 target cells and CL were measured in cell populations of human peripheral blood. The following results were obtained: (a) Peripheral blood mononuclear cells (MNC) showed NK activity and a response in CL, which could be induced by viable or paraformaldehyde-fixed K562 target cells as well as by latex particles. (b) Both T cells and non-T cells exhibited NK activity, but T cells gave no K562- or latex-induced CL responses. (c) Depletion of phagocytic cells from MNC abolished CL, but only marginally affected NK activity. (d) Reconstitution of phagocyte-depleted MNC with adherent cells revealed a superadditive enhanced CL response, but had no augmenting effect on NK activity. (e) Phagocyte-depleted cell populations, enriched for NK activity by density gradient centrifugation, did not respond in K562- and latex-induced CL. (f) MNC, highly enriched for NK activity by cell sorting with a cytofluorograf using the fluorescein isothiocyanate-labeled monoclonal antibody anti-Leu-11a, responded only with reduced CL, whereas the NK activity was enriched up to 45-fold. From these results it is concluded that NK cell-mediated cytolysis of K562 target cells and K562-induced CL are not functionally correlated, but represent properties of two distinct cell populations, namely NK cells and monocytes.     

NK cell activity and skin test antigen stimulation of NK like CMC in vitro are decreased to different degrees in pregnancy and sarcoidosis.

Peripheral blood mononuclear cells (PBMNC) isolated from normal subjects, pregnant women and patients with sarcoidosis were assayed for natural killer (NK) cell activity on day 0 and for NK like cell-mediated cytolysis (CMC) after 5 days of exposure, in vitro to Candida antigen, purified protein derivative (PPD), and human leucocyte interferon (IFN). Pregnant women and women with sarcoidosis had significantly decreased levels of NK cell activity compared to normal women. Pregnant women had the lowest mean NK cell activity. Cells from women with sarcoidosis and from pregnant women also had lower levels of killing than those from the normal women after in vitro stimulation of NK like CMC with Candida antigen, PPD and IFN. The lowest stimulations of NK like killing occurred in the cells from women with sarcoidosis. Skin test antigen stimulation of NK like CMC in vitro and the DTH response in vivo were strongly correlated for both Candida antigen and PPD in the sarcoidosis patients. There was no correlation between the level of NK cell activity in the PBMNC of sarcoid patients on day 0 and the amount of NK like CMC that was present in cells from those patients after 5 days of culture with Candida antigen, PPD or IFN. A significant correlation was found, however, between Candida antigen stimulation of NK like CMC and IFN stimulation of NK like CMC in both pregnant and sarcoid groups. Reduced NK cell activity on day 0 in a given patient thus did not necessarily indicate that skin test antigen or IFN stimulation of NK like CMC on day 5 would also be depressed. In addition, NK cell activity was often noted to be normal in patients with depressed in vitro stimulation of NK like CMC.     

Measurement of NK activity in whole blood by the CD69 up-regulation after co-incubation with K562, comparison with NK cytotoxicity assays and CD107a degranulation assay

In present study human peripheral blood NK cell activation after co-incubation with K569 cell line was investigated by CD69 expression. NK lytic activity was studied by two different assays: TDA (2,2':6',2″-terpyridine-6,6″-dicarboxylate) release assay (TRA) and flow cytometry assay (FCA) that display two approach to cytotoxicity measurement. We also investigated NK cell degranulation activity by estimation of CD107a (LAMPa) expression. Comparison of specific lysis value measured by both cytotoxicity assays showed high correlation coefficient between two methods (r=0.94447). Specific lysis value correlated significantly with CD69+ NK frequency and NK degranulation activity. We show that lymphocyte incubation with K562 results to increase CD69 expression on NK and NKT but not on T lymphocytes. Only a part of peripheral blood NK cells became CD69 positive after incubation with excess of K562 cells. CD69+ NK cell frequencies did not increase after elevation of K562/NK ratio or incubation period that confirmed existence of subset of NK cells able to response to K562. CD69 elevation on NK significantly correlated with NK cytotoxicity (r=0.726). CD69 increases were similar when whole blood or isolated PBMC was used in assay. We also found different capacity to activation in NK subsets that express CD62L at various densities. The results demonstrated that K562 induced CD69 expression displays NK lymphocyte functional condition that associated with cytotoxic function.     

Depression and reduced natural killer cytotoxicity: a longitudinal study of depressed patients and control subjects.

Cross-sectional studies have demonstrated that natural killer (NK) cell activity is reduced in depression. To extend these observations and examine further the association between severity of depressive symptoms and values of NK activity, this study used a longitudinal case-control design and assessed NK cytotoxicity at intake and at follow-up 6 months after discharge from the hospital in depressed patients and control subjects. From acute hospitalization to follow-up, depression scores significantly (P < 0.01) decreased following treatment in the depressed patients but did not change in the control subjects. NK activity significantly (P < 0.05) increased from intake to follow-up in the depressives while lytic activity did not change in the controls. At intake NK activity was significantly (P < 0.01) reduced in the depressed patients as compared to values in the controls, while at follow-up cytotoxicity was similar between the two groups. These longitudinal data suggest that a reduction of NK cytotoxicity is temporally associated with the state of acute depression.